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Article

Genome-Wide Association Analysis of Cowpea Mild Mottle Virus Resistance in Soybean Germplasms from Northeast China

by
Yameng Luan
1,
Siqi Yang
2,
Yuting Wang
1,
Yu Zhao
1,
Xiaoyun Wu
1,
Qingshan Chen
2,
Zhaoming Qi
2,
Xiaoxia Wu
3,
Weiqin Ji
1 and
Xiaofei Cheng
1,*
1
College of Plant Protection, Northeast Agricultural University, Harbin 150030, China
2
College of Agriculture, Northeast Agricultural University, Harbin 150030, China
3
College of Life Science, Northeast Agricultural University, Harbin 150030, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(3), 489; https://doi.org/10.3390/agronomy14030489
Submission received: 5 February 2024 / Revised: 17 February 2024 / Accepted: 26 February 2024 / Published: 28 February 2024
(This article belongs to the Special Issue Application of Modern Solutions against Plant Viral Disease)
Browse Figures
Versions Notes

Abstract

:
Cowpea mild mottle virus (CpMMV) is an important viral pathogen that seriously influences the yield and seed quality of soybeans worldwide. Resistance breeding is one of the most effective, economical, and environmentally safe strategies for controlling the disease caused by CpMMV. However, only few resistance genes have been identified in soybeans. In this study, the resistance of 169 soybean germplasms from Northeast China to a CpMMV strain isolated from soybean in China was evaluated, and a genome-wide association study (GWAS) was then performed to find possible resistance genes in these soybean germplasms. Nine resistant soybean germplasms were identified and two single nucleotide polymorphism sites (SNPs) were found to be closely associated with CpMMV resistance. A total number of 51 and 25 candidate genes neighboring the resistance-associated SNPs on chromosomes 6 and 12, respectively, were identified, among which one receptor-like kinase (RLK) on chromosome 6 and 2 toll-interleukin-1 receptor nucleotide-binding leucine-rich repeat receptors (TNLs) on chromosome 12 were recognized as the most probable resistance genes, respectively. Together, these data provide new insights on the resistance resources of soybeans to CpMMV, which will benefit the breeding of CpMMV-resistant soybean cultivars.

1. Introduction

Owing to its high protein and oil content, soybean [Glycine max (L.) Merr.] is recognized as a pivotal food and forage crop globally [1]. However, soybeans are susceptible to many phytopathogens, including fungi, bacteria, oomycetes, nematodes, and viruses [2]. Cowpea mild mottle virus (CpMMV) is emerging as a new viral threat to soybean production worldwide. CpMMV was first discovered in Ghana on cowpea [Vigna unguiculata (L.) Walp.] in the 1970s [3]. In the 2000s, an outbreak of CpMMV on soybeans was recorded in Brazil [4,5]. Soon, it was identified on soybeans in Iran, India, Venezuela, and other regions of Asia and the Americas [6,7,8,9,10]. In China, CpMMV was first reported in Taiwan province on cowpea and French bean (Phaseolus vulgaris L.) [11]. Later, it was recorded on soybeans in at least six provinces in the Huang-Huai-Hai region of China [12]. CpMMV shows high virulence to soybeans and induces a range of symptoms, including mosaic, ruffled, water-soak, necrosis, dwarfism, and stunting [13]. The global prevalence of CpMMV significantly compromises soybean productivity and economic value [5]. It was estimated that the infection of CpMMV can cause serious yield loss to soybeans, ranging from 174 to 638 kg·ha−1, depending on soybean cultivars [14,15].
CpMMV is a typical member of the genus Carlavirus in the family Betaflexiviridae. It is transmitted by seeds and whiteflies (Bemisia tabaci Gennadius) in a non-persistent manner in nature; however, it can also be transmitted mechanically in laboratory conditions [14,16,17]. CpMMV has a wide host range, including many legume crops such as soybean, cowpea, peanut (Arachis hypogaea L.), yardlong bean (Vigna unguiculata subsp. Sesquipedalis (L.) Verdc.), and common bean (Phaseolus vulgaris L.). The genome of CpMMV is comprised of a single-stranded positive-sense RNA molecule that is encapsulated in non-enveloped, flexuous, filamentous viral particles of approximately 1000 nm in length and 12–13 nm in diameter. The 3′ terminus of the CpMMV genome is polyadenylated, and the 5′ end is capped. The genome encodes a total number of six open reading frames (ORF) in the positive-sense strand [18,19,20]. The first ORF on the 5′ end of the genome encodes the viral RNA-dependent RNA polymerase (RdRp), the second to the fourth ORFs are partially overlapped and comprise the so-called triple-gene-block (TGB), which encode three movement-related proteins, namely TGBp1, TGBp2, and TGBp3, the fifth ORF encodes the viral coat protein (CP), and the final ORF on the 3′ end of the genome encodes a cysteine-rich ribonucleotide-binding protein (RBP) [18,19,20]. Except for the first ORF, which is translated directly from the viral genome, the other five ORFs are transcribed from at least two subgenomic RNAs (sgRNAs) [18,19,20].
Due to the high-seed transmission rate of CpMMV in some soybean cultivars and the high level of pesticide resistance of whiteflies, most traditional CpMMV management strategies have failed around the world [21,22,23,24]. The application of genetically resistant soybean cultivars is the most effective, economical, and environmentally friendly strategy for controlling CpMMV [25]. However, only a few resistant soybean varieties have been reported hitherto, namely F4C7-32 and JS335 from India, IA3023 from Puerto Rico, MLG0120 from Indonesia, and BRS133 from Brazil [21,22,23]. Moreover, only two dominant resistance genes have been preliminary mapped: the CpMMV resistance gene of soybean germplasms DS 12-5 and SL958 likely locates in the nucleotide-binding leucine-rich repeat receptors (NLRs) cluster of the linkage group H on chromosome 12 [22], while the resistance gene of soybean cultivar BRS 133 was mapped between markers Sat_308 and Satt303 on chromosome 18 [24]. Additionally, two distinct dominant genes determining the tolerance to CpMMV were identified in the soybean cultivars BRS 133 and BRSMT Pintado [13]. Previously, we reported an isolate of CpMMV from soybean in China (CpMMV-LZ), which is one of the most prevalent isolates of CpMMV in China [26]. In this study, we evaluated the resistance of 169 soybean germplasm resources from Northeast China to CpMMV-LZ and tried to discover resistance genes in these soybean germplasms by the genome-wide association study (GWAS), a widely used genetic analysis technique [27,28,29]. Two resistance-associated single nucleotide polymorphism sites (SNPs) were identified, and the most possible candidates were further determined.

