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BackgroundSelf-incompatibility (SI) systems prevent self-fertilization in several species of Poaceae, many of which are economically important forage, bioenergy and turf grasses. Self-incompatibility ensures cross-pollination and genetic diversity but restricts the ability to fix useful genetic variation. In most inbred crops it is possible to develop high-performing homozygous parental lines by self-pollination, which then enables the creation of F1 hybrid varieties with higher performance, a phenomenon known as heterosis. The inability to fully exploit heterosis in outcrossing grasses is partially responsible for lower levels of improvement in breeding programmes compared with inbred crops. However, SI can be overcome in forage grasses to create self-compatible populations. This is generating interest in understanding the genetical basis of self-compatibility (SC), its significance for reproductive strategies and its exploitation for crop improvement, especially in the context of F1 hybrid breeding.ScopeWe review the literature on SI and SC in outcrossing grass species. We review the currently available genomic tools and approaches used to discover and characterize novel SC sources. We discuss opportunities barely explored for outcrossing grasses that SC facilitates. Specifically, we discuss strategies for wide SC introgression in the context of the Lolium–Festuca complex and the use of SC to develop immortalized mapping populations for the dissection of a wide range of agronomically important traits. The germplasm available is a valuable practical resource and will aid understanding the basis of inbreeding depression and hybrid vigour in key temperate forage grass species.ConclusionsA better understanding of the genetic control of additional SC loci offers new insight into SI systems, their evolutionary origins and their reproductive significance. Heterozygous outcrossing grass species that can be readily selfed facilitate studies of heterosis. Moreover, SC introduction into a range of grass species will enable heterosis to be exploited in innovative ways in genetic improvement programmes.

Accurate identification of named accessions in germplasm collections is extremely important, especially for vegetatively propagated crops which are expensive to maintain. Thus, an inexpensive, reliable, and rapid genotyping method is essential because it avoids the need for laborious and time-consuming morphological comparisons. Single Nucleotide Polymorphism (SNP) marker panels containing large numbers of SNPs have been developed for many crop species, but such panels are much too large for basic cultivar identification. Here, we have identified a minimum set of SNP markers sufficient to distinguish apple cultivars held in the English and Welsh national collections providing a cheaper and automatable alternative to the markers currently used by the community. We show that SNP genotyping with a small set of well selected markers is equally efficient as microsatellites for the identification of apple cultivars and has the added advantage of automation and reduced cost when screening large numbers of samples.

The grass family (Poaceae) the fourth largest family of flowering plants encompasses the most economically important cereal forage and energy crops and exhibits a unique gametophytic self incompatibility (SI) mechanism that is controlled by at least two multi allelic and independent loci S and Z. Despite intense research efforts over the last six decades the genes underlying S and Z remain uncharacterised. Here we report a fine mapping approach to identify the male component of the S locus in perennial ryegrass (Lolium perenne L.) and provide multiple evidence that a domain of unknown function 247 (DUF247) gene is involved in its determination. Using a total of 10177 individuals from seven different mapping populations segregating for S we narrowed the S locus to a genomic region containing eight genes the closest recombinant marker mapping at a distance of 0.016 centimorgan. Of the eight genes co segregating with the S locus a highly polymorphic gene encoding for a protein containing a DUF247 was fully predictive of known S locus genotypes at the amino acid level in the seven mapping populations. Strikingly this genes showed a frame shift mutation in self compatible darnel (Lolium temulentum L.) whereas all of the self incompatible species of the Festuca Lolium complex were predicted to encode functional proteins. Our results represent a major step forward towards understanding the gametophytic SI system in one of the most important plant families and will enable the identification of additional components interacting with the S locus.

