ABSTRACT
Understanding the origins of variation in agricultural pathogens is of fundamental interest and practical importance, especially for diseases that threaten food security. Fusarium oxysporum is among the most important of soil-borne pathogens, with a global distribution and an extensive host range. The pathogen is considered to be asexual, with horizontal transfer of chromosomes providing an analog of assortment by meiotic recombination. Here, we challenge those assumptions based on the results of population genomic analyses, describing the pathogen's diversity and inferring its origins and functional consequences in the context of a single, long-standing agricultural system. We identify simultaneously low nucleotide distance among strains, and unexpectedly high levels of genetic and genomic variability. We determine that these features arise from a combination of genome-scale recombination, best explained by widespread sexual reproduction, and presence-absence variation consistent with chromosomal rearrangement. Pangenome analyses document an accessory genome more than twice the size of the core genome, with contrasting evolutionary dynamics. The core genome is stable, with low diversity and high genetic differentiation across geographic space, while the accessory genome is paradoxically more diverse and unstable but with lower genetic differentiation and hallmarks of contemporary gene flow at local scales. We suggest a model in which episodic sexual reproduction generates haplotypes that are selected and then maintained through clone-like dynamics, followed by contemporary genomic rearrangements that reassort the accessory genome among sympatric strains. Taken together, these processes contribute unique genome content, including reassortment of virulence determinants that may explain observed variation in pathogenic potential.
Subject(s)
Fusarium , Fusarium/genetics , Host Specificity , Genomics , Agriculture , Plant Diseases/geneticsABSTRACT
Here we present the approach used to develop the INCREASE "Intelligent Chickpea" Collections, from analysis of the information on the life history and population structure of chickpea germplasm, the availability of genomic and genetic resources, the identification of key phenotypic traits and methodologies to characterize chickpea. We present two phenotypic protocols within H2O20 Project INCREASE to characterize, develop, and maintain chickpea single-seed-descent (SSD) line collections. Such protocols and related genetic resource data from the project will be available for the legume community to apply the standardized approaches to develop Chickpea Intelligent Collections further or for multiplication/seed-increase purposes. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Characterization of chickpea seeds for seed-trait descriptors Basic Protocol 2: Characterization of chickpea lines for plant-trait descriptors specific for primary seed increase.
Subject(s)
Cicer , Fabaceae , Cicer/genetics , Genomics , Phenotype , Seeds/geneticsABSTRACT
Chickpea is the third most important grain legume worldwide. This is due in part to its high protein content that results from its ability to acquire bioavailable nitrogen when colonized by diverse, nitrogen fixing Mesorhizobium species. However, the diversity and distribution of mesorhizobia communities may depend on their adaptation to soil conditions. Therefore, this study was initiated in order to isolate and investigate the diversity and taxonomic identities of chickpea-nodulating Mesorhizobium species from low pH soils of Ethiopia. A total of 81 rhizobia strains were isolated from chickpea nodules harvested from low pH soils throughout Ethiopia, and their genomes were sequenced and assembled. Considering a representative set of the best-sequenced 81 genomes, the average sequence depth was 30X, with estimated average genome sizes of approximately 7 Mbp. Annotation of the assembled genome predicted an average of 7,453 protein-coding genes. Concatenation of 400 universal PhyloPhlAn conserved genes present in the genomes of all 81 strains allowed detailed phylogenetic analysis, from which eight well-supported species were identified, including M.opportunistum, M.australicum, Mesorhizobium sp. LSJC280BOO, M.wenxiniae, M.amorphae, M.loti and M.plurifarium, as well as a novel species. Phylogenetic reconstructions based on the symbiosis-related (nodC and nifH) genes were different from the core genes and consistent with horizontal transfer of the symbiotic island. The two major genomic groups, M.plurifarium and M.loti, were widely distributed in almost all the sites. The geographic pattern of genomic diversity indicated there was no relationship between geographic and genetic distance (r = 0.01, p > 0.01). In conclusion, low pH soils in Ethiopia harbored a diverse group of Mesorhizobium species, several of which were not previously known to nodulate chickpea.