2. Materials and Methods

2.1. CpMMV Isolate, Plant Materials and Culture Conditions

CpMMV-LZ was isolated from soybean leaves collected from Luzhuang Village, Fuyang City, Anhui Province, China, in August 2020 [26]. The soybean leaves that were infected by CpMMV-LV were used as the inoculum, which was preserved at −80 °C after quickly freezing in liquid nitrogen.
A total of 169 soybean germplasms were collected and used in this study, including 23 landraces, 134 elite cultivars, and 12 foreign varieties (Supplementary Table S1). All the soybean accessions were grown in an insect-proof net greenhouse under a 12 h light (26 °C) and 12 h darkness (24 °C) photoperiod with a 50% relative humidity.

2.2. Resistance Evaluation Assay

Soybean seedling leaves were sap-inoculated with CpMMV-LZ-infected inoculum, as previously described in [30]. In brief, the inoculum was homogenized in mortar in 0.01 M sodium phosphate buffer (pH 7.0). The unifoliate leaves of about 10-day-old soybean seedlings that were pre-dusted with 600-mesh carborundum powder were gently rubbed 3–4 times with the viral homogenate. The inoculated leaves were left on for 2–3 min and then rinsed with distilled water. The plants sap-inoculated with 0.01 M phosphate buffer were used as the negative control. Inoculated seedlings were moved to the insect-proof net greenhouse for symptom development. The symptoms were observed and recorded for a period of 20 days, and the severity of the disease was assessed according to previously described methods [31]. In short, the severity of viral symptoms was assessed and scored from 0 to 1.0. A score of ‘0’ represents no symptoms, while ‘0.2’ represents yellow blotches, ‘0.4’ represents mild mosaic and slight wrinkle, ‘0.6’ represents mosaic and severe wrinkle, ‘0.8’ represents leaf malformation and slight bud blight, and ‘1.0’ represents severe malformation and top necrosis. The disease index (DI) for each sample was calculated using the formula DI = Σ ( s × n ) N × S × 100% (‘s’ represents the score of the symptom of a certain infected plant, ‘n’ represents the number of affected plants in a certain category, ‘N’ represents the total number of plants, and S represents the value of the highest category). The software SPSS 20.0 was used for the calculation. Each treatment was triple-replicated, and the experiment was independently replicated three times. The frequency histogram depicting the distribution of disease index data was generated using the R software lme4 package [32].
The resistance of each soybean germplasm to CpMMV was evaluated based on the DI value, according to previously established standards [33]. Briefly, plants with DI values <20% were classified as high resistant (HR), 20–35% were classified as resistant (R), 35–50% were classified as moderately resistant (MR), while DI values between 50% and 70% were classified as susceptible (S), and DI values > 70% were classified as high susceptible (HS).

2.3. Genome-Wide Association Study

Single nucleotide polymorphism sites (SNPs) of the 169 soybean germplasm resources were retrieved from the soybean genome data stored at the Northeast Agricultural University with the ANOROAD software (Beijing, China). SNPs with a minor allele frequency (MAF) ≥ 0.05 and a missing rate ≤ 10% of the accessions were used for further analysis. The resulting high-quality SNPs were subjected to principal component analysis (PCA) using the TASSEL 5.0 software [34].
The MEGA 11 software was used to construct the phylogenetic tree of the 169 soybean germplasms based on the neighbor-joining method. The reliability of individual nodes in the phylogenetic tree was assessed by the Bootstrap resampling methods with 1000 replicates [35].
The model Y = μ + Line + Loc + (Line × Loc) + (Rep × Loc) + ε (where ‘Y’ was the DI value, ‘μ’ was the intercept, ‘Line’ was the genotypic effect, ‘Loc’ was the environment effect, ‘Rep’ was the replication, and ‘ε’ was the random effect) in SPSS 20 was used for calculating the best linear unbiased prediction (BLUP) values [32]. GWAS was conducted using the mixed linear model (MLM) based on the selected high-quality SNPs markers, and BLUP values of DI with the relatedness and relatedness matrix serving as covariates [36]. The BLUP values were generated from DI values using the R software lme4 package [32]. Bonferroni correction was used to calculate the p-values. The quantile–quantile plot and Manhattan plot were generated by package ggplot2 in R software [37].

2.4. Candidate Genes Prediction and Analysis

2.4.1. Candidate Genes Prediction and Functional Analysis

The candidate genes were predicted according to the reference genome of the soybean cultivar Williams 82 (Williams 82.a2.v1) from the Soybase (https://www.soybase.org/; accessed on 3 January 2022). The function of candidate genes was predicted using online databases QuickGO (https://www.ebi.ac.uk/QuickGO; accessed on 20 February 2022), Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html; accessed on 20 February 2022), NCBI (https://www.ncbi.nlm.nih.gov; accessed on 20 February 2022), and WeGo (http://wego.genomics.org.cn/cgi-bin/wego/index.pl; accessed on 20 February 2022).

2.4.2. Haplotype Analysis

The linkage disequilibrium (LD) analysis of candidate genes in 169 germplasm resources was conducted using the Haploview software [38]. The haplotype analysis was conducted by Dnasp5 software [39]. Haplotypes containing more than 5% of the population are considered predominant haplotypes.

2.5. Statistical Analysis

Statistical analyses were conducted using Microsoft Excel 2016 or SPSS 20.0. Differences in results with p values less than 0.05 were considered significant.

