Standards for fecal microbiota transplant: Tools and therapeutic advances.

Fecal microbiota transplantation (FMT) has been demonstrated to be efficacious in preventing recurrent Clostridioides difficile (C. difficile) infections, and is being investigated for treatment of several other diseases including inflammatory bowel disease, cancer, obesity, liver disease, and diabetes. To speed up the translation of FMT into clinical practice as a safe and standardized therapeutic intervention, additional evidence-based technical and regulatory guidance is needed. To this end in May of 2022, the International Alliance for Biological Standardization (IABS) and the BIOASTER Microbiology Technology Institute hosted a second webinar to discuss key issues still impeding the advancement and standardization of FMT. The goal of this two-day webinar was to provide a forum for scientific experts to share and discuss data and key challenges with one another. Discussion included a focus on the evaluation of safety, efficacy, clinical trial design, reproducibility and accuracy in obtained microbiome measurements and data reporting, and the potential for standardization across these areas. It also focused on increasing the application potential and visibility of FMT beyond treating C. difficile infections.

Legacy genetics of Arachis cardenasii in the peanut crop shows the profound benefits of international seed exchange.

The narrow genetics of most crops is a fundamental vulnerability to food security. This makes wild crop relatives a strategic resource of genetic diversity that can be used for crop improvement and adaptation to new agricultural challenges. Here, we uncover the contribution of one wild species accession, Arachis cardenasii GKP 10017, to the peanut crop (Arachis hypogaea) that was initiated by complex hybridizations in the 1960s and propagated by international seed exchange. However, until this study, the global scale of the dispersal of genetic contributions from this wild accession had been obscured by the multiple germplasm transfers, breeding cycles, and unrecorded genetic mixing between lineages that had occurred over the years. By genetic analysis and pedigree research, we identified A. cardenasii-enhanced, disease-resistant cultivars in Africa, Asia, Oceania, and the Americas. These cultivars provide widespread improved food security and environmental and economic benefits. This study emphasizes the importance of wild species and collaborative networks of international expertise for crop improvement. However, it also highlights the consequences of the implementation of a patchwork of restrictive national laws and sea changes in attitudes regarding germplasm that followed in the wake of the Convention on Biological Diversity. Today, the botanical collections and multiple seed exchanges which enable benefits such as those revealed by this study are drastically reduced. The research reported here underscores the vital importance of ready access to germplasm in ensuring long-term world food security.

TAR30, a homolog of the canonical plant TTTAGGG telomeric repeat, is enriched in the proximal chromosome regions of peanut (Arachis hypogaea L.).

Telomeres are the physical ends of eukaryotic linear chromosomes that play critical roles in cell division, chromosome maintenance, and genome stability. In many plants, telomeres are comprised of TTTAGGG tandem repeat that is widely found in plants. We refer to this repeat as canonical plant telomeric repeat (CPTR). Peanut (Arachis hypogaea L.) is a spontaneously formed allotetraploid and an important food and oil crop worldwide. In this study, we analyzed the peanut genome sequences and identified a new type of tandem repeat with 10-bp basic motif TTTT(C/T)TAGGG named TAndem Repeat (TAR) 30. TAR30 showed significant sequence identity to TTTAGGG repeat in 112 plant genomes suggesting that TAR30 is a homolog of CPTR. It also is nearly identical to the telomeric tandem repeat in Cestrum elegans. Fluorescence in situ hybridization (FISH) analysis revealed interstitial locations of TAR30 in peanut chromosomes but we did not detect visible signals in the terminal ends of chromosomes as expected for telomeric repeats. Interestingly, different TAR30 hybridization patterns were found between the newly induced allotetraploid ValSten and its diploid wild progenitors. The canonical telomeric repeat TTTAGGG is also present in the peanut genomes and some of these repeats are closely adjacent to TAR30 from both cultivated peanut and its wild relatives. Overall, our work identifies a new homolog of CPTR and reveals the unique distributions of TAR30 in cultivated peanuts and wild species. Our results provide new insights into the evolution of tandem repeats during peanut polyploidization and domestication.

Analysis of CACTA transposase genes unveils the mechanism of intron loss and distinct small RNA silencing pathways underlying divergent evolution of Brassica genomes.