Subject(s)
Cicer , Mesorhizobium , DNA, Bacterial , Ethiopia , Genomics , Hydrogen-Ion Concentration , Mesorhizobium/genetics , Phylogeny , RNA, Ribosomal, 16S , Sequence Analysis, DNA , Soil , SymbiosisABSTRACT
Evolutionary dynamics at the population level play a central role in creating the diversity of life on our planet. In this study, we sought to understand the origins of such population-level variation in mating systems and defensive acylsugar chemistry in Solanum habrochaites-a wild tomato species found in diverse Andean habitats in Ecuador and Peru. Using Restriction-site-Associated-DNA-Sequencing (RAD-seq) of 50 S. habrochaites accessions, we identified eight population clusters generated via isolation and hybridization dynamics of 4-6 ancestral populations. Detailed characterization of mating systems of these clusters revealed emergence of multiple self-compatible (SC) groups from progenitor self-incompatible populations in the northern part of the species range. Emergence of these SC groups was also associated with fixation of deleterious alleles inactivating acylsugar acetylation. The Amotape-Huancabamba Zone-a geographical landmark in the Andes with high endemism and isolated microhabitats-was identified as a major driver of differentiation in the northern species range, whereas large geographical distances contributed to population structure and evolution of a novel SC group in the central and southern parts of the range, where the species was also inferred to have originated. Findings presented here highlight the role of the diverse ecogeography of Peru and Ecuador in generating population differentiation, and enhance our understanding of the microevolutionary processes that create biological diversity.
Subject(s)
Gene Flow , Self-Incompatibility in Flowering Plants/genetics , Solanum lycopersicum/genetics , Solanum/genetics , Acetylation , Ecuador , Solanum lycopersicum/metabolism , Peru , Phylogeography , Self-Fertilization , Solanum/metabolismABSTRACT
The main purpose of this study was to screen and select strains from seven Mesorhizobium spp. for efficient phosphate solubilizing and other plant growth-promoting traits. Mesorhizobium species were tested for their ability to dissolve inorganic phosphate sources and multiple plant growth-promoting attributes. From a total of 62 Mesorhizobium strains, 47(76%) strains formed clear zones with an average PSI of 1.9-2.7 on Pikovskaya's agar plate. The selected strains also released soluble phosphorus [125-150 P (µgml-1)] from tri-calcium phosphate and low level of phosphorous i.e., 15.4 µg/ml and 14.5 µg/ml from inorganic ferrous and aluminum phosphates, respectively, in a liquid medium after 4 days of incubation. The release of soluble P was significantly (P < 0.01) correlated with a drop in pH of the medium. Moreover, screening for multiple plant growth-promoting attributes showed that 40, 28, 26, 21, and 38% of the strains were capable of producing indole-3-acetic acid, hydrogen cyanide, siderophores, ACC deaminase, and antagonism against Fusarium oxysporum f.sp. ciceris under in vitro conditions. The Mesorhizobium strains were endowed with the presence of ACC deaminase which was rarely reported elsewhere. All taken together, the acidic soils harbor numerous and more diverse phosphate solubilizing and plant growth-promoting Mesorhizobium spp. However, greenhouse and field conditions can be further studied within the context of improving chickpea production in Ethiopia.
