RESUMO
Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the 'pan-genome'1). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions2. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley-comprising landraces, cultivars and a wild barley-that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.
Assuntos
Cromossomos de Plantas/genética , Genoma de Planta/genética , Hordeum/genética , Internacionalidade , Mutação , Melhoramento Vegetal , Inversão Cromossômica/genética , Mapeamento Cromossômico , Loci Gênicos/genética , Genótipo , Hordeum/classificação , Polimorfismo Genético/genética , Padrões de Referência , Banco de Sementes , Inversão de Sequência , Sequenciamento Completo do GenomaRESUMO
Accurate characterisation of splice junctions (SJs) as well as transcription start and end sites in reference transcriptomes allows precise quantification of transcripts from RNA-seq data, and enables detailed investigations of transcriptional and post-transcriptional regulation. Using novel computational methods and a combination of PacBio Iso-seq and Illumina short-read sequences from 20 diverse tissues and conditions, we generated a comprehensive and highly resolved barley reference transcript dataset from the European 2-row spring barley cultivar Barke (BaRTv2.18). Stringent and thorough filtering was carried out to maintain the quality and accuracy of the SJs and transcript start and end sites. BaRTv2.18 shows increased transcript diversity and completeness compared with an earlier version, BaRTv1.0. The accuracy of transcript level quantification, SJs and transcript start and end sites have been validated extensively using parallel technologies and analysis, including high-resolution reverse transcriptase-polymerase chain reaction and 5'-RACE. BaRTv2.18 contains 39 434 genes and 148 260 transcripts, representing the most comprehensive and resolved reference transcriptome in barley to date. It provides an important and high-quality resource for advanced transcriptomic analyses, including both transcriptional and post-transcriptional regulation, with exceptional resolution and precision.
Assuntos
Hordeum , Transcriptoma , Perfilação da Expressão Gênica/métodos , Hordeum/genética , RNA-Seq , Análise de Sequência de RNA/métodos , Transcriptoma/genéticaRESUMO
The distribution of recombination events along large cereal chromosomes is uneven and is generally restricted to gene-rich telomeric ends. To understand how the lack of recombination affects diversity in the large pericentromeric regions, we analysed deep exome capture data from a final panel of 815 Hordeum vulgare (barley) cultivars, landraces and wild barleys, sampled from across their eco-geographical ranges. We defined and compared variant data across the pericentromeric and non-pericentromeric regions, observing a clear partitioning of diversity both within and between chromosomes and germplasm groups. Dramatically reduced diversity was found in the pericentromeres of both cultivars and landraces when compared with wild barley. We observed a mixture of completely and partially differentiated single-nucleotide polymorphisms (SNPs) between domesticated and wild gene pools, suggesting that domesticated gene pools were derived from multiple wild ancestors. Patterns of genome-wide linkage disequilibrium, haplotype block size and number, and variant frequency within blocks showed clear contrasts among individual chromosomes and between cultivars and wild barleys. Although most cultivar chromosomes shared a single major pericentromeric haplotype, chromosome 7H clearly differentiated the two-row and six-row types associated with different geographical origins. Within the pericentromeric regions we identified 22 387 non-synonymous SNPs, 92 of which were fixed for alternative alleles in cultivar versus wild accessions. Surprisingly, only 29 SNPs found exclusively in the cultivars were predicted to be 'highly deleterious'. Overall, our data reveal an unconventional pericentromeric genetic landscape among distinct barley gene pools, with different evolutionary processes driving domestication and diversification.
Assuntos
Hordeum , Cromossomos , Domesticação , Hordeum/genética , Desequilíbrio de Ligação/genéticaRESUMO
KEY MESSAGE: Selection over 70 years has led to almost complete fixation of a haplotype spanning ~ 250 Mbp of chomosome 5H in European two-rowed spring barleys, possibly originating from North Africa. Plant breeding and selection have shaped the genetic composition of modern crops over the past decades and centuries and have led to great improvements in agronomic and quality traits. Knowledge of the genetic composition of breeding germplasm is essential to make informed decisions in breeding programs. In this study, we characterized the structure and composition of 209 barley cultivars representative of the European two-rowed spring barley germplasm of the past 190 years. Utilizing high-density SNP marker data, we identified a distinct centromeric haplotype spanning a ~ 250 Mbp large region on chromosome 5H which likely was first introduced into the European breeding germplasm in the early to mid-twentieth century and has been non-recombining and under strong positive selection over the past 70 years. Almost all cultivars in our panel that were released after 2000 carry this new haplotype, suggesting that this region carries one or several genes conferring highly beneficial traits. Using the global barley collection of the German Federal ex situ gene bank at IPK Gatersleben, we found the new haplotype at high frequencies in six-rowed spring-type landraces from Northern Africa, from which it may have been introduced into modern European barley germplasm via southern European landraces. The presence of a 250 Mbp genomic region characterized by lack of recombination and high levels of fixation in modern barley germplasm has substantial implications for the genetic diversity of the modern barley germplasm and for barley breeding.
