Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (13): 2789-2801.doi: 10.3864/j.issn.0578-1752.2026.13.003

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

Whole Genome Association Study of Protein and Threonine Content in Mung Bean Seeds and Development of KASP Molecular Marker

CHEN ShengQi1(), LIU JinYang2(), HUANG JiaLi3, LIU XiaoCen3, ZHAO ShuoQian4, LIN Yun2, XUE ChenChen2, YAN Qiang2, CHEN JingBin2, WU RanRan2, CHEN Xin2,3(), CHENG JinPing1(), YUAN XingXing2,3()   

  1. 1 College of Agriculture, Nanjing Agricultural University, Nanjing 210095
    2 Institute of Economic Crops, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Genetic Improvement of Efficient Horticultural Crops, Nanjing 210014
    3 College of Life Sciences, Nanjing Agricultural University, Nanjing 210095
    4 College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
  • Received:2025-11-19 Accepted:2026-01-13 Online:2026-07-01 Published:2026-07-01
  • Contact: CHEN Xin, CHENG JinPing, YUAN XingXing

Abstract:

【Objective】Protein and threonine are the core nutrients of mung bean seeds, which have a critical impact on the nutritional quality of mung beans. Among them, mung bean protein is a high-quality protein source for vegetarians due to its easy digestion, absorption, and rich biological activity, which is of great value to human health. This study aims to explore genetic loci and candidate genes significantly associated with the protein content and relative content of threonine in mung bean seeds, providing a theoretical basis for genetic improvement of protein and threonine content in mung bean seeds. 【Method】This study used Kjeldahl nitrogen determination method and liquid chromatography-mass spectrometry to determine the protein content and relative content of threonine in 279 mung bean varieties planted in 2022. The general linear model of Tassel5 software was used for genome-wide association study to explore genetic loci related to protein content and relative content of threonine in mung bean seeds. By combining linkage disequilibrium analysis, transcriptome comparative analysis, and comparative genomics, key candidate genes related to nitrogen metabolism were identified. 【Result】Six key loci located on chromosome 3 were identified, which are related to the protein content and relative content of threonine in mung bean seeds, and can explain 6.06% to 12.20% of phenotypic variation. These 6 loci are closely linked and can be mainly divided into two haplotypes, with Hap2 being the dominant haplotype. Then, KASP molecular marker was designed for the key candidate locus SLG03_966208 (A/G), which can successfully classify 80 mung bean varieties into three genotypes: AA, GG, and AG, with a detection accuracy rate of 87.5%. The key candidate gene EVM0000757 was identified within the locus linkage region, and the homologous gene AT5G65750 in Arabidopsis was significantly associated with nitrogen metabolism. Its gene expression was significantly different between high and low protein varieties (P=1.84E-03, |log2FC|=1.39), making it a reliable candidate gene.【Conclusion】279 mung bean varieties were identified for their grain protein content and relative content of threonine. Six SNP loci were detected within the 939 296-1 039 749 bp region of chromosome 3. A KASP molecular marker for the SLG03_966208 (A/G) locus was developed, which can distinguish the high and low levels of protein and threonine content in mung bean grains. A key candidate gene related to nitrogen metabolism, EVM0000757, was identified.

Key words: mung bean, grain protein content, relative content of grain threonine content, genome-wide association study, KASP marker

Table 1

Statistical analysis of protein content and relative content of threonine in mung bean seeds"

性状
Trait
最大值
Maximum
最小值
Minimum
平均数±标准差
Mean±SD
变异系数
CV (%)
偏度系数
Skewness
峰度系数
Kurtosis
遗传力
H2 (%)
蛋白质Protein (%) 24.38 18.45 21.27±1.01 4.75 0.06 0.09 76.38
苏氨酸Threonine (PA) 3601595.23 834158.43 1509441.75±410080.96 27.17 1.14 2.60 -
PA:峰面积 PA: Peak area

Fig. 1

Frequency distribution histogram and correlation analysis of protein content and relative content of threonine in mung bean seeds *** represented significant at P<0.001 levels"

Fig. 2

Circular clustering heatmap of protein content in 279 mung bean seeds"

Fig. 3

Manhattan map of genome-wide association analysis of protein content and relative content of threonine in mung bean grains A: Manhattan plot of genome-wide association study for grain protein content; B: Manhattan plot of genome-wide association study for relative content of threonine in grains; C: Q-Q plot of genome-wide association study for grain protein content; D: Q-Q plot of genome-wide association study for relative content of threonine in grains"

