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    Identification and Candidate Gene Analysis of the ABNORMAL HULL 1 (ah1) Mutant in Rice (Oryza sativa L.)
    ZHANG BiDong, LIN Hong, ZHU SiYing, LI ZhongCheng, ZHUANG Hui, LI YunFeng
    Scientia Agricultura Sinica    2024, 57 (3): 429-441.   DOI: 10.3864/j.issn.0578-1752.2024.03.001
    Abstract198)   HTML35)    PDF (3242KB)(101)       Save

    【Objective】Rice is the staple grain crop worldwide, and the morphology of its grains directly influences its ultimate yield, nutritional excellence, and economic significance. Moreover, the intricate interplay between floral development and grain morphology adds further significance to this relationship. Thus, exploring novel rice floral development regulatory genes and molecular regulatory mechanisms lays the foundation for larger and more plump grains rice varieties. 【Method】Ethyl methyl sulfonate (EMS) was used to mutate XD1B (xian-type maintainer line), and a dwarf mutant abnormal hull 1 (ah1) with abnormal formation of glume and lodicule was identified. The agronomic traits of both the wild-type and mutant were observed and recorded. Spikelets from various flowering stage were collected to histological and morphological analysis. The F2 segregating population was established by ah1 and 56S (xian-type thermo-sensitive sterility line), and utlized for genetic analysis and gene mapping. RNA was isolated from young panicles of both the wild-type and mutant, then reverse transcribed into cDNA. The RT-qPCR analysis was performed to analyze the relative expression levels of the genes regulating floral development and the key genes in the ABA synthesis pathway. 【Result】The observation of agronomic traits revealed that the dwarfed plant was caused by the dramatic shortening of the internodes. At the same time, the mutant is also accompanied by severe spikelet abnormalities and low fruit setting rate. Histological and morphological analysis revealed that the ah1 mutant spikelets exhibited varying degrees of degeneration in floral organs such as palea, lemma, lodicules, and stamens. Some severely affected spikelets displayed altered floral organ characteristics and determinacy of floral meristems, often accompanied by extensive whitening. Based on the extent of degeneration, these spikelets could be classified as slight or severe mutant phenotypes. Genetic analysis showed a segregation ratio of 3﹕1 for the wild-type and mutant within the segregating population, indicating that the mutant traits of ah1 were controlled by a single recessive locus. The AH1 was mapped between the molecular markers RM6716 and RM128 on the chromosome 1, with a physical distance of approximately 8 Mb. Resequencing analysis of the mutant revealed that the LOC_Os01g53450 and LOC_Os01g51860 within this interval showed variation between wild-type and mutant, thus these two genes were provisionally identified as candidate genes. RT-qPCR analysis revealed significant alterations in the relative expression levels of floral organ development regulatory genes during the early developmental stages of mutant panicles; meanwhile, the relative expression levels of OsNCED1/OsNCED2/ OsNCED3/OsNCED4/OsNCED5, the ABA synthesis pathway key genes, were severe inhibited.【Conclusion】AH1 plays a crucial role in the morphological formation of floral organs, such as palea and lemma in rice. LOC_Os01g53450 and LOC_Os01g51860 were provisionally identified as candidate genes in this work.

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    Function Analysis of the Soybean Transcription Factor NAC1 in Tolerance to Low Phosphorus
    XIONG ChuWen, GUO ZhiBin, ZHOU QiangHua, CHENG YanBo, MA QiBin, CAI ZhanDong, NIAN Hai
    Scientia Agricultura Sinica    2024, 57 (3): 442-453.   DOI: 10.3864/j.issn.0578-1752.2024.03.002
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    【Objective】The productivity of acid soil crops is severely impacted by the limited availability of phosphorus. Soybean (Glycine max) is an important grain and oil crop, known for its preference for phosphorus. Phosphorus deficiency significantly affect both the yield and quality of soybean. While the NAC (NAM, ATAF1/2, CUC2) transcription factor family has been recognized for its involvement in regulating plant responses to various biotic and abiotic stresses, its role in soybean under low phosphorus stress remains largely unexplored. In this study, we focused on the low-phosphorus-tolerant wild soybean variety BW69 as our material, with the objective of cloning and analyzing the expression patterns and functions of the low-phosphorus-tolerant gene GsNAC1. This investigation lays the foundation for a deeper understanding the mechanisms behind the regulation of GsNAC1 response to low phosphorus stress. 【Method】The full-length sequence of GsNAC1 was cloned from BW69, and the characteristics of its encoded amino acid sequence were explored by bioinformatics analysis. In addition, the tissue expression patterns of GsNAC1 were examined through quantitative real-time PCR (qRT-PCR). The subcellular localization of GsNAC1 was observed using laser confocal microscopy. Furthermore, soybean genetic transformation experiments were conducted for further phenotype analysis, and RNA-seq analysis was performed to identify differentially expressed genes (DEGs) related to low phosphorus stress. 【Result】The GsNAC1 gene was successfully cloned, with a full-length coding region of 876 bp. Phylogenetic analysis showed a 62.46% sequence similarity between GsNAC1 and AtATAF1, and no difference was observed with the GmNAC1 sequence from the Williams 82 reference genome. Subcellular localization experiments further revealed that GsNAC1 was localized in the nucleus. Using qRT-PCR, it was discovered that GsNAC1 is expressed in roots, stems, leaves, apes, flowers and pods, with the highest relative expression level found in the roots. Notably, GsNAC1 exhibited significant upregulation in response to low pH and low phosphorus conditions. To assess the phenotypic effects, we performed experiments using both hydroponic and soil cultivation methods under low phosphorus conditions. The transgenic lines showed notable increases in root/shoot ratio, total root length, root surface area, root volume, and phosphorus content compared to the wild type (WT). Transcriptome analysis revealed that GsNAC1 may enhance tolerance to low phosphorus stress by promoting the expression of genes such as GmALMT6, GmALMT27, GmPAP27, and GmWRKY21. 【Conclusion】The expression of GsNAC1 was up-regulated by low pH and low phosphorus, and overexpression of GsNAC1 significantly enhanced the tolerance to low phosphorus stress in soybean, playing a promoting role in the response to low phosphorus stress. Besides, GsNAC1 may enhance the tolerance to low phosphorus stress in soybean by regulating the expression of downstream genes.

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    Progress on Genetic Transformation of Sorghum
    HAN LiJie, CAI HongWei
    Scientia Agricultura Sinica    2024, 57 (3): 454-468.   DOI: 10.3864/j.issn.0578-1752.2024.03.003
    Abstract183)   HTML10)    PDF (539KB)(124)       Save

    Sorghum is the fifth largest grain crop in the world and can be used for food, feed, brewing and bioenergy. Sorghum genetic transformation technology is an essential and important tool in the research of sorghum functional genomics and can also serve as an important complement to traditional breeding methods. In this review, we summarize the research progress of sorghum transformation in recent years, analyze the problems in sorghum genetic transformation and propose strategic solutions to them in order to provide a reference for further improvement of sorghum genetic transformation technology. By summarizing more than 50 literatures on sorghum tissue culture and genetic transformation in recent years, we introduced the current research status of sorghum genotypes, explant sources, and regeneration system construction for genetic transformation, and compared the advantages and disadvantages of four commonly used methods for sorghum genetic transformation: electroporation, pollen-mediated transformation, particle bombardment and Agrobacterium-mediated transformation, summarized the effects of the main components of genetic transformation vectors, including promoters, target genes, selective marker genes and reporter genes, on transformation efficiency, explained the current application status of sorghum genetic transformation, analyzed the main bottleneck problemns in sorghum genetic transformation technology, and studied countermeasures. Sorghum genotypes have a significant influence on tissue culture and P898012 and Tx430 are the most widely used. Gene bombardment and Agrobacterium-mediated transformation are the most commonly used methods for sorghum genetic transformation, and the advantages of Agrobacterium-mediated transformation are gradually emerging. In vector construction, CaMV35S and ubi1 are the most commonly used promoters, and antibiotic resistance genes (nptII, hpt), herbicide resistance genes (bar), and nutrient assimilation genes are the three commonly used selection markers. With the development of sorghum genetic transformation technology and CRISPR/Cas9-mediated gene editing technology, some genes with important agronomic traits have been successfully transferred into sorghum. However, strong genotype dependence, long tissue culture cycle, and poor genetic transformation stability are the main bottlenecks that limit the genetic transformation of sorghum. By introducing morphogenesis regulatory factors, somatic cell generation can be directly performed, which shortens the tissue culture cycle, improves the transformation efficiency, and expands the source of explants. This has become a major breakthrough in sorghum genetic transformation technology. The use of morphogenesis regulatory factors and adoption of cut-dip-budding (CDB) delivery system can further improve the sorghum genetic transformation technology. Combined with the application of CRISPR/Cas9 gene editing technology, they will surely provide an important technical basis for the sorghum molecular breeding and gene function identification.

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    CRISPR/Cas9-Mediated Editing of MODD Enhances Rice Dormancy
    GUO NaiHui, ZHANG WenZhong, SHENG ZhongHua, HU PeiSong
    Scientia Agricultura Sinica    2024, 57 (2): 227-235.   DOI: 10.3864/j.issn.0578-1752.2024.02.001
    Abstract344)   HTML45)    PDF (2256KB)(219)       Save

    【Objective】 Dormancy is an important agronomic trait of rice. Proper dormancy can inhibit the preharvest sprouting of rice and is a key factor to ensure yield and quality. However, the genes and regulatory networks of rice dormancy regulation still need further study. The MODD encoded a protein with unknown function, and it negatively regulate rice abscisic acid signaling and drought resistance, but its function in regulating rice dormancy is unknown. Studying the function of MODD in regulating rice dormancy will help to improve the rice dormancy regulatory network, and at the same time provide a new theoretical basis and germplasm resources for genetic breeding of preharvest sprouting resistance.【Method】 Based on the gene sequences published in the RGAP database, a CRISPR-Cas9 knockout vector for MODD was constructed, and the calli of Zhonghua 11 was transformed through agrobacterium mediated genetic transformation to obtain transgenic rice plants. The MODD knockout homozygous lines were screened and identified using PCR amplification, sequencing technology, and qRT-PCR technology. The amino acid sequences of the two mutant lines (KO-1 and KO-2) were obtained according to the CDS of the two mutant lines, and then the protein sequences of ZH11 and the two mutant lines (KO-1 and KO-2) were compared by DNAMAN. The homologous genes of MODD in rice were screened using Linux system. Take the seeds 35 days after heading and investigated the germination rate of ZH11 and knockout lines. The yeast hybridization and LUC experiments were used to verify the upstream gene of MODD. 【Result】 Six MODD homologous genes were found in rice, which were LOC_Os07g41160, LOC_Os03g30570, LOC_Os03g53630, LOC_Os04g35430, LOC_Os03g17050, LOC_Os06g01170. The knockout vector was successfully constructed and transferred it into ZH11, two homozygous mutant lines (KO-1 and KO-2) were obtained. The qRT-PCR results showed that the expression level of MODD in the two mutant line (KO-1 and KO-2) was significantly reduced. Protein sequence analysis showed that the frameshift mutations of KO-1 and KO-2 caused the early termination of protein translation. The germination rate of the two mutant lines (KO-1 and KO-2) was significantly lower than that of ZH11 by 15% and 15% respectively on the third day after water absorption; After that, the difference gradually expanded and reached the maximum on the 6th day, which was significantly lower than that of ZH11 by 35% and 35% respectively. The preharvest sprouting of two mutant lines (KO-1 and KO-2) was significantly lower than that of ZH11. The results of Y1H experiment showed that ABI5 could bind to the promoter region of MODD in yeast, and the binding range was further reduced to less than 300bp. LUC results showed that the fluorescence value of ABI5 was 2.6 times that of none alone, indicating that ABI5 could activate the expression of MODD.【Conclusion】 Knocking out MODD could increase seed dormancy, and MODD may regulate seed dormancy through ABA signaling pathway.

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    The Construction and Application of SSR and SNP Molecular ID for Maize Germplasm Resources of Jilin Province
    ZHANG MingQi, WANG Rui, ZHANG ChunXiao, SUN Bo, REN Jie, LI ShuFang, WANG Lu, ZHU ShaoXi, ZHANG JiangBin, SHI XinChen, WANG HaiJie, ZHANG YunLong, TIAN HongLi, ZHAO YiKun, KUANG Meng, WANG YuanDong, YI HongMei, LI XiaoHui, WANG FengGe
    Scientia Agricultura Sinica    2024, 57 (2): 236-249.   DOI: 10.3864/j.issn.0578-1752.2024.02.002
    Abstract340)   HTML37)    PDF (9255KB)(261)       Save

    【Objective】 Crop germplasm resources hold a crucial strategic position. The Maize Germplasm Resources Bank in Jilin Province safeguards a collection of germplasm resources distinctively representative of the Northern Spring Maize Region. Traditional germplasm resource management faces challenges in ascertaining accurate identity information. To address this issue, molecular marker technology has been employed to establish a process for the construction and classification of molecular IDs for germplasm resources, thereby enabling precise identification and bolstering categorical management. Thorough exploration of the exceptional resources within Jilin Province's Maize Germplasm Resources Bank is intended to advance the shared utilization of these valuable germplasm resources. 【Method】 A total of 2 918 maize germplasm resources were utilized from the Jilin Provincial Maize Germplasm Resources Bank as subjects of the study, the molecular IDs were constructed by using 40 pairs of SSR markers and 61 214 SNP markers recommended in maize variety identification standards. Based on the molecular ID information, the germplasm resources were categorized into core, closely related, heterogeneous, and population groups for management purposes. Furthermore, the core germplasms were analyzed on genetic diversity. 【Result】 In this investigation, the SSR molecular IDs were constructed for 2 918 maize germplasm resources, while the SNP molecular IDs were constructed for 2 502 maize germplasm resources, excluding heterogeneous germplasm. The standards for the construction of SSR and SNP molecular IDs were established for maize germplasm resources. The SSR molecular ID is composed of a combination of three-digit numbers and one-letter code converted from 40 SSR loci fingerprints, stored in the form of a QR code. The SNP molecular ID converts the fingerprints of 61 214 SNP loci into visual barcodes. Based on the features of sample homozygosity and fingerprint specificity, the samples were categorized into 1 561 cores, 705 closely related, 416 heterogeneous, and 236 population types of germplasm resources. Genetic diversity analysis indicates that domestic germplasm resources, represented by Lüdahonggu and Huanggai groups, constituting the main germplasm resources in the Jilin Provincial Maize Germplasm Resources Bank, accounting for 64.38% of all core germplasm resources. 【Conclusion】 This research outlines a methodology for constructing molecular IDs for maize germplasm resources. The SSR molecular IDs were constructed for 2 918 accessions stored in the Jilin Provincial Maize Germplasm Resources Bank and the SNP molecular IDs were constructed for 2 502 among them. The germplasm resources were categorized into core, closely related, heterogeneous, and population types to achieve the classification management.

