Noel Oi Cogan

Background: In crop species, QTL analysis is commonly used for identification of factors contributing to variation of agronomically important traits. As an important pasture species, a large number of QTLs have been reported for perennial ryegrass based on analysis of biparental mapping populations. Further characterisation of those QTLs is, however, essential for utilisation in varietal improvement programs. Results: A bibliographic survey of perennial ryegrass trait-dissection studies identified a total of 560 QTLs from previously published papers, of which 189, 270 and 101 were classified as morphology-, physiology- and resistance/tolerance-related loci, respectively. The collected dataset permitted a subsequent meta-QTL study and implementation of a cross-species candidate gene identification approach. A meta-QTL analysis based on use of the BioMercator software was performed to identify two consensus regions for pathogen resistance traits. Genes that are candidates for causal polymorphism underpinning perennial ryegrass QTLs were identified through in silico comparative mapping using rice databases, and 7 genes were assigned to the p150/112 reference map. Markers linked to the Lp DGL1, Lp Ph1 and Lp PIPK1 genes were located close to plant size, leaf extension time and heading date-related QTLs, respectively, suggesting that these genes may be functionally associated with important agronomic traits in perennial ryegrass. Conclusions: Functional markers are valuable for QTL meta-analysis and comparative genomics. Enrichment of such genetic markers may permit further detailed characterisation of QTLs. The outcomes of QTL meta-analysis and comparative genomics studies may be useful for accelerated development of novel perennial ryegrass cultivars with desirable traits.

Background: Single nucleotide polymorphisms (SNPs) provide essential tools for the advancement of research in plant genomics, and the development of SNP resources for many species has been accelerated by the capabilities of second-generation sequencing technologies. The current study aimed to develop and use a novel bioinformatic pipeline to generate a comprehensive collection of SNP markers within the agriculturally important pasture grass tall fescue; an outbreeding allopolyploid species displaying three distinct morphotypes: Continental, Mediterranean and rhizomatous. Results: A bioinformatic pipeline was developed that successfully identified SNPs within genotypes from distinct tall fescue morphotypes, following the sequencing of 414 polymerase chain reaction (PCR) – generated amplicons using 454 GS FLX technology. Equivalent amplicon sets were derived from representative genotypes of each morphotype, including six Continental, five Mediterranean and one rhizomatous. A total of 8,584 and 2,292 SNPs were identified with high confidence within the Continental and Mediterranean morphotypes respectively. The success of the bioinformatic approach was demonstrated through validation (at a rate of 70%) of a subset of 141 SNPs using both SNaPshot™ and GoldenGate™ assay chemistries. Furthermore, the quantitative genotyping capability of the GoldenGate™ assay revealed that approximately 30% of the putative SNPs were accessible to co-dominant scoring, despite the hexaploid genome structure. The sub-genome-specific origin of each SNP validated from Continental tall fescue was predicted using a phylogenetic approach based on comparison with orthologous sequences from predicted progenitor species. Conclusions: Using the appropriate bioinformatic approach, amplicon resequencing based on 454 GS FLX technology is an effective method for the identification of polymorphic SNPs within the genomes of Continental and Mediterranean tall fescue. The GoldenGate™ assay is capable of high-throughput co-dominant SNP allele detection, and minimises the problems associated with SNP genotyping in a polyploid by effectively reducing the complexity to a diploid system. This SNP collection may now be refined and used in applications such as cultivar identification, genetic linkage map construction, genome-wide association studies and genomic selection in tall fescue. The bioinformatic pipeline described here represents an effective general method for SNP discovery within outbreeding allopolyploid species.

Background: Field pea (Pisum sativum L.) and faba bean (Vicia faba L.) are cool-season grain legume species that provide rich sources of food for humans and fodder for livestock. To date, both species have been relative 'genomic orphans' due to limited availability of genetic and genomic information. A significant enrichment of genomic resources is consequently required in order to understand the genetic architecture of important agronomic traits, and to support germplasm enhancement, genetic diversity, population structure and demographic studies. Results: cDNA samples obtained from various tissue types of specific field pea and faba bean genotypes were sequenced using 454 Roche GS FLX Titanium technology. A total of 720,324 and 304,680 reads for field pea and faba bean, respectively, were de novo assembled to generate sets of 70,682 and 60,440 unigenes. Consensus sequences were compared against the genome of the model legume species Medicago truncatula Gaertn., as well as that of the more distantly related, but better-characterised genome of Arabidopsis thaliana L.. In comparison to M. truncatula coding sequences, 11,737 and 10,179 unique hits were obtained from field pea and faba bean. Totals of 22,057 field pea and 18,052 faba bean unigenes were subsequently annotated from GenBank. Comparison to the genome of soybean (Glycine max L.) resulted in 19,451 unique hits for field pea and 16,497 unique hits for faba bean, corresponding to c. 35% and 30% of the known gene space, respectively. Simple sequence repeat (SSR)-containing expressed sequence tags (ESTs) were identified from consensus sequences, and totals of 2,397 and 802 primer pairs were designed for field pea and faba bean. Subsets of 96 EST-SSR markers were screened for validation across modest panels of field pea and faba bean cultivars, as well as related non-domesticated species. For field pea, 86 primer pairs successfully obtained amplification products from one or more template genotypes, of which 59% revealed polymorphism between 6 genotypes. In the case of faba bean, 81 primer pairs displayed successful amplification, of which 48% detected polymorphism. Conclusions: The generation of EST datasets for field pea and faba bean has permitted effective unigene identification and functional sequence annotation. EST-SSR loci were detected at incidences of 14-17%, permitting design of comprehensive sets of primer pairs. The subsets from these primer pairs proved highly useful for polymorphism detection within Pisum and Vicia germplasm.