3. Results

3.1. Resistance Evaluation of Soybean Germplasms

A total number of 169 soybean germplasms were mechanically inoculated by CpMMV-LZ to search for resistant soybean germplasms. These soybean germplasms are part of the soybean germplasm collection of the Soybean Research Institute at Northeast Agricultural University, and their genomes have already been sequenced [40]. At 20 days post-inoculation (dpi), seedlings of some soybean germplasms displayed typical viral symptoms, such as mosaic, vein clearing, wrinkles, and systemic necrosis (Figure 1). According to the DI criteria [33], the 169 soybean germplasms were classified into 5 DI categories: highly resistant (HR), moderately resistant (MR), resistant (R), susceptible (S), and highly susceptible (HS). In detail, 94.67% (160/169) of tested soybean germplasms were categorized as S (147/169, 86.98%) or HS (13/169, 7.69%). Only 9 of the 169 soybean germplasms (5.33%) were scored as R to HR, among which 2 germplasms (Heihe 33 and Jiyu 94) were scored as HR, 4 germplasms (Hefeng 47, Yongjizaodou, Suinong 43, and Heinong 56) were scored as MR, and 3 germplasms (Злoтoвлacкa, T219H, and NO10) were scored as R (Supplementary Table S1).
We further calculated the DI values of the 169 soybean germplasms. Results showed that the DI value ranged from 16.67 to 85.00, with an average value of 59.53 and a median value of 58.33. The Shapiro–Wilk’s test confirmed the DI values of the 169 soybean germplasms had a normal distribution with a kurtosis of 0.07 and a skewness of 0.16 (Figure 2). Taken together, these data reveal that the germplasms display sufficient diversity in their resistance to CpMMV and are suitable for further GWAS.

3.2. SNP Characterization and Population Structure Analyses

A total of 52,357 high-quality SNPs with a minor allele frequency (MAF) ≥ 0.05 and a missing call rate ≤ 10% were retrieved from the genome sequences of these soybean germplasms and were used for subsequent GWAS. Among these SNPs, G to A substitution was the most abundant (18.67%), while C to G substitution was the scarcest (2.60%) (Figure 3). The number of SNPs per chromosome ranged from 664 (Chromosome 11) to 4044 (Chromosome 4) (Supplementary Table S2).
To evaluate the population structure of the 169 soybean germplasms, we performed population structure analyses based on the obtained 52,357 SNPs. Principal component analysis (PCA) showed that the 169 soybean germplasms could be divided into two major groups, namely PCA1 and PCA2. Despite partially overlapping, the differences between PCA1 and PCA2 were statistically significant (p ≤ 0.05; Figure 4a). We also constructed a neighbor-joining (NJ) phylogenetic tree based on the genotypes of the 169 soybean germplasms [35]. The results showed that the 169 soybean germplasms were divided into three major groups, namely groups A, B, and C (Figure 4b). Group A and B contained only 7 and 5 soybean germplasms, respectively, while Group C contained most (157 out of 169) of the soybean germplasms, which could be further divided into at least 12 clusters, namely C1 to C12 (Figure 4b). We then located the nine resistant soybean germplasms in the phylogenetic tree. We found that, although all nine resistant soybean germplasms located in Group A, they were placed in different groups, except for two resistant germplasms that were phylogenetically related (Figure 4b). Taken together, these data suggest that the 169 soybean germplasms contain sufficient genetic diversity for subsequent GWAS.

3.3. Identification of CpMMV Resistance-Associated SNPs by GWAS

We then performed a GWAS based on the phenotypic BLUP values of DI and the 52,357 SNPs to find the CpMMV resistance-associated SNPs. Two SNPs, one on chromosome 6 (SNP_Chr6_2025717; p = 1.36 × 10−7) and another on chromosome 12 (SNP_Chr12_14708858; p = 5.04 × 10−7), were found to be significantly associated with the resistance against CpMMV (Figure 5a). A subsequent quantile–quantile plot indicated that these two SNPs were highly associated with CpMMV resistance in these soybean germplasms, with an overall p value of 9.55 × 10−7 (Figure 5b). Taken together, these data suggest that there are at least two genes contribute to CpMMV resistance in these soybean germplasms.

3.4. Candidate Genes Prediction

Based on the GWAS results, we hypothesized that these were two CpMMV resistance genes genetically co-segregated with SNP_Chr6_2025717 and SNP_Chr12_14708858, respectively. To determine the suitable chromosomal region for candidate gene prediction, we performed a linkage disequilibrium (LD) analysis. The results showed that the mean LD rate (r2) is about 0.84 (Figure 6). Theoretically, the physical decay distance of LD is equal to half the maximum value of r2 (equal to ~240 kb). To avoid the loss of any candidate, we selected a 250-kb interval upstream and downstream of the significant SNP as the resistance-associated genetic region. According to the reference genome of soybean cultivar Williams 82, fifty-one and twenty-five genes were found in the genetic regions harboring SNP_Chr6_2025717 and SNP_Chr12_14708858, respectively (Supplementary Table S3). The resistance of plants against many phytopathogens, including viruses, was mostly governed by dominant nucleotide-binding leucine-rich repeat receptors (NLRs) [41]. Thus, protein domain prediction was performed to find the genes encoding NLRs in these candidates. We identified a cluster of three genes that encode NLR in the genetic regions neighboring the SNP_Chr12_14708858 on chromosome 12, namely Glyma.12G131900, Glyma.12G132000, and Glyma.12G132200. Further analyses showed that the protein encoded by Gmyma.12G131900 was homologous to the N protein that confers a hypersensitive response (HR) at the site of tobacco mosaic virus (TMV) infection, while the proteins encoded by Glyma.12G132000 and Glyma.12G132200 belonged to the toll-interleukin-1 receptor NLR (TNL) class, which were homologous to soybean GmRUN1 that might confer resistance to soybean mosaic virus (SMV) [42,43]. Interestingly, no NLR candidate was found in the genetic region neighboring SNP_Chr6_2025717 on chromosome 6 (Supplementary Table S3). Instead, we found a gene that encodes a protein containing a leucine-rich repeat (LRR) domain and a kinase domain in the resistance-associated genetic region (Glyma.06G026600), which was homologous to Arabidopsis thaliana cell-surface LRR receptor-like kinases (LRR-RLKs) (Supplementary Table S3).