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    The Effect of indica/Xian Pedigree Introgression in japonica/Geng Rice Breeding in China
    XU HAI, LI XIUKUN, LU JIAHAO, JIANG KAI, MA YUE, XU ZHENGJIN, XU QUAN
    Scientia Agricultura Sinica    2023, 56 (22): 4359-4370.   DOI: 10.3864/j.issn.0578-1752.2023.22.001
    Abstract391)   HTML64)    PDF (4344KB)(337)       Save

    【Objective】To demonstrate the impact of indica/Xian (XI) pedigree introgression on the yield and quality of japonica/Geng (GJ) rice varieties, providing a theoretical basis and genomic resources for optimizing XI pedigree introgression breeding programs in northern GJ rice.【Method】In this study, the whole genome sequence on Illumina platform was employed to elucidate the effects of XI pedigree introgression on the yield and quality of rice in Northeast China were analyzed using recombinant inbred lines (RIL) derived from the cross between XI and GJ varieties, and 74 major GJ varieties grown from Heilongjiang, Liaoning, Shandong, and Jiangsu provinces as test materials. Using CRISPR/Cas9 gene editing technology to knock out the unfavorable genes introduced by XI pedigree introgression. 【Result】Analysis of RIL revealed a significant positive correlation between XI pedigree introgression and panicle length, grain length, and a negative correlation with head rice ratio. XI pedigree introgression was significantly negatively correlated with Amylose content, and significantly positively correlated with protein content in Jiangsu. With the increase of latitude, the correlation efficiency between XI pedigree introgression and grain shape increased, while the correlation between XI pedigree introgression and panicle length and head rice ratio decreased. The genomic fragments of XI pedigree introgression are unevenly distributed across different chromosomes and are more abundantly present on chromosomes 1, 10, 11, and 12. The XI pedigree introgression of the major cultivars in Jiangsu and Liaoning provinces is significantly higher than that in Heilongjiang and Shandong provinces, and the XI pedigree introgression of the cultivars after 2000 is significantly higher than that before 2000. The XI pedigree introgression includes multiple resistance and fertility-related genes. The project identified an XI pedigree introgression fragment on chromosome 5 of YF47, including the XI type grain regulatory gene GS5 and XI type chalkiness regulatory gene Chalk5, which increased the 1000 grain weight of YF47 but affected its chalkiness-related traits. The project uses CRISPR/Cas9 technology to knock out the Chalk5 gene of YF47. The grain shape of the homozygous gene editing plants is similar to those of YF47, and its chalkiness character has been significantly improved. 【Conclusion】The XI pedigree introgression mainly increases the yield potential of GJ rice by increasing the number of grains per panicle, but has a negative impact on milling quality. Exploring the unfavorable alleles in varieties through high-throughput genome sequencing, combined with CRISPR/Cas9 gene editing, to break the genetic drag in breeding using the cross between XI and GJ, is an efficient breeding strategy that can quickly and accurately improve target traits.

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    Development and Application of Top-Down Approaches for Estimating Measurement Uncertainty of GMO Quantitative Results
    LI Jun, ZHAO Xin, CHEN Hong, LI FeiWu, LIANG JinGang, LI YunJing, WANG HaoQian, GAO HongFei, ZHANG Hua, CHEN ZiYan, WU Gang, SHEN Ping, XU LiQun, WU YuHua
    Scientia Agricultura Sinica    2023, 56 (22): 4371-4385.   DOI: 10.3864/j.issn.0578-1752.2023.22.002
    Abstract155)   HTML11)    PDF (567KB)(155)       Save

    【Objective】The enforcement of labeling regulations on genetically modified organisms (GMOs) requires establishing a standard system for accurate quantification of GMOs that includes the standard or guide for estimating measurement uncertainty (MU). It is urgent to establish a standardized top-down approach for estimating MU of quantitative results, which is conveniently adopted by the general testing laboratories. 【Method】There are two approaches for estimating the MU introduced by precision of quantitative method, one is to establish the equation of MU estimation using data obtained on 15 routine samples based on the "uncertainty function", the other is to evaluate the MU by repeatedly measuring a certified reference material (CRM) and calculating the intermediate precision. The uncertainty introduced by bias is evaluated using a CRM or a sample prepared by laboratory as bias control. The uncertainty of the nominal value of the sample prepared by laboratory is evaluated by using a simplified program based on the preparation process. The MU contributed by method precision and bias are combined into the standard uncertainty of the quantitative results, and then multiplied by the coverage factor k to obtain the expanded uncertainty. 【Result】The event-specific PCR method of genetically modified maize DBN9936 was took as an example. The MU of method precision was evaluated to be 0.76% using simulated DBN9936 routine samples, and to be 0.33% using a CRM (GBW (E) 100901). Compared with routine samples, the MU of method precision evaluated using a CRM is significantly underestimated. The uncertainty introduced by bias was evaluated to be 0.26% using a CRM (GBW (E) 100901) as a bias control. Using a laboratory prepared powder sample and a genomic DNA sample (nominal values of 3.0%) as bias control, the bias uncertainty was evaluated to be 0.20% and 0.19%, respectively. Since the simplified program ignored some uncertainty components, the uncertainty of the nominal value of laboratory prepared samples was estimated to be smaller. By combining the MU of method precision and bias, the expanded uncertainty using routine samples was obtained to be 1.26%, 1.20%, and 1.20%, respectively, the expanded uncertainty using a CRM was 0.84%, 0.78%, and 0.76%, respectively. 【Conclusion】This study established the top-down approaches for MU estimation of quantitative results, testing laboratories should prioritize routine samples to estimate the MU contributed by the method precision, and select CRMs as bias control in principle to evaluate bias uncertainty during GMO quantification.

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    The Molecular Mechanism of Pod Yield Difference Between Single- Seeding Precision Sowing and Multi-Seeds Sowing of Peanut Based on Transcriptome Analysis
    YANG Sha, LIU KeKe, LIU Ying, GUO Feng, WANG JianGuo, GAO HuaXin, MENG JingJing, ZHANG JiaLei, WAN ShuBo
    Scientia Agricultura Sinica    2023, 56 (22): 4386-4402.   DOI: 10.3864/j.issn.0578-1752.2023.22.003
    Abstract195)   HTML42)    PDF (5928KB)(341)       Save

    【Objective】In China, in order to ensure the emergence rate and quality of seedlings, the field often adopts multiple seed seeding. However, inter-plant competition in multi-seeds sowing often limits the growth and eventual yield of subsequent plants. In order to solve this contradiction, the team studied and established the high-yield cultivation technology of single-seed precision seeding. The combination of seed saving and yield increase effect of single-seed precision seeding technology can bring greater benefits and realize cost savings and increased efficiency. The differentially expressed genes in peanut leaves, roots and pods under different planting methods were used to explore the regulatory mechanism of single-seeding precision sowing to improve peanut pod yield, providing theoretical basis and technical support for further promoting peanut high yield and high efficiency. 【Method】Peanut variety Huayu 25 was used as the test material, while the yield related indexes of single-seed sowing and multi-seeds sowing were determined. Inverted three leaves, taproot and lateral root of peanut at 30 days after flowering and peanut pod at young fruit stage of chicken head were selected for transcriptome sequencing, and the yield differences of peanut under different sowing methods were revealed on the molecular level. 【Result】Compared with multi-seeds sowing, the pod number per plant, full fruit number per plant, fruit weight per plant and economic coefficient of single-seed sowing were significantly increased. After the transcriptome data is assembled, each library contains an average of 44.3 million readings. Through the analysis of differentially expressed genes, GO and KEGG pathways in different combinations, it was found that the expression levels of transcription factors, photosystem Ⅱ oxygen-releasing complex, chloroplast membrane, oxidation-reduction reaction and other genes involved in the processes of GA signal and light signal transduction were increased in the leaves of plants under single-seed sowing compared with multi-grain cave seeding. Genes related to phenylpropyl metabolism pathway induced by biological and abiotic stress were significantly enriched in roots, including cytochrome P450 gene, oxidation-reduction gene, stress response transcription factor and signal regulatory protein. The accumulation of starch and sucrose metabolism genes was more conducive to seed kernel enrichment during pod development. 【Conclusion】The up-regulated expression of photosynthetic related genes in peanut leaves at seedling stage could promote the increase of photosynthetic efficiency, which was closely related to the increase of yield. Single-seed sowing improved the ability of root system to resist biological and abiotic stress, and combined with the up-regulation of energy and material related genes in the early stage of pod development, it was beneficial for the development of underground peanut pod and increased peanut yield.

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    Analysis of Genetic and Breeding Selection Effects of A Major QTL-qSl-2D for Wheat Spike Length
    DONG JiZi, CHEN LinQu, GUO HaoRu, ZHANG MengYu, LIU ZhiXiao, HAN Lei, TIAN ZhaoSaShuang, XU NingHao, GUO QingJie, HUANG ZhenJie, YANG AoYu, ZHAO ChunHua, WU YongZhen, SUN Han, QIN Ran, CUI Fa
    Scientia Agricultura Sinica    2023, 56 (20): 3917-3930.   DOI: 10.3864/j.issn.0578-1752.2023.20.001
    Abstract259)   HTML33)    PDF (1330KB)(176)       Save

    【Objective】By analyzing the genetic and breeding selection effects of the stable major QTL for spike length in wheat, its genetic effects on yield-related traits were clarified, and the future breeding application potential was evaluated. The results could provide a basis for subsequent gene mining and molecular breeding of wheat. 【Method】A major QTL for spike length, named qSl-2D, was detected in multiple environments using a recombinant inbred lines population derived from the cross of Kenong9204 and Jing411, denoted as KJ-RIL; Two molecular markers closely linked to qSl-2D were developed by using the InDel sites in target interval. The genetic effects of yield-related traits based on KJ-RIL, MY-F2, NILs and natural mapping populations, were analyzed by combining genotype data of molecular markers or wheat 55K array, respectively. By genotyping the natural mapping population, the breeding selection effect of qSl-2D haplotype was parsed across different wheat regions and different ages. 【Result】QTL mapping results showed that qSl-2D could be detected in 7/10 sets of environmental data, and could explain 4.02%-10.10% of the phenotypic variation. The peak LOD of 5/10 sets of environmental data was positioned at 608.75 Mb. The results of genetic effect analysis showed that the enhancing allele of qSl-2D could significantly increase spike length in the four populations with different genetic backgrounds. In addition, it has positive effects on kernel number per spike and plant height, but has negative effects on thousand kernel weight, kernel weight per spike and yield per plant in most population backgrounds. Further analysis of plant height in KJ-RIL population showed that the enhancing allele had rod lowering effect on all internode lengths except the internode length below spike, which resulted in the insignificant increase in plant height. The results of qSl-2D haplotype analysis showed that the utilization rates of the long-spike haplotype Hap-AA-GG varied greatly in different wheat regions, with the highest utilization rate in the northern winter wheat region, accounting for 24%; while the short-spike haplotype Hap-CC-CC accounted for more than 30% in most wheat regions. Moreover, the utilization rate of qSl-2D long-spike haplotype showed a gradual decrease over time, while that of short-spike haplotype consistently maintained a higher selection trend. 【Conclusion】A stable major QTL-qSl-2D for spike length was identified, the enhancing allele of qSl-2D could significantly increase spike length under different genetic backgrounds, and had certain genetic effects on yield-related traits. The closely linked molecular markers developed in the target region can be used for the genetic improvement of wheat spike length and yield-related traits in wheat.

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    Genome-Wide Association Analysis of Yield Traits in Xinjiang Winter Wheat Germplasm
    MA YanMing, LOU HongYao, ZHANG ShengJun, WANG Wei, GUO Ying, NI ZhongFu, LIU Jie
    Scientia Agricultura Sinica    2023, 56 (18): 3487-3499.   DOI: 10.3864/j.issn.0578-1752.2023.18.001
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    Objective】To discover new high yield genes in wheat by association analysis, which can provide technical supports for the innovation and genetic improvement of high yield germplasm resources in wheat.【Method】Totally 188 bread wheat cultivars in Xinjiang were genotyped using the wheat 55K genotyping assay. GWAS was carried out to identify the signifcant single nucleotide polymorphisms (SNPs) which were associated with 9 wheat yield traits in 6 environments. The MLM algorithm in TASSEL5.0 was used to analyze the nine traits related to wheat yield traits.【Result】Totally 1309 SNPs explained 7.259%-70.792% of the phenotypic variation. 38 SNP loci were identifed, which were significantly correlated with 5 plant height weight SNP loci, 10 spike length weight SNP loci, 10 spikelet number SNP loci, 6 fertile spikelet number SNP loci, 6 spike grain number SNP loci, and 1 thousand grain weight SNP loci. These loci can explain 9.10%-23.81% of phenotypic variations. Comparing these 38 loci with the published wheat genome loci, only 3 functional genes were found, which annotated with gene function. There genes are: TraesCS2A01G448800 on chromosome 2A, which is close to the plant height associated site AX-108794050 and is related to the metabolic synthesis of transcription factor bHLH71; TraesCS2A01G448800, located on chromosome 1A at a distance similar to the spike length associated site AX-110689765, is related to protein coding; TraesCS4B01G031100, located on the 4B chromosome at a distance similar to the 1000 grain weight associated site AX-110399975, is associated with the encoding serine/threonine protein kinase SD1-8 and is involved in regulating cell proliferation and differentiation. 【Conclusion】38 QTL loci associated with wheat yield traits were detected. After verification, it was found that the associated excellent alleles have the effect of reducing plant height, increasing spike length, spikelet number, fertile spikelet number, grain number per spike, and thousand grain weight.

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    Genome-Wide Association Study of Nitrogen Use Efficient Traits in Sweetpotato Seeding Stage and Screening and Validation of Candidate Genes
    YU YongChao, FAN WenJing, LIU Ming, ZHANG QiangQiang, ZHAO Peng, JIN Rong, WANG Jing, ZHU XiaoYa, TANG ZhongHou
    Scientia Agricultura Sinica    2023, 56 (18): 3500-3510.   DOI: 10.3864/j.issn.0578-1752.2023.18.002
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    Objective】The objective of this paper was to analyze the genetic mechanisms of nitrogen use efficiency (NUE), and to explore the loci and candidate genes associated nitrogen (N) efficient traits, to provide support for the N-efficient molecular breeding and genetic improvement of sweetpotato.【Method】A total of 129 sweetpotato cultivars from all over the world were treated with N deficiency (0 mmol·L-1) and normal N (14 mmol·L-1). A hydroponic experiment was conducted to facilitate the genome-wide association study (GWAS) of six phenotypic traits (shoot biomass increment, root biomass increment, shoot N accumulation, root N accumulation, shoot N physiological utilization efficiency, and root N physiological utilization efficiency) of sweetpotato at the seedling stage. The N-efficient candidate genes were identified based on the GWAS and subsequently- verified using RT-qPCR.【Result】There were wide variations among the six traits related to NUE in sweetpotato under the normal N and N deficiency treatment conditions. The coefficient of variation (CV) of the shoot biomass increment under the N deficiency treatment condition was the greatest at 69.5%. The CV of the root N physiological utilization efficiency under N deficiency treatment condition was the smallest at 12.1%. All five traits were significantly correlated except for root N physiological utilization efficiency. The MLM model was used to conduct a GWAS of the six phenotypic trait values. A total of 134 QTL and 888 SNP loci were identified as being significantly associated with four out of the six traits, namely, shoot biomass increment, root biomass increment, root N accumulation, and shoot N physiological utilization efficiency. A total of 93 SNP markers across ten regions were significantly associated with shoot N physiological utilization efficiency with a high reliability. Six N efficiency candidate genes were obtained via gene annotation. RT-qPCR verified that the three candidate genes (itf01g08120.t1, itf01g22030.t1 and itf01g221000.t2) encoded glutamate dehydrogenase, NPH3 protein and TIP41-like protein, respectively, which warrants further research.【Conclusion】A total of 888 SNP loci associated with N utilization traits were detected in 129 sweetpotato cultivars. Among these, 93 SNP loci were significantly associated with shoot N physiological utilization efficiency, and six candidate genes were identified. Preliminary verification indicated that the itf01g08120.t1, itf01G2203.t1 and itf01g22100.t2 genes hold promising value for further research.

