Ian P. Armstead

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.

Alignment of the Genomes of Brachypodium distachyon and Temperate Cereals and Grasses Using Bacterial Artificial Chromosome Landing With Fluorescence in Situ Hybridization

As part of an initiative to develop Brachypodium distachyon as a genomic “bridge” species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice.

Synteny between a major heading-date QTL in perennial ryegrass (Lolium perenne L.) and the Hd3 heading-date locus in rice

The genetic control of induction to flowering has been studied extensively in both model and crop species because of its fundamental biological and economic significance. An ultimate aim of many of these studies has been the application of the understanding of control of flowering that can be gained from the study of model species, to the improvement of crop species. The present study identifies a region of genetic synteny between rice and Lolium perenne, which contains the Hd3 heading-date QTL in rice and a major QTL, accounting for up to 70% of the variance associated with heading date in L. perenne. The identification of synteny between rice and L. perenne in this region demonstrates the direct applicability of the rice genome to the understanding of biological processes in other species. Specifically, this syntenic relationship will greatly facilitate the genetic dissection of aspects of heading-date induction by enabling the magnitude of the genetic component of the heading-date QTL in L. perenne to be combined with the sequencing and annotation information from the rice genome.

Genetic analysis of adaptation differences between highland and lowland tropical maize using molecular markers

Abstract Molecular-marker loci were used to investigate the adaptation differences between highland and lowland tropical maize. An F2 population from the cross of two inbred lines independently derived from highland and lowland maize germplasm was developed, and extracted F3:4 lines were phenotype in replicated field trials at four thermally diverse tropical testing sites, ranging from lowland to extreme highland (mean growing season temperature range 13.2–24.6°C). Traits closely related with adaptation, such as biomass and grain yield, yield components, days from sowing to male and female flowering, total leaf number, plant height and number of primary tassel branches (TBN), were analyzed. A large line × environment interaction was observed for most traits. The genetic basis of this interaction was reflected by significant, but systematic, changes from lowland to highland sites in the correlation between the trait value and genomic composition (designated by the proportion of marker alleles with the same origin). Joint analysis of quantitative trait loci (QTLs) over sites detected 5–8 QTLs for each trait (except disease scores, with data only from one site). With the exception of one QTL for TBN, none of these accounted for more than 15% of the total phenotypic variation. In total, detected QTLs accounted for 24–61% of the variation at each site on average. For yield, yield components and disease scores, alleles generally favored the site of origin. Highland-derived alleles had little effect at lowland sites, while lowland-derived alleles showed relatively broader adaptation. Gradual changes in the estimated QTL effects with increasing mean site temperature were observed, and paralleled the observed patterns of adaptation in highland and lowland germplasm. Several clusters of QTLs for different traits reflected the relative importance in the adaptation differences between the two germplasm types, and pleiotropy is suggested as the main cause for the clustering. Breeding for broad thermal adaptation should be possible by pooling genes showing adaptation to specific thermal regimes, though perhaps at the expense of reduced progress for adaptation to a specific site. Molecular marker-assisted selection would be an ideal tool for this task, since it could greatly reduce the linkage drag caused by the unintentional transfer of undesirable traits.

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.).

A synteny-based draft genome sequence of the forage grass Lolium perenne.

Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species that is widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28,455 gene models, and we utilized macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family, to establish a synteny-based linear gene order. The gametophytic self-incompatibility mechanism enables the pistil of a plant to reject self-pollen and therefore promote out-crossing. We have used the sequence assembly to characterize transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterization of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and are potentially involved in the self-incompatibility mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of new varieties for more productive grasslands.

Introgression mapping in the grasses. I. Introgression of Festuca pratensis chromosomes and chromosome segments into Lolium perenne

Lolium perenne (4x)/ Festuca pratensis (2x) triploid hybrids (2n=3x=21) were produced and backcrossed to Lolium perenne. The BC1 progeny, which predominantly had 14 chromosomes, were analysed using genomic in situ hybridization (GISH) and genetic markers. GISH revealed that over 74% of the BC1 individuals carried one or more F. pratensis chromosome segments. By comparing the physical size of introgressed F. pratensis chromosome segments with the presence or absence of F. pratensis-specific polymorphisms, it was possible to determine the physical position of genetic markers. The potential of a new type of genetic mapping (‘introgression mapping’) for the alignment of physical and genetic maps, determining the genetic control of agronomically important characters and the production of novel germplasm for the development of new varieties is discussed.

Integration of gene-based markers in a pearl millet genetic map for identification of candidate genes underlying drought tolerance quantitative trait loci

BackgroundIdentification of genes underlying drought tolerance (DT) quantitative trait loci (QTLs) will facilitate understanding of molecular mechanisms of drought tolerance, and also will accelerate genetic improvement of pearl millet through marker-assisted selection. We report a map based on genes with assigned functional roles in plant adaptation to drought and other abiotic stresses and demonstrate its use in identifying candidate genes underlying a major DT-QTL.ResultsSeventy five single nucleotide polymorphism (SNP) and conserved intron spanning primer (CISP) markers were developed from available expressed sequence tags (ESTs) using four genotypes, H 77/833-2, PRLT 2/89-33, ICMR 01029 and ICMR 01004, representing parents of two mapping populations. A total of 228 SNPs were obtained from 30.5 kb sequenced region resulting in a SNP frequency of 1/134 bp. The positions of major pearl millet linkage group (LG) 2 DT-QTLs (reported from crosses H 77/833-2 × PRLT 2/89-33 and 841B × 863B) were added to the present consensus function map which identified 18 genes, coding for PSI reaction center subunit III, PHYC, actin, alanine glyoxylate aminotransferase, uridylate kinase, acyl-CoA oxidase, dipeptidyl peptidase IV, MADS-box, serine/threonine protein kinase, ubiquitin conjugating enzyme, zinc finger C- × 8-C × 5-C × 3-H type, Hd3, acetyl CoA carboxylase, chlorophyll a/b binding protein, photolyase, protein phosphatase1 regulatory subunit SDS22 and two hypothetical proteins, co-mapping in this DT-QTL interval. Many of these candidate genes were found to have significant association with QTLs of grain yield, flowering time and leaf rolling under drought stress conditions.ConclusionsWe have exploited available pearl millet EST sequences to generate a mapped resource of seventy five new gene-based markers for pearl millet and demonstrated its use in identifying candidate genes underlying a major DT-QTL in this species. The reported gene-based markers represent an important resource for identification of candidate genes for other mapped abiotic stress QTLs in pearl millet. They also provide a resource for initiating association studies using candidate genes and also for comparing the structure and function of distantly related plant genomes such as other Poaceae members.

EST-derived SSR markers used as anchor loci for the construction of a consensus linkage map in ryegrass ( Lolium spp.)

BackgroundGenetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm.ResultsA set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2.ConclusionsThe consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.