3.5. Haplotype Analyses

Haplotype polymorphism analyses showed that there were 18 haplotypes of Glyma.12G132000 in the 169 soybean germplasms (Table 1). The 3 predominant haplotypes, Gm12_Hap2a, Gm12_Hap3a, and Gm12_Hap5a, accounted for 75.74%, 7.69%, and 5.33% of the 169 soybean germplasms, respectively. Gm12_Hap2a belonged to the S haplotype, while Gm12_Hap3a and Gm12_Hap5a belonged to the MR haplotypes. There were 9, 14, and 5 different SNPs between haplotypes Gm12_Hap2a and Gm12_Hap3a, Gm12_Hap2a and Gm12_Hap5a, and Gm12_Hap3a and Gm12_Hap5a, respectively (Supplementary Table S4). Statistical analyses showed that Gm12_Hap2a was significantly different from Gm12_Hap3a and Gm12_Hap5a (Figure 7a). Taken together, these results indicate that Glyma.12G132000 may participate in the resistance to CpMMV.
Twenty-three haplotypes of Glyma.12G132200 were identified in the 169 soybean germplasms (Table 1). The 3 predominant haplotypes, Gm12_Hap2b, Gm12_Hap3b, and Gm12_Hap8b, accounted for 60.36%, 12.43%, and 5.92% of the 169 soybean germplasms, respectively. The three primary haplotypes were all belonging to R haplotypes, with average DI values of 49.76, 48.87, and 49.93, respectively. Statistical analyses showed that these haplotypes were significantly different from each other (p < 0.05) (Figure 7b). Moreover, there were 1, 7, and 8 different SNPs between haplotypes Gm12_Hap2b and Gm12_Hap3b, Gm12_Hap3b and Gm12_Hap8b, and Gm12_Hap2b and Gm12_Hap8b, respectively (Supplementary Table S5). Thus, these results suggest that Glyma.12G132200 may be involved in CpMMV resistance.
However, there was no SNP haplotype diversity of Gmyma.12G131900 in the 169 soybean germplasms, indicating that Gmyma.12G131900 may not directly contribute to soybean’s resistance against CPMMV infection.
Haplotype polymorphism analysis was also performed for Glyma.06G026600. The results showed that there were at least 16 haplotypes of Glyma.06G026600 in the 169 soybean germplasms. The 3 most prevalent haplotypes (Gm6_Hap1, Gm6_Hap2, and Gm6_Hap6) accounted for 64.50%, 6.51%, and 5.92% of the 169 soybean germplasms, respectively (Table 1). Gm6_Hap2 and Gm6_Hap6 were identified as MR haplotypes with an average DI value of 49.58 and 48.56, respectively (Supplementary Table S6). Gm6_Hap1 was an S haplotype with an average DI value of 52.30. Notably, significant differences were observed between the S haplotype (Gm6_Hap1) and MR haplotypes (Gm6_Hap2 and Gm6_Hap6), while no significant difference was found between the Gm6_Hap2 and Gm6_Hap6 (Figure 7c). Interestingly, only one distinct SNP was detected between S haplotype (Gm6_Hap1) and the MR haplotypes (Gm6_Hap2 and Gm6_Hap6), indicating that Glyma.06G026600 may be associated with the resistance to CpMMV infection (Supplementary Table S6).

4. Discussion

The global spread of CpMMV has caused serious yield and economic losses to soybean, cowpea, and other crops [5,15]. Several resistance screening studies have been carried out, but only a few resistant soybean germplasms have been identified [21,22,26]. In this study, we further evaluated the resistance of 169 soybean germplasms from Northeast China to CpMMV. Our results showed that only 9 of the 169 soybean germplasms (5.33%) displayed an R-to-HR level of resistance to CpMMV. These data indicate that there may be only a few resistant soybean germplasms, and most soybean cultivars are susceptible to CpMMV. Noticeably, we found that the infection with CpMMV caused severe symptoms on some soybean germplasm, which may cause a complete loss of yield (Figure 1). Thus, special attention should be paid to avoid the outbreak of CpMMV on soybean in China. Nevertheless, our data revealed that the nine R, MR, or HR soybean germplasms, namely, Heihe 33, Jiyu 94, Hefeng 47, Yongjizaodou, Suinong 43, and Heinong 56, Злoтoвлacкa, T219H, and NO10, can serve as important germplasm resources for breeding of CpMMV-resistant soybean varieties. The 169 germplasms only represent a small part of the soybean germplasm resources available in China. Thus, further studies are needed to find new CpMMV-resistant soybean resources.
At present, only two CpMMV resistance-associated genetic loci have been reported: the dominant resistance gene in the resistance varieties DS 12-5 and SL958 was mapped to the linkage group H between SSR markers Satt635 and Uo8405 on Chromosome 12 [22], and the resistance gene of the soybean cultivar BRS 133 was mapped on chromosome 18 of the soybean genome, between markers Sat_308 and Satt303 [24]. In this study, we further identified 2 CpMMV resistance genes that are closely associated with SNP_Chr6_2025717 on chromosome 6 and SNP_Chr12_14708858 on chromosome 12 from the 169 soybean germplasms by GWAS. The relationship between the dominant resistance gene in the resistance varieties DS 12-5 and SL958 and the resistance gene co-segregated with SNP_Chr12_14708858 on chromosome 12 is unknown at the present. Nevertheless, it will be interesting to investigate whether they are homologous to each other. The CpMMV resistance gene neighboring SNP_Chr6_2025717 on chromosome 6 represents a new resistance locus in soybean. Thus, this gene provides an alternative genetic resource for the breeding of CpMMV-resistant soybean varieties. Phylogenetic analysis showed that the nine resistant soybean resources located in different phylogenetic clusters, except for two soybean germplasms (Figure 4b), indicating that these two resistance genes may not have undergone artificial selection, but were randomly retained in a small number of soybean resources during the cultivation of soybean. Nevertheless, whether there are other resistance genes in soybeans is an interesting area for further investigation.
The resistance of plants to viruses can be determined by many mechanisms [44]. Among these mechanisms, TNLs play crucial roles in effector-triggered antiviral immunity (ETI) [45]. Lots of dominant resistance genes in plants against viruses belong to the TNL family, such as the N from Nicotiana glutinosa and RT4–4 from Phaseolus vulgaris that are involved in TMV and cucumber mosaic virus (CMV) resistances, respectively [43,46]. In the resistance-associated genetic region on chromosome 12, three candidate genes encoding NTLs were identified. However, there was no haplotype of Gmyma.12G131900, indicating that it is not directly involved in the resistance against CpMMV. Thus, it is possible that the resistance of some soybean germplasms tested in this study is controlled by one of the other two NTLs, which requires further investigation. For the resistance-associated genetic region on chromosome 6, we did not find any gene that encodes an NLR protein; instead, we only found an LRR-RLK. The product of this gene is homologous to MUSTACHES-LIKE (MUL) in A. thaliana [47]. It is possible that the resistance in the genetic region of chromosome 6 is controlled by an LRR-RLK; alternatively, it is possible that the resistance is controlled by other types of resistance genes, such as an atypical dominant resistance gene or a recessive resistance gene. It is also possible that the resistant soybean germplasms contain an insertion in the region that is missing in the soybean reference genome (Williams 82.a2.v1) that was used for candidate gene prediction. Nevertheless, further studies are needed to identify the real resistance gene in the two genetic regions and to uncover the mechanisms of the resistance.
Although GWAS is a widely-used and reliable method for discovering the genetic variants that are statistically associated with a specific trait, it also contains some limitations. For instance, the GWAS results can be significantly affected by the population size; they may also miss genetic variants with only a mild to modest effect; and rare mutations cannot be detected by the SNP-based GWAS [48]. Thus, further studies are needed to confirm whether the resistance of the nine resistant soybean germplasms is indeed due to the two genetic loci identified in this study or caused by other resistance that was missed by the GWAS. Nevertheless, our data provide valuable knowledge concerning CpMMV resistance in soybeans and genetic resources for the development of CpMMV-resistant soybean varieties.