Subject(s)
Cicer/microbiology , Mesorhizobium/metabolism , Phosphates/metabolism , Root Nodules, Plant/microbiology , Soil Microbiology , Antibiosis , Carbon-Carbon Lyases/metabolism , Cicer/growth & development , Ethiopia , Fusarium/physiology , Indoleacetic Acids/metabolism , Siderophores/metabolism , Soil/chemistryABSTRACT
Ancestral adaptations in crop wild relatives can provide a genetic reservoir for crop improvement. Here we document physiological changes to mild and severe drought stress, and the associated transcriptome dynamics in both wild and cultivated chickpea. Over 60% of transcriptional changes were related to metabolism, indicating that metabolic plasticity is a core and conserved drought response. In addition, changes in RNA processing and protein turnover were predominant in the data, suggestive of broad restructuring of the chickpea proteome in response to drought. While 12% of the drought-responsive transcripts have similar dynamics in cultivated and wild accessions, numerous transcripts had expression patterns unique to particular genotypes, or that distinguished wild from cultivated genotypes and whose divergence may be a consequence of domestication. These and other comparisons provide a transcriptional correlate of previously described species' genetic diversity, with wild accessions well differentiated from each other and from cultivars, and cultivars essentially indistinguishable at the broad transcriptome level. We identified metabolic pathways such as phenylpropanoid metabolism, and biological processes such as stomatal development, which are differentially regulated across genotypes with potential consequences on drought tolerance. These data indicate that wild Cicer reticulatum may provide both conserved and divergent mechanisms as a resource in breeding for drought tolerance in cultivated chickpea.
Subject(s)
Cicer/genetics , Dehydration/genetics , Gene Expression Regulation, Plant , Adaptation, Physiological/genetics , Cicer/physiology , Crops, Agricultural/genetics , Droughts , Gene Expression Profiling , Genotype , Plant Proteins/genetics , Plant Proteins/metabolism , Stress, Physiological/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
A defining challenge of the 21st century is meeting the nutritional demands of the growing human population, under a scenario of limited land and water resources and under the specter of climate change. The Vavilov seed bank contains numerous landraces collected nearly a hundred years ago, and thus may contain 'genetic gems' with the potential to enhance modern breeding efforts. Here, we analyze 407 landraces, sampled from major historic centers of chickpea cultivation and secondary diversification. Genome-Wide Association Studies (GWAS) conducted on both phenotypic traits and bioclimatic variables at landraces sampling sites as extended phenotypes resulted in 84 GWAS hits associated to various regions. The novel haploblock-based test identified haploblocks enriched for single nucleotide polymorphisms (SNPs) associated with phenotypes and bioclimatic variables. Subsequent bi-clustering of traits sharing enriched haploblocks underscored both non-random distribution of SNPs among several haploblocks and their association with multiple traits. We hypothesize that these clusters of pleiotropic SNPs represent co-adapted genetic complexes to a range of environmental conditions that chickpea experienced during domestication and subsequent geographic radiation. Linking genetic variation to phenotypic data and a wealth of historic information preserved in historic seed banks are the keys for genome-based and environment-informed breeding intensification.
Subject(s)
Cicer/genetics , Crops, Agricultural/genetics , Plant Breeding , Seeds , Biodiversity , Climate , Cluster Analysis , Conservation of Natural Resources , Genetic Association Studies , Genetic Markers , Genetic Variation , Genome, Plant , Genotype , Geography , Haplotypes , History, 20th Century , History, 21st Century , Likelihood Functions , Linkage Disequilibrium , Phenotype , Polymorphism, Single Nucleotide , Seed Bank/history , Seed Bank/organization & administrationABSTRACT
"Stay-green" crop phenotypes have been shown to impact drought tolerance and nutritional content of several crops. We aimed to genetically describe and functionally dissect the particular stay-green phenomenon found in chickpeas with a green cotyledon color of mature dry seed and investigate its potential use for improvement of chickpea environmental adaptations and nutritional value. We examined 40 stay-green accessions and a set of 29 BC2F4-5 stay-green introgression lines using a stay-green donor parent ICC 16340 and two Indian elite cultivars (KAK2, JGK1) as recurrent parents. Genetic studies of segregating populations indicated that the green cotyledon trait is controlled by a single recessive gene that is invariantly associated with the delayed degreening (extended chlorophyll retention). We found that the chickpea ortholog of Mendel's I locus of garden pea, encoding a SGR protein as very likely to underlie the persistently green cotyledon color phenotype of chickpea. Further sequence characterization of this chickpea ortholog CaStGR1 (CaStGR1, for carietinum stay-green gene 1) revealed the presence of five different molecular variants (alleles), each of which is likely a loss-of-function of the chickpea protein (CaStGR1) involved in chlorophyll catabolism. We tested the wild type and green cotyledon lines for components of adaptations to dry environments and traits linked to agronomic performance in different experimental systems and different levels of water availability. We found that the plant processes linked to disrupted CaStGR1 gene did not functionality affect transpiration efficiency or water usage. Photosynthetic pigments in grains, including provitaminogenic carotenoids important for human nutrition, were 2-3-fold higher in the stay-green type. Agronomic performance did not appear to be correlated with the presence/absence of the stay-green allele. We conclude that allelic variation in chickpea CaStGR1 does not compromise traits linked to environmental adaptation and agronomic performance, and is a promising genetic technology for biofortification of provitaminogenic carotenoids in chickpea.
