I. D. Thomas

An association mapping approach to identify flowering time genes in natural populations of Lolium perenne (L.)

We describe an association mapping approach using natural populations of perennial ryegrass (Lolium perenne L.) to identify molecular markers associated with heading date, an important trait affecting seasonal production, tillering, digestibility and grassland management regimes. Twenty-three natural populations originating from throughout Europe, with heading date phenotypes ranging from very early to very late, as well as three synthetic populations (varieties) were used for molecular marker genotyping using AFLP. In total, 589 polymorphic markers were identified. Hierarchical clustering, principal coordinate and other statistical analyses identified four outlying populations forming a clearly distinct sub-group. Removal of those four populations from the subsequent analysis reduced population sub-structure twofold. However, this made relatively little difference to the result of the association analysis. Linear regression identified three markers whose frequency of occurrence correlated with the heading date phenotype. Moreover, these markers were shown to be closely linked to each other within a major QTL on Chromosome 7, explaining 70% of the total variation in heading date. Pairwise linkage disequilibrium among them was also significant. These results suggest that association mapping approaches may be feasible in L. perenne, and that the use of natural populations could provide a useful source of genetic variation in traits of importance in crop improvement.

Association of Candidate Genes With Flowering Time and Water-Soluble Carbohydrate Content in Lolium perenne (L.)

We describe a candidate gene approach for associating SNPs with variation in flowering time and water-soluble carbohydrate (WSC) content and other quality traits in the temperate forage grass species Lolium perenne. Three analysis methods were used, which took the significant population structure into account. First, a linear mixed model was used enabling a structured association analysis to be incorporated with the nine populations identified in the structure analysis as random variables. Second, a within-population analysis of variance was performed. Third, a tree-scanning method was used, in which haplotype trees were associated with phenotypes on the basis of inferred haplotypes. Analysis of variance within populations identified several associations between WSC, nitrogen (N), and dry matter digestibility with allelic variants within an alkaline invertase candidate gene LpcAI. These associations were only detected in material harvested in one of the two years. By contrast, consistent associations between the L. perenne homolog (LpHD1) of the rice photoperiod control gene HD1 and flowering time were identified. One SNP, in the immediate upstream region of the LpHD1 coding sequence (C-4443-A), was significant in the linear mixed model. Within-population analysis of variance and tree-scanning analysis confirmed and extended this result to the 2118 polymorphisms in some of the populations. The merits of the tree-scanning method are compared to the single SNP analysis. The potential usefulness of the 4443 SNP in marker-assisted selection is currently being evaluated in test crosses of genotypes from this work with turf-grass varieties.

Comparison of ribosomal DNA sites inLolium species by fluorescencein situ hybridization

The position of the 18S-5.8S-26S and 5S rRNA genes have been physically mapped on the chromosomes of sevenLolium taxa. 18S-5.8S-26S sites were seen on two pairs of chromosomes in the inbreeding taxa. In the outbreeding taxa six sites were found in theL. multiflorum, seven inL. perenne and nine inL. rigidum var.rigidum. Two 5S sites were found in each of the taxa. In the inbreeders, the 5S sites were found adjacent to the 18S-5.8S-26S sites on chromosome 2. InL. multiflorum andL. perenne the 5S sites were on the short arm of chromosome 3. However, inL. rigidum var.rigidum the 5S rDNA site was found in either of the two positions.

Molecular genecology of temperature response in Lolium perenne: 2. Association of AFLP markers with ecogeography

Improved winter hardiness is an important breeding objective in the forage grass Lolium perenne. This is a complex trait with several components, including the ability to survive and grow at low temperature, to acclimate to cold, tolerate wind, snow cover and ice encasement. Marker‐assisted selection has the potential to increase the efficiency of breeding for improved cold tolerance. Here we describe a genecological approach to identifying molecular markers that are associated with adaptation to low winter temperatures. AFLP was used to assess the genetic diversity in 29 wild populations of ryegrass (Lolium perenne) representing a pan‐European temperature cline in terms of their geographical origin. A further 18 populations from a temperature cline in Bulgaria were also analysed. In addition, two varieties and five populations representing parents of mapping families currently in use at IGER were included in the analysis. Principal coordinate (PCoA) and cluster analyses of the molecular marker data showed that the Bulgarian altitude cline populations could be distinguished clearly from the other populations. Two regression analyses were carried out; one to identify AFLP markers that correlated in frequency with low mean January temperature of the geographical origin of the population, and another to identify AFLP markers correlating in frequency with the cold tolerance phenotype of the populations, as determined by LT50 values in freezing tests. In the first analysis six AFLP markers showed significant type II trends with mean January temperature, and in the second analysis 28 bands had a significant univariate relationship with the LT50 value of the accessions. In steps 2 and 3 of the stepwise analysis a further 4 and 5 bands, respectively, improved the fit significantly. The results of the two types of regression analysis are discussed in relation to ecogeography and cold tolerance phenotype of the populations.

