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Article

Genome-Wide Association Study on Candidate Genes Associated with Soybean Stem Pubescence and Hilum Colors

by
Miaomiao Zhou
1,†,
Junyan Wang
1,†,
Huatao Chen
2,3,4,
Qianru Jia
4,
Shengyan Hu
3,
Yawen Xiong
3,
Hongmei Zhang
4,
Wei Zhang
4,
Qiong Wang
2,4,* and
Chengfu Su
1,*
1
College of Agriculture, Qingdao Agricultural University, Qingdao 266109, China
2
Zhongshan Laboratory of Biological Breeding, Nanjing 210014, China
3
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
4
Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(3), 512; https://doi.org/10.3390/agronomy14030512
Submission received: 12 January 2024 / Revised: 21 February 2024 / Accepted: 26 February 2024 / Published: 1 March 2024
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
Versions Notes

Abstract

:
The colorations of stem pubescence and hilum are crucial criteria for discerning diverse soybean germplasms, governed by multiple genes that substantially influence the seed’s outward appearance quality and the resistance to abiotic stresses. This comprehensive study delved into the stem pubescence and hilum color traits across a natural population of 264 accessions during 2021 and 2022. The phenotypes of these two traits within our population were analyzed for the investigation of population genetics and evaluation of germplasm resources in the future. Numerous noteworthy SNPs associated with both traits were detected through a genome-wide association study (GWAS), with the most significant signals for 2021 and 2022 localized on chromosome 6. Seven candidate genes regulating stem pubescence color and four genes influencing hilum color were identified by analyzing the expression patterns, cold stress responses, and regulatory pathways of genes within the LD decay distance of SNPs. This study not only underscores the applicability of GWAS in unraveling the genetic basis of quality traits, but also contributes novel genetic reservoirs and research paradigms to the explorations of the soybean plant and seed color. These results provide foundational insights into the breeding improvement of seeds’ outward appearance quality and a comprehensive evaluation of soybean germplasm.

1. Introduction

Soybean [Glycine max (L.) Merr.] acts as an essential oil grain crop. Cultivated soybeans originated through the domestication of wild soybeans in China and are presently grown worldwide [1,2,3,4]. The rich protein, oil, and dietary fiber contents of soybeans make them crucial for the food industry and feed production [5,6]. With accelerating economic developments, the demand for soybeans is increasing, but soybean production remains insufficient to satisfy this demand. Obtaining optimal germplasms and investigating their traits is particularly important for researchers to improve soybean quality and yield [7,8].
The soybean traits of DUS (Distinctness, Uniformity, and Stability) are often utilized as important distinguishing indicators in various cultivars, encompassing yield traits (100-seed weight, seed number), quality traits (seed shape, seed color, and hilum color), as well as traits such as leaf shape and stem pubescence color, which have no direct commercial value [9,10]. In addition to high yields, researchers usually set high quality and efficiency goals for soybean breeding. Of those traits that are not directly related to yield, some have potential associations with plant resistance, which can ultimately affect soybean yield; some are related to market preferences for seed appearance quality. Currently, there is little research on these traits, and they may also play important but undiscovered roles in plant growth, yield, and stress resistance. The stem pubescence and hilum colors are regulated by some genes within the same pleiotropic loci. These loci overlap other loci responsible for soybean maturity, stem growth habits, leaf shape, and cold tolerance [11,12]. Soybean hilum and stem pubescence colors exert a direct or indirect influence on the growth, development, and even yield of plants [13]. In contrast to soybeans with gray pubescence, brown-pubescence soybeans possess elevated yields and improved stability in cold regions. However, in cold weather, some indicators of growth vitality, such as germination energy, are nearly unaffected by the colors of the seed coat. Soybeans with a brown hilum possess elevated growth vitality and cold tolerance than those with a yellow hilum. Nonetheless, due to the better appearance quality of yellow hilum soybeans, they account for a larger proportion of breeding and cultivating representatives [14]. The pubescence and hilum color alterations are phenotypes of cold damage, while also characterizing the capacity for cold resistance. For high-yield and cold-tolerant soybean breeding, it is critical to uncover the molecular mechanisms of the pubescence and hilum colors, and then specifically select soybean cultivars with brown pubescence and yellow hilum.
Currently, some research on stem pubescence and hilum colors has been conducted. It has been reported that T and Td are involved in controlling soybean pubescence color [15], multiple allele loci I/i-i/i-k/i regulate hilum color, and five genes (T, W1, I, R, O) induce the color change of seed coat [16,17]. The existence of T can lead to the appearance of a deeper hilum color and brown stem pubescence [17,18]. The T gene encodes F3′H (flavonoid 3′-hydroxylase), an endoplasmic reticulum membrane protein situated in the vacuoles of the hilum. It is involved in the color formation of soybean pubescence, seed coat, and hilum and could relieve cracking and abnormal browning in seed coat due to cold stress in soybeans [18,19,20]. Moreover, a transcription factor R2R3 MYB corresponding to the Td gene was uncovered, and a lower flavonoid content and lighter pubescence color were identified in mutants [21]. The soybean hilum and stem pubescence colors are intricate traits controlled by multiple genes, but the molecular mechanisms underlying their formation remain unclear.
With the progression of resequencing, genome-wide association analysis has widespread application in diverse crops like soybean, maize, and rice [22,23,24]. Many SNPs have been uncovered by studying traits such as the 100-seed weight, plant height, drought, and salt tolerance. However, qualitative traits such as stem pubescence color and hilum color have been reported less frequently [25].
This study employed 264 soybean accessions to observe the stem pubescence color and hilum color and conduct a genome-wide association analysis. Overall, this study explored novel loci and candidate genes, with the aim of providing fundamental insight into the genetic examination and innovation of the seed outward appearance quality of soybean germplasm. We summarized the responses of two traits in cold surroundings and hope to provide new ideas for the soybean molecular design breeding of cold resistance.