Self-incompatibility (SI) is a mechanism that many flowering plants employ to prevent fertilisation by self- and self-like pollen ensuring heterozygosity and hybrid vigour. Although a number of single locus mechanisms have been characterised in detail, no multi-locus systems have been fully elucidated. Historically, examples of the genetic analysis of multi-locus SI, to make analysis tractable, are either made on the progeny of bi-parental crosses, where the number of alleles at each locus is restricted, or on crosses prepared in such a way that only one of the SI loci segregates. Perennial ryegrass (Lolium perenne L.) possesses a well-documented two locus (S and Z) gametophytic incompatibility system. A more universal, realistic proof of principle study was conducted in a perennial ryegrass population in which allelic and non-allelic diversity was not artificially restricted. A complex pattern of pollinations from a diallel cross was revealed which could not possibly be interpreted easily per se, even with an already established genetic model. Instead, pollination scores were distilled into principal component scores described as Compatibility Components (CC1-CC3). These were then subjected to a conventional genome-wide association analysis. CC1 associated with markers on linkage groups (LGs) 1, 2, 3, and 6, CC2 exclusively with markers in a genomic region on LG 2, and CC3 with markers on LG 1. BLAST alignment with the Brachypodium physical map revealed highly significantly associated markers with peak associations with genes adjacent and four genes away from the chromosomal locations of candidate SI genes, S- and Z-DUF247, respectively. Further significant associations were found in a Brachypodium distachyon chromosome 3 region, having shared synteny with Lolium LG 1, suggesting further SI loci linked to S or extensive micro-re-arrangement of the genome between B. distachyon and L. perenne. Significant associations with gene sequences aligning with marker sequences on Lolium LGs 3 and 6 were also identified. We therefore demonstrate the power of a novel association genetics approach to identify the genes controlling multi-locus gametophytic SI systems and to identify novel loci potentially involved in already established SI systems., Frontiers in Plant Science, 8, ISSN:1664-462X

Perennial ryegrass (Lolium perenne L.) is one of the most widely grown forage grasses in temperate agriculture. In order to maintain and increase its usage as forage in livestock agriculture, there is a continued need for improvement in biomass yield, quality, disease resistance and seed yield. Genetic gain for traits such as biomass yield has been relatively modest. This has been attributed to its long breeding cycle, and the necessity to use population based breeding methods. Thanks to recent advances in genotyping techniques there is increasing interest in genomic selection from which genomically estimated breeding values (GEBV) are derived. In this paper we compare the classical RRBLUP model with state-of-the-art machine learning (ML) techniques that should yield themselves easily to use in GS and demonstrate their application to predicting quantitative traits in a breeding population of L. perenne. Prediction accuracies varied from 0 to 0.59 depending on trait, prediction model and composition of the training population. The BLUP model produced the highest prediction accuracies for most traits and training populations. Forage quality traits had the highest accuracies compared to yield related traits. There appeared to be no clear pattern to the effect of the training population composition on the prediction accuracies. The heritability of the forage quality traits was generally higher than for the yield related traits, and could partly explain the difference in accuracy. Some population structure was evident in the breeding populations, and probably contributed to the varying effects of training population on the predictions. The average linkage disequilibrium (LD) between adjacent markers ranged from 0.121 to 0.215. Higher marker density and larger training population closely related with the test population are likely to improve the prediction accuracy.

BACKGROUND AND SCOPE: Self-incompatibility (SI) in flowering plants ensures the maintenance of genetic diversity by ensuring outbreeding. Different genetic and mechanistic systems of SI among flowering plants suggest either multiple origins of SI or considerable evolutionary diversification. In the grasses, SI is based on two loci, S and Z, which are both polyallelic: an incompatible reaction occurs only if both S and Z alleles are matched in individual pollen with alleles of the pistil on which they alight. Such incompatibility is referred to as gametophytic SI (GSI). The mechanics of grass GSI is poorly understood relative to the well-characterized S-RNase-based single-locus GSI systems (Solanaceae, Rosaceae, Plantaginaceae), or the Papaver recognition system that triggers a calcium-dependent signalling network culminating in programmed cell death. There is every reason to suggest that the grass SI system represents yet another mechanism of SI. S and Z loci have been mapped using isozymes to linkage groups C1 and C2 of the Triticeae consensus maps in Secale, Phalaris and Lolium. Recently, in Lolium perenne, in order to finely map and identify S and Z, more closely spaced markers have been developed based on cDNA and repeat DNA sequences, in part from genomic regions syntenic between the grasses. Several genes tightly linked to the S and Z loci were identified, but so far no convincing candidate has emerged. RESEARCH AND PROGRESS: From subtracted Lolium immature stigma cDNA libraries derived from S and Z genotyped individuals enriched for SI potential component genes, kinase enzyme domains, a calmodulin-dependent kinase and a peptide with several calcium (Ca(2+)) binding domains were identified. Preliminary findings suggest that Ca(2+) signalling and phosphorylation may be involved in Lolium GSI. This is supported by the inhibition of Lolium SI by Ca(2+) channel blockers lanthanum (La(3+)) and verapamil, and by findings of increased phosphorylation activity during an SI response.

