• 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.
    • Characterization of Potato Virus Y Isolates and Assessment of Nanopore Sequencing to Detect and Genotype Potato Viruses

      Della Bartola, Michele; Byrne, Stephen; Mullins, Ewen; Department of Agriculture, Food and the Marine; 15/S/618 SCOPE (MDPI AG, 2020-04-23)
      Potato virus Y (PVY) is the most economically important virus infecting cultivated potato (Solanum tuberosum L.). Accurate diagnosis is crucial to regulate the trade of tubers and for the sanitary selection of plant material for propagation. However, high genetic diversity of PVY represents a challenge for the detection and classification of isolates. Here, the diversity of Irish PVY isolates from a germplasm collection and commercial sites was investigated using conventional molecular and serological techniques. Recombinant PVY isolates were prevalent, with PVYNTNa being the predominant genotype. In addition, we evaluated Nanopore sequencing to detect and reconstruct the whole genome sequence of four viruses (PVY, PVX, PVS, PLRV) and five PVY genotypes in a subset of eight potato plants. De novo assembly of Nanopore sequencing reads produced single contigs covering greater than 90% of the viral genome and sharing greater than 99.5% identity to the consensus sequences obtained with Illumina sequencing. Interestingly, single near full genome contigs were obtained for different isolates of PVY co-infecting the same plant. Mapping reads to available reference viral genomes enabled us to generate near complete genome sequences sharing greater than 99.90% identity to the Illumina-derived consensus. This is the first report describing the use of Oxford Nanopore’s MinION to detect and genotype potato viruses. We reconstructed the genome of PVY and other RNA viruses; indicating the technologies potential for virus detection in potato production systems, and for the study of genetic diversity of highly heterogeneous viruses such as PVY.
    • Genotyping by Sequencing and Plastome Analysis Finds High Genetic Variability and Geographical Structure in Dactylis glomerata L. in Northwest Europe Despite Lack of Ploidy Variation

      Hodkinson, Trevor R.; Perdereau, Aude; Klaas, Manfred; Cormican, Paul; Barth, Susanne; European Union; 289461 (MDPI AG, 2019-06-28)
      Large collections of the forage and bioenergy grass Dactylis glomerata were made in northwest (NW) Europe along east to west and north to south clines for genetic resource conservation and to inform breeding programmes of genetic diversity, genepools, and ploidy. Leaves were sampled for genetic analysis and seed and rhizome for ex-situ conservation. Genotyping by sequencing (GBS) was used to assay nuclear DNA diversity and plastome single nucleotide polymorphism (SNP) discovery was undertaken using a long-read PCR and MiSeq approach. Nuclear and plastid SNPs were analysed by principal component analysis (PCA) to compare genotypes. Flow cytometry revealed that all samples were tetraploid, but some genome size variation was recorded. GBS detected an average of approximately 10,000 to 15,000 SNPs per country sampled. The highest average number of private SNPs was recorded in Poland (median ca. 2000). Plastid DNA variation was also high (1466 SNPs, 17 SNPs/kbp). GBS data, and to a lesser extent plastome data, also show that genetic variation is structured geographically in NW Europe with loose clustering matching the country of plant origin. The results reveal extensive genetic diversity and genetic structuring in this versatile allogamous species despite lack of ploidy variation and high levels of human mediated geneflow via planting.
    • Genotyping by Sequencing and Plastome Analysis Finds High Genetic Variability and Geographical Structure in Dactylis glomerata L. in Northwest Europe Despite Lack of Ploidy Variation

      Hodkinson, Trevor R.; Perdereau, Aude; Klaas, Manfred; Cormican, Paul; Barth, Susanne; EU; 289461 (MDPI AG, 2019-06-28)
      Large collections of the forage and bioenergy grass Dactylis glomerata were made in northwest (NW) Europe along east to west and north to south clines for genetic resource conservation and to inform breeding programmes of genetic diversity, genepools, and ploidy. Leaves were sampled for genetic analysis and seed and rhizome for ex-situ conservation. Genotyping by sequencing (GBS) was used to assay nuclear DNA diversity and plastome single nucleotide polymorphism (SNP) discovery was undertaken using a long-read PCR and MiSeq approach. Nuclear and plastid SNPs were analysed by principal component analysis (PCA) to compare genotypes. Flow cytometry revealed that all samples were tetraploid, but some genome size variation was recorded. GBS detected an average of approximately 10,000 to 15,000 SNPs per country sampled. The highest average number of private SNPs was recorded in Poland (median ca. 2000). Plastid DNA variation was also high (1466 SNPs, 17 SNPs/kbp). GBS data, and to a lesser extent plastome data, also show that genetic variation is structured geographically in NW Europe with loose clustering matching the country of plant origin. The results reveal extensive genetic diversity and genetic structuring in this versatile allogamous species despite lack of ploidy variation and high levels of human mediated geneflow via planting.