2. Materials and Methods

2.1. Experimental Materials and Planting

A soybean population of 264 accessions encompassing 212 cultivars and 52 landraces, with the majority of these coming from the Huang-Huai region and southern China, was utilized in this study (Table S1). In 2021 and 2022, all materials were planted in the Luhe Experimental Field of the Jiangsu Academy of the Agricultural Sciences (Nanjing, China), employing a randomized block design with three replicates. Each material was planted in three rows, with twelve holes per row and each hole containing three seeds. The field was managed according to conventional field trial approaches [26].

2.2. Phenotypic Investigation and Analysis

The soybean pubescence color of the middle portions was observed and recorded from the florescence stage to the maturity stage. After harvesting, we classified the hilum color into five groups: yellow, light brown, brown, light black, and black. Six plants of each variety were randomly chosen. Partial materials lost during harvest or recording were replaced with “-”. The data were summarized and descriptively analyzed using Excel software version 2010, and a frequency histogram was plotted using Origin 2018 software.

2.3. Genome-Wide Association Study

This population of 264 materials has been resequenced, with an average sequencing depth of 12.4×, and a high-density physical map required for our study’s whole-genome association analysis was generated, including a total of 2,597,425 SNP markers. The linkage disequilibrium (LD) in this population was 120 kb. Detailed population information has been reported previously [27].
To control for false positives, the whole-genome association analysis was conducted utilizing a mixed linear model (MLM) according to the GAPIT version 3.0 package of R software [28], with the parameter PCA, Total = 3 SNP, MAF = 0.05, and Model = c (“MLM”). The threshold used to select was P ≤ 1 × 10−5 or −log10(P) ≥ 5.0. When the −log10(P) was ≥5.0, we considered the SNPs to be satisfying this criterion of significant association loci.

2.4. Candidate Gene Analysis

After the significant SNPs associated with stem pubescence color and hilum color were identified, we selected candidate genes within a range of 120 kb upstream and downstream of the SNPs. The soybean genome information was obtained from the Phytozome13 online database: (https://phytozome-next.jgi.doe.gov/info/Gmax_Wm82_a2_v1, accessed on 30 November 2023) [29]. Homologous Arabidopsis genes were queried using TAIR (https://www.arabidopsis.org/, accessed on 3 December 2023). The genes with relevant functions and expressing differently were selected as candidate genes, and the expression data were visualized using TBtools-II version.2.052 [30].

2.5. GO Enrichment Analysis for Candidate Genes

All candidate genes uncovered through the GWAS were tested for gene ontology enrichment. Gene ontology annotation analyses were performed by submitting gene information to Gene Ontology (GO), and significant GO terms were determined with TBtools-II version.2.052 [30].

3. Results

3.1. Phenotypic Analysis of Stem Pubescence and Hilum Colors throughout the Natural Population

We noted the pubescence color over different years and materials, summarizing the frequency data as outlined in Table 1. No significant difference in the frequency of the two types of pubescence color was found in the natural population over two years. One reason for this phenomenon is that the soybean stem pubescence color is stably inherited [31]. The seedling stage is the most sensitive period to cold damage; 15 degrees C is usually defined as the temperature threshold for soybeans to suffer from cold stress [32,33]. From 2021 to 2022, no significant temperature below the threshold was identified from flowering to maturity in the Huang-Huai region (Table S2), which was another potential reason for the trait’s stability during both growing seasons. Among them, there were more soybeans with gray pubescence compared to brown pubescence, and the two color types’ distribution ratio approaches 5:3. At the same time, the proportion of gray pubescence in cultivars was higher than that of landraces, amounting to 62.67%.
Hilum color is a qualitative trait orchestrated by complex genetic mechanisms, showing a discontinuous distribution in the population. When performing seed testing, we separated them into five types: yellow, light brown, brown, light black, and black. The frequency distribution histogram of hilum color is displayed in Figure 1A; the peak appears in the intermediate color (light brown and brown). The distribution is approximately bell-shaped with elongated tails, similar to the normal distribution of quantitative traits.
As the colors of the soybean hilum and stem pubescence are governed by the same gene, as noted in previous reports, some contact exists between the two traits. Therefore, this study defined ten phenotypes by arranging and integrating the two classes of pubescence color and five classes of hilum color collected in 2021 and 2022, including gray pubescence and yellow hilum; gray pubescence and light brown hilum; gray pubescence and brown hilum; gray pubescence and light black hilum; gray pubescence and black hilum; brown pubescence and yellow hilum; brown pubescence and light brown hilum; brown pubescence and brown hilum; brown pubescence and light black hilum; and brown pubescence and black hilum. The frequency relationship of each of these classes was characterized using a histogram (Figure 1B). The accessions with gray pubescence and brown hilum as well as those with gray pubescence and light brown hilum, were the most common, accounting for 28.86% and 26.02% in 2021 and both 26.88% in 2022 of the total population, respectively. The brown pubescence and brown hilum, brown pubescence and light black hilum, and brown pubescence and black hilum, accounted for 15.85%, 7.32%, and 9.76% in 2021 and 10.67%, 12.65%, and 12.25% in 2022, respectively, while the proportions of other types in the population were relatively small. Soybeans with darker stem pubescence (brown) are often accompanied by darker hilum color (black), while lighter gray pubescence is generally accompanied by lighter hilum color (brown), as seen in Figure 1B. However, the influence of hilum color on stem pubescence is not similar, potentially because the soybean hilum and pubescence colors are controlled by the same genes such as T; in contrast, some other genes also control hilum color in addition to these genes. This inference is consistent with previous research.