5. Conclusions

In conclusion, 9 CpMMV-resistant soybean germplasms were identified from the 169 soybean germplasms, 2 CpMMV resistance-associated SNPs on chromosome 6 and 12 were found by GWAS, and possible resistance genes were also predicted.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14030489/s1, Supplementary Table S1: The names and disease index categories of all 169 soybean germplasms; Supplementary Table S2: The number of SNPs on each chromosome; Supplementary Table S3: Information of candidate genes; Supplementary Table S4: The SNP haplotype of Glyma.12G132000; Supplementary Table S5: SNP haplotype of Glyma.12G132200; Supplementary Table S6: SNP haplotype of Glyma.06G02660.

Author Contributions

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

Funding

This research was funded by the Natural Science Foundation of Heilongjiang Province of China (grant no. TD2022C003), and the Leading Talent Support Program of the Northeast Agricultural University (grant no. NEAU2023QNLJ-010).

Data Availability Statement

All data are available within the Article and Supplementary Files. All constructs are available upon request.

Acknowledgments

We would like to express special appreciation to Dawei Xin from the Northeast Agricultural University for the help in data analysis.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Nadeem, M.; Chen, A.; Hong, H.; Li, D.; Li, J.; Zhao, D.; Wang, W.; Wang, X.; Qiu, L. GmMs1 encodes a kinesin-like protein essential for male fertility in soybean (Glycine max L.). J. Integr. Plant Biol. 2021, 63, 1054–1064. [Google Scholar] [CrossRef]
  2. Hartman, G.L.; Rupe, J.C.; Sikora, E.J.; Domier, L.L.; Davis, J.A.; Steffey, K.L. Compendium of Soybean Diseases and Pests, 5th ed.; APS Press: St. Paul, MN, USA, 2015; p. 201. [Google Scholar]
  3. Brunt, A.A.; Kenten, R.H. Cowpea mild mottle, a newly recognized virus infecting cowpeas (Vigna unguiculata) in Ghana. Ann. Appl. Biol. 1973, 74, 67–74. [Google Scholar] [CrossRef]
  4. Almeida, A.M.R.; Piuga, F.F.; Kitajima, E.W.; Gaspar, J.O.; Valentin, N.; Benato, L.C.; Marin, S.R.; Binneck, E.; De Oliveira, T.; Belintani, P. Necrose da haste da soja. ProBiota Sér Doc. 2003, 221. Available online: https://ainfo.cnptia.embrapa.br/digital/bitstream/CNPSO-2010/24143/1/doc221.pdf (accessed on 25 February 2024).
  5. Zanardo, L.G.; Silva, F.N.; Bicalho, A.A.C.; Urquiza, G.P.C.; Lima, A.T.M.; Almeida, A.M.R.; Zerbini, F.M.; Carvalho, C.M. Molecular and biological characterization of cowpea mild mottle virus isolates infecting soybean in Brazil and evidence of recombination. Plant Pathol. 2014, 63, 456–465. [Google Scholar] [CrossRef]
  6. Ghorbani, S.; Shahraeen, N.; Elahinia, S. Serodiagnosis of cowpea (Vigna unguiculata) viruses in Gilan province, Iran. Iran. J. Virol. 2008, 2, 30–33. [Google Scholar] [CrossRef]
  7. Tavassoli, M.; Shahraeen, N.; Ghorbani, S. Detection and some properties of cowpea mild mottle virus isolated from soybean in Iran. Pak. J. Biol. Sci. 2008, 11, 2624–2628. [Google Scholar] [CrossRef]
  8. Brito, M.; Fernández-Rodríguez, T.; Garrido, M.J.; Mejías, A.; Romano, M.; Marys, E. First report of cowpea mild mottle carlavirus on yardlong bean (Vigna unguiculata subsp. sesquipedalis) in Venezuela. Viruses 2012, 4, 3804–3811. [Google Scholar] [CrossRef] [PubMed]
  9. Rosario, K.; Capobianco, H.; Ng, T.F.F.; Breitbart, M.; Polston, J.E. RNA viral metagenome of whiteflies leads to the discovery and characterization of a whitefly-transmitted carlavirus in North America. PLoS ONE 2014, 9, e86748. [Google Scholar] [CrossRef] [PubMed]
  10. Celli, M.G.; Perotto, M.C.; Merino, M.C.; Nome, C.F.; Flores, C.R.; Conci, V.C. First report of cowpea mild mottle virus in chia (Salvia hispanica). Crop Prot. 2016, 89, 1–5. [Google Scholar] [CrossRef]
  11. Chang, C.; Chien, L.; Tsai, C.; Lin, Y.; Cheng, Y. First report of cowpea mild mottle virus in cowpea and French bean in Taiwan. Plant Dis. 2013, 97, 1001. [Google Scholar] [CrossRef] [PubMed]
  12. Wei, Z.; Mao, C.; Jiang, C.; Zhang, H.; Chen, J.; Sun, Z. Identification of a new genetic clade of cowpea mild mottle virus and characterization of its interaction with soybean mosaic virus in co-infected soybean. Front. Microbiol. 2021, 12, 650773. [Google Scholar] [CrossRef] [PubMed]
  13. Arias, C.A.A.; Almeida, A.M.R.; Mituti, T.; Kitajima, E.W. Inheritance of tolerance to cowpea mild mottle virus in soybean. Crop Breed. Appl. Biotechnol. 2015, 15, 132–138. [Google Scholar] [CrossRef]
  14. Zanardo, L.G.; Carvalho, C.M. Cowpea mild mottle virus (Carlavirus, Betaflexiviridae): A review. Trop. Plant Pathol. 2017, 42, 417–430. [Google Scholar] [CrossRef]
