Daniel Thorogood

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.

Self-incompatibility in ryegrass 12. Genotyping and mapping the S and Z loci of Lolium perenne L.

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.).

How far are we from unravelling self‐incompatibility in grasses?

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.

Allelic Variation in the Perennial Ryegrass FLOWERING LOCUS T Gene Is Associated with Changes in Flowering Time across a Range of Populations

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.

Identifying genetic components controlling fertility in the outcrossing grass species perennial ryegrass (Lolium perenne) by quantitative trait loci analysis and comparative genetics

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.

Identification and mode of action of self-compatibility loci in Lolium perenne L

The two-locus gametophytic incompatibility system in perennial ryegrass (Lolium perenne L.) is not always fully effective: obligate selfing of plants sieves self-compatible pollen mutants, and self-fertility becomes fixed in subsequent generations. Self-compatibility (SC) was investigated in an F2 family. In vitro self-pollinations were analysed and recorded and plants were classified as being either partially or fully compatible. Distorted segregation ratios of markers on linkage group (LG) 5 were found, which indicate the possible presence of a gametophytic SC locus. Interval linkage analysis of pollen compatibility after selfing confirmed that this distortion was due to a locus (T) analogous to the S5 locus of rye. However, even though markers in this region were, on average, less than 1 cM apart, the minimum number of plants possessing the unfavoured allele was never less than 6% for any marker locus. We proved that this was because of the presence of another SC locus, exhibiting gametophytic selection, segregating in this population and identified by interval mapping analysis of compatibility classes of in vitro self-pollinations. This locus was located on LG1, and probably corresponds to the S locus. We show that the T locus, a relic of a multilocus system, functions through interaction with the S locus: F2 segregation of incompatibility phenotypes and linked markers demonstrated that the S/t pollen genotype combination, expected to be compatible on selfing, was sometimes incompatible. Further evidence is presented to show that this interaction must be dependent on yet another locus located on LG2. A prime candidate would be the Z incompatibility locus.

Progress towards elucidating the mechanisms of self-incompatibility in the grasses: further insights from studies in Lolium

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.

Development of a Genomic Microsatellite Library in Perennial Ryegrass (Lolium perenne) and its Use in Trait Mapping

BACKGROUND AND AIMS 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. METHODS 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. KEY RESULTS AND CONCLUSIONS 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.

The genetic control of self-compatibility in an inbred line of Lolium perenne L

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.

A Gene Encoding a DUF247 Domain Protein Cosegregates with the S Self-Incompatibility Locus in Perennial Ryegrass

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 multiallelic and independent loci, S and Z. Despite intense research efforts over the last six decades, the genes underlying S and Z remain uncharacterized. 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 10,177 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 cM. Of the eight genes cosegregating 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 gene showed a frameshift 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 toward 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.