3.2. Genome-Wide Association Study on Soybean Stem Pubescence Color

The resequencing results and the phenotypic data of our natural population for stem pubescence were utilized in a genome-wide association study. When the significance threshold for identifying association sites was established to be at −log10(P) ≥ 5.0, a total of 12,476 reliable SNPs were identified between the two years, located on all chromosomes except 9 and 19. In 2021, a total of 12,171 SNPs of stem pubescence color were identified, and 12,391 SNPs were identified in 2022. The most abundant and significant of these were found on chromosome 6, while the fewest SNPs were found on chromosomes 2 and 7, with only one each over both years (Figure 2; Table S3).
Among these, 12,086 signals were consistently identified in two environments, with 12 SNPs located on chromosomes 1, 3, 4, 5, 6, 10, 12, 13, 15, 16, and 20 selected as candidate loci for the color of stem pubescence. The −log10(P) value of Chr06:18749716 was the highest of all SNPs, at 14.68 and 14.75 in 2021 and 2022, respectively (Table 2).

3.3. Genome-Wide Association Study on Hilum Color

A GWAS involving high-density SNPs was performed to identify the SNPs association with the hilum color trait. The threshold was established as −log10(P) = 5.0. Similarly, a total of 2826 SNPs for soybean hilum color were identified in this research, distributed across chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, and 20; repeat signals were detected only on chromosomes 4, 6, 7, 8, 9, 11, and 14. A total of 1906 and 1906 SNPs related to hilum color were uncovered in 2021 and 2022, respectively. A total of 1563 and 1734 SNPs were identified over both years on chromosome 6, with most of the SNPs distributed in clusters from 18.75 Mb to 34.52 Mb on chromosome 6 of the soybean genome. In addition to the most significant and the largest number of signals on chromosome 6, many signals were detected on chromosome 9 (Figure 3; Table S3). Among them, the densest and the most obvious signals were detected in the overlap region on chromosome 6 for both stem pubescence and hilum color traits. We speculate that this region may contain shared essential genes for these two traits.
Overall, we considered Chr06:18749528, Chr06:34516359, Chr07:38116634, Chr08:18381601, Chr09:45711122, Chr11:18166441, and Chr14:13707853 as lead SNPs for characterizing the range of candidate genes (Table 3), because of the most significant signal in their respective clusters over both years.

3.4. Candidate Genes of Soybean Stem Pubescence Color

Our study identified the upstream 120 kb and downstream 120 kb regions of lead SNPs as candidate gene selection ranges. These ranges, possessed with particularly dense SNPs, were expanded appropriately. The genomic data, such as functional annotations and homologous genes from the Phytozome13 online database, were searched further to identify potential candidate genes associated with stem pubescence color.
Seven candidate genes linked to stem pubescence color were characterized (Table 4), and the expression levels of each tissue acquired from transcriptome data were visualized and analyzed (Figure 4A). All genes exhibited high expression levels in the stem, and some genes, including Glyma.03G122000, Glyma.03G123200, Glyma.03G258700, Glyma.06G202300, and Glyma.15G182600 were highly expressed in the shoot apical meristem. At the same time, Glyma.03G122000, Glyma.04G030100, and Glyma.06G202300 all encoded cytochrome P450 family proteins, which are also highly expressed in pods and seeds.

3.5. Candidate Genes of Soybean Hilum Color

We characterized a total of four candidate genes governing hilum color using the same method (Table 5). Among these, the highest expression level of Glyma.06G202300 was in seeds, followed by the pod and stem apical meristem. Glyma.07G210800 is widely distributed across all tissues, with the highest expression level in seeds. Glyma.09G232400 encodes the AGC kinase protein, with high expression levels in pods, seeds, and stems. In contrast, Glyma.09G234700 encodes a vesicle transporter protein, and the expression levels in seeds are significantly higher than in other tissues.
Glyma.06G202300 is a candidate gene for both traits, encoding flavonoid 3′-hydroxylase and regulating and orchestrating soybean pubescence color, as reported in prior studies [12].
Also, we analyzed all candidate genes in this study via GO enrichment analysis; the results show that the candidate genes were enriched in various biological processes, especially in obsolete conjugation, conjugation with cellular fusion, the cell wall polysaccharide catabolic process, and the cell wall macromolecule catabolic process (Figure S1).

