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Epigenetic modifications including miRNA regulation, DNA methylation, and histone modifications play fundamental roles in establishing the interactions between host plants and parasitic nematodes. Over the past decade, an increasing number of studies revealed the key functions of various components of the plant epigenome in the regulation of gene expression and shaping plant responses to nematode infection. In this chapter, we provide a conceptual framework for methods used to investigate epigenetic regulation during plant-nematode interactions. We focus specifically on current and emerging methods used to study miRNA regulation and function. We also highlight various methods and analytical tools used to profile DNA methylation patterns and histone modification marks at the genome level. Our intention is simply to explain the advantages of various methods and how to overcome some limitations. With rapid development of single-cell sequencing technology and genome editing, advanced and new methodologies are expected to emerge in the near future to further improve our understanding of epigenetic regulation and function during plant-nematode interactions.
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MicroARNs , Tylenchoidea , Animales , Epigénesis Genética , Enfermedades de las Plantas/genética , Plantas/genética , Plantas/parasitología , Metilación de ADN , MicroARNs/genética , Tylenchoidea/fisiologíaRESUMEN
Drought conditions exhibit various physiological and morphological changes in crops and thus reduce crop growth and yield. In order to mitigate the negative impacts of drought stress on soybean (Glycine max L. Merr.) production, identification and selection of genotypes that are best adapted to limited water availability in a specific environmental condition can be an effective strategy. This study aimed to assess the inheritance of early stomatal closure traits in soybeans using a population of recombinant inbred lines (RILs) derived from a cross between N09-13890 and Ellis. Thirty soybean lines were subjected to progressive water-deficit stress using a dry-down experiment. The experiment was conducted from June to November 2022 at the West Tennessee Research and Education Center (WTREC), University of Tennessee in Jackson, TN, under controlled environment conditions. This study identified significant differences among soybean lines in their early stomatal closure thresholds. The fraction of transpirable soil water (FTSW) thresholds among 30 tested lines ranged from 0.18 to 0.80, at which the decline in transpiration with soil drying was observed. Almost 65% of the RILs had FTSW threshold values between 0.41 to 0.80. These results, indicating inheritance, are supportive of the expression of early stomatal closure trait in progeny lines at a high level in cultivar development for water-deficit stress conditions. Thus, identifying the differences in genotypes of water use and their response to water-deficit stress conditions can provide a foundation for selecting new cultivars that are best adapted to arid and semi-arid agricultural production systems.
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Recombination allows for the exchange of genetic material between two parents, which plant breeders exploit to make improved cultivars. This recombination is not distributed evenly across the chromosome. Recombination mostly occurs in euchromatic regions of the genome and even then, recombination is focused into clusters of crossovers termed recombination hotspots. Understanding the distribution of these hotspots along with the sequence motifs associated with them may lead to methods that enable breeders to better exploit recombination in breeding. To map recombination hotspots and identify sequence motifs associated with hotspots in soybean [Glycine max (L.) Merr.], two biparental recombinant inbred lines populations were genotyped with the SoySNP50k Illumina Infinium assay. A total of 451 recombination hotspots were identified in the two populations. Despite being half-sib populations, only 18 hotspots were in common between the two populations. While pericentromeric regions did exhibit extreme suppression of recombination, 27% of the detected hotspots were located in the pericentromeric regions of the chromosomes. Two genomic motifs associated with hotspots are similar to human, dog, rice, wheat, drosophila, and arabidopsis. These motifs were a CCN repeat motif and a poly-A motif. Genomic regions spanning other hotspots were significantly enriched with the tourist family of mini-inverted-repeat transposable elements that resides in <0.34% of the soybean genome. The characterization of recombination hotspots in these two large soybean biparental populations demonstrates that hotspots do occur throughout the soybean genome and are enriched for specific motifs, but their locations may not be conserved between different populations.