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    Function of Maize ZCN7 in Regulating Drought Resistance at Flowering Stage
    LI Yan, TAO KeYu, HU Yue, LI YongXiang, ZHANG DengFeng, LI ChunHui, HE GuanHua, SONG YanChun, SHI YunSu, LI Yu, WANG TianYu, ZOU HuaWen, LIU XuYang
    Scientia Agricultura Sinica    2023, 56 (16): 3051-3061.   DOI: 10.3864/j.issn.0578-1752.2023.16.001
    Abstract603)   HTML145)    PDF (1718KB)(646)       Save

    【Objective】The main producing areas of maize is mostly located on the arid or semi-arid region that relying on the rainfed farming in China. The maize production losses caused by drought is a great threaten to food security. As a cross-pollinating crop, maize is mostly sensitive to water stress during flowering time. Drought at flowering stage will lead to asynchronous development between the male and female flower and cause massive grain yield loss. Thus, mining drought resistance related genes at flowering stage is important for maize drought resistance improvement and breeding. 【Method】In the present study, the phylogenic tree of 24 ZCN genes in maize genome, which is homologs of Arabidopsis FT gene, was build. The gene expression patterns of ZCN7 were analysis using qRT-PCR and in vivo GFP fluorescence imaging. A maize natural population consisting of 118 diverse inbred lines were planted in three environments, Beijing in 2021 and 2022 and Urumqi in 2022, to identify the flowering time related traits under different water treatments. The genomic variants around ZCN7 were detected by PCR and Sanger sequencing. The candidate gene association analysis was performed based on mixed linear model and the significant associated variants with drought induced anthesis-silking interval was obtained. The gene expression level of ZCN7 in natural population at flowering time was also measured by qRT-PCR. The differences of drought resistance traits and ZCN7 expression were compared between different haplotypes of significant associated variant. The Ubi1:ZCN7 overexpression transgenic maize were obtained, and the phenotypic performance was identified under different water treatments. 【Result】The 24 ZCN genes in maize genome included 15 FT like genes, 6 TFL1 like genes and 3 MFT like genes. The protein sequence of ZCN genes varied from 111 nn to 193 nn. The ZCN7 showed close relationship with ZCN8 and the protein sequence identity was 83.3% between the two genes. ZCN7 showed highest gene expression in the leaf blade at V12 stage. And the ZCN7-promoter:GFP vector was transformed to Arabidopsis and the GFP showed enriched signal at the blade edge of mature leaf. The candidate gene association analysis revealed a SNP variant at 1001 bp upstream of ZCN7 start codon had highest association signal with drought induced anthesis-silking interval under drought. The A/A and G/G haplotypes of SNP-1001 included 78 and 27 inbred lines, respectively. The anthesis-silking interval of A/A haplotype lines were significantly lower than G/G lines. And the ZCN7 gene expression of A/A haplotype lines were significantly higher than G/G lines. In addition, the ZCN7 overexpression transgenic lines showed significantly decreased anthesis-silking interval than wild type lines. Under drought, the anthesis-silking intervals of OE1 and OE2 were 2.3 and 2.6 days shorter than wild type lines. And the grain yield per plant and kernel number per plant of transgenic lines were significantly higher than wild type lines under drought, while the hundred kernel weight, kernel length and kernel width showed no significant difference. 【Conclusion】The maize ZCN7 played positive role in drought resistance and its overexpression improved grain yield by reducing anthesis-silking interval under drought.

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    A Comprehensive Assessment of Proso Millet Varieties Tested in the State Multi-Region Trial by GYT Biplot Analysis
    MA ZhiXiu, CHAI ShaoHua, GUO Yan, SHI Xing, YANG QingHua, GAO JinFeng, GAO XiaoLi, FENG BaiLi, YANG Pu
    Scientia Agricultura Sinica    2023, 56 (16): 3062-3076.   DOI: 10.3864/j.issn.0578-1752.2023.16.002
    Abstract172)   HTML26)    PDF (1390KB)(467)       Save

    【Objective】GYT biplot analysis was employed to analyze agronomic traits, yields and cultivar-trait interactions of test proso millet varieties in different trial sites and comprehensively asses and classify these test varieties in term of their multiple traits, so as to provide a theoretical basis for reasonable arrangements and zonations of proso millet varieties in China.【Method】GGE biplot, GT biplot and GYT biplot along with the analysis of correlation were employed to comprehensively assess 20 varieties tested in the State Multi-region Trial for Proso Millet in 15 trial sites in 2019-2020 in terms of their growth period, plant heights, node numbers, main spike lengths, grain weights per spike, 1000-kernel weights and yields.【Result】It was showed by the analysis of correlation that in both non-waxy and waxy proso millet varieties, the yields were significantly and positively correlated with the growth period and negatively correlated with the main spike lengths, and the plant heights was significantly and positively correlated with the growth period and main spike lengths. In non-waxy proso millet varieties, the yields were also significantly negatively correlated with the node numbers, and the node numbers and 1000-kernel weights were significantly and positively correlated. In waxy proso millet varieties, the yields were significantly and positively correlated with the 1000-kernel weights, and the growth period were significantly and positively correlated with the plant heights and 1000-kernel weights. The analysis of genotype-trait interactive effects by GT biplot showed that the main components of GT biplot PC1 and PC2 explained 61.81% and 69.96% of genotype-trait interactive effects in no-waxy and waxy proso millets, respectively. The correlations between agronomic traits of the tested varieties displayed by GT biplot were basically consistent with Pearson correlation coefficients. The correlations between the yield-trait combinations of the tested varieties were analyzed by GYT biplot, and all the traits of these combinations were significantly and positively correlated. In terms of calculated ideal indexes, Yi 11-02-92-4, Gu 19-63, 0515-2-2, Yi 11-03-3-2-2 and Zhenglongmi 1 were identified as varieties with a good yield-traits combination performance. In terms of comprehensive performances, Chimi 3, Xinong 2018-N02, Xinong 2018-N10, Y1660, Xinong 18-W02 and Xinong 18-W06 poorly performed. It followed that Yi 11-02-92-4 and 0515-2-2 had wider adaptabilities and better yields than the other varieties in different planting areas, showing an absolute regional yield advantage. 【Conclusion】In assessing multiple traits of millet varieties, GYT biplot analysis was more reliable than GGE and GT biplot analysis, so that it was an effective method to scientifically assess merits of proso millet varieties. Among the varieties tested in the state multi-region trial for proso millet, the no-waxy variety with the best comprehensive performance was Yi 11-02-92-4 and suitable to be planted in spring planting areas of proso millet in Northeast China and spring and summer-planting areas of proso millet on the Loess Plateau. The waxy variety of 0515-2-2 with a better comprehensive performance was suitable to be planted in spring-planting areas of proso millet in northern China and spring- and summer-planting areas of proso millet on the Loess Plateau.

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    COXⅡ Functional Analysis in Tobacco sua-CMS Line
    WANG Jin, LIU YanFang, LIU WenWen, WANG YaQi, SONG ShiYang, ZHANG XingZi, LI FengXia
    Scientia Agricultura Sinica    2023, 56 (16): 3077-3087.   DOI: 10.3864/j.issn.0578-1752.2023.16.003
    Abstract136)   HTML13)    PDF (1930KB)(154)       Save

    【Objective】Cytoplasmic male sterility (CMS) is controlled by the cytoplasmic genome, mainly the mitochondrial genome, which plays an important role in breeding and production of hybrids. Sterile lines and hybrids are the main types of tobacco cultivation in China, accounting for more than 80% of the total tobacco cultivation area. The sterile type of tobacco in China is sua-CMS, which is also the only sterile type of tobacco production, but the control gene and sterility mechanism of sua-CMS are not clear. COXⅡ is the second subunit of cytochrome oxidase (COX), encoded by the mitochondrial genome. Nucleotide base variants occur at the terminal of the COXⅡ in sua-CMS lines, which resulted in the replacement of the C-terminal of COXⅡ Thr257 by Ser. The function analysis of COXⅡ in sua-CMS tobacco provides a foundation for the elucidation of the molecular mechanism of sterility in tobacco.【Method】In this study, four sets of sua-CMS lines and fertile controls were used as materials, and RNA blot hybridization and qRT-PCR methods were used to analyze the transcript and gene expression of COXⅡ between sua-CMS lines and fertile controls, and Western blot and COX enzyme activity experiments analyzed COX protein expression and COX enzyme activity, and ATP content were measured. The COXⅡ of fertile tobacco was overexpressed in sua-CMS line, and the effects of COXⅡ overexpression on COXⅡ expression, COX enzyme activity, ATP content and stamens development of tobacco were studied.【Result】COXⅡ multi-sequence alignment showed that Thr257 at C-terminal was highly conserved between species. Expression analysis showed that the COXⅡ transcript in sua-CMS line was significantly lower than that of the fertile control, and quantitative expression of four sets of sua-CMS lines and fertile controls showed that the expression amount of COXⅡ in sua-CMS lines was only 0.3%-0.4% of the fertile controls, the COX protein abundances and enzyme activity were significantly lower than that of the fertile control, and the ATP content of sua-CMS line was 52% of that of the fertile control. When COXⅡ was overexpressed in sua-CMS line, the expression levels of COXⅡ in transgenic plants increased by up to 171 times, the COX enzyme activity increased by 70%, the ATP content increased by 90%. However, all indicators including COXⅡ expression levels, COX enzyme activity and ATP content in transgenic plants were lower than those of fertile tobacco. We also observed significant changes in anther morphology in transgenic plants, and the differentiation of the anther wall appeared, but there was no pollen grains in the anthers.【Conclusion】This study shows that the mutant COXⅡ of sua-CMS line affects COX function, which is a main reason of anther energy deficiency, and it is also an important factor affecting the development of anthers.

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    Genetic Diversity Analysis and Comprehensive Evaluation of Sorghum Breeding Materials Based on Phenotypic Traits
    ZHANG YiZhong, ZHANG XiaoJuan, LIANG Du, GUO Qi, FAN XinQi, NIE MengEn, WANG HuiYan, ZHAO WenBo, DU WeiJun, LIU QingShan
    Scientia Agricultura Sinica    2023, 56 (15): 2837-2853.   DOI: 10.3864/j.issn.0578-1752.2023.15.001
    Abstract366)   HTML63)    PDF (644KB)(608)       Save

    【Objective】 The present study analyzed the genetic variation of phenotypic traits and genetic diversity of sorghum breeding materials. Additionally, the study explored a comprehensive method for the evaluation of germplasm materials and screening of excellent sorghum germplasm to provide an important basis for sorghum germplasm innovation and variety selection.【Method】 In total, 263 sorghum germplasms from different sources were used as the test materials, and 17 phenotypic traits were identified under different environments for two years. Genetic diversity of the phenotypic traits was calculated based on the Shannon-Wiener information diversity index. The sorghum germplasms were comprehensively evaluated using the correlation analysis, principal component analysis, cluster analysis, and stepwise regression. Excellent sorghum germplasms were screened according to the phenotypic comprehensive evaluation value (F value) and target traits.【Result】 Sorghum breeding materials exhibited high genetic diversity. The diversity index distribution of different traits ranged from 0.497 to 2.075, with the diversity index of spike shape being the smallest and that of spike stalk length being the largest. The coefficient of variation of seven plant height, stem diameter, panicle length, panicle stalk length, grain weight per spike, thousand grain weight, period of duration varied in different years; the smallest variation was observed in the period of duration, followed by the panicle length, whereas the largest variation was observed in grain weight per spike, followed by stem diameter. A comprehensive evaluation of the breeding materials showed that when the cumulative contribution percentage was >80%, the number of the total principal components was 11. F value of the sorghum breeding materials was calculated using the membership function method. The average F value was found to be 0.464, with the restorer line L28 having the highest F value (0.581) and the maintainer line 72B/DORADO having the lowest the F value (0.330). Through stepwise regression, a regression equation was established, with 12 traits (main vein color, ear type, ear shape, awn character, glume coating degree, grain shape, plant type, stem diameter, ear length, grain weight per ear, 1000-grain weight, and growth period) as independent variables. The equation could be used for a comprehensive evaluation of the phenotypic traits of breeding materials of sorghum breeding materials. Based on F value clustering, 263 materials were divided into six groups. Among these, 33 materials in group Ⅳ exhibited excellent agronomic characteristics and high F value, which could be used as parent materials for material innovation and cross breeding.【Conclusion】 Sorghum phenotypic traits exhibit rich genetic variation and high genetic diversity. A total of 33 excellent germplasms were obtained. Using multivariate statistical analysis is a feasible approach to comprehensively evaluate sorghum germplasm.

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    Status, Existing Problems and Strategy Discussion on Northward Expansion of Winter Rapeseed in China
    LIU ZiGang, WEI JiaPing, CUI JunMei, WU ZeFeng, FANG Yan, DONG XiaoYun, ZHENG GuoQiang
    Scientia Agricultura Sinica    2023, 56 (15): 2854-2862.   DOI: 10.3864/j.issn.0578-1752.2023.15.002
    Abstract253)   HTML28)    PDF (480KB)(338)       Save

    Since the 1950s, with the introduction and application of winter rapeseed (Brassica napus) in China, it has led to the rapid transformation, i.e., replacing winter turnip rape by winter oil rape, in the Yangtze River Basin being the main production areas of winter rapeseed in China. In the late 1980s, with the continuous breakthroughs in cold tolerance breeding, the planting area of winter oil rapeseed continued to extend northward. Winter oil rapeseed had replaced winter turnip rapeseed in the main production areas in China, such as the HuangHuai River Basin, Weihe River Basin, and Weibei Dry Plateau. In recent years, strong cold resistant cabbage type winter oil glycerol 4 and other varieties have been developed, which can replace winter turnip rapeseed in arid and cold regions of northern China, achieving doubled yield of winter rapeseed, “double low” quality, and suitable machine harvesting for lodging resistance. The essence of the successful northward migration of winter rapeseed was the northward migration of winter oil rapeseed in China, namely cold resistant varieties of winter oil rapeseed replaced winter turnip rapeseed in the original production area, which have greatly promoted the development of winter rapeseed industry. Nevertheless, the northward migration of winter turnip rapeseed faces completely different difficulties. Since 1955, the northward migration trial of winter turnip rapeseed has been terminated by the introduction of spring oil rapeseed into the planting regions of spring turnip rapeseed at the same time. In the subsequent trial, there is a deviation in the research direction on techlonogies of the northward migration, i.e., solely paying close attention to the cold resistance, while ignoring drought tolerace of varieties which have been migrated into the northward migration area with further reduction in precipitation. There was resulting in technology output not meeting the actual needs of the industry. As a result, the northward migration practice has been carried out for decades and has not yet formed a stable winter rapeseed planting area outside of traditional production areas. In recent years, the original planting area has also continued to shrink, and the northward migration of winter turnip rapeseed has had little effect. In practical condition, the main challenge faced by the northward migration of winter turnip rapeseed is water limitation and the combination of drought and cold stress. The low comparative efficiency is the leading factor in the predicament of industrial development. In recent years, the strong cold-tolerant varieties of winter oil rapeseed developed can stably overwinter in northern cold and arid regions, replacing the varieties of winter turnip rapeseed, significantly improving yield, quality, planting efficiency of winter rapeseed, etc., which is the hope for winter rapeseed in strong winter regions to break through industrial difficulties. We have reviewed the history of winter rape northward-extension in China, achievements and existing problems. The reasons for the dilemma of northward extension were analyzes, and suggestions were made. The revolution of replacing winter turnip rape with winter oilseed rape should be promoted to meet the challenges in winter rape industry development in north China.

