Time Course RNA-seq Reveals Soybean Responses against Root-Lesion Nematode and Resistance Players.

Pratylenchus brachyurus causes serious damage to soybean production and other crops worldwide. Plant molecular responses to RLN infection remain largely unknown and no resistance genes have been identified in soybean. In this study, we analyzed molecular responses to RLN infection in moderately resistant BRSGO (Chapadões-BRS) and susceptible TMG115 RR (TMG) Glycine max genotypes. Differential expression analysis revealed two stages of response to RLN infection and a set of differentially expressed genes (DEGs) in the first stage suggested a pattern-triggered immunity (PTI) in both genotypes. The divergent time-point of DEGs between genotypes was observed four days post-infection, which included the activation of mitogen-activated protein kinase (MAPK) and plant-pathogen interaction genes in the BRS, suggesting the occurrence of an effector-triggered immunity response (ETI) in BRS. The co-expression analyses combined with single nucleotide polymorphism (SNP) uncovered a key element, a transcription factor phytochrome-interacting factor (PIF7) that is a potential regulator of moderate resistance to RLN infection. Two genes for resistance-related leucine-rich repeat (LRR) proteins were found as BRS-specific expressed genes. In addition, alternative splicing analysis revealed an intron retention in a myo-inositol oxygenase (MIOX) transcript, a gene related to susceptibility, may cause a loss of function in BRS.

Genome-wide association study for resistance to the Meloidogyne javanica causing root-knot nematode in soybean.

KEY MESSAGE: A locus on chromosome 13, containing multiple TIR-NB-LRR genes and SNPs associated with M. javanica resistance, was identified using a combination of GWAS, resequencing, genetic mapping and expression profiling. Meloidogyne javanica, a root-knot nematode, is an important problem in soybean-growing areas, leading to severe yield losses. Some accessions have been identified carrying resistance loci to this nematode. In this study, a set of 317 soybean accessions was characterized for resistance to M. javanica. A genome-wide association study was performed using SNPs from genotyping-by-sequencing, and a region of 29.2 kb on chromosome 13 was identified. An analysis of haplotypes showed that SNPs were able to discriminate between susceptible and resistant accessions, with 25 accessions sharing the haplotype associated with resistance. Furthermore, five accessions that exhibited resistance without carrying this haplotype may carry different loci conferring resistance to M. javanica. We also conducted the screening of the SNPs in the USDA soybean germplasm, revealing that several soybean accessions previously reported as resistant to other nematodes also shared the resistance haplotype on chromosome 13. Two SNP-based TaqMan® assays were developed and validated in two panels of soybean cultivars and in biparental populations. In silico analysis of the region associated with resistance identified the occurrence of genes with structural similarity with classical major resistance genes (NBS-LRR genes). Specifically, several nonsynonymous SNPs were observed in Glyma.13g194800 and Glyma.13g194900. The expression profile of these candidate genes demonstrated that the two gene models were up-regulated in the resistance source PI 505,099 after nematode infection. Overall, the SNPs associated with resistance and the genes identified constitute an important tool for introgression of resistance to the root-knot nematode by marker-assisted selection in soybean breeding programs.

Association mapping of a locus that confers southern stem canker resistance in soybean and SNP marker development.

BACKGROUND: Southern stem canker (SSC), caused by Diaporthe aspalathi (E. Jansen, Castl. & Crous), is an important soybean disease that has been responsible for severe losses in the past. The main strategy for controlling this fungus involves the introgression of resistance genes. Thus far, five main loci have been associated with resistance to SSC. However, there is a lack of information about useful allelic variation at these loci. In this work, a genome-wide association study (GWAS) was performed to identify allelic variation associated with resistance against Diaporthe aspalathi and to provide molecular markers that will be useful in breeding programs. RESULTS: We characterized the response to SSC infection in a panel of 295 accessions from different regions of the world, including important Brazilian elite cultivars. Using a GBS approach, the panel was genotyped, and we identified marker loci associated with Diaporthe aspalathi resistance through GWAS. We identified 19 SNPs associated with southern stem canker resistance, all on chromosome 14. The peak SNP showed an extremely high degree of association (p-value = 6.35E-27) and explained a large amount of the observed phenotypic variance (R2 = 70%). This strongly suggests that a single major gene is responsible for resistance to D. aspalathi in most of the lines constituting this panel. In resequenced soybean materials, we identified other SNPs in the region identified through GWAS in the same LD block that clearly differentiate resistant and susceptible accessions. The peak SNP was selected and used to develop a cost-effective molecular marker assay, which was validated in a subset of the initial panel. In an accuracy test, this SNP assay demonstrated 98% selection efficiency. CONCLUSIONS: Our results suggest relevance of this locus to SSC resistance in soybean cultivars and accessions from different countries, and the SNP marker assay developed in this study can be directly applied in MAS studies in breeding programs to select materials that are resistant against this pathogen and support its introgression.

New insights into Phakopsora pachyrhizi infection based on transcriptome analysis in planta.

