A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST-RFLP loci in the F(1)(NA(6) x AU(6)) population. A comprehensive set of EST-SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA(6) genetic map contains 88 EST-RFLP and 71 EST-SSR loci with a total map length of 963 cM, while the AU(6) genetic map contains 67 EST-RFLP and 58 EST-SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

key variables: leaf appearance rate, leaf elongation rate, and leaf lifespan. The expression of each of these traits is Quantitative trait loci (QTLs) for a number of agronomically imunder both genetic and environmental control (Lemaire portant traits of perennial ryegrass (Lolium perenne L.) were identiand Chapman, 1996), and leaf development in Lolium fied by means of a reference molecular marker-based genetic map. Replicated phenotypic data was obtained for a number of field-assessed has been demonstrated to be under genetic control in a morphological and developmental traits as well as the winter hardinumber of studies (Edwards and Cooper, 1963; Rhodes, ness-associated characters of winter survival and electrical conductiv-1973; Hazard et al., 1996). Structural characteristics of ity. Marker-trait association analysis was performed by a number of plants such as tiller number, leaf number, and leaf size methods, and a high degree of congruence was observed between the are the result of these morphogenetic traits, and their respective results. QTLs were detected for morphological traits such measurement in breeding programs allows a dissection as plant height, tiller size, leaf length, leaf width, fresh weight at of the complex herbage yield trait as well as predictions harvest, plant type, spikelet number per spike and spike length, as well of the response to grazing. as the developmental traits of heading date and degree of aftermath Different ecoclimatic regions, or pastures under difheading. A number of traits were significantly correlated, and coinciferent grazing regimes, may provide alternative selecdent QTL locations were identified. No significant QTLs for winter survival in the field were identified. However, a QTL for electrical

Molecular genetic marker development in perennial ryegrass has largely been dependent on anonymous sequence variation. The availability of a large-scale EST resource permits the development of functionally-associated genetic markers based on SNP variation in candidate genes. Genic SNP loci and associated haplotypes are suitable for implementation in molecular breeding of outbreeding forage species. Strategies for in vitro SNP discovery through amplicon cloning and sequencing have been designed and implemented. Putative SNPs were identified within and between the parents of the F(1)(NA(6) x AU(6)) genetic mapping family and were validated among progeny individuals. Proof-of-concept for the process was obtained using the drought tolerance-associated LpASRa2 gene. SNP haplotype structures were determined and correlated with predicted amino acid changes. Gene-length LD was evaluated across diverse germplasm collections. A survey of SNP variation across 100 candidate genes revealed a high frequency of SNP incidence (c. 1 per 54 bp), with similar proportions in exons and introns. A proportion (c. 50%) of the validated genic SNPs were assigned to the F(1)(NA(6) x AU(6)) genetic map, showing high levels of coincidence with previously mapped RFLP loci. The perennial ryegrass SNP resource will enable genetic map integration, detailed LD studies and selection of superior allele content during varietal development.