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Glycine max , Fitomejoramiento , Humanos , Animales , Perros , Glycine max/genética , Genoma , Genotipo , Recombinación GenéticaRESUMEN
Gene co-expression network analysis is an efficient systems biology approach for the discovery of novel gene functions and trait-associated gene modules. To identify clusters of functionally related genes involved in soybean nodule formation and development, we performed a weighted gene co-expression network analysis. Two nodule-specific modules (NSM-1 and NSM-2, containing 304 and 203 genes, respectively) were identified. The NSM-1 gene promoters were significantly enriched in cis-binding elements for ERF, MYB, and C2H2-type zinc transcription factors, whereas NSM-2 gene promoters were enriched in cis-binding elements for TCP, bZIP, and bHLH transcription factors, suggesting a role of these regulatory factors in the transcriptional activation of nodule co-expressed genes. The co-expressed gene modules included genes with potential novel roles in nodulation, including those involved in xylem development, transmembrane transport, the ethylene signalling pathway, cytoskeleton organization, cytokinesis and regulation of the cell cycle, regulation of meristem initiation and growth, transcriptional regulation, DNA methylation, and histone modifications. Functional analysis of two co-expressed genes using TILLING mutants provided novel insight into the involvement of unsaturated fatty acid biosynthesis and folate metabolism in nodule formation and development. The identified gene co-expression modules provide valuable resources for further functional genomics studies to dissect the genetic basis of nodule formation and development in soybean.
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Redes Reguladoras de Genes , Glycine max , Glycine max/genética , Regulación de la Expresión Génica de las Plantas/genética , Perfilación de la Expresión Génica , Factores de Transcripción/genéticaRESUMEN
Switchgrass (Panicum virgatum L.) is a warm-season perennial grass species that is utilized as forage for livestock and biofuel feedstock. The stability of biomass yield and regrowth vigor under changing harvest frequency would help manage potential fluctuations in the feedstock market and would provide a continuous supply of quality forage for livestock. This study was conducted to (i) assess the genetic variation and (ii) identify the quantitative trait loci (QTL) associated with regrowth vigor after multiple cuttings in lowland switchgrass. A nested association mapping (NAM) population comprising 2000 pseudo F2 progenies was genotyped with single nucleotide polymorphism (SNP) markers derived from exome-capture sequencing and was evaluated for regrowth vigor in 2017 and 2018. The results showed significant variation among the NAM families in terms of regrowth vigor (p < 0.05). A total of 10 QTL were detected on 6 chromosomes: 1B, 5A, 5B, 6B, 7B, and 8A, explaining the phenotypic variation by up to 4.7%. The additive genetic effects of an individual QTL ranged from -0.13 to 0.26. No single QTL showed a markedly large effect, suggesting complex genetics underlying regrowth vigor in switchgrass. The homologs of candidate genes that play a variety of roles in developmental processes, including plant hormonal signal transduction, nucleotide biosynthesis, secondary metabolism, senescence, and responses to both biotic and abiotic stresses, were identified in the vicinity of QTL.
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DNA methylation has recently emerged as a powerful regulatory mechanism controlling the expression of key regulators of various developmental processes, including nodulation. However, the functional role of DNA methylation in regulating the expression of microRNA (miRNA) genes during the formation and development of nitrogen-fixing nodules remains largely unknown. In this study, we profiled DNA methylation patterns of miRNA genes during nodule formation, development, and early senescence stages in soybean (Glycine max) through the analysis of methylC-seq data. Absolute DNA methylation levels in the CG, CHH, and CHH sequence contexts over the promoter and primary transcript regions of miRNA genes were significantly higher in the nodules compared with the corresponding root tissues at these three distinct nodule developmental stages. We identified a total of 82 differentially methylated miRNAs in the nodules compared with roots. Differential DNA methylation of these 82 miRNAs was detected only in the promoter (69), primary transcript region (3), and both in the promoter and primary transcript regions (10). The large majority of these differentially methylated miRNAs were hypermethylated in nodules compared with the corresponding root tissues and were found mainly in the CHH context and showed stage-specific methylation patterns. Differentially methylated regions in the promoters of 25 miRNAs overlapped with transposable elements, a finding that may explain the vulnerability of miRNAs to DNA methylation changes during nodule development. Gene expression analysis of a set of promoter-differentially methylated miRNAs pointed to a negative association between DNA methylation and miRNA expression. Gene Ontology and pathways analyses indicate that changes in DNA methylation of miRNA genes are reprogrammed and contribute to nodule development through indirect regulation of genes involved in cellular processes and pathways with well-established roles in nodulation.
