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    Genetic Diversity Analysis of Winter Wheat Germplasm Resources in Shanxi Province Based on 55K SNP Array
    LEI MengLin, LIU Xia, WANG YanZhen, CUI GuoQing, MU ZhiXin, LIU LongLong, LI Xin, LU LaHu, LI XiaoLi, ZHANG XiaoJun
    Scientia Agricultura Sinica    2024, 57 (10): 1845-1856.   DOI: 10.3864/j.issn.0578-1752.2024.10.001
    Abstract23)   HTML2)    PDF (2921KB)(13)       Save

    【Objective】 Analyzing the evolutionary patterns of genetic diversity of wheat germplasm resources, providing richer and more diverse original parental materials for parental selection and variety selection in wheat breeding in Shanxi Province.【Method】 Using 323 landraces and 105 cultivated varieties as natural populations, a 55K SNP chip was used to perform whole genome scanning on 428 natural populations, analyzing genetic diversity, genetic structure, principal components, genetic clustering, and phylogenetic relationships among varieties. 【Result】 The distribution of SNP loci on 21 chromosomes ranged from 329 to 1 639, with an average of 1 152. The distribution range of 7 partially homologous groups is 2 154-3 852, with an average of approximately 3 456. The distribution pattern of the genome is: B genome>A genome>D genome. Genomic annotation polymorphism markers have the highest distribution among gene regions, accounting for about 50%. Analysis shows that SNP loci cover 21 chromosomes, 7 homologous groups and 3 genomes, but their distribution varies, with a polymorphism rate of 45.60%. The average observed heterozygosity of the entire population (0.0185) was lower than the expected heterozygosity (0.4992). The changes in the average shannon wiener index and polymorphism information content of the entire natural population were not significant. Comparing the diversity parameters of natural populations, it was found that the genetic diversity of the population is not high, the genetic diversity of cultivated varieties is slightly higher than that landraces. The population structure analysis of natural populations divides the population into two major groups. Group I has 307 materials, mainly landraces. Group Ⅱ has 121 materials, mainly cultivated varieties. The natural groups were divided into five groups by both principal component and cluster analysis. The average genetic distance between the varieties in group I is 0.21831, with a range of 0.00127-0.72461. The average genetic distance between varieties in group Ⅱ is 0.14619, with a range of 0.00038-0.76489. The varieties in group Ⅲ the average genetic distance between the varieties of group Ⅳ is 0.16521, with a range of 0.00049-0.43033. The average genetic distance between varieties of group Ⅳ is 0.17643, with a range of 0.00118-0.60496. The average genetic distance between varieties of group V is 0.12039, with a range of 0.00042-0.37032. It can be seen that the variation of genetic distance between wheat varieties is large in Shanxi Province. However, the average genetic distance value is low, the clustering classification differentiation is obvious. The genetic relationship between varieties in the middle of the group is relatively close. Comparison shows that the average genetic distance of group I and group Ⅳ is higher than that of group Ⅱ, group Ⅲ and group V. The genetic distance variation of group I and group Ⅳ is higher than that of group Ⅲ and group V. It can be seen that the genetic distance of cultivated varieties is generally greater than that of landraces.【Conclusion】 The 55K SNP chip was used to analyze the genetic diversity of Shanxi winter wheat germplasm resources, clarifying the distribution characteristics of genetic diversity at the genomic level between Shanxi wheat cultivated varieties and landraces. The introduction of exogenous genes into cultivated varieties is beneficial for improving genetic diversity, while the genetic diversity of landraces is relatively low. At the same time, the genetic relationships of very few varieties are polarized, so it should be rationally used differently in subsequent utilization.

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    Evaluation of Drought Resistance in Soybean Germplasm and Identification of Candidate Drought-Resistant Genes
    LI ShengYou, WANG ChangLing, YAN ChunJuan, ZHANG LiJun, SUN XuGang, CAO YongQiang, WANG WenBin, SONG ShuHong
    Scientia Agricultura Sinica    2024, 57 (10): 1857-1869.   DOI: 10.3864/j.issn.0578-1752.2024.10.002
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    【Objective】 In order to provide theoretical basis for molecular breeding of soybean drought resistance, the different evaluation indexes of drought resistance were comprehensively used to screen soybean germplasm with drought-resistance, and the candidate drought-tolerant genes were identified. 【Method】 In 2018, 2019, 2020 and 2021, a total of 188 soybean germplasm were used to determine pod number per plant, biomass per plant and yield per plant under well-watered and drought stressed conditions. Drought resistance index (DI), improved drought resistance index (IDI), weighted drought resistance coefficient (WDC) and weighted drought resistance index (WDI) were used to identify drought resistance of soybean germplasm. The single nucleotide polymorphisms (SNP) loci significantly associated with these parameters were detected by genome-wide association study (GWAS), and the candidate genes for drought resistance were screened by RNA-seq and qRT-PCR analysis of soybean seedling leaves under drought stress. 【Result】 The DI, IDI, WDC and WDI of 188 soybean germplasm varied widely, and five classification criteria for each drought resistance parameter were determined by hierarchical classification method. Among them, Liaodou 15, Liaodou 69, Liaodou 14, Jinzhangzi Huangdou, Zhonghuang 606, Kexin 3 and Koreane 4 were identified as first-grade drought resistant by all evaluation methods. By using GWAS for DI, IDI, WDC and WDI, a total of 15 significantly SNP loci were detected under multiple environments, and the contribution rate of these loci to phenotypic variation ranged from 12.46% to 25.60%. There are 226 annotated genes within 200 kb intervals of upstream and downstream for the significant SNP loci. According to RNA-seq and qRT-PCR analysis of drought-resistant cultivar Liaodou 14 and drought-sensitive cultivar Liaodou 21 under drought stress, a total of 32 annotated genes were significantly differentially expressed by drought stress. Among them, eight genes including Glyma.02G182900, Glyma.04G012400, Glyma.06G258900, Glyma.15G100900, Glyma.01G172600, Glyma.04G012300, Glyma.01G172200 and Glyma.04G010300, encodes calcium-dependent protein kinase, universal stress protein A-like protein, G-type lectin S-receptor-like serine/threonine-protein kinase, protein phosphatase 2C, isoflavone reductase, isoflavone reductase homolog, auxin-like protein, and bZIP transcription factor, respectively. 【Conclusion】 Seven germplasm were identified from 188 soybean germplasm by comprehensive application of different drought tolerance parameters. A total of 15 SNP loci significantly associated with drought tolerance parameters were identified by GWAS, and eight candidate genes were identified.

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    Development and Identification of an Interspecific Hexaploid Hybrid Between an A. hypogaea Cultivar and a Wild Species Arachis sp. 30119 in Peanut
    LIU Hua, ZENG FanPei, WANG Qian, CHEN GuoQuan, MIAO LiJuan, QIN Li, HAN SuoYi, DONG WenZhao, DU Pei, ZHANG XinYou
    Scientia Agricultura Sinica    2024, 57 (10): 1870-1881.   DOI: 10.3864/j.issn.0578-1752.2024.10.003
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    【Objective】 Wild Arachis species containing many elite disease and insect resistant genes are an important gene pool for the improvement of cultivated peanut A. hypogaea L., so the introduction of chromatin from wild species into cultivated peanut remains an important task for distant hybridization of peanut. Unfortunately, only a few wild species have been successfully involved in the development of interspecific hybrids, despite the existence of a huge amount of wild germplasm resource. The wild species A. sp. 30119 contains multiple disease resistance. However, no interspecific hybrid between it and the cultivated peanut has been reported.【Method】 After crossing allotetraploid peanut cultivar Baitu 131 with diploid wild species A. sp. 30119 and followed by the embryo rescue, the interspecific hybrid F1 (W1212) was obtained. However, interspecific hybrid F1 did not produce seed when selfed. In order to reveal the reason for infertility and continue the generation, the chromosome number of root tip cells was counted and chromosome pairing in pollen mother cells during meiosis of W1212 observed. The chromosome of W1212 was doubled through colchicine treatment during the in vitro culture W1212 plantlet. Finally, we harvested four one-seeded pods from the established W1212 plants; one of the pods undergone in vitro cultured and a number of plants were established from the culture and named as Am1212. The chromosome composition of Am1212 was analyzed by sequential GISH/FISH and SSR marker. Additionally, phenotypic characteristics of Am1212 were investigated. The metaphase chromosome numbers of 8 randomly selected F3 plants were analyzed by rDNA FISH to evaluate the genetic stability of Am1212.【Result】 The average chromosome configuration during meiosis of pollen mother cells of W1212 was 1Ⅲ+6Ⅱ+15Ⅰ, and the abnormalities in chromosome pairings lead to the high sterility of F1 plants. The pollen viability of peg-setting branches of W1212 was significantly improved after chromosome doubling treatment. Sequential GISH / FISH of Baitu 131, A. sp. 30119 and Am1212 indicated that A. sp. 30119 may be a diploid with A genome. Am1212 has 60 chromosomes consisting of all chromosomes of both Baitu 131 and A. sp. 30119, which confirmed its nature of a hexaploid hybrid, but 37.5% F3 plants derived from Am1212 exhibited chromosome number variation. After conducting the screening of SSR molecular markers and phenotypic investigation, we obtained 17 dominant or co-dominant markers for specifically tracking wild species chromosomes and clarified the genetic characteristics of Am1212.【Conclusion】 In this study, we successfully created a new hexaploid peanut Am1212 which incorporated A. sp. 30119 chromatin. However, Am1212 exhibited instability in chromosome numbers and inherited unacceptable agronomic traits from wild species, such as small pods. Therefore, it is necessary to develop more accurate and efficient chromosome manipulation techniques to minimize the unfavorable gene linkage and generate alien chromosomal lines that possess compensatory effects and favorable traits for future breeding purposes.

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    Genetic Analysis and Candidate Gene Identification on Fertility and Inheritance of Hybrid Sterility of XI and GJ Cross
    XU Na, TANG Ying, XU ZhengJin, SUN Jian, XU Quan
    Scientia Agricultura Sinica    2024, 57 (8): 1417-1429.   DOI: 10.3864/j.issn.0578-1752.2024.08.001
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    【Objective】The F1 hybrid sterility between XI/indica and GJ/japonica severely hinders the utilization of hybrid advantage between subspecies. Exploring the genetic mechanism and identifying new regulatory genes for XI/GJ hybrid sterility will provide theoretical basis for promoting genetic improvement of XI/GJ hybrid seed setting rate. 【Method】A series of stable genetic recombination inbred lines (RILs) containing 95 plant lines were derived from the cross between XI variety Habataki and GJ variety Sasanishiki after 10 generations inbred using single seed descent method. High throughput sequencing was performed on both parents and RILs on the Illumina platform, and the distribution of Habataki pedigree in RILs was analyzed at the whole genome level. The segregation distortion regions were identified, and hybrid sterile related gene loci were screened within the segregation distortion regions, then identified candidate genes through sequence alignment comparison. The targeted gene was knockout to verify the function using CRISPR gene editing technology. 【Result】The hybrid F1 plants derived from the cross between Habataki and Sasanishiki showed significant heterosis in panicles, grains per panicle, and thousand grain weight, but its seed setting rate significantly decreased. I2-KI microscopy revealed a significant decrease in F1 pollen fertility. High throughput sequencing of the entire genome of RILs revealed significant segregation distortion on Chr.1, Chr.3, Chr.5, Chr.6, Chr.7, and Chr.12, indicating that the genotype in this region tends towards the Habataki. Sequence alignment comparison revealed that Sc, S5, and HSA1 are target genes for the segregation distortion on Chr.3, Chr.6, and Chr.12. The CRISPR gene editing mutants with a knock-out Sc-Haba-3 allele in Habataki successfully improved the pollen fertility and seed setting rate of F1 hybrid with Sasanishiki. A complex structural variation was found between Sasanishiki and Habataki in the segregation distortion of Chr.1. A 24.7 kb segment containing 4 predicted genes in the Sasanishiki genome was replaced by a 64.8 kb segment containing 10 predicted genes in Habataki, the structural variation may involve in controlling the hybrid sterility of XI and GJ cross. 【Conclusion】This study detected multiple XI/GJ hybrid infertility related loci, and successfully improved F1 fertility by using CRISPR gene editing to knock out multiple copies of Sc in Habataki, locking in the target gene in the Sd region of Chr.1.

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    Construction of SSR Fingerprint Library and Comprehensive Evaluation for Approved Cotton Varieties in China
    WU YuZhen, HUANG LongYu, ZHOU DaYun, HUANG YiWen, FU ShouYang, PENG Jun, KUANG Meng
    Scientia Agricultura Sinica    2024, 57 (8): 1430-1443.   DOI: 10.3864/j.issn.0578-1752.2024.08.002
    Abstract79)   HTML13)    PDF (2278KB)(65)       Save

    【Objective】Cotton, a heterotetraploid crop with a complex genome structure, faces challenges in achieving high homozygosity due to frequent cross-pollination. The absence of effective technical supervision in the cotton seed market and the persistence of disordered varieties have a negative impact on the consistency of fiber quality. The objectives of this study are threefold: to establish a DNA fingerprint database for approved cotton varieties in China over the past 20 years, to explore a high-throughput SSR identification model for cotton varieties, and to provide a basis for the authentication of existing varieties and the specific identification of new cotton varieties. Additionally, we aim to analyze the genetic diversity and population differentiation among approved varieties. Ultimately, our goal is to provide a theoretical framework for identifying cotton varieties that are well-suited to different ecological regions and for developing varieties that can adapt to new environments. 【Method】Based on multiplex PCR technology and capillary electrophoresis detection method, using 60 SSR markers screened to construct a DNA fingerprint library of 1 015 standard samples of cotton approved varieties. Through the plant variety DNA fingerprint library management system, the SSR fingerprints of approved varieties were compared pairwise to analyze the genetic differences of approved varieties and screen the core SSR loci for variety identification. Cluster analysis and population structure analysis were used to analyze the genetic diversity of 1 015 cotton approved varieties and calculate the genetic differentiation index between populations. 【Result】60 SSR markers amplified 216 allelic variations in 1 015 approved varieties, with an average of 3.6 allelic variations and a mean PIC value of 0.37. When the SSR fingerprints of the 1 015 approved varieties were compared, a total of 513 591 pairwise results were generated, with a maximum of 58 different loci between samples. The percentage of different loci was mainly concentrated at 41%-70%, involving 428 115 groups, accounting for 83.36%. Among them, when the percentage of different loci was at 51%-60%, the largest number of groups was involved, accounting for 197 829 groups, accounting for 38.52%. When the percentage of different loci between varieties was greater than 20%, it accounted for more than 99% of all pairwise comparison groups, and the pairwise comparison results with a percentage of different loci lower than 20% only accounted for 0.58%. Based on the combination identification method, a set of cores SSR loci containing 10 SSR loci was selected, and the discrimination ability among the 1 015 varieties reached 99%. Clustering results and population structure analysis showed that the 1 015 varieties were clearly divided into five subpopulations. G1 (n=240) was an early-maturing cotton subpopulation, mainly distributed in northern and inland regions of China. This subpopulation had the most abundant genetic diversity among varieties, with an average genetic distance of 0.419 between varieties. G2 (n=277) was a medium-maturing cotton subpopulation, distributed in the Yangtze River Basin. This subpopulation had more hybrids, with an average genetic distance of 0.309 within the subpopulation. G3 (n=109) belonged to early-maturing and medium-maturing cotton subpopulations, distributed in Hebei'sHeilonggang region. This subpopulation had relatively simple genetic components, with the smallest average genetic distance among upland cotton subpopulations at only 0.150. G4 (n=254) belonged to a medium-early maturing cotton subpopulation, mainly distributed in the Yellow River Basin. The average genetic distance within this subpopulation was 0.307. G5 (n=37) consisted of 37 sea island cotton samples, with the smallest average genetic distance within the population at only 0.149. The genetic differentiation level between sea island cotton and upland cotton was the highest, with an average FST value of 0.503. Among upland cotton populations, the genetic differentiation level between G3 and other subpopulations was the highest, with FST values ranging from 0.193 to 0.242. The genetic differentiation level between the Yangtze River Basin and the Yellow River Basin was the lowest, with an FST value of 0.112. 【Conclusion】A DNA fingerprint library of standard samples of 1 015 approved varieties in China over the past 20 years was constructed. A set of cores SSR loci containing 10 SSR loci was selected to clearly identify more than 99% of the varieties. A high-throughput cotton identification model of "core loci + extended loci" was created. The 1 015 varieties were divided into five subpopulations, and upland cotton had obvious geographical distribution characteristics.

