Joanne Russell

A physical, genetic and functional sequence assembly of the barley genome

Barley (Hordeum vulgare L.) is among the world’s earliest domesticated and most important crop plants. It is diploid with a large haploid genome of 5.1 gigabases (Gb). Here we present an integrated and ordered physical, genetic and functional sequence resource that describes the barley gene-space in a structured whole-genome context. We developed a physical map of 4.98 Gb, with more than 3.90 Gb anchored to a high-resolution genetic map. Projecting a deep whole-genome shotgun assembly, complementary DNA and deep RNA sequence data onto this framework supports 79,379 transcript clusters, including 26,159 ‘high-confidence’ genes with homology support from other plant genomes. Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation. Our data provide a platform for both genome-assisted research and enabling contemporary crop improvement.

Direct comparison of levels of genetic variation among barley accessions detected by RFLPs, AFLPs, SSRs and RAPDs

Abstract RFLPs, AFLPs, RAPDs and SSRs were used to determine the genetic relationships among 18 cultivated barley accessions and the results compared to pedigree relationships where these were available. All of the approaches were able to uniquely fingerprint each of the accessions. The four assays differed in the amount of polymorphism detected. For example, all 13 SSR primers were polymorphic, with an average of 5.7 alleles per primer set, while nearly 54% of the fragments generated using AFLPs were monomorphic. The highest diversity index was observed for AFLPs (0.937) and the lowest for RFLP (0.322). Principal co-ordinate analysis (PCoA) clearly separated the spring types from the winter types using RFLP and AFLP data with the two-row winter types forming an intermediate group. Only a small group of spring types clustered together using SSR data with the two-row and six-row winter varieties more widely dispersed. Direct comparisons between genetic similarity (GS) estimates revealed by each of the assays were measured by a number of approaches. Spearman rank correlation ranked over 70% of the pairwise comparisons between AFLPs and RFLPs in the same order. SSRs had the lowest values when compared to the other three assays. These results are discussed in terms of the choice of appropriate technology for different aspects of germplasm evaluation.

How much effort is required to isolate nuclear microsatellites from plants

The attributes of codominance, reproducibility and high resolution have all contributed towards the current popularity of nuclear microsatellites as genetic markers in molecular ecological studies. One of their major drawbacks, however, is the development phase required to obtain working primers for a given study species. To facilitate project planning, we have reviewed the literature to quantify the workload involved in isolating nuclear microsatellites from plants. We highlight the attrition of loci at each stage in the process, and the average effort required to obtain 10 working microsatellite primer pairs.

Natural variation in a homolog of Antirrhinum CENTRORADIALIS contributed to spring growth habit and environmental adaptation in cultivated barley

As early farming spread from the Fertile Crescent in the Near East around 10,000 years before the present, domesticated crops encountered considerable ecological and environmental change. Spring-sown crops that flowered without the need for an extended period of cold to promote flowering and day length–insensitive crops able to exploit the longer, cooler days of higher latitudes emerged and became established. To investigate the genetic consequences of adaptation to these new environments, we identified signatures of divergent selection in the highly differentiated modern-day spring and winter barleys. In one genetically divergent region, we identify a natural variant of the barley homolog of Antirrhinum CENTRORADIALIS (HvCEN) as a contributor to successful environmental adaptation. The distribution of HvCEN alleles in a large collection of wild and landrace accessions indicates that this involved selection and enrichment of preexisting genetic variants rather than the acquisition of mutations after domestication.

Extreme Population-Dependent Linkage Disequilibrium Detected in an Inbreeding Plant Species, Hordeum vulgare

In human genetics a detailed knowledge of linkage disequilibrium (LD) is considered a prerequisite for effective population-based, high-resolution gene mapping and cloning. Similar opportunities exist for plants; however, differences in breeding system and population history need to be considered. Here we report a detailed study of localized LD in different populations of an inbreeding crop species. We measured LD between and within four gene loci within the region surrounding the hardness locus in three different gene pools of barley (Hordeum vulgare). We demonstrate that LD extends to at least 212 kb in elite barley cultivars but is rapidly eroded in related inbreeding ancestral populations. Our results indicate that haplotype-based sequence analysis in multiple populations will provide new opportunities to adjust the resolution of association studies in inbreeding crop species.

A chromosome conformation capture ordered sequence of the barley genome

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

Single-feature polymorphism discovery in the barley transcriptome

A probe-level model for analysis of GeneChip gene-expression data is presented which identified more than 10,000 single-feature polymorphisms (SFP) between two barley genotypes. The method has good sensitivity, as 67% of known single-nucleotide polymorphisms (SNP) were called as SFPs. This method is applicable to all oligonucleotide microarray data, accounts for SNP effects in gene-expression data and represents an efficient and versatile approach for highly parallel marker identification in large genomes.