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DNA methylation is a widespread epigenetic mark that affects gene expression and transposon mobility during plant development and stress responses. However, the role of DNA methylation in regulating the expression of microRNA (miRNA) genes remains largely unexplored. Here, we analyzed DNA methylation changes of miRNA genes using a pair of soybean (Glycine max) near-isogenic lines (NILs) differing in their response to soybean cyst nematode (SCN; Heterodera glycines). Differences in global DNA methylation levels over miRNA genes in response to SCN infection were observed between the isogenic lines. miRNA genes with significant changes in DNA methylation levels in the promoter and primary transcript-coding regions were detected in both lines. In the susceptible isogenic line (NIL-S), 82 differentially methylated miRNAs were identified in response to SCN infection whereas, in the resistant isogenic line (NIL-R), only 16 differentially methylated miRNAs were identified. Interestingly, gma-miR5032, gma-miR5043, gma-miR1520b, and gma-2107-ch16 showed opposite methylation patterns in the isogenic lines. In addition, the miRNA paralogs gma-miR5770a and gma-miR5770b showed hypermethylation and hypomethylation in NIL-S and NIL-R, respectively. Gene expression quantification of gma-miR5032, gma-miR5043, gma-miR1520b, and gma-miR5770a/b and their confirmed targets indicated a role of DNA methylation in regulating miRNA expression and, thus, their targets upon SCN infection. Furthermore, overexpression of these four miRNAs in NIL-S using transgenic hairy root system enhanced plant resistance to SCN to various degrees with a key role observed for miR5032. Together, our results provide new insights into the role of epigenetic mechanisms in controlling miRNA regulatory function during SCN-soybean interactions.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
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Metilación de ADN , Glycine max/genética , Interacciones Huésped-Parásitos/genética , MicroARNs , Enfermedades de las Plantas/parasitología , Tylenchoidea , Animales , Epigénesis Genética , MicroARNs/genética , Enfermedades de las Plantas/genética , Glycine max/parasitologíaRESUMEN
Soybean nodulation is a highly controlled process that involves complex gene regulation at both transcriptional and post-transcriptional levels. In the present study, we profiled gene expression changes, alternative splicing events, and DNA methylation patterns during nodule formation, development, and senescence. The transcriptome data uncovered key transcription patterns of nodule development that included 9669 core genes and 7302 stage-specific genes. Alternative splicing analysis uncovered a total of 2323 genes that undergo alternative splicing events in at least one nodule developmental stage, with activation of exon skipping and repression of intron retention being the most common splicing events in nodules compared to roots. Approximately 40% of the differentially spliced genes were also differentially expressed at the same nodule developmental stage, implying a substantial association between gene expression and alternative splicing. Genome-wide-DNA methylation analysis revealed dynamic changes in nodule methylomes that were specific to each nodule stage, occurred in a sequence-specific manner, and impacted the expression of 1864 genes. An attractive hypothesis raised by our data is that increased DNA methylation may contribute to the efficiency of alternative splicing. Together, our results provide intriguing insights into the associations between gene expression, alternative splicing, and DNA methylation that may shape transcriptome complexity and proteome specificity in developing soybean nodules.
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Empalme Alternativo , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Nodulación de la Raíz de la Planta , Empalme Alternativo/genética , Empalme Alternativo/fisiología , Metilación de ADN/genética , Metilación de ADN/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Genes de Plantas/genética , Genes de Plantas/fisiología , Nodulación de la Raíz de la Planta/genética , Nodulación de la Raíz de la Planta/fisiología , Nódulos de las Raíces de las Plantas/crecimiento & desarrollo , Nódulos de las Raíces de las Plantas/metabolismo , Glycine max/crecimiento & desarrollo , Glycine max/metabolismoRESUMEN
DNA methylation is a widespread epigenetic mark that contributes to transcriptome reprogramming during plant-pathogen interactions. However, the distinct role of DNA methylation in establishing resistant and susceptible responses remains largely unexplored. Here, we developed and used a pair of near-isogenic lines (NILs) to characterize DNA methylome landscapes of soybean roots during the susceptible and resistant interactions with soybean cyst nematode (SCN; Heterodera glycines). We also compared the methylomes of the NILs and their parents to identify introduced and stably inherited methylation variants. The genomes of the NILs were substantially differentially methylated under uninfected conditions. This difference was associated with differential gene expression that may prime the NIL responses to SCN infection. In response to SCN infection, the susceptible line exhibited reduced global methylation levels in both protein-coding genes and transposable elements, whereas the resistant line showed the opposite response, increased global methylation levels. Heritable and novel nonparental differentially methylated regions overlapping with genes associated with soybean response to SCN infection were identified and validated using transgenic hairy root system. Our analyses indicate that DNA methylation patterns associated with the susceptible and resistant interactions are highly specific and that novel and stably inherited methylation variants are of biological significance.
