Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (16): 2743-2757.doi: 10.3864/j.issn.0578-1752.2019.16.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 E-mail:zhangminron@126.com

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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

Table 1

Primer information of candidate genes"

基因ID
Gene ID		 基因名称
Gene name		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')
Glyma.18G176100 ABI3 CTCATCATGCAAATCCTACAGC CTATCTGATGCAACTCGACTCT
Glyma.13G123500 Gy5 CAGATATAGAGCACCCAGAGAC GGTGTTGAAGGATTGTGCTAAG
Glyma.18G175500 MAG2 CAAATTTGGGGCATGGCTAATA CTTGAAAATTGTTTCGGTTGCG
Glyma.18G226500 CRA1 CTGCAAGAAGTGGTCGTTTTTA ATTTCAGTTGTTCCATGTTCCG
Glyma.02g248300 PAT2 AAAGGCGTTGAAGCTGGATATA AATCTCCTATCTGGATCTCCGA
Glyma.11g112800 BBX21 CACAACAGGTTTCTTCTCACTG TGTTCTTTCTTCAATGGCAGTG
Glyma.14g062100 COI1 GTACATACACGACTCCAAGGAC TTTCCCTTCAGGTTTAACGACT
Glyma.05g062700 SAV1 GTGGGTCTCCAATGTTTTGTAC GCAAATTGTTTCAATGGGTGTG
Glyma.18g043000 KAN TTGGAGTTCACATTAGGGAGAC AAGTTTGTCACATGTTACCGTG
Glyma.03g227300 PHYA CAAAGTCTGATAGAGCAGCAAC CTCACTGTAAGCGATGACCTTA
ACT11 CGGTGGTTCTATCTTGGCATC GTCTTTCGCTTCAATAACCCTA

Fig. 1

Whole genome annotation results of Nandou 12 a: Whole genome annotation results of Nandou 12; b: Diagram of gene variations identified in four variations on Nandou 12 coding region; c: Classification of gene variations compared with COG database by blast; d: Classification of gene variations compared with KEGG database by blast"

Table 2

Top 10 genes that are highly polymorphic between Nandou 12 and Shiyuehuang by non-syn SNPs"

基因ID
Gene ID		 基因名称
Gene name		 注释
Annotation		 基因区域内的SNP数目
Genic SNP		 CDS中的SNP
SNP in CDS		 非同义替换SNP
Non-syn SNP
Glyma.18G176100 ABI3 ABI3-like转录因子家族成员,促进种子储藏蛋白的积累
A member of ABI3-like transcription factor family, promoting the accumulation of seed storage proteins		 72 31 20
Glyma.13G123500 Gy5 大豆球蛋白基因家族成员之一,编码一个11S球蛋白亚基 Encodes an 11S globulin subunit, a member of the soybean globulin gene family 46 33 18
Glyma.11G112800 BBX21 编码B-box锌指转录因子bbx21,与cop1基因相互作用调节避荫
Encodes a B-box zinc finger transcription factor BBX21, genetically interacts with COP1 to regulate shade avoidance.		 30 29 16
Glyma.18G175500 MAG2 参与种子贮藏蛋白从内质网到液泡的运输
Involved in transportation of seed storage proteins from the ER to the vacuole		 36 27 14
Glyma.13G335200 OBP3 编码一个主要在根中表达的包含转录因子的核定位Dof域 Encodes a nuclear localized Dof domain containing transcription factor expressed primarily in roots. 35 35 14
Glyma.02G057500 SAV1 编码一个油菜素类固醇生物合成途径的限速酶22α羟化酶 Encodes a 22α hydroxylase whose reaction is a rate-limiting step in brassinosteroid biosynthetic pathway 51 29 12
Glyma.08G171800 GA2OX4 编码作用于赤霉素C19的赤霉素2-氧化酶
Encodes a gibberellin 2-oxidase that acts on C19 gibberellins		 88 26 11
Glyma.20G048500 VPS26B 细胞内蛋白转运 Intracellular protein transport 32 25 11
Glyma.18G226500 CRA1 编码一个12S种子储存蛋白 Encodes a 12S seed storage protein 72 26 10
Glyma.14G062100 COI1 避荫 Shade avoidance 28 25 10

Fig. 2

Expression heatmap of mutant genes and validation of transcriptome sequencing data by qRT-PCR a: Expression heatmap of related to protein synthesis and accumulation gene during seed development; b: Expression pattern of shade regulation related genes in mature and young leaves under shading treatment; c, d: Validation of transcriptome sequencing data by qRT-PCR"

Fig. 3

Development and polymorphism analysis of CAPS and InDel markers a: Alignment of the partial sequence of containing the SNP which resulted in the creation of the restriction sites (BanⅡ) between Nandou 12 and Shiyuehuang in Gy1. Lines represent the resriction sites, while arrows indicate the position of digesting site; b: Sequence alignment results of BBX21 gene containing InDel in Nandou 12 and Shiyuehuang; c: Detection of CAPS labeled enzyme digestion targeting Gy1 gene; d: The results of InDel markers amplification for targeting BBX21 gene were analyzed by agarose gel electrophoresis. S: Shiyuehuang, N: Nandou 12, Figure exhibited partial verifying lines (code number: 1-10)"

Table 3

Phenotypic analysis of crude protein contents in the two parents and F2 population (%)"

性状
Trait		 亲本 Parent F2群体 F2 population
南豆12
Nandou 12		 十月黄
Shiyuehuang		 范围
Range		 均值
Mean		 方差
Variance		 标准差
SD		 变异系数
CV		 偏度
Skewness		 峰度
Kurtosis
蛋白质含量
Protein contents		 50.96 41.35 40.34—51.72 45.37 3.47 1.86 4.10 0.16 -0.09

Table 4

Putative QTL detected by composite interval mapping for crude protein contents"

数量性状座位
QTL		 染色体
Chromosome		 标记区间
Position (cM)		 置信区间
Confidence interval		 LOD 加性效应
Additive		 贡献率
R2 (%)
qSPC-1 Gm06(C2) 34.01 GmIn063—GmCA130 8.09 -0.23 5.39
qSPC-2 Gm07(M) 46.71 GmCA168—GmCA171 5.26 0.22 5.36
qSPC-3 Gm15(E) 26.51 GmIn149—GmIn150 4.53 0.21 4.68
qSPC-4 Gm15(E) 72.21 GmIn152—GmIn163 9.93 -0.29 9.32
qSPC-5 Gm20(I) 35.61 GmCA493—GmIn285 8.40 -0.47 12.04
qSPC-6 Gm20(I) 103.41 GmdCA522—GmCA537 15.06 -0.68 18.86

Fig. 4

Putative QTL locations of seed protein content on the genetic map Seed protein content"

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