4. Discussion

Although not as direct selection criteria for yield or quality during production, some soybean traits, such as plant height, stem pubescence color, hilum color, flower color, and number of primary stem nodes, are essential for growth and development, the abiotic stress response, and environmental adaptation [9,10]. When breeding novel soybean varieties, stem pubescence and hilum color are essential for distinguishing and evaluating marks for various accessions [34]. The colors of stem pubescence and hilum are related to plant cold resistance. Due to the consumer preference for soybeans with a lighter hilum color in some regions, hilum color has become a target for breeding [35]. Therefore, determining the genetic mechanism and transformation principles of soybean stem pubescence and hilum colors is critical for optimizing soybean yield and quality.
This study documented the stem pubescence and hilum colors of 264 soybean accessions from 2021 to 2022. The results indicate that the stem pubescence color positively influences hilum color; soybeans with darker stem pubescence color often have a darker hilum color. No significant changes in stem pubescence color were found in these two years. Significant lead SNPs on chromosomes 1, 3, 4, 5, 6, 10, 12, 13, 15, 16, and 20 as well as 6, 7, 8, 9, 11, and 14 for the two traits were detected through a GWAS. Among them, the most significant signal was detected on chromosome 6 (18.74–18.75 Mb) for both traits across both years. Therefore, it was considered the main locus of two traits, playing a primary role in trait formation. Seven and four genes were identified as candidate genes for the stem pubescence and hilum colors, respectively.

4.1. Glyma.06G202300 and Glyma.03G258700 Are Related to Both Traits

Previous studies have determined that the T gene governs seed coat and pubescence color, related to the cold stress response [33]. In an earlier study, Toda isolated the key gene sf3′h1 corresponding to the T gene in two NILs, To7B (TT, brown) and To7G (tt, gray). The sf3′h1 gene encodes flavonoid 3′- hydroxylase, located in the vacuoles of soybean hilum, and its absence leads to the soybean phenotype of gray pubescence [12,19]. The sf3′h1 is situated at 18.73 Mb on chromosome 6, falling within the candidate gene range of the two SNPs (Chr06:18749716 and Chr06:18749528) associated with stem pubescence and hilum color, also found in this research. Similarly, Glyma.03G258700, located on chromosome 3, which encodes an MYB transcription factor that regulates soybean pubescence color, was recently identified in the NIL population. Apart from the genes present on chromosomes 3 and 6, we speculate that there are many candidate genes orchestrating soybean pubescence and hilum color at other significant SNPs, directly or indirectly participating in soybean color formation.

4.2. Analysis of Candidate Genes Modulating Soybean Stem Pubescence Color

In addition to chromosomes 3 and 6, we identified candidate genes governing stem pubescence color on other chromosomes. Both Glyma.03G122000 and Glyma.04G030100 encode cytochrome P450 family proteins, which are primarily involved in the synthesis of flavonoids and play a role in the secondary metabolism, as these belong to the same protein family as F3′H and may have a similar role in soybean stem pubescence color regulation. The homologous gene AT2G40890 of Glyma.03G122000 was verified to encode β-coumarate-3-hydroxylase(C3′H), when the C3′H gene was interfered with; the stored cassava physiologic deterioration, including browning, was reduced significantly [36,37]. We speculate that it also alleviates the color changes in the hilum and pubescence due to cold damage in soybeans. Glyma.03G123200 encodes a bZIP transcription factor, commonly found in animals, plants, and microorganisms. The bZIP is related to the cold-activated stress response and alleviates potential plant damage by eliminating excess ROS in kiwifruit [38]; there may be similar cold damage repair mechanisms in deep-pubescence soybeans. The single zinc finger DNA binding protein is one of the plant-specific transcription factors believed to be associated with abiotic stress in cotton research [39]. Glyma.03G258800 encodes Dof-type zinc finger DNA binding family protein and may be involved in regulating soybean color alteration brought about by cold stress in soybeans. Glyma.15G182600 encodes sucrose synthase 4. While sucrose is necessary for anthocyanin synthesis, in cold environments [40], it may indirectly participate in plant pigment synthesis by regulating the sucrose synthesis pathway, resulting in color changes throughout specific areas of soybean, such as the browning of stem pubescence. In summary, these candidate genes are considered positive factors in the process of soybeans specific area coloring and cold response, which is reliable for use in subsequent research.