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Quistes , Glycine max , Animales , Metilación de ADN/genética , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genética , Glycine max/genéticaRESUMEN
BACKGROUND: Plants have evolved intimate interactions with soil microbes for a range of beneficial functions including nutrient acquisition, pathogen resistance and stress tolerance. Further understanding of this system is a promising way to advance sustainable agriculture by exploiting the versatile benefits offered by the plant microbiome. The rhizosphere is the interface between plant and soil, and functions as the first step of plant defense and root microbiome recruitment. It features a specialized microbial community, intensive microbe-plant and microbe-microbe interactions, and complex signal communication. To decipher the rhizosphere microbiome assembly of soybean (Glycine max), we comprehensively characterized the soybean rhizosphere microbial community using 16S rRNA gene sequencing and evaluated the structuring influence from both host genotype and soil source. RESULTS: Comparison of the soybean rhizosphere to bulk soil revealed significantly different microbiome composition, microbe-microbe interactions and metabolic capacity. Soil type and soybean genotype cooperatively modulated microbiome assembly with soil type predominantly shaping rhizosphere microbiome assembly while host genotype slightly tuned this recruitment process. The undomesticated progenitor species, Glycine soja, had higher rhizosphere diversity in both soil types tested in comparison to the domesticated soybean genotypes. Rhizobium, Novosphingobium, Phenylobacterium, Streptomyces, Nocardioides, etc. were robustly enriched in soybean rhizosphere irrespective of the soil tested. Co-occurrence network analysis revealed dominant soil type effects and genotype specific preferences for key microbe-microbe interactions. Functional prediction results demonstrated converged metabolic capacity in the soybean rhizosphere between soil types and among genotypes, with pathways related to xenobiotic degradation, plant-microbe interactions and nutrient transport being greatly enriched in the rhizosphere. CONCLUSION: This comprehensive comparison of the soybean microbiome between soil types and genotypes expands our understanding of rhizosphere microbe assembly in general and provides foundational information for soybean as a legume crop for this assembly process. The cooperative modulating role of the soil type and host genotype emphasizes the importance of integrated consideration of soil condition and plant genetic variability for future development and application of synthetic microbiomes. Additionally, the detection of the tuning role by soybean genotype in rhizosphere microbiome assembly provides a promising way for future breeding programs to integrate host traits participating in beneficial microbiota assembly.
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Bacterias/aislamiento & purificación , Glycine max/genética , Microbiología del Suelo , Bacterias/clasificación , Bacterias/genética , Genotipo , Microbiota , Filogenia , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Rizosfera , Suelo/química , Glycine max/crecimiento & desarrollo , Glycine max/microbiologíaRESUMEN
Soybean is an important oil-producing crop in the Fabaceae family and there are increasing demands for soybean oil and other soybean products. Genetic improvement of soybean is needed to increase its production. In order to provide genetic diversity and resources for identifying important genes, a new ethyl methane sulfonate (EMS) mutagenized soybean population was generated using the newly released germplasm, JTN-5203 (maturity group V). Treatment of soybean seeds with 60 mM EMS concentration was found to be suitable for inducing mutation. A total of 1,820 M1 individuals were produced from 15,000 treated seeds. The resulting M2 population was planted in the field for phenotyping. After harvest, seed traits including total oil, protein, starch, moisture content, fatty acid and amino acid compositions were measured by NIR. Phenotypic variations observed in this population include changes in leaf morphology, plant architecture, seed compositions, and yield. Of most interest, we identified plants with increased amounts of total protein (50% vs. 41% for control) and plants with higher amounts of total oil (25% vs. 21.2% control). Similarly, we identified plants with increases in oleic acid content and decreases in linoleic acid and linolenic acid. This EMS mutant population will be used for further studies including screening for various traits such as amino acid pathways, allergens, phytic acids, and other important soybean agronomic traits. In addition, these mutant individuals will be evaluated in the next generation to assess the heritability. Beneficial traits from these mutants can be exploited for future soybean breeding programs. This germplasm can also be used for discovering novel mutant alleles and for functional gene expression analysis using reverse genetics tools such as TILLING.
