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.

To identify the key genes controlling anthocyanin synthesis and accumulation, to uncover changes in anthocyanin content of the seed coat during seed development, and the primary anthocyanin components responsible for the red seed coat of Taixingaijiaohong (TXAJH); and to lay the groundwork for a thorough understanding of the regulatory mechanism of red seed coat formation.

Using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS), the anthocyanin composition and concentration of the yellow seed coat of soybean Suinong 14 (SN14) and the red seed coat of soybean TXAJH at various developmental stages were identified. The potential areas of red testa-related genes were first identified using bulked segregant analysis (BSA) on the recombinant inbred lines (RILs) made by crossing SN14 and TXAJH. Based on this discovery, we performed marker linkage analysis to restrict the candidate intervals and predict the candidate genes, and qRT-PCR to confirm the expression of the anticipated candidate genes.

When seed coats from the four developmental phases of SN14 and TXAJH were analyzed, a total of 12 anthocyanins were discovered. Cluster analysis of total anthocyanins revealed substantial changes in the seed coat's anthocyanin composition between TXAJH and SN14 as well as between TXAJH before and after color development. The anthocyanin content of the SN14 seed coat gradually decreased as the seed developed, whereas the TXAJH seed coat's content increased quickly and remained stable. After the development of the seed coat's color, the anthocyanin contents of SN14 and TXAJH showed highly significant differences, and at the mature stage, the TXAJH seed coat's anthocyanin content was more than 200 times that of SN14. The crimson coloring of the TXAJH seed coat was largely due to cyanidin-3-O-glucoside (Cy-3-glu), peonidin-3-O-glucoside (Pn-3-glu), and petunidin-3-O-glucoside (Pt-3-glu). The candidate interval for the red seed coat gene on chromosome 8 was discovered at 8.66 Mb by BSA-seq association analysis. 27 polymorphic markers were used in the marker linkage analysis, which produced 10 haplotypes and reduced the candidate interval to 702 kb. Nonsynonymous variations in 37 genes between the parents were found during this interval, these include the genes for encode the anthocyanin reductase 1 (Glyma.08g062000), the bHLH transcription factor (Glyma.08g061300 and Glyma.08g063900), and the MYB transcript factor (Glyma.08g059900). These genes may be involved in regulating the biosynthesis of anthocyanins, and anthocyanin reductase 1 can convert anthocyanins to proanthocyanidins (PA). The results of gene expression analysis revealed that candidate genes and genes related to the anthocyanin biosynthesis pathway had comparable expression patterns in SN14 and TXAJH, and both were expressed at lower levels in SN14 and at higher levels in TXAJH. It was discovered that there was a significant link between the principal constituents of seed coat anthocyanins and the level of candidate gene expression.

The anthocyanin makeup of SN14 and TXAJH's seed coats differed, and Cy-3-glu, Pn-3-glu, and Pt-3-glu may be to blame for the TXAJH's seed coat's red hue. According to predictions, Glyma.08g059900, Glyma.08g061300, Glyma.08g062000, and Glyma.08g063900 will likely be a candidate gene for the red seed coat, in which Glyma.08g059900, Glyma.08g061300, and Glyma.08g063900 may control a number of anthocyanin biosynthesis pathway genes.

An accurate and rapid indoor evaluation system was established by using soybeans with different resistance levels to Phomopsis seed decay as test materials. And then 170 soybean germplasm accessions were employed to screened out disease-resistant varieties, so as to provide methods and material basis for high-throughput assessment of Phomopsis seed decay in soybean and cultivation of resistant varieties.

In terms of establishing a reliable evaluation method for Phomopsis seed decay, Qihuang 34, Williams, Zhongzuo 09-560, z13-631-2, ZDD26268, Chenxiqingpidou 1 and Tongxianhuangdou were selected as experimental materials. For each soybean accession, the seeds with uniform size and undamaged seed coat were germinated in the dark after disinfection. At different germination stages, the pathogen of Phomopsis seed decay was inoculated for 24 h, 48 h, 72 h and 96 h. The mycelium coverage rate and seed decay rate of seed surface under different infection time were counted to determine the optimal identification period for evaluating Phomopsis seed decay in soybean. Then, the resistance of 170 different soybean germplasms in natural population was identified by using the coverage rate of mycelium on the surface of seeds and the decay rate of seeds as evaluation indexes. The high disease resistance varieties were screened based on 5 disease resistance levels.

