Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome‐assisted research in barley (Hordeum vulgare) including a whole‐genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole‐genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA‐coding exome reduces barley genomic complexity more than 50‐fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in‐solution hybridization‐based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full‐length cDNAs and de novo assembled RNA‐Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA‐coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping‐by‐sequencing and genetic diversity analyzes.     

Diversity and population structure of northern switchgrass as revealed through exome capture sequencing

Panicum virgatum L. (switchgrass) is a polyploid, perennial grass species that is native to North America, and is being developed as a future biofuel feedstock crop. Switchgrass is present primarily in two ecotypes: a northern upland ecotype, composed of tetraploid and octoploid accessions, and a southern lowland ecotype, composed of primarily tetraploid accessions. We employed high‐coverage exome capture sequencing (~2.4 Tb) to genotype 537 individuals from 45 upland and 21 lowland populations. From these data, we identified ~27 million single‐nucleotide polymorphisms (SNPs), of which 1 590 653 high‐confidence SNPs were used in downstream analyses of diversity within and between the populations. From the 66 populations, we identified five primary population groups within the upland and lowland ecotypes, a result that was further supported through genetic distance analysis. We identified conserved, ecotype‐restricted, non‐synonymous SNPs that are predicted to affect the protein function of CONSTANS (CO) and EARLY HEADING DATE 1 (EHD1), key genes involved in flowering, which may contribute to the phenotypic differences between the two ecotypes. We also identified, relative to the near‐reference Kanlow population, 17 228 genes present in more copies than in the reference genome (up‐CNVs), 112 630 genes present in fewer copies than in the reference genome (down‐CNVs) and 14 430 presence/absence variants (PAVs), affecting a total of 9979 genes, including two upland‐specific CNV clusters. In total, 45 719 genes were affected by an SNP, CNV, or PAV across the panel, providing a firm foundation to identify functional variation associated with phenotypic traits of interest for biofuel feedstock production.     

RNA‐Seq bulked segregant analysis enables the identification of high‐resolution genetic markers for breeding in hexaploid wheat

The identification of genetic markers linked to genes of agronomic importance is a major aim of crop research and breeding programmes. Here, we identify markers for Yr15, a major disease resistance gene for wheat yellow rust, using a segregating F₂ population. After phenotyping, we implemented RNA sequencing (RNA‐Seq) of bulked pools to identify single‐nucleotide polymorphisms (SNP) associated with Yr15. Over 27 000 genes with SNPs were identified between the parents, and then classified based on the results from the sequenced bulks. We calculated the bulk frequency ratio (BFR) of SNPs between resistant and susceptible bulks, selecting those showing sixfold enrichment/depletion in the corresponding bulks (BFR > 6). Using additional filtering criteria, we reduced the number of genes with a putative SNP to 175. The 35 SNPs with the highest BFR values were converted into genome‐specific KASP assays using an automated bioinformatics pipeline (PolyMarker) which circumvents the limitations associated with the polyploid wheat genome. Twenty‐eight assays were polymorphic of which 22 (63%) mapped in the same linkage group as Yr15. Using these markers, we mapped Yr15 to a 0.77‐cM interval. The three most closely linked SNPs were tested across varieties and breeding lines representing UK elite germplasm. Two flanking markers were diagnostic in over 99% of lines tested, thus providing a reliable haplotype for marker‐assisted selection in these breeding programmes. Our results demonstrate that the proposed methodology can be applied in polyploid F₂ populations to generate high‐resolution genetic maps across target intervals.     

Using a butterflyfish genome as a general tool for RAD‐Seq studies in specialized reef fish

Data from a large‐scale restriction site‐associated DNA sequencing (RAD‐Seq) study of nine butterflyfish species in the Red Sea and Arabian Sea provided a means to test the utility of a recently published draft genome (Chaetodon austriacus) and assess apparent bias in this method of isolating nuclear loci. We here processed double‐digest restriction site‐associated DNA (ddRAD) sequencing data to identify single nucleotide polymorphism (SNP) markers and their associated function with and without our reference genome to see whether it improves the quality of RAD‐Seq. Our analyses indicate (i) a modest gap between the number of nonannotated versus annotated SNPs across all species, (ii) an advantage of using genomic resources for closely related but not distantly related butterflyfish species based on the ability to assign putative gene function to SNPs and (iii) an enrichment of genes among sister butterflyfish taxa related to calcium transmembrane transport and binding. The latter result highlights the potential for this approach to reveal insights into adaptive mechanisms in populations inhabiting challenging coral reef environments such as the Red Sea, Arabian Sea and Arabian Gulf with further study.     