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DNA methylation is a dynamic and reversible type of epigenetic mark that contributes to cellular physiology by affecting transcription activity, transposon mobility and genome stability. When plants are infected with pathogens, plant DNA methylation patterns can change, indicating an epigenetic interplay between plant host and pathogen. In most cases methylation can change susceptibility. While DNA hypomethylation appears to be a common phenomenon during the susceptible interaction, the levels and patterns of hypomethylation in transposable elements and genic regions may mediate distinct responses against various plant pathogens. The effect of DNA methylation on the plant immune response and other cellular activities and molecular functions is established by localized differential DNA methylation via cis-regulatory mechanisms as well as through trans-acting mechanisms. Understanding the epigenetic differences that control the phenotypic variations between susceptible and resistant interactions should facilitate the identification of new sources of resistance mediated by epigenetic mechanisms, which can be exploited to endow pathogen resistance to crops.
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Metilación de ADN , Interacciones Huésped-Patógeno/fisiología , Enfermedades de las Plantas/genética , Plantas/microbiología , Plantas/virología , Animales , Elementos Transponibles de ADN , ADN de Plantas/metabolismo , Epigénesis Genética , Inestabilidad Genómica , Interacciones Huésped-Parásitos/fisiología , Nematodos/patogenicidad , Enfermedades de las Plantas/inmunología , Plantas/genética , Rhizobium/fisiología , SimbiosisRESUMEN
KEY MESSAGE: Genetic improvement of soybean protein meal is a complex process because of negative correlation with oil, yield, and temperature. This review describes the progress in mapping and genomics, identifies knowledge gaps, and highlights the need of integrated approaches. Meal protein derived from soybean [Glycine max (L) Merr.] seed is the primary source of protein in poultry and livestock feed. Protein is a key factor that determines the nutritional and economical value of soybean. Genetic improvement of soybean seed protein content is highly desirable, and major quantitative trait loci (QTL) for soybean protein have been detected and repeatedly mapped on chromosomes (Chr.) 20 (LG-I), and 15 (LG-E). However, practical breeding progress is challenging because of seed protein content's negative genetic correlation with seed yield, other seed components such as oil and sucrose, and interaction with environmental effects such as temperature during seed development. In this review, we discuss rate-limiting factors related to soybean protein content and nutritional quality, and potential control factors regulating seed storage protein. In addition, we describe advances in next-generation sequencing technologies for precise detection of natural variants and their integration with conventional and high-throughput genotyping technologies. A syntenic analysis of QTL on Chr. 15 and 20 was performed. Finally, we discuss comprehensive approaches for integrating protein and amino acid QTL, genome-wide association studies, whole-genome resequencing, and transcriptome data to accelerate identification of genomic hot spots for allele introgression and soybean meal protein improvement.
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Glycine max/genética , Semillas/genética , Proteínas de Soja/genética , Mapeo Cromosómico , Genómica , Fitomejoramiento , Sitios de Carácter CuantitativoRESUMEN
BACKGROUND: Achieving appropriate maturity in a target environment is essential to maximizing crop yield potential. In soybean [Glycine max (L.) Merr.], the time to maturity is largely dependent on developmental response to dark periods. Once the critical photoperiod is reached, flowering is initiated and reproductive development proceeds. Therefore, soybean adaptation has been attributed to genetic changes and natural or artificial selection to optimize plant development in specific, narrow latitudinal ranges. In North America, these regions have been classified into twelve maturity groups (MG), with lower MG being shorter season than higher MG. Growing soybean lines not adapted to a particular environment typically results in poor growth and significant yield reductions. The objective of this study was to develop a molecular model for soybean maturity based on the alleles underlying the major maturity loci: E1, E2, and E3. RESULTS: We determined the allelic variation and diversity of the E maturity genes in a large collection of soybean landraces, North American ancestors, Chinese cultivars, North American cultivars or expired Plant Variety Protection lines, and private-company lines. The E gene status of accessions in the USDA Soybean Germplasm Collection with SoySNP50K Beadchip data was also predicted. We determined the E allelic combinations needed to adapt soybean to different MGs in the United States (US) and discovered a strong signal of selection for E genotypes released in North America, particularly the US and Canada. CONCLUSIONS: The E gene maturity model proposed will enable plant breeders to more effectively transfer traits into different MGs and increase the overall efficiency of soybean breeding in the US and Canada. The powerful yet simple selection strategy for increasing soybean breeding efficiency can be used alone or to directly enhance genomic prediction/selection schemes. The results also revealed previously unrecognized aspects of artificial selection in soybean imposed by soybean breeders based on geography that highlights the need for plant breeding that is optimized for specific environments.