Subject(s)
Carotenoids/metabolism , Cicer , Cotyledon , Crop Production , Genetic Variation , Phenotype , Pigmentation/genetics , Cicer/genetics , Cicer/growth & development , Cotyledon/genetics , Cotyledon/growth & development , Photosynthesis/geneticsABSTRACT
Flowering time is a key trait in breeding and crop evolution, due to its importance for adaptation to different environments and for yield. In the particular case of chickpea, selection for early phenology was essential for the successful transition of this species from a winter to a summer crop. Here, we used genetic and expression analyses in two different inbred populations to examine the genetic control of domestication-related differences in flowering time and growth habit between domesticated chickpea and its wild progenitor Cicer reticulatum. A single major quantitative trait locus for flowering time under short-day conditions [Days To Flower (DTF)3A] was mapped to a 59-gene interval on chromosome three containing a cluster of three FT genes, which collectively showed upregulated expression in domesticated relative to wild parent lines. An equally strong association with growth habit suggests a pleiotropic effect of the region on both traits. These results indicate the likely molecular explanation for the characteristic early flowering of domesticated chickpea, and the previously described growth habit locus Hg. More generally, they point to de-repression of this specific gene cluster as a conserved mechanism for achieving adaptive early phenology in temperate legumes.
ABSTRACT
Although microorganisms are known to dominate Earth's biospheres and drive biogeochemical cycling, little is known about the geographic distributions of microbial populations or the environmental factors that pattern those distributions. We used a global-level hierarchical sampling scheme to comprehensively characterize the evolutionary relationships and distributional limitations of the nitrogen-fixing bacterial symbionts of the crop chickpea, generating 1,027 draft whole-genome sequences at the level of bacterial populations, including 14 high-quality PacBio genomes from a phylogenetically representative subset. We find that diverse Mesorhizobium taxa perform symbiosis with chickpea and have largely overlapping global distributions. However, sampled locations cluster based on the phylogenetic diversity of Mesorhizobium populations, and diversity clusters correspond to edaphic and environmental factors, primarily soil type and latitude. Despite long-standing evolutionary divergence and geographic isolation, the diverse taxa observed to nodulate chickpea share a set of integrative conjugative elements (ICEs) that encode the major functions of the symbiosis. This symbiosis ICE takes 2 forms in the bacterial chromosome-tripartite and monopartite-with tripartite ICEs confined to a broadly distributed superspecies clade. The pairwise evolutionary relatedness of these elements is controlled as much by geographic distance as by the evolutionary relatedness of the background genome. In contrast, diversity in the broader gene content of Mesorhizobium genomes follows a tight linear relationship with core genome phylogenetic distance, with little detectable effect of geography. These results illustrate how geography and demography can operate differentially on the evolution of bacterial genomes and offer useful insights for the development of improved technologies for sustainable agriculture.