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    The Genetic Basis of Flavonoid Contents in Wheat and Its Application in Functional Wheat Variety Breeding
    CHEN Jie, CHEN Wei
    Scientia Agricultura Sinica    2023, 56 (13): 2431-2442.   DOI: 10.3864/j.issn.0578-1752.2023.13.001
    Abstract488)   HTML63)    PDF (2776KB)(418)       Save

    Accompanying the elevated expenses on consumption, people’s urge upon food has been gradually changed from “eat to be fed” to “eat to be satisfied” and further to “eat to gain nutrition” and “eat to be healthy”. Accordingly, breeders considered the wheat breeding goals should be set as breeding wheat with better quality along with higher yield, wherein the phrase “functional wheat variety” was recently raised. Flavonoids comprise one of the most widely reported categories of metabolites, the contents of which have been included within the “functional wheat variety” breeding program for its connection with plant phenotypes and its contribution to human health. The combination of metabolomics approach and genetics design has been proved to be efficient in identifying the candidates that responsible for metabolite contents, that said its application in wheat was lagged behind due to the lately released wheat reference genome. Further, the deficient knowledge upon the genetic basis of metabolites has in turn constrained the application of breeding “functional wheat variety”. In the current manuscript, the research progresses on genetic basis of flavonoids are briefly summarized, and its application for wheat breeding is highlighted. Meanwhile, the metabolomics-assisted breeding frame is concepted. Ultimately, the “functional wheat variety” breeding program will be achieved through the combination of the fundamental researches and breeding applications.

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    Establishment and Standardization of Event-Specific Real-Time Quantitative PCR Detection Method of Stress-Resistant Soybean IND-ØØ41Ø-5
    LI YunJing, XIAO Fang, WU YuHua, LI Jun, GAO HongFei, ZHAI ShanShan, LIANG JinGang, WU Gang
    Scientia Agricultura Sinica    2023, 56 (13): 2443-2460.   DOI: 10.3864/j.issn.0578-1752.2023.13.002
    Abstract179)   HTML25)    PDF (2711KB)(497)       Save

    【Objective】Stress-resistant soybean IND-ØØ41Ø-5 has been authorized as a safety certificate for imported processed raw materials in China. This study aims to develop a specific, accurate, and sensitive real-time quantitative PCR (qPCR) assay for the quantification of the stress-resistant soybean IND-ØØ41Ø-5 event, providing a precise measurement technique for regulating its safety in China. 【Method】18 pairs of primers and probes were designed using Beacon designer 8.0 software for the 5′ end flanking sequence of the stress-resistant soybean IND-ØØ41Ø-5 event, in combination with one pair of primer and probe providing by Instituto de Agrobiotecnologia Rosario (INDEAR) S.A. Subsequently, the specific screening of the primers and probes was performed using real-time PCR technology, and one pair of candidate primer and probe was selected. The reaction system parameters, such as primer and probe concentration, were optimized during the establishing the qPCR method. A specificity test was performed using different test samples. The pure stress-resistant soybean IND-ØØ41Ø-5 genomic DNA was serially diluted into standard solution templates, and standard curves were plotted to investigate the linear dynamic range of the qPCR method. Test samples with copy number ratios of 5%, 1% and 0.1% were prepared by mixing IND-ØØ41Ø-5 genomic DNA with non-GM counterpart to evaluate the accuracy of the qPCR method. The limit of detection (LOD) was detected by using test samples with copy number ratios of 0.05% and 0.025%. The limit of quantification (LOQ) was determined after 16 tests on samples with a copy number ratio of 0.1%. Finally, the technical parameters of qPCR assay for the stress-resistant soybean IND-ØØ41Ø-5 event were determined. The specificity, LOD, LOQ and accuracy of the qPCR method were validated by eight qualified testing laboratories. The repeatability and reproducibility of the qPCR were evaluated by Cochran′s test and Grubbs' test, and the measurement uncertainty of the accuracy samples were pre-evaluated by linear least-square method. 【Result】The RBORD-F1/RBORD-R1/RBORD-P1 primer and probe combination was selected as a candidate to establish the qPCR for the stress-resistant soybean IND-ØØ41Ø-5 event, with an amplified fragment of 138 bp. The optimized reaction system had a final concentration of 0.4 μmol·L-1 for the primer and 0.2 μmol·L-1 for the probe. Standard curves of IND-ØØ41Ø-5 and Lectin gene assay showed good linearity with the dynamic range from 33 to 83190 copies of genomic DNA. The qPCR can accurately quantify 5%, 1%, and 0.1% content of IND-ØØ41Ø-5 test samples with less than 25% bias and relative standard deviation (RSD). The LOD was determined to be 0.05%, and the LOQ was 0.1%. After validation by eight qualified laboratories, the results indicated that the method was stable, specific and had good repeatability and reproducibility, with the LOD of 0.05% and LOQ of 0.1%. After pre-evaluating the measurement uncertainty, the content of IND-ØØ41Ø-5 in the five test samples was found to be (0.10±0.02)%, (0.53±0.09)%, (1.05±0.18)%, (2.05±0.34)% and (5.18±0.87)%, respectively. These results demonstrate the accuracy and reliability of the qPCR method established in this study for the quantification of stress-resistant soybean IND-ØØ41Ø-5 event components. 【Conclusion】This study successfully developed a specific, accurate, and sensitive qPCR assay for the quantification of stress-resistant soybean IND-ØØ41Ø-5 event using real-time PCR technology. The results show that method is capable of achieving precise measurement and reliable quantification of IND-ØØ41Ø-5 event components.

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    QTL Identification and Genetic Analysis of Plant Height in Wheat Based on 16K SNP Array
    YAO QiFu, CHEN HuangXin, ZHOU JieGuang, MA RuiYing, DENG Liang, TAN ChenXinYu, SONG JingHan, LÜ JiJuan, MA Jian
    Scientia Agricultura Sinica    2023, 56 (12): 2237-2248.   DOI: 10.3864/j.issn.0578-1752.2023.12.001
    Abstract397)   HTML51)    PDF (829KB)(688)       Save

    【Objective】There is a close relationship between plant height (PH) and yield. The aim of this study is to further explore quantitative trait loci (QTL) of PH with breeding value in wheat and analyze the genetic effects of major QTL for PH on other yield related traits toward to providing a theoretical basis for molecular breeding. 【Method】A recombinant inbred line population (MC) derived from a cross between the natural mutant msf and Chuannong 16 (CN16) was used for QTL analysis. During 2020 to 2022, planting and PH phenotype identification were conducted at five environments in Wenjiang, Chongzhou, and Ya’an of Sichuan Province. The high-quality genetic linkage map constructed using the 16K SNP array was used for QTL mapping of PH. Genotypes of flanking markers of major QTL for PH were used to analyze the genetic effects of positive alleles on yield related traits and evaluate the potentiality of QTL for yield improvement. 【Result】Eight QTL controlling PH were identified on chromosomes 1A, 3D, 4D, 5A, and 7B, respectively. Among them, two stable and major QTL, QPh.sau-MC-1A and QPh.sau-MC-5A, were located, which explained 9.09% to 25.56% and 3.91% to 13.09% of the phenotypic variation rate, respectively. Their positive alleles were all from CN16. The additive effect analysis showed that PH of the lines carrying positive alleles from QPh.sau-MC-1A and QPh.sau-MC-5A was significantly higher than that of the lines carrying only a single positive allele or none. Correlation analysis showed that PH has a significantly positive correlation with effective tiller number (ETN), a significantly negative correlation with flag leaf width (FLW), and no significant correlation with kernel number per spike (KNPS), kernel weight per spike (KWPS), thousand kernel weight (TKW), flag leaf length (FLL) and anthesis date (AD). Genetic effects analysis showed that positive allele of QPh.sau-MC-1A had a significant effect on improving ETN (56.51%), a significant effect on decreasing KNPS (-11.26%), KWPS (-13.04%), TKW (-5.47%), and FLW (-2.85%), and a significant effect on advancing AD (-0.61%). Positive allele of QPh.sau-MC-5A had a significant effect on improving ETN (10.57%), KNPS (4.32%), and TKW (2.92%), and a significant effect on delaying AD (1.07%). 【Conclusion】A major QTL QPh.sau-MC-5A for PH was mapped on chromosome 5A, and its positive allele significantly increased ETN, KNPS, and TKW, indicating that it may have a positive impact on yield.

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    Construction of DNA Molecular Identity Card of Core Germplasm of Broomcorn Millet in China Based on Fluorescence SSR
    XUE YaPeng, DING YiBing, WANG YuZhuo, WANG XiaoDan, CAO XiaoNing, SANTRA Dipak K, CHEN Ling, QIAO ZhiJun, WANG RuiYun
    Scientia Agricultura Sinica    2023, 56 (12): 2249-2261.   DOI: 10.3864/j.issn.0578-1752.2023.12.002
    Abstract210)   HTML13)    PDF (5282KB)(465)       Save

    【Objective】As an ancient minor grain crop, broomcorn millet ( Panicum miliaceum L. ) is abundant in germplasm. The construction of their DNA molecular identity based on fluorescent SSR markers would provide theoretical basis and molecular detection tool for digital management of resources. 【Method】Two hundred and thirty five broomcorn millet core accessions from China were used as experimental material, polymerase chain reaction were conducted several times using the broomcorn millet specific SSR markers which developed previously by the Broomcorn Millet Crop Molecular Breeding Research Group of the Agronomy College in Shanxi Agricultural University, core markers were obtained. With the given reference genome information of broomcorn millet, the core markers were mapped on chromosomes through BLAST sequence alignment. Fluorescence (FAM/HEX) was labeled on the 5' end of the SSR primer, the genotype of the material was given by capillary electrophoresis. Using binary coding means of expression, “0, 1” was written representing the presence or absence of amplified bands, and the discrimination of the material was detected by the software ID Analysis 4.0. Decimal (0-9) coding methods were used to calculate the size of the amplified fragments so as to obtain the character string molecular identity card of the accession. Genetic diversity, genetic clustering and principal component analysis were performed using the softwares Popgene, Powermarker, MEGA and NTSYS. The two-dimensional code DNA molecular identity card of the accession was given using the two-dimensional code online software (https://cli.im/). 【Result】PCR amplification results showed that all the 235 accessions could be separated by 7 fluorescent SSR markers (RYW3, RYW6, RYW11, RYW18, RYW37, RYW43 and RYW125) combined together. BLAST results showed that RYW18 and RYW37 were distributed on Chromosome 2, located at 0.60 cM and 0.80 cM, respectively. RYW125 is located on Chromosome 4 at 10.40 cM. RYW43 and RYW6 were distributed on Chromosome 5, located at 52.80 cM and 53.00 cM, respectively. RYW11 and RYW3 were located on Chromosome 6 at 2.10 cM and 20.70 cM, respectively. Genetic diversity analysis showed that 87 alleles were detected at 7 loci among all accessions, 3 (RYW11)-25 (RYW6) alleles were detected at each locus, with an average of 12.4286. Shannon diversity index (I) was detected and ranged from 0.2055 (RYW18) to 2.0587 (RYW6), with an average of 1.1398. The observed heterozygosity (Ho) was 0.0086 (RYW11)-0.9455 (RYW18). The expected observed heterozygosity (He) was 0.0795 (RYW18)-0.7469 (RYW11). Nei’s gene diversity index (Nei) was 0.0793 (RYW18)-0.7452 (RYW6). The polymorphism information content (PIC) was 0.0334 (RYW11)-0.8071 (RYW6), with an average of 0.5185. The results of cluster analysis and principal component analysis showed that 235 accessions were classified into 8 groups. The electrophoretic bands were number coding, and 7 marker combinations were used to construct the character string and two-dimensional code DNA molecular ID of all the accessions.【Conclusion】Two hundred and thirty five broomcorn millet core germplasms from China were used as material, polymerase chain reaction and capillary electrophoresis were conducted, 7 core SSR markers were screened. With the given reference genome information of broomcorn millet, the above markers were mapped on 4 chromosomes. Used the above SSR markers, genetic diversity analysis of all accessions was conducted and genetic diversity parameters were obtained. Based on Cluster analysis, all accessions were classified into 8 groups. Principal component analysis result resolved the deviation occured in Cluster analysis. According to the principle of most accessions were tell apart using the least markers, decimal (0-9) coding methods were used to calculate the size of the amplified fragments so as to obtain the character string molecular identity card of the accession. Combined the phenotype data with the above character string, two-dimensional code DNA molecular ID of all the accessions were developed.

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    Analysis and Comprehensive Evaluation of Phenotype Genetic Diversity in Kam Sweet Rice Germplasm Resources in Guizhou
    LI Huan, YAN XiaoQing, YANG ZhanLie, TAN JinYu, LI XiaoBing, CHEN NengGang, WU RongJu, CHEN HuiCha, RUAN RenChao
    Scientia Agricultura Sinica    2023, 56 (11): 2035-2046.   DOI: 10.3864/j.issn.0578-1752.2023.11.001
    Abstract324)   HTML58)    PDF (1446KB)(476)       Save

    【Objective】To analyze the phenotypic genetic diversity of traditional characteristic landraces of Kam Sweet Rice (KSR) in Guizhou, this study screened the comprehensive evaluation indicators for phenotype, and constructed a reliable mathematical model for comprehensive evaluation on phenotypes. This study provides valuable theoretical support for the discovery and breeding of exceptional KSR germplasm resources. 【Method】13 phenotypic traits from a total of 286 KSR accessions collected from the Southeast Guizhou were measured. A variety of multiple statistical methods, including Shannon-Wiener genetic diversity index, principal component analysis, subordinate function value analysis, and stepwise regression analysis, were used to analyze the phenotypic genetic diversity and comprehensively evaluate on KSR germplasm resources. 【Result】Firstly, the KSR germplasm showed high phenotypic genetic diversity, with the variation coefficients of the 13 phenotypic traits ranging from 6.79% (Grain width) to 30.73% (Panicle number per plant), and the diversity index (H') ranging from 2.484 (Ratio of length to width for grain) to 2.996 (Flag leaf width). Correlation analysis showed significant or highly significant correlations among the different traits. Principal component analysis showed that the 13 traits were integrated into 7 principal components, with contribution rates ranging from 8.44% to 23.14%, and the additive contributing rate came up to 90.29%. The phenotypic comprehensive evaluation D value calculated by subordinate function values analysis showed that the top 5 varieties had the best characteristics, and 11 phenotypic traits were significantly correlated with the D value. The stepwise regression analysis established a mathematical model for phenotypic evaluation of KSR, Y=-0.249+0.119X5+0.395X13+0.071X6-0.161X3+0.108X10+0.170X2+0.110X9 (F=2800.200, R2=0.986). Based on the model, 7 comprehensive evaluation indicators were screened out. At last, the 286 germplasm resources were systematically clustered into four categories based on the D value, displaying significant differences among the groups and outstanding characteristics. The group I, including 38 accessions, showed the best comprehensive traits and high yield potential; the group Ⅱ, including 103 accessions, showed general comprehensive traits and high seed setting rate; the group Ⅲ, including 94 accessions, showed poor comprehensive traits and long growth period; the group Ⅳ, including 51 accessions, had the worst comprehensive traits. 【Conclusion】The KSR germplasm resources in Guizhou have abundant phenotypic genetic diversity. It is feasible to use multiple statistical analysis methods for comprehensive evaluation on KSR germplasm diversity. The regression equation constructed under the same conditions can quantitatively evaluate the comprehensive performance of KSR germplasm resources. The filled grains per panicle, grain width, seed setting rate, panicle number per plant, flag leaf length, plant height and grain yield per plant can be used for identifying KSR germplasm resources. The outstanding germplasm resources with coordinated comprehensive traits such as Zaohe, Nuohe-12, 90 Tianhe, Goudong-1 and Nuohe-11 were screened out, which can be ultilized for genetic improvement of KSR and for rice breeding.

