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    Function of FCS-Like Zinc-Finger Protein OsFLZ18 in Regulating Rice Flowering Time
    MA YaMei,ZHANG ShaoHong,ZHAO JunLiang,LIU Bin
    Scientia Agricultura Sinica    2022, 55 (20): 3875-3884.   DOI: 10.3864/j.issn.0578-1752.2022.20.001
    Abstract569)   HTML115)    PDF (1565KB)(297)       Save

    【Objective】Flowering time is an important agronomic trait which determines the yield and regional adaptability of rice, but the underlining molecular regulatory mechanism need further study. FCS-like Zinc finger proteins (FLZs) are a class of plant specific regulatory proteins which play essential roles in plant growth and stress response, but their functions in regulating flowering time have not been reported. This study aims to investigate the potential function of FLZ proteins in rice flowering time control. The finding will broaden our understanding on the molecular regulatory mechanism of rice flowering time.and provide new theoretical basis and gene resource for rice breeding. 【Method】Based on the target sequences published in RGAP database, OsFLZ18 overexpression vector and CRISPR-Cas9 vector were generated and introduced into Japonica variety Nipponbare by Agrobacterium tumefaciens-mediated genetic transformation assay. Homozygous CRISPR knockout mutants were screened by PCR and sequencing analyses. The quantitative real-time PCR (qRT-PCR) assay was used to examine the spatial-temporal expression and diurnal rhythmic expression of OsFLZ18, as well as the effects of OsFLZ18 on the transcription of several known flowering time-related genes. Yeast two-hybrid assay (Y2H) was used to test the interaction between OsFLZ18 and the flowering time-related regulatory proteins.【Result】OsFLZ18 was ubiquitously expressed in various rice tissues, with the highest expression level in 14 day-old seedling, followed by leaf sheaths and leaf blades at the tillering stage, and stem and young panicles at reproductive stages. The OsFLZ18-CRISPR vector was constructed and transformed into Nipponbare. Two independent homozygous OE lines (OE-2, OE-3) with higher OsFLZ18 expression level and two homozygous mutants (CRISPR-21, CRISPR-25) were selected for further study. Phenotypic observation showed that the OE lines flowered later than the wild-type plants under both natural long-day and short-day conditions in Guangzhou, while the CRISPR lines had no obvious differences in heading date when compared to the wild-type plants. The expression levels of Ehd1, Hd3a and RFT1 were significantly decreased in OE-2 plants compared with those in the wild-type plants under artificial short-day conditions, but no significant difference in the expression level of Hd1 was observed between them. The results of Y2H experiment showed that OsFLZ18 interacted with OsMADS51, a positive regulator of rice flowering time. Furthermore, OsFLZ18 exhibits a diurnal rhythmic expression profile, showing lower expression levels in the daytime and higher expression levels at night with a peak at midnight. 【Conclusion】Overexpression of OsFLZ18 delays rice flowering time.

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    Identification of the Root-Specific Soybean GmPR1-9 Promoter and Application in Phytophthora Root-Rot Resistance
    YAN Qiang,XUE Dong,HU YaQun,ZHOU YanYan,WEI YaWen,YUAN XingXing,CHEN Xin
    Scientia Agricultura Sinica    2022, 55 (20): 3885-3896.   DOI: 10.3864/j.issn.0578-1752.2022.20.002
    Abstract348)   HTML60)    PDF (3529KB)(196)       Save

    【Objective】The objective of this study is to identify the root-specific promotors and the core regulatory sequence of soybean. Then evaluate the potential application of the synthetic promoter in Phytophthora root-rot resistance. 【Method】The genes which specifically expressed in roots with high expression levels were screened based on the transcriptome date of soybean root, stem and leaf tissues in the seedling stage. Based on the distribution of the cis elements, the promoter truncation approach was used to map the minimal promoter controlling root specific expression in soybean hairy roots. The obtained minimal promoter fragment was concatenated with the Phytophthora inducible promoter elements p4XD to construct the synthetic promoter. The synthetic promoter driven over-expression of Phytophthora resistance related gene GmNDR1 in soybean hairy roots, then the resistance level of transgenic tissue to Phytophthora and the expression profiles of GmNDR1 during the interaction had been analyzed. Furthermore, the transgenic Nicotiana benthamiana plants were generated to evaluate the resistance at plant level. 【Result】Though screening, six soybean PR1 homologues with significant root specific expression manner were identified, and GmPR1-9 had the highest promoter activity. Numbers of root specific expression related cis elements were identified in promoter sequence using the online tool PLACE. Truncation analysis of the promoter showed that serial 5’ end deletions L1, L2, L3, L4 and L5 had different GUS activities. The L5 (-166 to -1) fragment had 80% activity of the full-length promoter, and was able to drive GUS expression in roots of transgenic N. benthamiana. GUS enzyme activity was almost undetectable in three 3’ end deletions R1, R2 and R3, and the double terminal deletion mutant M1. When the fusion promoter p4XD-L5 driven GmNDR1 expression in soybean hairy roots, the resistance to P. sojae was significantly enhanced. The disease severity and lesion length were significantly reduced in the over-expression hairy roots when compared with control, and the relative biomass of Phytophthora decreased by 66.5% at 48 h post inoculation. GmNDR1 maintained high expression level in over-expression tissues, with 39.2 times of that in control tissues. The expressions were further up-regulated after inoculation, and reached the highest level at 36 h. In p4XD-L5::NDR1 transgenic N. benthamiana plants, the expression of GmNDR1 was significantly higher in roots than that in stems and leaves. Fifteen days after P. capsica inoculation, the plant height, root length and fresh weight of GmNDR1 over-expression plants were significantly higher, and meanwhile the leaf wilting rate and lesion length were significantly lower. 【Conclusion】This study obtained a soybean root specific promoter and identified the core regulation sequence. The strategy which driven the expression of GmNDR1 by the synthetic promoter p4XD-L5 combined the inducible and tissue-specific promoter core elements can significantly enhance the resistance of transgenic soybean hairy roots and Nicotiana benthamiana plants to Phytophthora pathogens.

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    Maize Transcription Factor ZmEREB93 Negatively Regulates Kernel Development
    PANG HaoWan,FU QianKun,YANG QingQing,ZHANG YuanYuan,FU FengLing,YU HaoQiang
    Scientia Agricultura Sinica    2022, 55 (19): 3685-3696.   DOI: 10.3864/j.issn.0578-1752.2022.19.001
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    【Objective】 Maize, a kind of crucial crop, is widely used in food supply, livestock feed, and industry. AP2/EREBP (APETALA2/ethylene response element-binding protein) transcription factor (TF) plays an important role in plant growth, development, and stress response. Previous study showed that ZmEREB93 might regulate seed size as a target gene of ZmBES1/BZR1-5 TF. ZmEREB93 was cloned and used to analyze its expression pattern and function, which lays foundation to clarify the function and mechanism of ZmEREB93 regulating seed size. 【Method】 The full length of ZmEREB93 was cloned from maize inbred line B73 by PCR. The characters of nucleotide and amino acid sequences were analyzed by informatic methods. Subsequently, the tissue expression specificity of ZmEREB93 was analyzed via quantitative real time PCR (qRT-PCR). The expression vector in plant and yeast was constructed and used for subcellular localization and transcription activation assay, respectively. ZmEREB93 was transformed into Arabidopsis mediated by agrobacterium transformation. The seed phenotype of transgenic lines was analyzed. Finally, the potential target genes of ZmEREB93 were screened by chromatin immunoprecipitation sequencing (Chip-seq) and co-expression analysis, and further confirmed by yeast one hybrid (Y1H). 【Result】 The ZmEREB93 gene was cloned by PCR. Sequence analysis showed that ZmEREB93 had no intron and a 618 bp ORF, encoding 205 amino acids with a highly conserved AP2 domain and belongs to the ERF subclade of AP2 family. The results of qRT-PCR showed that the ZmEREB93 gene highly expressed in kernels of 15 and 25 days after pollination (DAP), and slightly expressed in stem and root, but did not express in tassel, silk and bract. The expression level of ZmEREB93 was the highest in 25 DAP kernels reached 11 times of that in 15 DAP kernels. The results of transcriptional activation and subcellular localization assay exhibited that ZmEREB93 protein had no transcriptional activation activity in yeast cells and was localized in the nucleus, respectively. Compared to wild type, the seeds of transgenic lines were significantly smaller and showed lower thousand-seed-weight. Chip-seq and co-expression analysis suggested that the Zm00001d013611, Zm00001d006016, Zm00001d027448 and Zm00001d03991 genes were candidate target genes regulated by ZmEREB93 TF. The result of Y1H showed that ZmEREB93 directly bind to Zm00001d013611 promoter. 【Conclusion】 Maize ZmEREB93 TF specifically expressed in seeds and negatively regulated seed size.

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    Cloning and Functional Analyses of MsCIPK2 in Medicago sativa
    SU Qian,DU WenXuan,MA Lin,XIA YaYing,LI Xue,QI Zhi,PANG YongZhen
    Scientia Agricultura Sinica    2022, 55 (19): 3697-3709.   DOI: 10.3864/j.issn.0578-1752.2022.19.002
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    【Objective】 CIPKs are a group of important protein kinase involved in signaling pathway of plant in response to stress. They can form CBL-CIPK complex with CBL, to activate the expression of related responsive genes to cope with various abiotic stresses in cells. Exploration and study on the molecular mechanism of MsCIPK genes in alfalfa in response to abiotic stress will help to reveal the biological basis of stress resistance in alfalfa, and to provide new gene resources for alfalfa breeding with enhanced stress resistance. 【Method】 The MsCIPK2 gene was cloned by using PCR, the sequence was analyzed by bioinformatics tools, and the expression level of MsCIPK2, MsCBL2, MsCBL6, MsCBL7 and MsCBL10 genes in various tissues were analyzed by using qRT-PCR. The pCAMBIA1302-GFP-MsCIPK2 vector was transiently expressed in tobacco leaf epidermal cells, and the subcellular localization was observed under laser confocal microscope. Yeast two-hybrid assay was used to analyze interaction between MsCIPK2 and four MsCBLs proteins. Agrobacterium rhizogenes was used to generate alfalfa hairy roots over-expressing MsCIPK2. qRT-PCRs were used to analyze the expression levels of related genes in transgenic hairy root lines. 【Result】 The coding sequence of MsCIPK2 gene was obtained by using PCR, and it is 1 230 bp in length, encoding 409 amino acids. The deduced MsCIPK2 protein contained typical ATP binding site, activation loop, NAF motif and PPI motif as for the CIPK family genes. The expression level of MsCIPK2 gene was the highest in roots, and the lowest in the flowers of alfalfa. Subcellular localization results showed that MsCIPK2 protein was localized in the endoplasmic reticulum. Yeast two-hybrid assays showed that MsCIPK2 protein interacted with MsCBL2, MsCBL6, MsCBL7 and MsCBL10 proteins, showing stronger interaction with MsCBL10 than with other MsCBLs. The expression levels of MsCBL2, MsCBL6, and MsCBL10 were the highest in roots of alfalfa, and the expression level of MsCBL7 was the highest in pods. qRT-PCR results showed that the expression levels of abiotic stress-associated genes ATPase, P5CS, CYP705A5, COR47, HAK5 and RD2 were significantly up-regulated in hairy roots over-expressing MsCIPK2. Under the treatment of 200 mmol·L-1 NaCl and 20% PEG, when compared with the control hairy root line, hairy roots over-expressing MsCIPK2 had lower MDA content, and higher POD activity, proline content and soluble sugar content. 【Conclusion】 MsCIPK2 can interact with CBL protein, and responded to salt and drought stress in roots of alfalfa. Over-expression of MsCIPK2 can improve salt and drought stress resistance in alfalfa, and MsCIPK2 can be used as candidate gene for alfalfa breeding with improved abiotic stress resistance.

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    QTL Mapping of Thousand-Grain-Weight and Its Related Traits in Zhou 8425B × Xiaoyan 81 Population and Haplotype Analysis
    LinHan ZOU,XinYing ZHOU,ZeYuan ZHANG,Rui YU,Meng YUAN,XiaoPeng SONG,JunTao JIAN,ChuanLiang ZHANG,DeJun HAN,QuanHao SONG
    Scientia Agricultura Sinica    2022, 55 (18): 3473-3483.   DOI: 10.3864/j.issn.0578-1752.2022.18.001
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    【Objective】Zhou 8425B is one of the most important founder parents in China and Xiaoyan 81 is an elite cultivar with high yield and good quality. Thousand-grain weight (TGW) is an important factor that affects wheat yield. Identification of QTL associated with grain related traits from Zhou 8425B and Xiaoyan 81, and haplotype analysis of these QTL in wheat cultivars from different ecological regions would be beneficial for yield improvement by molecular marker-assisted selection.【Method】In this study, a RIL population (F8) derived from Zhou 8425B × Xiaoyan 81 was planted in Yangling during 2015 and 2016 cropping seasons to evaluate grain related traits. Using a high-density genetic map constructed by 90K SNP markers, QTL mapping of thousand-grain weight, grain length, grain width and thickness was performed under three environments. Simultaneously, the KASP markers linked to the identified QTL were developed and molecular detection was carried out among 479 wheat accessions worldwide. Moreover, haplotype analysis of target QTL was performed in 106 current wheat commercial cultivars from Yellow and Huai River Valley Winter Wheat Region selected from 479 wheat accessions.【Result】A total of 22 QTL on 8 chromosomes were detected, and the phenotypic variation explanation (PVE) ranged from 4.77% to 19.95%. Among them, 12 QTL are major QTL (PVE>10%) and Qkgw.nwafu-6B is a new QTL. QTL on chromosomes 4A, 6A, 6B, and 7D were detected in multiple environments, of which, the QTL on chromosomes 4A and 7D are same as previously reported ones. Compared to TaGW2-6A using molecular detection, both Zhou 8425B and Xiaoyan 81 carried the same allele of TaGW2. Based on haplotype result of Qkgw.nwafu-6A, Zhou 8425B and Xiaoyan 81 were placed in different groups. Therefore, Qkgw.nwafu-6A tends to be a new one. Haplotype analysis showed that there were five haplotypes for Qkgw.nwafu-6A and there were eight haplotypes for Qkgw.nwafu-6B. 6A_h1 and 6B_h6 accounted for over 20% in different ecological regions. In addition, a co-segregated KASP marker was developed for Qkgw.nwafu-6B and was significantly associated with the grain weight in the 479 wheat accessions.【Conclusion】Qkgw.nwafu-6A and Qkgw.nwafu-6B are possible new QTL associated with thousand-grain-weight, and 6A_h1 and 6B_h6 are likely favorable haplotypes. A molecular marker KASP_IWA349 co-segregated with Qkgw.nwafu-6B was developed and will be useful for marker assisted selection.

