大豆种质抗旱性评价及抗旱候选基因的挖掘

Evaluation of Drought Resistance in Soybean Germplasm and Identification of Candidate Drought-Resistant Genes

【目的】综合运用不同抗旱评价指标,筛选大豆优异抗旱种质,发掘重要抗旱基因,为大豆抗旱分子育种提供理论基础。【方法】利用188份大豆种质资源,于2018、2019、2020和2021年测定正常供水和干旱胁迫下的单株荚数、单株生物量和单株产量,利用抗旱指数、改进抗旱指数、加权抗旱系数和加权抗旱指数等参数鉴定大豆种质的抗旱性,通过全基因组关联分析(genome-wide association study,GWAS)检测与大豆抗旱参数显著相关的SNP(single nucleotide polymorphisms)标记,并结合干旱胁迫下大豆幼苗叶片基因表达谱分析,筛选抗旱候选基因。【结果】188份大豆种质资源的抗旱指数、改进抗旱指数、加权抗旱系数和加权抗旱指数存在广泛变异,分别利用逐级分类法将供试材料划分为5个等级。其中,辽豆15、辽豆69、辽豆14、金杖子黄豆、中黄606、科新3号、Koreane 4等7份种质在不同评价方法中均被鉴定为一级抗旱。对抗旱指数、改进抗旱指数、加权抗旱系数和加权抗旱指数进行GWAS分析,共定位到15个显著关联的SNP位点能够在多环境下稳定表达,这些位点对表型变异的贡献率为12.46%—25.60%。在显著SNP位点上、下游各200 kb区间内共有注释基因226个。通过对抗旱品种辽豆14和干旱敏感型品种辽豆21在干旱胁迫下进行转录组测序和实时荧光定量PCR分析,鉴定出32个注释基因受干旱胁迫诱导显著差异表达。其中,Glyma.02G182900、Glyma.04G012400、Glyma.06G258900、Glyma.15G100900、Glyma.01G172600、Glyma.04G012300、Glyma.01G172200和Glyma.04G010300等8个候选基因分别编码钙依赖性蛋白激酶、通用应激蛋白A、G型凝集素S受体样丝氨酸/苏氨酸蛋白激酶、蛋白磷酸酶2C、异黄酮还原酶、异黄酮还原酶同源物、生长素蛋白和bZIP转录因子。【结论】综合运用不同抗旱参数,在188份大豆种质资源中筛选出7份抗旱种质资源,鉴定了15个与抗旱参数显著关联的SNP位点,并鉴定出抗旱候选基因8个。

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