Causal variant loci and protein-coding genes for soybean bacterial pustule resistance in the flowering stage

Pollyanna Capobiango da Fonseca Dalton de Oliveira de Oliveira Ferreira Túlio Morgan Tiago Antônio de Oliveira e Mendes Felipe Lopes da Silva About the authors

Abstract

Bacterial pustule is an important soybean disease caused by Xanthomonas citri pv. glycines, but information about genetic resistance to this pathogen is scarce. This study aimed to investigate soybean genetic resistance to bacterial pustule in the flowering stage through association analysis, characterization of candidate SNP markers, and identification of protein-coding genes potentially regulating defense processes. Therefore, 109 soybean cultivars genotyped with a 6k Illumina platform were assessed for disease severity. A genome-wide analysis revealed a total of 13 SNPs significantly associated. Through protein annotation, we identified three markers located inside the coding regions of uncharacterized protein LOC100779077, histone-lysine N-methyltransferase SUVR4, and ABC-transporter B family member-9. Nucleotide polymorphism on the first two of these markers produces a non-synonymous polymorphism with polarity shift from hydrophobic to polar amino acid. It is convenient to prioritize these three candidate markers for validation procedures with the purpose of using them in marker-assisted soybean breeding programs.

Keywords:
Bacterial pustule; Glycine max (L.) Merri; GWAS; SNP; candidate genes

INTRODUCTION

Bacterial pustule caused by the gram-negative phytobacterium Xanthomonas citri pv. glycines is a common disease infecting soybean cultivars (Goradia et al. 2009GoradiaLHartmanGLDanielSL2009 Evaluation of glyphosate-tolerant soybean cultivars for resistance to bacterial pustule. European Journal of Plant Pathology 124:331-335). Small yellowish halos are the symptoms produced by its infection, which coalesce with disease progression, leading to necrosis and premature loss of soybean leaves (Park et al. 2008ParkHJHanSWOhCLeeSRaDLeeSHHeuS2008 Avirulence gene diversity of Xanthomonas axonopodis pv. glycines isolated in Korea. Journal of Microbiology and Biotechnology 18:1500-1509). Severity as high as 70% occurs under certain environmental conditions, such as high temperature and humidity, which favor colonization and pathogen development (Zinsou et al. 2015ZinsouVAAfoudaLACZoumarou-WallisNPate-BataTDossouLGötzMWinterS2015 Occurrence and characterization of Xanthomonas axonopodis pv. glycines, causing bacterial pustules on soybean in Guinea Savanna of Benin. African Crop Science Journal 23:203-221).

The recessive resistance gene rxp, located in soybean chromosome 17 and linkage group D2 (Narvel 2001NarvelJM2001 Molecular mapping of rxp conditioning reaction to bacterial pustule in soybean. Journal of Heredity 92:267-270), is the main studied source of resistance to Xanthomonas citri pv. glycines. Several studies have shown that resistance to bacterial pustule is a complex trait, for example, the study of glyphosate-resistant soybean and characterization of bacterial pustule aggressiveness conducted by Goradia et al. (2009GoradiaLHartmanGLDanielSL2009 Evaluation of glyphosate-tolerant soybean cultivars for resistance to bacterial pustule. European Journal of Plant Pathology 124:331-335). Therefore, the identification of genes and the SNP haplotypes controlling the trait is of interest to be introduced in elite breeding lines used in crop breeding. The genome-wide association study (GWAS), based on the linkage disequilibrium of the phenotype with an SNP marker in a great number of individuals (Uffelmann et al. 2021UffelmannEHuangQQMunungNSVriesJOkadaYMartinAMartinHCLappalainenTPosthumaD2021 Genome-wide association studies. Nature Reviews Methods Primers 1:59), is a statistical tool that allows the identification of significant single-base polymorphisms (SNPs), which can provide information about disequilibrium blocks, proximal genes and QTLs, for example. Numerous studies have been conducted using GWAS for different pathosystems, such as Phytophthora root and stem rot (Ludke et al. 2019LudkeWHSchusterISilvaFLDallaTMontecelliNSoaresBDAOliveiraABVolpatoL2019 SNP markers associated with soybean partial resistance to Phytophthora sojae. Crop Breeding and Applied Biotechnology 19:31-39) and cyst nematode (Vuong et al. 2015VuongTDSonahHMeinhardtCGDeshmukhRKadamSNelsonRLShannonJGNguyenHT2015 Genetic architecture of cyst nematode resistance revealed by genome-wide association study in soybean. BMC Genomics 16:1-13) in soybean.