SNP discovery in nonmodel organisms: strand bias and base‐substitution errors reduce conversion rates

Single nucleotide polymorphisms (SNPs) have become the marker of choice for genetic studies in organisms of conservation, commercial or biological interest. Most SNP discovery projects in nonmodel organisms apply a strategy for identifying putative SNPs based on filtering rules that account for random sequencing errors. Here, we analyse data used to develop 4723 novel SNPs for the commercially important deep‐sea fish, orange roughy (Hoplostethus atlanticus), to assess the impact of not accounting for systematic sequencing errors when filtering identified polymorphisms when discovering SNPs. We used SAMtools to identify polymorphisms in a velvet assembly of genomic DNA sequence data from seven individuals. The resulting set of polymorphisms were filtered to minimize ‘bycatch’—polymorphisms caused by sequencing or assembly error. An Illumina Infinium SNP chip was used to genotype a final set of 7714 polymorphisms across 1734 individuals. Five predictors were examined for their effect on the probability of obtaining an assayable SNP: depth of coverage, number of reads that support a variant, polymorphism type (e.g. A/C), strand‐bias and Illumina SNP probe design score. Our results indicate that filtering out systematic sequencing errors could substantially improve the efficiency of SNP discovery. We show that BLASTX can be used as an efficient tool to identify single‐copy genomic regions in the absence of a reference genome. The results have implications for research aiming to identify assayable SNPs and build SNP genotyping assays for nonmodel organisms.     

Comparative genomics analysis in Prunoideae to identify biologically relevant polymorphisms

Prunus is an economically important genus with a wide range of physiological and biological variability. Using the peach genome as a reference, sequencing reads from four almond accessions and one sweet cherry cultivar were used for comparative analysis of these three Prunus species. Reference mapping enabled the identification of many biological relevant polymorphisms within the individuals. Examining the depth of the polymorphisms and the overall scaffold coverage, we identified many potentially interesting regions including hundreds of small scaffolds with no coverage from any individual. Non‐sense mutations account for about 70 000 of the 13 million identified single nucleotide polymorphisms (SNPs). Blast2GO analyses on these non‐sense SNPs revealed several interesting results. First, non‐sense SNPs were not evenly distributed across all gene ontology terms. Specifically, in comparison with peach, sweet cherry is found to have non‐sense SNPs in two 1‐aminocyclopropane‐1‐carboxylate synthase (ACS) genes and two 1‐aminocyclopropane‐1‐carboxylate oxidase (ACO) genes. These polymorphisms may be at the root of the nonclimacteric ripening of sweet cherry. A set of candidate genes associated with bitterness in almond were identified by comparing sweet and bitter almond sequences. To the best of our knowledge, this is the first report in plants of non‐sense SNP abundance in a genus being linked to specific GO terms.     

A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.)

Cicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi‐type chickpea genome using next‐generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520‐Mb assembly covers 70% of the predicted 740‐Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27 571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA‐Seq reads identified several tissue‐specific and stress‐responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole‐genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties.     

Next‐generation sequencing for identification of candidate genes for Fusarium wilt and sterility mosaic disease in pigeonpea (Cajanus cajan)

To map resistance genes for Fusarium wilt (FW) and sterility mosaic disease (SMD) in pigeonpea, sequencing‐based bulked segregant analysis (Seq‐BSA) was used. Resistant (R) and susceptible (S) bulks from the extreme recombinant inbred lines of ICPL 20096 × ICPL 332 were sequenced. Subsequently, SNP index was calculated between R‐ and S‐bulks with the help of draft genome sequence and reference‐guided assembly of ICPL 20096 (resistant parent). Seq‐BSA has provided seven candidate SNPs for FW and SMD resistance in pigeonpea. In parallel, four additional genotypes were re‐sequenced and their combined analysis with R‐ and S‐bulks has provided a total of 8362 nonsynonymous (ns) SNPs. Of 8362 nsSNPs, 60 were found within the 2‐Mb flanking regions of seven candidate SNPs identified through Seq‐BSA. Haplotype analysis narrowed down to eight nsSNPs in seven genes. These eight nsSNPs were further validated by re‐sequencing 11 genotypes that are resistant and susceptible to FW and SMD. This analysis revealed association of four candidate nsSNPs in four genes with FW resistance and four candidate nsSNPs in three genes with SMD resistance. Further, In silico protein analysis and expression profiling identified two most promising candidate genes namely C.cajan_01839 for SMD resistance and C.cajan_03203 for FW resistance. Identified candidate genomic regions/SNPs will be useful for genomics‐assisted breeding in pigeonpea.     