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    Genome-Wide Association Study of Grain Main Quality Related Traits in Winter Wheat
    DONG YiFan, REN Yi, CHENG YuKun, WANG Rui, ZHANG ZhiHui, SHI XiaoLei, GENG HongWei
    Scientia Agricultura Sinica    2023, 56 (11): 2047-2063.   DOI: 10.3864/j.issn.0578-1752.2023.11.002
    Abstract277)   HTML35)    PDF (3371KB)(550)       Save

    【Objective】The quality of wheat grain was an important factor affecting the processing quality and nutritional. Mining loci and candidate genes significantly associated with wheat grain quality traits provided a basis for broadening the understanding of the genetic mechanism of quality traits and molecular marker-assisted quality. 【Method】By measuring five quality traits, including protein content (GPC), wet gluten content (WGC), starch content (GSC), settling value (SV) and grain hardness (GH), in 259 winter wheat varieties (lines) from domestic and abroad, and conducting genome-wide association analysis in combination with 90K SNP chip, the significant association loci located were subjected to haplotype analysis. 【Result】All five traits conformed to normal distribution and showed rich variation among different environments, and the coefficient of variation of sedimentation value was the largest (20.11%-24.42%). All traits have shown highly significant differences (P<0.001) among genotype, environment, and genotype×environment, with a broad-sense heritability of 0.77-0.84. A total of 44 loci significantly associated (P<0.001) with five traits were detected by genome-wide association analysis, distributed in 19 linkage groups other than chromosomes 1D and 3D. Eighteen loci were stable in two or more environments, involving all five traits including protein content (12), wet gluten content (9), starch content (11), sedimentation value (12) and grain hardness (7), explaining 4.27%-10.98% of the genetic variation. Thirteen of them were multi-effect loci, with the largest number of multi-effect loci (7) associated with traits such as protein content, wet gluten content, settling value and starch content. The GENE-0762_631, IAAV7742 and RAC875_c66845_466 loci located on 2B, 2D and 3A chromosomes were detected simultaneously at two environmental and BLUP values with a range of 4.32%-7.07% phenotypic contribution. Through haplotype analysis of multi-effect loci present in multiple environments with high phenotypic contribution, four different haplotypes, Hap1, Hap2, Hap3 and Hap4, which were significantly associated with traits such as protein content, sedimentation value and starch content, were uncovered at the D_GDS7LZN02F4FP5_176 locus of chromosome 5D, among them Hap1 was a high starch content haplotype (P<0.001), while Hap2 and Hap3 were both haplotypes with high protein content and sedimentation value (P<0.05), and the four haplotypes accounted for 74.22%, 16.21%, 6.92% and 2.65%, respectively. The distribution frequencies of haplotypes from different sources of winter wheat were analyzed, in which the distribution frequencies of haplotype Hap2 with high protein content and sedimentation value were from high to low in the Huanghuai winter wheat regions>northern winter wheat region>abroad varieties>middle and lower reaches of the Yangtze River winter wheat region>southwest winter wheat region. Candidate genes were mined for stable genetic loci, and 10 candidate genes that might be related to wheat grain quality were screened. 【Conclusion】In the study, 18 stable loci significantly associated with grain quality traits were detected, 4 different haplotypes were identified, and 10 candidate genes related to grain quality were screened.

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    Physiological Changes and Integrity of ATP Synthase Subunits mRNA in Naturally Aged Cotton Seeds
    SONG Ci, GU FengXu, XING ZhenZhen, ZHANG JunMing, HE WenXue, WANG TianBo, WANG YuLu, CHEN JunYing
    Scientia Agricultura Sinica    2023, 56 (10): 1827-1837.   DOI: 10.3864/j.issn.0578-1752.2023.10.001
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    【Objective】Seed aging is a complex biological process, previous studies have been used to elucidate the events. However, the mechanism of seed aging is still unclear. The naturally aged cotton seeds were used as experimental materials, and the physiological and biochemical changes as well as the changes in ATP synthase mRNA integrity that occurred in cotton seed during storage were investigated in order to provide a foundation for further illuminating the aging mechanism of cotton seeds.【Method】In this study, a collection of seeds (cultivar Xinluzao 74) that had been stored for 3 and 5 years served as the experimental materials, the newly harvested seeds were used as the control (CK). The germination percentage, water absorption and viability of cotton seeds were valued by germination test between paper, low constant temperature over method, and TTC staining method, respectively; The acid value and respiratory rate of cotton seeds were determined by the acid-base titration method, and the ATP synthase activity was detected with plant ATP synthase ELISA Kit. The mRNA integrity of ATP synthase subunit α, β, γ, ε, and δ in cotton embryo was analyzed by reverse transcription blocking-double primer amplification method.【Result】Our data suggest that seed vigor dramatically decreased over storage time. After 3 and 5 years of storage, the germination percentage of cotton seeds was significantly decreased from 98.7% to 84.0% and 58.0%, respectively (P<0.05). At the initial stage of seed imbibition (the first 4 h), the water absorption rate of seeds was significantly decreased by 11.0% and 26.9%, respectively. The results of TTC staining showed that only the radicle was slightly stained in seeds preserved 5 years but not the cotyledons and other organs stained; The acid value of seeds was significantly increased by 28.4% and 40.0%, respectively (P<0.05), this indicated that severe hydrolysis of lipid occurred in seeds. Seed respiration rate and ATP synthase activity showed an increasing trend during imbibition, but the increasement was significantly decreased (P<0.05); The respiration rate of seeds was reduced by 33.3% and 49.2% after 24 hours of imbibition, and the activity of ATP synthase was decreased by 17.9% and 73.4% after 12 hours of imbibition, respectively. The results of reverse transcription blocking-double primer amplification showed that the R value of ATP synthase subunits α, β, γ, and δ mRNAs stored in seeds were significantly decreased, but the subunit ε mRNA was significantly increased. These results indicated that the integrity of the ATP synthase subunits mRNA decreased to varying degrees during the natural storage process.【Conclusion】These results showed that a prolonged storage time could reduce seed vigor; The integrity loss of ATP synthase subunit mRNAs stored in seed embryos would cause ATP synthase subunit to be impaired and ATP synthase activity declined, thus lead to a decreased production of ATP and affect seed germination capacity. This might be one of the important reasons for cotton seed aging.

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    Molecular Marker Assisted Identification and Application of Maize Germplasms for Maize Rough Dwarf Disease Resistance
    WANG JiangHao, WANG LiWei, ZHANG DongMin, GUO Rui, ZHANG QuanGuo, LI XingHua, WEI JianFeng, SONG Wei, WANG BaoQiang, LI RongGai
    Scientia Agricultura Sinica    2023, 56 (10): 1838-1847.   DOI: 10.3864/j.issn.0578-1752.2023.10.002
    Abstract399)   HTML59)    PDF (2438KB)(269)       Save

    【Objective】Molecular markers tightly linked to three maize rough dwarf disease (MRDD) resistant loci were employed to identify resistant inbred lines, then the classification of heterotic groups and analysis of combining ability of these inbred lines were carried out, which proved a highly efficient way for maize MRDD resistance breeding.【Method】A recombinant inbred lines (RILs) population consisting of 263 F9 lines was developed through single seed descent method from a segregating F2 population by crossing a resistant inbred line K36 to a susceptible inbred line S221. The MRDD resistances of the RILs were identified in different growing environments. Meanwhile the RILs were genotyped by employing three pairs of molecular markers, 5FR, 6W53 and IDP25K which were closely linked to the three resistant loci, qMrdd2, Rmrdd6 and qMrdd8. The excellent lines with disease resistance and good agronomic traits were selected out after field evaluation. Totally 24 maize inbred lines including the elite lines were genotyped using Maize 56K SNP array, then the genetic distances between the selected lines and other elite inbred lines were calculated according to Roger's algorithm and cluster analysis was conducted to classify the heterotic groups. Meanwhile, hybrid combinations were generated and the combining abilities were tested to screen the combinations with strong disease resistance and heterosis.【Result】The inbred line K36 were homozygous resistant at the three loci, qMrdd2, Rmrdd6 and qMrdd8 while S221 were homozygous susceptible. All the 263 RILs were genotyped into 21 patterns in terms of genetic composition of the three resistant loci. The lowest DSI (0.281) appeared when all the three loci were homozygous resistant while the highest DSI (0.776) appeared when the three loci were homozygous susceptible, which were consistent with the resistant and susceptible parents (0.257, 0.623). The order of DSI from low to high value for one homozygous resistant locus was Rmrdd6 (0.396), qMrdd8 (0.478) and qMrdd2 (0.654) when the other two loci were homozygous susceptible, which showed that Rmrdd6 and qMrdd2 performed the strongest and the weakest resistance while qMrdd8 was in the middle. The variation range of genetic distance between JR2136 with the genotype of three homozygous resistant loci and other 23 inbred lines was 0.2234-0.2895, with an average value of 0.2612. The inbred line with the smallest genetic distance was C413, and the largest was Chang7-2. According to the results of cluster analysis, JR2136 was classified into Reid group, hybrid combinations with inbred lines H92 and H521 belonging to Huanggai group performed strong disease resistance and heterosis.【Conclusion】The resistance of K36 to MRDD was controlled by three loci, qMrdd2, Rmrdd6 and qMrdd8, and it had quantitative genetic characteristics and gene additive effect. Maize varieties with homozygous resistant genotypes demonstrated the strongest disease resistance. The developed molecular markers closely linked with the three resistant loci have proved valuable tools in disease-resistant breeding and screening of resistant germplasm resources. It is feasible to use molecular markers for assisted selection and gene aggregation to select highly heterotic combinations with strong disease resistance.

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    Principle, Optimization and Application of Mixed Models in Genome- Wide Association Study
    TAN LiZhi, ZHAO YiQiang
    Scientia Agricultura Sinica    2023, 56 (9): 1617-1632.   DOI: 10.3864/j.issn.0578-1752.2023.09.001
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    Genome-wide association study (GWAS) is an effective method to locate genomic loci that are significantly associated with traits. With the accumulated phenotypic data, the continuous development of high-throughput genotyping technology, and the improved statistical methods, it promotes the wide application of GWAS in area of human disease and animal and plant genetics. False positives are one of the important concerns that impair the reliability of genome-wide association results. To control the false positives, in addition to correcting the P-values, GWAS models have been continuously improved from the naive methods like ANOVA (for quantitative trait) or Chi-square test (for quality trait), to general linear model (GLM), which incorporates fixed-effect covariates, to the mixed linear model (MLM), which incorporates random effects. Fitting individual genetic effects into random effects defined by the genomic relationships matrix (GRM) is commonly adapted currently. Since the parameter estimation of MLM consumes a lot of computational resources, researchers have tried to optimize solving models and constructing GRM (which also improves computing efficiency), and the time complexity gradually decreased from O(MN3) to O(MN) for MLM-based methods, achieving a great leap in computational speed and statistical efficacy. For inflations caused by unbalanced case-control data, researchers further correct the generalized mixed linear model (GLMM). This paper comprehensively introduces the basic principles and development of GWAS, with specific emphasis on the model improvement and optimization details. We also list the applications of MLM in GWAS in agriculture, including progress on animals, plants and microbes, as well as the application of haplotype in GWAS. Finally, we give prospects on the future developments of GWAS from the viewpoints of further model optimization and experimental design.

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    Overexpression of Wheat TaCYP78A5 Increases Flower Organ Size
    PENG HaiXia, KA DeYan, ZHANG TianXing, ZHOU MengDie, WU LinNan, XIN ZhuanXia, ZHAO HuiXian, MA Meng
    Scientia Agricultura Sinica    2023, 56 (9): 1633-1645.   DOI: 10.3864/j.issn.0578-1752.2023.09.002
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    【Objective】The function and mechanism of TaCYP78A5 regulating flower organ size was preliminarily analyzed by means of expression pattern analysis, transgenic overexpression and cytological observation. The results provide genetic resources and theoretical basis for crop genetic improvement. 【Method】According to the sequence information of CYP78A family members of different species in EnsemblePlants genome database, sequence alignment and evolutionary analysis were carried out for the homologous genes of TaCYP78A5 in wheat and other species. The gene and protein structures and the expression patterns of different organs of wheat TaCYP78A5 were analyzed by bioinformatics. Through the strategy of constitutive overexpression and local specific overexpression in reproductive organs of TaCYP78A5 in Arabidopsis, it is clear that TaCYP78A5 has the function of regulating flower organ size. The cytological characteristics of flower organs of different transgenic Arabidopsis were observed under microscope, and the cytological mechanism of TaCYP78A5 regulating flower organ size was analyzed. The function of TaCYP78A5 in regulating wheat spike size and other spike traits was clarified by using the strategy of transgenic overexpression in wheat. The correlation analysis of haplotype data and spike phenotype data of 323 wheat accessions was used to explore the effect of TaCYP78A5 expression on spike size and other spike traits of different wheat accessions. 【Result】The gene and protein sequence similarity of wheat TaCYP78A5 and Arabidopsis AtCYP78A5 is low, but the gene and protein structure similarity is high. Wheat TaCYP78A5 and Arabidopsis AtCYP78A5 are widely expressed in many organs, but highly expressed in flower organs. Compared with wild type, the constitutive overexpression of TaCYP78A5 in Arabidopsis could lead to the enlargement of flower organs and a significant increase in petal area of 13.5%-35.4%. Moreover, the specific overexpression of TaCYP78A5 only in the ovule was enough to cause the enlargement of the flower organ of Arabidopsis, and the petal area increased significantly by 9%-22.1%. On the contrary, the flower organ of Arabidopsis cyp78a5 mutant was significantly smaller than that of wild type, and the petal area was significantly reduced by 27%. The constitutive overexpression of TaCYP78A5 in Arabidopsis resulted in a significant increase in the size of petal epidermal cells by 49%-54% compared with wild type, and a significant decrease in the number of cells by 11%-19% compared with wild type. Locally specific overexpression of TaCYP78A5 in Arabidopsis also resulted in a significant increase in the size of petal epidermal cells by 20%-49% compared with wild type, and a significant decrease in the number of cells by 8%-24% compared with wild type. The constitutive overexpression of TaCYP78A5 in wheat resulted in the increase of wheat spike length by 7.9%-8.9%, glume area by 9.6%-14.7%, and grain number per spike by 12.4%-23.8%. The spikelet number per spike and grain number per spikelet showed different degrees of change. The results of haplotype analysis showed that among 323 wheat accessions, wheat accessions with higher TaCYP78A5-A expression level had longer spike length, more grains per spikelet and fewer spikelets per spike than wheat accessions with lower TaCYP78A5-A expression level, but there was no significant difference in grain number per spike. 【Conclusion】TaCYP78A5 promoted the growth of flower organs in a non cellular self-made mode. The overexpression of TaCYP78A5 in wheat and Arabidopsis could lead to the enlargement of flower organs.