4.3. Analysis of Candidate Genes for Soybean Hilum Color

AT3G19130 is the homologous gene of Glyma.07G210800, and alongside 2′, 3′- cAMP, it is involved in generating stress granules. These stress granules could balance cellular energy distribution by inhibiting the translation of the related genes to improve plant stress tolerance [41,42]. Glyma.09G232400 encodes an AGC protein kinase family protein; AGC protein kinases are considered to closely relate to plant growth and development [43]. Garcia et al. reviewed the roles of AGC protein kinases in plant growth and stress responses [44]. SNARE proteins are essential components in signal transduction, linked to the unique intracellular transport of plants. They also participate in plant growth, development and abiotic stress responses. Glyma.09G234700 encodes a SNARE protein for vesicular transport, which can regulate plant cold tolerance via material transportation [45]. Temperature variation affects hilum color, and different colors correspond to variability in cold resistance. These three genes are all related to the abiotic stress response, but further research is required to uncover how they influence hilum color.
Currently, research related to stem pubescence and hilum colors has rarely been reported, and the candidate genes discovered in this study have not been thoroughly examined. In addition to appearance quality and cold resistance, the important roles of these two traits are also played in soybean yield and at maturation time [35,46,47]; we believe that research on these traits and candidate genes has broad prospects. And due to the market’s preference for light hilum color, cultivating soybean varieties with a light hilum color and deep pubescence color is of great significance for improving the commercial value of soybeans. Although these two traits are jointly controlled by some single gene, we still believe that it is necessary to explore candidate genes for controlling a single trait, either stem pubescence color or hilum color. Cultivars can adapt to cold environments and meet market requirements for soybean appearance by inhibiting the expression of hilum color genes and increasing the expression of pubescence color genes. In the future, genes can be overexpressed or knocked out in soybeans to explore their impacts on soybean stem pubescence color or hilum color. Through protein–protein interaction technology, such as GST-pull down or protein–nucleic acid interaction technology, such as RNA Immunoprecipitation, to identify the upstream and downstream interacting proteins or genes, the regulatory mechanism of this gene on these traits can be figured out further. Also, markers such as KASPs can be developed for significant SNPs associated with two traits, and research into the function and molecular mechanisms could be carried out on the candidate genes to promote genetic improvement and the breeding for hilum and pubescence colors. Additionally, our study first used a GWAS to detect the SNPs of hilum color and stem pubescence color, confirming the reliability of GWAS for quality traits. Concurrently, it provided novel genetic resources for the molecular mechanism research of stem pubescence and hilum colors and contributed new research avenues for soybean stress resistance molecular breeding.

5. Conclusions

Our study analyzed the stem pubescence and hilum colors in 264 soybean population accessions. The results indicate that our population tends toward brown hilum and grey pubescence soybeans, and from a comprehensive phenotype perspective, the hilum color often exhibits a phenomenon that is one shade darker than the color of the stem pubescence. Through a GWAS, a total of 12,476 and 2826 SNPs were detected; most of them were clustered on chromosome 6. We selected twelve and seven lead SNPs to characterize the candidate soybean genes governing stem pubescence and hilum color. Among the candidate genes, three genes modulate stem pubescence color: Glyma.03G122000, Glyma.04G030100, and Glyma.06G202300, which encode cytochrome P450 family proteins. Interestingly, Glyma.06G202300 has been confirmed to be involved in soybean stem pubescence and hilum colors. In addition, novel candidate genes listed in our study may play a role in the mechanism and regulatory pathways of soybean color formation. More in-depth functional research on these genes must be conducted to examine new mechanisms orchestrating soybean color. In summary, this study analyzed the distribution of soybean stem pubescence and hilum color and discussed the roles of these in response to cold stress, provided fundamental insights for genetic resource evaluation, and contributed new ideas to the study of color-related characteristics. Novel loci and genes regulating these two traits were identified in this study, providing novel genetic resources for soybean quality and cold resistance breeding.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14030512/s1: Figure S1: GO enrichment analysis of candidate genes; Table S1: Detailed information for 264 accessions; Table S2: Temperature from June to September in Luhe of 2021 and 2022; Table S3: Total significant SNPs and most significant SNPs associated with the color of soybean hilum and stem pubescence.

Author Contributions

Conceptualization, C.S. and Q.W.; methodology, W.Z. and M.Z.; software, Q.W.; validation, Q.J., J.W. and S.H.; formal analysis, M.Z.; investigation, H.Z., S.H. and J.W.; resources, H.C.; data curation, M.Z. and Y.X.; writing—original draft preparation, M.Z. and Q.J.; writing—review and editing, H.C. and C.S.; visualization, W.Z., M.Z. and Y.X.; project administration, H.C. and C.S.; funding acquisition, H.C., C.S. and H.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Open Competition Project of the Seed Industry Revitalization of Jiangsu Province (JBGS [2021] 060), the Jiangsu Agriculture Science and Technology Innovation Fund (JASTIF) CX(22)5002, the Natural Science Foundation of Shandong Province of China (Grant#ZR2021MC071), the National Key Research and Development Program (2022YFD2300101-1), the Seed-Industrialized Development Program in Shandong Province (2021LZGC003), the Qingdao Science and Technology Benefit the People Demonstration Project (23-2-8-xdny-10-nsh), and the Natural Science Foundation of Jiangsu Province (BK20220740).