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    Deciphering of the Genetic Diversity After Field Late Blight Resistance Evaluation of Potato Breeds
    XiaoChuan LI,ChaoHai WANG,Ping ZHOU,Wei MA,Rui WU,ZhiHao SONG,Yan MEI
    Scientia Agricultura Sinica    2022, 55 (18): 3484-3500.   DOI: 10.3864/j.issn.0578-1752.2022.18.002
    Abstract329)   HTML50)    PDF (3315KB)(192)       Save

    【Objective】To evaluate the late blight resistance in field. To use SNP markers to analyze the genetic diversity of late blight resistance of potato germplasm and discern the genetic segments that may affect the phenotype of potato late blight resistance, and to provide a theoretical basis for the innovation and utilization of potato late blight resistant germplasm.【Method】Field resistance to late blight was evaluated in potato germplasm at multiple locations and over the course of several years. SNP markers were detected using a dd-RAD simplified genome sequencing strategy. The population genetic structure was analyzed using Admixture, principal component analysis was conducted using GCTA, a phylogenetic tree was constructed using fastTree, population genetic diversity parameters were calculated using the populations command in the Stacks package, selective sweep parameters were calculated using vcftools, protein sequences were aligned using Clustal Omega, and a proteins phylogenetic tree was drawn using MEGA6. Genome-wide association analysis was constructed using GEMMA 0.98.1 and QQ and manhattan plots were drawn using CMplot.【Result】Through years of evaluation of late blight field resistance in multiple locations, potato germplasm of 101 late blight resistant varieties (lines) and 21 susceptible varieties were obtained. A total of 8 697 602 relatively evenly distributed SNPs were obtained using dd-RAD simplified genome sequencing on these germplasms. This germplasm can be further divided into 6 populations through structural analysis, principal component analysis and phylogenetic analysis. The average nucleotide diversity (π) within the 6 populations ranged from 0.2055 to 0.2572 and the fixation index (Fst) among the six populations ranged from 0.156909 to 0.187336, revealing a relatively large genetic diversity for these germplasms. The expected heterozygosity (He) within the 6 populations ranged from 0.187 to 0.2297 and the observed heterozygosity (Ho) ranged from 0.0829 to 0.1186. The values of Ho were less than those of He in all six populations. Meanwhile, the inbreeding coefficient (Fis) for the six populations ranged from 0.2412 to 0.3554, indicating inbreeding events during the breeding process. To identify the genetic segments that may affect the phenotype of potato late blight resistance, π ratios and Fst among different late blight resistance germplasms in the whole potato genome were calculated using 20 kb as the window length and 5 kb as the step length. The 745 genetic segments which had a π ratio value in the lowest 5% and a Fst value in highest 10% were further analyzed by performing a selective sweep analysis. These selected segments contain a total of 507 genes, including 4 NBS-LRR genes. A genome-wide association analysis was also conducted, yielding 9 SNP highly associated with late blight resistance. Of the 69 genes located in the genome within 50 kb around the 9 SNPs, 15 genes were predicted to be involved in stress response, and 12 genes were predicted to be involved in removing peroxide radicals.【Conclusion】Large amount of SNPs, which are relatively evenly distributed in the potato genome, can be genotyped by dd-RAD simplified genome sequencing. Potato late blight field resistance germplasm has a large genetic diversity, but had inbreeding events in the process of breeding. Population structure analysis can reveal the genetic relationship between potato germplasms, which can further provide a theoretical basis for parental selection in breeding. Selective sweep and genome-wide association analyses help to isolate genetic segments that may affect late blight resistance traits.

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    Effects of Seedlings Enriched with Zinc on Cadmium Accumulations and Related Transporter Genes Expressions in Different Rice Cultivars
    JIANG XiaoTing,HUANG GaoXiang,XIONG XiaoYing,HUANG YunPei,DING ChangFeng,DING MingJun,WANG Peng
    Scientia Agricultura Sinica    2022, 55 (17): 3267-3277.   DOI: 10.3864/j.issn.0578-1752.2022.17.001
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    【Objective】 Rice seedlings enriched with zinc (Zn) is a new method to antagonize cadmium (Cd) within rice, studying the various effects and underlying mechanisms among different rice cultivars is useful to provide a basis for the application of this method. 【Method】 Hydroponic and pot experiments were conducted to test the effects of the Zn enrichment in four cultivars including the Wuyunjing21, Zhejingyou1578, Huanghuazhan, and Huiliangyousimiao. Dynamics of Zn accumulation in different cultivars, and variations of Cd uptake and transport in rice tissues were investigated. Furthermore, the expressions of the Cd-related transporter genes of the root were analyzed to explore the underlying mechanisms responsible for the various effects in different cultivars. 【Result】 Results showed that the Zn cultivation didn’t affect the growth of all rice seedlings. Zn accumulations varied obviously among different cultivars, the Zhejingyou1578 had the highest Zn level (230 μg Zn per plant), followed by the Wuyunjing21 (124 μg Zn per plant), the Huanghuazhan and Huiliangyousimiao had the low levels (85.1-95.0 μg Zn per plant). Zn enrichment greatly influenced the expression of Cd-related transporters in the Zhejingyou1578, the OsZIP7 was down-regulated by 48.7% and OsZIP1 was up-regulated by 81.3%. The expressions of the OsIRT1 and OsZIP7 in the Wuyunjing21 were also down-regulated by 35.9% and 35.0%, respectively, and the OsZIP1 was up-regulated by 31.1%. However, the Cd-related transporters in the Huanghuazhan and Huiliangyousimiao were insensitive to Zn enrichment. As a result, Zn enrichment significantly reduced Cd concentrations in the root and shoot by 26.7% and 36.7% of the Wuyunjing21, and 32.0% and 40.0% of the Zhejingyou1578 with a 12.0% inhibition on the Cd transport, respectively. Furthermore, brown rice Cd were reduced by 37.5%, 36.7% and 25.3% in the Wuyunjing21, Zhejingyou1578, and Huanghuazhan, respectively, while no difference occurred in the Huiliangyousimiao, revealing various effects on reducing Cd among different cultivars induced by the Zn enrichment. Correlation analysis showed that the brown rice Cd was negatively correlated with the shoot Cd which was negatively correlated with the root and shoot Zn, revealing a significant antagonism between Zn and Cd within rice. However, the root and shoot Zn were negatively correlated with the OsZIP7 and OsIRT1 and positively correlated with the OsZIP1 expression, indicating that the Zn enrichment within seedlings influenced the brown rice Cd mainly through regulating the expressions of the OsZIP1, OsZIP7 and OsIRT1. 【Conclusion】 The regulations on Cd-related transporters through Zn enrichment were varied among different cultivars, and the Zhejingyou1578 and Wuyunjing21 were more sensitive, so that the reductions of brown rice Cd were higher than other cultivars.

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    Leaf Stomatal Close and Opening Orchestrate Rhythmically with Cell Wall Pectin Biosynthesis and Degradation
    ZHANG XiaoPing,SA ShiJuan,WU HanYu,QIAO LiYuan,ZHENG Rui,YAO XinLing
    Scientia Agricultura Sinica    2022, 55 (17): 3278-3288.   DOI: 10.3864/j.issn.0578-1752.2022.17.002
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    【Objective】 Comparing on differential expression proteins between stomatal closing and opening at different leaf stomata-densities, it is to be revealed how pectin metabolism regulates stomata closing and opening. The result will play an essential role in understanding how stomata functions to environment adaptation.【Method】 Vectors, either over- or inhibiting-expression of StEPF-2 (Solanum tuberosum EPIDERMAL PATTERNING FACTOR 2) in vivo were constructed. The fusing genes were transformed into Solanum tuberosum cultivar Kexin 1. Transgenic potato lines, either rise or lower at leaf stomatal density were generated. Gene and protein expression profiles of leaves at various stomatal densities were assayed via RNA-seq and iTRAQ. Comparing differentiation expression proteins, pectin metabolic enzymes driving stomatal movement under light and darkness were identified and confirmed by the Pulldown and LC-MS/MS. A pectin metabolism pathway regulating stomatal movement was to be proposed.【Result】 At least 14 protein families, driving stomata closing and opening involved in pectin metabolism of the guard cell wall during stomatal mature. Five protein families were detected and confirmed only in the stomatal-closed leaves under darkness, including polygalacturonase inhibitor proteins (PGIP) and rhamnose synthase (RHM) for RG side-chain biosynthesis. Four protein families, polygalacturonase (PG), pectate lyase-like (PLL), pectinmethylesterase (PME) and α-galactosidase (AGAL) were identified only in leaves at various stomatal densities under light. Additionally, five protein families were concurrently identified in both leaves of stomata closing and opening, including pectinacetylesterase (PAE) and subtilase (SBT). 【Conclusion】 Under light, PMEs catalyze pectin demethylesterification, afterwards, pectin was exo- and endo-hydrolyzed by PG, PLL and AGAL. Pectin losing structure was split under turgor, results in stomatal opening. Reversely, under darkness, PGI inhibited pectin hydrolysis. Pectin side-chain biosynthesis was promoted by RHM. Therefore, stomata kept closing due to structurally-complete pectin with voluntary expending function.

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    Mining of Genetic Locus of Maize Stay-Green Related Traits Under Multi-Environments
    CHANG LiGuo,HE KunHui,LIU JianChao
    Scientia Agricultura Sinica    2022, 55 (16): 3071-3081.   DOI: 10.3864/j.issn.0578-1752.2022.16.001
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    【Objective】 Functional stay-green is generally considered a desirable trait in major crop varieties including maize. Finding new loci and candidate genes related to stay-green, and providing new theoretical basis for the genetic research on stay-green. 【Method】Using 150 recombinant inbred lines (RIL) populations derived from the cross between Xu 178 and K12, QTL mapping of three stay-green related traits (visual stay green (VSG), green leaf number at silking stage (GLNS) and green leaf number at mature stage (GLNM) were performed by the composite interval mapping(CIM)method of Windows QTL Cartographer V2.5. Besides, an association population, which composed of 139 natural materials genotyped with 50790 high-quality SNP markers, was used to dissect genetic locus of three traits by genome-wide association study (GWAS) based on the mixed linear model MLM). 【Result】Based on CIM, three traits (GLNM, GLNS and VSG)were mapped using phenotypic values in a single environment and best linear unbiased prediction (BLUP) value. A total of 37 QTLs were detected on all chromosomes except Chromosome 10, and the LOD score ranged from 2.58-11.36, with a phenotypic variation contribution rate of 4.34%-22.40%. Among them, 14, 12 and 11 loci were detected for GLNM, GLNS and VSG traits, respectively. Four of the QTLs, qGLNS2-1, qVSG1-1, qVSG1-2 and qVSG7-1, were genetically stable and were detected simultaneously in three or more different single environments. GWAS was performed on three stay-green related traits using MLM, and a total of 44 significant SNPs above the threshold line were detected. According to the physical position of SNP markers in the B73 reference genome, a total of 15 SNP were found to fall into the QTL interval mapped by linkage analysis. 【Conclusion】Combined with the results of QTL mapping and genome-wide association study, a total of 4 genetically stable colocalization genetic regions were detected (the corresponding physical position intervals on the B73 reference genome version 4 are 6.2-8.2 Mb on chromosome 1, 209.1-221.4 Mb on chromosome 2, 96.8-102.1 Mb on chromosome 6, and 4.9-11.4 Mb on chromosome 7), and four important candidate genes (Zm00001d006119, Zm00001d018975, Zm00001d006535 and Zm00001d036763) related to photosynthesis and stress response were mined.

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    The Functional Analysis of High Mobility Group MsHMG-Y Involved in Flowering Regulation in Medicago sativa L.
    ZHANG YunXiu,JIANG Xu,WEI ChunXue,JIANG XueQian,LU DongYu,LONG RuiCai,YANG QingChuan,WANG Zhen,KANG JunMei
    Scientia Agricultura Sinica    2022, 55 (16): 3082-3092.   DOI: 10.3864/j.issn.0578-1752.2022.16.002
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    【Objective】Flowering is an important signal indicating the transformation from vegetative growth to reproductive growth and has a significant effect on plant biomass. Alfalfa is one of the upmost forage crops worldwide, its yield and quality are closely related to flowering time. The optimum harvest time for alfalfa is during the early flowering stage, which could give the highest yield and the best quality. In the current study, an alfalfa flowering related gene, Medicago sativa High Mobility Group Y (MsHMG-Y), was cloned. The gene structure and expression pattern of MsHMG-Y were studied. Function of MsHMG-Y in alfalfa flowering regulation was analyzed. This work could provide theoretical support for mechanism study underlying flowering regulation. 【Method】MsHMG-Y was cloned by homology cloning strategy and the amino acid sequence was analyzed by multiple sequence alignment. The phylogenetic tree was also constructed. qRT-PCR analysis was used to detect the expression level of MsHMG-Y in different tissues and different flowering stages. The expression pattern of MsHMG-Y under light, gibberellin (GA3), salicylic acid (SA) or methyl jasmonate (MeJA) treatment were analyzed. The phenotype of MsHMG-Y-overexpressing alfalfa was analyzed, and the expression levels of flowering activators and suppressors were also analyzed. 【Result】Phylogenetic analysis showed that MsHMG-Y has the closest relationship with MtHMG-Y in Medicago truncatula. Spatial expression pattern analysis showed that MsHMG-Y was expressed in flowers, stems and leaves, with the highest expression level in flowers and the lowest expression level in leaves in both paternal and maternal alfalfa. In paternal alfalfa with early flowering phenotype, the expression level of MsHMG-Y was the highest at early flowering stage. The highest expression level of MsHMG-Y was detected at flower bud differentiation stage in maternal alfalfa with late flower phenotype. Photoperiod analysis showed that MsHMG-Y was down-regulated after 16-hour light treatment. After 28 hours of light treatment, the expression level of MsHMG-Y was continuously lower than that in the control group, indicating that MsHMG-Y was down-regulated after light treatment. After 50 μmol·L-1 GA3, 100 μmol·L-1 SA or 100 μmol·L-1 MeJA treatment, the expression level of MsHMG-Y was up-regulated compared with the mock treatment. In detail, the expression level of MsHMG-Y was the highest at 1 h under GA3 treatment, which was 3.5 folds higher than control. Under SA treatment, the expression level of MsHMG-Y was the highest at 6 h, which was 24 folds higher than the mock treatment. The expression level of MsHMG-Y was the highest at 3 h under MeJA treatment, which was 11 folds higher than the control. These results indicated that the expression of MsHMG-Y was inducible by the above three hormones. MsHMG-Y-overexpressing alfalfa has late flowering phenotype. The expression levels of flowering activator genes were down-regulated in MsHMG-Y-overexpressing alfalfa, while the expression levels of flowering inhibitor genes were up-regulated. Among these genes, expression of flowering activator genes MsPHYA, MsGI and MsFTa1 was significantly down-regulated by 4.9 folds, 3.9 folds and 2.8 folds respectively, and the expression level of flowering inhibitor genes MsTEM and MsSVP was increased by 2.5 folds and 1.9 folds, respectively. 【Conclusion】The expression of MsHMG-Y is inducible by photoperiod and exogenous hormone treatment, including GA3, SA and MeJA. Overexpression of MsHMG-Y in alfalfa resulted in delayed flowering time. MsHMG-Y plays an important role in regulatory mechanism underlying late flowering in alfalfa.