In comparison with retrotransposons, DNA transposons make up a smaller proportion of most plant genomes. However, these elements are often proximal to genes to affect gene expression depending on the activity of the transposons, which is largely reflected by the activity of the transposase genes. Here, we show that three AT-rich introns were retained in the TNP2-like transposase genes of the Bot1 (Brassica oleracea transposon 1) CACTA transposable elements in Brassica oleracea, but were lost in the majority of the Bot1 elements in Brassica rapa. A recent burst of transposition of Bot1 was observed in B. oleracea, but not in B. rapa. This burst of transposition is likely related to the activity of the TNP2-like transposase genes as the expression values of the transposase genes were higher in B. oleracea than in B. rapa. In addition, distinct populations of small RNAs (21, 22 and 24 nt) were detected from the Bot1 elements in B. oleracea, but the vast majority of the small RNAs from the Bot1 elements in B. rapa are 24 nt in length. We hypothesize that the different activity of the TNP2-like transposase genes is likely associated with the three introns, and intron loss is likely reverse transcriptase mediated. Furthermore, we propose that the Bot1 family is currently undergoing silencing in B. oleracea, but has already been silenced in B. rapa. Taken together, our data provide new insights into the differentiation of transposons and their role in the asymmetric evolution of these two closely related Brassica species.

Genome-Wide Reinforcement of DNA Methylation Occurs during Somatic Embryogenesis in Soybean.

Somatic embryogenesis is an important tissue culture technique that sometimes leads to phenotypic variation via genetic and/or epigenetic changes. To understand the genomic and epigenomic impacts of somatic embryogenesis, we characterized soybean (Glycine max) epigenomes sampled from embryos at 10 different stages ranging from 6 weeks to 13 years of continuous culture. We identified genome-wide increases in DNA methylation from cultured samples, especially at CHH sites. The hypermethylation almost exclusively occurred in regions previously possessing non-CG methylation and was accompanied by increases in the expression of genes encoding the RNA-directed DNA methylation (RdDM) machinery. The epigenomic changes were similar between somatic and zygotic embryogenesis. Following the initial global wave of hypermethylation, rare decay events of maintenance methylation were observed, and the extent of the decay increased with time in culture. These losses in DNA methylation were accompanied by downregulation of genes encoding the RdDM machinery and transcriptome reprogramming reminiscent of transcriptomes during late-stage seed development. These results reveal a process for reinforcing already silenced regions to maintain genome integrity during somatic embryogenesis over the short term, which eventually decays at certain loci over longer time scales.

Construction and comparison of three reference-quality genome assemblies for soybean.

We report reference-quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single-nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan-gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40-42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.

Workshop report: Toward the development of a human whole stool reference material for metabolomic and metagenomic gut microbiome measurements.

INTRODUCTION: To date, there has been little effort to develop standards for metabolome-based gut microbiome measurements despite the significant efforts toward standard development for DNA-based microbiome measurements. OBJECTIVES: The National Institute of Standards and Technology (NIST), The BioCollective (TBC), and the North America Branch of the International Life Sciences Institute (ILSI North America) are collaborating to extend NIST's efforts to develop a Human Whole Stool Reference Material for the purpose of method harmonization and eventual quality control. METHODS: The reference material will be rationally designed for adequate quality assurance and quality control (QA/QC) for underlying measurements in the study of the impact of diet and nutrition on functional aspects of the host gut microbiome and relationships of those functions to health. To identify which metabolites deserve priority in their value assignment, NIST, TBC, and ILSI North America jointly conducted a workshop on September 12, 2019 at the NIST campus in Gaithersburg, Maryland. The objective of the workshop was to identify metabolites for which evidence indicates relevance to health and disease and to decide on the appropriate course of action to develop a fit-for-purpose reference material. RESULTS: This document represents the consensus opinions of workshop participants and co-authors of this manuscript, and provides additional supporting information. In addition to developing general criteria for metabolite selection and a preliminary list of proposed metabolites, this paper describes some of the strengths and limitations of this initiative given the current state of microbiome research. CONCLUSIONS: Given the rapidly evolving nature of gut microbiome science and the current state of knowledge, an RM (as opposed to a CRM) measured for multiple metabolites is appropriate at this stage. As the science evolves, the RM can evolve to match the needs of the research community. Ultimately, the stool RM may exist in sequential versions. Beneficial to this evolution will be a clear line of communication between NIST and the stakeholder community to ensure alignment with current scientific understanding and community needs.