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Glycine max/crecimiento & desarrollo , Modelos Genéticos , Genes de Plantas , Banco de Semillas , Selección GenéticaRESUMEN
BACKGROUND: A landmark in soybean research, Glyma1.01, the first whole genome sequence of variety Williams 82 (Glycine max L. Merr.) was completed in 2010 and is widely used. However, because the assembly was primarily built based on the linkage maps constructed with a limited number of markers and recombinant inbred lines (RILs), the assembled sequence, especially in some genomic regions with sparse numbers of anchoring markers, needs to be improved. Molecular markers are being used by researchers in the soybean community, however, with the updating of the Glyma1.01 build based on the high-resolution linkage maps resulting from this research, the genome positions of these markers need to be mapped. RESULTS: Two high density genetic linkage maps were constructed based on 21,478 single nucleotide polymorphism loci mapped in the Williams 82 x G. soja (Sieb. & Zucc.) PI479752 population with 1083 RILs and 11,922 loci mapped in the Essex x Williams 82 population with 922 RILs. There were 37 regions or single markers where marker order in the two populations was in agreement but was not consistent with the physical position in the Glyma1.01 build. In addition, 28 previously unanchored scaffolds were positioned. Map data were used to identify false joins in the Glyma1.01 assembly and the corresponding scaffolds were broken and reassembled to the new assembly, Wm82.a2.v1. Based upon the plots of the genetic on physical distance of the loci, the euchromatic and heterochromatic regions along each chromosome in the new assembly were delimited. Genomic positions of the commonly used markers contained in BARCSOYSSR_1.0 database and the SoySNP50K BeadChip were updated based upon the Wm82.a2.v1 assembly. CONCLUSIONS: The information will facilitate the study of recombination hot spots in the soybean genome, identification of genes or quantitative trait loci controlling yield, seed quality and resistance to biotic or abiotic stresses as well as other genetic or genomic research.
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Mapeo Cromosómico , Ligamiento Genético , Glycine max/genética , Sitios de Carácter Cuantitativo/genética , Cromosomas de las Plantas , Genoma de Planta , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The soybean cyst nematode (SCN; Heterodera glycines) induces the formation of a multinucleated feeding site, or syncytium, whose etiology includes massive gene expression changes. Nevertheless, the genetic networks underlying gene expression control in the syncytium are poorly understood. DNA methylation is a critical epigenetic mark that plays a key role in regulating gene expression. To determine the extent to which DNA methylation is altered in soybean (Glycine max) roots during the susceptible interaction with SCN, we generated whole-genome cytosine methylation maps at single-nucleotide resolution. The methylome analysis revealed that SCN induces hypomethylation to a much higher extent than hypermethylation. We identified 2,465 differentially hypermethylated regions and 4,692 hypomethylated regions in the infected roots compared with the noninfected control. In addition, 703 and 1,346 unique genes were identified as overlapping with hyper- or hypomethylated regions, respectively. The differential methylation in genes apparently occurs independently of gene size and GC content but exhibits strong preference for recently duplicated paralogs. Furthermore, a set of 278 genes was identified as specifically syncytium differentially methylated genes. Of these, we found genes associated with epigenetic regulation, phytohormone signaling, cell wall architecture, signal transduction, and ubiquitination. This study provides, to our knowledge, new evidence that differential methylation is part of the regulatory mechanisms controlling gene expression changes in the nematode-induced syncytium.