Assuntos
Hordeum , Haplótipos , Hordeum/genética , Melhoramento Vegetal , Fenótipo , CromossomosRESUMO
BACKGROUND: Plant breeding and crop research rely on experimental phenotyping trials. These trials generate data for large numbers of traits and plant varieties that needs to be captured efficiently and accurately to support further research and downstream analysis. Traditionally scored by hand, phenotypic data is nowadays collected using spreadsheets or specialized apps. While many solutions exist, which increase efficiency and reduce errors, none offer the same familiarity as printed field plans which have been used for decades and offer an intuitive overview over the trial setup, previously recorded data and plots still requiring scoring. RESULTS: We introduce GridScore which utilizes cutting-edge web technologies to reproduce the familiarity of printed field plans while enhancing the phenotypic data collection process by adding advanced features like georeferencing, image tagging and speech recognition. GridScore is a cross-platform open-source plant phenotyping app that combines barcode-based systems with a guided data collection approach while offering a top-down view onto the data collected in a field layout. GridScore is compared to existing tools across a wide spectrum of criteria including support for barcodes, multiple platforms, and visualizations. CONCLUSION: Compared to its competition, GridScore shows strong performance across the board offering a complete manual phenotyping experience.
Assuntos
Produtos Agrícolas , Melhoramento Vegetal , Coleta de Dados , FenótipoRESUMO
Flowering plants reproduce sexually by combining a haploid male and female gametophyte during fertilization. Male gametophytes are localized in the anthers, each containing reproductive (meiocyte) and non-reproductive tissue necessary for anther development and maturation. Meiosis, where chromosomes pair and exchange their genetic material during a process called recombination, is one of the most important and sensitive stages in breeding, ensuring genetic diversity. Most anther development studies have focused on transcript variation, but very few have been correlated with protein abundance. Taking advantage of a recently published barley anther transcriptomic (BAnTr) dataset and a newly developed sensitive mass spectrometry-based approach to analyse the barley anther proteome, we conducted high-resolution mass spectrometry analysis of barley anthers, collected at six time points and representing their development from pre-meiosis to metaphase. Each time point was carefully staged using immunocytology, providing a robust and accurate staging mirroring our previous BAnTr dataset. We identified >6100 non-redundant proteins including 82 known and putative meiotic proteins. Although the protein abundance was relatively stable throughout prophase I, we were able to quantify the dynamic variation of 336 proteins. We present the first quantitative comparative proteomics study of barley anther development during meiotic prophase I when the important process of homologous recombination is taking place.
Assuntos
Hordeum , Proteoma , Flores , Hordeum/genética , Hordeum/metabolismo , Meiose , Prófase Meiótica I , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteoma/metabolismoRESUMO
We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly inulin- and neoseries-type fructans, showing differential abundance. Clustering revealed two profile groups where the 'high' set contained greater amounts of sugar monomers, sucrose, and overall fructans, but lower fructosylraffinose. A genome-wide association study (GWAS) identified a significant association for the variability of two fructan types: neoseries-DP7 and inulin-DP9, which showed increased strength when applying a novel compound ratio-GWAS approach. Gene models within this region included three known fructan biosynthesis genes (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase). Two other genes in this region, 6(G)-fructosyltransferase and vacuolar invertase1, have not previously been linked to fructan biosynthesis and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data, several single nucleotide polymorphisms related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes. Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide the first scientific evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation and reveal novel gene candidates likely to be involved in the differential biosynthesis of various types of fructan in barley.
