Overcoming challenges in variant calling: exploring sequence diversity in candidate genes for plant development in perennial ryegrass (Lolium perenne).

Revealing DNA sequence variation within the Lolium perenne genepool is important for genetic analysis and development of breeding applications. We reviewed current literature on plant development to select candidate genes in pathways that control agronomic traits, and identified 503 orthologues in L. perenne. Using targeted resequencing, we constructed a comprehensive catalogue of genomic variation for a L. perenne germplasm collection of 736 genotypes derived from current cultivars, breeding material and wild accessions. To overcome challenges of variant calling in heterogeneous outbreeding species, we used two complementary strategies to explore sequence diversity. First, four variant calling pipelines were integrated with the VariantMetaCaller to reach maximal sensitivity. Additional multiplex amplicon sequencing was used to empirically estimate an appropriate precision threshold. Second, a de novo assembly strategy was used to reconstruct divergent alleles for each gene. The advantage of this approach was illustrated by discovery of 28 novel alleles of LpSDUF247, a polymorphic gene co-segregating with the S-locus of the grass self-incompatibility system. Our approach is applicable to other genetically diverse outbreeding species. The resulting collection of functionally annotated variants can be mined for variants causing phenotypic variation, either through genetic association studies, or by selecting carriers of rare defective alleles for physiological analyses.

Genomic prediction of crown rust resistance in Lolium perenne.

BACKGROUND: Genomic selection (GS) can accelerate genetic gains in breeding programmes by reducing the time it takes to complete a cycle of selection. Puccinia coronata f. sp lolli (crown rust) is one of the most widespread diseases of perennial ryegrass and can lead to reductions in yield, persistency and nutritional value. Here, we used a large perennial ryegrass population to assess the accuracy of using genome wide markers to predict crown rust resistance and to investigate the factors affecting predictive ability. RESULTS: Using these data, predictive ability for crown rust resistance in the complete population reached a maximum of 0.52. Much of the predictive ability resulted from the ability of markers to capture genetic relationships among families within the training set, and reducing the marker density had little impact on predictive ability. Using permutation based variable importance measure and genome wide association studies (GWAS) to identify and rank markers enabled the identification of a small subset of SNPs that could achieve predictive abilities close to those achieved using the complete marker set. CONCLUSION: Using a GWAS to identify and rank markers enabled a small panel of markers to be identified that could achieve higher predictive ability than the same number of randomly selected markers, and predictive abilities close to those achieved with the entire marker set. This was particularly evident in a sub-population characterised by having on-average higher genome-wide linkage disequilibirum (LD). Higher predictive abilities with selected markers over random markers suggests they are in LD with QTL. Accuracy due to genetic relationships will decay rapidly over generations whereas accuracy due to LD will persist, which is advantageous for practical breeding applications.

Quantitative trait loci associated with different polar metabolites in perennial ryegrass - providing scope for breeding towards increasing certain polar metabolites.

BACKGROUND: Recent advances in the mapping of biochemical traits have been reported in Lolium perenne. Although the mapped traits, including individual sugars and fatty acids, contribute greatly towards ruminant productivity, organic acids and amino acids have been largely understudied despite their influence on the ruminal microbiome. RESULTS: In this study, we used a targeted gas-chromatography mass spectrometry (GC-MS) approach to profile the levels of 25 polar metabolites from different classes (sugars, amino acids, phenolic acids, organic acids and other nitrogen-containing compounds) present in a L. perenne F2 population consisting of 325 individuals. A quantitative trait (QTL) mapping approach was applied and successfully identified QTLs regulating seven of those polar metabolites (L-serine, L-leucine, glucose, fructose, myo-inositol, citric acid and 2, 3-hydroxypropanoic acid).Two QTL mapping approaches were carried out using SNP markers on about half of the population only and an imputation approach using SNP and DArT markers on the entire population. The imputation approach confirmed the four QTLs found in the SNP-only analysis and identified a further seven QTLs. CONCLUSIONS: These results highlight the potential of utilising molecular assisted breeding in perennial ryegrass to modulate a range of biochemical quality traits with downstream effects in livestock productivity and ruminal digestion.

Using variable importance measures to identify a small set of SNPs to predict heading date in perennial ryegrass.