  15. Barreto da Silva, F.; Muller, C.; Bello, V.H.; Watanabe, L.F.M.; Rossitto De Marchi, B.; Fusco, L.M.; Ribeiro-Junior, M.R.; Minozzi, G.B.; Vivan, L.M.; Tamai, M.A.; et al. Effects of cowpea mild mottle virus on soybean cultivars in Brazil. PeerJ 2020, 8, e9828. [Google Scholar] [CrossRef]
  16. Marubayashi, J.M.; Yuki, V.A.; Wutke, E.B. Transmission of the cowpea mild mottle virus by whitefly Bemisia tabaci biotype B for plants of beans and soy. Summa Phytopathol. 2010, 36, 158–160. [Google Scholar] [CrossRef]
  17. Sutrawati, M.; Hidayat, S.H.; Suastika, G.; Purnomo Wahyu Sukarno, B.; Nurmansyah, A. Seed-transmission of cowpea mild mottle virus on several varieties of soybean in Indonesia. Biodiversitas 2021, 22, 4182–4185. [Google Scholar] [CrossRef]
  18. Martelli, G.P.; Adams, M.J.; Kreuze, J.F.; Dolja, V.V. Family Flexiviridae: A case study in virion and genome plasticity. Ann. Rev. Phytopathol. 2007, 45, 73–100. [Google Scholar] [CrossRef]
  19. Menzel, W.; Winter, S.; Vetten, H.J. Complete nucleotide sequence of the type isolate of cowpea mild mottle virus from Ghana. Arch. Virol. 2010, 155, 2069–2073. [Google Scholar] [CrossRef] [PubMed]
  20. Lefkowitz, E.J.; Dempsey, D.M.; Hendrickson, R.C.; Orton, R.J.; Siddell, S.G.; Smith, D.B. Virus taxonomy: The database of the International Committee on Taxonomy of Viruses (ICTV). Nucleic Acids Res. 2018, 46, D708–D717. [Google Scholar] [CrossRef]
  21. Suryanto, A.; Kuswanto, K.; Sitompul, S.; Kasno, A. Estimation of number and genes actions of CpMMV (cowpea mild mottle virus) disease resistance genes on soybean crop. IOSR J. Agric. Vet. Sci. 2014, 7, 51–56. [Google Scholar] [CrossRef]
  22. Cheruku, D.; Lal, S.K.; Talukdar, A.; Mandal, B.; Singh, R. Inheritance and mapping of resistance against cowpea mild mottle virus strain D1 in soybean. Plant Breed. 2017, 136, 155–160. [Google Scholar] [CrossRef]
  23. Brace, R.C.; Fehr, W.R.; Graham, M. Inheritance and molecular mapping of an allele providing resistance to a cowpea mild mottle-like virus in soybean. Crop Sci. 2012, 52, 2109–2114. [Google Scholar] [CrossRef]
  24. Oliveira, M.A.R.D.; Carpentieri-Pípolo, V.; Nora, T.D.; Vieira, E.S.N.; Schuster, I. Rbc2, a new dominant gene for resistance of soybean to cowpea mild mottle virus: Inheritance and mapping. Crop Breed. Appl. Biotechnol. 2018, 18, 169–175. [Google Scholar] [CrossRef]
  25. Gómez, P.; Rodríguez-Hernández, A.M.; Moury, B.; Aranda, M.A. Genetic resistance for the sustainable control of plant virus diseases: Breeding, mechanisms and durability. Eur. J. Plant Pathol. 2009, 125, 1–22. [Google Scholar] [CrossRef]
  26. Yang, S.; Liu, Y.; Wu, X.; Cheng, X.; Wu, X. Synonymous codon pattern of cowpea mild mottle virus sheds light on its host adaptation and genome evolution. Pathogens 2022, 11, 419. [Google Scholar] [CrossRef] [PubMed]
  27. Xiong, H.; Chen, Y.; Pan, Y.B.; Wang, J.; Lu, W.; Shi, A. A genome-wide association study and genomic prediction for Phakopsora pachyrhizi resistance in soybean. Front. Plant Sci. 2023, 14, 1179357. [Google Scholar] [CrossRef] [PubMed]
  28. Che, Z.; Zhang, S.; Pu, Y.; Yang, Y.; Liu, H.; Yang, H.; Wang, L.; Zhang, Y.; Liu, B.; Zhang, H.; et al. A novel soybean malectin-like receptor kinase-encoding gene, GmMLRK1, provides resistance to soybean mosaic virus. J. Exp. Bot. 2023, 74, 2692–2706. [Google Scholar] [CrossRef] [PubMed]
  29. Xie, H.; Lin, C.; Lu, W.; Han, Z.; Wei, D.; Huo, X.; Li, T.; Zhang, J.; He, Y.; Chen, C.; et al. OsBLS6.2: A rice bacterial leaf streak resistance gene identified by GWAS and RNA-seq. Crop J. 2023, 11, 1862–1871. [Google Scholar] [CrossRef]
  30. Hull, R. Mechanical inoculation of plant viruses. Curr. Protoc. Microbiol. 2009, 13, 16B-6. [Google Scholar] [CrossRef]
  31. Zhi, H.; Gai, J. Performances and germplasm evaluation of quantitative resistance to soybean mosaic virus in soybeans. Sci. Agric. Sin. 2004, 3, 247–253. [Google Scholar]
  32. Merk, H.L.; Yarnes, S.C.; Van Deynze, A.; Tong, N.; Menda, N.; Mueller, L.A.; Mutschler, M.A.; Loewen, S.A.; Myers, J.R.; Francis, D.M. Trait diversity and potential for selection indices based on variation among regionally adapted processing tomato germplasm. J. Am. Soc. Hortic. Sci. 2012, 137, 427–437. [Google Scholar] [CrossRef]
  33. Zubaidah, S.; Kuswantoro, H. Foliar symptoms recovery: Developing scoring technique for assessment of soybean resistance to CpMMV (cowpea mild mottle virus). J. Biol. Res. 2016, 21, 85–89. [Google Scholar] [CrossRef]
  34. Bradbury, P.J.; Zhang, Z.; Kroon, D.E.; Casstevens, T.M.; Ramdoss, Y.; Buckler, E.S. TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics 2007, 23, 2633–2635. [Google Scholar] [CrossRef]
