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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (2): 310-320.doi: 10.3724/SP.J.1006.2023.24015

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

Fine mapping of qPRO-20-1 related to high protein content in soybean

YANG Shuo1,3(), WU Yang-Chun4, LIU Xin-Lei2, TANG Xiao-Fei2, XUE Yong-Guo2, CAO Dan2, WANG Wan3, LIU Ting-Xuan3, QI Hang3, LUAN Xiao-Yan2,*, QIU Li-Juan1,3,*()   

  1. 1College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang, China
    2Heilongjiang Academy of Agricultural Sciences, Harbin 150030, Heilongjiang, China
    3National Key Facility for Gene Resources and Genetic Improvement / Key Laboratory of Crop Germplasm Utilization, Ministry of Agriculture and Rural Affairs / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    4College of Life Sciences, Jilin Agricultural University, Changchun 130000, Jinlin, China
  • Received:2022-01-10 Accepted:2022-06-07 Online:2022-07-08 Published:2022-07-08
  • Contact: LUAN Xiao-Yan,QIU Li-Juan E-mail:857813782@qq.com;qiulijuan@caas.cn
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(31960408);Central Public-interest Scientific Institution Basal Research Fund(S2022ZD02);Agricultural Science and Technology Innovation Program

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Abstract:

Protein content is a crucial quality trait of soybean, which is controlled by multiple genes. It is of great significance to locate soybean protein content-related loci and mine candidate genes for directional breeding of soybean varieties with high protein content. In this study, an F2 population consisting of 265 individual plants was constructed by crossing the excellent variety Heinong 88 as the female parent with the high-protein germplasm P73-6B as the male parent. The genotypes of F2 population were identified by using high-density SNP chip of “ZDX1” and the physical map was constructed. Combined with the protein content phenotypic data, the initial mapping interval of a 2.46 Mb QTL was located on chromosome 20 using the IciMapping 4.2 software. Using 11 polymorphic SSR markers screened out, the mapping range was narrowed from 2.46 Mb to 100.8 kb. Adding four SNP markers (Gm20_28349696, Gm20_30805913, Gm20_31341532, and Gm20_31483719), the interval was further reduced to 95.8 kb. The relative expression levels of the four genes contained in the interval in nine different tissues in both databases Phytozome v13.1 and PPRD RNA-seq yielded two candidate genes (Glyma.20g081800 and Glyma.20g082000). These results provide a theoretical basis for soybean protein content gene cloning and protein regulation mechanism research, as well as elite material and molecular marker for breeding high protein soybean.

Key words: soybean, protein content, QTL mapping, candidate genes

Table 1

SSR markers for fine mapping"

引物名称
Primer name		 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (5°-3°)
BARCSOYSSR_20_0608 GTGTTCCACTCCACGTTTCC CATTTCCCCTTTCACAATCG
BARCSOYSSR_20_0613 AACCGAGTTTGGTTCGATTC TGCTGCTTGATGATGAGGAC
BARCSOYSSR_20_0616 CCATCTTATGGACTTGTTTGGA GCCAAGAATGACCATTATGC
BARCSOYSSR_20_0618 TCACTAATCACAACAACCCAAA CGACCGGTGTGTTTAAGGTC
BARCSOYSSR_20_0619 TCAGTCGCAGATTGATCAGG CCCAATTGTATCCATCAACG
BARCSOYSSR_20_0629 AACCTAGCATTGCAACCTGC TCATCACCCCTTATCCGTTC
BARCSOYSSR_20_0636 AAAACGAGGCCTTAATCGAAA AAACCAAAGAATACCGTGAAAAA
BARCSOYSSR_20_0638 CGAAATGCCACCTTTTCAAT AGCAAACTAAGGTCGTTTTCG
BARCSOYSSR_20_0644 GCAGTTGTGCGTGGGAGAGAG GCGACATAGCTAATTAAGTAAGTT
BARCSOYSSR_20_0647 GCGTGGTGCACGATCATATAGA GCGTCTCCTTCGCTATCTCAAAC
BARCSOYSSR_20_0649 CCAGGAATGCAGGTTTCTCT CGTGACTCTTCTTCCTTTCCA

Fig. 1

Histogram of frequency distribution of protein content in parents and F2 population Mean value and standard deviation of parental phenotypes are indicated by vertical dotted lines. Curve represents density plot. ***, P < 0.001."