White clover (Trifolium repens L.) is a forage legume of considerable economic importance in temperate agricultural systems. It has a strong self-incompatibility system. The molecular basis of self-incompatibility in T. repens is unknown, but it is under the control of a single locus, which is expressed gametophytically. To locate the self-incompatibility locus (S locus) in T. repens, we carried out cross-pollination experiments in an F(1) mapping population and constructed a genetic linkage map using amplified fragment length polymorphism and simple sequence repeat markers. As the first step in a map-based cloning strategy, we locate for the first time the S locus in T. repens on a genetic linkage map, on the homoeologous linkage group pair 1 (E), which is broadly syntenic to Medicago truncatula L. chromosome 1. On the basis of this syntenic relationship, the possibility that the S locus may or may not possess an S-RNase gene is discussed.

Self-incompatibility (SI) in Lolium perenne is controlled gametophytically by the S-Z two-locus system. S and Z loci mapped to L. perenne linkage groups 1 and 2, respectively, with their corresponding putative-syntenic regions on rice chromosome 5 (R5) and R4. None of the gene products of S and Z have yet been identified. SI cDNA libraries were developed to enrich for SI expressed genes in L. perenne. Transcripts were identified from the SI libraries that were orthologous to sequences on rice R4 and R5. These represent potential SI candidate genes. Altogether ten expressed SI candidate genes were identified. A rapid increase in gene expression within two minutes after pollen-stigma contact was revealed, reaching a maximum between 2 and 10 min. The potential involvement of these genes in the SI reactions is discussed.

Perennial ryegrass (Lolium perenne) is one of the key forage and amenity grasses throughout the world. In the UK it accounts for 70 % of all agricultural land use with an estimated farm gate value of 6 billion pounds per annum. However, in terms of the genetic resources available, L. perenne has lagged behind other major crops in Poaceae. The aim of this project was therefore the construction of a microsatellite-enriched genomic library for L. perenne to increase the number of genetic markers available for both marker-assisted selection in breeding programmes and gene isolation.Primers for 229 non-redundant microsatellite markers were designed and used to screen two L. perenne genotypes, one amenity and one forage. Of the 229 microsatellites, 95 were found to show polymorphism between amenity and forage genotypes. A selection of microsatellite primers was selected from these 95 and used to screen two mapping populations derived from intercrossing and backcrossing the two forage and amenity grass genotypes.The utility of the resulting genetic maps for analysis of the genetic control of target traits was demonstrated by the mapping of genes associated with heading date to linkage groups 4 and 7.

Mutational load and resource allocation factors and their effects on limiting seed set were investigated in ryegrass by comparative mapping genomics and quantitative trait loci (QTL) analysis in two perennial ryegrass (Lolium perenne) mapping families sharing common genetic markers. Quantitative trait loci for seed-set were identified on chromosome (LG) 7 in both families and on LG4 of the F2/WSC family. On LG7, seed-set and heading date QTLs colocalized in both families and cannot be unequivocally resolved. Comparative genomics suggests that the LG7 region is syntenous to a region of rice LG6 which contains both fertility (S5(n)) and heading date (Hd1, Hd3a) candidate genes. The LG4 region is syntenous to a region of rice LG3 which contains a fertility (S33) candidate gene. QTL maxima for seed-set and heading date on LG4 in the F2/WSC family are separated by c. 8 cm, indicating distinct genetic control. Low seed set is under the control of recessive genes at both LG4 and LG7 locations. The identification of QTLs associated with seed set, a major component of seed yield in perennial ryegrass, indicates that mutational load associated with these genomic regions can be mitigated through marker-assisted selection.

Fourier transform infrared spectroscopy (FT-IR) was used to obtain ‘holistic’ metabolic fingerprints from a wide range of plants to differentiate species, population, single plant genotype, and chromosomal constitution differences. Sample preparation simply entailed the maceration of fresh leaves with water, and these samples were then dried and analysed by reflectance FT-IR where spectral acquisition was typically 10 s. All samples gave reproducible, characteristic biological infrared absorption spectra and these were analysed by chemometric methods. FT-IR is not biased to any particular chemical species and thus the whole tissue profiles produced measure the total biochemical makeup of the test sample; that is to say it represents a plant phenotype. We show that by simple cluster analysis these phenotypic measurements can be related to the genotypes of the plants and can reliably differentiate closely related individuals. We believe that this approach provides a valuable new tool for the rapid metabolomic profiling of plants, with applications to plant breeding and the assessment of substantial equivalency for genetically-modified plants.