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    Effect of 14-Hydroxylated Brassinosteroids Growth Regulator on Growth and Yield of Rapeseed
    HE YongQiang, ZHANG JinKui, XU JinSong, DING XiaoYu, CHENG Yong, XU BenBo, ZHANG XueKun
    Scientia Agricultura Sinica    2024, 57 (8): 1444-1454.   DOI: 10.3864/j.issn.0578-1752.2024.08.003
    Abstract85)   HTML12)    PDF (740KB)(81)       Save

    【Objective】As a new Plant growth regulator, 14-hydroxylated brassinosteroids(14-HBR) increase biological activity by 50% than the traditional Brassicin sterols, while the relation effect of 14-HBR on rapeseed growth, yield and seed coating pesticide were not clear. 【Method】In this study, 14-HBR regulator and pesticide are used to treat the seeds in mid-duration winter rapeseed variety (Yangguang 2009) and early-duration variety (Yangguang 131), investigated germination, seedling growth, insect resistance, and yield, to reveal the interaction effects of environment, variety genotype and 14-HBR. 【Result】0.0075 and 0.015 mg·L-1 14-HBR treated seed increased significantly germination potential in medium duration rapeseed, but decreased germination potential and germination rate significantly as mixed with pesticide. 14-HBR treated seed had no significant effect on germination rate and germination potential in short duration rapeseed. The 14-HBR showed better biological activity in seedling growth and yield than that of Brassinolide, 0.0075 and 0.015 mg·L-1 Brassinolide increased by an average of 5.19% and 8.15%, 0.0075 and 0.015 mg·L-1 14-HBR increased by an average of 11.98% and 5.50%, respectively. 14-HBR mixed with seed pesticide of Clothianidin and Thiamethoxam, also increased seedlings weight and yield. The yield of Thiamethoxam and Clothianidin seed treatments increased by 4.7% and 4.6% independently. The yield of mixed with 0.0075 and 0.015 mg·L-1 14-HBR to Clothianidin increased 6.8% and 3.3%, mixed to Thiamethoxam increased by 3.5% and 8.2%, respectively. 14-HBR did not affect insecticidal activity of Thiamethoxam and Clothianidin to peach bud nymphs and phyllotreta striolata fabricius. 【Conclusion】The study revealed seed treatment with 14-HBR regulator has a positive effect on early growth of rapeseed and increased yield of rapeseed significantly, interacted with planting environment, pesticide type and varieties genotype as traditional regulators, it is necessary to optimize seed treatment technology of 14-HBR regulator to obtain higher harvest yield.

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    Screening of High-Efficient sgRNA for Targeted Knockout of GhAGL16 Gene in Cotton
    LEI JianFeng, YOU YangZi, ZHANG JinEn, DAI PeiHong, YU Li, DU ZhengYang, LI Yue, LIU XiaoDong
    Scientia Agricultura Sinica    2024, 57 (6): 1023-1033.   DOI: 10.3864/j.issn.0578-1752.2024.06.001
    Abstract198)   HTML20)    PDF (2457KB)(163)       Save

    【Objective】 As an important negative transcriptional regulator in cotton MADS-box gene family, AGL16 plays an important role in resisting drought and salt stress. Virus-induced gene editing (VIGE) was used to screen sgRNAs that knockout the cotton GhAGL16 and verify the specificity of these sgRNAs, which laid a foundation for the creation of cotton agl16 mutants.【Method】 Three sgRNAs could knockout GhAGL16 were predicted based on the actual GhAGL16 genomic sequence cloned on subgroup A and D in cotton YZ-1; Three CLCrV-AtU6-26::GhAGL16-sgRNAs vectors were constructed based on the cotton leaf crumple virus (CLCrV)-mediated VIGE system; The expression of Cas9 in Cas9 over-expression (Cas9-OE) plants was detected by qPCR to determine whether Cas9 was stably genetically expressed; Three CLCrV-AtU6-26::GhAGL16-sgRNAs vectors were transformed respectively into Cas9-OE cotton cotyledons and detected the mutations of the three targets by PCR/RE; The secondary structures of three GhAGL16-sgRNAs were analyzed by bioinformatics; Hi-TOM high-throughput sequencing was performed on mutant plants to determine the efficiency of gene editing. Meanwhile, the off-target rate of GhAGL16-sgRNA2 mutant plants were identified to detect the specificity of gene editing.【Result】 Three sgRNAs capable of simultaneously knocking out GhAGL16-A and D subgroups were successfully constructed. The detection results of Cas9 expression showed that Cas9 was stably expressed in different Cas9-OE cotton plants. PCR/RE mutation detection results showed that GhAGL16-sgRNA2 could be effectively used for the knockout of GhAGL16. Different mutation types with base deletions appeared at the target sites of cotton subgroups A and D, while GhAGL16-sgRNA1 and GhAGL16-sgRNA3 were two invalid sgRNAs. The secondary structure analysis results of three GhAGL16-sgRNAs indicated that GhAGL16-sgRNA1 and GhAGL16-sgRNA3 might have a phenomenon that the guide sequence was easy to pair with other sequences and difficult to unwind, which interfered with the recognition of the target site by the guide sequences and lead to the invalid sgRNA. To further quantify the editing efficiency of GhAGL16-sgRNA2 on GhAGL16, the mutation detection results of each Cas9-OE plant transformed with CLCrV-AtU6-26::GhAGL16-sgRNA2 showed that six of the nine Cas9-OE plants were mutated, with a mutation efficiency of 66.67%. In addition, Hi-TOM high-throughput sequencing results showed that the editing efficiency of GhAGL16-sgRNA2 for GhAGL16 was 13.69%-54.42%. The off-target identification results showed that no off-target phenomenon was detected at the four predicted off-target sites, indicating that GhAGL16-sgRNA2 not only has high gene editing efficiency, but also has specific gene editing specificity.【Conclusion】 A sgRNA that can effectively knocking out the GhAGL16 was obtained by transforming Cas9-OE cotton using the CLCrV-mediated VIGE system, providing an ideal sgRNA for creating cotton agl16 mutants.

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    Analysis of Transposable Element Associated Epigenetic Regulation under Drought in Maize
    GAO ChenXi, HAO LuYang, HU Yue, LI YongXiang, ZHANG DengFeng, LI ChunHui, SONG YanChun, SHI YunSu, WANG TianYu, LI Yu, LIU XuYang
    Scientia Agricultura Sinica    2024, 57 (6): 1034-1048.   DOI: 10.3864/j.issn.0578-1752.2024.06.002
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    【Objective】 Drought is one of the serious abiotic stresses influencing maize production worldwide. Understanding the molecular mechanisms underlying drought tolerance is of great importance in maize improvement. 【Method】 In the present study, representative maize inbred lines were selected for field drought experiment and the drought tolerance was estimated based on leaf relative water content and anthesis-silking interval. Two inbred lines with contrasting drought tolerance was used for genome resequencing and transposable element insertions were identified. The DNA methylation level of leaf and root tissues under different water treatments of the two lines were measured using Whole Genome Bisulfite Sequencing (WGBS). And the gene expression profiles of these samples were detected by RNA sequencing. The integrative atlas of transposable element insertion/deletion variants, differentially methylated regions and differentially expressed genes in the two lines were constructed. In addition, the transposable element insertion/deletion variant mediated epigenetic regulation of ZCN7, which has been conferred the drought tolerance function in our previous study, was analyzed. 【Result】 The field experiment showed inbred line H082183 showed highest drought tolerance, in which the leaf relative water content and anthesis-silking interval had no significant difference between drought and well-watered treatments. While the Lü28 displayed lowest leaf relative water content and largest anthesis-silking interval under drought. Thus, these two lines were selected for further analysis. A total of 333 754 and 333 296 transposable element insertions were identified in the genome of H082183 and Lü28, respectively. And 89 954 transposable element insertions were polymorphism between two lines. The transposable element insertions, introns and promoters showed higher CG and CHG methylation level than exons and untranslated regions. Furthermore, 41 352 differentially methylated regions were identified between H082183 and Lü28. And 60% of the differentially methylated regions were located in the transposable element insertion\deletion variants and 5 kb flanking regions. The gene expression level showed negatively correlated with CG and CHG methylation. Differentially expression analysis between H082183 and Lü28 obtained 4 196 and 3 500 differentially expressed genes in leaf and root under drought, respectively. The 19.5% and 19.7% of these differentially expressed genes were located in differentially methylated regions. Three LTR transposable element insertions were identified in the 34 kb region of ZCN7 in Lü28 but absent in the genome H082183. And the DNA methylation levels of CG and CHG in this genomic region were significantly higher in Lü28 than H082183 under both drought and well-watered environments, which conferred higher ZCN7 expression in the drought tolerant line H082183. 【Conclusion】 Our results highlight the important role of interplay of transposable element insertions, DNA methylation and gene expression under drought. And gene expression regulation mechanism of ZCN7 relied on the transposable element insertion/deletion variants mediated DNA methylation was proposed.

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    Seedling Characterization and Genetic Analysis of Low Phosphorus Tolerance in Shanxi Varieties
    WEI NaiCui, TAO JinBo, YUAN MingYang, ZHANG Yu, KAI MengXiang, QIAO Ling, WU BangBang, HAO YuQiong, ZHENG XingWei, WANG JuanLing, ZHAO JiaJia, ZHENG Jun
    Scientia Agricultura Sinica    2024, 57 (5): 831-845.   DOI: 10.3864/j.issn.0578-1752.2024.05.001
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    【Objective】In arid and semi-arid regions, the water and nutrients are scarce in the soil. The phosphorus use efficiency between different wheat genotypes varies greatly. Therefore, identification of low phosphorus-tolerant germplasm and mapping of related loci is helpful for genetic improvement of wheat. 【Method】Using 282 Shanxi wheat varieties as materials, twelve seedling morphological indicators were investigated under three phosphorus concentrations, including SDW, RDW, DW, SFW, RFW, FW, MRL, TRL, RS, RV, RD, and RN. Principal component analysis, membership function analysis, and cluster analysis were used to comprehensively evaluate the low phosphorus tolerance characteristics of different varieties at the seedling stage. On this basis, the trait evolution trend and biomass allocation at seedling stage were analyzed. At the same time, GWAS was used to identify significant loci related to the low phosphorus-related traits. 【Result】The response of different traits to low phosphorus at the seedling stage was different. Lower phosphorus concentrations led to changes in biomass allocation strategy, and shoot growth was less affected by change in phosphorus concentrations than root growth. The decrease in phosphorus concentration inhibited the growth of shoot, and SDW and SFW were significantly reduced. In contrast, low phosphorus promoted root growth, and the indicators of RDW, RFW, MRL, TRL, RV and RN increased significantly. According to the correlation analysis between D-value and morphological indicators, it was found that MRL and RD could be used as selection indicators for low phosphorus tolerance at seedling stage. Based on D-value clustering analysis, 9 low phosphorus tolerant varieties were selected, including Jinmai 46, Jinmai 61, Youmangdahongjing, Hongtumai, Hongheshang, Baikehong, Baixianmai, Huoshaotou, Baishanmai. Analysing trends in trait evolution showed that cultivars were not directly selected for their ability to tolerate low phosphorus. The ability to tolerate low phosphorus decreased first and then increased over time. Before 2010, there was a decreasing trend in the ability of varieties to tolerate low phosphorus, and after 2010, there was an increase in the ability of varieties to tolerate low phosphorus. GWAS stably detected eight loci with R2>10% in three environments, in which 1A_545074550, 2B_489279799, 6A_166899658 and 6A_273060644 were not reported previously.【Conclusion】The MRL and RD can be used as selection indicators for low phosphorus tolerance at seedling stage. A total of nine varieties were selected through comprehensive evaluation of ability in Shanxi wheat to tolerate low phosphorus during seedling stage. Association analysis detected four novel loci associated with low phosphorus tolerance on chromosomes 1A, 2B and 6A, and the results provide germplasm resources and QTL for future low phosphorus tolerance wheat breeding.

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    Current Situation of Breeding and Popularization of Short-Growth- Period Winter Rapeseed Varieties for Rice-Rice-Rapeseed Mode
    LI RongDe, HE Ping, LUO LiXia, SHI MengYa, HOU Qian, MA ZhenGuo, GUO RuiXing, CHENG HongTao
    Scientia Agricultura Sinica    2024, 57 (5): 846-854.   DOI: 10.3864/j.issn.0578-1752.2024.05.002
    Abstract248)   HTML21)    PDF (449KB)(150)       Save

    The self-sufficiency rate of edible vegetable oil is less than 31 percent in China, with a high degree of import dependence. Rapeseed is the only winter oilseed crop with a wide range of suitable planting region, and it is an important source of edible vegetable oil in China. Planting more rapeseed is an important measure to guarantee national edible oil supply security. Making full use of the winter fields in the southern double cropping rice area to promote “rice-rice-rapeseed” production is an important approach to expand the planting area of rapeseed. The area suitable for the “rice-rice-rapeseed” production mode is mainly distributed in the double cropping rice area of Hunan, Jiangxi, Guangxi and Hubei provinces in China, with a potential area of about 1.87 million hm2. According to the conditions of temperature and light resources, three suitable areas for the “rice-rice-rapeseed” production include the ample area, tightly balanced area and the constrained area. All the areas require early-maturing rapeseed varieties with a growth period of around 180 days, which are suitable for being sown in mid- to late October and harvested in mid- to late April. Among a total of 75 new rapeseed lines participated in the early-maturity group of the national rapeseed variety trials from 2013 to 2022, the average growth period ranged from 169.3 to 185.5 days, and the average yield was 1 635.90-2 228.55 kg·hm-2, with 22 varieties out yielded the check variety. There are 72 early-maturing winter oilseed rape varieties with a growth period less than 190 days were registered by the end of May 2023. These varieties are suitable to be used in the “rice-rice-rapeseed” mode, and most of them are hybrid varieties with low erucic acid and low glucosinolate quality. 11 varieties, Yangguang 131, Fengyou 730, Fengyou 320, Fengyou 847, Ganyouza 906, Shengguang 127, Xiangyou 420, Jingyou 69, Fengyou 112, Huayouza 652, Ganyouza 1009, are the most promoted and applied early-maturing winter rapeseed varieties in the “rice-rice-rapeseed” production area, with more than 135 hm2 each in 2022. The present varieties can basically meet the early-maturation demand in the ample area. In the tightly balanced and constraint areas, however, the growth period of these varieties is too long. To expand the production and efficiency of rapeseed industry, it is urgently needed to strengthen the policy and financial security in the future, carry out joint breeding projects for short-growth-period winter rapeseed varieties to further improve the yield in the ample area and shorten the growth period in the tightly balanced and constrained areas. Meanwhile, to strengthen the research and promotion of supporting cultivation technology for elite varieties, match well early rice, late rice, and rapeseed varieties are also good measures to support the expansion of rapeseed production in the “rice-rice-rapeseed” production area. In addition, improving agricultural socialized services, expanding agricultural insurance and increasing subsidies for rapeseed planting to ensure production benefit will enhance the farmers’ enthusiasm for the “rice-rice-rapeseed” production.