Table 2

Statistical analysis of protein content of parents and F2 population"

群体
Population		 母本
Female parent
(mean±SD, %)		 父本
Male parent
(mean±SD, %)		 F2分离群体F2 segregation population
变异范围
Range		 平均数±标准差
(mean±SD, %)		 变异系数
CV (%)		 峰度
Kurtosis		 偏度
Skewness
黑农88×P73-6B
Heinong 88×P73-6B		 43.67±0.67 49.64±2.50 39.22-53.25 47.01±3.98 8.48 2.94 0.26

Fig. 2

A protein QTL mapped on chromosome 20 of soybean by F2 population consisting of Heinong 88 and P73-6B"

Table 3

Protein QTLs identified in F2 population of Heinong 88 × P73-6B"

群体
Population		 染色体
Chr.		 标记区间
Marker interval		 LOD得分
LOD score		 表型贡献率
R2		 加性效应
Additive
effect		 显性效应
Dominant
effect
F2 20 Gm20_28349696-Gm20_30805913 12.23 19.31 -1.79 0.19

Fig. 3

Fine mapping of protein content QTL qPRO-20-1 A: construct a genetic map to locate the QTL site qPRO-20-1 between SNP 2839696-SNP 30805913 using Heinong 88×P73-6B F2 200K chip sequencing data. B: the genotypes of five exchange monocultures (297, 272, 68, 72, and 209) were identified using a total of 11 markers BARCSOYSSR_20_0608, BARCSOYSSR_20_0613, BARCSOYSSR_20_0616, BARCSOYSSR_20_0618, BARCSOYSSR_20_0619, BARCSOYSSR_20_0629, BARCSOYSSR_20_0636, BARCSOYSSR_20_0638, BARCSOYSSR_20_0644, BARCSOYSSR_20_0647, BARCSOYSSR_20_0649; the genotypes were verified and primed. C: the interval was finely localized to the SNP marker 30805913-BARCSOYSSR_20_0636, BARCSOYSSR_20_0649 using the chip data bits SNP locus markers 28349696, 30805913, 31343532, 31483719, and the three SSR markers (BARCSOYSSR_20_0636, BARCSOYSSR_20_0647, BARCSOYSSR_20_0649). D: four candidate genes of Glyma.20g081800, Glyma.20g081900, Glyma.20g082000, and Glyma.20g082100 were obtained."

Fig. 4

Mapping QTL on chromosome 20 for soybean seed protein content in F2 population from the cross of Heinong 88 and P73-6B using seven molecular markers"

Table 4

QTL verified in the population of Heinong 88 × P73-6B F2"

群体
Population		 染色体
Chr.		 标记区间
Marker interval		 LOD得分
LOD score		 表型贡献率
R2		 加性效应
Additive effect		 显性效应
Dominant effect
F2 20 Gm20_30805913-BARCSOYSSR_20_0649 12.75 21.44 -2.03 0.56

Fig. 5

Correlation analysis of genotype and protein phenotype of markers Gm20_30805913 and BARCSOYSSR_20_0649 on both sides of qPRO-20-1 locus in F2 population A: genotype and phenotype correlation of Gm20_30805913; B: genotype and phenotype correlation of BARCSOYSSR_20_0649. The abscissa is the genotype, the high protein is denoted as b, the low protein is denoted as a, and the vertical ordinate is protein content. ***: P < 0.001."

Table 5

Gene annotation of genes in the location interval"

基因
Gene name		 基因注释
Gene annotation
Glyma.20g081800 具有RNA结合(RRM-RBD-RNP基序)域的核运输因子2 (NTF2)家族蛋白
Nuclear transport factor 2 (NTF2) family protein with RNA binding (RRM-RBD-RNP motifs) domain
Glyma.20g081900 PTHR32246:SF16与钙相关的脂质结合蛋白
PTHR32246:SF16-calcium-dependentlipid-binding domain-containing protein-related
Glyma.20g082000 未知
Unknown
Glyma.20g082100 PantherFam黄嘌呤-尿嘧啶/维生素C渗透酶家族成员
PantherFam Xanthine-uracil/Vitamin C permease family member

Fig. 6

Relative expression profiles of four genes by Phytozome v13.1"

Fig. 7

Relative expression of four genes in nine different tissues of soybean obtained with the PRRD database"

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