An increased disease incidence has been observed in cultivars considered resistant, mostly in the flowering stage of the soybean development. Because the plant developmental stage can actively influence specific or broad-spectrum resistance, such a factor is crucial in our understanding of the genetic basis of plant-pathogen interactions and plant defense mechanisms (Develey-Rivière and Galiana 2007Develey-RivièreMPGalianaE2007 Resistance to pathogens and host developmental stage: A multifaceted relationship within the plant kingdom. New Phytologist 175:405-416). This study aimed to investigate soybean genetic resistance to bacterial pustule in the flowering stage through the phenotyping of cultivars with recommended strains and an association analysis, followed by the characterization of associated SNPs and indication of nearby protein-coding genes of likely great importance for the development of resistant cultivars in a breeding program.

MATERIAL AND METHODS

Location and experimental design

A total of 109 soybean elite cultivars from various Brazilian breeding programs were sown in 200 mL plastic cups filled with substrate Tropstrato HT Hortaliças (Vida Verde, Mogi Mirim/SP, Brazil) and placed in a greenhouse located at the Diogo Alves de Melo Experimental Field, Viçosa, MG (lat 20° 45’ S, long 42° 52’ W, alt 648 m asl). The trial was arranged in a completely randomized design from May to July 2018, with three replications of each soybean genotype for each strain. Greenhouse conditions were kept with about 70% of humidity and 28 °C.

Bacterial strains, inoculation and evaluation

Strains XAG2440p (pathovar) and XAG2447 of Xanthomonas citri pv. glycines were obtained from the Instituto Biológico de Campinas. They were cultured in 523 medium plates (Kado and Heskett 1970KadoCHeskettM1970 Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopathology 60:969-979) at 28 °C for 48h and suspended on a 10mM MgCl2 solution until OD600 of 0.3 prior to inoculation. Phenotyping trials were independent for each strain and the same inoculation protocols were applied. A humid chamber was simulated inside the greenhouse from 24h prior to inoculation to 24h after inoculation, to achieve higher humidity levels, which favored soybean colonization by the pathogen. Inoculation was conducted in the soybean flowering stage R3-R4 (Fehr and Caviness 1977FehrWRCavinessCE1977 Stages of soybean development. Iowa State University, Ames, p. 1-12). A 2.9 cm x 0.65 mm needle was used for the perforation of 28 spots per leaflet, and 3.5 mL of inoculum suspension was pulverized on each plant with the aid of an atomizer. The evaluation of symptoms was proceeded with assignment of scores ranging from 1 to 5 (Resistant-1; Moderately resistant-2; Moderately susceptible-3; Susceptible-4; Highly susceptible-5), as described by Goradia et al. (2009GoradiaLHartmanGLDanielSL2009 Evaluation of glyphosate-tolerant soybean cultivars for resistance to bacterial pustule. European Journal of Plant Pathology 124:331-335), nine days after inoculation.

Genotyping and association analysis

All 109 cultivars were genotyped by 6,000 SNPs. The data were obtained with the use of SoySNP6K BeadChip (Akond et al. 2013AkondMLiuSSchoenerLAndersonJAKantartziSKMeksemKSongQWangDWenZLightfootDAKassemMA2013 A SNP-based genetic linkage map of soybean using the SoySNP6K Illumina infinium beadchip genotyping array. Journal of Plant Genome Sciences 1:80-89), a high-throughput genotyping platform by Illumina (Illumina Inc., San Diego, USA). The genotyping was run by Deoxi Biotechnology Ltda®, in Araçatuba, São Paulo, Brazil, and the same data set was used in other studies, including GWAS for agronomic traits (Contreras-Soto et al. 2017Contreras-SotoRIMoraFDe OliveiraMARHigashiWScapimCASchusterI2017 A genome-wide association study for agronomic traits in soybean using SNP markers and SNP-based haplotype analysis. PLoS ONE 12:1-22). Tassel 5 software (Bradbury et al. 2007BradburyPJZhangZKroonDECasstevensTMRamdossYBucklerES2007 TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics 23:2633-2635) was used for data processing, with a maximum of 10% of missing genotypes, and minor allele frequency higher than 5%.