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    Genome-Wide Association Study of Ear Related Traits in Maize Hybrids
    LI Ting,DONG Yuan,ZHANG Jun,FENG ZhiQian,WANG YaPeng,HAO YinChuan,ZHANG XingHua,XUE JiQuan,XU ShuTu
    Scientia Agricultura Sinica    2022, 55 (13): 2485-2499.   DOI: 10.3864/j.issn.0578-1752.2022.13.001
    Abstract583)   HTML88)    PDF (4472KB)(326)       Save

    【Objective】Ear traits are important components of grain yield in maize. Dissecting their genetic basis and mining significant SNPs using genome-wide association study (GWAS) can provide references for cloning functional genes and breeding high-yield maize varieties. 【Method】A total of 115 superior inbred lines from Shaan A group and Shaan B group, as well as four domestic backbone lines were selected as parents. Based on NCⅡ genetic design, an association population consisting of 442 hybrids was constructed, which was planted in two different environments to collect phenotype data of ear traits. Meanwhile, all parental lines were sequenced by the tunable genotyping by sequencing (tGBS) protocols. According to the genotype of inbred lines, altogether 19 461 high-quality SNPs were inferred in the association population. Then, GWAS was performed using 19 461 SNPs and phenotype data by three models including additive, dominance and epistasis, respectively. Combining with the transcriptome data of maize ear related tissues in the public database and the annotation information of genes, candidate genes were predicted. 【Result】Phenotypic data analysis showed that eight ear traits followed a continuous distribution, and there were 3.78%-45.25% of phenotypic variation. Analysis of variance indicated that environment and genotype effects reached an extremely significant level (P<0.001), and the range of broad-sense heritability was from 54.15% to 68.89%. And there were significantly positive or negative correlations among ear traits of hybrids. In total, 16, 3, 79 significant SNPs/pairs were identified under additive, dominant, and epistatic models, respectively. The significant loci detected by the three models cumulatively explained 38.21%-60.69% of the phenotypic variation of each trait. The cumulative phenotypic variation of significant SNP detected by additive model and epistatic model was 0.00-41.26% and 15.18%-45.36%, respectively. Effect analysis of significant SNPs identified by additive and dominant models showed most SNPs with additive or partial dominance effects, and only two with over-dominance effects. Further, only seven single-SNPs and five interaction pairs explained more than 5% of the phenotypic variation, and 17 candidate genes were predicted based on the SNP locations and gene expression information. 【Conclusion】Ear traits of maize hybrids were mainly affected by additive and epistasis effects, but less by dominance effects. Multiple SNPs identified by additive and dominant models showed additive and partially dominance effects, and aggregating favorable alleles of these SNPs could improve the target traits.

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    QTL Mapping for Traits Related to Seed Number Per Pod in Peanut (Arachis hypogaea L.)
    HAO Jing,LI XiuKun,CUI ShunLi,DENG HongTao,HOU MingYu,LIU YingRu,YANG XinLei,MU GuoJun,LIU LiFeng
    Scientia Agricultura Sinica    2022, 55 (13): 2500-2508.   DOI: 10.3864/j.issn.0578-1752.2022.13.002
    Abstract374)   HTML46)    PDF (1382KB)(166)       Save

    【Objective】Peanut (Arachis hypogaea L.) is one of the important vegetable oil and cash crop. High yield is always the predominant objective in peanut breeding and determined by seed number per unit area and seed weight. Seed number per unit area is produced by planting density per unit area×number of pods per plant×number of seeds per pod. Therefore, the genetic dissection of the number of seeds per pod is helpful to explore the gene/locus related to this trait, which provides an important theoretical basis for the molecular breeding of yield in peanut.【Method】A RIL population, derived from Silihong×Jinonghei 3, were planted at Qingyuan experimental station of Hebei Agricultural University in Baoding city, Hebei province in 2018(E1) and 2020(E2). Phenotypic values of traits associated with the number of one seed pods per plant, two seeds pods per plant and multiple pods per plant were investigated at harvest stage. By using the genetic linkage map constructed by laboratory of Peanut innovation team, Hebei Agricultural University and software of QTL Icimapping V4.1(the Inclusive composite interval Mapping (ICIM)), QTL mapping for the number of seeds per pod was carried out under two environments.【Result】The results showed that the rates of one seed pods per plant and two seeds pods per plant were normal distribution, while the rate of multiple pods per plant was skewed normal distribution. A total of 11 QTLs were detected for the three traits, which could explain the phenotypic variation of 4.66%-22.34% and the additive effects of -9.35-9.42. Among of them, 5 QTLs for the rate of multiple pods per plant with explained 3.19% to 22.34% of phenotypic variation were obtained. The additive effect of one QTL from Jinonghei 3 was negative (-4.77), while the additive effect of the other four QTLs from Silihong was positive (3.59-9.42). Two QTLs for the rate of one seed pods per plant were mapped with explained 4.97%-6.43% of phenotypic variation. The additive effects of the two QTLs from Jinonghei 3 were negative (-4.45 and -4.54). Four QTLs for the rate of two seed pods per plant were located with explained 4.97%-6.43% of phenotypic variation. The additive effects of the four QTLs from Jinonghei 3 were negative (-9.35--3.84). Among of these QTLs, 6 QTLs were major QTLs, of which qRMSPA05 was repeatedly detected, and the heritable phenotypic variation was 16.58%-17.34%, and the additive effect was 7.69-8.12.【Conclusion】Six major QTLs and one major stable QTL for multiple pods per plant were identified, which will be helpful for improving the yield traits in peanut. The results can be used as important candidate segments for genetic improvement, and molecular marker assisted selection and fine mapping.

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    Genome-Wide Association Analysis of Yield and Combining Ability Based on Maize Hybrid Population
    LI ZhouShuai,DONG Yuan,LI Ting,FENG ZhiQian,DUAN YingXin,YANG MingXian,XU ShuTu,ZHANG XingHua,XUE JiQuan
    Scientia Agricultura Sinica    2022, 55 (9): 1695-1709.   DOI: 10.3864/j.issn.0578-1752.2022.09.001
    Abstract518)   HTML93)    PDF (3116KB)(314)       Save

    【Objective】By analyzing the yield of the hybrids from the inbred lines bred from the Shaan A and Shaan B group, the combining ability of the inbred lines were evaluated, genome-wide association analysis, and mining associated loci for yield and its combining ability conducted. It will provide references for improving maize inbred lines selected from Shaan A group and Shaan B group and applying them in varieties breeding. 【Method】Based on NCⅡ genetic design, 85 excellent inbred lines from Shaan A group and Shaan B group were used to construct a hybrid population containing 246 F1. Then, the yield of the hybrid population was tested in three environments to evaluate their general combining ability (GCA) and special combining ability (SCA). Using the 6H90K maize array to detect the parental genotypes, 63 879 high-quality SNPs were obtained, which were used to analyze the genetic characteristics of parental lines. According to the parental genotypes, 55 951 high-quality SNPs were inferred in the hybrid population for genome-wide association analysis of hybrid yield, GCA, and SCA using additive model and non-additive model. Meanwhile, candidate genes around the significant SNPs were screened and annotated based on the maize B73 reference genome.【Result】The yield in the three environments accorded to the normal distribution with wide variation, the broad-sense heritability of yield was 59.04%, and the environmental effect was significant. There was significant positive correlation between hybrid yield and combining ability, and the correlation between hybrid yield and SCA (r=0.95) was higher than that between hybrid yield and GCA (r=0.62). The genetic characteristic of Shaan A group and Shaan B group was different, and inbred lines from Shaan A group have higher general combining ability. Totally, five, seven and nine significant SNPs were detected (-log10(P)>3.86) for GCA, hybrid yield and SCA, respectively. Among them, four SNPs were co-located in hybrid yield and SCA. Ultimately, 17 associated SNPs were anchored. Dominant allele analysis of different trait-associated loci showed that four GCA-associated SNPs were controlled by additive effects, and the F1 BLUE-associated loci could be divided into 4 types mainly by the dominant effect, and the heterozygous genotype is the favorite allele or sub-optimal allele for yield in F1. Through functional annotation, the candidate genes were specifically expressed in maize growth and kernel establishment, for example, GRMZM2G165828 and GRMZM2G057557 were related to maize kernel development. 【Conclusion】Based on this study, we consider that GCA and SCA jointly affect the yield of hybrids, and the effect of SCA is greater. Moreover, GCA and SCA may have different genetic basis, and GCA can be increased with the accumulation of favorable alleles. Using the genome-wide association analysis in the F1 hybrid population can carry out genetic analysis related to combining ability, mine the genetic loci related to yield and combining ability, and accelerated the application of the associated loci in molecular breeding.

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    Establishment of ALSV-Induced Gene Silencing in Chinese Soybean Cultivars
    DONG YongXin,WEI QiWei,HONG Hao,HUANG Ying,ZHAO YanXiao,FENG MingFeng,DOU DaoLong,XU Yi,TAO XiaoRong
    Scientia Agricultura Sinica    2022, 55 (9): 1710-1722.   DOI: 10.3864/j.issn.0578-1752.2022.09.002
    Abstract635)   HTML66)    PDF (4153KB)(237)       Save

    【Objective】 The establishment of apple latent spherical virus (ALSV)-induced gene silencing on domestic soybean cultivars will offer a simple, time-saving and operable system for gene function and genetic breeding of soybean cultivars in China.【Method】The infectious clones of ALSV which was used to infect plant through Agro-infiltration was constructed. A 327 bp cDNA fragment of phytoene desaturase (GmPDS) cDNA was amplified from soybean cultivar Williams 82 and inserted into the pALSV2 vector. The pALSV1 and pALSV2-GmPDS were used to co-infect Nicotiana benthamiana plant through Agro-infiltration, and the ALSV virus particle was purified from systemic infected leaves of N. benthamiana plants at 17 days post infiltration (dpi). The purified virions were mechanically inoculated onto the first-round true leaves of soybean and photobleaching phenotype on the upper non-inoculated leaves was monitored from 10 to 30 dpi. Using the empty ALSV vector as a control, the gene expression levels of ALSV coat protein (CP) gene and GmPDS in ALSV:GmPDS infected soybean were examined by RT-PCR and quantitative RT-PCR analysis, respectively. 【Result】At 20 dpi, soybean cultivar Nannong 1138-2 inoculated with ALSV:GmPDS showed photobleaching phenotype in the systemic infected leaves, however Williams 82 did not show gene silencing phenotype of PDS in the systemic infected leaves. The results of qRT-PCR confirmed that the RNA expression level of GmPDS in Nannong 1138-2 was significantly reduced but ALSV:GmPDS was unable to silence PDS in Williams 82. By using the same method, the gene silencing efficiency of other 9 domestic soybean cultivars mediated by ALSV:GmPDS was examined. Soybean cultivars including Nannong 47, Andou 203, Xiangdou 4, Zhonghuang 13, Shanning 29 and Qihuang 34 all displayed photobleaching phenotype in plant systemic infected leaves after inoculation with ALSV:GmPDS, while GmPDS was not silenced in Hedou 12, Zhonghuang 311 and Shanning 16.【Conclusion】In this study, an Agrobacterium-mediated ALSV viral VIGS vector was constructed. ALSV virions was propagated and purified from N. benthamiana plant. The purified virions were rub inoculated onto the first-round true leaves of soybean. Finally, an efficient gene silencing system was established on a number of domestic soybean cultivars in China.

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    Optimization of Callus Genetic Transformation System and Its Application in FtCHS1 Overexpression in Tartary Buckwheat
    ZHAO HaiXia,XIAO Xin,DONG QiXin,WU HuaLa,LI ChengLei,WU Qi
    Scientia Agricultura Sinica    2022, 55 (9): 1723-1734.   DOI: 10.3864/j.issn.0578-1752.2022.09.003
    Abstract429)   HTML43)    PDF (2457KB)(205)       Save

    Objective】To develop a novel tool for functional verification and molecular breeding in tartary buckwheat, this study focused on establishing and optimizing an efficient callus genetic transformation system. 【Method】Callus induction factors including different explants, ratios of diverse growth regulators, and Agrobacterium tumefaciens types were systematically evaluated using “Xiqiao No. 2” as the derived plant. We further overexpressed FtCHS1, a key enzyme gene involved in the biosynthesis of tartary buckwheat flavonoids in obtained calli to validate the optimized genetic callus transformation system. The positive transgenic lines were confirmed by PCR and fluorescent observation. Subsequently, the content of anthocyanins and metabolites in flavonol branch pathway were determined by UV spectrophotometry and High Performance Liquid Chromatography (HPLC), respectively. Furthermore, quantitative real-time PCR was performed to analyze expression levels of genes involved in flavonoid synthesis, in order to compare the differences between the FtCHS1-overexpressed calli and the control. 【Result】The optimal explant was hypocotyls and the optimal induction medium was the Murashige and Skoog (MS) medium supplemented with the addition of 0.8 mg·L-1 6-BA (6-Benzylaminopurine) and 3.5 mg·L-1 2,4-D (2,4-Dichlorophenoxyacetic acid). The induction rate of calli grown on the above medium reached up to 72%. Moreover, the optimized subculture medium containing MS with the additives of 3 mg·L-1 6-BA and 1 mg·L-1 KT (Kinetin) increased the percentage and coefficient of callus proliferation to 98% and 1.09, respectively. Additionally, the best Agrobacterium tumefaciens in the transformation process was GV3101, and the transformation efficiency was up to 31.3%. The functional analysis of FtCHS1 overexpressing in transgenetic calli demonstrated that: (1) The accumulations of kaempferol and quercetin in transgenic calli overexpressing FtCHS1 were dramatically higher than those in control groups (P<0.01), and anthocyanin, rutin and myricetin contents were also remarkably higher (P<0.05); (2) Overexpression of the exogenous FtCHS1 did not affect the expression levels of 5 endogenous orthologous genes FtCHSs in the transgenic calli (P>0.05), whereas genes encoding key enzymes of the flavonoid synthesis pathway, such as FtCHI, FtF3H, FtFLS1, FtFLS2, FtFLS3, and FtDFR1, were up-regulated (P<0.05); (3) FtMYB5 and FtMYB6, the transcription factor genes that specifically positively regulated the flavonol synthesis, were up-regulated, while FtMYB8, a suppressor gene of anthocyanin synthesis, was down-regulated (P<0.05). 【Conclusion】In this study, the callus genetic transformation system of tartary buckwheat was successfully established from “Xiqiao No. 2”. FtCHS1 overexpression in the transgenic calli up-regulated genes related to flavonoid synthesis, resulting in flavonoids accumulation.

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    The Progress and Prospects of the Theoretical Research on the Safe Conservation of Germplasm Resources in Genebank
    YIN GuangKun,XIN Xia,ZHANG JinMei,CHEN XiaoLing,LIU YunXia,HE JuanJuan,HUANG XueQi,LU XinXiong
    Scientia Agricultura Sinica    2022, 55 (7): 1263-1270.   DOI: 10.3864/j.issn.0578-1752.2022.07.001
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    The safe conservation of germplasm resources is the core of high-level protection and a prerequisite for sustainable utilization. Low-temperature genebank is the most important way to conserve crop germplasm resources. These aims, extending the conservation longevity, maintaining the genetic integrity and preventing the accidental loss, always were the research difficulties and hotspots in the safe conservation. This review systematically sorted out the research history of germplasm resources in genebanks in the past 100 years, and pointed out: (1) Low moisture content and low storage temperature are key factors for prolonging seed longevity, forming the low temperature and moisture theory, which is the theoretical basis for safe conservation in genebanks; (2) The declining viability of seeds in genebanks has the characteristic of the critical node, which is the lower limit for maintaining genetic integrity, forming the critical node theory, which is the theoretical basis for the safe conservation of germplasm resources; (3) The conserved germplasm should be backed up by the physical space or storage method, forming the back up conservation mechanism, which is the a safeguard measure to prevent the irreversible loss of resources caused by accidents. This review focused on the connotation, significance and practical guidance of the double-low theory, the critical node theory and the duplication conservation mechanism, in order to further promoting the development of the safe conservation of germplasm resources.