Prior knowledge on heading date enables the selection of parents of synthetic cultivars that are well matched with respect to time of heading, which is essential to ensure plants put together will cross pollinate. Heading date of individual plants can be determined via direct phenotyping, which has a time and labour cost. It can also be inferred from family means, although the spread in days to heading within families demands roguing in first generation synthetics. Another option is to predict heading date from molecular markers. In this study we used a large training population consisting of individual plants to develop equations to predict heading date from marker genotypes. Using permutation-based variable selection measures we reduced the marker set from 217,563 to 50 without impacting the predictive ability. Opportunities exist to develop a cheap assay to sequence a small number of regions in linkage disequilibrium with heading date QTL in thousands of samples. Simultaneous use of these markers in non-linkage based marker-assisted selection approaches, such as paternity testing, should enhance the utility of such an approach.

The genome sequence of Barbarea vulgaris facilitates the study of ecological biochemistry.

The genus Barbarea has emerged as a model for evolution and ecology of plant defense compounds, due to its unusual glucosinolate profile and production of saponins, unique to the Brassicaceae. One species, B. vulgaris, includes two 'types', G-type and P-type that differ in trichome density, and their glucosinolate and saponin profiles. A key difference is the stereochemistry of hydroxylation of their common phenethylglucosinolate backbone, leading to epimeric glucobarbarins. Here we report a draft genome sequence of the G-type, and re-sequencing of the P-type for comparison. This enables us to identify candidate genes underlying glucosinolate diversity, trichome density, and study the genetics of biochemical variation for glucosinolate and saponins. B. vulgaris is resistant to the diamondback moth, and may be exploited for "dead-end" trap cropping where glucosinolates stimulate oviposition and saponins deter larvae to the extent that they die. The B. vulgaris genome will promote the study of mechanisms in ecological biochemistry to benefit crop resistance breeding.

Markers associated with heading and aftermath heading in perennial ryegrass full-sib families.

BACKGROUND: Heading and aftermath heading are important traits in perennial ryegrass because they impact forage quality. So far, genome-wide association analyses in this major forage species have only identified a small number of genetic variants associated with heading date that overall explained little of the variation. Some possible reasons include rare alleles with large phenotypic affects, allelic heterogeneity, or insufficient marker density. We established a genome-wide association panel with multiple genotypes from multiple full-sib families. This ensured alleles were present at the frequency needed to have sufficient statistical power to identify associations. We genotyped the panel via partial genome sequencing and performed genome-wide association analyses with multi-year phenotype data collected for heading date, and aftermath heading. RESULTS: Genome wide association using a mixed linear model failed to identify any variants significantly associated with heading date or aftermath heading. Our failure to identify associations for these traits is likely due to the extremely low linkage disequilibrium we observed in this population. However, using single marker analysis within each full-sib family we could identify markers and genomic regions associated with heading and aftermath heading. Using the ryegrass genome we identified putative orthologs of key heading genes, some of which were located in regions of marker-trait associations. CONCLUSION: Given the very low levels of LD, genome wide association studies in perennial ryegrass populations are going to require very high SNP densities. Single marker analysis within full-sibs enabled us to identify significant marker-trait associations. One of these markers anchored proximal to a putative ortholog of TFL1, homologues of which have been shown to play a key role in continuous heading of some members of the rose family, Rosaceae.

Using a Candidate Gene-Based Genetic Linkage Map to Identify QTL for Winter Survival in Perennial Ryegrass.

Important agronomical traits in perennial ryegrass (Lolium perenne) breeding programs such as winter survival and heading date, are quantitative traits that are generally controlled by multiple loci. Individually, these loci have relatively small effects. The aim of this study was to develop a candidate gene based Illumina GoldenGate 1,536-plex assay, containing single nucleotide polymorphism markers designed from transcripts involved in response to cold acclimation, vernalization, and induction of flowering. The assay was used to genotype a mapping population that we have also phenotyped for winter survival to complement the heading date trait previously mapped in this population. A positive correlation was observed between strong vernalization requirement and winter survival, and some QTL for winter survival and heading date overlapped on the genetic map. Candidate genes were located in clusters along the genetic map, some of which co-localized with QTL for winter survival and heading date. These clusters of candidate genes may be used in candidate gene based association studies to identify alleles associated with winter survival and heading date.

