中国农业科学 ›› 2019, Vol. 52 ›› Issue (16): 2743-2757.doi: 10.3864/j.issn.0578-1752.2019.16.001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于全基因组重测序的大豆分子标记开发及籽粒蛋白质含量QTL定位

王嘉,曾召琼,梁建秋,于晓波,吴海英,张明荣()   

  1. 四川省南充市农业科学院大豆研究所/国家大豆产业技术体系南充综合试验站,四川南充 637000
  • 收稿日期:2019-03-25 接受日期:2019-05-07 出版日期:2019-08-16 发布日期:2019-08-21
  • 通讯作者: 张明荣
  • 作者简介:王嘉,E-mail:wangjia0724@126.com
  • 基金资助:
    国家重点研发计划(2017YFD0101500)

Development New Molecular Markers for Quantitative Trait Locus (QTL) Analysis of the Seed Protein Content Based on Whole Genome Re-Sequencing in Soybean

WANG Jia,ZENG ZhaoQiong,LIANG JianQiu,YU XiaoBo,WU HaiYing,ZHANG MingRong()   

  1. Soybean Research Institute, Nanchong Academy of Agricultural Sciences/Nanchong comprehensive experimental station of National Soybean Industry Technology System, Nanchong 637000, Sichuan
  • Received:2019-03-25 Accepted:2019-05-07 Online:2019-08-16 Published:2019-08-21
  • Contact: MingRong ZHANG

摘要:

【目的】基于全基因组重测序结果,开发与高蛋白、耐荫、抗倒伏等性状紧密相关的分子标记,同时利用开发的分子标记构建遗传连锁图谱,并对籽粒蛋白质含量进行QTL定位,为后续高蛋白、耐荫、抗倒育种研究提供参考和分子标记资源。【方法】以大面积栽培品种南豆12和地方品种十月黄为亲本,构建F2分离群体。对亲本材料进行覆盖度约为40×的全基因组重测序,用BWA、GATK及Breakdancer等软件比对,检测亲本材料在全基因组范围内的突变类型,挖掘相关变异基因。结合种子不同发育时期和荫蔽处理获得的转录组数据,结合qRT-PCR对发生突变的储藏蛋白、环境适应相关基因进行表达规律分析。同时,基于重测序数据,挖掘亲本间存在于基因编码区的SNP位点,对其进行酶切位点分析,将SNP标记转化为CAPS或dCAPS标记。此外,搜索亲本间存在的插入/缺失变异位点,在插入/缺失位点两侧高度保守的区域设计引物开发InDel标记。对开发的CAPS标记和InDel标记进行多态性筛选,选取具有多态性的CAPS分子标记和InDel标记,对F2材料进行基因分型。根据分型结果,利用JoinMap 4.0软件进行遗传连锁图谱的构建。依据构建的遗传图谱,结合近红外分析获得F2材料的籽粒蛋白质含量数据,使用Windows QTL Cartographer V2.5软件对大豆籽粒蛋白质含量进行QTL分析。【结果】测序结果显示,南豆12大量储藏蛋白、环境适应相关的重要基因或同源基因发生突变。转录组数据分析结果显示部分变异基因呈现不同的表达模式且差异显著,qRT-PCR分析进一步验证了该结果。此外,经检测开发的540个CAPS分子标记中有332个具有酶切多态性,300对InDel引物中有201对引物能扩增出多态性。基于533个多态性分子标记构建了一张包含20个连锁群的遗传连锁图谱,覆盖长度2 973.87 cM,标记间平均遗传距离5.58 cM。利用此图谱对大豆籽粒蛋白质含量进行QTL定位,共检测到QTL位点6个,可解释4.68%—18.25%的表型变异。【结论】基于亲本间的变异位点,共开发了533个多态性分子标记(包含8个基因特异性分子标记),检测到6个大豆籽粒蛋白质含量QTL位点,其中,主效QTL位点1个(qSPC-6)。

关键词: 大豆, 全基因组重测序, 套作, 高蛋白, 耐荫, 抗倒伏, 分子标记

Abstract:

【Objective】 Based on the results of genome-wide re-sequencing, molecular markers closely related to high protein, shade tolerance, lodging resistance and other traits were developed. At the same time, At the same time, genetic linkage maps were constructed using the developed molecular markers, and seed protein content was mapped by QTL, providing reference and molecular marker resources for subsequent research on high protein, shade tolerance and lodging resistance breeding. 【Method】 A F2 segregating population derived from the cross of Nandou 12 and Shiyuehuang consists of 672 individuals, and two parents were re-sequenced. With the published genome as a reference, the obtained data were assembled with BWA, and explored for the SNP and InDel by GATK and SV by Breakdancer. Carry out expression pattern analysis toward mutational storage proteins and genes related to environmental adaptation by combining with the transcriptome data obtained from different development stages and shade processing of seeds and qRT-PCR, At the same time, based on the resequencing data, excavate the SNP sites in the gene coding region between the parents, analyze the restriction enzyme cutting site and transform the SNP markers into CAPS or dCAPS markers. In addition, search the insertion/deletion mutation site and design primer development InDel marker in highly conserved regions on both sides of the insertion/deletion site. Perform polymorphism screening on the CAPS markers and InDel markers developed, select the CAPS molecular markers and InDel markers with polymorphism and carry out genotyping toward F2 materials. Utilize JoinMap 4.0 software to construct the genetic linkage map according to the genotyping result. Obtain the seed protein content data of F2 material according to the genetic map constructed by combining with the near-infrared analysis and use Windows QTL Cartographer V2.5 to carry out QTL analysis toward soybean seed protein content. 【Result】 The results showed that a large number of storage proteins and important genes or homologous genes related to environmental adaptation mutated in Nandou 12. The results of transcriptome data analysis showed that some variant genes showed different expression patterns and significant differences and the results were further validated by qRT-PCR analysis. In addition, 332 of the 540 CAPS molecular markers had polymorphic, and 201 of 300 pairs of InDel primers could amplify polymorphism. A genetic linkage map containing 20 linkage groups was constructed based on polymorphic molecular markers, covering 2973.87 cM with an average genetic distance of 5.58 cM. Using this map to map the seed protein content of soybean, six QTL loci were detected, which could explain 4.68%-18.25% phenotypic variation.【Conclusion】 Based on the variation loci among parents, 533 polymorphic molecular markers (including 8 gene-specific molecular markers) were developed. Six QTL loci were detected for seed protein content in soybean, including one major QTL locus (qSPC-6).

Key words: soybean, whole genome re-sequencing, relay intercropping, high protein, shade tolerant, lodging resistance, molecular marker