The genotypic values in soybean cultivars used in this study were predicted by the Linear Mixed Model with Software Selegen-REML/BLUP (Resende 2016ResendeMDV2016 Software Selegen-REML/BLUP: A useful tool for plant breeding. Crop Breeding and Applied Biotechnology 16:330-339). Its results were applied in a genome-wide association analysis through the Genome Association Prediction Tool (Lipka et al. 2012LipkaAETianFWangQPeifferJLiMBradburyPJGoreMABucklerESZhangZ2012 GAPIT: Genome association and prediction integrated tool. Bioinformatics 28:2397-2399), GAPIT package, in R software (R Core Team 2018R Core Team2018 R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Available at: <Available at: https://www.R-project.org >/. Accessed on June, 16, 2021
https://www.R-project.org...
). The population structure and kinship coefficient matrix used in the association analysis were inferred as described by Contreras-Soto et al. (2017Contreras-SotoRIMoraFDe OliveiraMARHigashiWScapimCASchusterI2017 A genome-wide association study for agronomic traits in soybean using SNP markers and SNP-based haplotype analysis. PLoS ONE 12:1-22). Manhattan plots were obtained using the qqman 0.1.4 R package (Turner 2018TurnerSD2018 Qqman: an R package for visualizing GWAS results using Q-Q and manhattan plots. Journal of Open Source Software 3:731).

Haplotype block and annotation of nearby genes

Statistically significant SNPs were mapped into linkage disequilibrium blocks with the aid of Haploview 4.2 (Barrett et al. 2005BarrettJCFryBMallerJDalyMJ2005 Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics 21:263-265), and the extensions of the blocks were estimated. The proteins within each block were annotated with Augustus 3.2.2 (Stanke and Morgenstern 2005StankeMMorgensternB2005 AUGUSTUS: A web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Research 33: W465-W467.) using Arabidopsis genome as a reference, and Gene Ontology was retrieved with Goanna tool from AgBase (McCarthy et al. 2006McCarthyFMWangNMageeGBNanduriBLawrenceMLCamonEBBarrellDGHillDPDolanMEWilliamsWPLutheDSBridgesSMBurgessSC2006 AgBase: A functional genomics resource for agriculture. BMC Genomics 7:1-13).

Flanking sequences of each significant SNP associated with bacterial pustule, with 2000 bp downstream and upstream, were compared with the annotation reference (Williams 82 assembly V2.1, NCBI RefSeq assembly accession: GCF_000004515.5) (O’Leary et al. 2016O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse B, Smith-White B, Ako-Adjei D, Astashyn A, Badretdin A, Bao Y, Blinkova O, Brover V, Chetvernin V, Choi J, Cox E, Ermolaeva O, Farrell CM, Goldfarb T, Gupta T, Haft D, Hatcher E, Hlavina W, Joardar VS, Kodali VK, Li W, Maglott D, Masterson P, McGarvey KM, Murphy MR, O’Neill K, Pujar S, Rangwala SH, Rausch D, Riddick LD, Schoch C, Shkeda A, Storz SS, Sun H, Thibaud-Nissen F, Tolstoy I, Tully RE, Vatsan AR, Wallin C, Webb D, Wu W, Landrum MJ, Kimchi A, Tatusova T, DiCuccio M, Kitts P, Murphy TD and Pruitt KD2016 Reference sequence (RefSeq) database at NCBI: Current status, taxonomic expansion, and functional annotation. Nucleic Acids Research 44: D733-D745.) using translated BLAST (blastx) (Johnson et al. 2008JohnsonMZaretskayaIRaytselisYMerezhukYMcGinnisSMaddenTL2008 NCBI BLAST: a better web interface. Nucleic Acids Research 36:5-9). Then, the protein sequences of Open Reading Frames (ORFs), protein polarity and stability were identified with the use of the web tool Expert Protein Analysis System (ExPASy) (Gasteiger et al. 2003GasteigerEGattikerAHooglandCIvanyiIAppelRDBairochA2003 ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Research 31:3784-3788). The sequence alignment was carried out using Clustal Omega Multiple Sequence Alignment (Sievers et al. 2011SieversFWilmADineenDGibsonTJKarplusKLiWLopezRMcWilliamHRemmertMSödingJThompsonJDHigginsDG2011 Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7:1-6) with BLOSUM 62 matrix to verify the identity of the sequence compared with database information.