Subject(s)
Cicer/microbiology , Gene Transfer, Horizontal , Genome, Bacterial , Mesorhizobium/genetics , Microbial Consortia/genetics , Biological Evolution , Conjugation, Genetic , Mesorhizobium/classification , Metagenomics/methods , Nitrogen Fixation/physiology , Phylogeny , Phylogeography , Soil/classification , Soil Microbiology , Symbiosis/geneticsABSTRACT
Domestication and subsequent breeding have eroded genetic diversity in the modern chickpea crop by Ë100-fold. Corresponding reductions to trait variation create the need, and an opportunity, to identify and harness the genetic capacity of wild species for crop improvement. Here we analyze trait segregation in a series of wild x cultivated hybrid populations to delineate the genetic underpinnings of domestication traits. Two species of wild chickpea, C. reticulatum and C. echinospermum, were crossed with the elite, early flowering C. arietinum cultivar ICCV96029. KASP genotyping of F2 parents with an FT-linked molecular marker enabled selection of 284 F3 families with reduced phenological variation: 255 F3 families of C. arietinum x reticulatum (AR) derived from 17 diverse wild parents and 29 F3 families of C. arietinum x echinospermum (AE) from 3 wild parents. The combined 284 lineages were genotyped using a genotyping-by-sequencing strategy and phenotyped for agronomic traits. 50 QTLs in 11 traits were detected from AR and 35 QTLs in 10 traits from the combined data. Using hierarchical clustering to assign traits to six correlated groups and mixed model based multi-trait mapping, four pleiotropic loci were identified. Bayesian analysis further identified four inter-trait relationships controlling the duration of vegetative growth and seed maturation, for which the underlying pleiotropic genes were mapped. A random forest approach was used to explore the most extreme trait differences between AR and AE progenies, identifying traits most characteristic of wild species origin. Knowledge of the genomic basis of traits that segregate in wild-cultivated hybrid populations will facilitate chickpea improvement by linking genetic and phenotypic variation in a quantitative genetic framework.
Subject(s)
Cicer/genetics , Genes, Plant/genetics , Plant Breeding/methods , Bayes Theorem , Cicer/growth & development , DNA, Plant/genetics , Domestication , Genetic Association Studies , Genetic Linkage/genetics , Hybridization, Genetic/genetics , Quantitative Trait Loci/genetics , Quantitative Trait, Heritable , Seeds/growth & developmentABSTRACT
Chickpea ( L.) production in arid regions, such as those predominant in Pakistan, faces immense challenges of drought and heat stress. Addressing these challenges is made more difficult by the lack of genetic and phenotypic characterization of available cultivated varieties and breeding materials. Genotyping-by-sequencing offers a rapid and cost-effective means to identify genome-wide nucleotide variation in crop germplasm. When combined with extended crop phenotypes deduced from climatic variation at sites of collection, the data can predict which portions of genetic variation might have roles in climate resilience. Here we use 8113 single nucleotide polymorphism markers to determine genetic variation and compare population structure within a previously uncharacterized collection of 77 landraces and 5 elite cultivars, currently grown in situ on farms throughout the chickpea growing regions of Pakistan. The compiled landraces span a striking aridity gradient into the Thal Desert of the Punjab. Despite low levels of variation across the collection and limited genetic structure, we found some differentiation between accessions from arid, semiarid, irrigated, and coastal areas. In a subset of 232 markers, we found evidence of differentiation along gradients of elevation and isothermality. Our results highlight the utility of exploring large germplasm collections for nucleotide variation associated with environmental extremes, and the use of such data to nominate germplasm accessions with the potential to improve crop drought tolerance and other environmental traits.