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    Identification of the Bru1 Genomic Region for Brown Rust Resistance and Functional Analysis of Candidate Resistance Genes in Sugarcane
    WU QiBin, XIE WanJie, ZHONG Hui, FENG ChunYan, PAN HaoRan, QI YiYing, ZHANG JiSen, WANG HengBo
    Scientia Agricultura Sinica    2024, 57 (5): 855-867.   DOI: 10.3864/j.issn.0578-1752.2024.05.003
    Abstract147)   HTML13)    PDF (5262KB)(105)       Save

    【Objective】Sugarcane brown rust, caused by Puccinia melanocephala H. Sydow & P. Sydow, is one of the most destructive fungal diseases in sugarcane industry, leading to a reduction in sucrose content by 10% to 36%. Previous studies revealed that the major gene(s) for brown rust resistance was located in the Bru1 genomic region. The cloning of crucial candidate genes and their functional investigation should provide important candidate gene resources for breeding new sugarcane cultivars resistant to brown rust.【Method】In this study, the contig-level genomic information of sugarcane cultivar ROC22 was obtained by utilizing the PacBio Sequel Ⅱ sequencing platform, and the Bru1 region associated with brown rust candidate resistance gene was identified, annotated, cloned, and analyzed for tissue specificity, expression patterns in resistant and susceptible sugarcane cultivars, subcellular localization, and transient overexpression.【Result】The results demonstrated that, firstly, using tightly associated molecular markers R12H16 and 9O20-F4 within the Bru1 region, a total of 33 genes were annotated from this region, and five candidate resistance genes (Brrg99, Brrg103, Brrg108, Brrg115, and Brrg116) were selected based on the typical/conserved domains of the resistance genes. Secondly, the full-length sequence cDNA sequence of the Brrg116 gene with an open reading frame of 729 bp and encoding 242 amino acid residues, was cloned from sugarcane cultivar ROC22. The gene sequence was aligned with the genomic databases of Saccharum spontaneum, S. officinarum, and the closely related diploid species sorghum. A high degree of sequence similarity was observed between S. spontaneum and S. officinarum, exceeding 98%. In contrast, its similarity with sorghum was 93.77%. Phylogenetic tree analysis suggests that this gene may originate from S. spontaneum species during sugarcane domestication. qRT-PCR analysis showed its constitutive expression in various tissues of sugarcane cultivars, particularly with the highest expression level in the +1 leaf, which was 5.2 times higher than in the bud. Furthermore, significantly differential expression of Brrg116 was observed at 6 h and 72 h post-inoculation with the brown rust pathogen in resistant and susceptible sugarcane cultivars. Subcellular localization analysis indicated that the encoded protein of this gene was located on the cell membrane. Finally, the Brrg116 gene was transiently overexpressed in Nicotiana benthamiana leaves, followed by inoculation with Fusarium solani var. coeruleum. The color of Nicotiana benthamiana leaves showed a gradual deepening phenotype by 3,3'-diaminobenzidine (DAB) staining. The genes related to hypersensitive response, salicylic acid, and ethylene synthesis had a sustained upregulation pattern, evidencing that the expression of these genes can enhance plant disease resistance.【Conclusion】Brrg116 was constitutively expressed in different sugarcane tissues and in response to brown rust pathogen infection, it showed a rapidly induced expression pattern in the resistant sugarcane cultivar. Overexpression of Brrg116 could trigger defense responses through hormone signaling pathways such as salicylic acid and ethylene. It is thus hypothesized that this gene may play a significant regulatory role in enhancing plant disease resistance.

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    Genome-Wide Association Studies for Canopy Activity Related Traits and Its Genetic Effects on Yield-Related Traits
    LI FaJi, CHENG DunGong, YU XiaoCong, WEN WeiE, LIU JinDong, ZHAI ShengNan, LIU AiFeng, GUO Jun, CAO XinYou, LIU Cheng, SONG JianMin, LIU JianJun, LI HaoSheng
    Scientia Agricultura Sinica    2024, 57 (4): 627-637.   DOI: 10.3864/j.issn.0578-1752.2024.04.001
    Abstract186)   HTML31)    PDF (1826KB)(134)       Save

    【Objective】Canopy activity is an important indicator of wheat growth and development. Identification the loci for canopy activity related traits and their relationships with grain yield (GY) related traits can provide theoretical support for the dissection of genetic structure of yield trait and assisted wheat breeding.【Method】A total of 166 wheat varieties originating from both domestic and international sources were planted in Anyang of Henan province and Suixi of Anhui province in cropping seasons. With the integrated physical map containing 326 570 SNP markers from the wheat 90K and 660K chips, genome-wide association studies for normalized difference vegetation index at seedling stage (NDVI-S) and 10 days after flowering (NDVI-10), and chlorophyll content in flag leaf at 10 days after flowering (Chl-10) were carried out. The results were compared with the previous study for GY related traits using the same material. 【Result】Analysis of variance (ANOVA) showed highly significant effects (P<0.01) of genotypes, environments and genotype×environment interactions on NDVI-S, NDVI-10 and Chl-10, with broad-sense heritabilities (h2 b) of 0.81, 0.81 and 0.91, respectively. Thirteen, 12 and 15 loci were detected to be significantly correlated with NDVI-S, NDVI-10 and Chl-10, respectively, among which 12, 11 and 12 were new, and five loci were associated with two or more traits. The number of favorable alleles for NDVI-S, NDVI-10 and Chl-10 ranged from 4 to 11, 3 to 11 and 4 to 12, respectively, in the 166 wheat varieties, and the phenotypic values increased with the accumulation of favorable alleles. NDVI-S showed significant (P<0.01) and positive correlations with thousand-kernel weight, kernel length and kernel width. Chl-10 was significant positively correlated with GY and flag leaf width (P<0.01), whereas significant negatively correlated with spike number per unit area, plant height and uppermost internode length (P<0.01). Seven pleiotropic loci were detected co-related with both GY and canopy activity related traits.【Conclusion】NDVI-S can be directly used for selection of yield traits. The stable and pleiotropic loci detected in this study can be used for marker-assisted selection.

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    Red and Blue Light Promotes Cotton Callus Induction and Proliferation
    LI KaiLi, WEI YunXiao, CHONG ZhiLi, MENG ZhiGang, WANG Yuan, LIANG ChengZhen, CHEN QuanJia, ZHANG Rui
    Scientia Agricultura Sinica    2024, 57 (4): 638-649.   DOI: 10.3864/j.issn.0578-1752.2024.04.002
    Abstract192)   HTML17)    PDF (1658KB)(106)       Save

    【Objective】Upland cotton (Gossypium hirsutum) genetic transformation faces series of challenges such as a prolonged cycle and low efficiency, with the relatively slow proliferation rate of callus tissue being a critical factor contributing to the extended transformation period. This study aims to investigate the optimal light conditions for upland cotton callus growing. The establishment of this research endeavor is poised to accelerate callus proliferation and ultimately provide a technical foundation for shortening the period of cotton genetic transformation. 【Method】The hypocotyls of the upland cotton line WC were used as explants to induce callus tissues under four different lights: red, blue, red-blue (1:1), and white light (CK). The study aimed to investigate the varied effects of the different lights on callus induction and proliferation, determine the optimal light condition by comparing with the callus proliferation rate, morphological characteristics et al. under different light treatments. 【Result】Different light treatments had a significant impact on the callus induction and growth. The red-blue light treatment exhibited a most positive effect on both callus inducing and proliferation, which observed on the 7th and 15th day. The fresh weight of callus at 7 d under the red-blue light (0.39 g) was the heaviest, followed by the blue light (0.34 g), then the white light (0.24 g) and the red light (0.23 g). The same fresh weight order of the callus was observed at 15 d, with the treatment of the red-blue light (1.15 g) > blue light (0.98 g) > white light (0.69 g) > red light (0.51 g). The callus weight under red-blue light was 1.65 times heavier than the control, increased by 16.5% and 125.5% compared to the treatment of only blue or red light, respectively. In line with this, the callus proliferation rate in the second week under red-blue light was as high as 14.67%, which is twice to that of the red-light treatment (7.17%). The expression level of those genes promoted to cell proliferation and somatic embryo regeneration was consistent with the phenotype, the highest level was under the red-blue light treatment. Furthermore, the activity of the catalase (CAT) was significantly increased under the red-blue light treatment, while the content of reactive oxygen species (ROS) was lower than control. 【Conclusion】Employing different light conditions could result in the varied proliferation rate of cotton callus. The optimal light treatment is by red-blue light in a 1﹕1 ratio, followed by blue light and then white light. However, employing red light only does not favor the callus growth. The treatment of red-blue light (1﹕1) induces the expression of phytochromes and cryptochromes in the callus, increases the expression level of those genes promoted to callus growth. It also enhances the activity of catalase, reduces the content of ROS, and finally promotes the callus proliferation.

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    Genetic Analysis and Gene Mapping of Glossy Leaf in Brassica napus
    GUAN ZhiLin, JIN FengWei, LIU TingTing, WANG Yi, TAN YingYing, YANG ChunHui, LI RuiTong, WANG Bo, LIU KeDe, DONG Yun
    Scientia Agricultura Sinica    2024, 57 (4): 650-662.   DOI: 10.3864/j.issn.0578-1752.2024.04.003
    Abstract153)   HTML47)    PDF (1887KB)(85)       Save

    【Objective】Epicuticular waxes is a hydrophobic layer covering plant leaves and stems, playing an important role in stress resistance and affecting photosynthesis, growth and development. Rapeseed (Brassica napus L.) is one of the main oil crops in the world, but the mechanism of epicuticular waxes formation on its leaves is still unclear. This study aims to reveal the genetic mechanism of epicuticular wax on leaves through a mutant, which will help to achieve high and stable yield of rapeseed.【Method】In this study, leaf of glossy leaf mutant M8 and common waxy leaf inbred lines Zhongshuang 11 (ZS11) and the C20 were characterized, and its photosynthetic rate were measured by portable plant photosynthesis measurement system. Two F1 hybrids and F2 segregating populations were constructed by crossing the M8 with ZS11 and C20, respectively, and were used to analyze the heredity of leaf characters in rapeseed. The F2 population hybridized by M8 and ZS11 was constructed to analyze the genetic regulation of the glossy leaf trait in rapeseed by bulked segregation analysis (BSA) combined with Target-sequencing (Target-seq). Combined with comparative genome and transcriptome database, candidate genes were predicted and then verified by RT-PCR.【Result】The leaf stomatal conductance and photosynthetic efficiency were higher in the glossy leaf mutant M8, compared with ZS11 in rapeseed. The genetic analysis results showed that the glossy leaf was controlled by one pair of recessive genes and glossy leaf was recessive compared with waxy leaf. It was finally fine mapped in the physical region of 0.134-0.699 Mb on chromosome A08 by map based cloning method. Further analysis revealed that there was a large segmental deletion in the 0.22-0.58 Mb region of chromosome A08 in the M8 glossy leaf mutant compared to ZS11, and BnaA08G0006900ZS (BnaA08.SAGL1) in this region of the genome was identified as the candidate gene for the glossy leaf trait. This gene encodes a Kelch-F-box protein, and its deletion may lead to the observed glossy leaf trait in the M8 mutant, as it was highly expressed in ZS11 but none in M8. 【Conclusion】Compared with wild-type waxy leaves, the photosynthetic rate of the new glossy leaf mutant M8 was higher, and the glossy leaf phenotype was controlled by a recessive gene. In the picture, the glossy leaf phenotype regulatory gene was identified as BnaA08.SAGL1, and deletion of the gene resulted in glossy leaf in rapeseed.

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    QTL Mapping and Molecular Marker Development of Traits Related to Grain Weight in Wheat
    ZHANG ZeYuan, LI Yue, ZHAO WenSha, GU JingJing, ZHANG AoYan, ZHANG HaiLong, SONG PengBo, WU JianHui, ZHANG ChuanLiang, SONG QuanHao, JIAN JunTao, SUN DaoJie, WANG XingRong
    Scientia Agricultura Sinica    2023, 56 (21): 4137-4149.   DOI: 10.3864/j.issn.0578-1752.2023.21.001
    Abstract466)   HTML48)    PDF (4065KB)(293)       Save

    【Objective】The yield of wheat, the second-highest-yielding food product in the world, has a major impact by grain weight. This research used materials from a recombinant inbred line (RIL) population derived from Heshangtou (HST) and Longchun 23 (LC23). Based on 55K SNP genotype data, QTL mapping was performed for traits related to grain weight of wheat, and co-segregation markers of major grain length QTL were developed and verified to provide reference for molecular marker assisted selection breeding.【Method】The wheat 55K SNP microarray was used to genotype parents and RIL populations, and a high density genetic linkage map was constructed, and its correlation with Chinese spring reference genome IWGSC RefSeq v1.0 was analyzed. QTL mapping of traits related to grain weight in multiple environments based on inclusive composite interval mapping method. The analysis of variance of major effect QTLs were performed to judge the additive interaction effect among different QTLs, and to analyse its effect on traits related to grain weight. At the same time, the corresponding kompetitive allele specific PCR marker was developed according to the closely linked SNP loci of major QTL for grain length, and verified in 242 wheat accessions worldwide.【Result】In this study, a high density genetic map of Heshangtou/Longchun 23 RIL population was constructed, with full length 4 543 cM, including 22 linkage groups, covering 21 chromosomes of wheat, and the average genetic distance was 1.7 cM. There was a significant correlation between genetic map and physical map, and the Pearson correlation coefficient were 0.77-0.99 (P<0.001). A total of 51 QTLs related to grain weight were detected, among them, 4 stable major QTLs were found in multi-environments (three or more environments) and distributed on 2D, 5A, 6B and 7D chromosomes. According to the physical interval and functional markers, it is inferred that stable major QTLs Qtkw.nwafu-2D.1 and Qtkw.nwafu-7D are photoperiod gene Ppd-D1 and flowering gene FT-D1, respectively. The analysis of variance shows that there is a significant interaction between them. The favorite alleles polymerization of Qtkw.nwafu-2D.1 and Qtkw.nwafu-7D can significantly increase thousand grain weight and grain width of wheat. In addition, the corresponding KASP molecular detection marker AX-111067709 was developed based on the co-segregated SNP of the major locus Qgl.nwafu-5A for grain length, which was significantly correlated with grain length and grain weight traits in a diversity panel comprising of 242 wheat accessions, and could increase grain length by 3.33% to 4.59% and grain weight 5.70% to 10.35% in different environments (P<0.001).【Conclusion】There are several genetic loci that affect traits linked to grain weight in Heshangtou (HST) and Longchun 23 (LC23), and Qtkw.nwafu-2D.1 and Qtkw.nwafu-7D dramatically increased thousand grain weight and grain width through additive interaction effects. Qgl.nwafu-5A is significantly correlated with grain weight and grain length, and its co-segregated molecular marker AX-11106770 can be used in molecular marker assisted selection breeding.