  35. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  36. Zhang, Z.; Ersoz, E.; Lai, C.-Q.; Todhunter, R.J.; Tiwari, H.K.; Gore, M.A.; Bradbury, P.J.; Yu, J.; Arnett, D.K.; Ordovas, J.M.; et al. Mixed linear model approach adapted for genome-wide association studies. Nat. Genet. 2010, 42, 355–360. [Google Scholar] [CrossRef]
  37. Turner, S. Annotated Manhattan plots and QQ plots for GWAS using R, Revisited. Nat. Prec. 2011. [Google Scholar] [CrossRef]
  38. Barrett, J.C. Haploview: Visualization and analysis of SNP genotype data. Cold Spring Harb. Protoc. 2009, 2009, pdb.ip71. [Google Scholar] [CrossRef]
  39. Librado, P.; Rozas, J. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 2009, 25, 1451–1452. [Google Scholar] [CrossRef]
  40. Qi, Z.; Guo, C.; Li, H.; Qiu, H.; Li, H.; Jong, C.; Yu, G.; Zhang, Y.; Hu, L.; Wu, X. Natural variation in fatty acid 9 is a determinant of fatty acid and protein content. Plant Biotechnol. J. 2023. [Google Scholar] [CrossRef]
  41. Ngou, B.P.M.; Ding, P.; Jones, J.D.G. Thirty years of resistance: Zig-zag through the plant immune system. Plant Cell 2022, 34, 1447–1478. [Google Scholar] [CrossRef]
  42. Diao, P.; Sun, H.; Bao, Z.; Li, W.; Niu, N.; Li, W.; Wuriyanghan, H. Expression of an antiviral gene GmRUN1 from soybean is regulated via intron-mediated enhancement (IME). Viruses 2021, 13, 2032. [Google Scholar] [CrossRef]
  43. Whitham, S.; Dinesh-Kumar, S.P.; Choi, D.; Hehl, R.; Corr, C.; Baker, B. The product of the tobacco mosaic virus resistance gene N: Similarity to toll and the interleukin-1 receptor. Cell 1994, 78, 1101–1115. [Google Scholar] [CrossRef]
  44. Wu, X.; Valli, A.; García, J.A.; Zhou, X.; Cheng, X. The tug-of-war between plants and viruses: Great progress and many remaining questions. Viruses 2019, 11, 203. [Google Scholar] [CrossRef]
  45. Yuan, M.; Jiang, Z.; Bi, G.; Nomura, K.; Liu, M.; Wang, Y.; Cai, B.; Zhou, J.-M.; He, S.Y.; Xin, X.F. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature 2021, 592, 105–109. [Google Scholar] [CrossRef]
  46. Seo, Y.S.; Rojas, M.R.; Lee, J.Y.; Lee, S.W.; Jeon, J.S.; Ronald, P.; Lucas, W.J.; Gilbertson, R.L. A viral resistance gene from common bean functions across plant families and is up-regulated in a non-virus-specific manner. Proc. Natl. Acad. Sci. USA 2006, 103, 11856–11861. [Google Scholar] [CrossRef]
  47. Xun, Q.; Wu, Y.; Li, H.; Chang, J.; Ou, Y.; He, K.; Gou, X.; Tax, F.E.; Li, J. Two receptor-like protein kinases, MUSTACHES and MUSTACHES-LIKE, regulate lateral root development in Arabidopsis thaliana. New Phytol. 2020, 227, 1157–1173. [Google Scholar] [CrossRef]
  48. Tam, V.; Patel, N.; Turcotte, M.; Bossé, Y.; Paré, G.; Meyre, D. Benefits and limitations of genome-wide association studies. Nat. Rev. Genet. 2019, 20, 467–484. [Google Scholar] [CrossRef]
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Figure 1. The typical symptoms of CpMMV on different soybean germplasms. Photos were taken at 20 days post-inoculation. (a) Healthy soybean; (b) yellow blotches; (c) yellowing; (d) mosaic and wrinkle; (e) leaf malformation and bud blight; (f) malformation and necrosis.
Figure 1. The typical symptoms of CpMMV on different soybean germplasms. Photos were taken at 20 days post-inoculation. (a) Healthy soybean; (b) yellow blotches; (c) yellowing; (d) mosaic and wrinkle; (e) leaf malformation and bud blight; (f) malformation and necrosis.
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Figure 2. The distribution of the DI values of 169 soybean germplasms. The X- and Y-axis are DI values and corresponding frequency. The orange curve is theoretical frequency distribution curve of normal distribution and the blue columns indicate the number of soybean germplasms with indicated DI values.
Figure 2. The distribution of the DI values of 169 soybean germplasms. The X- and Y-axis are DI values and corresponding frequency. The orange curve is theoretical frequency distribution curve of normal distribution and the blue columns indicate the number of soybean germplasms with indicated DI values.
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Figure 3. Histogram of substitution types. The X- and Y-axis are the type of substitution and frequency, respectively. The number on the top of each column indicates the percentage of each type of substitution.
Figure 3. Histogram of substitution types. The X- and Y-axis are the type of substitution and frequency, respectively. The number on the top of each column indicates the percentage of each type of substitution.
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Figure 4. Population structure of the 169 soybean accessions. (a) The principal component plot. The magenta and cyan dots representing two distinct subgroups (PCA1 and PCA2). The X- and Y-axis are two principal components (PC1 and PC2). The magenta and cyan spots indicates the locations of the soybean germplasms. (b) Neighbor-joining (NJ) phylogenetic tree of the 169 soybean germplasms constructed using the SNP data. The tree was collapsed at ≥80% bootstrap confidences. The branches of the nine resistant soybean germplasms are shown in red.