Subject(s)
Cicer/genetics , Genetic Variation , Climate , Droughts , Genetics, Population , Pakistan , Plant Breeding , Polymorphism, Single NucleotideABSTRACT
Domesticated species are impacted in unintended ways during domestication and breeding. Changes in the nature and intensity of selection impart genetic drift, reduce diversity, and increase the frequency of deleterious alleles. Such outcomes constrain our ability to expand the cultivation of crops into environments that differ from those under which domestication occurred. We address this need in chickpea, an important pulse legume, by harnessing the diversity of wild crop relatives. We document an extreme domestication-related genetic bottleneck and decipher the genetic history of wild populations. We provide evidence of ancestral adaptations for seed coat color crypsis, estimate the impact of environment on genetic structure and trait values, and demonstrate variation between wild and cultivated accessions for agronomic properties. A resource of genotyped, association mapping progeny functionally links the wild and cultivated gene pools and is an essential resource chickpea for improvement, while our methods inform collection of other wild crop progenitor species.
Subject(s)
Cicer/genetics , Crops, Agricultural/genetics , Agriculture , Cicer/classification , Cicer/physiology , Ecology , Environment , Genetic Variation , Genome, Plant , Genomics , Genotype , Seeds/classification , Seeds/genetics , Seeds/physiologyABSTRACT
The Vavilov Institute of Plant Genetic Resources (VIR), in St. Petersburg, Russia, houses a unique genebank, with historical collections of landraces. When they were collected, the geographical distribution and genetic diversity of most crops closely reflected their historical patterns of cultivation established over the preceding millennia. We employed a combination of genomics, computational biology and phenotyping to characterize VIR's 147 chickpea accessions from Turkey and Ethiopia, representing chickpea's center of origin and a major location of secondary diversity. Genotyping by sequencing identified 14,059 segregating polymorphisms and genome-wide association studies revealed 28 GWAS hits in potential candidate genes likely to affect traits of agricultural importance. The proportion of polymorphisms shared among accessions is a strong predictor of phenotypic resemblance, and of environmental similarity between historical sampling sites. We found that 20 out of 28 polymorphisms, associated with multiple traits, including days to maturity, plant phenology, and yield-related traits such as pod number, localized to chromosome 4. We hypothesize that selection and introgression via inadvertent hybridization between more and less advanced morphotypes might have resulted in agricultural improvement genes being aggregated to genomic 'agro islands', and in genotype-to-phenotype relationships resembling widespread pleiotropy.
Subject(s)
Cicer/genetics , Crops, Agricultural , Genome, Plant , Genomic Islands , Polymorphism, Single Nucleotide , Quantitative Trait, Heritable , Cicer/classification , Computational Biology , Databases, Genetic , Ethiopia , Genetic Pleiotropy , Genome-Wide Association Study , Genotype , Phenotype , Phylogeny , Quantitative Trait Loci , Russia , TurkeyABSTRACT
Fusarium oxysporum species complex (FOSC) isolates were obtained from celery with symptoms of Fusarium yellows between 1993 and 2013 primarily in California. Virulence tests and a two-gene dataset from 174 isolates indicated that virulent isolates collected before 2013 were a highly clonal population of F. oxysporum f. sp. apii race 2. In 2013, new highly virulent clonal isolates, designated race 4, were discovered in production fields in Camarillo, California. Long-read Illumina data were used to analyze 16 isolates: six race 2, one of each from races 1, 3, and 4, and seven genetically diverse FOSC that were isolated from symptomatic celery but are nonpathogenic on this host. Analyses of a 10-gene dataset comprising 38 kb indicated that F. oxysporum f. sp. apii is polyphyletic; race 2 is nested within clade 3, whereas the evolutionary origins of races 1, 3, and 4 are within clade 2. Based on 6,898 single nucleotide polymorphisms from the core FOSC genome, race 3 and the new highly virulent race 4 are highly similar with Nei's Da = 0.0019, suggesting that F. oxysporum f. sp. apii race 4 evolved from race 3. Next generation sequences were used to develop PCR primers that allow rapid diagnosis of races 2 and 4 in planta.