RESULTS AND DISCUSSION

Soybean cultivar screening for bacterial pustule

The set of 109 cultivars was inoculated with XAG2440p and XAG2447, Xanthomonas citri pv. glycines strains, using the same design and methodology in separated trials. Considering the inoculation of XAG2447, the average score of the cultivars ranged from 1 to 1.67, and 76.15% of the cultivars were classified as resistant. The remaining cultivars showed a few symptoms and were consequently classified as moderately resistant, including FUNDACEP 57 RR, CD 249 RR STS, CD 2630 RR, and CD 233 RR. In contrast, the XAG2440p inoculation resulted in a wider range of scores, and 17.43% of the cultivars were categorized as resistant or moderately resistant; 35.78%, as moderately susceptible; and 46.79%, as susceptible or highly susceptible, with TMG 7161 RR and TMG115 RR having the least severe symptoms. Also, the reference resistant cultivar of the Brazilian Department of Agriculture, BRS 133, was found to be resistant with XAG2447 screening, but susceptible to XAG2440p strain inoculation. Random residual effects are normally distributed.

The different responses for XAG2440p and XAG2447 could be due to some pathogen specificities. For example, the pathogen type III secretion system (TTSS) could be more intense on XAG2440p. TTSS is encoded by avirulence gene avrBs3 homolog avrXg1 (Athinuwat et al. 2009AthinuwatDPrathuangwongSCursinoLBurrT2009 Xanthomonas axonopodis pv. glycines soybean cultivar virulence specificity is determined by avrBs3 homolog avrXg1. Phytopathology 99:996-1004), associated with the pathogen aggressiveness, and may be the reason for more severe symptoms caused by XAG2440p infection. A second explanation would be a mutation in the extracellular diffusible factor (DSF). The RpfF gene is responsible for the expression of the DSF signaling molecule in Xanthomonas citri pv. glycines, and it is related to pathogen colonization (Thowthampitak et al. 2008ThowthampitakJShafferBTPrathuangwongSLoperJE2008 Role of rpfF in virulence and exoenzyme production of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule of soybean. Phytopathology 98:1252-1260). Thereby, the virulence of the rpfF mutants, as it could be the case of XAG2447, is reduced by the interruption of quorum sensing signaling. Thus, plant scientists should be aware of the variability of symptoms caused by XAG2440p and XAG2447 strains in the flowering stage in the development of future cultivars.

The genome-wide association (GWA) analysis

After the genotyping data were processed, 3807 polymorphic SNPs that meet MAF > 0.05 and call rate < 0.1 in the population were kept for statistical analysis. The distribution of these SNPs was on average 190.35 markers per chromosome, with a minimum of 140 markers in chromosome 1 and a maximum of 266 in chromosome 13. Markers of greater effects, with a threshold of −log10 (p-value) ≥ 2.53, were selected, so that we would have a higher number of SNPs to be used in subsequent searches for putative nearby genes and proteins associated with bacterial pustule. Since the set of soybean genotypes inoculated with XAG2447 strain showed only resistant or moderately resistant phenotypes, the GWAS was solely conducted with strain XAG2440p phenotyping data, which resulted in 13 non-redundant SNP markers across six chromosomes significantly associated with bacterial pustule (Table 1). Out of the 13 SNPs, five are located in chromosome 18, three in chromosome 13, two in chromosome 3, and one in chromosomes 5, 10, and 15, as illustrated in the Manhattan plot (Figure 1).

Table 1
Significantly associated markers identified in the association analysis of soybean bacterial pustule with strain XAG2440p

Figure 1
Manhattan plot of GWAS for soybean flowering stage resistance to XAG2440p strain of Xanthomonas citri pv. glycines.

The most significant SNPs associated with bacterial pustule were Gm18_4188718_A_C in chromosome 18 and Gm10_48426970_A_G, in chromosome 10. The linkage disequilibrium (LD) block determination by Haploview showed Gm03_46592189_A_G and Gm03_46889507_T_C within a disequilibrium block ranging from Gm03_46337339_T_C to Gm03_47039930_A_G, with 702 kb (Figure 2). The following Arabidopsis correspondent and defense-related genes were identified within the LD block: VIRF INTERACTING PROTEIN 4 (AT5G28040; GO:0042742), RPI3 (AT3G04790; GO:0042742), PEPTIDE-N-GLYCANASE 1 (AT5G49570; GO:0010188), PATHOGENESIS-RELATED 4 (AT3G04720; GO:0042742, GO:0050832, GO:0080027), and NON-RESPONDING TO OXYLIPINS 7 (AT1G64790; GO:0042742, GO:0045087), among other defense-related genes. Chromosome 3 was the only one to form a LD block with significant associated SNP within it.