Data Availability Statement

The data supporting the reported results will be available upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Chowdhury, M.; Telli, H. Migration; Springer: Berlin/Heidelberg, Germany, 2004; pp. 47–59. [Google Scholar] [CrossRef]
  2. Wang, L.; Zhang, M. A telomere-to-telomere gap-free assembly of soybean genome. Mol. Plant 2023, 16, 1711–1714. [Google Scholar] [CrossRef] [PubMed]
  3. Sedivy, E.J.; Wu, F. Soybean domestication: The origin, genetic architecture and molecular bases. New Phytol. 2017, 214, 539–553. [Google Scholar] [CrossRef] [PubMed]
  4. Hammer, K. Das Domestikations syndrom. Kulturpflanze 1984, 32, 11–34. [Google Scholar] [CrossRef]
  5. Singer, W.M.; Lee, Y.C. Soybean genetics, genomics, and breeding for improving nutritional value and reducing antinutritional traits in food and feed. Plant Genome 2023, 16, e20415. [Google Scholar] [CrossRef] [PubMed]
  6. Hartman, G.L.; West, E.D. Crops that feed the World 2. Soybean—Worldwide production, use, and constraints caused by pathogens and pests. Food Secur. 2011, 3, 5–17. [Google Scholar] [CrossRef]
  7. Han, X.R.; Wang, X.D. Strategy for Ensuring China’s Food Security through Planting Industry in the New Era. Strateg. Study CAE 2023, 25, 1–11. [Google Scholar] [CrossRef]
  8. Qiu, L.J.; Chen, P.Y. The worldwide utilization of the Chinese soybean germplasm collection. Plant Genet. Resour. 2011, 9, 109–122. [Google Scholar] [CrossRef]
  9. Xie, Y.H. Difference analysis on DUS traits of soybean standard varieties. Soybean Sci. 2007, 2, 284–286. [Google Scholar] [CrossRef]
  10. Ramteke, R.; Murlidharan, P. Characterization of soybean (Glycine max) varieties as per DUS guidelines. Indian J. Agric. Sci. 2012, 84, 572–577. [Google Scholar] [CrossRef]
  11. Tsegaw, M.; Zegeye, W.A. Progress and Prospects of the Molecular Basis of Soybean Cold Tolerance. Plants 2023, 12, 459. [Google Scholar] [CrossRef]
  12. Toda, K.; Yang, D. A single-base deletion in soybean flavonoid 3′-ydroxylase gene is associated with gray pubescence color. Plant Mol. Biol. 2002, 50, 187–196. [Google Scholar] [CrossRef]
  13. Morrison, M.J.; Voldeng, H.D. Soybean Pubescence Color Influences Seed Yield in Cool-Season Climates. Agron J. 1994, 86, 796–799. [Google Scholar] [CrossRef]
  14. Lou, Z.Z.; Deng, D. Correlation Analysis between Seed Color and Seed Vigor of Glycine max. J. Chuxiong Norm. Univ. 2023, 38, 147–154. [Google Scholar] [CrossRef]
  15. Cober, E.R.; Ablett, G.R. Imperfect Yellow Hilum Color in Soybean is Conditioned by II rr TT. Crop Sci. 1998, 38, 940–941. [Google Scholar] [CrossRef]
  16. Qiu, H.M.; Chen, L. Research progress on genetic regulatory mechanism of seed color in soybean (Glycine max). Acta Agron. Sin. 2021, 47, 2299–2313. [Google Scholar] [CrossRef]
  17. Takahashi, R.; Asanuma, S. Association of T gene with chilling tolerance in soybean. Crop Sci. 1996, 36, 559–562. [Google Scholar] [CrossRef]
  18. Takahashi, R.; Benitez, E.R. Soybean maturity and pubescence color genes improve chilling tolerance. Crop Sci. 2005, 45, 1387–1393. [Google Scholar] [CrossRef]
  19. Toda, K.; Kuroiwa, H. The soybean F3′H protein is localized to the tonoplast in the seed coat hilum. Planta 2012, 236, 79–89. [Google Scholar] [CrossRef] [PubMed]
  20. Yamaguchi, N.; Suzuki, C. A pubescence color gene enhances tolerance to cold-induced seed cracking in yellow soybean. Breed Sci. 2021, 71, 467–473. [Google Scholar] [CrossRef] [PubMed]
  21. Yan, F.; Githiri, S.M. Loss-of-Function Mutation of Soybean R2R3 MYB Transcription Factor Dilutes Tawny Pubescence Color. Front. Plant Sci. 2019, 10, 1809. [Google Scholar] [CrossRef] [PubMed]
  22. Yang, Y.; Zhao, T.T. Identification of candidate genes for soybean seed coat-related traits using QTL mapping and GWAS. Front. Plant Sci. 2023, 14, 1190503. [Google Scholar] [CrossRef]
  23. Lv, Y.; Ma, J. Combining GWAS, Genome-Wide Domestication and a Transcriptomic Analysis Reveals the Loci and Natural Alleles of Salt Tolerance in Rice (Oryza sativa L.). Front. Plant Sci. 2022, 13, 912637. [Google Scholar] [CrossRef]
  24. Zhang, X.; Guan, Z. Combined GWAS and QTL analysis for dissecting the genetic architecture of kernel test weight in maize. Mol. Genet. Genom. 2020, 295, 409–420. [Google Scholar] [CrossRef]
  25. Korte, A.; Farlow, A. The advantages and limitations of trait analysis with GWAS: A review. Plant Methods 2013, 9, 29. [Google Scholar] [CrossRef]
  26. Karaagaoglu, E. An Experimental Design Technique: Randomized Block Design. Turk J. Biochem. 2013, 38, 1–4. [Google Scholar] [CrossRef]
  27. Zhang, W.; Xu, W.J. Comparative selective signature analysis and high-resolution GWAS reveal a new candidate gene controlling seed weight in soybean. Theor. Appl. Genet. 2021, 134, 1329–1341. [Google Scholar] [CrossRef] [PubMed]