A Gene Encoding a DUF247 Domain Protein Cosegregates with the S Self-Incompatibility Locus in Perennial Ryegrass.

The grass family (Poaceae), the fourth largest family of flowering plants, encompasses the most economically important cereal, forage, and energy crops, and exhibits a unique gametophytic self-incompatibility (SI) mechanism that is controlled by at least two multiallelic and independent loci, S and Z. Despite intense research efforts over the last six decades, the genes underlying S and Z remain uncharacterized. Here, we report a fine-mapping approach to identify the male component of the S-locus in perennial ryegrass (Lolium perenne L.) and provide multiple evidence that a domain of unknown function 247 (DUF247) gene is involved in its determination. Using a total of 10,177 individuals from seven different mapping populations segregating for S, we narrowed the S-locus to a genomic region containing eight genes, the closest recombinant marker mapping at a distance of 0.016 cM. Of the eight genes cosegregating with the S-locus, a highly polymorphic gene encoding for a protein containing a DUF247 was fully predictive of known S-locus genotypes at the amino acid level in the seven mapping populations. Strikingly, this gene showed a frameshift mutation in self-compatible darnel (Lolium temulentum L.), whereas all of the self-incompatible species of the Festuca-Lolium complex were predicted to encode functional proteins. Our results represent a major step forward toward understanding the gametophytic SI system in one of the most important plant families and will enable the identification of additional components interacting with the S-locus.

A synteny-based draft genome sequence of the forage grass Lolium perenne.

Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species that is widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28,455 gene models, and we utilized macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family, to establish a synteny-based linear gene order. The gametophytic self-incompatibility mechanism enables the pistil of a plant to reject self-pollen and therefore promote out-crossing. We have used the sequence assembly to characterize transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterization of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and are potentially involved in the self-incompatibility mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of new varieties for more productive grasslands.

Comparative transcriptome analysis within the Lolium/Festuca species complex reveals high sequence conservation.

BACKGROUND: The Lolium-Festuca complex incorporates species from the Lolium genera and the broad leaf fescues, both belonging to the subfamily Pooideae. This subfamily also includes wheat, barley, oat and rye, making it extremely important to world agriculture. Species within the Lolium-Festuca complex show very diverse phenotypes, and many of them are related to agronomically important traits. Analysis of sequenced transcriptomes of these non-model species may shed light on the molecular mechanisms underlying this phenotypic diversity. RESULTS: We have generated de novo transcriptome assemblies for four species from the Lolium-Festuca complex, ranging from 52,166 to 72,133 transcripts per assembly. We have also predicted a set of proteins and validated it with a high-confidence protein database from three closely related species (H. vulgare, B. distachyon and O. sativa). We have obtained gene family clusters for the four species using OrthoMCL and analyzed their inferred phylogenetic relationships. Our results indicate that VRN2 is a candidate gene for differentiating vernalization and non-vernalization types in the Lolium-Festuca complex. Grouping of the gene families based on their BLAST identity enabled us to divide ortholog groups into those that are very conserved and those that are more evolutionarily relaxed. The ratio of the non-synonumous to synonymous substitutions enabled us to pinpoint protein sequences evolving in response to positive selection. These proteins may explain some of the differences between the more stress tolerant Festuca, and the less stress tolerant Lolium species. CONCLUSIONS: Our data presents a comprehensive transcriptome sequence comparison between species from the Lolium-Festuca complex, with the identification of potential candidate genes underlying some important phenotypical differences within the complex (such as VRN2). The orthologous genes between the species have a very high %id (91,61%) and the majority of gene families were shared for all of them. It is likely that the knowledge of the genomes will be largely transferable between species within the complex.

Vernalization mediated changes in the Lolium perenne transcriptome.

Vernalization is a key requirement for the induction of flowering in perennial ryegrass (Lolium perenne L.). The transcriptome of two genotypes with contrasting vernalization requirement was studied during primary (vernalization and short day conditions) and secondary induction (higher temperature and long day conditions) using an RNA-Seq approach. This revealed transcripts with expression profiles indicative of a role in floral induction, both in the promotion and repression of flowering. We observed similarities and specific differences between the two genotypes related to cold response, carbohydrate metabolism, and photoperiod regulation. Components of the photoperiod pathway showed regulation during vernalization, pointing to possible interactions between elements of the photoperiod and vernalization pathways. The results provide a global picture of the processes ongoing during the transition from vegetative to reproductive phase of perennial ryegrass genotypes with and without a vernalization requirement.