The soybean accessions showed the most significant differences in disease resistance levels after 96 h of germination when mycelium coverage rate and seed decay rate of soybean surface were used as evaluation indexes. Further comparison of the incidence of 24 h, 48 h, 72 h and 96 h after infection showed that the difference in disease resistance between different varieties after infection for 72 h was the most obvious. Therefore, it was the most suitable period, 72 h of infection at the bud stage after 96 h of germination, for evaluating the resistance level of different soybean varieties to Phomopsis seed decay. The resistance of 170 soybean varieties to Phomopsis seed decay was identified and classified into five disease resistance grades, namely, high resistance, medium resistance, medium susceptibility, susceptibility and high susceptibility. Among them, there were 30 varieties of grade I (high resistance to disease), 51 varieties of grade Ⅱ (medium resistance to disease), 71 varieties of grade Ⅲ (medium disease susceptibility), 4 varieties of grade Ⅳ (disease susceptibility) and 14 varieties of grade V (high disease susceptibility), idicating that there are extensive variations in the resistance to Phomopsis seed decay of soybean germplasm resources in China.

In this study, the most optimum stage of disease identification was considered as soybean seeds after 96 h germination to infect the Phomopsis longicolla for 72 h. After that, the mycelium coverage rate and seed decay rate of soybean surface were counted as evaluation parameters. The evaluation system has high accuracy and reliability, which can provide an effective method for high-throughput identification of different varieties in the laboratory. And 30 highly resistant varieties were further screened to provide a material basis for the breeding of resistant varieties.

Hardness, a structural feature of seed physical dormancy, is an important trait in soybean domestication. Although hardness is beneficial for seeds to survive in unfavorable environments, it will seriously reduce the emergence rate of soybean in the field, and detrimental to yield and processing quality. Analyzing the QTL and candidate genes using bulked segregant analysis sequencing (BSA-Seq), can provide a theoretical reference for understanding the molecular mechanism of hard seededness in soybean.

The hard seed mutant M_zp661 was obtained from the seeds of Zhongpin 661 induced by ethyl methane sulfonate (EMS), and was crossed with cultivated soybean Zhonghuang 13 (male parent) to construct recombinant inbred line (RIL) population. The progeny lines were investigated for seed hardness, water absorption capacity and anatomical structure of seed coats. Two types of extreme lines in the RIL population, with hard seeds or with imbibed seeds, were selected to construct DNA mixed pools respectively, and then BSA-Seq technology was used to detect genotype differences in extreme-mixed pools and parents. Euclidean distance (ED), delta SNP-index, and delta InDel-index methods were applied to associate hard seed genetic loci of soybean. Combining with bioinformatics analysis, transcriptome data of different soybean tissues and gene annotation information, candidate genes within significant association regions were predicted.

In the progenies of M_zp661, all areas of imbibitive seeds had the penetration ability, and the seed volume increased continuously with the soaking time. However, no changes were observed for hard seeds over 36 hours. With the prolonged of soaking time, the seed coat of hard seeds began to shrink locally and gradually spread to other parts, and finally cotyledons recovered their imbibition ability. The hard seed not only has smooth and compact seed coat, but also has regular network structure of cuticle and thicker palisade layer, while numbers of stomata and loose structures, tiny cracks and thinner palisade layer were existed in the imbibed seeds. These results suggest that the seed hardness of M_zp661 may be caused by the impermeability of the seed coat. ED, delta SNP-index and delta InDel-index association analysis methods not only identified the reported seed physical dormancy locus qHS1, but also simultaneously detected the candidate region Chr.06: 45897227-47746047, which contains a total of 189 genes. Further, transcriptome data and gene annotation predicted that Glyma.06G275300, which is specifically and highly expressed in seeds, might be the candidate gene for this associated region to regulate soybean seed hardness.

Seed hardness of soybean mutant M_zp661 was caused by the impermeability of the seed coat, and Glyma.06G275300 was predicted as a candidate gene affecting the structure of seed coat using BSA-Seq.