大豆荫蔽胁迫响应基因GmYUC2的克隆、自然变异分析及KASP分子标记开发

Clone, natural variation analysis and development of KASP molecular markers for GmYUC2 geneto respond shade stress in soybean

关键词:  大豆, GmYUC2基因, 耐荫性, 自然变异, KASP分子标记

Key words:  soybean, GmYUC2 gene, shade-tolerant, natural variation, KASP molecular marker

摘要:

YUC2是生长素生物合成中的限速酶之一,参与植物对荫蔽胁迫的响应。该研究以大豆极耐荫品种‘贡豆7号'为材料进行GmYUC2基因克隆,基于394份中国南方大豆自然变异群体的重测序数据及3个环境下的耐荫指数(STI)表型鉴定数据,对大豆荫蔽胁迫响应基因GmYUC2序列进行自然变异分析及开发KASP分子标记。结果表明:(1)从‘贡豆7号'中克隆到GmYUC2基因,其CDS序列长1 148 bp,编码382个氨基酸,编码蛋白均含有FMO-like、Pyr-redox-2和Pyr-redox-3结构域。(2)GmYUC2基因存在4个SNP变异位点,其中3个SNP变异位于内含子中,1个SNP变异位于5_prime_UTR区,SNP2与SNP3之间连锁程度最高(r²=0.988 8),其次为SNP1与SNP4(r²=0.921 934),SNP1与SNP2之间连锁程度最低(r²=0.475 691)。(3)基于这4个SNP变异位点在394份群体中鉴定出4种单倍型,其中Hap1单倍型与参考基因组Wm82一致,Hap2、Hap3和Hap4均由Hap1经过一步突变而来,Hap2所对应种质的STI极显著小于Hap1,说明携有Hap2单位型大豆种质耐荫性比携有Hap1的强。(4)在荫蔽条件下,极耐荫种质和不耐荫种质中的GmYUC2基因表达量均上调,并且强耐荫种质中该基因的表达量显著高于极不耐荫种质。(5)利用GmYUC2基因的3个SNP变异位点开发的KASP分子标记对18份大豆材料进行验证,基因型和表型的鉴定结果具有88.89%的高度一致性。该研究成功克隆了GmYUC2基因,并发现其在大豆响应荫蔽胁迫过程中起重要作用,开发出的KASP分子标记可用于大豆资源苗期耐荫性鉴定。该研究为进一步深入解析大豆GmYUC2基因的主要功能和表达调控机制提供参考,为高效准确地进行大豆耐荫性的分子标记辅助育种提供了理论基础。

Abstract:

YUC2 was one of the rate-limiting enzymes in auxin biosynthesis, and it involved in plant response to shade stress. In this study, Gongdou 7, an extremely shade-tolerant soybean variety was used for clone of GmYUC2 gene. Based on the resequencing data and the phenotypic identification data of shade tolerance index(STI)in three environments of 394 natural variation populations of soybean in southern China, this research analyzed the natural variation of GmYUC2 gene to respond shade stress in soybean, and developed KASP molecular markers. The results were as follows:(1)The length of CDS sequence for GmYUC2 gene cloned from Gongdou 7 was 1 148 bp, it encoded 382 amino acids, the encoded proteins all contained domains including FMO-like, Pyr-redox-2 and Pyr-redox-3.(2)There existed four SNP variation sites in gene GmYUC2, three SNP variants were located in the intron and one SNP variant was located in the 5_prime_UTR region, SNP2 and SNP3 had the highest linkage degree(r²=0.988 8), followed by SNP1 and SNP4(r²=0.921 934), SNP1 and SNP2 had the lowest linkage degree(r²=0.475 691).(3)Four haplotypes were identified in 394 populations based on these four SNP sites, the Hap1 haplotype was consistent with the reference genome Wm82, and Hap2, Hap3, and Hap4 were all directly mutated from Hap1, the STI of soybean germplasm corresponding to Hap2 was significantly lower than that of Hap1, indicating that the soybean germplasm carrying Hap2 haplotype had better shade-tolerance than that carrying Hap1.(4)Under shady conditions, the expression of gene GmYUC2 was up-regulated in both extremely shade-tolerant germplasm and negative shade-tolerant germplasm, and the expression of gene GmYUC2 was significantly higher in extremely shade-tolerant germplasm than that in negative shade-tolerant germplasm.(5)The KASP molecular marker developed by three SNP mutation sites of gene GmYUC2 was used to verify 18 soybean materials, the identification results of genotype and phenotype were highly consistent with 88.89%. The study cloned GmYUC2 gene successfully, and found GmYUC2 gene played an important role in soybean response to shade stress, the KASP molecular marker developed could be used to identify shade-tolerance of soybean resources at seedling stage. This study provides a reference for further analysis of the main function and expression regulation mechanism of gene GmYUC2, and provides a theoretical basis for molecular marker-assisted breeding of shade tolerance in soybean.