• The Characterisation of Genetic Diversity of a Collection of Perennial Ryegrass (Lolium perenne L.).

      McGrath, Sarah Katherine; Teagasc Walsh Fellowship Programme (2008)
      Perennial ryegrass (Lolium perenne L.) is a member of the Poaceae family, is native to Europe, the Near East and North Africa and is grown in all the temperate climate areas of the world as a forage and turf grass. Due to its persistence, palatability and nutritive value for ruminants, it is a principal component of pastures, and the most important forage species in Ireland. The primary aim of this thesis was to characterise the level of diversity in a large genetic resource collection of L. perenne germplasm held at Teagasc, Oak Park. Molecular markers, both chloroplast and nuclear SSRs, biochemical characters (water soluble carbohydrate, crude protein, and dry matter), and morphological characters (vegetative and flowering) were used to characterise this diversity, as well as population differentiation, and geographic patterns. Levels of diversity in all systems were found to be high in this collection. Primers to amplify microsatellite markers from the chloroplast genome of Lolium perenne were designed and optimized using de novo sequencing and in silico sequences. With one exception, each locus was polymorphic with a range from two to nine alleles in L. perenne. The newly developed primer pairs cross-amplified in different species of Lolium and in 50 other grass species representing nine grass subfamilies. These markers were then used to characterise chloroplast genetic diversity at allelic and haplotypic level in 104 accessions of Lolium perenne, other Lolium species, Festuca species and ´Festulolium cultivars. Furthermore, genetic relationships between the accessions and biogeographic distribution of haplotypes were investigated using a range of population genetic diversity measures and an Analysis of Molecular Variance (AMOVA). An extremely high number of 511 haplotypes was detected in 1,575 individuals possibly attributable to natural and anthropogenic migration. Much of the L. perenne European ecotype diversity (61%) could be attributed to within population variance. Plastid gene pools and maternal lineages for L. perenne could be clearly identified. Evidence was found showing a most likely migration route of L. perenne into Ireland from southern regions of Europe northwards. 5 Morphological variation of 13 vegetative and reproductive traits was characterised for 2,481 individuals from 50 L. perenne accessions, a mixture of Irish and European ecotypes and cultivars. Considerable levels of among and within population variation was found across traits. Principal component analysis and UPGMA dendrograms were able to separate ecotypes from cultivars. Cultivars generally had later dates of ear emergence, better spring and summer growth, longer rachis length and more spikelets per spike than ecotypes. Correlation and regression analysis were used to assess relationships between traits and strong positive relationships were seen between reproductive characters, i.e. rachis length with spikelets per spike, florets per spikelet and glume length. The strong relationship between rachis length and the other reproductive characters suggested that rachis length could be used as a predictor for reproductive performance. Later flowering was correlated with improved spring and summer growth. Water soluble carbohydrate (WSC; glucose and fructose determined by HPLC), crude protein (determined via LECO analysis), and dry matter contents were recorded for 1,320 individuals, pooled into 132 samples from 33 L. perenne ecotypes and cultivars at five different harvest time points across the 2004 growing season. While, in general, the cultivars had higher WSC contents than the ecotypes, individual ecotypes did show potential to be used in breeding programmes, as they showed higher values than all other accessions at particular cutting points. In correlation analyses, positive relationships were shown between dry matter and glucose both early and late in the growing season, and this was in agreement with the amount of leaves compared to stem at these times in the growing season. PCA analysis allowed the separation either between cultivars and ecotypes, or between tetraploid cultivars and the rest of the accessions at four out of five cutting points. In the ANOVA analysis, cutting point was the most significant factor influencing the variation in the traits. Eight nuclear SSR markers were used to characterize genetic diversity in 928 individuals from 40 diploid ecotypes and cultivars of L. perenne. High levels of genetic diversity (0.82, Nei’s gene diversity, over all accessions) and high numbers of alleles (22.25 average number of alleles per locus) was found. An average polymorphic information content (PIC) value of 0.81 across all loci was found. When deviations from Hardy-Weinberg equilibrium were tested, the majority of populations 6 had an excess of homozygotes. Very low levels of linkage disequilibrium were found between pairs of loci tested. AMOVA analysis and F statistics were used to test partitioning of variation, and most variation was found within populations (e.g. 31% for glume length in ecotypes). UPGMA, PCA and STRUCTURE analysis all gave similar patterns of relationships between populations, where relationships with high bootstrap support on the UPGMA dendrogram were also seen in the other analyses. The overall results of the thesis are discussed in the context of plant breeding programmes and natural population genetic variation. Strategies for incorporation of the results of the thesis (and the novel markers developed within) into plant breeding programmes are suggested.
    • Exploration of flowering control in Lolium perenne L.