Horizontal Transfer of Non-LTR Retrotransposons from Arthropods to Flowering Plants.

Even though lateral movements of transposons across families and even phyla within multicellular eukaryotic kingdoms have been found, little is known about transposon transfer between the kingdoms Animalia and Plantae. We discovered a novel non-LTR retrotransposon, AdLINE3, in a wild peanut species. Sequence comparisons and phylogenetic analyses indicated that AdLINE3 is a member of the RTE clade, originally identified in a nematode and rarely reported in plants. We identified RTE elements in 82 plants, spanning angiosperms to algae, including recently active elements in some flowering plants. RTE elements in flowering plants were likely derived from a single family we refer to as An-RTE. Interestingly, An-RTEs show significant DNA sequence identity with non-LTR retroelements from 42 animals belonging to four phyla. Moreover, the sequence identity of RTEs between two arthropods and two plants was higher than that of homologous genes. Phylogenetic and evolutionary analyses of RTEs from both animals and plants suggest that the An-RTE family was likely transferred horizontally into angiosperms from an ancient aphid(s) or ancestral arthropod(s). Notably, some An-RTEs were recruited as coding sequences of functional genes participating in metabolic or other biochemical processes in plants. This is the first potential example of horizontal transfer of transposons between animals and flowering plants. Our findings help to understand exchanges of genetic material between the kingdom Animalia and Plantae and suggest arthropods likely impacted on plant genome evolution.

Heterochromatin evolution in Arachis investigated through genome-wide analysis of repetitive DNA.

MAIN CONCLUSION: The most conspicuous difference among chromosomes and genomes in Arachis species, the patterns of heterochromatin, was mainly modeled by differential amplification of different members of one superfamily of satellite DNAs. Divergence in repetitive DNA is a primary driving force for genome and chromosome evolution. Section Arachis is karyotypically diverse and has six different genomes. Arachis glandulifera (D genome) has the most asymmetric karyotype and the highest reproductive isolation compared to the well-known A and B genome species. These features make A. glandulifera an interesting model species for studying the main repetitive components that accompanied the genome and chromosome diversification in the section. Here, we performed a genome-wide analysis of repetitive sequences in A. glandulifera and investigated the chromosome distribution of the identified satellite DNA sequences (satDNAs). LTR retroelements, mainly the Ty3-gypsy families "Fidel/Feral" and "Pipoka/Pipa", were the most represented. Comparative analyses with the A and B genomes showed that many of the previously described transposable elements (TEs) were differently represented in the D genome, and that this variation accompanied changes in DNA content. In addition, four major satDNAs were characterized. Agla_CL8sat was the major component of pericentromeric heterochromatin, while Agla_CL39sat, Agla_CL69sat, and Agla_CL122sat were found in heterochromatic and/or euchromatic regions. Even though Agla_CL8sat belong to a different family than that of the major satDNA (ATR-2) found in the heterochromatin of the A, K, and F genomes, both satDNAs are members of the same superfamily. This finding suggests that closely related satDNAs of an ancestral library were differentially amplified leading to the major changes in the heterochromatin patterns that accompanied the karyotype and genome differentiation in Arachis.

MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs.

Legumes and many nonleguminous plants enter symbiotic interactions with microbes, and it is poorly understood how host plants respond to promote beneficial, symbiotic microbial interactions while suppressing those that are deleterious or pathogenic. Trans-acting siRNAs (tasiRNAs) negatively regulate target transcripts and are characterized by siRNAs spaced in 21-nucleotide (nt) "phased" intervals, a pattern formed by DICER-LIKE 4 (DCL4) processing. A search for phased siRNAs (phasiRNAs) found at least 114 Medicago loci, the majority of which were defense-related NB-LRR-encoding genes. We identified three highly abundant 22-nt microRNA (miRNA) families that target conserved domains in these NB-LRRs and trigger the production of trans-acting siRNAs. High levels of small RNAs were matched to >60% of all ∼540 encoded Medicago NB-LRRs; in the potato, a model for mycorrhizal interactions, phasiRNAs were also produced from NB-LRRs. DCL2 and SGS3 transcripts were also cleaved by these 22-nt miRNAs, generating phasiRNAs, suggesting synchronization between silencing and pathogen defense pathways. In addition, a new example of apparent "two-hit" phasiRNA processing was identified. Our data reveal complex tasiRNA-based regulation of NB-LRRs that potentially evolved to facilitate symbiotic interactions and demonstrate miRNAs as master regulators of a large gene family via the targeting of highly conserved, protein-coding motifs, a new paradigm for miRNA function.