  28. Wang, J.; Zhang, Z. GAPIT version 3: Boosting power and accuracy for genomic association and prediction. Genom. Proteom. Bioinform. 2021, 19, 629–640. [Google Scholar] [CrossRef] [PubMed]
  29. Zhao, Z.X.; Guo, C. Genome-Wide Analysis of Tandem Repeats in Plants and Green Algae. G3 2014, 4, 67–78. [Google Scholar] [CrossRef] [PubMed]
  30. Chen, C.J.; Chen, H. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  31. Yadav, S.P.; Sharma, S.P. Variation for hilum colour and its stability during four crop seasons in soybean (Glycine max). Indian J. Agric. Sci. 2001, 71, 23–26. [Google Scholar]
  32. Jian, W.; Qiang, Q. Deciphering the myth of cold tolerance in soybean: An overview of molecular breeding applications. Not. Bot. Horti Agrobot. 2023, 51, 13160. [Google Scholar] [CrossRef]
  33. Toda, K.; Takahashi, R. Difference in chilling-induced flavonoid profiles, antioxidant activity and chilling tolerance between soybean near-isogenic lines for the pubescence color gene. J. Plant Res. 2011, 124, 173–182. [Google Scholar] [CrossRef] [PubMed]
  34. Shao, Y.B.; Sun, B.C. Breeding Report of an Extremely Early Mature Soybean Variety Mengdou 343. China Seed Ind. 2023, 11, 152–153. [Google Scholar] [CrossRef]
  35. Hideki, K.; Setsuzo, Y. Correlation of cold-weather tolerance with pubescence color and flowering time in yellow hilum soybeans in hokkaido. Breed Sci. 2004, 54, 303–311. [Google Scholar] [CrossRef]
  36. Ma, Q.; Xu, J. Knockdown of p-Coumaroyl Shikimate/ Quinate 3′-Hydroxylase Delays the Occurrence of Post-Harvest Physiological Deterioration in Cassava Storage Roots. Int. J. Mol. Sci. 2022, 23, 9231. [Google Scholar] [CrossRef]
  37. Jeon, H.S.; Jang, E. Pathogen-induced autophagy regulates monolignol transport and lignin formation in plant immunity. Autophagy 2023, 19, 597–615. [Google Scholar] [CrossRef]
  38. Liu, X.; Bulley, S.M. Kiwifruit bZIP transcription factor AcePosF21 elicits ascorbic acid biosynthesis during cold stress. Plant Physiol. 2023, 192, 982–999. [Google Scholar] [CrossRef] [PubMed]
  39. Ju, L.Z.; Zhao, T. Genome-Wide Identification and Analysis of Dof Gene Family in Upland Cotton. Cotton Sci. 2020, 32, 279–291. [Google Scholar] [CrossRef]
  40. Das, P.K.; Shin, D.H. Sugar-hormone cross-talk in anthocyanin biosynthesis. Mol. Cells 2012, 34, 501–507. [Google Scholar] [CrossRef]
  41. Ruiz-Solaní, N.; Salguero-Linares, J. Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence. Plant Cell 2023, 35, 3325–3344. [Google Scholar] [CrossRef]
  42. Xie, Z.; Zhao, S. Phenolic acid-induced phase separation and translation inhibition mediate plant interspecific competition. Nat. Plants 2023, 9, 1481–1499. [Google Scholar] [CrossRef] [PubMed]
  43. Jiang, Y.; Liu, X. Genome-Wide Identification of the AGC Protein Kinase Gene Family Related to Photosynthesis in Rice (Oryza sativa). Int. J. Mol. Sci. 2022, 23, 12557. [Google Scholar] [CrossRef] [PubMed]
  44. Garcia, A.V.; Al-Yousif, M. Role of AGC kinases in plant growth and stress responses. Cell Mol. Life Sci. 2012, 69, 3259–3267. [Google Scholar] [CrossRef] [PubMed]
  45. Feng, Y.J.; Jiang, S. Advances in the Research of R-SNARE in Plants. Chin. J. Cell Biol. 2023, 45, 786–794. [Google Scholar] [CrossRef]
  46. Cober, E.R.; Voldeng, H.D. Maturity and pubescence color are associated in short-season soybean. Crop Sci. 1997, 37, 424–427. [Google Scholar] [CrossRef]
  47. Morrison, M.J.; Voldeng, H.D. Yield of cool-season soybean lines differing in pubescence color and density. Agron. J. 1997, 89, 218–221. [Google Scholar] [CrossRef]
Agronomy 14 00512 g001
Figure 1. Distributions of soybean hilum color (A) and ten comprehensive types (B) over 264 accessions. Comprehensive types are named “pubescence color & hilum color”.
Figure 1. Distributions of soybean hilum color (A) and ten comprehensive types (B) over 264 accessions. Comprehensive types are named “pubescence color & hilum color”.
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Figure 2. Manhattan plots (A) and quantile–quantile plots (B) for the GWAS on soybean stem pubescence color in 2021 and 2022. Chromosomes are depicted in different colors. The horizontal solid line corresponds to a −log10(P) ≥ 5.0 threshold and SNPs above this line are plotted as orange dots. The dashed line corresponds to a significant threshold and SNPs above this line are plotted as red dots.
Figure 2. Manhattan plots (A) and quantile–quantile plots (B) for the GWAS on soybean stem pubescence color in 2021 and 2022. Chromosomes are depicted in different colors. The horizontal solid line corresponds to a −log10(P) ≥ 5.0 threshold and SNPs above this line are plotted as orange dots. The dashed line corresponds to a significant threshold and SNPs above this line are plotted as red dots.