Genome‐wide association mapping of natural variation in odour‐guided behaviour in Drosophila

A defining goal in the field of behavioural genetics is to identify the key genes or genetic networks that shape behaviour. A corollary to this goal is the goal of identifying genetic variants that are responsible for variation in the behaviour. These goals are achieved by measuring behavioural responses to controlled stimuli, in the present case the responses of Drosophila melanogaster to olfactory stimuli. We used a high‐throughput behavioural assay system to test a panel of 157 Drosophila inbred lines derived from a natural population for both temporal and spatial dynamics of odour‐guided behaviour. We observed significant variation in response to the odourant 2,3‐butanedione, a volatile compound present in fermenting fruit. The recent whole genome sequencing of these inbred lines allowed us to then perform genome‐wide association analyses in order to identify genetic polymorphisms underlying variation in responses. These analyses revealed numerous single nucleotide polymorphisms associated with variation in responses. Among the candidate genes identified were both novel and previously identified olfaction‐related genes. Further, gene network analyses suggest that genes influencing variation in odour‐guided behaviour are enriched for functions involving neural processing and that these genes form a pleiotropic interaction network. We examined several of these candidate genes that were highly connected in the protein‐ and genetic interaction networks using RNA interference. Our results showed that subtle changes influencing nervous system function can result in marked differences in behaviour .     

Towards a whole‐genome sequence for rye (Secale cereale L.)

We report on a whole‐genome draft sequence of rye (Secale cereale L.). Rye is a diploid Triticeae species closely related to wheat and barley, and an important crop for food and feed in Central and Eastern Europe. Through whole‐genome shotgun sequencing of the 7.9‐Gbp genome of the winter rye inbred line Lo7 we obtained a de novo assembly represented by 1.29 million scaffolds covering a total length of 2.8 Gbp. Our reference sequence represents nearly the entire low‐copy portion of the rye genome. This genome assembly was used to predict 27 784 rye gene models based on homology to sequenced grass genomes. Through resequencing of 10 rye inbred lines and one accession of the wild relative S. vavilovii, we discovered more than 90 million single nucleotide variants and short insertions/deletions in the rye genome. From these variants, we developed the high‐density Rye600k genotyping array with 600 843 markers, which enabled anchoring the sequence contigs along a high‐density genetic map and establishing a synteny‐based virtual gene order. Genotyping data were used to characterize the diversity of rye breeding pools and genetic resources, and to obtain a genome‐wide map of selection signals differentiating the divergent gene pools. This rye whole‐genome sequence closes a gap in Triticeae genome research, and will be highly valuable for comparative genomics, functional studies and genome‐based breeding in rye.     

Epistatic partners of neurogenic genes modulate Drosophila olfactory behavior

The extent to which epistasis affects the genetic architecture of complex traits is difficult to quantify, and identifying variants in natural populations with epistatic interactions is challenging. Previous studies in Drosophila implicated extensive epistasis between variants in genes that affect neural connectivity and contribute to natural variation in olfactory response to benzaldehyde. In this study, we implemented a powerful screen to quantify the extent of epistasis as well as identify candidate interacting variants using 203 inbred wild‐derived lines with sequenced genomes of the Drosophila melanogaster Genetic Reference Panel (DGRP). We crossed the DGRP lines to P[GT1]‐element insertion mutants in Sema‐5c and neuralized (neur), two neurodevelopmental loci which affect olfactory behavior, and to their coisogenic wild‐type control. We observed significant variation in olfactory responses to benzaldehyde among F₁ genotypes and for the DGRP line by mutant genotype interactions for both loci, showing extensive nonadditive genetic variation. We performed genome‐wide association analyses to identify the candidate modifier loci. None of these polymorphisms were in or near the focal genes; therefore, epistasis is the cause of the nonadditive genetic variance. Candidate genes could be placed in interaction networks. Several candidate modifiers are associated with neural development. Analyses of mutants of candidate epistatic partners with neur (merry‐go‐round (mgr), prospero (pros), CG10098, Alhambra (Alh) and CG12535) and Sema‐5c (CG42540 and bruchpilot (brp)) showed aberrant olfactory responses compared with coisogenic controls. Thus, integrating genome‐wide analyses of natural variants with mutations at defined genomic locations in a common coisogenic background can unmask specific epistatic modifiers of behavioral phenotypes.     

Genome‐guided investigation of plant natural product biosynthesis

The medicinal plant Madagascar periwinkle, Catharanthus roseus (L.) G. Don, produces hundreds of biologically active monoterpene‐derived indole alkaloid (MIA) metabolites and is the sole source of the potent, expensive anti‐cancer compounds vinblastine and vincristine. Access to a genome sequence would enable insights into the biochemistry, control, and evolution of genes responsible for MIA biosynthesis. However, generation of a near‐complete, scaffolded genome is prohibitive to small research communities due to the expense, time, and expertise required. In this study, we generated a genome assembly for C. roseus that provides a near‐comprehensive representation of the genic space that revealed the genomic context of key points within the MIA biosynthetic pathway including physically clustered genes, tandem gene duplication, expression sub‐functionalization, and putative neo‐functionalization. The genome sequence also facilitated high resolution co‐expression analyses that revealed three distinct clusters of co‐expression within the components of the MIA pathway. Coordinated biosynthesis of precursors and intermediates throughout the pathway appear to be a feature of vinblastine/vincristine biosynthesis. The C. roseus genome also revealed localization of enzyme‐rich genic regions and transporters near known biosynthetic enzymes, highlighting how even a draft genome sequence can empower the study of high‐value specialized metabolites.