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    Characteristics of High-Quality Rice Varieties and Taste Sensory Evaluation Values in China
    ZHU DaWei,ZHANG LinPing,CHEN MingXue,FANG ChangYun,YU YongHong,ZHENG XiaoLong,SHAO YaFang
    Scientia Agricultura Sinica    2022, 55 (7): 1271-1283.   DOI: 10.3864/j.issn.0578-1752.2022.07.002
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    【Objective】In order to clarify the quality characteristics of high quality japonica rice in north and south China and high quality indica rice, and explore the physical and chemical indexes related to rice taste quality, this study investigated the differences of quality and taste sensory evaluation value of high quality rice in China. It would provide theoretical basis for quality evaluation and improvement of high quality rice varieties in China. 【Method】 A total of 122 high quality rice grain varieties from 30 provinces of China in the third national high-quality rice variety taste quality evaluation activities were used as materials. According to suitable planting area, all the samples were divided into three types as northern japonica, southern japonica and indica rice with the sample numbers of 38, 15 and 69, respectively. To clarify the relationship between physical chemical parameters and taste sensory evaluation value, rice appearance quality, rice flour pasting properties, protein content and taste sensory evaluation value were measured. According to the taste sensory evaluation value, each type of rice was divided into three categories as high (≥90), medium (80-90) and low taste (<80). The differences of rice qualities among different types and taste categories were analyzed. 【Result】Northern japonica rice could be divided into high and medium taste categories. The length-width ratio, protein content, setback viscosity and hardness of high taste category rice were lower than those of medium taste category rice, by 0.2, 0.32 g/100 g, 134 cP, 16 g, respectively (P<0.05). However, the peak viscosity, stickiness and spring value of high taste category rice were higher than those of medium taste category rice, by 145 cP, 70 g and 0.04%, respectively (P<0.05). Southern japonica rice could be divided into high, medium and low taste categories. The chalkiness degree, peak, and breakdown viscosity, and stickiness of high taste category rice were higher than those of low taste category rice, by 0.65%, 314 cP, 259 cP, 261 g, respectively (P<0.05). However, the apparent amylose content, protein content, final and setback viscosity of high taste category rice were lower than those of low taste category rice, by 4.0 g/100 g, 9.2 g/100 g, 260 cP and 574 cP, respectively (P<0.05). Indica rice could be divided into high, medium and low taste categories. The high taste category rice had a longer and thinner grain shape and a better appearance quality than the low taste category rice (P<0.05). The protein content, setback viscosity and hardness of high taste category rice were higher than those of low taste category rice, by 0.45 g/100 g, 157 cP, 46 g, respectively (P<0.05). However, the breakdown viscosity and stickiness of high taste category rice were lower than those of low taste category rice, by 115 cP and 107 g, respectively (P<0.05). 【Conclusion】Northern japonica rice type with high taste sensory evaluation value had a crystal-clear appearance (chalkiness degree was less than 1%), low protein content (about 6 g/100 g), moderate hardness, and high elasticity (about 0.6%). Southern japonica rice type with high taste sensory evaluation value had a good appearance quality, low apparent amylose content (about 13 g/100 g) and setback viscosity (about -250 cP), and high cooked rice stickiness (about -1200 g). Indica rice type with high taste sensory evaluation value had a long and thin grain (length-width ratio about 4.0) and crystal-clear appearance, and low ratio of hardness to stickiness (about 0.25).

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    Genetic Variations of Potassium Harvest Index in 437 Wheat Varieties
    LIU Shuo,ZHANG Hui,GAO ZhiYuan,XU JiLi,TIAN Hui
    Scientia Agricultura Sinica    2022, 55 (7): 1284-1300.   DOI: 10.3864/j.issn.0578-1752.2022.07.003
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    【Objective】The present study aimed to investigate the variation of potassium (K) harvest index (KHI) among 437 wheat varieties, analyze the relationships between KHI and yield, grain K content, K absorption of different organs and K utilization efficiency; and clarify the effects of the release year of wheat varieties, plant height and awn types on KHI. The present study provided useful information for breeding wheat cultivars with high yield and K use efficiency. 【Method】Field experiments were conducted in Luoyang of Henan Province and Yangling of Shaanxi Province during the 2018-2019 and 2019-2020 growth seasons. Four hundred and thirty-seven wheat varieties with different release years, plant heights and awn types were used as materials. An augmented randomized complete block design was applied and 14 blocks were set up, with 31 experimental varieties and 5 control varieties in each block. Each wheat variety was planted with 6 rows and 3 m long. At maturity, a complete row in the middle of each plot was selected for grain harvest, and the grains were oven-dried and weighed. For tissue K concentration measurement, six tillers were blindly selected in five sampling sites in each plot. Grain, straw and glume were separated, oven-dried, and digested using H2SO4-H2O2. K concentrations of different organs were measured with a flame photometer, and parameters including KHI, grain, straw and glume K uptake, grain K utilization efficiency (GKUE) and shoot K utilization efficiency (SKUE) were calculated. 【Result】There were significant differences in KHI among different wheat varieties (P<0.01) under all the four environments (19Luoyang, 20Luoyang, 19Yangling and 20Yangling) and KHI of 437 wheat varieties varied from 0.04 to 0.40. The average KHI of Yangling was higher than that of Luoyang. Seven wheat varieties including Yangmai 18, Yannong 5158, Chuanmai 104, Huamai 5, Zhengmai 1860, Dromedaris and Space 6 had higher KHI and yield. There were significant positive correlations between KHI and wheat yield, grain K concentration and grain K uptake in three environments (P<0.05). There were significant negative correlations between KHI and straw K uptake, glume K uptake and shoot total K uptake (P<0.05). Grain yield, grain K concentration and uptake increased along with the increase of KHI, however, straw, glume and the shoot total K uptake decreased along with the increase of KHI. There was a significant positive correlation between KHI and GKUE or SKUE (P<0.001). The KHI of the wheat varieties released before 1970 and from 1970 to 1990 was significantly lower than that of the varieties released after 1990 (P<0.05). The wheat varieties released between 1990 to 2010 had similar KHI with the varieties released after 2010 (P>0.05). There was a significant negative correlation between plant height and KHI. There was no significant difference in KHI between the wheat varieties with and without awn. 【Conclusion】There was a distinct inter-variety variation in wheat KHI. Increasing wheat KHI may positively influence the grain yield of wheat. KHI may be also a good indicator of K utilization efficiency of wheat. To further improve KHI of wheat, novel breeding technologies should be developed to improve the remobilization efficiency of K from vegetative organs to grain. Breeding dwarf or semi-dwarf wheat varieties is beneficial to improve KHI.

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    Research Progress on the Physiology and Its Molecular Mechanism of Seed Desiccation Tolerance
    SONG SongQuan,LIU Jun,TANG CuiFang,CHENG HongYan,WANG WeiQing,ZHANG Qi,ZHANG WenHu,GAO JiaDong
    Scientia Agricultura Sinica    2022, 55 (6): 1047-1063.   DOI: 10.3864/j.issn.0578-1752.2022.06.001
    Abstract917)   HTML118)    PDF (971KB)(442)       Save

    Dehydration tolerance (DT) is defined as the ability of an organism or tissue to survive the removal of all, or almost all the cellular water without irreversible damage. DT of seeds is an adaptive mechanism to ensure the survival and reproduction of plant species in the long-term evolution process, and plays a key role in the conservation of plant seeds and germplasm resources. However, the DT of seeds is a complex trait, and its molecular mechanism is not now largely understood. Therefore, in the present paper, the research progresses on the physiological and molecular mechanisms of seed DT were reviewed. It was found that the DT of orthodox seeds was gradually formed during development, and reached the peak at physiological maturity. Recalcitrant seeds do not undergo the development stage of maturity dehydration, and are very sensitive to dehydration throughout development. Mature orthodox seeds maintained their resistance to re-dehydration at the initial stage of imbibition. With the time course of germination, the DT decreased gradually, and finally lost completely. The DT of seeds and embryos can be re-established during the early stage of germination, and of different tissues is different. The DT of seeds and embryos was inversely correlated with the decrease in mitochondrial respiratory activity. Respiratory activity of recalcitrant axis mitochondria was higher than that of orthodox embryo ones. During dehydration, the H2O2 content, the production rate of superoxide anion radical (·O2-) and the content of thiobarbituric acid reactive substance in desiccation-tolerant embryos (axes) were significantly lower than those of desiccation-sensitive embryos (axes), while the reactive oxygen species scavenging system in desiccation-tolerant embryos (axes), including enzymatic and non-enzymatic activities, was significantly higher than that in desiccation-sensitive embryos (axes). During the maturation of seeds, the accumulation of late embryogenesis abundant (LEA) proteins, small heat shock proteins and non-reducing oligosaccharides is closely related to the formation of DT. The AFL subfamily of B3 transcription factors (including ABI3 (ABA INSENSITIVE 3), FUS3 (FUSCA3) and LEC2 (LEAFY COTYLEDON 2)) increase the DT of seeds and embryos by positively regulating the accumulation of storage materials and protective proteins. The level of DNA methylation increased significantly throughout seed development and then decreased gradually during seed germination. Compared with embryos during the early stage of development and seedlings, mature embryos had a higher level of genomic methylation. In seeds, the parallel ABA and DOG1 (DELAY OF GERMINATION 1) signaling pathways activate synthesis of raffinose family oligosaccharides, and expression of LEA and HSP (heat shock protein) genes, thus regulating the onset of DT and transit to dormancy. Finally, the scientific issues that require to be further studied in this field are proposed, including the re-establishment of their model research system by using seeds and their tissues with different DT. Germinability, DT and dormancy characteristics of seeds are initiated and completed during development, and the relationship among them is still now unclear. There are both core ABA signaling pathway and DOG1 signaling pathway in seeds, and they converge at the ABI3 or downstream of ABI3. Which pathway will response preferentially and how these two pathways coordinate during dehydration of seeds? This paper will provide a reference for comprehensively understanding of the physiology and molecular mechanism of seed DT, increasing the stress resistance and yield of plant crops, improving the storage conditions of the resource bank and long-term preserving plant seed (germplasm) resources.

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    Genome-Wide Association Analysis of Lead Tolerance in Wheat at Seedling Stage
    ZHI Lei,ZHE Li,SUN NanNan,YANG Yang,Dauren Serikbay,JIA HanZhong,HU YinGang,CHEN Liang
    Scientia Agricultura Sinica    2022, 55 (6): 1064-1081.   DOI: 10.3864/j.issn.0578-1752.2022.06.002
    Abstract508)   HTML82)    PDF (2881KB)(356)       Save

    【Objective】With the advancement of industrialization, the pollution of arable land by heavy metals, especially lead, has become a worldwide problem. Wheat is an important food crop, and its safe cultivation is critical to maintaining food security. Screening wheat varieties with strong tolerance to lead, low lead accumulation and mining relevant regulatory genes or QTL regions would lay foundation for further elucidating the genetic mechanism of lead tolerance in wheat. 【Method】The tolerance to lead of 102 wheat varieties (advanced lines) were evaluated with a 140 mg·kg-1 lead nitrate solution at the seedling stage, by the weighted membership function value (D Value) of the lead tolerance coefficients of maximum root length, root biomass and growth rate under three replicates, combining with the 335 438 high-quality SNPs (single nucleotide polymorphism)markers by wheat 660K SNP chip, a genome-wide association study (GWAS) for lead tolerance in wheat was conducted, to mine the candidate genes for lead tolerance in wheat. 【Result】The lead among between wheat varieties (advanced lines) under different replicates showed rich variation, with the coefficient of variation of 44.8%-46.2%, and the correlation coefficient was between 0.87-0.97 (P<0.001). It was found that varieties with strong lead tolerance showed low lead accumulation characteristics. The genotyping results showed that the SNP polymorphic information content (PIC) was 0.28-0.32, the population structure analysis showed that these wheat materials could be divided into 7 subgroups; a total of 20 SNPs and 8 key chromosomal segments that were significantly associated with lead tolerance in wheat (P≤0.001) were detected by two GWAS methods respectively distributed on chromosomes 1B, 2A, 2D, 3A, 3B, 5A, and 7A. A single locus explains 15.33%-19.75% of the phenotypic variation, and 10 and 8 key chromosomal segments were detected in two and more environments. Analysis of candidate genes for significant and stable association sites and intervals revealed that the functions of the candidate genes were mainly related to transmembrane transport, protein modification and oxidative stress response, including 7 genes related to transporters, including TraesCS1B02G433800, TraesCS7A02G118800, TraesCS7A02G117900 etc. Two genes involved in ubiquitination and deubiquitination (TraesCS2A02G550900 and TraesCS7A02G477300), three genes encoding transmembrane proteins (TraesCS2D02G570500, TraesCS3B02G039900 and TraesCS3B02G466000), one gene related to peroxidase (TraesCS7A02G474200). 【Conclusion】Seven wheat materials with strong lead tolerance were screened, 20 SNPs in 8 candidate regions significantly related to lead tolerance in wheat were detected, and 13 candidate genes related to lead tolerance in wheat were finally screened.

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    Comprehensive Evaluation of Phenotypic Characters of Nature Population in Upland Cotton
    WANG XiuXiu,XING AiShuang,YANG Ru,HE ShouPu,JIA YinHua,PAN ZhaoE,WANG LiRu,DU XiongMing,SONG XianLiang
    Scientia Agricultura Sinica    2022, 55 (6): 1082-1094.   DOI: 10.3864/j.issn.0578-1752.2022.06.003
    Abstract553)   HTML74)    PDF (731KB)(257)       Save

    【Objective】To analyze the fiber quality, yield, and early maturity of Upland cotton germplasm, screen the evaluation indicators comprehensively, establish a reliable evaluation model, and provide theoretical support for developing new Upland cotton varieties.【Method】 A total of 630 Upland cotton accessions selected from various sources were used to investigate 17 traits in 8 environments in three major cotton-growing regions in China. The best linear unbiased prediction (BLUP) was estimated for phenotypic traits by utilizing R package “lme4” and used for further analysis, including correlation analysis, principal component analysis, affiliation function method, cluster analysis and stepwise regression to evaluate population characteristics.【Result】This upland cotton population had high genetic diversity with a diversity index ranging from 1.961 to 2.084, and significant regional specificity existed. The boll number, fiber elongation, spinning consistent index, and short fiber index of this population had considerable variation, while that of fiber length, fiber strength, and growth period traits was lower. Correlation analysis showed significant or highly significant correlations among most traits, and there was a strong correlation among some fiber quality traits. The 17 traits were converted into 6 independent composite indices through principal component analysis with a contribution range of 5.860%-31.044% and a cumulative contribution of 82.642%. Principal component analysis can classify the fiber quality traits, yield traits and agronomic traits in this upland cotton population. The comprehensive evaluation value (F value) of phenotypes was calculated using the affiliation function method, the phenotypes of 17 traits were all significantly correlated with F values. The result demonstrated that the accessions with high F values (mean value of 0.668) had significantly higher yield traits (boll number, boll weight, lint percentage, and seed index), fiber quality traits (fiber length, fiber length uniformity, fiber strength, spinning consistent index, and short fiber index), plant height and fertility traits than those with low F values (mean value of 0.396). Regression equations for eight traits (boll number, boll open date, boll weight, flowering date, lint percentage, plant height, sympodial brand node, and spinning consistent index) as independent variables were established using stepwise regression. Based on the F value, the 630 germplasm were clustered into four categories, the first category was the high-quality fiber type, containing 118 accessions; the second category was the high-yield type, comprising 250 accessions; the third category was the early-maturing type, comprising 51 accessions; the characteristics of the fourth category was between the second and third categories. Finally, 23 better fiber quality and 135 high-yield germplasms were selected for breeding and production.【Conclusion】The phenotypic traits of Upland cotton are geographically specific; it is feasible to use multivariate statistical analysis to comprehensively evaluate Upland cotton germplasm; the whole population can be classified into four categories (high-quality, high-yield, early maturity and other types).