Asian soybean rust (ASR) is one of the most destructive diseases affecting soybeans. The causative agent of ASR, the fungus Phakopsora pachyrhizi, presents characteristics that make it difficult to study in vitro, limiting our knowledge of plant-pathogen dynamics. Therefore, this work used leaf lesion laser microdissection associated with deep sequencing to determine the pathogen transcriptome during compatible and incompatible interactions with soybean. The 36,350 generated unisequences provided an overview of the main genes and biological pathways that were active in the fungus during the infection cycle. We also identified the most expressed transcripts, including sequences similar to other fungal virulence and signaling proteins. Enriched P. pachyrhizi transcripts in the resistant (PI561356) soybean genotype were related to extracellular matrix organization and metabolic signaling pathways and, among infection structures, in amino acid metabolism and intracellular transport. Unisequences were further grouped into gene families along predicted sequences from 15 other fungi and oomycetes, including rust fungi, allowing the identification of conserved multigenic families, as well as being specific to P. pachyrhizi. The results revealed important biological processes observed in P. pachyrhizi, contributing with information related to fungal biology and, consequently, a better understanding of ASR.

Evaluation of genetic variation among Brazilian soybean cultivars through genome resequencing.

BACKGROUND: Soybean [Glycine max (L.) Merrill] is one of the most important legumes cultivated worldwide, and Brazil is one of the main producers of this crop. Since the sequencing of its reference genome, interest in structural and allelic variations of cultivated and wild soybean germplasm has grown. To investigate the genetics of the Brazilian soybean germplasm, we selected soybean cultivars based on the year of commercialization, geographical region and maturity group and resequenced their genomes. RESULTS: We resequenced the genomes of 28 Brazilian soybean cultivars with an average genome coverage of 14.8X. A total of 5,835,185 single nucleotide polymorphisms (SNPs) and 1,329,844 InDels were identified across the 20 soybean chromosomes, with 541,762 SNPs, 98,922 InDels and 1,093 CNVs that were exclusive to the 28 Brazilian cultivars. In addition, 668 allelic variations of 327 genes were shared among all of the Brazilian cultivars, including genes related to DNA-dependent transcription-elongation, photosynthesis, ATP synthesis-coupled electron transport, cellular respiration, and precursors of metabolite generation and energy. A very homogeneous structure was also observed for the Brazilian soybean germplasm, and we observed 41 regions putatively influenced by positive selection. Finally, we detected 3,880 regions with copy-number variations (CNVs) that could help to explain the divergence among the accessions evaluated. CONCLUSIONS: The large number of allelic and structural variations identified in this study can be used in marker-assisted selection programs to detect unique SNPs for cultivar fingerprinting. The results presented here suggest that despite the diversification of modern Brazilian cultivars, the soybean germplasm remains very narrow because of the large number of genome regions that exhibit low diversity. These results emphasize the need to introduce new alleles to increase the genetic diversity of the Brazilian germplasm.

Early transcriptional response of soybean contrasting accessions to root dehydration.

Drought is a significant constraint to yield increase in soybean. The early perception of water deprivation is critical for recruitment of genes that promote plant tolerance. DeepSuperSAGE libraries, including one control and a bulk of six stress times imposed (from 25 to 150 min of root dehydration) for drought-tolerant and sensitive soybean accessions, allowed to identify new molecular targets for drought tolerance. The survey uncovered 120,770 unique transcripts expressed by the contrasting accessions. Of these, 57,610 aligned with known cDNA sequences, allowing the annotation of 32,373 unitags. A total of 1,127 unitags were up-regulated only in the tolerant accession, whereas 1,557 were up-regulated in both as compared to their controls. An expression profile concerning the most representative Gene Ontology (GO) categories for the tolerant accession revealed the expression "protein binding" as the most represented for "Molecular Function", whereas CDPK and CBL were the most up-regulated protein families in this category. Furthermore, particular genes expressed different isoforms according to the accession, showing the potential to operate in the distinction of physiological behaviors. Besides, heat maps comprising GO categories related to abiotic stress response and the unitags regulation observed in the expression contrasts covering tolerant and sensitive accessions, revealed the unitags potential for plant breeding. Candidate genes related to "hormone response" (LOX, ERF1b, XET), "water response" (PUB, BMY), "salt stress response" (WRKY, MYB) and "oxidative stress response" (PER) figured among the most promising molecular targets. Additionally, nine transcripts (HMGR, XET, WRKY20, RAP2-4, EREBP, NAC3, PER, GPX5 and BMY) validated by RT-qPCR (four different time points) confirmed their differential expression and pointed that already after 25 minutes a transcriptional reorganization started in response to the new condition, with important differences between both accessions.

Identification of the soybean HyPRP family and specific gene response to Asian soybean rust disease.