The genome and transcriptome of perennial ryegrass mitochondria.

BACKGROUND: Perennial ryegrass (Lolium perenne L.) is one of the most important forage and turf grass species of temperate regions worldwide. Its mitochondrial genome is inherited maternally and contains genes that can influence traits of agricultural importance. Moreover, the DNA sequence of mitochondrial genomes has been established and compared for a large number of species in order to characterize evolutionary relationships. Therefore, it is crucial to understand the organization of the mitochondrial genome and how it varies between and within species. Here, we report the first de novo assembly and annotation of the complete mitochondrial genome from perennial ryegrass. RESULTS: Intact mitochondria from perennial ryegrass leaves were isolated and used for mtDNA extraction. The mitochondrial genome was sequenced to a 167-fold coverage using the Roche 454 GS-FLX Titanium platform, and assembled into a circular master molecule of 678,580 bp. A total of 34 proteins, 14 tRNAs and 3 rRNAs are encoded by the mitochondrial genome, giving a total gene space of 48,723 bp (7.2%). Moreover, we identified 149 open reading frames larger than 300 bp and covering 67,410 bp (9.93%), 250 SSRs, 29 tandem repeats, 5 pairs of large repeats, and 96 pairs of short inverted repeats. The genes encoding subunits of the respiratory complexes - nad1 to nad9, cob, cox1 to cox3 and atp1 to atp9 - all showed high expression levels both in absolute numbers and after normalization. CONCLUSIONS: The circular master molecule of the mitochondrial genome from perennial ryegrass presented here constitutes an important tool for future attempts to compare mitochondrial genomes within and between grass species. Our results also demonstrate that mitochondria of perennial ryegrass contain genes crucial for energy production that are well conserved in the mitochondrial genome of monocotyledonous species. The expression analysis gave us first insights into the transcriptome of these mitochondrial genes in perennial ryegrass.

Early response mechanisms of perennial ryegrass (Lolium perenne) to phosphorus deficiency.

BACKGROUND AND AIMS: Improving phosphorus (P) nutrient efficiency in Lolium perenne (perennial ryegrass) is likely to result in considerable economic and ecological benefits. To date, research into the molecular and biochemical response of perennial ryegrass to P deficiency has been limited, particularly in relation to the early response mechanisms. This study aimed to identify molecular mechanisms activated in response to the initial stages of P deficiency. METHODS: A barley microarray was successfully used to study gene expression in perennial ryegrass and this was complemented with gas chromatography-mass spectrometry metabolic profiling to obtain an overview of the plant response to early stages of P deficiency. KEY RESULTS: After 24 h of P deficiency, internal phosphate concentrations were reduced and significant alterations were detected in the metabolome and transcriptome of two perennial ryegrass genotypes. Results indicated a replacement of phospholipids with sulfolipids and the utilization of glycolytic bypasses in response to P deficiency in perennial ryegrass. CONCLUSIONS: The transcriptome and metabolome of perennial ryegrass undergo changes in response to reductions in P supply after 24 h.

Transcriptional and metabolic profiles of Lolium perenne L. genotypes in response to a PEG-induced water stress.

Metabolic profiling was carried out in the forage grass Lolium perenne L. (perennial ryegrass) to uncover mechanisms involved in the plants response to water stress. When leaf and root materials from two genotypes, with a contrasting water stress response, were analysed by GC-MS, a clear difference in the metabolic profiles of the leaf tissue under water stress was observed. Differences were principally due to a reduction in fatty acid levels in the more susceptible Cashel genotype and an increase in sugars and compatible solutes in the more tolerant PI 462336 genotype. Sugars with a significant increase included: raffinose, trehalose, glucose, fructose and maltose. Increasing the ability of perennial ryegrass to accumulate these sugars in response to a water deficit may lead to more tolerant varieties. The metabolomics approach was combined with a transcriptomics approach in the water stress tolerant genotype PI 462336, which has identified perennial ryegrass genes regulated under water stress.