Figure 2
(A) Chromosome 3 LD blocks. (B) Zoomed LD block from Gm03_46337339_T_C (SNP753) to Gm03_47039930_A_G (SNP760), containing chromosome 3 markers Gm03_46592189_A_G (SNP757) and Gm03_46889507_T_C (SNP758) significantly associated with bacterial pustule, LD in r2.

The two markers most significantly associated with bacterial pustule (Gm18_4188718_A_C and Gm10_48426970_A_G) showed low MAF values, which reflect low allele frequency in the studied cultivars, due to recent mutation events or their low fitness and consequent reduced reproductive success. On the other hand, the Gm03_46592189_A_G and Gm03_46889507_T_C markers had the highest values of MAF. The equality of MAF values of markers in chromosome 3 is not surprising, since those alleles are segregating together in a linkage disequilibrium block of 702 kb. Several genes and transcription factors coding for defense-related proteins are found in this region, which makes it promising and worthy of further investigation.

Candidate genes identification and protein annotation

No significant similarity was found between Gm05_33176582_G_A, Gm10_48426970_A_G, and Gm13_34818193_C_T flanking sequences of 2000 bp upstream and downstream and reference soybean gene annotation at NCBI. Markers Gm03_46889507_T_C and Gm18_122382_A_G are also located in intergenic regions, distant less than 2000 bp from the nearest gene. The Gm18_4188718_A_C, Gm18_3954704_C_T, Gm13_34946643_T_C, Gm18_54979_G_A, and Gm18_4324818_G_T markers are located in intragenic regions (Table 2).

Table 2
Nearby significant SNPs sequence annotation and its gene position (inside or outside the gene and coding region). The SNP order follows the most significant SNPs

Markers Gm13_898111_T_G, Gm03_46592189_A_G and Gm15_6925513_T_C are located inside protein coding regions of genes. They code, respectively, for uncharacterized protein LOC100779077, histone-lysine N-methyltransferase SUVR4, and ABC transporter B family member 9. Markers Gm13_898111_T_G and Gm03_46592189_A_G, located in ORFs 5'3', frames 2 and 3, second and first bases of the codon, respectively, resulted in an amino acid change with allele variation, in addition to a polarity shift. Marker Gm13_898111_T_G is at 1334 bp from the initiation of the ORF and allele variation T/G modified the amino acid produced to Phenylalanine (F)/Cysteine (C), a shift from hydrophobic to a polar amino acid. Considering marker Gm03_46592189_A_G, located 208 bp from the beginning of the ORF, the nucleotide change A/G altered the amino acid produced to Threonine (T)/Alanine (A), a shift from a polar amino acid to a hydrophobic one, similarly to Gm03_46592189_A_G. Marker Gm15_6925513_T_C is located at 237 bp or 79 codons from the beginning of the ORF, 3'5' frame 1, the third base of the respective codon. Nucleotide base variation T/C did not change the translated amino acid, which continues to be the polar Lysine (K).

Since LOC100779077 is an uncharacterized protein, we cannot infer about its function or connection to a specific plant defense mechanism based on previous studies. Histone-lysine N-methyltransferase SUVR4 is highly related to chromatin structure modulation (Thorstensen et al. 2006ThorstensenTFischerASandvik SVJohnsenSSGriniPEReuterGAalenRB2006 The Arabidopsis SUVR4 protein is a nucleolar histone methyltransferase with preference for monomethylated H3K9. Nucleic Acids Research 34:5461-5470), possibly regulating transposons damage with post-translational silencing histone H3K9 through methylation, an epigenetic control (Veiseth et al. 2011Veiseth SV.RahmanMAYapKLFischerAEgge-JacobsenWReuterGZhouMMAalenRBThorstensenT2011 The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis. PLoS Genetics 7:1-14). Histone lysine methyl transferases are known for regulating ETI and affecting plant immunity pathways, correlated with lipid, cuticular wax, and carotenoid biosynthesis, for example (Lee et al. 2016LeeSFuFXuSLeeSYYunD-JMengisteT2016 Global regulation of plant immunity by histone lysine methyl transferases. The Plant Cell 28:1640-1661). ABC transporter B family member 9 is an ATP-binding cassette transporter of molecules across membranes, and a protein that can participate in resistance and parasitism processes (Hwang et al. 2016HwangJUSongWYHongDKoDYamaokaYJangSYimSLeeEKhareDKimKPalmgrenMYoonHSMartinoiaELeeY2016 Plant ABC transporters enable many unique aspects of a terrestrial plant’s lifestyle. Molecular Plant 9:338-355). During infection, for example, ABC transporters in bacteria can use efflux pumps to carry virulence factors or quorum sensing signaling molecules and overcome plant defenses (Du et al. 2018DuDWang-KanXNeubergerAvan VeenHWPosKMPiddockLJVLuisiBF2018 Multidrug efflux pumps: structure, function and regulation. Nature Reviews Microbiology 16:523-539). Furthermore, ABC transporters play an important role in the transportation of secondary metabolites, often related to plant defense, including alkaloids and phenolic compounds (Yazaki 2006YazakiK2006 ABC transporters involved in the transport of plant secondary metabolites. FEBS Letters 580:1183-1191). In potatoes, the ABC transporter subfamily, composed of pleiotropic drug resistance proteins, is related to the pathogen response regulation through foliar secretion exudates with antimicrobial activity (Ruocco et al. 2011RuoccoMAmbrosinoPLanzuiseSWooSLLoritoMScalaF2011 Four potato (Solanum tuberosum) ABCG transporters and their expression in response to abiotic factors and Phytophthora infestans infection. Journal of Plant Physiology 168:2225-2233).

