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

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.

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.

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.

279 mung bean varieties were identified for their grain protein content and relative content of threonine. Six SNP loci were detected within the 939296-1039749 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.