Comprehensive definition of genome features in Spirodela polyrhiza by high-depth physical mapping and short-read DNA sequencing strategies.

Spirodela polyrhiza is a fast-growing aquatic monocot with highly reduced morphology, genome size and number of protein-coding genes. Considering these biological features of Spirodela and its basal position in the monocot lineage, understanding its genome architecture could shed light on plant adaptation and genome evolution. Like many draft genomes, however, the 158-Mb Spirodela genome sequence has not been resolved to chromosomes, and important genome characteristics have not been defined. Here we deployed rapid genome-wide physical maps combined with high-coverage short-read sequencing to resolve the 20 chromosomes of Spirodela and to empirically delineate its genome features. Our data revealed a dramatic reduction in the number of the rDNA repeat units in Spirodela to fewer than 100, which is even fewer than that reported for yeast. Consistent with its unique phylogenetic position, small RNA sequencing revealed 29 Spirodela-specific microRNA, with only two being shared with Elaeis guineensis (oil palm) and Musa balbisiana (banana). Combining DNA methylation data and small RNA sequencing enabled the accurate prediction of 20.5% long terminal repeats (LTRs) that doubled the previous estimate, and revealed a high Solo:Intact LTR ratio of 8.2. Interestingly, we found that Spirodela has the lowest global DNA methylation levels (9%) of any plant species tested. Taken together our results reveal a genome that has undergone reduction, likely through eliminating non-essential protein coding genes, rDNA and LTRs. In addition to delineating the genome features of this unique plant, the methodologies described and large-scale genome resources from this work will enable future evolutionary and functional studies of this basal monocot family.

Genome resources for climate-resilient cowpea, an essential crop for food security.

Cowpea (Vigna unguiculata L. Walp.) is a legume crop that is resilient to hot and drought-prone climates, and a primary source of protein in sub-Saharan Africa and other parts of the developing world. However, genome resources for cowpea have lagged behind most other major crops. Here we describe foundational genome resources and their application to the analysis of germplasm currently in use in West African breeding programs. Resources developed from the African cultivar IT97K-499-35 include a whole-genome shotgun (WGS) assembly, a bacterial artificial chromosome (BAC) physical map, and assembled sequences from 4355 BACs. These resources and WGS sequences of an additional 36 diverse cowpea accessions supported the development of a genotyping assay for 51 128 SNPs, which was then applied to five bi-parental RIL populations to produce a consensus genetic map containing 37 372 SNPs. This genetic map enabled the anchoring of 100 Mb of WGS and 420 Mb of BAC sequences, an exploration of genetic diversity along each linkage group, and clarification of macrosynteny between cowpea and common bean. The SNP assay enabled a diversity analysis of materials from West African breeding programs. Two major subpopulations exist within those materials, one of which has significant parentage from South and East Africa and more diversity. There are genomic regions of high differentiation between subpopulations, one of which coincides with a cluster of nodulin genes. The new resources and knowledge help to define goals and accelerate the breeding of improved varieties to address food security issues related to limited-input small-holder farming and climate stress.

High-density genetic map using whole-genome resequencing for fine mapping and candidate gene discovery for disease resistance in peanut.