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    Screening of Low Phosphorus Tolerant Germplasm in Cotton at Seedling Stage and Comprehensive Evaluation of Low Phosphorus Tolerance
    KAYOUMU MiReZhaTiJiang, WUMAIERJIANG XiErAiLi, LI XiaoTong, WANG XiangRu, GUI HuiPing, ZHANG HengHeng, ZHANG XiLing, DONG Qiang, SONG MeiZhen
    Scientia Agricultura Sinica    2023, 56 (21): 4150-4162.   DOI: 10.3864/j.issn.0578-1752.2023.21.002
    Abstract260)   HTML29)    PDF (2662KB)(253)       Save

    【Objective】To establish an evaluation system for low phosphorus tolerance in cotton varieties (lines), screen low phosphorus tolerant cotton germplasm and evaluate different types of phosphorus efficiency, and lay the foundation for studying the physiological mechanisms of low phosphorus tolerance in cotton and mining low phosphorus tolerance genes.【Method】Using 140 cotton cultivars (lines) from different cotton regions at home and abroad, 21 traits such as biomass, root-related indexes and phosphorus efficiency-related indexes were measured under low (10 μmol·L-1 KH2PO4) and normal (500 μmol·L-1 KH2PO4) phosphorus treatments in a hydroponic experiment. The index of low phosphorus stress tolerance was calculated for each index. Using the integrated affiliation function method, principal component analysis, regression analysis and cluster analysis were conducted to classify the low phosphorus tolerance of each cotton variety and to comprehensively evaluate the low phosphorus tolerance and phosphorus efficiency type of each cotton variety.【Result】Compared with the normal phosphorus treatment, the mean values of total phosphorus accumulation, total phosphorus content, aboveground dry weight and total dry matter weight of the tested cotton varieties decreased more under the low phosphorus treatment, while the mean values of root average diameter, specific root area, root tips number and phosphorus use efficiency increased. Under low phosphorus treatment, the coefficients of variation of each index ranged from 6.04% to 47.79%,the coefficients of variation of root indexes such as specific root tips density, root tips number, specific root length and root average diameter were higher than those of normal phosphorus treatment, and the coefficients of variation were 47.49%, 42.13%, 40.19% and 19.16%, respectively; the principal component analysis of the 21 indexes of low phosphorus stress tolerance showed that the cumulative variance contribution of the six principal components reached 77.21%, and the comprehensive low phosphorus tolerance value (D) was calculated using the affiliation function method. The D-value regression equation was established by multiple regression analysis to determine the six low phosphorus tolerance indices and perform systematic clustering to classify different cotton varieties (lines) into three categories: low phosphorus tolerant, intermediate and low phosphorus sensitive.【Conclusion】Total dry matter weight, phosphorus use efficiency, root fresh weight, total root length, root surface area and total phosphorus accumulation were identified as indicators for the evaluation of low phosphorus tolerance in cotton.

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    Identification and Evaluation of Drought Tolerance and Screening of Drought-Tolerant Germplasm for Core Germplasms in Proso Millet at Adult Stage
    WANG Qian, DONG KongJun, XUE YaPeng, LIU ShaoXiong, WANG RuoNan, YANG JiaQi, LU Ping, WANG RuiYun, YANG TianYu, LIU MinXuan
    Scientia Agricultura Sinica    2023, 56 (21): 4163-4174.   DOI: 10.3864/j.issn.0578-1752.2023.21.003
    Abstract183)   HTML24)    PDF (989KB)(224)       Save

    【Objective】Drought is one of the major constraints influencing on the growth, development, and yield of proso millet. To screen excellent drought-tolerant accessions and identification indicators will promote the drought-tolerant varieties breeding and drought tolerance molecular mechanisms analysis of proso millet.【Method】In this study, 200 core germplasm accessions of proso millet were used to identify field drought tolerance at adult stage in 2021-2022 at Dunhuang, Gansu province, with two treatments of normal irrigation and drought stress. Eleven morphological indicators, such as leaf area (LA), main stem diameter (MSD), number of main stem nodes (NMSN), main panicle length (MPL), peduncle length (PL), plant height (PH), straw weight per plant (SWPP), panicle weight per plant (PWPP), grain weight per plant (GWPP), thousand grain weight (TGW), and yield per plot (YPP) were determined. Comprehensive drought tolerance coefficient (CDTC value), drought resistance index (DRI value), and drought resistance comprehensive evaluation value (D value) were combined to identify the drought tolerance of proso millet at adult stage.【Result】There were significant differences in all eleven indexes among different accessions under different water treatments, including leaf area, main stem diameter, number of main stem nodes, main panicle length, peduncle length, plant height, straw weight per plant, panicle weight per plant, grain weight per plant, thousand grain weight, and yield per plot. The growth of proso millet was inhibited under drought stress treatments. Compared with the normal irrigation treatments, all the eleven indexes under drought treatments were significantly reduced, and yield per plot was more sensitive to drought treatments. The correlation analysis found that certain degrees of correlation existed among the drought tolerance coefficients of all traits, the correlation between panicle weight per plant and grain weight per plant was strongest, with a correlation coefficient of 0.943. Eleven evaluation indexes were converted into six comprehensive indexes by principal component analysis, with a cumulative variance contribution of 80.667%. The drought tolerance ranking of proso millet accessions based on CDTC value, DRI value and D value was generally consistent. The 200 proso millet accessions were classified into four categories according to D value cluster analysis, 10 of cluster Ⅰ were highly drought tolerant, 70 of cluster Ⅱ were drought tolerant, 81 of cluster Ⅲ were drought sensitive, and 39 of cluster Ⅳ were highly drought sensitive. Plant height, grain weight per plant, panicle weight per plant and main panicle length were highly correlated with D value, with correlation coefficients of 0.756, 0.697, 0.696 and 0.679, respectively. The regression equation for the drought tolerance evaluation was constructed by stepwise regression analysis: Y=-1.509+0.362X1+0.174X2+0.349X3+0.389X4+0.307X5+ 0.251X6+0.218X7.【Conclusion】The drought resistance comprehensive evaluation value method could be suitable for evaluating the drought resistance of proso millet at adult stage. Ten accessions with highly drought tolerance, such as Balinzuogedashu (00000525), Gaotaiwumizi (00002677) and Minlehongmizi (00002687). Plant height, panicle weight per plant and main panicle length could be used as primary evaluation indexes for drought tolerance of proso millet at adult stage.

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    QTL Analysis for Seeding Traits Related to Low Nitrogen Tolerance in Foxtail Millet
    QIN Na, FU SenJie, ZHU CanCan, DAI ShuTao, SONG YingHui, WEI Xin, WANG ChunYi, YE ZhenYan, LI JunXia
    Scientia Agricultura Sinica    2023, 56 (20): 3931-3945.   DOI: 10.3864/j.issn.0578-1752.2023.20.002
    Abstract156)   HTML16)    PDF (750KB)(197)       Save

    【Objective】The analysis of quantitative trait loci (QTL) related to low nitrogen tolerance traits of millet (Setaria italica L.) laid a foundation for fine mapping, cloning and functional research of low nitrogen tolerance genes. At the same time, it also provided technical support for revealing the genetic mechanism of low nitrogen tolerance of millet and breeding low nitrogen tolerance varieties. 【Method】The recombinant inbred line (RIL) population consisting of 120 family lines was used as experimental materials, that was constructed from parents Yugu 28, a low nitrogen tolerant variety, and Qiyehuang, a low nitrogen sensitive variety. The RIL populations were treated with low nitrogen and normal nitrogen at seedling stage, and seven traits were analyzed of hydroponic for 21 days, which inculding seedling length, maximum root length, root dry weight, seedling dry weight, plant dry weight, relative chlorophyll content and plant nitrogen content. At the same time, we used composite interval mapping (CIM) to locate and analyze QTLs for traits related to low nitrogen tolerance, and predicted the candidate genes in the confidence intervals of QTLS. 【Result】The traits associated with low nitrogen tolerance of RIL populations exhibited continuous distribution with apparent transgressive segregation both under low nitrogen and normal nitrogen levels, which conformed to the typical genetic characteristics of quantitative traits and were suitable for QTL genetic analysis. Correlation analysis showed that seeding length was positively correlated with maximum root length, root dry weight, seeding dry weight, plant dry weight and relative chlorophyll content, and maximum root length was negatively correlated with plant nitrogen content. A total of thirty-four QTLs related to seeding length, maximum root length, root dry weight, seeding dry weight, plant dry weight, relative chlorophyll content and plant nitrogen content were located under low nitrogen and normal nitrogen levels, which distributed on chromosomes from 1 to 9. They explained individually 5.15%-52.42% phenotypic variation. Ten QTLs were simultaneously detected under both two nitrogen levels, eleven and thirteen QTLs were only identified under single low nitrogen and normal nitrogen conditions, respectively. A total of fifteen QTLs were major QTL, and five major QTLs were repeatedly detected under both two nitrogen levels, which including qRDW3, qMRL1.1, qMRL1.2, qSL5 and qSPAD1. Five QTL overlaps were detected with gathering multiple QTLs under two nitrogen levels. Six candidate genes related to nitrogen metabolism were identified from the confidence interval of the five QTL overlaps, suggesting that genes related to nitrogen assimilation, absorption and utilization probably control the expression of these genes. 【Conclusion】Thirty-four QTLs were scattered on sixteen clusters of nine chromosomes. Based on gene annotation, a total of 6 candidate genes related to nitrogen metabolism were screened in foxtail millet, indicating the different traits involved in common genetic mechanisms, and the favorable alleles for low nitrogen tolerance can be polymerized by marker-assisted selection.

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    Genome-Wide Association Study of Nitrogen Efficiency Related Traits at Seedling Stage in Brassica juncea
    ZENG Jian, WANG RuMeng, GONG Pan, YANG Xiao, YIN XiaoQi, LI JiangHe, CHEN ShiLong, YAO Lei, SONG HaiXing, KANG Lei, ZHANG ZhenHua
    Scientia Agricultura Sinica    2023, 56 (20): 3946-3959.   DOI: 10.3864/j.issn.0578-1752.2023.20.003
    Abstract149)   HTML24)    PDF (2886KB)(187)       Save

    【Objective】The genome-wide association analysis was performed to identify SNP loci significantly associated with nitrogen use efficiency (NUE) traits in Brassica juncea at seedling stage and to predict the relevant candidate genes, providing a theoretical basis for revealing the molecular mechanism of nitrogen use efficiency in rapeseed and creating nitrogen-efficient germplasm. 【Method】The population of 153 Brassica juncea resources was used as the analysis population. Two treatments, low N and normal N, were established using three replicates for each treatment, and two replicated nutrient culture trials were conducted over a two-year period (2021 and 2022). The relative values of root-shoot ratio and shoot nitrogen concentration (low/normal N) were calculated and utilized as NUE traits for a genome-wide association study (GWAS) aimed at exploring candidate genes for NUE. 【Result】NUE traits of Brassica juncea resources exhibited abundant variation, ranging from 0.21-2.44 with coefficients of variation of 22.92%-26.19%. The GWAS identified 45 significant SNP loci, among which 16 overlapped between the first relative root-shoot ratio (RRSR1) and the second relative root-shoot ratio (RRSR2), accounting for a phenotype variance range of 10.69%-15.39%. Additionally, 29 significant SNP loci were shared between the first relative shoot nitrogen concentration (RSNC1) and the second relative shoot nitrogen concentration (RSNC2), explaining a phenotype variance range of 13.22%-23.96%. 15 candidate genes for NUE were identified within 200 kb upstream and downstream regions of significant SNP loci, including 5 genes related to nitrate transport (BjuNPF5.8, BjuNRT2.7, BjuNPF2.3, BjuCLCb and BjuNRT1.3), 3 genes associated with nitrogen metabolism (BjuASN3, BjuGLU2 and BjuADCS), 4 genes involved in plant growth and development (BjuCOBL8, BjuPYL6, BjuSAUR72 and BjuUP3) and 3 genes participated in stress response (BjuNTP7, BjuJUB1 and BjuPYL6). 【Conclusion】45 SNP loci were detected significantly associated with NUE traits and 15 candidate genes for NUE were identified in this study.

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    Population Genetic Analysis of Puccinia striiformis tritici in Main Winter-Increasing Areas Based on Virulent Phenotypes and Genotypes
    GAO XinPei, ZHAO Jun, LIU BoFan, GUO Yi, KANG ZhenSheng, ZHAN GangMing
    Scientia Agricultura Sinica    2023, 56 (14): 2629-2642.   DOI: 10.3864/j.issn.0578-1752.2023.14.001
    Abstract319)   HTML37)    PDF (1592KB)(426)       Save

    【Objective】To clarify the virulence structure and genetic diversity of Pst populations in the major winter-increasing areas of China, and to provide reference for the prevention and control of Pst and the rational layout of wheat resistance genes in the winter-increasing areas and the wheat production in Huang-huai-hai. 【Method】A total of 148 Pst isolates were collected and isolated from the major winter-increasing areas such as Sichuan Basin, Hubei and southern Henan, and the virulence phenotype was identified by using Chinese differentials and single-gene lines, and 17 pairs of KASP-SNP primers were used to mark the isolates and complete the genotype analysis. 【Result】Based on the Chinese differentials, 14 known races and 63 unknown pathotypes were identified, among which CYR34 (16.2%), G22-14 (12.2%), CYR32 (6.8%), CYR33 (5.4%) were the dominant races (pathotypes); based on the single-gene lines, 113 races (pathotypes) were identified, among which race1 (7.4%), race2 (3.4%), race3 (3.4%) were the dominant races (pathotypes). The Guinong 22 group was the largest epidemic group of Pst population in China’s winter-increasing area, and all tested Pst isolates did not infect single-gene lines varieties carrying Yr5 and Yr15. The virulence phenotype and genotype of CYR34 and G22-14 showed diversification by single-gene lines virulence identification and molecular marker, indicating that there was high differentiation within these two dominant races. The clustering based on the virulence data of two sets of differentials showed that the Pst populations in Sichuan Basin and southern Hubei were similar, while the Pst populations in northwestern Hubei and southern Henan were similar; the genetic clustering based on KASP-SNP molecular data showed that there was genotype differentiation between the Pst populations in Sichuan Basin, southern Hubei and northwestern Hubei, southern Henan; Structure analysis showed that Sichuan Basin, southern Hubei population mainly had two genetic backgrounds, northwestern Hubei, southern Henan population mainly had one genetic background; population genetic differentiation analysis showed that Sichuan Basin Pst population and southern Henan Pst population had the largest Fst value, which was 0.118, with the largest genetic difference and obvious genetic differentiation; northwestern Hubei population and southern Henan population had the smallest degree of genetic differentiation, Fst value was 0.010; gene flow analysis obtained Nm value between northwestern Hubei population and southern Henan population was 25.236, Nm>4, there was a high-level gene flow between them, northwestern Hubei and southern Henan population and Sichuan Basin population had Nm values of 2.923 and 1.864 respectively, both had a low-level gene flow; genetic diversity analysis results showed that Sichuan Basin, southern Hubei region Pst population had a high-level of genetic diversity, northwestern Hubei, southern Henan Pst population had a low-level of genetic diversity. The above conclusions all support that Sichuan Basin, southern Hubei population has genetic differentiation with northwestern Hubei, southern Henan population. 【Conclusion】Single-gene lines can accurately identify Chinese Pst races; Pst populations in China’s major winter-increasing areas have different sources.