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    Construction of A High-Density Genetic Map and QTL Mapping for Yield Related Traits in Upland Cotton
    JIA XiaoYun, WANG ShiJie, ZHU JiJie, ZHAO HongXia, LI Miao, WANG GuoYin
    Scientia Agricultura Sinica    2023, 56 (4): 587-598.   DOI: 10.3864/j.issn.0578-1752.2023.04.001
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    【Objective】By constructing a high-density SNP genetic map, QTL mapping for cotton yield related traits of multiple populations was carried out to obtain stable and accurate QTL. It will provide references for the excavation of yield trait regulatory genes and the development of effective molecular markers. 【Method】Jifeng 1271 with high and stable yield and Jifeng173 with super fiber quality were used to construct an F2 population composing of 200 plants. A high-density SNP genetic map was constructed by GBS (Genotyping by sequencing), and QTL mapping was carried out for lint percentage, seed index and boll weight of F2, F2:3 and F2:4 populations. Genes in major and stable QTL were annotated, and the expression patterns in different tissues were analyzed for candidate genes selection. 【Result】A total of 383.07 Gb data was obtained by GBS, including 26.93 Gb for the maternal cultivar Jifeng 1271, 27.30 Gb for the paternal inbred line Jifeng 173 and 328.84 Gb for the 200 F2 plants, and Q30 scores were 90.55%, 89.95% and 95.77%, respectively. And 1 305 642 SNP markers were developed in the F2 population, including 410 726 aa×bb type SNP that were used for genetic map construction. A high-density genetic map containing 16 088 SNP and spanning 4 282.81 cM was constructed, and the average genetic distance between adjacent SNP was 0.27 cM. Collinearity analysis proved high quality of the genetic map. A total of 108 yield related QTL were mapped, including 34 lint percentage QTL, 36 seed index QTL and 38 boll weight QTL. 30 QTL overlapped with or were close to the published QTL, and the other 78 QTL were new. 10 major QTL and 16 stable QTL were found, and 5 major QTL could be mapped in 2 or 3 populations. qLP-A13-4 could be mapped in 3 populations, and the R2 reached 13.78%. qLP-A13-6 could be mapped in 2 populations, and the R2 reached 10.01%. qLP-D10-2 could be mapped in 2 populations, and the R2 reached 10.92%. qSI-D10-1 could be mapped in 2 populations and the R2 reached 12.31%. qBW-D5-3 could be mapped in 2 populations and the R2 reached 15.54%. A total of 3 415 genes were annotated in these major and stable QTL. By KEGG and GO analysis, the annotated genes are mainly involved in plant hormone signal transduction, TCA cycle, biosynthesis of secondary metabolites and amino acids, carbon fixation in photosynthetic organisms. Using the transcriptome data of TM-1 and NDM8, 8 genes were highly expressed in fiber, ovule or seed, which may be important candidate genes for boll weight and yield by regulating lint percentage or seed index. 【Conclusion】An intra-specific high-density genetic map was constructed for upland cotton. 108 yield related QTL were mapped, 5 major QTL could be mapped in at least 2 populations, and 8 candidate genes with high expression level in fiber, ovule or seed were identified.

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    Correlation Between Stomatal Characteristics and Cold Resistance of Brassica napus L.
    FAN JunQiang, WU JunYan, LIU LiJun, MA Li, YANG Gang, PU YuanYuan, LI XueCai, SUN WanCang
    Scientia Agricultura Sinica    2023, 56 (4): 599-618.   DOI: 10.3864/j.issn.0578-1752.2023.04.002
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    【Objective】 The characteristics of the stomatal movement of winter Brassica napus with different cold resistance in northern China in a low-temperature environment were analyzed, and the relationship between cold resistance and stomatal movement was clarified, which provided a basis for analyzing the cold resistance mechanism of winter Brassica napus and cultivating strong cold resistance varieties. 【Method】 The physiological indexes of 14 winter Brassica napus varieties under semi-lethal temperature and low temperature were determined to judge the difference in cold resistance. At the same time, 12 stomatal indexes of the lower epidermis of the leaves of the plants treated at 24℃ for 12 h, 0℃ for 1 h, and 0℃ for 12 h were determined. Stomatal evaluation indexes significantly related to cold resistance evaluation indexes were screened by correlation analysis. 【Result】 (1) According to the results of semi-lethal temperature determination, the cold resistance of the tested varieties was identified as gau-1 (-8.06)>gau-24 (-7.83)>gau-30 (-7.58)>gau-39 (-7.44)>ts309 (-7.28)>ts312 (-7.08)>nts158 (-6.81)>npz269 (-6.62)>Tianyou 14 (-5.98)>16-2444 (-5.4)>17-2251 (-5.13)>Tianyou 2266 (-4.8)>Tianyou 2238 (-4.6)>Tianyou 2288 ( -4.38 ). According to the comprehensive evaluation value of physiological indexes, the results of cold resistance were gau-1 (0.990)>gau-24 (0.876)>gau-30 (0.693)>gau-39 (0.644)>ts309 (0.534)>ts312 ( 0.463)>nts158 (0.439)>npz269 (0.388)>Tianyou 14 (0.352)>16-2444 (0.307)>17-2251 (0.282)>Tianyou 2266 (0.236)>Tianyou 2238 (0.126)>Tianyou 2288 ( 0.000). The cold resistance measured by semi-lethal temperature was consistent with the comprehensive evaluation results of physiological indexes. (2) 12 stomatal movement-related indicators changed significantly after low-temperature treatment. The correlation analysis showed that there was no significant correlation between the 12 stomatal indexes and the semi-lethal temperature at room temperature and 0℃ after 1 h treatment. After 12 h treatment at 0℃, the stomatal pore length, area of stomatal pore, circumference of stomatal pore, stomatal apparatus length, area of stomatal apparatus, Circumference of stomatal apparatus, and stomatal closure rate were significantly correlated with cold resistance. The semi-lethal temperature was significantly correlated with the comprehensive evaluation value (Z) of stomata, and the correlation coefficient was-0.572. 【Conclusion】 Low-temperature treatment can significantly affect the stomatal movement of winter Brassica napus, and the characteristics of stomatal closure tend to be more obvious with the extension of low-temperature treatment time. At the same time, the relative changes of stomata of different temperature-sensitive varieties were different, and the varieties with strong cold resistance had stronger ability to keep stomata open after low-temperature treatment.

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    Cloning of MYBL2 Gene from Brassica and Its PCR Identification in Genomes A, B and C
    ZOU Ting, LIU LiLi, XIANG JianHua, ZHOU DingGang, WU JinFeng, LI Mei, LI Bao, ZHANG DaWei, YAN MingLi
    Scientia Agricultura Sinica    2023, 56 (3): 416-429.   DOI: 10.3864/j.issn.0578-1752.2023.03.002
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    【Objective】In Arabidopsis, MYBL2 negatively regulates the biosynthesis of anthocyanins and proanthocyanidins. The MYBL2 genes from six Brassica species with different leaf colors were cloned. By analyzing the sequence and expression pattern of MYBL2, the function of MYBL2 in the biosynthesis of anthocyanins in Brassica species was explored. 【Method】The sequences of MYBL2 from the six Brassica species with different leaf colors were obtained using homology-based cloning method and multi sequence alignment and phylogenetic tree analysis were performed. The purple leaf materials of B. rapa, B. napus, B. juncea and B. carinata were treated with shading, and the expression level of MYBL2 gene was analyzed by transcriptome sequencing and qRT-PCR. The qRT-PCR in B. oleracea, B. napus, B. juncea and B. carinata with purple and green leaves were also performed to evaluate the expression level of MYBL2. Based on the nucleotide variation sites of the cloned MYBL2-1 and MYBL2-2 sequences, PCR markers which could distinguish the genomic origin of alleles were developed. 【Result】A total of 56 copies of 9 homologs of MYBL2-1 and MYBL2-2 were cloned from 19 samples of six species of Brassica. The BcaMYBL2-1 gene was obtained for the first time. The total length of BcaMYBL2-1 sequence was 867 bp, including two introns of 168 bp and 102 bp respectively, encoding 198 amino acids, with a molecular weight of 22.69 kD and an isoelectric point (pI) of 8.72. Sequence alignment and evolutionary analysis showed that BcaMYBL2-1 was derived from B genome. Among the MYBL2-1 and MYBL2-2 copies of six species in Brassica, only BraA07.MYBL2-1, BolC06.MYBL2-1 and BcaMYBL2-1 exhibited sequence differences in different leaf color materials. After shading treatment, the leaf color of purple leaf material becomes lighter than that of the unshaded part. In Chinese cabbage Zibao 5, the expression of BraA07.MYBL2-1 and BraA02.MYBL2-2 in the shaded part were 0.7 and 0.4 times of that in the unshaded part, respectively. In Brassica napus with purple leaves and white flowers, the expression of BnaA07.MYBL2-1, BnaC06.MYBL2-1, BnaA02.MYBL2-2 and BnaC02.MYBL2-2 in the shaded part were 0.4, 0.5, 0.4 and 0.4 times of that in the unshared part, respectively. In purple leaf mustard, the expression of BjuA07.MYBL2-1, BjuB03.MYBL2-1, BjuA02.MYBL2-2 and BjuB05.MYBL2-2 in the shaded part were 0.4, 0.3, 0.4 and 0.2 times of that in the unshared part, respectively. In B. carinata with purple leaf, the expression of BcaMYBL2-1, BcaB03.MYBL2-1 and BcaC03.MYBL2-2 in the shaded part were 0.3, 0.4 and 0.5 times of that in the unshared part, respectively. However, the expression of BcaB05.MYBL2-2 in the shaded part was 2.4 times of that in the unshared part. Comparing the expression of MYBL2 gene in different leaf color materials of Brassica, the results showed that the expression of most of the homologous genes of MYBL2 gene in purple leaf materials was higher than that in green leaf materials except kale. In kale, the expression of BolC06.MYBL2-1 and BolC02.MYBL2-2 in green kale were 2.5 and 3.5 times that in purple kale, respectively. In Brassica napus, the expression of BnaA07.MYBL2-1, BnaC06.MYBL2-1 and BnaC02.MYBL2-2 in purple leaf and white flower were 7.5, 8.6 and 26.0 times of that in green leaf and white flower, while the expression of BnaA02.MYBL2-2 in green leaf and white flower was 13.0 times of that in purple leaf and white flower. The expression levels of BjuA07.MYBL2-1, BjuB03.MYBL2-1, BjuA02.MYBL2-2 and BjuB05.MYBL2-2 in Brassica juncea were 8.3 times, 11.8 times, 23.2 times and 14.6 times of those in Sichuan yellow respectively. In B. carinata with purple leaf, BcaMYBL2-1 and BcaB03.MYBL2-1 were 7.1 and 27.6 times as much as W-BCDH76, respectively. However, BcaB05.MYBL2-2 and BcaC03.MYBL2-2 genes of W-BCDH76 were 2.8 and 5.0 times as much as those of B. carinata with purple leaf, respectively. Five pairs of primers were obtained, which can effectively identify the MYBL2 from A, B and C genomes of Brassica. 【Conclusion】After shading treatment, MYBL2 gene expression was closely related to light. The regulation mechanism of MYBL2 gene in Brassica plants involved in anthocyanin biosynthesis was different from that of Arabidopsis plants in which MYBL2 gene negatively regulated anthocyanin biosynthesis.

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    Mapping and Analysis of QTL for Embryo Size-Related Traits in Tetraploid Wheat
    CHEN JiHao, ZHOU JieGuang, QU XiangRu, WANG SuRong, TANG HuaPing, JIANG Yun, TANG LiWei, $\boxed{\hbox{LAN XiuJin}}$, WEI YuMing, ZHOU JingZhong, MA Jian
    Scientia Agricultura Sinica    2023, 56 (2): 203-216.   DOI: 10.3864/j.issn.0578-1752.2023.02.001
    Abstract397)   HTML68)    PDF (1615KB)(185)       Save

    【Objective】 This study is to excavate embryo-related quantitative trait loci (QTL) with potential breeding value, to explore the genetic relationship between embryo and other agronomic traits in tetraploid wheat, and finally to aim at laying an important foundation for the fine mapping and breeding utilization of embryo-related traits in the future. 【Method】A total of 121 F8 recombinant inbred lines (RIL) constructed by crossing tetraploid durum wheat (Ailanmai) and wild emmer wheat (LM001) were used. This RIL population was planted in five different environments including Chongzhou (2018-2020), Wenjiang (2020), and Ya'an (2020) in Sichuan Province for phenotypic evaluation of embryo length (EL), embryo width (EW), embryo length/embryo width (EL/EW), embryo length/kernel length (EL/KL), embryo width/kernel width (EW/KW), and embryo area (EA). QTL mapping was performed based on a genetic linkage map constructed based on the wheat 55K SNP. 【Result】 The embryo size-related traits showed approximately normal distribution in the RIL population satisfying the genetic characteristics of quantitative traits. A total of 27 QTL for embryo size-related traits were detected in five environments over three years. Among them, seven ones controlling EL could contribute 7.75%-21.74% of phenotypic variation. Seven QTLs controlling EW could explain 7.67%-33.29% of phenotypic variation. Five stable and major QTLs (QEL.sicau-AM-3B, QEW.sicau-AM-2B, QEW/KW.sicau-AM-2B, QEL/EW.sicau- AM-2B-1 and QEA.sicau-AM-2B) were identified, and they explained 11.88%-18.99%, 21.77%-29.41%, 8.80%-24.92%, 12.79%- 25.69% and 10.47%-15.22% of phenotypic variation, respectively. In addition, four QTL-rich regions were identified in the embryo size-related loci mentioned above. The QTL controlling EL/KL and EL was located on chromosome 1B, that for EW, EL/EW, EW/KW, and EA was located on 2B, that controlling EL and EA was on 3B, and that controlling EL/EW and EW/KW was on 6B. Embryo size was significantly and positively correlated with kernel size. Further, the major QTL for EL, QEL.sicau-AM-3B was co-located with that for kernel length identified previously, but that for EW QEW.sicau-AM-2B was independent of that for kernel width. Four genes likely involved in regulation of embryo size were identified in intervals where major QTL were mapped. 【Conclusion】Five stable and major QTLs were identified: QEL.sicau-AM-3B, QEW.sicau-AM-2B, QEW/KW.sicau-AM-2B, QEL/EW.sicau-AM-2B-1, QEA.sicau-AM-2B, among which QEW.sicau-AM-2B may be novel.