Table 2

SNP loci significantly associated with protein and threonine identified through GWAS"

序号Number 位点SNP 染色体Chromosome 位置Position (bp) -log10_(P) 表型贡献率R2 (%)
1 SLG03_939296 3 939296 5.64 6.16
2 SLG03_942729 3 942729 6.29 6.79
3 SLG03_942730 3 942730 6.29 6.79
4 SLG03_1038622 3 1038622 5.16 6.06
5 SLG03_966208 3 966208 7.01 11.21
6 SLG03_1039749 3 1039749 7.70 12.20

Fig. 4

Haplotype analysis of loci significantly correlated with protein content and relative content of threonine in mung bean seeds A: Haplotype analysis of six associated loci; B: Linkage disequilibrium analysis among six associated loci; C: Boxplots of grain protein content for different haplotypes of the six loci; D: Boxplots of relative threonine content for different haplotypes of the six loci. * and ** indicate significant differences at the P<0.05 and P<0.01 levels, respectively. The same as below"

Fig. 5

KASP molecular marker typing results of 80 mung bean varieties at the SLG03_966208 site A: KASP marker genotyping diagram of the SLG03_966208 locus; B: Box plot of grain protein content in mung bean varieties carrying different alleles at the SLG03_966208 locus; C: Box plot of grain threonine relative content in mung bean varieties carrying different alleles at the SLG03_966208 locus"

Fig. 6

Linkage disequilibrium analysis and candidate gene expression differential analysis of all loci within ±100 kb of the SLG03_966208 locus A: Local Manhattan plot of protein content in 279 mung bean seeds; B: Linkage disequilibrium analysis of SNP loci; C: Heatmap of expression differences of candidate genes within the linkage region between high and low protein mung bean varieties"

Table 3

Analysis of significant QTL associated candidate genes"

染色体
Chromosome
位置
Position (bp)
基因
Gene
拟南芥同源基因
Homologous gene of Arabidopsis
功能注释
Functional annotation
参考文献
References
LG03 880773-881672 EVM0013103 AT3G09110 非特征蛋白LOC106770734 Uncharacterized protein LOC106770734
LG03 899614-909356 EVM0002414 AT3G55320 ABC转运蛋白B家庭成员20 ABC transporter B family member 20
LG03 910014-911736 EVM0023300 AT2G39470 叶绿体腔定位光合NDH亚基1
Photosynthetic NDH subunit of lumenal location 1, chloroplastic
LG03 914126-915697 EVM0026632 AT2G39490 F-box蛋白At2g39490 F-box protein At2g39490
LG03 916012-918173 EVM0019126 AT3G55340 核仁素2 Nucleolin 2
LG03 921375-922998 EVM0031542 AT5G59790 类FLC上游蛋白 Protein upstream of FLC-like
LG03 925340-928379 EVM0016950 AT3G55350 类ALP蛋白 Protein ALP1-like
LG03 929960-930569 EVM0003649 AT2G39500 非特征蛋白LOC108325383,部分
Uncharacterized protein LOC108325383, partial
LG03 937645-938435 EVM0002963 AT2G37430 类锌指蛋白ZAT11 Zinc finger protein ZAT11-like
LG03 943232-949840 EVM0000757 AT5G65750 2-酮戊二酸脱氢酶E1组分 2-Oxoglutarate dehydrogenase E1 component [27]
LG03 950896-951478 EVM0004529 - 非特征蛋白类At4g04775 Uncharacterized protein At4g04775-like
LG03 952343-952875 EVM0028246 AT3G05850 非特征蛋白LOC106770420 Uncharacterized protein LOC106770420
LG03 959895-962941 EVM0006824 AT5G59840 ras相关蛋白RABE1c ras-related protein RABE1c
LG03 964662-966659 EVM0003873 AT4G35550 WUSCHEL相关同源盒13 WUSCHEL-related homeobox 13
LG03 970064-975466 EVM0008260 AT5G13680 延伸复合蛋白1亚型X1 Elongator complex protein 1 isoform X1
LG03 977401-984719 EVM0013687 AT3G55400 甲硫氨酸-tRNA连接酶,叶绿体/线粒体
Methionine--tRNA ligase, chloroplastic/mitochondrial
LG03 985197-986871 EVM0005089 - 假设蛋白VIGAN_08013300,部分
Hypothetical protein VIGAN_08013300, partial
LG03 988811-990132 EVM0031142 AT3G55390 类CASP蛋白4C2 CASP-like protein 4C2
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