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    Pigment Identification and Gene Mapping in Red Seed Coat of Soybean
    CAO Jie, GU YongZhe, HONG HuiLong, WU HaiTao, ZHANG Xia, SUN JianQiang, BAO LiGao, QIU LiJuan
    Scientia Agricultura Sinica    2023, 56 (14): 2643-2659.   DOI: 10.3864/j.issn.0578-1752.2023.14.002
    Abstract241)   HTML23)    PDF (3120KB)(526)       Save

    【Objective】To identify the key genes controlling anthocyanin synthesis and accumulation, to uncover changes in anthocyanin content of the seed coat during seed development, and the primary anthocyanin components responsible for the red seed coat of Taixingaijiaohong (TXAJH); and to lay the groundwork for a thorough understanding of the regulatory mechanism of red seed coat formation.【Method】Using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS), the anthocyanin composition and concentration of the yellow seed coat of soybean Suinong 14 (SN14) and the red seed coat of soybean TXAJH at various developmental stages were identified. The potential areas of red testa-related genes were first identified using bulked segregant analysis (BSA) on the recombinant inbred lines (RILs) made by crossing SN14 and TXAJH. Based on this discovery, we performed marker linkage analysis to restrict the candidate intervals and predict the candidate genes, and qRT-PCR to confirm the expression of the anticipated candidate genes.【Result】When seed coats from the four developmental phases of SN14 and TXAJH were analyzed, a total of 12 anthocyanins were discovered. Cluster analysis of total anthocyanins revealed substantial changes in the seed coat's anthocyanin composition between TXAJH and SN14 as well as between TXAJH before and after color development. The anthocyanin content of the SN14 seed coat gradually decreased as the seed developed, whereas the TXAJH seed coat's content increased quickly and remained stable. After the development of the seed coat's color, the anthocyanin contents of SN14 and TXAJH showed highly significant differences, and at the mature stage, the TXAJH seed coat's anthocyanin content was more than 200 times that of SN14. The crimson coloring of the TXAJH seed coat was largely due to cyanidin-3-O-glucoside (Cy-3-glu), peonidin-3-O-glucoside (Pn-3-glu), and petunidin-3-O-glucoside (Pt-3-glu). The candidate interval for the red seed coat gene on chromosome 8 was discovered at 8.66 Mb by BSA-seq association analysis. 27 polymorphic markers were used in the marker linkage analysis, which produced 10 haplotypes and reduced the candidate interval to 702 kb. Nonsynonymous variations in 37 genes between the parents were found during this interval, these include the genes for encode the anthocyanin reductase 1 (Glyma.08g062000), the bHLH transcription factor (Glyma.08g061300 and Glyma.08g063900), and the MYB transcript factor (Glyma.08g059900). These genes may be involved in regulating the biosynthesis of anthocyanins, and anthocyanin reductase 1 can convert anthocyanins to proanthocyanidins (PA). The results of gene expression analysis revealed that candidate genes and genes related to the anthocyanin biosynthesis pathway had comparable expression patterns in SN14 and TXAJH, and both were expressed at lower levels in SN14 and at higher levels in TXAJH. It was discovered that there was a significant link between the principal constituents of seed coat anthocyanins and the level of candidate gene expression.【Conclusion】The anthocyanin makeup of SN14 and TXAJH's seed coats differed, and Cy-3-glu, Pn-3-glu, and Pt-3-glu may be to blame for the TXAJH's seed coat's red hue. According to predictions, Glyma.08g059900, Glyma.08g061300, Glyma.08g062000, and Glyma.08g063900 will likely be a candidate gene for the red seed coat, in which Glyma.08g059900, Glyma.08g061300, and Glyma.08g063900 may control a number of anthocyanin biosynthesis pathway genes.

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    Identification of Adult Plant Stripe Rust Resistance Candidate Genes of YrZ501-2BL by Gene Association and Transciptome Analysis in Wheat (Triticum aestivum L.)
    ZHANG Xu, HAN JinYu, LI ChenChen, ZHANG DanDan, WU QiMeng, LIU ShengJie, JIAO HanXuan, HUANG Shuo, LI ChunLian, WANG ChangFa, ZENG QingDong, KANG ZhenSheng, HAN DeJun, WU JianHui
    Scientia Agricultura Sinica    2023, 56 (8): 1429-1443.   DOI: 10.3864/j.issn.0578-1752.2023.08.001
    Abstract468)   HTML78)    PDF (5768KB)(296)       Save

    【Objective】Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), significantly reduced wheat production worldwide. Identification of stripe rust resistance genes is the foundation of improving wheat resistance breeding and revealing its genetic mechanism.【Method】A multi-omics approach combined with genome-wide association study (GWAS) was used for dissecting adult plant stripe rust resistance for wheat advanced breeding lines collected from International Maize and Wheat Improvement Center (CIMMYT) and International Centre for Agricultural Research in the Dry Areas (ICARDA) bread-wheat breeding programs. In the present study, a diversity panel of 411 wheat lines from CIMMYT and ICARDA was used for genome-wide association study and a major locus on chromosome arm 2BL was identified. In order to verify the stability of the locus, the resistant line Z501 with the resistance allele of the locus was crossed by the susceptible line Jinmai 79, and the locus tentatively named YrZ501 was successfully confirmed using linkage mapping based on F2:3 genetic population of Jinmai 79×Z501. Then we performed candidate gene analysis based on gene annotation, comparative genome, transcriptome and gene-based association analysis. 【Result】Combining GWAS and linkage mapping results, the YrZ501-2BL was located in the physical interval of 0.26 Mb (575.706-576.587 Mb) on chromosome 2B. According to the annotation information of Chinese Spring reference genome IWGSC v1.1, there were six high confidence genes of 12 genes in this region. Using online website, the target interval in the Chinese spring reference genome was compared with other published different ploidy wheat genomes. The six high-confidence genes within this interval can basically be found homologous in other wheat lines, and the genes arranged in the same order, indicating that the interval may not have large fragment insertions, deletions and inversions. The above results showed that we can perform candidate gene prediction analysis based on the reference genome information. After analysis of their transcriptomic data between the resistant parent Z501 and susceptible parent Jimai 79, only three genes, TraesCS2B02G406400, TraesCS2B02G406500 and TraesCS2B02G406600 showed variable expression levels and were induced by stripe rust infection. Further, they encode GATA transcription factor, SH3 domain-containing protein 2 and zinc finger protein, respectively. Gene-based association analysis revealed that there was a significant SNP (G1369A) in TraesCS2B02G406500 that was associated with stripe rust responses. Although this SNP (G1369A) did not cause amino acid coding changes (both TCG and TCA encode serine), it may be associated with alternative splicing. Moreover, it showed significant differences of the stripe rust responses between the different haplotypes (G1369A). Further analysis revealed two other variants G1377A and G1431A, that caused amino acid changes, i. e. valine (GTT) to isoleucine (ATT) and valine (GTG) to methionine (ATG), respectively. However, the two SNPs were rare variants as they accounting for only 0.87% of the 455 re-sequencing wheat accessions and they were not tested for significance. In summary, TraesCS2B02G406500 was preliminarily considered as an important candidate gene of YrZ501-2BL. In addition, the corresponding AQP markers were developed based on the SNPs among the YrZ501 candidate regions, which can be used to marker-assisted selection in molecular breeding application of wheat stripe rust resistance.【Conclusion】A candidate causal gene TraesCS2B02G406500 associated with stripe rust resistance was successfully identified on wheat chromosome 2B using an integrated method of multi-omics and association analysis, which laid a solid foundation for further gene cloning and functional verification.

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    Development of A Set of Matrix Reference Materials in Different Mass Fractions of Genetically Modified Maize MON87427
    LI Jun, SHAN LuYing, XIAO Fang, LI YunJing, GAO HongFei, ZHAI ShanShan, WU Gang, ZHANG XiuJie, WU YuHua
    Scientia Agricultura Sinica    2023, 56 (8): 1444-1455.   DOI: 10.3864/j.issn.0578-1752.2023.08.002
    Abstract244)   HTML26)    PDF (568KB)(106)       Save

    【Objective】The GMO (genetically modified organism) reference materials (RMs) are the material basis for GMO safety supervision and labeling policy implementation. During GMO detection the utilization of RMs guarantees the traceability of quantitative results within laboratory and the comparability of quantitative results between laboratories. GM (genetically modified) maize MON87427 has been approved to be imported as raw material in China, it’s urgent to develop certified reference materials (CRMs) for safety supervision and quantification. 【Method】 The GM maize MON87427 hybrid seeds and non-GM counterparts provided by the developer were used as raw materials to perform washing, drying, freeze-grinding, particle size measurement, and moisture content measurement, sequentially. The matrix RMs of MON87427a, MON87427b and MON87427c were produced by blending the seed powder of the GM maize MON87427 and a non-GM counterpart in GMO mass fractions of 60.0 mg·g-1, 99.5 mg·g-1 and 1 000.0 mg·g-1 on a dry basis. The real-time quantitative PCR was used to conduct an initial assessment of homogeneity before packing. The MON87427/zSSIIb duplex digital PCR (ddPCR) was used to evaluate the homogeneity and stability of the RMs as well as the collaborative characterization by 8 qualified laboratories. The data processing of homogeneity, stability, collaborative characterization together with uncertainty evaluation of RMs were carried out according to the standard "General and Statistical Principles for Characterization of Reference Materials" (JJF 1343). 【Result】 This batch of RMs contains three RMs of MON87427a, MON87427b, and MON87427c in different mass fractions, with more than 80% of particle size of less than 200 μm and less than 5% of moisture content. The RMs were packed in brown glass bottles, nitrogen flushed before capping, bottling amount was not less than 1.0 g/bottle, a total of 400 bottles were produced for each mass fraction RM. The calculated F values of the homogeneity test were all less than the critical value of F0.05 (14, 30) (2.04) for the three RMs, displaying good homogeneity within and between bottles, and the minimum intake was determined to be 100 mg. The RMs can be stored stably at 25℃, 37℃, and 60℃ for 14 days, the property value of the RMs does not change significantly after 14 days of transportation at room temperature; the long-term stability can reach 12 months at 4℃ and -20℃; The property value of the samples taken from the same bottle of RM after 5 opening-capping cycles, does not deviate significantly from that of the first taken sample. The collaborative characterization data by eight qualified laboratories displayed normal distribution without outliers and outlying standard deviations. The standard value and expanded uncertainty of MON87427a, MON87427b, Mon87427c were certified to be (2.92±0.44)%, (4.89±0.57)%, (52.1±3.4)%. 【Conclusion】The developed GM maize MON87427 matrix RMs in different mass fractions have good homogeneity, and can be stably transported and stored. This batch of RMs meets the requirements of qualitative and quantitative detection of MON87427 event, providing reliable CRMs for the safety supervision and implementation of quantitative labeling policy for GMO-derived products.

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    Identification of Excellent Wheat Germplasms and Classification of Source-Sink Types
    NAN Rui, YANG YuCun, SHI FangHui, ZHANG LiNing, MI TongXi, ZHANG LiQiang, LI ChunYan, SUN FengLi, XI YaJun, ZHANG Chao
    Scientia Agricultura Sinica    2023, 56 (6): 1019-1034.   DOI: 10.3864/j.issn.0578-1752.2023.06.001
    Abstract395)   HTML81)    PDF (833KB)(210)       Save

    【Objective】 The aim of this study is to screen the evaluation indexes of wheat source-sink and classify the source-sink types. In addition, the relationships between different source-sink types and the agronomic traits, yield and grain quality of wheat were also clarified, which provides a better understanding of wheat source-sink metabolism and wheat breeding. 【Method】 In this study, the related agronomic traits of source metabolism and sink metabolism of 190 wheat varieties which from different regions were measured. Then, the source-sink metabolic capacity of wheat was evaluated by principal component analysis, and the superior wheat materials were selected according to the composite score. Furthermore, the hierarchical clustering was conducted based on the source size (leaf area), source activity, sink number and sink activity. Then, based on the clustering results, the source-sink types of wheat were classified and the source-sink characteristics of different region of wheats were analyzed. Meanwhile, the agronomic, yield and quality traits of different wheat source-sink types were compared and analyzed. 【Result】 For better understanding the results, six indicators related to source activity were converted into three independent indicators (photochemical quenching coefficient, maximum photosynthetic potential, chlorophyll content), and five indicators related to sink activity were converted into two independent indicators (maximum filling rate, filling duration) based on the principal component analysis. The cumulative contributions of three source activity indicators and two sink activity indicators were 82.80% and 92.90%, respectively. Then the top 10 wheat varieties were screened based on the source activity, source size (leaf area), sink activity, and sink number (number of spike grains). According to the source-library relationship, all the wheat varieties were divided into three major categories and eight subcategories, including sufficient source-weak sink type (medium source-weak sink type, strong source-medium sink type), weak source-sufficient sink type (medium source-strong sink type, weak source-medium sink type) and source-sink balance type (weak source-weak sink type limited by sink activity, weak source-weak sink type limited by source activity and grain number per spike, medium source-medium sink type, and strong source-strong sink type). 76.84% of wheat lines were contained in three subcategories (weak source-weak sink type, the medium source-strong sink type, and strong source-strong sink type), other wheat lines were distributed in other subcategories evenly. Most wheat varieties of China showed similar source-sink relationship, which presented the medium level of source activity, leaf area and grain number per spike, while the sink activity was high. But the sink activity of wheat varieties which were cultivated at the middle and lower reaches of the Yangtze River is low. The plant height, length of uppermost internode and spikelet number in different categories indicated that the sufficient source-weak sink type>source-sink balance type>weak source-sufficient sink type, and the dry protein content, dry wet gluten content and sedimentation value in different categories showed that source-weak sink type > source-sink balance type > weak source-sufficient sink type. Grain water absorption showed that the stronger the sink activity had the higher the water absorption rate. The yield of per plant was different among different source-bank groups, but it was positively correlated with the source activity and the number of grains per spike in the three subcategories which contained most wheat varieties. 【Conclusion】 In this study, photochemical quenching coefficient, maximum photosynthetic potential and chlorophyll content could be used as the main indexes to evaluate the activity of wheat source. The maximum filling rate and filling duration could be used as the main indexes to evaluate the activity of wheat sink. In practical production, the wheat yield could be improved by increasing the number of grains per spike and the activity of source. When the supply capacity of source is stronger than the absorption capacity of sink, the plant height, peduncle length, spikelet number, dry protein content, dry wet gluten content and sedimentation value would be increased. Strong sink activity would help us to improve the water absorption of wheat grains.