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    Genetic Analysis of Flag Leaf Traits in Wheat Under High and Low Nitrogen
    WU YaRui,LIU XiJian,YANG GuoMin,LIU HongWei,KONG WenChao,WU YongZhen,SUN Han,QIN Ran,CUI Fa,ZHAO ChunHua
    Scientia Agricultura Sinica    2022, 55 (1): 1-11.   DOI: 10.3864/j.issn.0578-1752.2022.01.001
    Abstract442)   HTML86)    PDF (524KB)(263)       Save

    【Objective】 Flag leaf is an important place for wheat photosynthetic carbon fixation, which plays an important role in wheat yield. The genetic characteristics and the genetic mechanism were analyzed under high and low nitrogen for flag leaf traits of wheat, which will provide a reference for excellent plant-type breeding and high-yield breeding. 【Method】 188 recombinant inbred line (RIL) populations derived from a cross between Kenong9204 and Jing411 was used in this study, which were planted in low nitrogen (LN) and high nitrogen (HN), respectively. The flag leaf traits of 188 RILs were investigated in 6 different environments, then the genetic analysis was conducted to determine the number of genes controlling each trait, and to estimate the genetic effect value and the heritability. In addition, the relationship between flag leaf characters and yield related traits of wheat was also studied.【Result】 Under LN environment: The optimal genetic model of flag leaf length was 2MG-CE (two pairs of interaction major genes) in E3. The additive × additive epistatic interaction value was 1.098, and the heritability of major genes was 31.35%. The flag leaf length was polygenic in another LN environment. The width of flag leaf was polygenic in all the LN environment. The optimal genetic model for flag leaf area (except E5) was 2MG-CE. The additive × additive epistatic interaction value was 1.884 and the heritability of major genes was 36.7%, while it was polygenic inheritance in E5. Under HN environment: The optimal genetic model for flag leaf length (except E4) was 2MG-CE, the additive × additive epistatic interaction value was 1.133, and the heritability of major genes was 32.6%. The optimal genetic model was 2MG-ER (two pairs of recessive epistatic major genes) in E4, which the additive effect value was 1.431 and 1.108 for the first and the second major genes respectively, and the heritability of the major gene was 51.77%. The optimal genetic model for flag leaf width (except E2) was 2MG-CE, the additive × additive epistatic interaction value was 0.119, and the heritability of major genes was 37.29%, while it showed polygenic inheritance in E2. The optimal genetic model for flag leaf area was 2MG-CE, which the additive × additive epistatic interaction value was 3.067 and the heritability of the main gene was 44.42%. The genetic models of flag leaf traits were different in different environments, which the genetic model was more stable under HN than that in LN. The correlation analysis of flag leaf and yield traits showed that flag leaf traits were significantly positively correlated with grain number per spike, grain weight per spike and yield per plant, and the influence degree was different in the 6 environments. 【Conclusion】 Flag leaf traits are easily affected by environment, and the performance of flag leaf traits is different in HN and LN. Flag leaf traits exhibited different major gene inheritance and polygene inheritance in LN, while they showed major gene inheritance which controlled by two pairs of interactions genes in most of HN environment, which might be major QTLs. Yield per plant and grain weight per spike could be increased by improving flag leaf traits.

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    Construction and Application of Detection Model for Amylose and Amylopectin Content in Sorghum Grains Based on Near Infrared Spectroscopy
    ZHANG BeiJu,CHEN SongShu,LI KuiYin,LI LuHua,XU RuHong,AN Chang,XIONG FuMin,ZHANG Yan,DONG LiLi,REN MingJian
    Scientia Agricultura Sinica    2022, 55 (1): 26-35.   DOI: 10.3864/j.issn.0578-1752.2022.01.003
    Abstract541)   HTML38)    PDF (870KB)(219)       Save

    【Objective】 Sorghum is one of the main raw materials for wine making and feed. The ratio of amylose content to amylopectin content in its grains is closely related to liquor quality and feed quality. Traditional chemical detection methods of sorghum components are no longer suitable for high-throughput testing. Modified PLS is used to perform spectral preprocessing, score processing and result monitoring on the near-infrared spectra of sorghum samples to establish sorghum grain amylose and amylopectin. The prediction model of amylose content aims to obtain a fast, efficient and low-cost detection method, laying the foundation for genetic improvement and quality analysis of sorghum. 【Method】 From 450 sorghum resources, 112 representative varieties were selected as calibration set and verification set. The chemical values of amylose and amylopectin content in 112 sorghum varieties were measured, and near-infrared spectra with wavelengths of 850-1 048 nm were collected, and the spectrum was scanned data matrix and chemical data calculated score (PL1) processing and interpreting the differences between the spectra, and eliminating abnormal species with Global H (GH) greater than 3 to reduce modeling errors. Modified PLS regression technology is used for modeling, and different calibration models are established through different scattering processing and derivative processing methods. Determine the best model according to the cross-validation standard deviation (SECV) and cross-validation correlation coefficient (1-VR), and perform result monitoring and non-parametric testing to evaluate the predictive performance of the model.【Result】 The near-infrared prediction model SECV of amylose is 2.7732, 1-VR is 0.9503, and the correlation coefficient (RSQ) is 0.9688. Bias=0.229<2.7732(SECV)×0.6, that is, the deviation (Bias) is less than 0.6 times of the calibration model SECV; the predicted standard deviation (SEP)=1.266<2.7732(SECV)×1.3=3.60516, that is, the SEP is less than the calibration. The model SECV is 1.3 times, 11.01(SD)-10.81(SD)=0.2<11.02(SD)×0.2=2.204, that is, the difference between the standard deviation (SD) of the chemical data and the near-infrared prediction data is less than 20% of the chemical data SD. The near-infrared prediction model SECV of amylopectin is 1.7516, 1-VR is 0.8818, and RSQ is 0.9127. Bias=-0.014<1.7516(SECV)×0.6 means that Bias is less than 0.6 times of SECV of calibration model, SEP=1.316<1.7516(SECV)×1.3=2.2708 means SEP is less than 1.3 times of SECV of calibration model, 5.30-5.29=0.01<5.30×0.2=1.06, that is, the difference between the chemical data and the near-infrared prediction data SD is less than 20% of the chemical data SD. Using 30 sorghum grains outside the model to conduct a two-pair sample non-parametric test on the validity of the model, the results showed that the difference between the measured and predicted values of amylose content and amylopectin content was not significant (P=0.262>0.05; P=0.992>0.05).【Conclusion】 The established near-infrared model has high accuracy and good stability, can accurately and quickly detect the content of amylose and amylopectin in sorghum, and can be used for the genetic improvement of sorghum and the detection of sorghum quality.

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    Construction of High-Density Genetic Map and QTL Analysis of Grain Shape in Rice RIL Population
    ZHANG YaDong,LIANG WenHua,HE Lei,ZHAO ChunFang,ZHU Zhen,CHEN Tao,ZHAO QingYong,ZHAO Ling,YAO Shu,ZHOU LiHui,LU Kai,WANG CaiLin
    Scientia Agricultura Sinica    2021, 54 (24): 5163-5176.   DOI: 10.3864/j.issn.0578-1752.2021.24.001
    Abstract713)   HTML95)    PDF (1849KB)(310)       Save

    【Objective】Rice grain shape is an important agronomic trait directly related to yield, which affects the appearance quality and commercial value of rice. Research on new rice grain shape genes is of great value for revealing the genetic mechanism of rice grain shape, and it can provide some new genetic resources for molecular breeding. 【Method】In the present study, a RIL population which constructed by an extra-large grain japonica rice variety TD70 and a small-grain indica rice variety Kasalath was used as the research material. The phenotypic data of grain shape, such as grain length, grain width, grain thickness and thousand grain weight were investigated. Using the Genotyping-By-Sequencing approach to re-sequence the parents and RILs to obtain SNP information. The sliding window method (the number of SNP/InDel is 15) was used for genotype calling and recombination breakpoint determination. Based on these results, a high-density Bin map was constructed. Meanwhile, the compound interval mapping method of QTL IciMapping software was used to map the QTLs related to grain shape. 【Result】A high-density genetic map containing 12 328 Bin markers was constructed. The number of Bin markers on each chromosome is 763 to 1367, and the average physical distance between markers was 30.26 kb. The frequency distribution of each trait for RIL population was continuous, which were consistent with the characteristics of quantitative characters, so it was suitable for the detection of QTL. QTL analysis of RIL population in 2018 showed that 40 grain-shape QTL were detected, including 12 grain length QTL, 9 grain width QTL, 8 grain thickness QTL, and 11 thousand-grain weight QTL. QTL analysis was performed of RIL population in 2019, and 56 grain-related QTL were detected, including 15 grain length QTL, 11 grain width QTL, 13 grain thickness QTL, and 17 thousand-grain weight QTL. Based on the two-year mapping results, we have mapped a total of 96 grain shape QTL. We found that 11 QTL could be detected for two consecutive years; among them, 7 QTL have been cloned and 4 new QTL were distributed on 1, 3, 4 and 5 chromosomes. Among the 4 new QTL, qGL-1-2 and qGL-5-2 was related to grain length, qGT-3-2 related grain thickness and qGW-4-1 related to grain width. 【Conclusion】We constructed a molecular genetic linkage map containing 12 328 Bin markers and used the map to analyze the grain shape loci of extra-large grain rice resources. Four new QTLs related to grain shape were obtained, which can be used for subsequent fine mapping, cloning and functional studies.

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    Analyzing Genetic Effects for Plant Height and Panicle Traits by Means of the Mixed Inheritance Model of Major Gene Plus Polygene in Foxtail Millet
    GUO ShuQing,SONG Hui,YANG QingHua,GAO JinFeng,GAO XiaoLi,FENG BaiLi,YANG Pu
    Scientia Agricultura Sinica    2021, 54 (24): 5177-5193.   DOI: 10.3864/j.issn.0578-1752.2021.24.002
    Abstract506)   HTML46)    PDF (2625KB)(223)       Save

    【Objective】Plant height and panicle traits are key yield-dependent traits in foxtail millet. The objective was to probe into inheritance patterns of plant height and panicle traits and provide a reference basis for genetically improving related traits and mapping their genes. 【Method】Yugu 18, a high performing foxtail millet variety, was arranged as the male parent to cross two foxtail millet varieties, Huangruangu and Hongjiugu, and thus two F7 populations of which each was composed of recombinant inbred lines involving 250 family lines(YYRIL and YRRIL)were established. Phenotypic data of Five agronomic traits of the two populations, plant height, panicle length, internode length under panicle, spikelet number per panicle and grain weight per panicle, were genetically examined in two different environments using the mixed inheritance model of major gene plus polygene. 【Result】In these two environments, all the five agronomic traits showed continuous variations with their kurtosis and skewness values standing at the absolute value of less than 1 and thus presenting a distribution close to a normal distribution, were characterized by typical inheritance of quantitative traits; some of these traits saw super-parent separation phenomena. The correlation analysis among the traits showed that the plant height appeared significantly and positively correlated with the panicle length, and an extremely significantly positive correlation between spikelet number per panicle and grain weight per panicle was also found in the two environments. The analysis by the inheritance model showed that the best inheritance models for the plant height of the YYRIL and YRRIL population were the PG-AI and PG-A polygene models, and the heritability of the polygenes standing at 95.15% and 91.27%, respectively. The best inheritance models for the spikelet number per panicle of the two populations were the PG-AI, with the heritability of the polygenes standing at 70.07%-71.58%. The best inheritance models for the internode length under panicle of the two populations were the 4MG-CEA and 3MG-CEA of which both were models for equally additive major genes. In YYRIL, the heritability of the major genes for the internode length under panicle stood at 9.69%, and the four pairs of major genes had an equal additive effect value of -0.34, taking negative effect; and in the YYRIL, the heritability of the major genes for the internode length under panicle stood at 45.78%, and the 3 major gene pairs in question had an equal additive effect value of 1.17, taking positive effect. In the YYRIL, the best inheritance model for the panicle length was the MX2-ED-A, a model for two pairs of dominant epistatic major genes and additive polygenes, with the heritability of the major genes and polygenes standing at 43.56% and 50.56%, respectively. the two pairs of panicle length-dependent major genes separately had the additive effect values of -1.21 and 1.68 and the polygenes had a lower additive effect value of -0.0017; in the YRRIL, the best inheritance model for the panicle length was the MX2-AE-A, a mixed inheritance model for two pairs of accumulative effect major genes and additive polygenes; the major genes and polygenes for the panicle length had heritability values standing at 46.40% and 46.91%, respectively. The first pair of panicle length-dependent major genes had an additive effect value of 1.53, taking positive effect; The additive and epistatic interactions effect value of the first×the second pairs of major genes were 0.60. The polygenes had an additive effect value of -0.47, taking the lower negative inheritance effect. In the YYRIL, the best inheritance model for the grain weight per panicle was the MX2-ED-A; the grain weight per panicle followed the inheritance model for two pairs of dominant epistatic major genes + additive polygenes with the heritability of the major genes and polygenes standing at 69.09% and 12.08%; the additive effect values of the two pairs of grain-weight per panicle-dependent major genes were separately 0.58 and 5.82, with the additive effect of the second pair of major genes dominating, and the additive effect value of polygenes stood at a value of -3.81. In the YRRIL, the best inheritance model for the grain weight per panicle was the 3MG-PEA, an inheritance model for three pairs of partially equal additive major genes; the heritability of the grain weight per panicle-dependent major genes stood at 81.10% and the additive effect values of the three pairs of major genes separately were -2.68, -2.68and 2.66, all taking negative effect. 【Conclusion】In foxtail millet, the plant height and spikelet number per panicle had similar inheritance models, were all under polygenic control with a higher heritability and environmentally affected to a slight content; the inheritance of the internode length under panicle was genetically controlled by major genes, which had a lower heritability and were environmentally affected to a great extent, and thus environmental factors should be taken into full account in production; the panicle length was genetically controlled jointly by major genes and polygenes; the grain weight per panicle was genetically controlled by major genes with a high heritability in both of the two population and probably carried major QTL.

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    Overexpression of OsPR1A Enhanced Xa21-Mediated Resistance to Rice Bacterial Blight
    LIU YuQing,YAN GaoWei,ZHANG Tong,LAN JinPing,GUO YaLu,LI LiYun,LIU GuoZhen,DOU ShiJuan
    Scientia Agricultura Sinica    2021, 54 (23): 4933-4942.   DOI: 10.3864/j.issn.0578-1752.2021.23.001
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    【Background】Previous studies revealed that the expression of pathogenesis-related protein OsPR1A was regulated by the upstream resistance gene Xa21. The rapid induction of OsPR1A protein at early stage after inoculation was crucial in Xa21-mediated rice bacterial blight resistance. The expression of OsPR1A was induced by Xanthomonas oryzae pv. oryzae (Xoo). OsPR1A was well known as a marker gene to demonstrate the reaction between rice and pathogen, however, no direct evidence was obtained for the biological function of OsPR1A. 【Objective】 In this paper, transgenic plants overexpression OsPR1A were obtained and the phenotype and agronomic traits were investigated. The relationship between OsPR1A expression and resistance were surveyed to further explore the function of OsPR1A in the process of rice resistance to bacterial blight.【Method】The construct of OsPR1a-OX was transferred to rice recipient 4021 via Agrobacterium-mediated protocol. Positive homozygous transgenic lines were identified by PCR and western blot (WB) respectively. At the mature stage, the phenotype and agronomic traits of OsPR1A overexpression rice plants were investigated, including plant height, spike length, tiller number, seed setting rate and grain size. Rice seedlings of TP309, 4021 and OsPR1A overexpression plants grown for two weeks were inoculated with Xoo at 31℃. The length of lesions was measured at 0, 2, 4, 6, 8, 10, and 12 days post-inoculation (dpi) respectively. At 0, 4 and 6 dpi, the rice leaves of TP309, 4021 and OsPR1A overexpression plants were collected to extract total protein, and the expression profiling of OsPR1A were surveyed by WB.【Result】The OsPR1a-OX transformation vector was constructed and transformed into recipient 4021. Two homozygous OsPR1A overexpression lines (#704 and #709) were identified. At the mature stage, the phenotype and agronomic traits of the OsPR1A overexpression plants were investigated. Compared with the control 4021, #704 and #709 lines showed lower plant height, shorter panicle length, fewer tiller number, lower seed-setting rate. The grain size in transgenic rice plants were larger, which might be related to the lower seed-setting rate. At 31℃, the lesion length of OsPR1A overexpression plants was significantly shorter than that of the control 4021 (P<0.05). At 0, 4, and 6 dpi, the abundance of OsPR1A expression of overexpression plants was higher than that of 4021 and TP309, and the high level of OsPR1A protein might contribute to the resistance of Xoo.【Conclusion】OsPR1A overexpression transgenic plants were obtained by the Agrobacterium-mediated method. Overexpression of OsPR1A affected the normal development of rice plants and also enhanced the resistance to bacterial blight mediated by Xa21.