In plants, lifespan is agronomically important as it is a major determinant of productivity. The onset of leaf senescence is associated with the decrease in photosynthetic capability that reduces herbage yield and biomass production. This research aims to identify genes/quantitative trait loci (QTL) that can prolong photosynthesis and delay the senescence processes in Lolium perenne. This study is vital for understanding the mechanism of senescence and also for practical purposes such as improvement of productivity, silage and hay quality and stress tolerance. A linkage map of an F2 Lolium perenne population derived from a cross between genotypes of the cultivars Aurora and Perma was developed by IBERS consisting of seven linkage groups. This same population was used for the characterisation of leaf senescence related traits. Four different phenotyping activities were conducted, namely: (1) detached leaf senescence under natural light; (2) dark-induced senescence of detached leaves; (3) attached leaves grown in a controlled environment and (4) attached leaves grown under unheated green-house conditions with natural light.

Leaf elongation rate (LER) is a key component of forage grass dry matter yield. Leaf lamina initiate rapid expansion during the winter at a time that coincides with lengthening days but this extension is not associated with any large environmental shifts such as an increase in light intensity or temperature. The rate increase precedes any anatomical indication of floral initiation in apical meristems (double ridge formation) by around 2–3 weeks. We measured LER in a perennial ryegrass (Lolium perenne L.) pair-cross mapping family that has been extensively mapped with more than 1,200 SNP markers generated by Illumina’s Infinium platform. These have been anchored to the seven Lolium linkage groups as defined by Triticeae nomenclature with seven micro-satellite markers originally developed by ViaLactia Biosciences. Measurements were made in the winter/spring during extending day length, after full natural vernalisation. The plants’ leaves were beginning to expand rapidly with leaf extension rates varying between approximately 0.4 mm and 1.1 mm h−1. We obtained a significant correlation between LER and both apical meristem elongation and ear emergence (the date at which inflorescences emerge above the flag-leaf ligule) measured in two separate seasons. A QTL for LER was revealed with a peak some distance away from the major floral initiation and ear emergence date QTL on linkage group 7. This is the first evidence of genetic linkage that determines the often observed association between LER in herbage grasses in the spring with floral initiation determined in large part by the action of Arabidopsis flowering initiation gene homologues, Flowering Time Locus (FT) and Constans (CO).

Self-incompatibility (SI) is a mechanism that prevents plants from self-pollination through molecular recognition between pollen and pistil; but neither the genes nor the proteins involved are known. Determining haplotype diversity of the SI locus region is an indirect way of predicting incompatibility genotype which would enable plant breeders to develop strategies to ensure precision crossing for exploiting heterotic combinations. In the case of ryegrass (Lolium perenne), SI is known to be a gametophytic reaction involving two loci, S and Z. This paper reports on an assay of marker allele diversity around the Z locus for a population produced from twelve generations of mixed and half-sib family selection. Using conventional genotyping of marker length polymorphisms, as well as high resolution melt (HRM) curve analysis, the 55 plants of the population were classified into thirteen groups according to their genotypes for each marker. HRM genotyping proved to be more discriminating than STS markers and could enhance the precision of Z genotype prediction. Although half-sib family selection is expected to lead to allele fixing, this initial analysis was encouraging in that it indicated high haplotype diversity around the Z-locus suggesting maintenance of a high degree of cross-compatibility in the breeding population. This would be expected as SI alleles are subjected to frequency dependent selection.