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    Investigation of Low Nitrogen Tolerance of ZmCCT10 in Maize
    LI YiPu, TONG LiXiu, LIN YaNan, SU ZhiJun, BAO HaiZhu, WANG FuGui, LIU Jian, QU JiaWei, HU ShuPing, SUN JiYing, WANG ZhiGang, YU XiaoFang, XU MingLiang, GAO JuLin
    Scientia Agricultura Sinica    2023, 56 (6): 1035-1044.   DOI: 10.3864/j.issn.0578-1752.2023.06.002
    Abstract350)   HTML43)    PDF (2363KB)(172)       Save

    【Objective】 The lack of soil nitrogen impacts the yield and quality of maize, which is a major problem of maize production in China. ZmCCT10 encodes the transcription factor, which is pleiotropic. ZmCCT10 is a very important co-factor regulating the growth, development and responding to abiotic stress of maize. The molecular mechanism of maize tolerance to low nitrogen is the basis for breeding maize varieties with low nitrogen tolerance and high nitrogen efficiency. 【Method】 In this study, we compared the traits those relate to low-nitrogen tolerance, expression pattern of ZmCCT10 and transcriptome results of ZmCCT10 near-isogenic lines under low-nitrogen stress and complete nutrient conditions. To analysis the characteristics of ZmCCT10 in response to low-nitrogen stress and the molecular mechanisms involved in low-nitrogen tolerance were explored. 【Result】 This study indicated that different alleles of ZmCCT10 showed significant differences in root length traits, biomass and nitrogen physiological traits under low nitrogen stress. The Y331-ΔTE haplotype without transposon insertion of ZmCCT10 had significantly longer total root length, main radicle length and lateral root length than Y331 after low nitrogen stress. What is more, root dry weight, shoot dry weight, nitrogen accumulation and nitrate reductase activity were also significantly higher than Y331. The expression levels of ZmCCT10 in roots and leaves were significantly higher than those in the control treatment. The expression level of ZmCCT10 in roots reached a peak at 3 hours after stress treatment. In leaves, the expression of ZmCCT10 continued to increase and peaked 6 hours after stress treatment. Root samples were collected under 0.04 mmol·L-1 low nitrogen stress after 3h for transcriptome sequencing. The correlation coefficients between biological replicates are more than 0.9. GO enrichment analysis showed that the expression levels of amine synthesis process and cellular nitrogen compound catabolic process were significantly different in near-isogenic lines after low nitrogen stress. Combined with the amount and expression pattern of differential genes, ZmCCT10-regulated candidate genes involved in low-nitrogen tolerance were selected. qRT-PCR confirmed that the expression levels of ZmMPK5, ZmNS2 and other genes were significantly different after stress in near-isogenic lines. 【Conclusion】 ZmCCT10 is a candidate gene involved in low nitrogen tolerance in maize and it participates in the low-nitrogen tolerance response of maize as transcriptional regulation.

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    Application of Yr52 Gene in Wheat Improvement for Stripe Rust Resistance
    FANG TaoHong,ZHANG Min,MA ChunHua,ZHENG XiaoChen,TAN WenJing,TIAN Ran,YAN Qiong,ZHOU XinLi,LI Xin,YANG SuiZhuang,HUANG KeBing,WANG JianFeng,HAN DeJun,WANG XiaoJie,KANG ZhenSheng
    Scientia Agricultura Sinica    2022, 55 (11): 2077-2091.   DOI: 10.3864/j.issn.0578-1752.2022.11.001
    Abstract455)   HTML77)    PDF (1980KB)(164)       Save

    【Objective】 The objective of this study is to evaluate the application value of the high temperature adult plant (HTAP) resistance gene Yr52 in wheat production for improving stripe rust resistance. And wheat lines with good agronomic characters and high disease resistance were developed and selected. It laid a foundation for making full use of the existing HTAP resistance resources and improving the yield related traits.【Method】 The stripe rust resistance gene Yr52 was introgressed to Lunxuan 987 (LX987), Bainong Aikang 58 (AK58) and Han 6172 (H6172) by backcrossing and self-crossing combined with marker-assisted selection breeding. Adult-plant resistance of donor parent, receptor cultivars and their progeny lines were evaluated in the disease nursery fields at Mianyang, Sichuan and Yangling, Shaanxi by mixed endemic physiological races CYR32, CYR33 and CYR34. In comparison to the Chinese Spring reference genome, the flanking SSR markers Xcfa2040 (6.8 cm-Yr52) and Xbarc182 (1.2 cm-Yr52) of Yr52 were combined to search for markers of 35K SNP chip in the physical interval of target genes, and developed into derived cleaved amplified polymorphic sequences (dCAPS) and kompetitive allele specific PCR (KASP) markers. The resistance gene Yr52 was detected in BC2F5:6 progeny lines.【Result】 The evaluation of adult plant resistance and agronomic traits indicated that nineteen BC2F5:6 lines with LX987 background was obtained: Among of them, 11 were high resistance (IT=0-3, DS=1%-20%), 8 were moderate resistance (IT=4-6, DS=15%-30%), the average thousand kernel weight (TKW), kernels per spike (KPS), productive tiller number per line (PTN), plant height (PH) and spike length (SL) was 45.33 g, 46, 7, 113.26 cm and 10.05 cm, respectively. Four BC2F5:6 families with AK58 background: all showed high resistance (IT=0-3, DS=5%-25%); the average TKW, KPS, PTN, PH and SL were 44.67 g, 48, 7, 96.54 cm and 10.17 cm, respectively. Five BC2F5:6 lineages with H6172 background showed high resistance to stripe rust (IT=0-3, DS=5%-20%). The average TKW, KPS, PTN, PH and SL were 43.74 g, 49, 8, 109.72 cm and 10.06 cm, respectively. The detection rate of three simple sequence repeat (SSR) molecular markers Xbarc182, Xcfa2040 and Xwmc557 linked to Yr52, in offspring population were 78.57%, 66.67% and 66.67%, respectively. One dCAPS marker Xdcaps-Yr52-1 and one KASP marker Xkasp-Yr52-1 were successfully developed, and the detection rates were 73.68% and 41.67%, respectively. The agronomic traits of lines with high (IT=0-3) and medium (IT=4-6) resistance levels were compared. The results showed that the average TKW (P>0.05), PTN (P>0.05) and KPS (P<0.05) of lines with IT=0-3 were higher than those of families with moderate resistance level lines (IT=4-6). Five lines with disease resistance and stable agronomic traits were selected by evaluated of PH=80-105 cm, PTN≥6,KPS≥45, TKW≥42 g, SL≥8 cm.【Conclusion】 Yr52 was found to be resistant to all of the present predominant races in the adult plant stage. After introgression Yr52 into the main susceptible Chinese wheat varieties, the progeny lines with good disease resistance and agronomic characters could be used for breeding resistance varieties with multi-gene polymerization, it is enriching for the diversity of disease resistance genes and achieving durable utilization. The development of molecular markers will facilitate detect the utilization of Yr52 gene in resistance identification of germplasm in the future.

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    Accurate Identification and Comprehensive Evaluation of Panicle Phenotypic Traits of Landraces and Cultivars of Sorghum bicolor (L.) Moench in China
    XU Xiao,REN GenZeng,ZHAO XinRui,CHANG JinHua,CUI JiangHui
    Scientia Agricultura Sinica    2022, 55 (11): 2092-2108.   DOI: 10.3864/j.issn.0578-1752.2022.11.002
    Abstract527)   HTML64)    PDF (4958KB)(239)       Save

    【Objective】 Through the research on the phenotypic diversity and genetic variation of the sorghum germplasm resources in the panicle, we will screen for superior sorghum germplasm, enrich the genetic information of sorghum panicle-related traits, and provide a reference for the conservation and efficient use of existing germplasm resources and the selection and breeding of new varieties.【Method】 Using 320 sorghum accessions from different parts of China as test materials, we accurately identified 12 panicle traits (grain length, grain width, thousand-grain weight, grain hardness, grain density, corneous endosperm rate, kernel weight per panicle, main panicle length, panicle neck length, panicle neck diameter, primary branches length, primary branches number) in two different ecological environments. A comprehensive evaluation of sorghum germplasm resources using correlation analysis, principal component analysis, cluster analysis, and other methods. We screened elite sorghum germplasms with different outstanding characteristics according to the comprehensive evaluation value F and target traits. 【Result】 The frequency distribution of each quantitative trait showed a trend of high in the middle and low on both sides. The two-year frequency distribution and curve trend of grain hardness, kernel weight per panicle and grain density, and corneous endosperm rate were similar at the Baoding and Jinzhong test sites. Most of the traits only showed normal distribution in one year or a single location. Except for the main panicle length and number of primary branches, the other traits differed between the two test sites in the same year. The mean diversity index (H') distribution of the 12 panicle traits ranged from 1.72 to 2.11, among which the average diversity index of grain hardness was the highest, and the average diversity index of primary branches length was the lowest. The coefficients of variation of grain hardness, corneous endosperm rate, kernel weight per panicle, primary branches length, and the number of primary branches were all higher than 30.00%. The cumulative contribution rate of the extracted four principal components was 65.39%. Cluster analysis classified the 320 accessions into three groups, class I can be used as the germplasm for screening process (broom) sorghum; class II is suitable for selecting excellent germplasm for grain (brewing) sorghum; class III was the germplasm with poor panicle traits. We screen 29 superior germplasm with outstanding characteristics according to the comprehensive score value F and target traits. 【Conclusion】 The variability of sorghum germplasm resources in panicle traits was rich and diverse; the coefficient of variation of corneous endosperm rate and primary branches length was high; grain length, grain width, grain hardness, grain density, and kernel weight per panicle were significantly affected by environmental conditions. We screened 29 superior germplasm.

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    Establishment of High Efficient Extraction and Detection Technology of Sesamin and Screening of High Sesamin Germplasm
    ZHONG YanPing,SHI LiSong,ZHOU Rong,GAO Yuan,HE YanQing,FANG Sheng,ZHANG XiuRong,WANG LinHai,WU ZiMing,ZHANG YanXin
    Scientia Agricultura Sinica    2022, 55 (11): 2109-2120.   DOI: 10.3864/j.issn.0578-1752.2022.11.003
    Abstract272)   HTML35)    PDF (3898KB)(83)       Save

    【Objective】 Establish an efficient extraction and detection technology for sesamin content evaluation in sesame seeds, then apply it to examine sesamin content variation and identify high-sesamin content materials in sesame germplasm, which will lay the foundation for advancing basic research and breeding of sesame with high sesamin content. 【Method】 Using sesame seeds as raw materials, the single factor tests, including diameters of steel balls, crushing time, crushing amplitude, and seeds weight, were investigated. The seeds were extracted with 80% ethanol via ultrasonic mechanical crushing and cavitation, and the sesamin content was determined by High Performance Liquid Chromatography (HPLC). Based on the Box-Behnken center combined test design principle, we carried out the response surface test with four factors and three levels and established the quadratic polynomial regression equation model with sesamin content as response value. We drew the response surface diagram and contour diagram and determined the main factors affecting sesamin content and the interaction between factors. Finally, the optimal extraction conditions of sesamin were determined, and 1 151 local resources and innovative germplasm were analyzed. The accessions with high sesamin content≥9 g·kg-1 were selected and identified at the Oil and Product Quality Supervision and Testing Center of the Ministry of Agriculture and Rural Affairs. 【Result】 The regression model established in this experiment was extremely significant (P<0.05), and the lack of fit was insignificant (P=0.1768). The model had a good fitting degree and could be used to predict sesamin content. The influence of the four factors on sesamin content was as follows: crusher amplitude>diameters of steel balls>crushing time>seeds weight. The interaction between the crusher amplitude and diameters of steel balls was significant, while that between crusher amplitude and crushing time was close to the significance level. The optimal conditions for ultrasonic-assisted extraction of sesamin were as follows: steel ball diameter of 6.5 mm, crushing time of 225 s, crushing amplitude of 1 335 r/min, and seeds weight of 0.20 g. Under these conditions, the sesamin content detected was 4.601 g·kg-1, which was consistent with the predicted value of 4.633 g·kg-1. Fifteen varieties with specific high sesamin content were identified from 1 151 sesame germplasm for breeding purposes. The highest content was 14.36 g·kg-1, and the average content was 12.35 g·kg-1.【Conclusion】 An efficient extraction technology of sesamin was established,compared with the traditional method, the method only needed 0.2 g seeds, which improved the extraction efficiency and reduced sample consumption, the operation was simple, and the sesamin content in sesame seeds could be accurately detected.

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    Genome-Wide Association Study of Grain Quality Related Characteristics of Spring Wheat
    YAN YongLiang,SHI XiaoLei,ZHANG JinBo,GENG HongWei,XIAO Jing,LU ZiFeng,NI ZhongFu,CONG Hua
    Scientia Agricultura Sinica    2021, 54 (19): 4033-4047.   DOI: 10.3864/j.issn.0578-1752.2021.19.001
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    【Objective】Identify SNPs and candidate genes that are significantly related to wheat grain quality traits, and reveal their genetic mechanism. 【Method】In this study, 298 introduced varieties (lines), Xinjiang landrace (lines) and Xinjiang bred varieties (lines) were used for an association population. Seven grain quality traits, including protein content (PRC), wet gluten content (WGC), sedimentation value (SV), starch content (STC), grain hardness (GH), flour yield (FY) and test weight (TW), were measured under five environments. Based on phenotypes of seven quality traits and genotypes of 55K SNP markers in this population, the Q+K association mixed model was used for genome-wide association analysis to obtain significantly associated SNP loci.【Result】The coefficients of variation of the seven grain quality traits of introduced varieties (lines), landraces and bred varieties (lines) under different environments were 1.3%-13.4%, 1.1%-18.6% and 1.0%-13.9%, respectively. Among them, the protein content, wet gluten content and sedimentation value of introduced varieties (lines) have the highest coefficient of variation (CV); Xinjiang bred varieties (lines) have the largest CV of starch content, grain hardness and flour yield. Whereas, for other six grain quality traits, including protein content, wet gluten content, sedimentation value, starch content, grain hardness, and the coefficients of variation of Xinjiang landraces are all between those of the introduced varieties (lines) and Xinjiang bred varieties (lines). Population structure analysis showed that 298 wheat varieties (lines) can be divided into 3 subgroups. Subgroup 1 contains 128 (43.0%) the materials mainly from landrace (lines); Subgroup 2 has 24 (8.1%) materials, mainly including introduced varieties (lines) and landraces; Subgroup 3 contains 146 (48.9%) materials, mainly introduced varieties (lines). The linkage disequilibrium analysis showed that the LD attenuation distances of the A, B and D genomes and the whole genome respectively were 10, 10, 6 and 8 Mb, according to the LD attenuation distance of the whole genome, the loci in the 8 Mb interval after the physical map were identified as a candidate loci. A total of 85 loci were simultaneously detected in two or more environments, that were significantly associated with 7 wheat grain quality traits detected by GWAS, with a contribution rate of 3.7%-10.9%. Stable SNPs associated with multiple traits were detected on chromosomes 1B, 1D, 2D, 3A, 3D, 4A, 4B, 5A, 6A, 6D, 7A and 7D. Among them, AX-109452823-AX-110545157 on chromosome 7A is related to protein content, starch content, wet gluten content, sedimentation value, flour yield and grain hardness, and was detected across four environments. Candidate genes at stable loci associated with multiple traits were searched, and 10 candidate genes that might be related to wheat grain quality were screened. Among them, TraesCS4A01G299800 (cationic amino acid transporter), TraesCS7A01G059500 (tryptophan decarboxylase), TraesCS7A01G331200, TraesCS7D01G418700 (xyloglucan endoglucosylase/hydrolase) play important roles in regulating the amino acid content in wheat grains.【Conclusion】The 85 loci were simultaneously detected in two or more environments, and 10 candidate genes related to wheat grain quality traits were predicted.