      Byrne, Stephen; Mur, Luis AJ; Donnisson, Iain; Guiney, Emma (Teagasc, 01/08/2009)
      Flowering or heading in Lolium perenne (perennial ryegrass) is induced by a period of vernalization, followed by long days at higher temperatures. When heading occurs there is a reduction in the feed quality of the forage and therefore extending the period of vegetative growth or eliminating heading during the growing season will improve the potential of perennial ryegrass in agriculture. Conversely, a better control of flowering time and increased heading will lead to higher seed yield for commercial producers. The aim of this project was to investigate the underlying genetic control of flowering time in perennial ryegrass. An F1 population was created by crosspollinating two lines with different heading dates and a genetic linkage map was constructed using Simple Sequence Repeat (SSR) markers. The population and genetic linkage map was then used to identify Quantitative Trait Loci (QTL) associated with heading date, spike length and spikelets per spike. A number of QTL were identified for all traits, some of which had not previously been identified in perennial ryegrass. A Suppression Subtractive Hybridization (SSH) study was also employed to identify genes differentially expressed between an extremely late flowering line and earlier flowering sibling line. Expression analysis of a number of identified genes through floral induction was performed using real time RT-PCR. This revealed a number of transcripts with expression profiles indicative of a role to play in floral induction.
    • An Immortalized Genetic Mapping Population for Perennial Ryegrass: A Resource for Phenotyping and Complex Trait Mapping

      Velmurugan, Janaki; Milbourne, Dan; Connolly, Vincent; Heslop-Harrison, J. S.; Anhalt, Ulrike C. M.; Lynch, M. B.; Barth, Susanne; Teagasc Walsh Fellowship Programme (Frontiers, 2018-05-31)
      To address the lack of a truly portable, universal reference mapping population for perennial ryegrass, we have been developing a recombinant inbred line (RIL) mapping population of perennial ryegrass derived via single seed descent from a well-characterized F2 mapping population based on genetically distinct inbred parents in which the natural self-incompatibility (SI) system of perennial ryegrass has been overcome. We examined whether it is possible to create a genotyping by sequencing (GBS) based genetic linkage map in a small population of the F6 generation of this population. We used 41 F6 genotypes for GBS with PstI/MspI-based libraries. We successfully developed a genetic linkage map comprising 6074 SNP markers, placing a further 22080 presence and absence variation (PAV) markers on the map. We examined the resulting genetic map for general and RIL specific features. Overall segregation distortion levels were similar to those experienced in the F2 generation, but segregation distortion was reduced on linkage group 6 and increased on linkage group 7. Residual heterozygosity in the F6 generation was observed at a level of 5.4%. There was a high proportion of chromosomes (30%) exhibiting the intact haplotype of the original inbred parents of the F1 genotype from which the population is derived, pointing to a tendency for chromosomes to assort without recombining. This could affect the applicability of these lines and might make them more suitable for situations where repressed recombination is an advantage. Inter- and intra-chromosomal linkage disequilibrium (LD) analysis suggested that the map order was robust. We conclude that this RIL population, and subsequent F7 and F8 generations will be useful for genetic analysis and phenotyping of agronomic and biological important traits in perennial ryegrass.
    • An Immortalized Genetic Mapping Population for Perennial Ryegrass: A Resource for Phenotyping and Complex Trait Mapping

      Velmurugan, Janaki; Milbourne, Dan; Connolly, Vincent; Heslop-Harrison, J. S.; Anhalt, Ulrike C. M.; Lynch, M. B.; Barth, Susanne; Teagasc Walsh Fellowship Programme; Teagasc (Frontiers, 2018-05-31)
      To address the lack of a truly portable, universal reference mapping population for perennial ryegrass, we have been developing a recombinant inbred line (RIL) mapping population of perennial ryegrass derived via single seed descent from a well-characterized F2 mapping population based on genetically distinct inbred parents in which the natural self-incompatibility (SI) system of perennial ryegrass has been overcome. We examined whether it is possible to create a genotyping by sequencing (GBS) based genetic linkage map in a small population of the F6 generation of this population. We used 41 F6 genotypes for GBS with PstI/MspI-based libraries. We successfully developed a genetic linkage map comprising 6074 SNP markers, placing a further 22080 presence and absence variation (PAV) markers on the map. We examined the resulting genetic map for general and RIL specific features. Overall segregation distortion levels were similar to those experienced in the F2 generation, but segregation distortion was reduced on linkage group 6 and increased on linkage group 7. Residual heterozygosity in the F6 generation was observed at a level of 5.4%. There was a high proportion of chromosomes (30%) exhibiting the intact haplotype of the original inbred parents of the F1 genotype from which the population is derived, pointing to a tendency for chromosomes to assort without recombining. This could affect the applicability of these lines and might make them more suitable for situations where repressed recombination is an advantage. Inter- and intra-chromosomal linkage disequilibrium (LD) analysis suggested that the map order was robust. We conclude that this RIL population, and subsequent F7 and F8 generations will be useful for genetic analysis and phenotyping of agronomic and biological important traits in perennial ryegrass.
    • Markers associated with heading and aftermath heading in perennial ryegrass full-sib families

      Arojju, Sai Krishna; Barth, Susanne; Milbourne, Dan; Conaghan, Patrick; Velmurugan, Janaki; Hodkinson, Trevor R; Byrne, Stephen L.; Department of Agriculture, Food and the Marine, Ireland; Teagasc Walsh Fellowship Programme; EU Marie-Sklodowska-Curie Fellowship; et al. (Biomed Central, 16/07/2016)
      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.
    • Quantitative trait loci associated with different polar metabolites in perennial ryegrass - providing scope for breeding towards increasing certain polar metabolites

      Foito, Alexandre; Hackett, Christine A; Stewart, Derek; Velmurugan, Janaki; Milbourne, Dan; Byrne, Stephen L.; Barth, Susanne; Department of Agriculture, Food and the Marine, Ireland; RSF 06–346 (Biomed Central, 10/10/2017)
      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.