Figure 4. Population structure of the 169 soybean accessions. (a) The principal component plot. The magenta and cyan dots representing two distinct subgroups (PCA1 and PCA2). The X- and Y-axis are two principal components (PC1 and PC2). The magenta and cyan spots indicates the locations of the soybean germplasms. (b) Neighbor-joining (NJ) phylogenetic tree of the 169 soybean germplasms constructed using the SNP data. The tree was collapsed at ≥80% bootstrap confidences. The branches of the nine resistant soybean germplasms are shown in red.
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Figure 5. Results of GWAS. (a) Circular Manhattan plot for CpMMV resistance. The outer circle represents the single nucleotide polymorphism sites (SNPs) density of each chromosome. The line that crosses the circle represents the -log10 (p value) for each genetic variant. The most significantly associated SNPs are highlighted in green (p ≤ 0.01) and red (p ≤ 0.001), respectively. Chr1 to Chr20 indicate the twenty soybean chromosomes. (b) Quantile–quantile plot showing the significant SNP associations (p < 0.001) with CpMMV resistance. The X-axis is expected value of −log10(p), and Y-axis is observed value of −log10(p). The red horizontal line represents the significant p-value threshold (p = 9.55 × 10−7).
Figure 5. Results of GWAS. (a) Circular Manhattan plot for CpMMV resistance. The outer circle represents the single nucleotide polymorphism sites (SNPs) density of each chromosome. The line that crosses the circle represents the -log10 (p value) for each genetic variant. The most significantly associated SNPs are highlighted in green (p ≤ 0.01) and red (p ≤ 0.001), respectively. Chr1 to Chr20 indicate the twenty soybean chromosomes. (b) Quantile–quantile plot showing the significant SNP associations (p < 0.001) with CpMMV resistance. The X-axis is expected value of −log10(p), and Y-axis is observed value of −log10(p). The red horizontal line represents the significant p-value threshold (p = 9.55 × 10−7).
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Figure 6. Linkage disequilibrium (LD) of soybean germplasm resources. The X-axis is the mean LD rate estimated as r2 and the Y-axis is physical distance (bp).
Figure 6. Linkage disequilibrium (LD) of soybean germplasm resources. The X-axis is the mean LD rate estimated as r2 and the Y-axis is physical distance (bp).
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Figure 7. Variance of haplotypes. Variance analysis of the predominant haplotypes, Glyma.12G132000 (a), Glyma.12G132200 (b), and Glyma.06G026600 (c). Y-axis indicates the DI values. Statistical analyses were performed using SPSS 20.0; * and ** represent p < 0.05 and 0.01, respectively.
Figure 7. Variance of haplotypes. Variance analysis of the predominant haplotypes, Glyma.12G132000 (a), Glyma.12G132200 (b), and Glyma.06G026600 (c). Y-axis indicates the DI values. Statistical analyses were performed using SPSS 20.0; * and ** represent p < 0.05 and 0.01, respectively.
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Table 1. Haplotypes of candidate genes in soybean natural population.
Table 1. Haplotypes of candidate genes in soybean natural population.
Candidate GenesHaplotype NumberHaplotype NamesGermplasm NumberHaplotype Ratios
Glyma.06G02660016Gm6_Hap110964.50%
Gm6_Hap2116.51%
Gm6_Hap6105.92%
Glyma.12G13200018Gm12_Hap2a12875.74%
Gm12_Hap3a137.69%
Gm12_Hap5a95.33%
Glyma.12G13220023Gm12_Hap2b10260.36%
Gm12_Hap3b2112.43%
Gm12_Hap8b105.92%
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Luan, Y.; Yang, S.; Wang, Y.; Zhao, Y.; Wu, X.; Chen, Q.; Qi, Z.; Wu, X.; Ji, W.; Cheng, X. Genome-Wide Association Analysis of Cowpea Mild Mottle Virus Resistance in Soybean Germplasms from Northeast China. Agronomy 2024, 14, 489. https://doi.org/10.3390/agronomy14030489

AMA Style

Luan Y, Yang S, Wang Y, Zhao Y, Wu X, Chen Q, Qi Z, Wu X, Ji W, Cheng X. Genome-Wide Association Analysis of Cowpea Mild Mottle Virus Resistance in Soybean Germplasms from Northeast China. Agronomy. 2024; 14(3):489. https://doi.org/10.3390/agronomy14030489

Chicago/Turabian Style

Luan, Yameng, Siqi Yang, Yuting Wang, Yu Zhao, Xiaoyun Wu, Qingshan Chen, Zhaoming Qi, Xiaoxia Wu, Weiqin Ji, and Xiaofei Cheng. 2024. "Genome-Wide Association Analysis of Cowpea Mild Mottle Virus Resistance in Soybean Germplasms from Northeast China" Agronomy 14, no. 3: 489. https://doi.org/10.3390/agronomy14030489

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