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    Genome-Wide Identification of VQ Gene Family in Fagopyrum tataricum and Its Expression Profiles in Response to Leaf Spot Pathogens
    ZHENG FengSheng,WANG HaiHua,WU QingTao,SHEN Quan,TIAN JianHong,PENG XiXu,TANG XinKe
    Scientia Agricultura Sinica    2021, 54 (19): 4048-4060.   DOI: 10.3864/j.issn.0578-1752.2021.19.002
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    【Objective】VQ gene family plays important roles in plant growth, development and responses to biotic or abiotic stress. The aim of this study is to comprehensively identify Fagopyrum tataricum L. Gaertn. VQ (FtVQ) gene family on genome-wide scale and analyze its expression profiles under challenge of leaf spot pathogens Alternaria. alternata and Nigrospora osmanthi, and treatment of defense-related hormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), thus providing a solid foundation not only for further elucidation possible roles of members of the VQ genes in defense response to leaf spot pathogens and underlying mechanisms in tartary buckwheat, but also for mining gene resources of breeding for crop disease resistance. 【Method】Based on the Hidden Markov Model profile of the conserved VQ domain (PF05678), HMMER 3.0 software was used to identify FtVQ genes from F. tataricum cv. Pinku1 genome database. Bioinformatic tools such as DNAMAN, MapInspect, MEGA, MEME, OrthoFinder and PLACE were used to analyze gene structure, chromosomal location of genes, cis-elements of gene promoters, physicochemical properties of proteins, conserved motifs of proteins, subcellular localization of proteins, and phylogenetic relationships. Quantitative real-time PCR (qPCR) was employed to analyze the expression profiles of leaf FtVQ genes of tartary buckwheat plants under infection of the pathogens and treatment of the hormones. 【Result】A total of 28 VQ genes were identified in the genomes of tartary buckwheat, with the gene size ranging from 566 to 1454 bp. The FtVQ genes contain no introns, and distribute unevenly on chromosomes 1-8. According to their physical locations on the chromosomes, the FtVQ genes were named from FtVQ1 to FtVQ28. Each of the FtVQ proteins has a highly conserved VQ motif FxxxVQx (L/F/I/V/A/Y) TG, where x represents any amino acid. Analysis of subcellular localization showed that 21 FtVQ proteins were predicted to the nucleus, and the others to the chloroplasts or cytoplasm. Based upon their amino acid sequence and presence of various conserved motifs, the FtVQ proteins were classified into five subfamilies (Ⅰ-Ⅴ). Each subfamily shared relatively conserved gene structures and protein motifs. The analysis of gene duplication revealed that F. tataricum genome had 8 pairs of paralogous pairs, all of which were segmental duplicated genes, suggesting that segmental duplication played major roles in FtVQ gene expansion. The ratio of nonsynonymous to synonymous substitutions (Ka/Ks) of paralogous pairs was less than 1, suggesting that they underwent purifying pressure during the evolution process. Prediction of cis-elements showed that pathogen-, SA-, JA-, or ET-responsive elements, such as BIHD1OS, CGTCA, ERELEA4, W-box and W-box-like sequences, were present within the promoters of all the FtVQ genes. Especially, FtVQ10, FtVQ14, FtVQ15, FtVQ22, FtVQ23 and FtVQ27 contained more elements in their promoter regions. 55% to 70% of the detectable FtVQ genes were differentially expressed genes (DEGs), and 72.7% to 85.7% of the DEGs were significantly induced on the level of transcription under the infection of leaf spot fungi A. alternata and N. osmanthi, or the treatment of SA, methyl jasmonate and ethephon. 【Conclusion】The tartary buckwheat genome contains 28 members of VQ gene family. Some FtVQ genes may be involved tartary buckwheat defense response to leaf spot pathogens.

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    Cloning and Function Analysis of MsMAX2 Gene in Alfalfa (Medicago sativa L.)
    MA Lin,WEN HongYu,WANG XueMin,GAO HongWen,PANG YongZhen
    Scientia Agricultura Sinica    2021, 54 (19): 4061-4069.   DOI: 10.3864/j.issn.0578-1752.2021.19.003
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    【Background】 Branching is one of the key yield components, which plays an important role in alfalfa (Medicago sativa L.) breeding. Exploring and functional characterization of key branching-related genes are of significance in accelerating breeding of alfalfa with high yield and quality. MAX2 is an important branching-related gene, which is involved in the regulation of branching in several plant species. 【Objective】 Our research on the functional characterization of MsMAX2 in alfalfa will lay a foundation for the molecular mechanism of MsMAX2 in regulating branch development in alfalfa. 【Method】 The gene sequence of MAX2 in alfalfa was isolated by using homologous cloning. Sequence characteristics and phylogenetic tree of MsMAX2 were analyzed by using bioinformatics tools including Expy Protparatam, DNAMAN, and MEGA-X. The real-time quantitative PCR (qPCR) was applied to detect the tissue-specific expression pattern of MsMAX2 in alfalfa. The subcellular localization of the MsMAX2 protein was determined by using transient expression system in tobacco. The biological function of MsMAX2 was clarified by transformation in the Arabidopsis mutant via Agrobacterium-mediated transformation. Proteins interacting with MsMAX2 were determined by using yeast two-hybrid assay. 【Result】The length of MsMAX2 CDS is 2 136 bp, encoding a protein of 711 amino acids, and it belongs to the F-box protein super-family. Phylogenetic analysis showed that the evolution of MAX2 homologs was highly similar to the differentiation of species, indicating that MsMAX2 was a functionally conserved gene. It was showed that MsMAX2 was expressed in the neck at the highest level, followed by in the leaves of seedling, the inflorescences on pollination day and the roots; the expression level of MsMAX2 was relatively low in other tissues, indicating it functions in multiple tissues. Subcellular localization assay showed that the MsMAX2 protein was localized in the nucleus. Complementation assay in Arabidopsis max2 mutant showed that the multi-branch phenotype was recovered by the ectopic expression of MsMAX2. Yeast two hybrid assay demonstrated that the interaction between MsMAX2 and hormone receptor D14 depended on the existence of strigolactones. 【Conclusion】The MsMAX2 was obtained from alfalfa and it was highly expressed in the neck and the encoding MsMAX2 protein was localized in nucleus. When the MsMAX2 was over-expressed in the Arabidopsis max2 mutant, its multi-branch phenotype was recovered, indicating that MsMAX2 regulates branch development in alfalfa plant, and its function was conserved.

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    Gene Mapping and Candidate Gene Analysis of Grain Width Mutant gw87 in Rice
    ZHANG XiangYu,GUO Jia,WANG San,CHEN CongPing,SUN ChangHui,DENG XiaoJian,WANG PingRong
    Scientia Agricultura Sinica    2021, 54 (12): 2487-2498.   DOI: 10.3864/j.issn.0578-1752.2021.12.001
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    【Objective】Phenotypic identification, genetic analysis, gene mapping and candidate gene analysis of grain width mutant gw87 (grain width87) were performed in rice, so as to lay a good foundation for further understanding molecular mechanism and utilization potentiality of this gene regulating rice grain size.【Method】Through ethyl methanesulfonate mutagenesis, we isolated gw87 mutant showing significant increase of grain width and 1000-grain weight from indica restorer line material 676R. To identify phenotypic and physiological characteristics of the mutant, we performed observation of phenotypes, investigation of agronomic traits, sensitivity analysis of exogenous brassinolide (BL), determination of chlorophyll contents and photosynthetic parameters by using gw87 and its wild type. To analyze its genetic behavior, we investigated F1 phenotypes and F2 segregation of the crossing combination between gw87 and its wild-type parent 676R. To find candidate gene of gw87, we conducted high-throughput sequencing and MutMap analysis of 30 mutant plants from the F2 population of gw87×676R, and molecular mapping using the F2 population crossing gw87 and japonica cultivar Nipponbare. Meanwhile, we confirmed the candidate gene by sequencing its DNA and cDNA sequences. In addition, we detected expression levels of brassinolide synthesis pathway genes OsDWARF4, D11 and D2 using qRT-PCR analysis.【Result】Compared with the wild-type parent 676R, the gw87 mutant showed significant reduction of productive panicle number per plant, main panicle length and seed setting rate, but significant increase of grain width and 1000-grain weight. Meanwhile, the mutant displayed decreased plant height and shortened internodes, in which the first internode was distorted and its length is shortened the most. Besides, leaf length of gw87 was decreased but the width was increased. Sensitivity assays of exogenous BL suggested that the sensitivity of gw87 seedlings to exogenous BL was reduced. Determination of photosynthetic pigments and photosynthetic parameters showed that chlorophyll content and net photosynthetic rate were decreased. Genetic analysis revealed that the mutant phenotype of gw87 was governed by a single recessive nuclear gene. High-throughput sequencing and MutMap analysis suggested that the gw87 locus was located in the middle of chromosome 5, and only one base mutation causing amino acid change of the encoded protein occurred in this chromosome region. Molecular marker linkage analysis demonstrated that the gw87 gene was mapped to a 101 kb genomic region between InDel markers X2 and X3. The MutMap and molecular mapping revealed that the candidate gene was LOC_Os05g32270 that encodes a transcription factor containing the AP2/EREBP DNA-binding domain. Subsequently, we confirmed the candidate gene by sequencing its DNA and cDNA. In the gw87 mutant, a single nucleotide G-to-A substitution occurred at position 1041 in its DNA sequence, and the 76-bp intron sequence adjacent to this mutated nucleotide was spliced into exon in its cDNA, which caused shift of the reading frame and then premature termination of the protein translation. In addition, qRT-PCR analysis showed that the expression of BR synthetic genes were significantly up-regulated in gw87, indicating that BR signal was weakened in the mutant.【Conclusion】gw87 is a new allelic mutant of smos1, shb, rla1, and ngr5. However, unlike these mutants, gw87 exhibited significant increase of grain width and 1000-grain weight relative to its wild type. The reason for the differentiation could be that different mutation sites in the LOC_Os05g32270 gene, resulting in different functional activities of the encoded protein.

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    Genetic Diversity and Comprehensive Evaluation of Phenotypic Traits in Sea-Island Cotton Germplasm Resources
    YANG Tao,HUANG YaJie,LI ShengMei,REN Dan,CUI JinXin,PANG Bo,YU Shuang,GAO WenWei
    Scientia Agricultura Sinica    2021, 54 (12): 2499-2509.   DOI: 10.3864/j.issn.0578-1752.2021.12.002
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    【Objective】The study is an attempt to the genetic diversity relationship of phenotypic traits of Sea-island cotton germplasm and screen Sea-island cotton germplasm with excellent traits, which provides a theoretical basis for in depth research on quality traits of Sea-island cotton.【Method】Genetic diversity analysis and comprehensive evaluation were carried out with 12 phenotypic traits of 175 Sea-island cotton. Principal component analysis, weights and the use of membership function to generate comprehensive evaluation value D for comprehensive evaluation of Sea-island cotton germplasm resources.【Result】The results (showed that) Sea-island cotton have rich types and consistent with the range of phenotypic traits was 6.40%-28.10%,which the resource genetic diversity index is 1.97 to 2.05, the diversity analysis significant differences in genetic diversity of Sea-island cotton resources between Xinjiang and outside Xinjiang (P<0.05); 3 comprehensive factors were converted from principal component analysis 12 traits, which the contribution rates are: 49.34%, 18.03% and 10.63%, with a total contribution rate of 78%; The larger load of the first principal component is the number of envoys, the height of envoys, the number of effective branches, the number of bolls, the number of effective bolls, the weight of seed cotton per plant and the weight of lint per plant, representing growth and effective yield factors; The larger load of the second component is lint, single boll seed cotton weight and single boll lint weight, which represents the single boll yield factor; The third component load is the plant height and the number of boll shedding, representing plant height and boll shedding factor; The statistical analysis of Sea-island cotton resources evident that the difference was not significant in extreme germplasm at domestic , and there are more materials with medium-performing Sea-island cotton resources in China. In China, the proportion of extreme germplasm outside Xinjiang is relatively high, and the difference was not significant between Xinjiang and foreign intermediate germplasm; Cluster analysis 175 Sea-island cotton resources then divided into 4 groups. Group I has discrepancy germplasm with short stalk of gravity, low yield; Group II has high lint percent and extensive yield of lint yield per plant, and has higher potential yielding germplasm; Group III has perferable comprehensive traits and excellent germplasm; group IV is short-stalked, high-lint, and extremely particular germplasm; comprehensive evaluation screens out 2 excellent comprehensive traits varieties XH30 and 270; By stepwise regression screening Sea-island cotton germplasm screening phenotype of five key indicators (plant height first node height, number of fruit branches, lint weight per boll and cotton weight per seed). 【Conclusion】The genetic diversity of the tested Sea-island cotton germplasm resources is relatively high, but the genetic diversity is low. The plant height, fruit branch number and lint yield per plant are normally distributed among the genotypes, those left traits are skewed. The tested germplasm divided into 4 categories; Plant height, height of the first node, fruit branch number, yield of single boll Lint yield, seed cotton yield per plant, the research provides a theoretical reference to selection of excellent germplasm for sea-island cotton germplasm resources that 5 indicators can be used as comprehensive indicators for the evaluation of core germplasm.

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    Precise Evaluation of 48 Maize Inbred Lines to Major Diseases
    ZHAO ZiQi,ZHAO YaQi,LIN ChangPeng,ZHAO YongZe,YU YuXiao,MENG QingLi,ZENG GuangYing,XUE JiQuan,YANG Qin
    Scientia Agricultura Sinica    2021, 54 (12): 2510-2522.   DOI: 10.3864/j.issn.0578-1752.2021.12.003
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    【Objective】 In order to provide valuable resources to the maize community for enhancing disease resistance, we selected 30 core maize inbred lines from Shaan A and Shaan B heterotic groups, and 18 major maize germplasms worldwide for disease resistance evaluation and resistance gene identification.【Method】Gibberella stalk rot, Gibberella ear rot, northern leaf blight (NLB) and southern leaf blight (SLB) were artificially inoculated, while gray leaf spot (GLS) was naturally infected in replicated field trials at different locations in 2019 and 2020. A randomized complete block design was adopted with two to three replicates being evaluated at each location. Sorghum grain inoculum was artificially inoculated to the whorl of each plant at the seedling stage for NLB and SLB. Gibberella stalk rot was artificially inoculated using corn grain inoculum buried next to the root of each plant at heading time, while Gibberella ear rot was artificially inoculated by injecting conidial suspension through the silk channel and the husk separately. The best linear unbiased predictions (BLUPs) were estimated and correlation coefficients were calculated between each pair of the disease resistance. Disease resistance genes were genotyped using functional markers. 【Result】Nine highly resistant (HR) lines for NLB, two HR lines for SLB, ten resistant (R) lines for GLS, five HR lines for stalk rot, and five R lines for ear rot resistance were identified out of the 48 maize germplasms. Eight inbred lines showed multiple disease resistance to the three foliar diseases, such as 1145, CML170, and so on. We identified seven elite inbred lines showing good performance for all the five diseases, including 1145, CML170, KA105, KB020, X178, Shen137, and Zheng58. NLB, SLB, GLS, and Gibberella stalk rot resistance were significantly positively correlated, while Gibberella ear rot was not correlated with the other four diseases. 1145, KA081, and Shen137 contain the resistant qRfg1 allele. KB109 harbors the resistant Rcg1 allele. Many inbred lines carry the resistant Htn1 and ZmCCoAOMT2 alleles.【Conclusion】1145, CML170, KA105, KB020, X178, Shen137, and Zheng58 could be selected for multiple disease resistance improvement in breeding program. Shen137 is the best line for marker-assisted selection for qRfg1, Htn1, and ZmCCoAOMT2 loci.

