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The Characterisation of Genetic Diversity of a Collection of Perennial Ryegrass (Lolium perenne L.).
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2008
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Thesis McGrath Sarah.pdf
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McGrath SK. 2008. The characterisation of genetic diversity of a collection of perennial ryegrass (lolium perenne L.). Ireland: Trinity College Dublin (University of Dublin) (Ireland)
Abstract
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.
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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
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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.