Whole-genome resequencing (WGRS) of mapping populations has facilitated development of high-density genetic maps essential for fine mapping and candidate gene discovery for traits of interest in crop species. Leaf spots, including early leaf spot (ELS) and late leaf spot (LLS), and Tomato spotted wilt virus (TSWV) are devastating diseases in peanut causing significant yield loss. We generated WGRS data on a recombinant inbred line population, developed a SNP-based high-density genetic map, and conducted fine mapping, candidate gene discovery and marker validation for ELS, LLS and TSWV. The first sequence-based high-density map was constructed with 8869 SNPs assigned to 20 linkage groups, representing 20 chromosomes, for the 'T' population (Tifrunner × GT-C20) with a map length of 3120 cM and an average distance of 1.45 cM. The quantitative trait locus (QTL) analysis using high-density genetic map and multiple season phenotyping data identified 35 main-effect QTLs with phenotypic variation explained (PVE) from 6.32% to 47.63%. Among major-effect QTLs mapped, there were two QTLs for ELS on B05 with 47.42% PVE and B03 with 47.38% PVE, two QTLs for LLS on A05 with 47.63% and B03 with 34.03% PVE and one QTL for TSWV on B09 with 40.71% PVE. The epistasis and environment interaction analyses identified significant environmental effects on these traits. The identified QTL regions had disease resistance genes including R-genes and transcription factors. KASP markers were developed for major QTLs and validated in the population and are ready for further deployment in genomics-assisted breeding in peanut.

Genetic and epigenetic divergence of duplicate genes in two legume species.

Soybean (Glycine max) and common bean (Phaseolus vulgaris) share a polyploidy event ~59 MYA, followed by a Glycine-specific whole genome duplication (WGD) ~8-13 MYA. Duplicated genes were classified into five categories: singletons, dispersed, proximal, tandem, or WGD/segmental and found strong correlations between gene category and functional annotation. Photosynthesis and transcriptional regulation-related Gene Ontology terms were significantly over-represented in singletons and WGD genes, respectively, aligning with the gene balance hypothesis. We found that the divergence of gene expression and DNA methylation between WGD-derived paralogs increased with age and that WGD genes, initially retained via dosage constraints, subsequently underwent expression divergence, associated with other factors such as DNA methylation. Genes derived from different modes of duplication differed in breadth, level, and specificity of expression in both species. Orthologous genes and ungrouped genes (genes not in an ortholog group) differed in expression patterns. The protein divergence rates of WGD paralog pairs containing an ungrouped gene were higher than those for which both copies had orthologs. We propose that many ungrouped genes are derived from divergent and redundant gene copies, concordant with the neofunctionalization hypothesis. Tandemly duplicated genes were distinct from WGD-derived genes, indicating that mode of duplication contributes to the evolutionary fate of duplicated genes.

Segmental allopolyploidy in action: Increasing diversity through polyploid hybridization and homoeologous recombination.

PREMISE OF THE STUDY: The genetic bottleneck of polyploid formation can be mitigated by multiple origins, gene flow, and recombination among different lineages. In crop plants with limited origins, efforts to increase genetic diversity have limitations. Here we used lineage recombination to increase genetic diversity in peanut, an allotetraploid likely of single origin, by crossing with a novel allopolyploid genotype and selecting improved lines. METHODS: Single backcross progeny from cultivated peanut × wild species-derived allotetraploid cross were studied over successive generations. Using genetic assumptions that encompass segmental allotetraploidy, we used single nucleotide polymorphisms and whole-genome sequence data to infer genome structures. KEY RESULTS: Selected lines, despite a high proportion of wild alleles, are agronomically adapted, productive, and with improved disease resistances. Wild alleles mostly substituted homologous segments of the peanut genome. Regions of dispersed wild alleles, characteristic of gene conversion, also occurred. However, wild chromosome segments sometimes replaced cultivated peanut's homeologous subgenome; A. ipaënsis B sometimes replaced A. hypogaea A subgenome (~0.6%), and A. duranensis replaced A. hypogaea B subgenome segments (~2%). Furthermore, some subgenome regions historically lost in cultivated peanut were "recovered" by wild chromosome segments (effectively reversing the "polyploid ratchet"). These processes resulted in lines with new genome structure variations. CONCLUSIONS: Genetic diversity was introduced by wild allele introgression, and by introducing new genome structure variations. These results highlight the special possibilities of segmental allotetraploidy and of using lineage recombination to increase genetic diversity in peanut, likely mirroring what occurs in natural segmental allopolyploids with multiple origins.

Widespread and frequent horizontal transfers of transposable elements in plants.