The Arabidopsis FLOWERING LOCUS T (FT) gene and its orthologues in other plant species (e.g. rice OsFTL2/Hd3a) have an established role in the photoperiodic induction to flowering response. The genomic and phenotypic variation associated with the Lolium perenne (perennial ryegrass) orthologue of FT, designated LpFT3, was assessed in a diverse collection of 9 European germplasm populations, which together comprised an association panel of 864 plants. Sequencing and genotyping of a series of amplicons derived from the 9 populations, containing the complete exon and intron sequences as well as 5' and 3' non-coding sequences of LpFT3 identified a total of 7 haplotypes. Genotyping assays designed to detect the genomic variation showed that 3 haplotypes were present in approximately equal proportions and represented 84% of the total, with a fourth representing a further 11%. Of the 3 major haplotypes, 2 were predicted to code for identical protein products and the third contained two amino acid substitutions. Association analysis using either a mixed model with a relationship matrix to correct for population structure and relatedness, or structured association with further correction using genomic control indicated significant associations between LpFT3 and variation in flowering time. These associations were corroborated in a validation population segregating for the same major alleles. The most 'diagnostic' region of genomic variation was situated 5' of the coding sequence. Analysis of this region identified that the inter-haplotype variation was closely associated with sequence motifs which were apparently conserved in the 5' region of orthologues of LpFT3 from other plant species. These may represent cis-regulatory elements involved in influencing the expression of this gene. The Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) gene and its orthologs in other plant species (e.g. rice [Oryza sativa] OsFTL2/Hd3a) have an established role in the photoperiodic induction of flowering response. The genomic and phenotypic variations associated with the perennial ryegrass (Lolium perenne) ortholog of FT, designated LpFT3, was assessed in a diverse collection of nine European germplasm populations, which together constituted an association panel of 864 plants. Sequencing and genotyping of a series of amplicons derived from the nine populations, containing the complete exon and intron sequences as well as 5' and 3' noncoding sequences of LpFT3, identified a total of seven haplotypes. Genotyping assays designed to detect the genomic variation showed that three haplotypes were present in approximately equal proportions and represented 84% of the total, with a fourth representing a further 11%. Of the three major haplotypes, two were predicted to code for identical protein products and the third contained two amino acid substitutions. Association analysis using either a mixed model with a relationship matrix to correct for population structure and relatedness or structured association with further correction using genomic control indicated significant associations between LpFT3 and variation in flowering time. These associations were corroborated in a validation population segregating for the same major alleles. The most "diagnostic" region of genomic variation was situated 5' of the coding sequence. Analysis of this region identified that the interhaplotype variation was closely associated with sequence motifs that were apparently conserved in the 5' region of orthologs of LpFT3 from other plant species. These may represent cis-regulatory elements involved in influencing the expression of this gene

The vast range of natural adaptation and wide distribution of the grass family, together with an inherent affinity with open landscapes has naturally led to a capability and desire to improve this natural resource to be incorporated into, and optimised in, the lifestyles of urban-dwelling man.

The segregation of self-compatibility from L. temulentum was examined in backcross generations derived from hybrids between this species and the self-incompatible species, L. perenne and L. multiflorum, the latter being used as recurrent parents. Segregation patterns for self-compatibility were determined by percentage self seed set and by in-vitro self-pollination scores in the resulting backcross generations. Self-compatibility in L. temulentum is controlled in a gametophytic manner by a single gene mutation of either the Z locus or a locus tightly linked to it. Self-compatibility can be fixed in the homozygous form (i.e. 100 per cent pollen-tube growth on selfed stigmas) by selfing half compatible backcross plants. The S locus is still present in L. temulentum and functions when combined with a functional Z locus derived from either L. perenne or L. multiflorum. This has evolutionary significance for the relationship of the self-compatible and -incompatible Lolium species.

The segregation of interspecific recombinant Festuca pratensis (Fp) alleles, introgressed into the germolasm of Lolium multiflorum (Lm), at four loci (PGI/2, AcP/2, GOT/3 and BAP) is described. Heterozygous (Lm/Fp) plants were backcrossed to L. multiflorum (2n=2×=14) and subsequent BC2 Lm/Fp sibling genotypes intercrossed. In crosses between BC1 heterozygous plants (Lm/Fp) used as males and L. multiflorum, there was a reduced transmission of the F. pratensis (Donor Parent) derived alleles in the populations with PGI/2 and AcP/2 marker loci compared to the reciprocal cross but the reduction was not significantly different in those with GOT/3 and BAP markers. Two PGI/2 marked BC2 half-sib families in particular exhibited a more extreme deficiency of Fp/Fp progeny plants than anticipated from the BC2 segregations indicating possible linkage to zygotic lethals. Deficiencies of F. pratensis alleles were, in most cases, less marked in BC2 half-sib families indicating that a further round of recombination had reduced the size of the introgressed chromosome segment or that deleterious linkages had been broken. A tendency towards heterozygote advantage was found in one BAP marked halfsib family. The significance for forage grass breeding of reduced transmission rates of Donor Parent alleles in early back-cross generations especially through the male gametes is discussed.