Subject(s)
Apium/microbiology , Fusarium/genetics , Genetic Variation , Plant Diseases/microbiology , California , Evolution, Molecular , Fusarium/isolation & purification , Fusarium/pathogenicity , High-Throughput Nucleotide Sequencing , Sequence Analysis, DNA , VirulenceABSTRACT
Chickpea (Cicer arietinum) is among the founder crops domesticated in the Fertile Crescent. One of two major forms of chickpea, the so-called kabuli type, has white flowers and light-colored seed coats, properties not known to exist in the wild progenitor. The origin of the kabuli form has been enigmatic. We genotyped a collection of wild and cultivated chickpea genotypes with 538 single nucleotide polymorphisms (SNPs) and examined patterns of molecular diversity relative to geographical sources and market types. In addition, we examined sequence and expression variation in candidate anthocyanin biosynthetic pathway genes. A reduction in genetic diversity and extensive genetic admixture distinguish cultivated chickpea from its wild progenitor species. Among germplasm, the kabuli form is polyphyletic. We identified a basic helix-loop-helix (bHLH) transcription factor at chickpea's B locus that conditions flower and seed colors, orthologous to Mendel's A gene of garden pea, whose loss of function is associated invariantly with the kabuli type of chickpea. From the polyphyletic distribution of the kabuli form in germplasm, an absence of nested variation within the bHLH gene and invariant association of loss of function of bHLH among the kabuli type, we conclude that the kabuli form arose multiple times during the phase of phenotypic diversification after initial domestication of cultivated chickpea.
Subject(s)
Alleles , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cicer/genetics , Domestication , Genetic Variation , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cicer/anatomy & histology , Crops, Agricultural/genetics , Ecotype , Flowers/anatomy & histology , Flowers/physiology , Gene Expression Regulation, Plant , Haplotypes/genetics , Phylogeny , Polymorphism, Single Nucleotide/genetics , Principal Component Analysis , Seeds/anatomy & histologyABSTRACT
The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae. To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factor-induced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:ß-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor- and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.
Subject(s)
Ethylenes/metabolism , High-Throughput Nucleotide Sequencing/methods , Medicago truncatula/genetics , Medicago truncatula/microbiology , Plant Proteins/metabolism , Plant Roots/genetics , Signal Transduction/drug effects , Transcriptome/genetics , Biosynthetic Pathways/drug effects , Biosynthetic Pathways/genetics , Cluster Analysis , Ethylenes/pharmacology , Feedback, Physiological , Gene Expression Regulation, Plant/drug effects , Gene Ontology , Gene Regulatory Networks , Genes, Plant , Medicago truncatula/drug effects , Plant Proteins/genetics , Plant Roots/drug effects , Plant Roots/growth & development , Plant Roots/microbiology , Rhizobium/drug effects , Rhizobium/physiology , Signal Transduction/genetics , Symbiosis/genetics , Time Factors , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Transcriptome/drug effectsABSTRACT
BACKGROUND: Genetic markers are tools that can facilitate molecular breeding, even in species lacking genomic resources. An important class of genetic markers is those based on orthologous genes, because they can guide hypotheses about conserved gene function, a situation that is well documented for a number of agronomic traits. For under-studied species a key bottleneck in gene-based marker development is the need to develop molecular tools (e.g., oligonucleotide primers) that reliably access genes with orthology to the genomes of well-characterized reference species. RESULTS: Here we report an efficient platform for the design of cross-species gene-derived markers in legumes. The automated platform, named CSGM Designer (URL: http://tgil.donga.ac.kr/CSGMdesigner), facilitates rapid and systematic design of cross-species genic markers. The underlying database is composed of genome data from five legume species whose genomes are substantially characterized. Use of CSGM is enhanced by graphical displays of query results, which we describe as "circular viewer" and "search-within-results" functions. CSGM provides a virtual PCR representation (eHT-PCR) that predicts the specificity of each primer pair simultaneously in multiple genomes. CSGM Designer output was experimentally validated for the amplification of orthologous genes using 16 genotypes representing 12 crop and model legume species, distributed among the galegoid and phaseoloid clades. Successful cross-species amplification was obtained for 85.3% of PCR primer combinations. CONCLUSION: CSGM Designer spans the divide between well-characterized crop and model legume species and their less well-characterized relatives. The outcome is PCR primers that target highly conserved genes for polymorphism discovery, enabling functional inferences and ultimately facilitating trait-associated molecular breeding.