Vertical, transgenerational transmission of genetic material occurs through reproduction of living organisms. In addition to vertical inheritance, horizontal gene transfer between reproductively isolated species has recently been shown to be an important, if not dominant, mechanism in the evolution of prokaryotic genomes. In contrast, only a few horizontal transfer (HT) events have been characterized so far in eukaryotes and mainly concern transposable elements (TEs). Whether these are frequent and have a significant impact on genome evolution remains largely unknown. We performed a computational search for highly conserved LTR retrotransposons among 40 sequenced eukaryotic genomes representing the major plant families. We found that 26 genomes (65%) harbor at least one case of horizontal TE transfer (HTT). These transfers concern species as distantly related as palm and grapevine, tomato and bean, or poplar and peach. In total, we identified 32 cases of HTTs, which could translate into more than 2 million among the 13,551 monocot and dicot genera. Moreover, we show that these TEs have remained functional after their transfer, occasionally causing a transpositional burst. This suggests that plants can frequently exchange genetic material through horizontal transfers and that this mechanism may be important in TE-driven genome evolution.

Divergent cytosine DNA methylation patterns in single-cell, soybean root hairs.

Chromatin modifications, such as cytosine methylation of DNA, play a significant role in mediating gene expression in plants, which affects growth, development, and cell differentiation. As root hairs are single-cell extensions of the root epidermis and the primary organs for water uptake and nutrients, we sought to use root hairs as a single-cell model system to measure the impact of environmental stress. We measured changes in cytosine DNA methylation in single-cell root hairs as compared with multicellular stripped roots, as well as in response to heat stress. Differentially methylated regions (DMRs) in each methylation context showed very distinct methylation patterns between cell types and in response to heat stress. Intriguingly, at normal temperature, root hairs were more hypermethylated than were stripped roots. However, in response to heat stress, both root hairs and stripped roots showed hypomethylation in each context, especially in the CHH context. Moreover, expression analysis of mRNA from similar tissues and treatments identified some associations between DMRs, genes and transposons. Taken together, the data indicate that changes in DNA methylation are directly or indirectly associated with expression of genes and transposons within the context of either specific tissues/cells or stress (heat).

Decreased Nucleotide and Expression Diversity and Modified Coexpression Patterns Characterize Domestication in the Common Bean.

Using RNA sequencing technology and de novo transcriptome assembly, we compared representative sets of wild and domesticated accessions of common bean (Phaseolus vulgaris) from Mesoamerica. RNA was extracted at the first true-leaf stage, and de novo assembly was used to develop a reference transcriptome; the final data set consists of ∼190,000 single nucleotide polymorphisms from 27,243 contigs in expressed genomic regions. A drastic reduction in nucleotide diversity (∼60%) is evident for the domesticated form, compared with the wild form, and almost 50% of the contigs that are polymorphic were brought to fixation by domestication. In parallel, the effects of domestication decreased the diversity of gene expression (18%). While the coexpression networks for the wild and domesticated accessions demonstrate similar seminal network properties, they show distinct community structures that are enriched for different molecular functions. After simulating the demographic dynamics during domestication, we found that 9% of the genes were actively selected during domestication. We also show that selection induced a further reduction in the diversity of gene expression (26%) and was associated with 5-fold enrichment of differentially expressed genes. While there is substantial evidence of positive selection associated with domestication, in a few cases, this selection has increased the nucleotide diversity in the domesticated pool at target loci associated with abiotic stress responses, flowering time, and morphology.

Highly distinct chromosomal structures in cowpea (Vigna unguiculata), as revealed by molecular cytogenetic analysis.

Cowpea (Vigna unguiculata (L.) Walp) is an important legume, particularly in developing countries. However, little is known about its genome or chromosome structure. We used molecular cytogenetics to characterize the structure of pachytene chromosomes to advance our knowledge of chromosome and genome organization of cowpea. Our data showed that cowpea has highly distinct chromosomal structures that are cytologically visible as brightly DAPI-stained heterochromatic regions. Analysis of the repetitive fraction of the cowpea genome present at centromeric and pericentromeric regions confirmed that two retrotransposons are major components of pericentromeric regions and that a 455-bp tandem repeat is found at seven out of 11 centromere pairs in cowpea. These repeats likely evolved after the divergence of cowpea from common bean and form chromosomal structure unique to cowpea. The integration of cowpea genetic and physical chromosome maps reveals potential regions of suppressed recombination due to condensed heterochromatin and a lack of pairing in a few chromosomal termini. This study provides fundamental knowledge on cowpea chromosome structure and molecular cytogenetics tools for further chromosome studies.