Five out of 13 SNPs are within intragenic regions, related to protein LOC100797777, protein DJ-1 homolog D, dynamin-related protein 3A isoform X2, pheophytinase (chloroplastic), and KH domain-containing protein SPIN1 isoform X2. Protein DJ1 family is related to the plant stress response in yeast homolog Hsp31 through detoxifying capabilities (Melvin et al. 2017MelvinPBankapalliKD’SilvaPShivaprasadPV2017 Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants. Plant Molecular Biology 94:381-397). It probably has the same function in plants, since it is a highly conserved protein (Xu et al. 2010XuXMLinHMapleJBjorkblomBAlvesGLarsenJPMollerSG2010 The Arabidopsis DJ-1a protein confers stress protection through cytosolic SOD activation. Journal of Cell Science 123:1644-1651). Dynamin-related protein 3A is associated with peroxisome replication in Arabidopsis (Lingard et al. 2008LingardMJGiddaSKBinghamSRothsteinSJMullenRTTreleaseRN2008 Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A cooperate in cell cycle-associated replication of peroxisomes. The Plant Cell Online 20:1567-1585), an organelle related to molecular signaling, which accumulates endogenous reactive oxygen species (ROS) (Nyathi and Baker 2006NyathiYBakerA2006 Plant peroxisomes as a source of signalling molecules. Biochimica et Biophysica Acta - Molecular Cell Research 1763:1478-1495). In the event of host cell infection, ROS act on cell response and plant-pathogen interaction, and ozone plays an important role in the triggering of a hypersensitive response (Waszczak et al. 2018WaszczakCCarmodyMKangasjarviJ2018 Reactive oxygen species in plant signaling. Annual Review of Plant Biology 69:209-236). For the remaining markers, no association with plant defense response was found.

Markers Gm13_898111_T_G, Gm03_46592189_A_G, and Gm15_6925513_T_C and other defense-related candidate genes, identified to be associated with soybean bacterial pustule in the flowering stage, should be investigated simultaneously and separately. While focusing on the comprehension of resistance mechanisms and host-pathogen interactions through gene expression, we might be able to identify molecular markers to improve the marker-assisted selection used in molecular breeding programs.

The biological information related to the small effects of single SNPs obtained from the association analysis was important for the identification of candidate genes regulating the soybean response in the flowering stage to Xanthomonas citri pv. glycines complex pathosystem defense process. The study of resistance considering the possible existence of a relationship between the developmental stage and defense mechanisms can improve our knowledge about key resistant genes and their role in plant-pathogen interactions, which is essential for a target-specific and effective molecular breeding program. Since GWAS is based on single tests for each marker, further understanding of gene-gene and gene-protein interactions, post-transcriptional gene regulation, and epigenetics control would allow for unprecedented advancement in GWAS interpretation, and its applications. Therefore, the complementary use of other methodologies, such as a network analysis, may provide biological insights to elucidate the results of soybean pustule resistance association.

ACKNOWLEDGMENTS

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Brasil - Finance Code 001. This work was supported by CNPq (National Council for Scientific and Technological Development) and UFV (Universidade Federal de Viçosa).

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Publication Dates

  • Publication in this collection
    04 May 2022
  • Date of issue
    2022

History

  • Received
    10 Aug 2021
  • Accepted
    22 Feb 2022
  • Published
    30 Mar 2022
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