Assuntos
Hexosiltransferases , Hordeum , Sequência de Aminoácidos , Frutanos , Estudo de Associação Genômica Ampla , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Hordeum/genética , Hordeum/metabolismo , Vacúolos/metabolismoRESUMO
Phylogenetically related groups of species contain lineage-specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1-deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1-H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll-like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non-invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage-specific.
Assuntos
Variação Genética , Proteínas de Plantas/genética , Poaceae/genética , Alelos , Sequência de Aminoácidos , Evolução Biológica , Geografia , Haplótipos , Hordeum/citologia , Hordeum/genética , Imageamento por Ressonância Magnética , Família Multigênica , Filogenia , Proteínas de Plantas/metabolismo , Poaceae/citologia , Reprodução , Sementes/citologia , Sementes/genética , Alinhamento de Sequência , Especificidade da Espécie , Triticum/citologia , Triticum/genéticaRESUMO
BACKGROUND: The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. RESULTS: A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts - BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427-433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20-28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5' and 3' UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. CONCLUSION: A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.
Assuntos
Perfilação da Expressão Gênica/métodos , Hordeum/genética , Proteínas de Plantas/genética , Processamento Alternativo , Bases de Dados Genéticas , Regulação da Expressão Gênica de Plantas , Análise de Sequência de RNA , Sequenciamento do ExomaRESUMO
Despite conservation of the process of meiosis, recombination landscapes vary between species, with large genome grasses such as barley (Hordeum vulgare L.) exhibiting a pattern of recombination that is very heavily skewed to the ends of chromosomes. We have been using a collection of semi-sterile desynaptic meiotic mutant lines to help elucidate how recombination is controlled in barley and the role of the corresponding wild-type (WT) meiotic genes within this process. Here we applied a combination of genetic segregation analysis, cytogenetics, and immunocytology to genetically map and characterize the meiotic mutant desynaptic5 (des5). We identified an exonic insertion in the positional candidate ortholog of Disrupted Meiotic cDNA 1 (HvDMC1) on chromosome 5H of des5. des5 exhibits a severe meiotic phenotype with disturbed synapsis, reduced crossovers, and chromosome mis-segregation. The meiotic phenotype and reduced fertility of des5 is similarly observed in Hvdmc1RNAi transgenic plants and HvDMC1p:GusPlus reporter lines show DMC1 expression specifically in the developing inflorescence. The des5 mutation maintains the reading frame of the gene and exhibits semi-dominance with respect to recombination in the heterozygote indicating the value of non-knockout mutations for dissection of the control of recombination in the early stages of meiosis.
Assuntos
Hordeum/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Hordeum/metabolismo , Mutação/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de SequênciaRESUMO
Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-ß-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-ß-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels.
Assuntos
Evolução Molecular , Genes de Plantas , Glucosiltransferases/genética , Família Multigênica , Poaceae/enzimologia , Poaceae/genética , Substituição de Aminoácidos , Aminoácidos/genética , Cromossomos de Plantas/genética , Sequência Conservada/genética , Modelos Moleculares , Filogenia , Seleção Genética , Homologia Estrutural de ProteínaRESUMO
BACKGROUND: (1,3;1,4)-ß-Glucan is an important component of the cell walls of barley grain as it affects processability during the production of alcoholic beverages and has significant human health benefits when consumed above recommended threshold levels. This leads to diametrically opposed quality requirements for different applications as low levels of (1,3;1,4)-ß-glucan are required for brewing and distilling and high levels for positive impacts on human health. RESULTS: We quantified grain (1,3;1,4)-ß-glucan content in a collection of 399 2-row Spring-type, and 204 2-row Winter-type elite barley cultivars originating mainly from north western Europe. We combined these data with genotypic information derived using a 9 K Illumina iSelect SNP platform and subsequently carried out a Genome Wide Association Scan (GWAS). Statistical analysis accounting for residual genetic structure within the germplasm collection allowed us to identify significant associations between molecular markers and the phenotypic data. By anchoring the regions that contain these associations to the barley genome assembly we catalogued genes underlying the associations. Based on gene annotations and transcript abundance data we identified candidate genes. CONCLUSIONS: We show that a region of the genome on chromosome 2 containing a cluster of CELLULOSE SYNTHASE-LIKE (Csl) genes, including CslF3, CslF4, CslF8, CslF10, CslF12 and CslH, as well as a region on chromosome 1H containing CslF9, are associated with the phenotype in this germplasm. We also observed that several regions identified by GWAS contain glycoside hydrolases that are possibly involved in (1,3;1,4)-ß-glucan breakdown, together with other genes that might participate in (1,3;1,4)-ß-glucan synthesis, re-modelling or regulation. This analysis provides new opportunities for understanding the genes related to the regulation of (1,3;1,4)-ß-glucan content in cereal grains.
