关键词: 大豆, R6期, 氨基酸, 综合评价, 限制性两阶段多位点全基因组关联分析

Abstract:

【Objective】This study aimed to dissect the genetic diversity of amino acid contents in soybean seeds at the R6 stage, comprehensively evaluate their nutritional quality, screen elite germplasm resources, and map key genetic loci and candidate genes regulating amino acid accumulation, thereby providing a theoretical basis for molecular breeding in vegetable soybean.【Method】The contents of 17 amino acids in seeds of 133 soybean germplasms at the R6 stage were determined using an automatic amino acid analyzer. A comprehensive evaluation system for amino acid quality was established by integrating principal component analysis (PCA) with the membership function method. The test materials were systematically evaluated, and elite germplasms were screened via cluster analysis. Combined with high-density genotyping data, restricted two-stage multi-locus genome-wide association analysis (RTM-GWAS) was performed to associate the total amino acid content (TAA) and comprehensive evaluation value (F value), aiming to identify significant genetic loci and mine candidate genes. On the basis of the QTL-allele matrix, the breeding potential of hybrid combinations for TAA was predicted in the tested population, and the optimal hybrid combinations were screened out for subsequent breeding application.【Result】The 133 soybean accessions exhibited abundant genetic diversity for amino acid contents. Among the 17 detected amino acids, glutamic acid had the highest content, while methionine and cysteine had the lowest contents. PCA revealed that the cumulative contribution rate of the first three principal components was 95.92%, adequately representing most phenotypic variation of the 17 amino acid traits. Combined with the F value, cluster analysis classified the tested germplasms into three distinct groups, and nine germplasms with superior comprehensive performance were ultimately identified. A total of 55 loci significantly associated with amino acid contents were detected by RTM-GWAS. Among these, 8 loci overlapped with previously reported genomic regions, and 7 were stably detected across two or more environments. Chr.17_3312682 was a stable locus co-localized with both the F value and TAA, with 29 candidate genes annotated in its flanking genomic region. Notably, Glyma.17g044500 encodes a protein with aspartate esterase activity and is highly expressed in seeds, suggesting that it is a key candidate gene regulating amino acid accumulation in soybean seeds at the R6 stage. Based on the QTL-allele matrix, the TAA breeding potential was predicted for all possible cross combinations within the tested population, and the top 10 optimal combinations were identified, with their pure-line progenies projected to achieve the TAA content of 29.36%.【Conclusion】This study systematically elucidated the genetic diversity of amino acid contents in soybean seeds at the R6 stage, established a reliable comprehensive evaluation system for amino acid quality, classified 133 germplasms into three major groups, and identified 9 elite germplasm resources with superior amino acid quality. Furthermore, a stably associated locus (Chr.17_3312682) was identified via RTM-GWAS, and subsequent analysis identified Glyma.17g044500 as the key candidate gene regulating amino acid accumulation in soybean seeds at the R6 stage.

Key words: soybean (Glycine max (L.) Merr.), R6 stage, amino acids, comprehensive evaluation, restricted two-stage multi-locus genome-wide association analysis