Soybean [Glycine max (L.) Merril], one of the most important crop species in the world, is very susceptible to abiotic and biotic stress. Soybean plants have developed a variety of molecular mechanisms that help them survive stressful conditions. Hybrid proline-rich proteins (HyPRPs) constitute a family of cell-wall proteins with a variable N-terminal domain and conserved C-terminal domain that is phylogenetically related to non-specific lipid transfer proteins. Members of the HyPRP family are involved in basic cellular processes and their expression and activity are modulated by environmental factors. In this study, microarray analysis and real time RT-qPCR were used to identify putative HyPRP genes in the soybean genome and to assess their expression in different plant tissues. Some of the genes were also analyzed by time-course real time RT-qPCR in response to infection by Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease. Our findings indicate that the time of induction of a defense pathway is crucial in triggering the soybean resistance response to P. pachyrhizi. This is the first study to identify the soybean HyPRP group B family and to analyze disease-responsive GmHyPRP during infection by P. pachyrhizi.

Expression patterns of GmAP2/EREB-like transcription factors involved in soybean responses to water deficit.

Soybean farming has faced several losses in productivity due to drought events in the last few decades. However, plants have molecular mechanisms to prevent and protect against water deficit injuries, and transcription factors play an important role in triggering different defense mechanisms. Understanding the expression patterns of transcription factors in response to water deficit and to environmental diurnal changes is very important for unveiling water deficit stress tolerance mechanisms. Here, we analyzed the expression patterns of ten APETALA2/Ethylene Responsive Element Binding-like (AP2/EREB-like) transcription factors in two soybean genotypes (BR16: drought-sensitive; and Embrapa 48: drought-tolerant). According to phylogenetic and domain analyses, these genes can be included in the DREB and ERF subfamilies. We also analyzed a GmDRIP-like gene that encodes a DREB negative regulator. We detected the up-regulation of 9 GmAP2/EREB-like genes and identified transcriptional differences that were dependent on the levels of the stress applied and the tissue type analyzed (the expression of the GmDREB1F-like gene, for example, was four times higher in roots than in leaves). The GmDRIP-like gene was not induced by water deficit in BR16 during the longest periods of stress, but was significantly induced in Embrapa 48; this suggests a possible genetic/molecular difference between the responses of these cultivars to water deficit stress. Additionally, RNAseq gene expression analysis over a 24-h time course indicates that the expression patterns of several GmDREB-like genes are subject to oscillation over the course of the day, indicating a possible circadian regulation.

The use of microRNAs as reference genes for quantitative polymerase chain reaction in soybean.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a robust and widely applied technique used to investigate gene expression. However, for correct analysis and interpretation of results, the choice of a suitable gene to use as an internal control is a crucial factor. These genes, such as housekeeping genes, should have a constant expression level in different tissues and across different conditions. The advances in genome sequencing have provided high-throughput gene expression analysis and have contributed to the identification of new genes, including microRNAs (miRNAs). The miRNAs are fundamental regulatory genes of eukaryotic genomes, acting on several biological functions. In this study, miRNA expression stability was investigated in different soybean tissues and genotypes as well as after abiotic or biotic stress treatments. The present study represents the first investigation into the suitability of miRNAs as housekeeping genes in plants. The transcript stability of 10 miRNAs was compared to those of six previously reported housekeeping genes for the soybean. In this study, we provide evidence that the expression stabilities of miR156b and miR1520d were the highest across the soybean experiments. Furthermore, these miRNAs genes were more stable than the most commonly protein-coding genes used in soybean gene expression studies involving RT-qPCR.

Size of AT(n) insertions in promoter region modulates Gmhsp17.6-L mRNA transcript levels.

During earlier experiments, an SSR molecular marker (176 Soy HSP) showing high correlation (70%) with resistance/susceptibility to javanese root-knot nematode Meloidogyne javanica was identified in soybean. After being sequenced, results indicated that the SSR 176 Soy HSP marker was inserted in the promoter region of Gmhsp17.6-L gene. It was also detected in this region that resistant genotypes presented insertions between AT(31) and AT(33) in size and susceptible genotypes, AT(9). Gmhsp17.6-L gene coding region presented a perfect match in amino acid sequence in all soybean genotypes. A ribonuclease protection assay showed that Gmhsp17.6-L gene mRNA transcripts were present in all genotypes. A real-time relative quantification (qPCR) indicated in the resistant individuals higher mRNA transcripts levels, which presented in the sequencing more AT(n) insertions. These results suggest that the number of AT(n) insertions inside this promoter region could modulate up or down gene levels. Those findings can lead to the possibility of manipulating, between some limits, the mRNA transcripts levels using different sizes of AT(n) insertions.

Genetic relationships between Chinese, Japanese, and Brazilian soybean gene pools revealed by simple sequence repeat (SSR) markers

An understanding of the relationship of geographically different soybean gene pools, based on selectively neutral DNA markers would be useful for the selection of divergent parental cultivars for use in breeding. We assessed the relationships of 194 Chinese, 59 Japanese, and 19 Brazilian soybean cultivars (n = 272) using 12 simple sequence repeat (SSR) markers. Quantification Theory III and clustering analyses showed that the Chinese and Japanese cultivars were genetically quite distant to each other but not independent, while Brazilian cultivars were distantly related to the cultivars from the other two countries and formed a cluster that was distant from the other two gene pool clusters. Our results indicated that the Brazilian soybean gene pool is different from the Chinese and Japanese pool. Exchanges of these gene pools might be useful to increase the genetic variability in soybean breeding.