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    Development of DNA Molecular ID in Camellia oleifera Germplasm Based on Transcriptome-Wide SNPs
    LIN Ping, WANG KaiLiang, YAO XiaoHua, REN HuaDong
    Scientia Agricultura Sinica    2023, 56 (2): 217-235.   DOI: 10.3864/j.issn.0578-1752.2023.02.002
    Abstract415)   HTML78)    PDF (10200KB)(181)       Save

    【Objective】Camellia oleifera is a traditional woody oil plant and been widely cultivated in China. In order to facilitate the protection and precise management of C. oleifera cultivars and avoid the phenomenon of homonyms and synonyms, single-nucleotide polymorphism (SNP) marker database of C. oleifera cultivars was established, and a set of core SNPs were selected to construct molecular fingerprint and ID for each cultivar. 【Method】The RNA of developing seeds of 221 C. oleifera clones was extracted and RNA-seq were performed. Using C. oleifera var. ‘Nanyongensis’ genome sequence as reference, high-quality SNPs for C. oleifera were screened and the genotyping of accessions was carried out. Furthermore, the genetic diversity of C. oleifera population and subpopulations were analyzed using SNP data, including observed heterozygosity, expected heterozygosity and polymorphism information content (PIC) of the SNPs, etc. The SNP loci were further filtered by their polymorphism and location information to obtain the optimal combination of core SNP loci. Sanger-seq was performed to verify the core SNP loci. The fingerprints of each clone were formed according to the genotypes of the core SNPs. The molecular IDs of C. oleifera clones were finally constructed by combining the basic information and fingerprint of C. oleifera clones. 【Result】A total of 1 849 953 high-quality SNP loci were obtained from the transcriptomes of C. oleifera. The average values of observed heterozygosity, expected heterozygosity, fixed index, PIC and minor allele frequency of the C. oleifera population were 0.2966, 0.2462, -0.2048, 0.2073, and 0.1648, respectively. The genetic differentiation among the subpopulations of C. oleifera was minor with the high level gene flow, while the main variation was inside of the subpopulation. Filtered by PIC, LD, etc., 31 core SNP loci were screened out to distinguish all C. oleifera clones. The genotypes of all accessions in the eight core loci were further detected using Sanger-seq, and the verified rates were over 91.36%. All C. oleifera clones used in this study can be distinguished using the DNA fingerprints constructed by the 31 core SNPs. Based on the fingerprint of 31 SNP markers and the basic information of C. oleifera clones, a molecular ID of each clone, which composed of 66 digits, was formed finally. 【Conclusion】According to the polymorphism information of SNP markers, 31 core SNP loci were catched. And all C. oleifera clones were accurately distinguished. Furthermore, The DNA fingerprints of 221 C. oleifera clones were constructed by the 31 SNP markers. A unique molecular identity code for each germplasm was constructed using the DNA fingerprints and the converted serial codes from information of the C. oleifera clones. Finally, the bar codes and quick response (QR) codes are generated as the molecular ID, which can be quickly identified by the code scanning equipment.

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    Evaluation of Resistance to Leaf Scald Disease in Different Sugarcane Genotypes
    DU JinXia,LI YiSha,LI MeiLin,CHEN WenHan,ZHANG MuQing
    Scientia Agricultura Sinica    2022, 55 (21): 4118-4130.   DOI: 10.3864/j.issn.0578-1752.2022.21.003
    Abstract353)   HTML57)    PDF (2126KB)(175)       Save

    【Objective】Sugarcane leaf scald disease is an important bacterial disease affecting sugarcane yield. Selection of disease-resistant genotypes can effectively reduce the incidence of this disease. This study aimed to explore the leaf-scald resistance of sugarcane genotypes, standardize resistance evaluation method, and provide a basis for the selection and utilization of germplasm resources of sugarcane. 【Method】Xanthomonas albilineans JG43 isolated from Guitang 46, was used as inoculum on 70 different sugarcane genotypes using the decapitation method by placing 500 mL of bacterial suspension on the surface previously cut above the apical meristem with scissors dipped in the inoculum suspension of 108 CFU/mL. The disease incidence (IC) was calculated at 14, 28, 42, 56, and 70 days post-inoculation (Dpi). The disease index (DI) and the area under the disease progress curve (AUDPC) were calculated according to the disease severity of leaf scald in sugarcane. Variance, principal component, and discriminant analysis were performed using SPSS 25.0 software. Among them, a general linear model procedure (PROC) and the square sum model of type III were used to analyze the variance, with IC, DI and AUDPC as dependent variables, genotype, block and days post-inoculation as fixed factors. After the original data were processed by standardization (Z-score), principal component analysis was carried out by KOM and Bartlett sphere test. The Euclidean metric was calculated for cluster analysis using the WPGMA method of DPS 9.50 software. The discriminant analysis was performed to evaluate the clustering results according to Fisher’s criterion. 【Result】Some genotypes displayed white pencil lines at 14 dpi, then gradually expanded to the edge at 28 dpi. The leaves began yellowing or albinism from the edge to the veins at 42 dpi, then curled inward and died at 56 dpi. The severely infected plant withered and eventually died at 70 dpi. Variance analysis exhibited highly significant effects for IC, DI, and AUDPC among genotype (Gen), days post-inoculation (Dpi), and their interactions effect (Gen × Dpi) (P<0.01). Approximately 42% of the total sum of square was attributed to Dpi effect, indicating significant differences among genotypes resistance across the days post-inoculation. At 56 dpi, the disease reached a steady plateau, and the data in this period could be better divided among sugarcane genotypes. The results of discriminant and cluster analysis showed that 70 genotypes were divided into five different groups, including 15 highly resistant, 14 resistant, 15 moderate, 11 susceptible, and 15 highly susceptible genotypes. 【Conclusion】The resistance of sugarcane genotypes to leaf scald was assessed using the decapitation method, the IC, DI and AUDPC at 56 dpi were used as the evaluation indicators. The combined method of clustering and discriminant analysis could improve the accuracy of clustering results. Fifteen genotypes of high resistance to leaf scald were assessed and used for the sugarcane breeding program in China, including Zhongzhe 9, Zhongzhe 4, Zhongzhe 2, GUC19, GUC8, Yunrui 03-103, Yunrui 05-649, Yunrui 05-182, Yunrui 05-367, Yunrui 89-159, Funong 11601, Funong 09-4059, Guitang 02-467, Guitang 08-297, ROC22.

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    Analysis of Cross Compatibility Variation Among Diverse Sesamum Species and Biological Characteristics of the Interspecific Hybrid Progenies
    JU Ming, MIAO HongMei, HUANG YingYing, MA Qin, WANG HuiLi, WANG CuiYing, DUAN YingHui, HAN XiuHua, ZHANG HaiYang
    Scientia Agricultura Sinica    2022, 55 (20): 3897-3909.   DOI: 10.3864/j.issn.0578-1752.2022.20.003
    Abstract324)   HTML50)    PDF (4302KB)(155)       Save

    【Objective】 The research aims to explore the cross compatibility between different Sesamum species and analyze the biological characteristics of interspecies hybrid progeny so as to supply the foundation for efficient application of wild sesame species. 【Method】 A sesame cultivar Yuzhi 11 (S. indicum, 2n=26) and 4 wild Sesamum species including S. latifolium (2n=32), S. calycinum (2n=32), S. angustifolium (2n=32), and S. radiatum (2n=64) were applied to construct interspecies cross combinations using diallel hybridization method by artificial pollination in the field. Embryo rescue method was also used to obtain interspecific hybrid F1. Interspecific hybrid compatibility was compared based on hybrid capsule formation rate. Botanical characters of hybrids were observed during flowering and mature stages. Pollen fertility was assessed using Alexander staining method. Chromosome number and karyotype characteristics of root somatic cells of hybrids were observed using smear chromosome preparation technique. Specific and polymorphic SSR primers in Sesamum were used to analyze the molecular difference in interspecific hybrids.【Result】 Twenty positive and reciprocal cross combinations were constructed for the 5 Sesamum species. A total of 2091 flowers were pollinated and 370 hybrid capsules were harvested. As to the female parents with more chromosomes, hybrid capsules were more easily obtained. The cross compatibility among the 5 Sesamum species significantly varied from 1.18% (S. radiatum×S. calycinum) to 63.33% (S. calycinum×S. angustifolium). F1 plants of 9 combinations produced hybrid seeds, while the ratio of pollen sterility of F1 progeny ranged from 35.21%-100.00%. The cross S. calycinum×S. angustifolium presented the highest sterility ratio to 87.68%. Hybrid progeny exhibited the obvious heterosis over parents in plant height, plant type, and some key agronomic traits. As to the positive and reciprocal hybrid F1 derived from sesame cultivar and the wild species, leaf shape, flower shape, and flower color showed partial characters of both parents. The cross compatibility between sesame cultivar (n=13) and the 3 Sesamum species with chromosome group n=16 ranked as S. angustifolium>S. calycinum>S. latifolium. The cross compatibility between wild species S. radiatum (n=32) and the 3 species with n=16 ranked as S. calycinum>S. angustifolium>S. latifolium. Among the 5 Sesamum species, the genetic relationship between S. calycinum and S. angustifolium is relatively closest. The chromosome number of root tip cells of some hybrid plants is consistent with the theoretical value calculated from the parents. Screening results of the 3 pairs of polymorphic SSR primers indicated that 99.66% of obtained F1 plants are true hybrid. Chromosome karyotype and SSR marker screening results reflected the genetic difference and characters of Sesamum species. 【Conclusion】Among the 5 Sesamum species, the cross compatibility varies significantly and the heterosis of interspecific hybrid is obvious. Of which only S. calycinum and S. angustifolium have the relatively closest genetic relationship and could be directly applied for elite germplasm creation and interspecific hybrid breeding in Sesamum. Reproductive isolation barriers exist in other cross combinations. Some techniques including embryo rescue and molecular marker application should be used to achieve the utilization of wild Sesamum species for sesame breeding.

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    Genome-Wide Association Study of Yield Component Traits in Upland Cotton (Gossypium hirsutum L.)
    WANG Juan, MA XiaoMei, ZHOU XiaoFeng, WANG Xin, TIAN Qin, LI ChengQi, DONG ChengGuang
    Scientia Agricultura Sinica    2022, 55 (12): 2265-2277.   DOI: 10.3864/j.issn.0578-1752.2022.12.001
    Abstract520)   HTML121)    PDF (6687KB)(270)       Save

    【Objective】The loci, elite alleles and candidate genes associated with yield component traits, such as boll weight, lint percentage, number of bolls per plant and seed index, were explored using a genome-wide association analysis (GWAS), which provided a theoretical reference for the molecular breeding of cotton yield.【Method】The GWAS based on a mixed linear model was performed on 408 upland cotton accessions grown in six different environments using the Cotton SNP 80K chip for the four yield component traits, and the significant SNP loci (SNPs) and elite allele were also detected. Finally, on the basis of the gene expression levels of the transcriptome, candidate genes related to the target traits were mined within a 1 Mb genome range of the flanking sequences of the significant SNPs. 【Result】The four yield component traits showed wide phenotypic variations in different environments, with the maximum coefficient of variation for number of bolls per plant being 16.67%-22.66%. The heritability of each trait was between 48.4% and 92.2%. The correlations among traits were significant or highly significant, except between boll weight and lint percentage. A total of 23 significant SNPs distributed in seven different genomic regions associated with the four traits were identified across the 408 cotton accessions in the BLUP. The numbers of loci associated with boll weight, lint percentage, number of bolls per plant and seed index were 5, 1, 9 and 8, respectively, and three loci (TM21094, TM21102, and TM57382) were associated with multiple target traits simultaneously. Seven elite allele types, TM21099(TT), TM57382(GG), TM78920(CC), TM53448(TT), TM59015(AA), TM43412(GG) and TM69770(AA), were identified. A total of 158 candidate genes potentially related to yield formation were selected through an analysis of gene expression patterns in RNA-Seq data. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that the functions and metabolic pathways of most genes were varied.【Conclusion】In this study, 23 significant SNPs associated with four yield component traits were identified across 408 cotton accessions, and 158 candidate genes were predicted using RNA-Seq.

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    Comprehensive Evaluation of Potato Tuber Texture
    LI WenLi, YUAN JianLong, DUAN HuiMin, JIANG TongHui, LIU LingLing, ZHANG Feng
    Scientia Agricultura Sinica    2022, 55 (12): 2278-2293.   DOI: 10.3864/j.issn.0578-1752.2022.12.002
    Abstract420)   HTML74)    PDF (3410KB)(180)       Save

    【Objective】 The comprehensive evaluation of texture qualities of potato tubers not only are beneficial to the subdivision of processing quality traits and accurately locate purpose of potato, but also assist breeders screening new varieties, and accelerate the development of potato products.【Method】Potato cultivated varieties were selected as the research samples. Puncture, TPA compression and shear methods were chosen to analyze the texture parameters. These included puncture distance, puncture initial force, puncture speed, compression deformation, compression speed, compression interval time, compression initial force, shear initial force and speed. The texture indexes of eight varieties were measured under the optimal texture analyzer parameters setting, then the correlation among the texture parameters and the evaluation of optimal texture parameters were analyzed. 【Result】 Optimal parameters of fresh tuber puncture: Cylindrical metal probe (TMS 2 mm Steel), 2 mm puncture distance and, 2.5 N initial force, 50 mm·min-1 detection speed. The optimal test factors of TPA compression (fresh/steamed): The cylinder sample for fresh and steamed tubers both ranged in the diameter and height from 10 mm to 15 mm, no significant difference was examined among three probe selection in the fresh tubers. Cylindrical aluminum probe (TMS 36.0 mm Aluminum Cylinder) was the optimal type for steamed tubers probe. The optimal parameters (fresh/steamed): 50% and 60% deformation, 60 mm·min-1 and 80 mm·min-1 detection speed, 6 s and 10 s interval time, both 0.7 N initial force. The optimal shear parameters (fresh/steamed): The length, width and height of the cuboid sample were 30 mm, 15 mm, and 10 mm, respectively. The probe type was light single knife probe (TMS Perspex Knife Edge), with both 60 mm·min-1 detection speed, and 1 N and 0.5 N initial force. They’re existed significant correlation between springiness and the peel crispness, no significant correlation among the other texture parameters of TPA compression and shear. They’re existed significant positive correlation among puncture, TPA compression and shear texture parameters (0.410-0.959) in fresh tubers. There also existed significant positive correlation between TPA compression and shear texture parameters (0.441-0.952) in steamed tubers. 【Conclusion】 Puncture, TPA compression and shear methods were suitable for the samples evaluation of the quality of fresh tubers. The indexes of peel hardness, peel brittleness, TPA hardness, cohesiveness, chewiness, shear hardness can be chosen as important parameters to compare differences of texture. TPA compression and shear methods were suitable for the sample’s evaluation of the quality of steamed tubers. The indexes of TPA hardness, adhesiveness, cohesiveness, springiness, chewiness, shear hardness can be chosen as important parameters to compare differences of texture.