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Figure 3. Manhattan plots (A) and quantile–quantile plots (B) for the GWAS on soybean hilum color in 2021 and 2022. Chromosomes are depicted in different colors. The horizontal solid line corresponds to a −log10(P) ≥ 5.0 threshold and SNPs above this line are plotted as orange dots. The dashed line corresponds to a significant threshold and SNPs above this line are plotted as red dots.
Figure 3. Manhattan plots (A) and quantile–quantile plots (B) for the GWAS on soybean hilum color in 2021 and 2022. Chromosomes are depicted in different colors. The horizontal solid line corresponds to a −log10(P) ≥ 5.0 threshold and SNPs above this line are plotted as orange dots. The dashed line corresponds to a significant threshold and SNPs above this line are plotted as red dots.
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Figure 4. Heat map of candidate genes’ relative expression levels for (A) stem pubescence color and (B) hilum color.
Figure 4. Heat map of candidate genes’ relative expression levels for (A) stem pubescence color and (B) hilum color.
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Table 1. Statistical analysis of stem pubescence color across the soybean natural population.
Table 1. Statistical analysis of stem pubescence color across the soybean natural population.
YearsMaterialsBrownGreyTotal
2021Cultivar84141225
Landrace121224
2022Cultivar86143229
Landrace131326
Table 2. Significant SNPs associated with the color of soybean stem pubescence in 2021 and 2022.
Table 2. Significant SNPs associated with the color of soybean stem pubescence in 2021 and 2022.
Lead SNPChromosomePosition−log10(P)
20212022
Chr01:1686559911686559912.24 11.52
Chr03:334903673334903677.46 7.46
Chr03:452337203452337208.65 8.72
Chr04:2543721425437216.82 6.84
Chr05:295262615295262617.22 7.30
Chr06:1874971661874971614.68 14.75
Chr10:25135631025135636.82 6.84
Chr12:1690295912169029598.95 9.17
Chr13:1437485013143748507.80 7.81
Chr15:1786202015178620207.22 7.30
Chr16:14402531614402538.74 8.79
Chr20:1969267620196926768.929.01
Table 3. Significant SNPs associated with the color of soybean hilum in 2021 and 2022.
Table 3. Significant SNPs associated with the color of soybean hilum in 2021 and 2022.
Lead SNPChromosomePosition−log10(P)
20212022
Chr06:187495286187495286.37 7.96
Chr06:345163596345163596.37 5.49
Chr07:381166347381166347.35 6.49
Chr08:183816018183816015.63 5.28
Chr09:457111229457111226.15 5.45
Chr11:1816644111181664415.64 5.58
Chr14:1370785314137078535.305.11
Table 4. Candidate genes for the color of soybean stem pubescence.
Table 4. Candidate genes for the color of soybean stem pubescence.
Gene IDHomologsFunctional Annotation
Glyma.03G122000AT2G40890cytochrome P450, family 98, subfamily A, polypeptide 3
Glyma.03G123200AT2G40950Basic-leucine zipper (bZIP) transcription factor family protein
Glyma.03G258700AT4G38620myb domain protein 4
Glyma.03G258800AT3G61850Dof-type zinc finger DNA binding family protein
Glyma.04G030100AT1G19630cytochrome P450, family 722, subfamily A, polypeptide 1
Glyma.06G202300AT5G07990Cytochrome P450 superfamily protein
Glyma.15G182600AT3G43190sucrose synthase 4
Table 5. Candidate genes for the color of soybean hilum.
Table 5. Candidate genes for the color of soybean hilum.
Gene IDHomologsFunctional Annotation
Glyma.06G202300AT5G07990Cytochrome P450 superfamily protein
Glyma.07G210800AT3G19130RNA-binding protein 47B
Glyma.09G232400AT5G62310AGC kinase family protein
Glyma.09G234700AT1G26670Vesicle transport v-SNARE family protein
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Zhou, M.; Wang, J.; Chen, H.; Jia, Q.; Hu, S.; Xiong, Y.; Zhang, H.; Zhang, W.; Wang, Q.; Su, C. Genome-Wide Association Study on Candidate Genes Associated with Soybean Stem Pubescence and Hilum Colors. Agronomy 2024, 14, 512. https://doi.org/10.3390/agronomy14030512

AMA Style

Zhou M, Wang J, Chen H, Jia Q, Hu S, Xiong Y, Zhang H, Zhang W, Wang Q, Su C. Genome-Wide Association Study on Candidate Genes Associated with Soybean Stem Pubescence and Hilum Colors. Agronomy. 2024; 14(3):512. https://doi.org/10.3390/agronomy14030512

Chicago/Turabian Style

Zhou, Miaomiao, Junyan Wang, Huatao Chen, Qianru Jia, Shengyan Hu, Yawen Xiong, Hongmei Zhang, Wei Zhang, Qiong Wang, and Chengfu Su. 2024. "Genome-Wide Association Study on Candidate Genes Associated with Soybean Stem Pubescence and Hilum Colors" Agronomy 14, no. 3: 512. https://doi.org/10.3390/agronomy14030512

APA Style

Zhou, M., Wang, J., Chen, H., Jia, Q., Hu, S., Xiong, Y., Zhang, H., Zhang, W., Wang, Q., & Su, C. (2024). Genome-Wide Association Study on Candidate Genes Associated with Soybean Stem Pubescence and Hilum Colors. Agronomy, 14(3), 512. https://doi.org/10.3390/agronomy14030512

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