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    Characterizations of Transcriptional and Haplotypic Variations of SiTOC1 in Foxtail Millet
    ZHANG LinLin,ZHI Hui,TANG Sha,ZHANG RenLiang,ZHANG Wei,JIA GuanQing,DIAO XianMin
    Scientia Agricultura Sinica    2021, 54 (11): 2273-2286.   DOI: 10.3864/j.issn.0578-1752.2021.11.003
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    【Objective】 Identification of allelic variations of heading date adaptation related genes and laying foundation for breeding of wide-adapted varieties in foxtail millet. 【Method】In this trial, a vital regulator of heading time in foxtail millet, SiTOC1, was identified using genome-wide association analysis. Spatio-temporal transcription (multi-omics database for Setaria italica,MDSi), sub-cellular localization and 24 hours rhythm expression pattern of SiTOC1 was analyzed. Sequence variations of both promoter and encoding regions in SiTOC1 and relationships between haplotypic variations and heading date were characterized in 99 foxtail millet accessions. 【Result】A significant GWAS signal (Position: 31 456 761 bp) was detected on Chromosome 1 and only one TOC1 homologue was identified (SiTOC1). SiTOC1 highly expressed in root, stem and leaf, and located into cell nucleus. An elevated expression of SiTOC1 was identified at dusk across whole day transcription survey under short-day environment. Many haplotypic variations of SiTOC1 were identified but REC and CCT domains of SiTOC1 were conserved in foxtail millet accessions, and two main haplotypes including H-2 and H-6 in protein encoding regions combined with two co-segregated haplotypes including Hp-591C and Hp-591A were identified. Nearly 2.5 times higher expression of Hp-591C haplotype combined with 9,11 and 12 days delay of heading time through Hainan, Changzhi and Urumuqi were observed. 【Conclusion】The major haplotype Hp-591A identified at 591 bp in the promoter region of SiTOC1 matures earlier than Hp-591C and could be selected as a main effective locus for molecular breeding of foxtail millet.

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    Genetic Research Advances on Maize Stalk Lodging Resistance
    WANG XiaQing,SONG Wei,ZHANG RuYang,CHEN YiNing,SUN Xuan,ZHAO JiuRan
    Scientia Agricultura Sinica    2021, 54 (11): 2261-2272.   DOI: 10.3864/j.issn.0578-1752.2021.11.002
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    Maize stalk lodging has a great adverse effect on yield, quality and mechanized harvesting, and is one of the main problems to be solved urgently in current maize production and breeding. Strengthening the research on the lodging resistance of maize stalk will have great significance for improving the lodging resistance of maize. In this paper, we summarize the main factors affecting maize stalk lodging resistance, and their genetic mechanisms. The stalk lodging resistance is closely related to the stalk strength. The greater the stalk strength, the stronger the lodging resistance. The stalk strength is affected by the developmental stage, the internal and external structures of the stalk, and the components of the stalk cell wall. The meristem zone has vigorously dividing cells and is easily broken. After entering the reproductive growth, the rind and sclerenchyma tissue of the stalk are thickened, the vascular bundles are mature, and thus the stalk strength is enhanced. The main components of the stalk cell wall, including cellulose, hemicellulose, lignin, soluble sugars, inorganic substances, can improve the strength of the stalk. To date, based on the high-throughput phenotyping platforms, various maize linkage and natural populations, and mapping methods, researchers have identified a series of QTLs and candidate genes that affect stalk morphology, strength, and cell wall components. The studies have shown that the haplotype-based mapping method is better than SNP-based mapping method. Meta-QTL analysis integrates the mapping results of different genetic populations and can improve the versatility of QTLs. The genetic basis of stalk strength is complex, which is determined by polygenes with minor effect and additive effect. Candidate genes in the QTLs involve cell wall metabolism, transcription factors, protein kinases, and so on. MAIZEWALL is an important database of genes related to maize cell wall. So far, the database contains 1 156 candidate genes related to maize cell wall biology, which provides a powerful resource for research in this field. A series of genes affecting cell wall components, stalk morphology and stalk strength in maize have been identified. Their functions of these genes are related to cellulose synthesis pathways, such as genes of cellulose synthase, Cobra, glycosyltransferase and ribose transport; phenylpropane pathway genes, such as genes regulating bm1-bm5; plant hormones genes, such as genes related to gibberellin, auxin and brassinosteroid; transcription factors such as NAC, MYB; miRNA (ZmmiR528) and F-box genes (stiff1). In the future research, it is needed to explore the mechanical mechanism of stalk lodging at different developmental stages. Develop diverse natural populations and breeding materials for genetic analysis. Employ a various of mapping strategies to improve the efficiency of identification of the QTL and genes related to lodging resistance. Design various molecular markers based on the favorable alleles to improve the molecular marker assisted selection for lodging resistance. These efforts will promote the research of the genetic mechanism of stalk lodging resistance, and provide a reference for the molecular breeding of new varieties with strong lodging resistance.

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    Genome-Wide Association Analysis of Superoxide Dismutase (SOD) Activity in Wheat Grain
    WANG JiQing,REN Yi,SHI XiaoLei,WANG LiLi,ZHANG XinZhong,SULITAN· GuZhaLiAYi,XIE Lei,GENG HongWei
    Scientia Agricultura Sinica    2021, 54 (11): 2249-2260.   DOI: 10.3864/j.issn.0578-1752.2021.11.001
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    【Objective】The activity of superoxide dismutase (SOD) in wheat grains has a significant effect on the color and nutritional quality of wheat flour. Identification of associated loci and candidate genes for SOD activity in wheat grains is important for discovering the genetic mechanism of SOD activity in wheat grains and genetic improvement of wheat flour color. 【Method】The SOD activity of 212 common wheat varieties (lines) planted in 3 environments was detected by photoreduction method of nitro-blue tetrazolium (NBT), and the genome-wide association study (GWAS) of SOD activity in wheat grains was carried out by 16 705 high-quality SNP markers of 90K SNP chip, and candidate genes of significantly associated loci of stable inheritance were identified. 【Result】The phenotypic variation of SOD activity among wheat varieties (lines) was significant in different environments, with the coefficient of variation ranging from 4.34% to 5.23%, the correlation coefficient ranging from 0.60 to 0.90 (P<0.001). Polymorphic information content (PIC) ranging from 0.24 to 0.29 and the whole genome linkage disequilibrium (LD) attenuation distance of 7 Mb. The analysis of population structure showed that the tested materials could be divided into 3 subgroups. GWAS analysis showed that 29 loci (P≤0.001) were significantly associated with SOD activity, which were distributed on chromosomes 1A, 1B, 2A, 2B, 2D, 3B, 3D, 4B, 4D, 5A, 5B, 5D, 6A, 6B, 6D and 7B. A single locus can explain the phenotypic variation(R2) between 5.47% and 32.43%, of which 14 loci were detected in 2 or more environments. Nine significant associated loci were detected in three environments, distributed on chromosomes 1B, 2B, 4B, 5A, 5B, 6B and 6D, with a contribution rate of 6.21%-16.62%. SOD genes of TraesCS2B01G567600, TraesCS3D01G069900, TraesCS3D01G070200, TraesCS5B01G525700, TraesCS6A01G021400 and TraesCS6D01G431500, and SOD-activity-related candidate genes of TraesCS5A01G263500 and TraesCS6B01G707800 were used to identify the candidate genes of significantly associated loci of stable inheritance. The functions of the candidate genes were mainly related to the inhibition of cell reactive oxygen species accumulation and the participation in antioxidant regeneration. 【Conclusion】Twenty-nine SNP loci associated with SOD activity in wheat grains were detected, and 7 SOD genes and 2 candidate genes related to SOD activity were screened out.

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    OsCSC11 Mediates Dry-Hot Wind/Drought-Induced Ca2+ Signal to Regulate Stamen Development in Rice
    REN ZhiJie,LI Qian,SUN YuJia,KONG DongDong,LIU LiangYu,HOU CongCong,LI LeGong
    Scientia Agricultura Sinica    2021, 54 (10): 2039-2052.   DOI: 10.3864/j.issn.0578-1752.2021.10.001
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    【Objective】When rice is occasionally stressed by dry-hot wind (DHW) or drought at flowering stage, the yield greatly decreases due to the rapid water loss in developing gamete cells. During this process, calcium as a universal second messenger mediates cellular signal transduction in response to drought or other stresses. However, the underlying molecular mechanism remains elusive. This study attempts to dissect the physiological and molecular function of Oryza sativa Calcium-permeable Stress-responsive Cation channels (OsCSCs) which will provide a new strategy for studying the stress responsive mechanism to DHW in crops.【Method】Based on the methods of genetics, electrophysiology and bioinformatics, a typical receptor-like-channel named OsCSC11 was identified. The expression pattern of OsCSC11 were analyzed by qRT-PCR and GUS staining. Further, subcellular localization of OsCSC11-GFP was observed in Arabidopsis protoplasts and onion epidermal cells. Meanwhile, the oscsc11 mutants were generated by CRISPR/Cas9 gene editing tool. Finally, the phenotype and physiological functions were analyzed by cytological method. 【Result】Sequence alignment results in DUF221 family revealed that OsCSC11 includes a typical conserved domain and some unique motifs, which belongs to an independent subfamily. OsCSC11 is mainly expressed in anthers and leaves in rice. Further analysis showed that the activity of full-length protein of OsCSC11 which is in a resting state can be activated by the hypertonic solution. However, OsCSC11ΔTM1-3(TM1-3 was truncated in OsCSC11) has constitutive channel activity that specifically mediates divalent cations of calcium and magnesium. Thus, we speculated that TM1-3 is a receptor domain in CSCs/OSCAs channel for sensing DHW stresses, and the rest part of OsCSC11 (OsCSC11ΔTM1-3) generates calcium signal. OsCSC11 and OsCSC11ΔTM1-3 are both localized in the plasma membrane which may be related to the receptor function. In loss of function mutants oscsc11-1 and oscsc11-2, anthers become smaller and wrinkler compared to wild type, and pollen sterility rate reached to 60%-70% and water content dramatically reduced.【Conclusion】OsCSC11 functions in regulating anther water status and pollen development through mediating calcium influx and possibly involves in the primary sensing step under DHW stresses.

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    Genome-wide Association Analysis of Wheat Grain Size Related Traits Based on SNP Markers
    ZHANG Fang,REN Yi,CAO JunMei,LI FaJi,XIA XianChun,GENG HongWei
    Scientia Agricultura Sinica    2021, 54 (10): 2053-2063.   DOI: 10.3864/j.issn.0578-1752.2021.10.002
    Abstract538)   HTML60)    PDF (1525KB)(521)       Save

    【Objective】Grain traits are important factors affecting wheat yield. the significant locus of controlling wheat grain traits was explored by genome-wide association analysis of wheat grain traits, which provided a theoretical reference for the study of genetic improvement of wheat grain traits. 【Method】The genome-wide association analysis (GWAS) based on mixed linear model MLM (Q+K) was performed on 121 wheat grown in Xinjiang using wheat 50 K SNP chips for 6 traits which including grain length, grain width, grain length-width ratio, grain area, grain perimeter and 1000-grain weight.【Result】Six grain traits showed wide phenotypic variation in different environments, in which the maximum coefficient of variation of 1000-grain weight was 13.91-17.79 and the heritability of each grain trait was between 0.85-0.90. The polymorphism information content PIC value was 0.09-0.38, and the minimum allele frequency MAF value was 0.05-0.50. Group structure analysis shows that the natural groups used in the experiment can be divided into 4 subgroups. GWAS results showed that a total of 592 significant association sites (P<0.001) were detected in 6 traits, of which 29 SNPs were repeatedly detected in 2 or more environments, distributed in 1A(5), 1B(2), 1D, 2A(5), 3B, 5A, 5D, 6B(4), 6D, 7B and 7D(7) chromosomes, can explain 9.3% to 22.7% of the phenotypic variation. Six markers associated with stable grain length were detected, which distributed on 1A, 2A, and 7D chromosomes to explain the phenotypic variation of 9.9%-22.7%. Two markers associated with stable grain width were detected, which distributed on 3 B and 5 D chromosomes to explain the phenotypic variation of 9.6%-12.2%. Six markers associated with stable grain length-width ratio were detected, which distributed on 2A(2), 5A, 7B, and 7D(2) chromosomes to explain the phenotypic variation of 10.1%-19.4%. Three markers associated with stable grain area were detected, which distributed on 1A, 1B and 1D chromosome to explain the phenotypic variation of 9.9%-18.2%. Six markers with stable correlation with grain perimeter were detected, which distributed on 1A(2), 2A, 6D and 7D(2) chromosomes to explain the phenotypic variation of 9.3%-22.6%. Six markers associated with stable 1000-grain weight were detected, which distributed on 1B, 2A and 6B chromosomes to explain the phenotypic variation 9.7%-12.9%. Five dominant loci of pleiotropism with were found to control wheat grain traits, which distributed 1A, 2A(2) and 7D(2) chromosomes, explaining the phenotypic variation of 9.9%-22.7%.【Conclusion】In this study, the genetic diversity of the materials was abundant, a total of 29 multi-environment stability loci were found in natural population with 2 or more environmental associated with 6 grain traits.

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    Creation of New Maize Variety with Fragrant Rice Like Flavor by Editing BADH2-1 and BADH2-2 Using CRISPR/Cas9
    ZHANG Xiang,SHI YaXing,LU BaiShan,WU Ying,LIU Ya,WANG YuanDong,YANG JinXiao,ZHAO JiuRan
    Scientia Agricultura Sinica    2021, 54 (10): 2064-2072.   DOI: 10.3864/j.issn.0578-1752.2021.10.003
    Abstract509)   HTML40)    PDF (3919KB)(505)       Save

    【Objective】Fragrance is an important trait for quality improvement of crops. The 2-acetyl-1-pyrroline (2-AP) is the major component of the aroma flavor. BADH2 controls fragrance in plants, and its null or weak alleles lead to 2-AP accumulation. In this study, the fragrance related genes were modified in JING724, a maize elite inbred line invented by Beijing Academy of Agriculture and Forestry Sciences, using CRISPR/Cas9 to improve its trait of fragrance. 【Method】To find BADH gene family of target species, OsBADH2 protein sequence was used to search against protein databases of Arabidopsis, rice and maize with the Ensembl online BLAST tool. All BADH family members were verified by protein domain information in UniProt database. Furthermore, phylogenetic analysis conducted in MEGA software was used to search for maize BADH2 homologs as gene-editing targets. Based on principles of CRISPR/Cas9, we designed highly specific sgRNA to target the candidate genes. The CRISPR/Cas9 vector containing this sgRNA was introduced into the maize variety JING724 by Agrobacterium-mediated transformation. We obtained transgenic maize plants with PMI resistance. Sanger sequencing was used to confirm the CRISPR/Cas9-mediated mutations. Finally, we used gas chromatography-mass spectrometry (GC-MS) to measure the 2-AP content in T1 seeds of the gene-editing lines. 【Result】Phylogenetic analysis showed that there were two BADH2 homologs in the maize gnome, subsequently they were named ZmBADH2-1 and ZmBADH2-2. ZmBADH2-1 is located in chromosome 4, whereas ZmBADH2-2 was in chromosome 1. Both genes have 15 exons and 14 introns. The 4th exon of ZmBADH2-1 shares high nucleotide identity with that of ZmBADH2-2. A specific target sequence, which is located in the 4th exons of both genes, was designed and introduced into a CRISPR/Cas9 vector. Using this vector, 10 gene-editing lines were acquired after maize transformation. PCR amplification and sanger sequencing revealed that, in each of the 10 gene-editing lines, different type of insertions or deletions were introduced into the target sites of both ZmBADH2 genes successfully. Genotypes of mutations included biallelic and multi-allelic mutations. Mess spectra analysis showed that Zmbadh2-1/Zmbadh2-2 double mutants had 2-AP, which is the same substance of flavor with that in fragrant rice. Using GC-MS, we found that 2-AP contents in grains gathered from four randomly selected T0 gene-editing lines were 438.29, 404.63, 348.65 and 161.82 μg·kg-1, respectively. On the contrary, no 2-AP was detected in JING724 wild type. 【Conclusion】With site specific mutations introduced into ZmBADH2-1 and ZmBADH2-2 simultaneously using CRISPR/Cas9, new maize variety with fragrant rice like flavor was created successfully.

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