The “stay-green” trait can prolong the effective photosynthesis duration in soybeans and increase dry matter accumulation, thereby holding significant potential for improving yield. Mining stay-green related QTL and elucidating their molecular mechanisms can provide a theoretical basis and technical support for enhancing soybean yield.

A soybean nested association mapping population was evaluated for stay-green traits across multiple environments. Genome-wide association study was conducted using genotyping data. Candidate genes were screened via SNP variation, tissue-specific expression, and functional annotation analyses, haplotype, promoter cis-acting elements, and protein structure prediction analyses were performed to characterize the candidate genes. Additionally, the application effect of genomic selection for the stay-green trait was evaluated.

Six significant QTL intervals were co-localized on chromosomes 3, 4, 5, and 16. Among these, qSG5-1 (Chr.5: 41600128..42273303, 613.18 kb) was repeatedly mapped across multiple environments and represents a novel QTL for stay-green regulation in soybean. Linkage disequilibrium analysis allocated two significantly associated regions within qSG5-1: qSG5-1.1 (Chr.5: 41798499..41996276, 197.78 kb) and qSG5-1.2 (Chr.5: 41996989..42273303, 276.32 kb), containing 29 and 37 genes, respectively. SNP variation analysis identified 53 genes containing variants that cause nonsynonymous mutations, alternative splicing, stopgain, or stoploss. Of these, eight genes were transcriptionally active in stems and leaves. Functional annotation suggested that Glyma.05G245200 and Glyma.05G247900 were involved in protein folding and oxidative metabolism, respectively, which highlights they might regulate cell cycle, growth metabolism, and nutrient remobilization during senescence. Besides, two major haplotypes of these genes exhibited highly significant phenotypic differences as Glyma.05G245200 harbored nonsynonymous mutations which changed C617T into A206V and C44T into P15L, and caused subtle alterations in its protein structure. Likewise, Glyma.05G247900 also contained a nonsynonymous mutation which changed A275G into D92G that did not alter its protein conformation. Analysis of cis-acting elements revealed that the presence of light and abscisic acid (ABA)-responsive elements in their promoters hints they might regulate soybean growth, senescence, and the stay-green trait by participating in light and hormonal signaling. These genes may serve as candidate genes for soybean stay-green and the prediction accuracy of genome-wide selection for stay-green across different marker sets ranged from 0.27 to 0.36.

This study identified a novel QTL, qSG5-1, and two candidate genes, Glyma.05G245200 and Glyma.05G247900, associated with the stay-green trait in soybean.