Comparative analysis of population genetic structure in Athyrium distentifolium (Pteridophyta) using AFLPs and SSRs from anonymous and transcribed gene regions

To examine the performance and information content of different marker systems, comparative assessment of population genetic diversity was undertaken in nine populations of Athyrium distentifolium using nine genomic and 10 expressed sequence tag (EST) microsatellite (SSR) loci, and 265 amplified fragment length polymorphism (AFLP) loci from two primer combinations. In range‐wide comparisons (European vs. North American populations), the EST‐SSR loci showed more reliable amplification and produced more easily scorable bands than genomic simple sequence repeats (SSRs). Genomic SSRs showed significantly higher levels of allelic diversity than EST‐SSRs, but there was a significant correlation in the rank order of population diversities revealed by both marker types. When AFLPs, genomic SSRs, and EST‐SSRs are considered, comparisons of different population diversity metrics/markers revealed a mixture of significant and nonsignificant rank–order correlations. However, no hard incongruence was detected (in no pairwise comparison of populations did different marker systems or metrics detect opposingly significant different amounts of variation). Comparable population pairwise estimates of FST were obtained for all marker types, but whilst absolute values for genomic and EST‐SSRs were very similar (FST = 0.355 and 0.342, respectively), differentiation was consistently higher for AFLPs in pairwise and global comparisons (global AFLP FST = 0.496). The two AFLP primer combinations outperformed 18 SSR loci in assignment tests and discriminatory power in phenetic cluster analyses. The results from marker comparisons on A. distentifolium are discussed in the context of the few other studies on natural plant populations comparing microsatellite and AFLP variability.

Evaluating genetic relationships between indigenous coconut (Cocos nucifera L.) accessions from Sri Lanka by means of AFLP profiling

PCR-based DNA profiling of coconut palms indigenous to Sri Lanka was conducted using amplified fragment length polymorphism (AFLPs). A total of 322 amplification products were generated from the 42 genotypes with eight pairs of primers (EcoRI and MseI). Overall most variation was detected in the tall (Typica) rather than the intermediate (Aurantiaca) and dwarf (Nana) forms. A hierarchical analysis of molecular variance (AMOVA) was used to quantify and partition levels of variability into between- and within-form components. This revealed that for the inbreeding dwarf and intermediate forms most variation was observed between, rather than within, forms. In contrast, the outbreeding tall forms exhibited as much variation within as between forms. These observations have important implications for the maintenance and collection of coconut germplasm. This study also provided insights into the genetic (as opposed to phenotypic) relatedness of coconut accessions. Morphologically the Aurantiaca group of accessions are considered to be intermediate between the tall and dwarf accessions. Estimation of genetic relatedness based on AFLP analysis identified the Aurantiaca group as being more similar to the dwarf rather than the tall group. In addition, putative duplicate accessions were identified in the Aurantiaca group. Information emerging from this study will facilitate the management of coconut germplasm and optimise the choice of genetically divergent parents for crossing.

The USDA barley core collection: genetic diversity, population structure, and potential for genome-wide association studies.

New sources of genetic diversity must be incorporated into plant breeding programs if they are to continue increasing grain yield and quality, and tolerance to abiotic and biotic stresses. Germplasm collections provide a source of genetic and phenotypic diversity, but characterization of these resources is required to increase their utility for breeding programs. We used a barley SNP iSelect platform with 7,842 SNPs to genotype 2,417 barley accessions sampled from the USDA National Small Grains Collection of 33,176 accessions. Most of the accessions in this core collection are categorized as landraces or cultivars/breeding lines and were obtained from more than 100 countries. Both STRUCTURE and principal component analysis identified five major subpopulations within the core collection, mainly differentiated by geographical origin and spike row number (an inflorescence architecture trait). Different patterns of linkage disequilibrium (LD) were found across the barley genome and many regions of high LD contained traits involved in domestication and breeding selection. The genotype data were used to define ‘mini-core’ sets of accessions capturing the majority of the allelic diversity present in the core collection. These ‘mini-core’ sets can be used for evaluating traits that are difficult or expensive to score. Genome-wide association studies (GWAS) of ‘hull cover’, ‘spike row number’, and ‘heading date’ demonstrate the utility of the core collection for locating genetic factors determining important phenotypes. The GWAS results were referenced to a new barley consensus map containing 5,665 SNPs. Our results demonstrate that GWAS and high-density SNP genotyping are effective tools for plant breeders interested in accessing genetic diversity in large germplasm collections.