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    Effects of Physicochemical Properties of Wheat (Triticum aestivum L.) Starch with Different HMW-GSs Combinations on Dough Stability
    NIU HongZhuang,LIU Yang,LI XiaoPing,HAN YuXuan,WANG KeKe,YANG Yan,YANG QianHui,MIN DongHong
    Scientia Agricultura Sinica    2021, 54 (23): 4943-4953.   DOI: 10.3864/j.issn.0578-1752.2021.23.002
    Abstract411)   HTML61)    PDF (1939KB)(296)       Save

    【Objective】Wheat dough is made of starch wrapped around filaments of gluten proteins. The rheological properties of dough have influence on the processing qualities of food. To explore the effect of starch physicochemical properties on the rheological properties of dough can provide a theoretical basis for clarifing the function of starch in dough.【Method】12 wheat varieties or strains were used as experimental materials, and the 12 test materials had three high molecular weight glutenin subunits (HMW-GSs) combinations. The subunits of Xinmai 26, Jimai 44, Xinong 615, and Gaocheng 8901 were 1, 7+8, and 5+10; the subunits of Xinong 221, Xinong 979, Xinong 633, and Xinong 059 were 1, 7+8, and 2+12; the subunits of 15(85)2A, 14(417)0-0-10, Xiaoyan 22, and Zhoumai 18 were 1, 7+9, and 2+12. Starch was extracted from the seeds of the test materials and the morphology of starch grains was observed by scanning electron microscope. The physicochemical properties of starch (including starch grain distribution, amylose content, relative crystallinity, short-range order degree, and thermal characteristics) and dough mixograph factors (including formation time and stability time) were determined and analyzed.【Result】For the starch granules of the 12 materials, the large starch granules were irregular oval, while the small starch granules were irregular oval or polyhedron. The starch grains of Xinmai 26, Jimai 44, Gaocheng 8901, Xinong 221, Xinong 979, and Xinong 059 were closely arranged, while the starch grains of Xinong 615, Xinong 633, 15(85)2A, 14(417)0-0-10, Xiaoyan 22, and Zhoumai 18 were dispersed. Xinong 979, 15(85)2A, Xiaoyan 22, and Zhoumai 18 had larger diameter of A-type starch grains. The higher the content of A-type starch grains, the lower the content of B-type starch grains, and the smaller the ratio of B-type starch grains to A-type starch grains. When the amylose content between two materials was similar, their dough stability time was similar. The X-ray diffraction patterns of the starches of the 12 materials are similar and all belong to A-type crystal structure. The Fourier transform infrared spectrum analysis of starch showed that Xinmai 26 had the highest 1 045/1 022 cm-1 value and the lowest 1 022/995 cm-1value, while the value of 1 022/995 cm-1 in Zhoumai 18 was the highest, but the value of 1 045/1 022 cm-1 was lower. This indicates that a higher 1 045/1 022 cm-1 value of the test material usually corresponds to a lower 1 022/995 cm-1 value. The starch properties of the 12 test materials were different, and the stability time of the dough was also significantly different. The materials Xinmai 26, Jimai 44, Xinong 221, Xinong 979, and Xinong 633 had longer dough stability time, and the dough stability time of Xinmai 26 was much longer than that of other materials.【Conclusion】 Among the 12 test materials, amylose content (r=0.88, P<0.01) and short-range order of starch (r=0.83, P<0.01) were significantly positively correlated with the dough stability time. The A-type starch content (r=0.61, P<0.05), starch relative crystallinity (r = 0.84, P<0.01), and enthalpy of starch gelatinization (r=0.71, P<0.01) were significantly negatively correlated with the dough stability time.

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    Comprehensive Evaluation of Low-Fertility Tolerance of Different Sorghum Cultivars in Middle-Late-Maturing Area
    ZHANG Yan,WANG JinSong,DONG ErWei,WU AiLian,WANG Yuan,JIAO XiaoYan
    Scientia Agricultura Sinica    2021, 54 (23): 4954-4968.   DOI: 10.3864/j.issn.0578-1752.2021.23.003
    Abstract443)   HTML59)    PDF (810KB)(289)       Save

    【Objective】Soil fertility is essential to crop growth and yield. The arable land per capita in China is low, and approximately 21.95% of the arable land has serious barriers for crop production. To ensure food security, it is necessary to exploit cultivated land reserves, along with the efforts to increase the productivity of arable land. Therefore, it is important to cultivate crops that can tolerate low-fertility in marginal lands, to avoid land competition with other main crops and meet increasing food demand. To better use marginal land for sorghum production, a field experiment was conducted in Yuci, Shanxi Province in 2019, to study the variation in low-fertility tolerance ability of different sorghum cultivars. This study aimed to identify the sorghum cultivars with strong tolerance to low-fertility and the index to evaluate low-fertility tolerance, and the results would provide evidence for sorghum selection in marginal land cultivation.【Method】In this study, a field experiment including 23 sorghum cultivars was conducted with contrasting soil fertility treatments, including high soil fertility (control) treatment and low soil fertility treatment (stress). A total of 15 parameters including yield and yield components, dry matter accumulation of shoot and nutrient uptake were investigated. The low-fertility index of each parameter was calculated. Principal component analysis (PCA) and cluster analysis were used to evaluate low-fertility tolerance and identify low-fertility tolerant cultivar. Low-fertility index and comprehensive evaluation value were used, combined with regression analysis and correlation analysis, to identify the optimal indicators for low-fertility tolerance.【Result】The results showed that the yield, grain number per spike, harvest index, dry matter accumulation at harvest and after heading, leaf area index (LAI), panicle length and width of sorghum were decreased by the low-fertility stress, compared with those in high-fertility plots. The decreasing rate was as follows: dry matter accumulation after heading>yield>grain number per spike>dry matter accumulation of shoot>LAI>panicle width>harvest index. The accumulation of N, P, K in both grains and shoot above ground was also decreased. The N accumulation was the most sensitive to low-fertility stress. The 15 parameters were categorized into five comprehensive indexes (the cumulative contribution rate was 89.28%) by principal component analysis, and the comprehensive evaluation value (D) of the low-fertility tolerance was calculated. According to the D value and the cluster analysis, the 23 sorghum cultivars were divided into four tolerance levels. Six, seven, seven and three cultivars were classified as stronger (0.633≤D≤0.755), strong (0.467≤D≤0.592), weak (0.310≤D≤0.421) and weaker (0.166≤D≤0.246) tolerance to low fertility, respectively. The cultivars Jiniang 2, Liaoza 19, Jinza 18, Jinza 22, Jinfeng 301 and Jinza 28 had the highest D value. Stepwise regression analysis was used to establish the optimal regression equation to evaluate the sorghum low-fertility tolerance. The yield, dry matter accumulation of shoot, N accumulation above ground, grain P accumulation, grain K accumulation and panicle width were selected, which had significant effects on the tolerance to low-fertility stress. The low-fertility index of yield, dry matter accumulation of shoot, N accumulation in shoot above ground, P accumulation in shoot above ground, grain N accumulation and grain P accumulation were significantly correlated with D value. The correlation coefficients were 0.845, 0.836, 0.766, 0.778, 0.761 and 0.757, respectively. 【Conclusion】There were large variation in low-fertility tolerance of the 23 sorghum cultivars. The cultivars of Jiniang 2, Liaoza 19, Jinza 18, Jinza 22, Jinfeng 301 and Jinza 28 were significantly tolerant to low-fertility land. The low-fertility index of grain yield, dry matter accumulation of shoot, N accumulation above ground and grain P accumulation were recommended in rapid screening of low-fertility tolerant sorghum cultivars.

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    Establishment and Application of A Duplex ddPCR Method to Quantify the NK603/zSSIIb Copy Number Ratio in Transgenic Maize NK603
    XIAO Fang,LI Jun,WANG HaoQian,ZHAI ShanShan,CHEN ZiYan,GAO HongFei,LI YunJing,WU Gang,ZHANG XiuJie,WU YuHua
    Scientia Agricultura Sinica    2021, 54 (22): 4728-4739.   DOI: 10.3864/j.issn.0578-1752.2021.22.002
    Abstract401)   HTML46)    PDF (2378KB)(343)       Save

    【Objective】Transgenic maize NK603 approved for import is an important target of genetically modified organism (GMO) regulation in China. The implementation of GMO regulation requires reference materials (RMs) and standardized detection methods. Establishment of a duplex droplet digital PCR (ddPCR) would provide accurate measurement technology for quantification of NK603 event and development of NK603 RMs. 【Method】 A standard plasmid molecule pUC57-NK603 was constructed by DNA synthetic technique; the primer/probe set of NK603 event was combined with different maize reference genes one by one to select reference gene PCR assay with identical amplification ability to NK603 event-specific PCR assay; main reaction parameters, such as annealing temperature and primer/probe concentration,were optimized in the course of establishing duplex ddPCR; the standard plasmid solution was serially diluted to investigate the limit of detection (LOD), limit of quantification (LOQ) and dynamic range of the duplex ddPCR assay; blinded samples with mass fractions of 100%, 10% and 6% were prepared by mixing NK603 powder with non-GM counterpart, to evaluate the accuracy of quantitative results of the duplex ddPCR. 【Result】 The reference gene zSSIIb was determined to combine with NK603 event to establish the NK603/zSSIIb duplex ddPCR method with the standard plasmid molecule pUC57-NK603 as a quality control after analyzing the fluorescence amplitude of positive droplets, separation between positive and negative droplets, raindrop number, and consistency between measured copy number ratio and expected copy number ratio of NK603 event to reference gene. The primer/probe concentration was optimized to be 400 nmol·L-1/200 nmol·L-1 for both NK603 event and zSSIIb gene, and the annealing temperature was determined to be 60°C. The LOD of NK603/zSSIIb duplex ddPCR was estimated to be 2 copies of DNA template, the LOQ was 48 copies of DNA template, both NK603 assay and zSSIIb assay showed good linearity between measured values and theoretical values over the dynamic range from 10 to 60 000 copies of DNA template. The NK603/zSSIIb duplex ddPCR achieved accurate quantitative results of NK603 content in blind maize samples with less than 25% of coefficient of variation; the quantitative results of ddPCR were not significantly different from those of real-time quantitative PCR (qPCR), moreover, the duplex ddPCR showed an advantage over qPCR in term of precision. 【Conclusion】 The selection of reference genes affects the accuracy of quantitative results by ddPCR. Establishment of ddPCR methods should use samples with accurate GMO content as quality controls to evaluate the applicability of reference genes. The NK603/zSSIIb duplex ddPCR method was successfully established using the synthetic standard plasmid molecule pUC57-NK603 as a quality control. NK603 certified reference materials (CRMs) have been successfully developed in China by applying the established NK603/zSSIIb duplex ddPCR assay to characterize the property values.

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    Quantitative Trait Locus Mapping of Bruchids Resistance Based on A Novel Genetic Linkage Map in Cowpea (Vigna unguiculata)
    WANG Yan,FAN BaoJie,CAO ZhiMin,ZHANG ZhiXiao,SU QiuZhu,WANG Shen,WANG XueQing,PENG XiuGuo,MEI Li,WU YuHua,LIU ShaoXing,TIAN ShengMin,XU JunJie,JIANG ChunZhi,WANG WeiJuan,LIU ChangYou,TIAN Jing
    Scientia Agricultura Sinica    2021, 54 (22): 4740-4749.   DOI: 10.3864/j.issn.0578-1752.2021.22.003
    Abstract379)   HTML46)    PDF (1724KB)(306)       Save

    【Objective】Bruchids are the main storage pests of cowpea. The discovery of bruchids resistance genes is helpful for breeding resistant varieties and reducing the harm of bruchids to cowpea production.【Method】In this study, a RIL population of 282 lines derived from the cross between Zhongjiang No.1 (a bruchid-susceptible cultivar) and Pant-lobia-1 (a bruchid-resistant cultivar) was used for phenotype identification of bruchids resistance by artificial inoculation of Callosobruchus chinensis and Callosobruchus maculates. The two parents were used to screen polymorphic markers from 3 992 SSR markers of mung bean, adzuki bean and cowpea. Genotypes of 282 lines were analyzed using the polymorphic SSR markers. Based on the phenotype identification and genotype analysis, a genetic linkage map was constructed and the bruchids resistance gene(s) of cowpea was located using Inclusive Composite Interval Mapping (ICIM-ADD). 【Result】The results showed that Zhongjiang No. 1 and F1 seeds were 100% susceptible to Callosobruchus chinensis and Callosobruchus maculates, and the damage rates of Pant-lobia-1 were 22.5% and 42.5%, respectively. It was speculated that the resistance of Pant-lobia-1 to bruchids was recessive inheritance. 182 polymorphic markers were obtained from 3 992 SSR markers of mung bean, adzuki bean and cowpea. Using those polymorphic SSR markers, a genetic linkage map with 11 linkage groups was constructed. The map covered a total length of 1 065.23 cM with an average interval of 5.85 cM between adjacent markers. Two bruchid-resistant QTLs from linkage groups 1 and 5 were discovered, which were temporarily named as vubr1-1 and vubr5-1. QTL vuvr1-1 was located between markers XD11-44 and HAAS_VR_2274, which genetic distance was 7.6 cM, explaining 7.16% and 6.92% of the phenotypic variation in the two bruchid-resistant tests respectively. QTL vubr5-1 was located between markers XD1-14 and CP185, which genetic distance was 2.9 cM, explaining 6.96% and 6.37% of the phenotypic variation in the two bruchid-resistant tests respectively. 【Conclusion】A genetic linkage map containing 11 linkage groups and 182 polymorphic markers was constructed. Two QTLs linked with bruchids resistance were identified on linkage groups 1 and 5.