Assuntos
Genômica , Hordeum/genética , Hordeum/metabolismo , Estações do Ano , beta-Glucanas/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Glucosiltransferases/genética , Glicosídeo Hidrolases/genética , Hordeum/enzimologia , Hordeum/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Barley genomic resources are increasing rapidly, with the publication of a barley pangenome as one of the latest developments. Two-row spring barley cultivars are intensely studied as they are the source of high-quality grain for malting and distilling. Here we provide data from a European two-row spring barley population containing 209 different genotypes registered for the UK market between 1830 to 2014. The dataset encompasses RNA-sequencing data from six different tissues across a range of barley developmental stages, phenotypic datasets from two consecutive years of field-grown trials in the United Kingdom, Germany and the USA; and whole genome shotgun sequencing from all cultivars, which was used to complement the RNA-sequencing data for variant calling. The outcomes are a filtered SNP marker file, a phenotypic database and a large gene expression dataset providing a comprehensive resource which allows for downstream analyses like genome wide association studies or expression associations.
Assuntos
Genoma de Planta , Hordeum , Estudo de Associação Genômica Ampla , Genômica , Genótipo , Hordeum/genética , RNARESUMO
We report a novel approach for establishing the number and position of CO events in individual homozygous inbred plants by combining low level EMS mutagenesis, speed breeding, whole genome shotgun sequencing and sliding window analysis of the induced molecular variant data. We demonstrate the approach by exploring CO frequency and distribution in self-fertilised progeny of the inbred barley cultivar Bowman and compare these observations to similar data obtained from a Bowman nearly isogenic line (BW230 Hvmlh3) containing a mutation in the DNA mismatch repair gene HvMLH3. We have previously shown that Hvmlh3 decreases both plant fertility and recombination by ~50%. We compare our results to those from previously published traditional genetic analysis of F3 families derived from multiple F2 lines containing WT or mutant alleles of HvMLH3, revealing a high level of correspondence between analyses. We discuss possible applications of the approach in streamlining the assessment of recombination in plant meiosis research.
RESUMO
RNA sequencing (RNA-seq) data is by now the most common method to study differential gene expression. Here we present a pipeline from RNA-seq generation to analysis with examples based on our own barley anther and meiocyte transcriptome. The bioinformatics pipeline will give everyone, from a beginner to a more experienced user, the possibility to analyze their datasets and identify significantly differentially expressed genes. It also allows differential alternative splicing analysis which will become increasingly common due to the high regulatory impact on the gene expression. We describe use of the Galaxy interface for RNA-seq read quantification and the 3D RNA-seq app for the downstream data analysis.
Assuntos
Hordeum , Sequência de Bases , Análise de Dados , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Hordeum/genética , Hordeum/metabolismo , RNA/genética , Análise de Sequência de RNA , TranscriptomaRESUMO
(1,3;1,4)-ß-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-ß-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-ß-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located -331 bp to -382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression.
RESUMO
Faced with terrestrial threats, land plants seal their aerial surfaces with a lipid-rich cuticle. To breathe, plants interrupt their cuticles with adjustable epidermal pores, called stomata, that regulate gas exchange, and develop other specialised epidermal cells such as defensive hairs. Mechanisms coordinating epidermal features remain poorly understood. Addressing this, we studied two loci whose allelic variation causes both cuticular wax-deficiency and misarranged stomata in barley, identifying the underlying genes, Cer-g/ HvYDA1, encoding a YODA-like (YDA) MAPKKK, and Cer-s/ HvBRX-Solo, encoding a single BREVIS-RADIX (BRX) domain protein. Both genes control cuticular integrity, the spacing and identity of epidermal cells, and barley's distinctive epicuticular wax blooms, as well as stomatal patterning in elevated CO2 conditions. Genetic analyses revealed epistatic and modifying relationships between HvYDA1 and HvBRX-Solo, intimating that their products participate in interacting pathway(s) linking epidermal patterning with cuticular properties in barley. This may represent a mechanism for coordinating multiple adaptive features of the land plant epidermis in a cultivated cereal.