The maximum temperature (34°C) at which perennial ryegrass plants from one breeding population successfully shed pollen was determined and the seed set of ten clones taken from the same population and self-pollinated both in an unheated glasshouse and at a constant 34°C during anthesis was compared. Seed set by selfing of all ten clones was markedly increased by heat treatment, from a mean of 2.3% to 30.7%. Heat treatment of anthers, but not stigmas, greatly enhanced pollen tube growth on stigmas from the same genotype. Germination of progeny was not affected by the heat treatment. Heat treatment of ten clones from a breeding population with contrasting morphology and growth rate was not so successful. Only one clone shed pollen freely at 34°C and showed a marked improvement in subsequent seed set. The advantages to the breeder of routinely producing full-sib families of elite perennial ryegrass parents by selfing are outlined.

Differences were found in the expression of resistance of perennial ryegrass, Lolium perenne, cultivars to crown rust, Puccinia coronata Corda, when plants were incubated at either 10°C or 25°C. In an extreme example, all the genotypes of ‘Carrick’ were resistant at 10°C but susceptible at 25°C. The variation between cultivars was due both to a general increase in expression of susceptibility and the proportion of plants which became susceptible. A BC1 line containing a stay-green gene derived from Festuca pratensis was insensitive to temperature, while a sister line was sensitive.

The segregating generations derived from a cross of a self-compatible with an incompatible inbred line of Lolium perenne were analysed for the incompatibility reaction as revealed by the pollen/stigma fluorescence test. All F1 plants showed a half-compatible reaction whilst segregation occurred in the F2 into half- and fully-self-compatible classes. The latter plants bred true in the F3 whilst the former once again showed the same pattern of segregation. These results indicate that the control of self-compatibility is by a single gene (Sc) which is gametophytic in action. Further analysis of an intra-F2 pollination diallel and of reciprocal cross-pollinations between F2 and F1 plants reveal that this gene is additional to the SZ incompatibility system.

The genetic and physiological mechanisms involved in limiting self-fertilization in angiosperms, referred to as self-incompatibility (SI), have significant effects on population structure and have potential diversification and evolutionary consequences. Up to now, details of the underlying genetic control and physiological basis of SI have been elucidated in two different gametophytic SI (GSI) systems, the S-RNase SI and the Papaver SI systems, and the sporophytic SI (SSI) system (Brassica). In the grass family (Poaceae), which contains all the cereal and major forage crops, SI has been known for half a century to be controlled gametophytically by two multiallelic and independent loci, S and Z. But still none of the gene products for S and Z is known and only limited information on related biochemical responses is available. Here we compare current knowledge of grass SI with that of other well-characterized SI systems and speculate about the relationship between SSI and grass SI. Additionally, we discuss comparative mapping as a tool for the further investigation of grass SI.

Ryegrasses and fescues of the Lolium/Festuca complex form the basis of many temperate grassland agricultural and turf systems and the ability to manipulate flowering and fertility within these grasses is of considerable importance. Genetic studies can identify multiple quantitative trait loci affecting these traits which can be used in the development of marker-assisted selection protocols for combining favourable alleles. By applying a knowledge of plant comparative genetics and genomics, it is possible to increase the information content of these studies by ‘cross-referencing’ between the Lolium/Festuca grasses and other crop and model species (e.g. rice, Triticeae cereals, Arabidopsis). Syntenic genomic regions, new genetic markers and candidate genes can, thus, be identified.

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.

Perennial ryegrass (Lolium perenne L.) is an outcrossing, wind-pollinated species exhibiting a gametophytic two-locus system of self-incompatibility (S and Z). The two incompatibility loci were genotyped in a cross between a doubled-haploid plant crossed as the female parent with a normal heterozygous plant. The S and Z loci were found to segregate in the expected 1:1 ratio and also segregated independently. The two loci were mapped to linkage groups one and two respectively, in accordance with the Triticeae consensus map. In addition, there were notable associations between the segregation of particular alleles mapping to the S locus region of linkage group 1 and those mapping to the WG889/CDO920 loci region of linkage group 3 which resulted in significant segregation distortions. No such associations were found between the Z locus and this region or any other region of the genome. The L. perenne S and Z loci showed conserved synteny with the equivalent loci in rye (Secale cereale L.).