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    The Genetic Contribution of the Important Breeding Parent Chuanmai 44 to Its Derivatives
    LUO JiangTao,ZHENG JianMin,DENG QingYan,LIU PeiXun,PU ZongJun
    Scientia Agricultura Sinica    2021, 54 (20): 4255-4264.   DOI: 10.3864/j.issn.0578-1752.2021.20.001
    Abstract430)   HTML63)    PDF (1888KB)(311)       Save

    【Objective】Common wheat variety Chuanmai 44 has the characteristics of high yield, stable yield and wide adaptability. Ten new varieties have been selected and approved in breeding program using Chuanmai 44 as parent. It indicates Chuanmai 44 is an important breeding parent. To clarify the genetic base of Chuanmai 44 as a vital parent in breeding exercise and identify important genes or QTL within it will be helpful in breeding new elite varieties using Chuanmai 44. 【Method】Fluorescence in situ hybridization was applied to Chuanmai 44 and its ten derived varieties to identify whether there were wheat-alien translocations, and to analyze the chromosome diversity among them. The 660K SNP array data of Chuanmai 44 and its derived varieties were used to calculate the genetic contribution of Chuanmai 44 to its derived varieties and clarify the high transmission genomic segments. Functional molecular markers within cloned genes and linked molecular markers for yield-related traits were used to identify important genes or QTL in Chuanmai 44 for breeding. 【Result】 Chuanmai 44 did not harbor the 6VS/6AL and 1RS/1BL translocation chromosomes which both frequently existed in wheat varieties in Sichuan. Only two out of its ten derivatives, Changmai 32 and Changmai 34, contained 1RS/1BL translocation, which is inherited from another parent Changmai 19. The existence of 1RS/1BL translocation in the two varieties may explain their weak gluten phenotype. Except wheat-relative translocation, the karyotypes of Chuanmai 44 and its 10 derivative varieties also showed polymorphisms on some chromosomes. For instance, there were two types of chromosome 4A among derivatives, and 80% of them showed the same as Chuanmai 44. Chromosomes 5A, 6B and 7B had 4, 2 and 2 karyotypes, respectively. These three chromosomes in the derivative population of Chuanmai 44 showed the same karyotype with Chuanmai 44 in a frequency of 40%. 660K SNP chip analysis identified 1127 genomic segments with high transmission frequency (>50%) within its derived varieties. These genomic segments located on all 21 chromosomes and their mean length was 1.57 Mb. B genome owned the most number and the largest length of the high transmission frequency segments. Chromosomes 4A, 2B and 5B were the three chromosomes with the longest high transmission frequency segments. Chromosomes 4A, 2B and 3B were the three chromosomes with the most number of high transmission frequency segments. Combing the genotype data of 61 functional markers of cloned wheat gene and 13 SNP markers linked with yield-related QTL and the distribution of Chuanmai 44 high transmission genomic regions, we discovered that there are 9 genes markers and 3 QTL markers are anchored in the high transmission rate section of Chuanmai 44. The twelve markers responding to two favorable alleles and three QTL, including TaSdr, NAM-A1, QTKW.sicau-2AS.1, QTKW.Sicau-4AL, QSL.sicau-5AL.2, which exhibited positive effect on preharvest sprouting resistance, effective tiller number, thousand grain weight and spike length, respectively. 【Conclusion】The length of genomic segments retained within its derived varieties was short. It suggested that Chuanmai 44 as a breeding parent had high genetic combining ability, and its chromosomes were easy to recombine with different homologous chromosomes in resulting hybrids, which is beneficial to reduce linkage drag. Therefore, it plays an important role as a skeleton parent in breeding excercise. TaSdr, NAM-A1, QTKW.sicau-2AS.1, QTKW.Sicau-4AL and QSL.sicau-5AL.2 were the important loci in Chuanmai 44, which should be widely used in further breeding program under molecular marker assisted selecting.

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    IbMKP6, A Mitogen-Activated Protein Kinase, Confers Low Temperature Tolerance in Sweetpotato
    JIN Rong,LIU Ming,ZHAO Peng,ZHANG QiangQiang,ZHANG AiJun,TANG ZhongHou
    Scientia Agricultura Sinica    2021, 54 (20): 4265-4273.   DOI: 10.3864/j.issn.0578-1752.2021.20.002
    Abstract266)   HTML36)    PDF (1109KB)(324)       Save

    【Objective】Studying the function of mitogen activated protein kinase (MAPK) IbMPK6 in respect of low temperature stress tolerance in sweetpotato, that will help us to understand the mechanism of adaption to low temperature stress and play a fundamental role in molecular breeding of sweetpotato. 【Method】Agrobacterium tumefaciens strain EHA105 harbored the plasmid 35S::IbMPK6-GFP were transformed intosweetpotato cv. Xushu29 embryogenic callus. Molecular examination and qRT-PCR were used to screen and select transgenic lines. For low temperature stress assay, selected transgenic lines were performed to observe the phenotype and determine the physiological indexes such as Fv/Fm, the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) after low temperature treatment and recovery treatment. Diaminobenzidine (DAB) staining and nitro blue tetrazolium (NBT) staining analysis were performed to observe reactive oxygen species (ROS) accumulation. The expression level of the key transcription factor IbCBF3 and downstream gene IbCOR27 involved in low temperature signal transduction pathway were identified before and after low temperature treatment. 【Result】Twelve transgenic lines were generated and three transgenic lines (L3, L8 and L11) with a high expression level of IbMPK6 were selected for low temperature tolerance assay. Under low temperature stress, the level of Fv/Fm in transgenic lines L3, L8 and L11 was 0.79, 0.79 and 0.80, while that in WT was 0.05. After temperature recovery treatment, Fv/Fm in transgenic lines has recovered to former levels, whereas the level of Fv/Fm in WT was only 0.70, which was significantly lower than that in transgenic lines. MDA content of three transgenic (lines L3, L8 and L11) increased by 0.02, 0.04 and 0.02 μmol·g-1, and it of WT increased by 0.05 μmol·g-1 after low temperature stress treatment, respectively. After recovery treatment, MDA content in transgenic lines was 0.01 μmol·g-1 on average, whereas it of WT was 0.03 μmol·g-1. The results of DAB and NBT staining showed that the leaves of WT were stained deeper than those of transgenic lines, indicated that hydrogen peroxide and superoxide anion were accumulation less in transgenic lines than in WT. Furthermore, H2O2 level in WT was significantly higher than that in transgenic lines under low temperature stress condition and after recovery treatment. Low temperature regulated the expression level of IbCBF3 and IbCOR27 genes, but the expression level in transgenic lines was higher than that in WT. 【Conclusion】Overexpression of IbMPK6 in sweetpotato resulted in enhanced tolerance to low temperature stress, via alleviating the damage of membrane and photosynthetic system, and decreasing ROS accumulation. IbMPK6 involved in low temperature signaling transduction pathway by up-regulating the expression level of cold related genes IbCBF3 and IbCOR27.

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    Effects of PvEG261 Gene on the Fusarium Wilt and Drought- Resistance in Common Bean
    XUE RenFeng,FENG Ming,HUANG YuNing,Matthew BLAIR,Walter MESSIER,GE WeiDe
    Scientia Agricultura Sinica    2021, 54 (20): 4274-4285.   DOI: 10.3864/j.issn.0578-1752.2021.20.003
    Abstract273)   HTML34)    PDF (2209KB)(128)       Save

    【Objective】By analyzing the sequence and expression pattern characteristics of PvEG261 from common beans, and studying its resistance to Fusarium wilt and drought, the foundation was laid for the signal regulation network analysis of Fusarium wilt and drought-resistance and molecular breeding in common beans. 【Method】 Bioinformatics analysis was performed on the open reading frame (ORF) of PvEG261 to predict the physical and chemical properties, secondary structure, signal peptide sequence of the protein encoded by the PvEG261, and search for highly homologous protein sequence in NCBI database through Blastp tool online for sequence alignment and phylogenetic tree construction; the tissue expression specificity of PvEG261 and the expression pattern in response to Fusarium wilt pathogen and drought stress were analyzed by qRT-PCR; PvEG261 overexpression vector was constructed and transformed into Agrobacterium rhizogenes K599 to induce the generation of hairy transgenic roots in common beans. Meanwhile, the PvEG261 silencing vector was constructed, and the transcription product in vitro was inoculated on the seedlings of common bean to interfere with PvEG261 expression. Through inoculation with the pathogen and drought treatment, the phenotypes of control, PvEG261-overexpressed and silenced plants were observed, disease and drought-resistance were both identified, and hydrogen peroxide (H2O2) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and peroxidase (POD) activity as physiological and biochemical indicators were all assayed. 【Result】 The cDNA sequence of PvEG261 was 471 bp, which encodes a protein composed of 156 amino acids. The structure prediction indicated that it contained 10 strand structures, the predicted molecular mass of the encoding product was 38.89 kD, and the theoretical pI was 5.21. PvEG261 belonged to the members of dirigent gene superfamily, it contained a signal peptide sequence of 10 amino acids, and belonged to a secreted protein. The relationship between PvEG261 and cowpea DIR22 protein is the closest, which reached 91.61%. The results of qRT-PCR showed that the expression in the root tissues increased significantly after inoculation with Fusarium wilt pathogen and drought treatment, and the gene has obvious tissue expression specificity, with the highest expression level in the roots. After inoculation with pathogen and drought treatment, the disease and drought-resistance of the overexpressed plants were significantly improved in comparison with the control, the plant disease scores and the wilting degree caused by water shortage were significantly reduced, and the H2O2 content, POD and SOD activity in the roots were all significantly higher than the control plant, while the MDA content was dramatically lower than the control plant. The disease and wilting degree of the gene silenced plants were significantly increased. The H2O2 content, POD and SOD activity in the roots were significantly lower than the control plant, and the MDA content was significantly higher than the control plants. 【Conclusion】 PvEG261 responded to Fusarium wilt pathogen infection and drought stress, and positively regulated the Fusarium wilt and drought-resistance in common beans.

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    Transforming and Upgrading Off-Season Breeding in Hainan Through Molecular Plant Breeding
    ZHANG Xingping,QIAN Qian,ZHANG JiaNan,DENG XingWang,WAN JianMin,XU Yunbi
    Scientia Agricultura Sinica    2021, 54 (18): 3789-3804.   DOI: 10.3864/j.issn.0578-1752.2021.18.001
    Abstract1205)   HTML140)    PDF (659KB)(960)       Save

    Generation advancement at non-target environments is a cost-effective and efficient approach to accelerating plant breeding. Hainan, as a southernmost province of China where crops can grow all year round, has been used as a core off-season breeding station, largely for seed increasing and an annually added crop generation advancement, while its favorable climate and environments are not fully exploited. Molecular biotechnologies such as marker-assisted selection, when integrated with other modern breeding technologies, will facilitate the transformation and upgrading of the Hainan off-season breeding. The transition will be made from a simple generation advancement to a full pipeline breeding including germplasm introduction and evaluation, selection, purity testing, germplasm exchange and variety right protection, and from one added winter breeding season to breeding all year round. Geographical and ecological advantages of Hainan as an off-season breeding station should be transitioned into a breeding pipeline by integration of generation advancement with functional breeding system to accelerate breeding procedures, improve breeding efficiency and promote advanced breeding industry. Considering the geographical and ecological advantages of Hainan and current status of off-season breeding, following issues are discussed in this article on transformation and upgrading of Hainan off-season breeding enterprise: necessity, possibility, challenges and opportunities. Such transition depends on the change of mind-set on non-target environment selection, favorable government policy regulation, breeding platform development, biosafety containment, well-established variety right protection, and resource-sharing and exchanging regulation. Breeding theories required for the transition include quantitative and population genetics, genotype by environment interaction, and molecular design and big- data management. Molecular design should be implemented at both macro-scale involving individuals, populations and species and micro-scale involving genes, metabolisms and networks. The breeding platform needs to be established for high-throughput precision phenotyping, envirotyping, information management and networking, and decision support system. As a key support technology for molecular breeding, genotyping by target sequencing and liquid chip (GBTS-LC) has been developed, by which GenoPlexs can be used to multiplex up to 5 000 target primers while GenoBaits can capture in solution up to 40K target loci each with multiple SNPs. Compared to other genotyping platforms, GBTS-LC has several advantages to make it a replacement of solid SNP chips, including wide platform suitability, high marker flexibility, high genotyping efficiency, easy information accumulation, less-demanding support system, wide application fields. For breeding in Hainan but testing across China, an integrative breeding system can be established with high-efficient breeding facilities supported by speeding breeding, gene transfer, genome editing, DH technology, and genomic selection. Other proposals are also suggested, including establishment of open-source breeding programs for resource-sharing, development of methodologies, technologies and platforms, implementation of germplasm introduction, monitoring and evaluation, construction of germplasm fingerprinting, and intensification of variety right protection, seed quality ensurance and see purity control. It is expected that this article will stimulate public awareness and relevant government policy development for the transformation and upgrading of breeding in Hainan, and thus enhance the scientific and technological progress and advancement of breeding and seed industry as a whole.

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    Generation of ospin9 Mutants in Rice by CRISPR/Cas9 Genome Editing Technology
    WU ShiYang,YANG XiaoYi,ZHANG YanWen,HOU DianYun,XU HuaWei
    Scientia Agricultura Sinica    2021, 54 (18): 3805-3817.   DOI: 10.3864/j.issn.0578-1752.2021.18.002
    Abstract795)   HTML70)    PDF (2333KB)(499)       Save

    【Objective】Auxin efflux protein family PIN-FORMED (PIN) is a key protein family in controlling polar auxin transport (PAT). OsPIN9 is one of the monocot-specific PIN genes in rice, while its biological function still needs to be further elucidated. In this study, OsPIN9 was edited and ospin9 homozygous mutants were obtained using CRISPR/Cas9 genome editing technology. The resultant ospin9 mutant lines could provide a basis for further research on the function of OsPIN9.【Method】The specific target sequence was designed according to OsPIN9 genome sequence and OsPIN9 genome editing vector was constructed. Nippobare (Oryza sativa japonica) was used as the material and the hygromycin-resistant rice was obtained by Agrobacterium-mediated transformation. The positive transgenic lines were screened by PCR. The mutation sites were confirmed by the combination of PCR and subsequent analysis of sequencing results, the homozygous mutants were obtained and the difference of amino acid sequence and tertiary structure of OsPIN9 protein was analyzed between WT and ospin9 mutants. The expression of OsPINs genes in mutant roots was performed by quantitative real-time PCR (qRT-PCR), and the phenotype of ospin9 mutants was analyzed at the seedling stage. The effects of 1-naphthaleneacetic acid (NAA) treatment on seedling development were also analyzed under 0.05 μmol·L -1 NAA for 7 d.【Result】The target site sequence was designed based on the sequence of exon1 of OsPIN9 and, subsequently, the OsPIN9 genome editing recombinant vector was constructed. A total of 18 independent transgenic lines were obtained by transformation. Sequencing analysis revealed that three different mutation types were present in 7 T0 generation lines, including 3 lines with T insertion, 3 lines with G insertion and 1 line with C insertion, and all the mutation sites happened at the 18 th base of the target sequence. Two homozygous mutation lines were further identified in the T1 generation. BLAST analysis showed that the two types of OsPIN9 mutations caused frame-shift mutation and premature termination of translation, and the mutation protein was shortened from 426 aa in WT to 172 aa, thus leading to the complete disappearance of the transmembrane helices. qPCR analysis indicated that the transcription abundance of OsPIN9 significantly decreased in ospin9 mutants compared with WT, OsPIN1a and OsPIN5b were up-regulated, while OsPIN5a was down-regulated in ospin9 mutants. Both the shoot height and the number of adventitious roots of ospin9 mutants were reduced significantly than that of WT, while its root length was comparable to that of WT. The plant growth was inhibited and the adventitious root number was still less than that of WT under NAA treatment, but no significant difference was found between ospin9 mutants and WT plants. 【Conclusion】 Auxin efflux carrier OsPIN9 was directionally edited by using CRISPR/Cas9 technology, and two transgene-free homozygous ospin9 mutants were obtained. The mutation of OsPIN9 affected the expression level of other OsPINs genes, the shoot and root development were inhibited in ospin9 mutants at the seedling stage and NAA treatment partially rescued the development of adventitious roots in ospin9 mutants.