Assuntos
Hordeum , Dióxido de Carbono/metabolismo , Regulação da Expressão Gênica de Plantas , Hordeum/genética , Hordeum/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Epiderme Vegetal/metabolismo , Ceras/metabolismoRESUMO
Plant breeding relies on the meiotic recombination or crossing over to generate the new combinations of the alleles along and among the chromosomes. However, crossing over is constrained in the crops such as barley by a combination of the low frequency and biased distribution. In this study, we attempted to identify the genes that limit the recombination by performing a suppressor screen for the restoration of fertility to the semi-fertile barley mutant desynaptic10 (des10), carrying a mutation in the barley ortholog of MutL-Homolog 3 (HvMLH3), a member of the MutL-homolog (MLH) family of DNA mismatch repair genes. des10 mutants exhibit reduced recombination and fewer chiasmata, resulting in the loss of obligate crossovers (COs) leading to chromosome mis-segregation. We identified several candidate suppressor lines and confirmed their restored fertility in an Hvmlh3 background in the subsequent generations. We focus on one of the candidate suppressor lines, SuppLine2099, which showed the most complete restoration of fertility. We characterized this line by using a target-sequence enrichment and sequencing (TENSEQ) capture array representing barley orthologs of 46 meiotic genes. We found that SuppLine2099 contained a C/T change in the anti-CO gene RecQ-like helicase 4 (RECQL4) resulting in the substitution of a non-polar glycine to a polar aspartic acid (G700D) amino acid in the conserved helicase domain. Single nucleotide polymorphism (SNP) genotyping of F3 populations revealed a significant increase in the recombination frequency in lines with Hvrecql4 in the Hvmlh3 background that was associated with the restoration of fertility. The genotyping also indicated that there was nearly double the recombination levels in homozygous Hvrecql4 lines compared to the wild type (WT). However, we did not observe any significant change in the distribution of CO events. Our results confirm the anti-CO role of RECQL4 in a large genome cereal and establish the possibility of testing the utility of increasing recombination in the context of traditional crop improvement.
RESUMO
Programmed meiotic DNA double-strand breaks (DSBs), necessary for proper chromosomal segregation and viable gamete formation, are repaired by homologous recombination (HR) as crossovers (COs) or non-crossovers (NCOs). The mechanisms regulating the number and distribution of COs are still poorly understood. The regulator of telomere elongation helicase 1 (RTEL1) DNA helicase was previously shown to enforce the number of meiotic COs in Caenorhabditis elegans but its function in plants has been studied only in the vegetative phase. Here, we characterised barley RTEL1 gene structure and expression using RNA-seq data previously obtained from vegetative and reproductive organs and tissues. Using RNAi, we downregulated RTEL1 expression specifically in reproductive tissues and analysed its impact on recombination using a barley 50k iSelect SNP Array. Unlike in C. elegans, in a population segregating for RTEL1 downregulated by RNAi, high resolution genome-wide genetic analysis revealed a significant increase of COs at distal chromosomal regions of barley without a change in their total number. Our data reveal the important role of RTEL1 helicase in plant meiosis and control of recombination.
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Barley (Hordeum vulgare) is one of the most important crops worldwide and is also considered a research model for the large-genome small grain temperate cereals. Despite genomic resources improving all the time, they are limited for the cv Golden Promise, the most efficient genotype for genetic transformation. We have developed a barley cv Golden Promise reference assembly integrating Illumina paired-end reads, long mate-pair reads, Dovetail Chicago in vitro proximity ligation libraries and chromosome conformation capture sequencing (Hi-C) libraries into a contiguous reference assembly. The assembled genome of 7 chromosomes and 4.13Gb in size, has a super-scaffold N50 after Chicago libraries of 4.14Mb and contains only 2.2% gaps. Using BUSCO (benchmarking universal single copy orthologous genes) as evaluation the genome assembly contains 95.2% of complete and single copy genes from the plant database. A high-quality Golden Promise reference assembly will be useful and utilized by the whole barley research community but will prove particularly useful for CRISPR-Cas9 experiments.