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    Cloning and Drought Resistance Analysis of Transcription Factor GhMYB108 in Gossypium hirsutum
    LIU RuiDa, GE ChangWei, WANG MinXuan, SHEN YanHui, LI PengZhen, CUI ZiQian, LIU RuiHua, SHEN Qian, ZHANG SiPing, LIU ShaoDong, MA HuiJuan, CHEN Jing, ZHANG GuiYin, PANG ChaoYou
    Scientia Agricultura Sinica    2022, 55 (10): 1877-1890.   DOI: 10.3864/j.issn.0578-1752.2022.10.001
    Abstract549)   HTML92)    PDF (7239KB)(392)       Save

    【Objective】As one of the largest transcription factor families in plants, MYB genes play an important role in resisting stress. The MYB transcription factor GhMYB108 was cloned and analyzed to verify its role in drought stress response, which laid a foundation for further study on the molecular mechanism of GhMYB108 regulating drought tolerance in G. hirsutum.【Method】Through the analysis of unpublished drought transcriptome data, GhMYB108 was identified to drought response. The target gene was amplified from the root cDNA by polymerase chain reaction (PCR). Through bioinformatics analysis of gene structure characteristics, the sequence information and phylogenetic relationship of these genes were predicted. The obtained gene promoter sequences were analyzed by Plant Care website. The genes expression characteristics under different stress conditions were analyzed using Real time fluorescence quantitative PCR (qRT-PCR). The location of GhMYB108 protein was determined by subcellular localization. The transcriptional activity was tested in yeast cell; The GhMYB108 gene was silenced using Virus induced gene silencing (VIGS), and the gene silencing efficiency was detected by qRT-PCR. The phenotypic changes before and after drought treatment were observed and the survival rate was counted. The relevant physiological and biochemical indexes were measured by Solarbio Kit; The relationship between GhMYB108 and ABA was analyzed by spraying ABA and Fluridone on cotton leaves.【Result】GhMYB108 (Gh_A10G1563) was cloned from G. hirsutum, with 879 bp length and 292 amino acids. Its protein relative molecular weight and isoelectric point is 33.288 kD and 6.037, respectively. Multiple sequence alignment and conserved domain analysis showed that GhMYB108 contains two highly conserved MYB binding domains, which belongs to a typical R2R3 MYB transcription factor. Phylogenetic analysis of different species showed that GhMYB108 was highly homology with ATMYB108, ATMYB78 and ATMYB2, belonging to the same subfamily. Previous studies found that ATMYB108, ATMYB78 and ATMYB2 were related to drought and ABA signaling pathway. GhMYB108 located in the nucleus and had transcriptional activation activity. The expression level of GhMYB108 was the highest in roots and the lowest in stems, and was induced by abiotic stresses including natural drought, 18% PEG 6000 simulated drought, salt stress and low temperature. The GhMYB108 silenced plants showed a critical phenotype under natural drought conditions. Compared with the control, the silenced plants showed more serious wilting and decreased survival rate. Some physiological and biochemical indexes also changed significantly, such as accelerated leaf water loss rate, increased malondialdehyde content, decreased leaf relative water content and proline content, and decreased CAT and POD activities. Through DAB and NBT staining, the hydrogen peroxide (H2O2) and superoxide anion (O2-) were significantly accumulated in plants. By spraying the hormone ABA or Fluridone on cotton leaves, we found that GhMYB108 could be positively regulated by ABA signal.【Conclusion】GhMYB108 positively regulates cotton drought resistance and is positively regulated by ABA signal.

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    Research Progress of Nitrogen Efficiency Related Genes in Rice
    SANG ShiFei, CAO MengYu, WANG YaNan, WANG JunYi, SUN XiaoHan, ZHANG WenLing, JI ShengDong
    Scientia Agricultura Sinica    2022, 55 (8): 1479-1491.   DOI: 10.3864/j.issn.0578-1752.2022.08.001
    Abstract613)   HTML104)    PDF (904KB)(409)       Save

    Over the last few years, China government has put forward a strategy to achieve the goal of “zero growth of chemical fertilizer”. It is particularly important to reduce the input of nitrogen fertilizer in agricultural production and enhance the nitrogen use efficiency in crops. Nitrogen is mostly absorbed from the soil by plant roots in the form of nitrate nitrogen (NO3-) and ammonium nitrogen (NH4-). It is transported from roots in plants to synthesize essential life substances, such as amino acids and nucleotides. Nitrogen is used as a basic element for crop growth and yield formation. However, excessive application of nitrogen fertilizer destroys the physical and chemical properties of the soil, causes undesirable changes to soil salinization, and pollutes the ecological environment, and pollutes the ecological environment. By reducing the quantity of nitrogen fertilizer, will destabilize the yield potential of field crops including rice and wheat which is being used as a staple food in China. It can threaten food security of the country. To improve the nitrogen use efficiency (NUE) and stabilize the food security, mining nitrogen-efficient genes, such as NRT1.1B, OsGRF4 etc., genetic improvement of current existing varieties through molecular design breeding will help to cultivate new nitrogen efficiently rice lines. Tapping the productive potential of current rice varieties will improve the level of sustainable agricultural development in our country. In this article, based on the nitrogen-efficient genes excavated in the current rice research, this article reviews the PTR (polypeptide transporter) family, NRT (nitrate transporter) family, AMT family (ammonium transporter family), NLP family and other types of rice nitrogen-efficient genes. The future prospects of gene utilization have been prospected. Based on the nitrogen-efficient genes excavated in the current rice research, they are divided into four categories: NRT/PTR, AMT (ammonium transporter), NLP and other types, and summarize their functions and characteristics, and analysis the utilization prospect and existing problems of nitrogen-efficient genes with potential breeding value.

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    Unconditional and Conditional QTL Analysis of Wheat Spike Length in Common Wheat Based on 55K SNP Array
    TANG HuaPing, CHEN HuangXin, LI Cong, GOU LuLu, TAN Cui, MU Yang, TANG LiWei, LAN XiuJin, WEI YuMing, MA Jian
    Scientia Agricultura Sinica    2022, 55 (8): 1492-1502.   DOI: 10.3864/j.issn.0578-1752.2022.08.002
    Abstract431)   HTML52)    PDF (985KB)(161)       Save

    【Objective】This study is to excavate spike length (SL)-related quantitative trait loci (QTL) with potential breeding value, explore the genetic relationship between SL and other important agronomic traits in wheat, and aim at laying a foundation for fine mapping and molecular-assisted selection breeding. 【Method】A total of 126 F7 recombinant inbred lines (RIL) constructed by crossing 20828 and SY95-71 were used in this study. The RIL population including their parents were planted in seven different environments for phenotypic evaluation: Wenjiang, Chongzhou, Ya'an of Sichuan Province in China, and Khulna in Bangladesh during 2016-2017 and 2017-2018 growing seasons. Unconditional QTL mapping was performed using a genetic linkage map constructed using the wheat 55K SNP array, and QTLs’ effects were further analyzed. Conditional QTL analysis was performed to analyze the relationship between SL and other agronomic traits including plant height (PH), spike extension length (SEL), spikelet number per spike (SNS) and thousand-kernel weight (TKW). 【Result】Thirteen QTLs controlling SL were identified using unconditional QTL mapping, and they were located on chromosomes 1A, 1D, 2B, 2D, 4B, 6D, and 7A. The LOD values ranged from 2.79 to 6.19, and the phenotypic variation rate ranged from 5.35% to 12.77%. Three stable and major QTLs (QSl-sau-2SY-2B, QSl-sau-2SY-2D.5 and QSl-sau-2SY-4B) were identified, and they explained 6.54% to 11.72%, 10.16% to 12.57%, and 5.35% to 10.92% of phenotypic variation rate, respectively. Furthermore, these three major QTLs could be also detected in multi-environment analysis. Moreover, aggregation analysis suggested that the SL of lines polymerizing the positive allels at these three major QTLs was significantly longer than that of those with any two ones or those carrying only one. Meanwhile, it was found that QSl-sau-2SY-2B had no significant effect on PH, SEL, SNS and TKW. QSl-sau-2SY-2D.5 had a significant effect on improving TKW (3.98%), but no significant effect on PH, SEL and SNS. QSl-sau-2SY-4B had a significant effect on decreasing PH (-12.28%) and SEL (-22.26%), but no significant effect on SNS and TKW. The conditional QTL analysis showed that QSl-sau-2SY-2B was independent of PH and SEL, whereas, affected by SNS and TKW. QSl-sau-2SY-2D.5 was independent of SEL, SNS and TKW, but affected by PH. QSl-sau-2SY-4B was independent of SEL and TKW, but affected by PH and SNS. 【Conclusion】In this study, three stable and major QTLs were identified for SL: QSl-sau-2SY-2B, QSl-sau-2SY-2D.5, and QSl-sau-2SY-4B, among which QSl-sau-2SY-2B may be a novel QTL independent of PH and SEL.

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    Characteristics and Cold Tolerance of Upland Cotton Genetic Standard Line TM-1
    WANG JunJuan, LU XuKe, WANG YanQin, WANG Shuai, YIN ZuJun, FU XiaoQiong, WANG DeLong, CHEN XiuGui, GUO LiXue, CHEN Chao, ZHAO LanJie, HAN YingChun, SUN LiangQing, HAN MingGe, ZHANG YueXin, FAN YaPeng, YE WuWei
    Scientia Agricultura Sinica    2022, 55 (8): 1503-1517.   DOI: 10.3864/j.issn.0578-1752.2022.08.003
    Abstract696)   HTML44)    PDF (6422KB)(182)       Save

    【Objective】We systematically investigated the major agronomic traits and cold tolerance of accession TM-1 at the bud and seedling stages. The relative expressions of cold tolerance-related genes were analyzed by the qRT-PCR method. The cold tolerance mechanism of TM-1 was further discussed, which provides the theoretical basis for the breeding utilization of TM-1.【Method】The major agronomic traits of TM-1 were manually investigated in the field using variety CRI35 as the control. The fiber quality was assessed by an international calibrated cotton standard (HVICC), and the insect resistance (Bt) was detected by kanamycin screening and molecular detection technologies. For the cold tolerance testing, two contrasting accessions, cold-resistant accession Yu 2067 and cold-sensitive variety Hengmian 3 were set as controls, respectively. The cold resistance of TM-1 at bud stage and cotyledon stage was identified, treated at 4℃ and then recovered under normal conditions for 7 days, and the relative cotyledon spreading rate and the cold injury levels of plants were investigated, and cold injury indexes and cold resistance indexes were calculated. The portable chlorophyll meter was used for in vivo testing the leaf relative chlorophyll content (represented by SPAD value). The expressions of cold tolerance-related genes in leaves were measured by qRT-PCR method. 【Result】 The leaves of TM-1 were large and dark green. The pre-frost seed cotton yield was 2 791.50 kg·hm-2, and the plant height was 94.60 cm. The growth period was about 135 days, and the yield, plant height, fruit branch number per plant, boll number per plant were higher than CRI35, while other agronomic traits were similar to CRI35. TM-1 had medium fiber quality. The test results of kanamycin and test paper showed that TM-1 did not contain the Bt like CRI35. Identification results of cold tolerance at bud stage showed that compared with the control treatment, the relative chlorophyll content and plant height of TM-1 decreased significantly. Low-temperature stress significantly inhibited hypocotyl elongation and chlorophyll synthesis in cotton leaves. Under low-temperature treatment, the taproots of TM-1 were damaged, but the lateral roots were more developed than those of the control. The cold tolerance level of TM-1 reached high cold resistance at the bud stage. Identification of cold tolerance at the cotyledon stage showed that the relative chlorophyll content and plant height of TM-1 decreased significantly compared with the control. The cold tolerance index of TM-1 at the cotyledon stage was 85.32%, which was significantly higher than Yu 2067, and the tolerance level of TM-1 reached cold resistance at the cotyledon stage. After the treatment of low-temperature stress for 24 h at the trefoil stage, nine genes were up-regulated in the TM-1 leaves, and their up-regulated expression folds were significantly higher than those of cold-sensitive accession. Dehydrin gene was up-regulated in TM-1 leaves, and the expression fold was similar to that in the leaves of Yu 2067, which was 4.69 times that in the leaves of Hengmian 3. The expression fold of the LEA3 gene in TM-1 leaves was significantly higher than that of Yu 2067 and Hengmian 3. 【Conclusion】 Accession TM-1 has stable agronomic characters and the medium fiber quality. It can be used as an ideal receptor for transferring exotic genes because without Bt. TM-1 can also be used as an important parent for cotton breeding and a gene source for cloning genes because of its good cold tolerance.

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    Mapping of QTLs for Chlorophyll Content in Flag Leaves of Rice on High-Density Bin Map
    ZHAO Ling, ZHANG Yong, WEI XiaoDong, LIANG WenHua, ZHAO ChunFang, ZHOU LiHui, YAO Shu, WANG CaiLin, ZHANG YaDong
    Scientia Agricultura Sinica    2022, 55 (5): 825-836.   DOI: 10.3864/j.issn.0578-1752.2022.05.001
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    【Objective】Finding new loci and genes related to rice chlorophyll content, and providing new theoretical basis for the research on the genetic mechanism of rice chlorophyll content. 【Method】 A recombinant inbred line (RIL) population containing 186 lines was constructed by crossing the japonica rice TD70 and the indica rice Kasalath with obvious difference in the chlorophyll content of the flag leaf. The two parents and RIL population were re-sequenced to construct a high-density genetic linkage map with 12 328 recombination Bin markers. The RILs and two parents were planted in fields at the Jiangsu Academy of Agricultural Sciences, in Nanjing in 2011 and 2020. The contents of chlorophyll of flag leaves were directly measured using the chlorophyll meter SPAD-502 on the 3rd day after heading. QTLs that control the chlorophyll content of the flag leaf at the heading stage of rice were detected by IciMappingv3.4 software with inclusive compound interval mapping method. The photosynthesis parameters of 20 SPAD extreme strains in the RIL population were measured with a portable photosynthesis system. 【Result】19 QTLs controlling chlorophyll content of flag leaves were detected on 9 chromosomes except Chr.8, Chr.9 and Chr.10 in two years. The phenotype variation explained (PVE) of single QTL ranged from 3.09% to 13.13%, LOD value ranged from 2.74 to 14.08. After comparing the physical positions, 10 QTLs were found to locate in the same interval or adjacent to previously QTLs. qCHL2-1 and qCHL5-1 were detected every year showing their genetic stability. qCHL2-1 was mapped between the 7.63-7.71 Mb on chromosome 2, and the two-year LOD values are 14.08 and 7.93 with the PVE 13.13% and 7.94%, respectively. qCHL5-1 was mapped between the 23.44-23.49 Mb on chromosome 5, and the two-year LOD values are 4.31 and 3.76, respectively. After the annotation and sequences analysis of genes located in the region of qCHL2-1and qCHL5-1, two genes, Os02g0236000 and Os05g0476700, were found to be associated with chlorophyll content of flag leaves in the rice. There are differences in sequences of the two genes between TD70 and Kasalath. Os02g0236000 is the AAT1 gene encoding the Aspartate Aminotransferase, which is an important enzyme in nitrogen metabolism and related to protein and amino acid content of rice. Os05g0476700 encodes protein relating to spotted leaf, which might associate with leaf color. Based on the mutation of AAT1 at CDS+273 bp, the haplotypes of ATT1 were classified in RIL population. Among the 20 extreme SPAD RIL lines, there were significant differences between different haplotype of ATT1 in SPAD value, chlorophyll content, water use efficiency, transpiration rate, stomatal conductance and net photosynthetic rate of flag leaf. 【Conclusion】19 QTLs associated with chlorophyll content in flag leaf at heading stage of rice were detected and two stable QTL loci, qCHL2-1and qCHL5-1 were identified. Two candidate genes were obtained after annotation and sequence comparison. One of them, ATT1, was considered as the most possible candidate gene after effort analysis of different haplotypes in photosynthetic efficiency. The QTLs and gene we obtained could be used for subsequent functional studies of flag leaf chlorophyll regulation and molecular marker breeding.

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