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    Evaluation and Screening of Nitrogen Efficiency of Wheat Germplasm Resources at Mature Stage
    ZHAO Rui,ZHANG XuHui,ZHANG ChengYang,GUO JingLei,WANG Yu,LI HongXia
    Scientia Agricultura Sinica    2021, 54 (18): 3818-3833.   DOI: 10.3864/j.issn.0578-1752.2021.18.003
    Abstract520)   HTML90)    PDF (3812KB)(478)       Save

    【Objective】 In order to provide theoretical foundation for the study of physiological mechanism of nitrogen efficiency in wheat and material basis for the breeding of nitrogen efficiency varieties, the evaluation method for nitrogen efficiency of wheat at mature stage was established to explore and screen nitrogen efficiency germplasm resources. 【Method】 In the field experiment, 108 different genotype wheat varieties were used for the treatment of 4 nitrogen application(0, 180, 240, 360 kg·hm-2). Eleven indexes of major agronomic traits, including plant height, spike length, flag leaf length, flag leaf width, stem diameter, fertile spikelet number, grain number per spike, thousand kernel weight, grain length, grain width and grain weight per spike were measured for two consecutive years. The nitrogen tolerance and genotype difference of wheat varieties were assessed by the method of Fuzzy membership function, principal component analysis and cluster analysis. 【Result】 The results of two-year data show that plant height, spike length, flag leaf length, flag leaf width, stem diameter, fertile spikelet number, spike grain number and grain weight per spike under low nitrogen stress were all inhibited to varying degrees, among which flag leaf length was the most sensitive to nitrogen stress. Four principal components were extracted by principal component analysis, and the contribution rate was 39.766%, 16.661%, 9.361% and 9.275%, respectively. The cumulative contribution rate reached 75.064%. According to the comprehensive evaluation D value of low nitrogen tolerance, the tested wheat varieties were divided into 5 types: strong low nitrogen tolerance, low nitrogen tolerance, intermediate, low nitrogen light sensitive and low nitrogen sensitive. Five low-nitrogen tolerant wheats including Wenmai19, Xinong 529, Shi 4185, Longmai 212 and Fengkang 2 and two strong low-nitrogen tolerant wheats (Zhongmai 875 and Xinong 158) were selected. Different from low nitrogen stress, high nitrogen stress only inhibited 5 traits including stem diameter, thousand kernel weight, grain length, grain width and grain weight per spike, and 6 traits including plant height, spike length, flag leaf length, flag leaf width, fertile spikelet number and spike grain number increased with the increase of nitrogen application. Principal component analysis extracts 4 principal components, the contribution rates are 31.348%, 20.387%, 12.452% and 9.850%, respectively, and the cumulative contribution rate is 74.037%. Based on the D value of comprehensive evaluation of high nitrogen tolerance, the tested wheat varieties were divided into four categories: high nitrogen tolerance type, intermediate type, high nitrogen light sensitive type and high nitrogen sensitive type. Nine wheat varieties with high nitrogen tolerance were identified, including Lankao Aizao 8, Liangxing 99, Nongda 179, Yunnong 9901, Lankao 926 and Zhengnong 46. Based on the D value of comprehensive evaluation of nitrogen and grain yield, 108 wheat varieties were divided into four nitrogen efficiency types, dual-efficiency type (Xinong 158 and Longmai 212 et al.), low-nitrogen efficiency type (Xinong 585 and Shi 4185 et al.), high-nitrogen efficiency type (Changfeng 1 and Zhongsimai 10 et al.) and dual-inefficiency type (Jinfeng 7183 and Fanmai 5 et al.). 【Conclusion】 Nitrogen supply level has a significant influence on wheat yield-related traits and indexes. The nitrogen efficiency of wheat germplasm resources at maturity might be accurately evaluated combined with three evaluation methods, based on the difference of nitrogen absorption and utilization efficiency between wheat germplasm.

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    Selection of PMS Rice Varieties and Application in Flooding Irrigation for Cadmium Reduction
    ZENG XiaoShan,TANG GuoHua,XIE HongJun,ZHU MingDong,AO HeJun,CHEN Bo,LI FangTing,HAO Ming,XIAO Yan,FU HuiRong,ZHANG Jian,YU YingHong
    Scientia Agricultura Sinica    2021, 54 (17): 3561-3572.   DOI: 10.3864/j.issn.0578-1752.2021.17.001
    Abstract498)   HTML92)    PDF (534KB)(469)       Save

    【Objective】 With the extension of mechanization and large-scale rice production mode, late harvest of rice grains brought by improper weather, conflicts in using mechanics and drying facilities usually gives rise to severe yield loss and quality deterioration, which has been deeply concerned by the farmers and greatly challenged the middle and late rice production in China. On the other hand, cadmium accumulation in rice grains has become a top food safety issue for breeders and producers. Flooding irrigation can effectively reduce cadmium content in rice. This research aims to select PMS (post-maturation sustainability) and low cadmium accumulation rice varieties by continuous flooding irrigation treatment. We expect to establish an effective evaluation system for PMS, select and utilize rice varieties with both PMS and low cadmium accumulation for production and research. 【Method】 Continual flooding irrigation were carried out in the experimental field during booting stage to yellow ripening stage in 2018. The mature plant lodging, seed dormancy and grain shattering of the 244 materials from China were evaluated through field observation and data investigation after ripening. The appraisal time and value of PMS rice varieties were defined using significant analysis. The 1000-grain weight, milling quality, appearance quality, gelatinization temperature, gel consistency and amylose content were tested for PMS rice. In 2019, 132 rice varieties promoting in Hunan were evaluated and identified using the appraisal time and value of post-harvest lodging. 1000-grain-weight were investigated between flooding and dry-wet alternate irrigation. The yield and effect of cadmium accumulation reduction of PMS rice varieties were tested in demonstration paddy field. 【Result】 The threshold for PMS was optimized as follows: lodging resistance is defined as the angle between rice stalk and vertical inclination <45° at the 14th day after maturity; rice shattering <5.0% at 7 days after maturity; and spike germination rate <10.0% at the same day of maturity. 21 PMS rice varieties were obtained in 2019. No significant difference existed in 1000-grain weight between flooding and dry-wet alternate irrigation, and also in yield in demonstration paddy field. The cadmium content of brown rice in flooding irrigation was lower than 0.20 mg·kg -1, which is significantly lower than that of dry-wet alternate irrigation management. 【Conclusion】 PMS rice varieties can withstand long-term flooding irrigation. There is no significant effect on yield and quality in delayed harvest after maturity. Application of PMS rice varieties in mild and moderate-level cadmium contaminated paddy, safety production can be realized in combination with flooding irrigation.

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    Genome-Wide Identification of WOX Family and Expression Analysis of Callus Induction Rate in Tartary Buckwheat
    HOU SiYu,WANG XinFang,DU Wei,FENG JinHua,HAN YuanHuai,LI HongYing,LIU LongLong,SUN ZhaoXia
    Scientia Agricultura Sinica    2021, 54 (17): 3573-3586.   DOI: 10.3864/j.issn.0578-1752.2021.17.002
    Abstract502)   HTML74)    PDF (4840KB)(366)       Save

    【Objective】 This study aimed to identify the whole genome WOX (WUSCHEL-related home obox) gene family in Tartary buckwheat and reveal the correlation with sequence characteristics of its gene family members, gene expression pattern and the rate of callus induction. It provides a theoretical basis for breaking through the regeneration and genetic transformation problem of Tartary buckwheat. 【Method】 The protein and nucleic acid sequence of the WOX gene family members in Tartary buckwheat were obtained by homology blast and the sequence of Arabidopsis WOX genes were served as reference. Based on protein homology and conserved domain analysis, all members of Tartary buckwheat WOX gene family were identified. The TBtools software was used to further demonstrate the characteristics of the WOX genes in Tartary buckwheat, including gene structure, conserved domain and cis-acting element. Genomic collinearity of WOX gene family members between Tartary buckwheat and Arabidopsis thaliana was analysed. Based on proximity method, the MEGA X software was used to perform phylogenetic tree of these WOX genes in Tartary buckwheat, Arabidopsis and rice. The hypocotyl explants of 70 Tartary buckwheat varieties were cultured with MS+2,4-D 3.0 mg·L -1+6-BA 1.0 mg·L-1 for callus induction and the callus emergence rate of different genotypes was evaluated. The FtWOX gene expression level was performed by qPCR to compare the different Tartary buckwheat varieties with high and low callus yield. The correlation between callus rate and FTWOXS gene family members was analysed based on Pearson correlation coefficient. 【Result】 A total of 30 WOX genes were identified in Tartary buckwheat and they were unevenly distributed on 8 chromosomes. The 30 Tartary buckwheat WOX genes could be divided into three groups by phylogenetic tree. The WOX genes contained different conserved domains in different groups, and the main conserved domains were HD(Homeodomain), START and MEKHLA. The conserved motif analysis showed that the conserved motif number of FtWOX genes may contain 2 to 10 motifs, and the gene structure analysis showed that the number of exons contained in the genes between 2 to 18. Promoter elements analysis showed 26 different kinds of cis-acting elements in the 30 WOX genes. The phylogenetic analysis showed that 30 Tartary buckwheat, 15 Arabidopsis thaliana and 12 rice WOX gene family members could be divided into three categories, of which the third group is unique to Tartary buckwheat. The collinearity analysis showed that six WOX genes were genomic collinearity between Tartary buckwheat and Arabidopsis thaliana. Expression pattern and correlation analysis show that the expression level of FtWOX1/FtWOX12/FtWOX22/FtWOX23/ FtWOX24 has positive correlation with the callus induction. 【Conclusion】 Collectively, these data suggest that the Tartary buckwheat FtWOX members showed abundant sequence variation characteristics. The expression level and callus rate of WOX gene in different Tartary buckwheat genotypes were significantly different and correlated to some extent, suggesting that different Tartary buckwheat WOX genes had potential functional diversity.

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    Identification and Gene Functional Analysis of Yellow Green Leaf Mutant ygl3 in Rice
    XU ZiYi,CHENG Xing,SHEN Qi,ZHAO YaNan,TANG JiaYu,LIU Xi
    Scientia Agricultura Sinica    2021, 54 (15): 3149-3157.   DOI: 10.3864/j.issn.0578-1752.2021.15.001
    Abstract643)   HTML101)    PDF (1886KB)(418)       Save

    【Objective】 To enrich and deepen people’s understanding of the molecular mechanism of plant leaf color, the phenotype identification and gene cloning of the yellow green leaf mutant ygl3 (yellow green leaf 3) were carried out to clarify the molecular function of YGL3 and lay the foundation for elucidating the molecular mechanism of YGL3 regulating rice leaf color.【Method】 Two stable genetic allelic yellow green leaf mutants, ygl3-1 and ygl3-2, were isolated from the CRISPR-Cas9 knockout mutant library of Zhonghua 11. The phenotype of the mutant was identified, and the chlorophyll contents of the wild-type and ygl3 were determined. The chloroplast structure of the wild-type and ygl3 was observed by transmission electron microscope. qRT-PCR was used to analyze the tissue expression of YGL3, and BioXM2.6 software was used for sequence alignment of YGL3 and its homologs. Yeast two hybrid was used to screen the interacting proteins of YGL3.【Result】 Compared with the wild type, the leaves of ygl3 were yellowing, and the contents of chlorophyll, carotenoid and total photosynthetic pigment at seedling stage in ygl3 were significantly decreased. Transmission electron microscopy showed that the chloroplast morphology of ygl3 was abnormal, and the thylakoid lamellar structure was less, whereas the chloroplast morphology of the wild type was normal and the thylakoid lamellar structure was orderly arranged. CRISPR-Cas9 knock-out site identification showed that the LOC_Os01g73450 gene had a single base insertion, which resulted in the early termination of protein translation. The gene encoding 351 amino acids was mutated into a truncated protein with 55 amino acids. Compared with the wild type, the expression level of LOC_Os01g73450 was significantly down-regulated in the mutants. qRT-PCR showed that YGL3 was expressed in roots, panicles, seeds, leaf sheaths and leaves. YGL3 was highly expressed in leaves. YGL3 encodes a plastid localized UMP kinase. The YGL3 protein was conserved in Zea mays, Sorghum bicolor and Arabidopsis thaliana. YGL3 shared the high sequence homology (59.4% amino acid identity) to Arabidopsis. qRT-PCR showed that chlorophyll synthesis genes, including HEMC, HEMC and URO-D, were significantly down-regulated in ygl3, whereas the expression levels of HEMB, HEMF and HEML were no significant difference between the wild type and ygl3. Yeast two hybrid screen showed that YGL3 interacted with RNA editing factor MORF8.【Conclusion】 The phenotype of the yellow leaf mutant ygl3 resulted from the LOC_Os01g73450 mutation. YGL3 was an allele of the yellow green leave gene YL2/YGL8. YGL3 was highly expressed in leaves, and YGL3 interacted with MORF8 in yeasts.

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    Characterization of Wheat Cultivar Zhongmai 895 with High Yield Potential, Broad Adaptability, and Good Quality
    ZHANG Yong,YAN Jun,XIAO YongGui,HAO YuanFeng,ZHANG Yan,XU KaiJie,CAO ShuangHe,TIAN YuBing,LI SiMin,YAN JunLiang,ZHANG ZhaoXing,CHEN XinMin,WANG DeSen,XIA XianChun,HE ZhongHu
    Scientia Agricultura Sinica    2021, 54 (15): 3158-3167.   DOI: 10.3864/j.issn.0578-1752.2021.15.002
    Abstract549)   HTML50)    PDF (507KB)(480)       Save

    Characterization of leading cultivars will provide crucially important information for cultivar development. The objective of this research is to characterize high yield potential, broad adaptation, good disease resistance and stress tolerance, as well as good quality in wheat cultivar Zhongmai 895. The dataset of two populations of Zhongmai 871/Zhongmai 895 recombinant inbred lines and Yangmai 16/Zhongmai 895 doubled haploids, as well as regional yield and pilot trial related with Zhongmai 895 were used in this research. The high yield potential of Zhongmai 895 was largely due to the increased spike number which could easily reach 640/m2, guaranteed by erect plant type with small leaves, and short plant height around 75 cm related to the outstanding lodging resistance, on the basis of two QTL for tiller angle, five QTL for leaf angle, and combination of semi dwarfing genes Rht2 and Rht24. It was characterized with high and stable thousand grain weight (48 g) related to the fast grain filling rate which provided excellent tolerance to high temperature during grain filling period, contributing to the perfect performance in late sowing environment, together with the contribution from high water and fertilizer use efficiency, high activity of root system, and slow leaf senescence, on the basis of four QTL for high thousand grain weight and grain filling rate, five genes for drought tolerance with vernalization gene combination of vrn-A1x, Vrn-B1a, and vrn-D1w. The performance of the broad adaptability was further contributed by the good resistance to stripe rust, powdery mildew and Fusarium head blight in the production, due to the presence of one major QTL for stripe rust resistance, two QTL for slow powdery mildew, and one new QTL for Fusarium head blight resistance. Zhongmai 895 was one of the few cultivars performing good dual qualities for Chinese dry white noodles and steamed bread with stable medium gluten strength, bright and yellow flour colour across environments in the Yellow-Huai River Valleys Winter Wheat Region, with Glu-A3d, and performed high nutrition quality with stable and high phenolic acid concentration of 748 μg·g-1. This research provides very important information and experience for developing new cultivars with high yield potential and broad adaptability.

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