ABSTRACT
BACKGROUND: As our world becomes warmer, agriculture is increasingly impacted by rising soil salinity and understanding plant adaptation to salt stress can help enable effective crop breeding. Salt tolerance is a complex plant phenotype and we know little about the pathways utilized by naturally tolerant plants. Legumes are important species in agricultural and natural ecosystems, since they engage in symbiotic nitrogen-fixation, but are especially vulnerable to salinity stress. RESULTS: Our studies of the model legume Medicago truncatula in field and greenhouse settings demonstrate that Tunisian populations are locally adapted to saline soils at the metapopulation level and that saline origin genotypes are less impacted by salt than non-saline origin genotypes; these populations thus likely contain adaptively diverged alleles. Whole genome resequencing of 39 wild accessions reveals ongoing migration and candidate genomic regions that assort non-randomly with soil salinity. Consistent with natural selection acting at these sites, saline alleles are typically rare in the range-wide species' gene pool and are also typically derived relative to the sister species M. littoralis. Candidate regions for adaptation contain genes that regulate physiological acclimation to salt stress, such as abscisic acid and jasmonic acid signaling, including a novel salt-tolerance candidate orthologous to the uncharacterized gene AtCIPK21. Unexpectedly, these regions also contain biotic stress genes and flowering time pathway genes. We show that flowering time is differentiated between saline and non-saline populations and may allow salt stress escape. CONCLUSIONS: This work nominates multiple potential pathways of adaptation to naturally stressful environments in a model legume. These candidates point to the importance of both tolerance and avoidance in natural legume populations. We have uncovered several promising targets that could be used to breed for enhanced salt tolerance in crop legumes to enhance food security in an era of increasing soil salinization.
Subject(s)
Adaptation, Physiological/genetics , Ecological and Environmental Phenomena , Genomics , Medicago truncatula/genetics , Medicago truncatula/physiology , Salinity , Evolution, Molecular , Gene Frequency , Genetic Loci/genetics , Molecular Sequence Annotation , Recombination, Genetic , Selection, Genetic , Soil/chemistry , Species SpecificityABSTRACT
Lentil (Lens culinaris Medik.) is a global food crop with increasing importance for food security in south Asia and other regions. Lens ervoides, a wild relative of cultivated lentil, is an important source of agronomic trait variation. Lens is a member of the galegoid clade of the Papilionoideae family, which includes other important dietary legumes such as chickpea (Cicer arietinum) and pea (Pisum sativum), and the sequenced model legume Medicago truncatula. Understanding the genetic structure of Lens spp. in relation to more fully sequenced legumes would allow leveraging of genomic resources. A set of 1107 TOG-based amplicons were identified in L. ervoides and a subset thereof used to design SNP markers for mapping. A map of L. ervoides consisting of 377 SNP markers spread across seven linkage groups was developed using a GoldenGate genotyping array and single SNP marker assays. Comparison with maps of M. truncatula and L. culinaris documented considerable shared synteny and led to the identification of a few major translocations and a major inversion that distinguish Lens from M. truncatula, as well as a translocation that distinguishes L. culinaris from L. ervoides. The identification of chromosome-level differences among Lens spp. will aid in the understanding of introgression of genes from L. ervoides into cultivated L. culinaris, furthering genetic research and breeding applications in lentil.