Physical mapping of rDNA sites in possible diploid progenitors of polyploid Festuca species

Abstract.Festuca species form a polyploid series but only two of the diploid species have been firmly proposed as progenitors of any polyploid. The number and distribution of rDNA sites on the chromosomes of F. scariosa (section Scariosae) and the four diploid species that comprise section Montanae are presented with their relative DNA amounts and key morphological features. Comparisons of the results with those of some polyploid Festuca species from section Bovinae published previously indicate that F. scariosa and F. altissima could be diploid progenitors of the polyploids. It is unlikely that any one of the other three Montanae species is a progenitor of these polyploids.

Molecular genecology of temperature response in Lolium perenne: 1. preliminary analysis to reduce false positives

Molecular genecology is the study of geographical clines in frequencies of molecular markers and their relationship to ecological clines in environmental conditions. This study outlines the principles underlying the selection of populations, focusing on avoiding ‘false positives’— noncausal correlations between allele frequency and the environment. The principles are illustrated by identifying a set of populations of Lolium perenne for the study of temperature responses. The selected set of populations encompasses a 20 °C range in mean January temperature. Their freezing tolerance shows a linear trend with winter temperature, LT50 decreasing by 0.25 °C for each 1 °C reduction in mean January temperature.

Evaluating drought resistance in germplasm of meadow fescue

SummaryThe drought resistance of 25 accessions of meadow fescue (Festuca pratensis Huds.) from seven countries was investigated in four experiments: two in the glasshouse using pot-grown plants from which water was withheld for various periods, and two in controlled environments under osmotic stress.There were significant differences between populations (‘broad-sense heritabilities’ or ‘repeatabilities’) in all four experiments. In the glasshouse there was a large residual effect of yield potential on production during and after slight-to-moderate drought, and different susceptibilities appeared only after very severe drought. The most consistently high-yielding accessions were from the Bergamo alps in Italy. Recovery after drought was strongly correlated with tiller survival. Continued production under moderate drought was considered important in wetter climates, whereas survival and recovery under severe drought was often associated with low production or flowering in the seeding year, and more typical of summer-drought climates.Leaf growth rates of plants subjected to zero or moderate osmotic stress were correlated with yields of irrigated or moderately-stressed plants in the glasshouse, although there was no differential susceptibility to mild stress. Under severe osmotic stress there were very large differences in survival between populations, but there was no relationship with survival under glasshouse conditions.The inconsistency of population rankings across experiments shows that no one technique gives a full evaluation of drought resistance, and emphasizes the complex nature of the phenomenon. It was possible, however, to identify (a) regions meriting further collections, and (b) ecotypes that had desirable responses in all experiments and could contribute to variety improvement.

Genetic resources in Trifolium and their utilization in plant breeding

Abberton, M. T., Thomas, I. (2011). Genetic resources in Trifolium and their utilization in plant breeding. Plant Genetic Resources: Characterization and Utilization, 9, (1), 38-44. RONO: 03131

A statistical comparison of various factors on embryogenic proliferation, morphogenesis and regeneration in Lolium temulentum cell suspension colonies

Cell suspension colonies from four embryogenic Lolium temulentum lines were selected and plated individually in 25 embryoid maturation treatments which varied in various factors reported to stimulate embryogenesis or improve regeneration. Using a numerical scoring system to compare the cultures against a control, treatments were identified which increased growth, suppressed morphogenesis or encouraged premature shoot formation.No treatment significantly improved the proportion of colonies with globular or mature embryoids, but some prevented maturation and increased the proportion with translucent embryogenic proliferation. Other treatments accelerated maturation causing increased de-differentiation of embryogenic tissues. These treatments also tended to discourage the differentiation of discreet embryoids.Colonies were later transferred en masse to a regeneration medium and scored using another numerical system. Embryoid maturation conditions were then identified which increased or suppressed subsequent shoot regeneration. The two scoring systems enabled cultures of the four lines to be characterised in detail and identified somatic variation in embryogenic development, morphogenesis and de-differentiation.

A forage grass and small grain legume plant collecting expedition in South East Poland, 1990

SummaryThe Plant Genetic Resources Unit (PGRU) of the Institute of Grassland and Environmental Research, Welsh Plant Breeding Station (IGER, WPBS), UK, and the Plant Genetic Resources Section of the Plant Breeding and Acclimatization Institute (PBAI), Poland, carried out a joint collecting expedition in Poland between 25 August and 7 September 1990. The expedition was unique in that it was the first time that vegetative sampling had been applied to the perennial forage grass and legume populations of south east Poland. The expedition focused on seminatural vegetation in agriculturally managed situations and detailed collection site data on management systems was obtained from landowners.Samples were collected from 62 sites. Vegetative collections of Lolium spp. (37 populations) and Trifolium spp. (56 populations), and seed collections of Festuca spp. (32 populations) were made by the IGER team (Table 2). The PBAI team made 59 separate seed collections, mainly of Leguminosae.The expedition covered four geographical subregions of south east Poland. (See Fig. 1). These were the Nizina Mazowiecka south of Warszawa, the Wyzyna Malopolska with the town of Kielce at its centre and the Beskidy Zachodnie and Beskidy Wschodnie regions of the Carpathian mountains.A diverse range of habitats was sampled covering a broad range of altitude agricultural management systems and ecological conditions. Polish agriculture is faced with the problems of a rapid orientation to a market economy and it is likely that the diverse range of habitats encoutered will be reduced as agricultural practices change. This will lead to genetic erosion of the unique forage grass and legume populations to be found in Poland.