Background: Lentil (Lens culinaris Medik.) is a cool-season grain legume which provides a rich source of protein for human consumption. In terms of genomic resources, lentil is relatively underdeveloped, in comparison to other Fabaceae species, with limited available data. There is hence a significant need to enhance such resources in order to identify novel genes and alleles for molecular breeding to increase crop productivity and quality. Results: Tissue-specific cDNA samples from six distinct lentil genotypes were sequenced using Roche 454 GS-FLX Titanium technology, generating c. 1.38 × 10⁶ expressed sequence tags (ESTs). De novo assembly generated a total of 15,354 contigs and 68,715 singletons. The complete unigene set was sequence-analysed against genome drafts of the model legume species Medicago truncatula and Arabidopsis thaliana to identify 12,639, and 7,476 unique matches, respectively. When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space. A total of 25,592 lentil unigenes were subsequently annoated from GenBank. Simple sequence repeat (SSR)-containing ESTs were identified from consensus sequences and a total of 2,393 primer pairs were designed. A subset of 192 EST-SSR markers was screened for validation across a panel 12 cultivated lentil genotypes and one wild relative species. A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism. Conclusions: A substantial collection of ESTs has been developed from sequence analysis of lentil genotypes using second-generation technology, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

With the exception of the oyster herpesvirus OsHV-1, all herpesviruses characterized thus far infect only vertebrates. Some cause neurological disease in their hosts, while others replicate or become latent in neurological tissues. Recently a new herpesvirus causing ganglioneuritis in abalone, a gastropod, was discovered. Molecular analysis of new herpesviruses, such as this one and others, still to be discovered in invertebrates, will provide insight into the evolution of herpesviruses.

Background: The agriculturally important pasture grass tall fescue (Festuca arundinaceaSchreb. syn.Lolium arundinaceum(Schreb.) Darbysh.) is an outbreeding allohexaploid, that may be more accurately described as a species complex consisting of three major (Continental, Mediterranean and rhizomatous) morphotypes. Observation of hybrid infertility in some crossing combinations between morphotypes suggests the possibility of independent origins from different diploid progenitors. This study aims to clarify the evolutionary relationships between each tall fescue morphotype through phylogenetic analysis using two low-copy nuclear genes (encoding plastid acetyl-CoA carboxylase [Acc1] andcentroradialis[CEN]), the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) and the chloroplast DNA (cpDNA) genome-locatedmatKgene. Other taxa within the closely relatedLolium-Festucaspecies complex were also included in the study, to increase understanding of evolutionary processes in a taxonomic group characterised by multiple inter-specific hybridisation events.Results: Putative homoeologous sequences from both nuclear genes were obtained from each polyploid species and compared to counterparts from 15 diploid taxa. Phylogenetic reconstruction confirmedF. pratensisandF. arundinaceavar.glaucescensas probable progenitors to Continental tall fescue, and these species are also likely to be ancestral to the rhizomatous morphotype. However, these two morphotypes are sufficiently distinct to be located in separate clades based on the ITS-derived data set. All four of the generated data sets suggest independent evolution of the Mediterranean and Continental morphotypes, with minimal affinity between cognate sequence haplotypes. No obvious candidate progenitor species for Mediterranean tall fescues were identified, and only two putative sub-genome-specific haplotypes were identified for this morphotype.Conclusions: This study describes the first phylogenetic analysis of theFestucagenus to include representatives of each tall fescue morphotype, and to use low copy nuclear gene-derived sequences to identify putative progenitors of the polyploid species. The demonstration of distinct tall fescue lineages has implications for both taxonomy and molecular breeding strategies, and may facilitate the generation of morphotype and/or sub-genome-specific molecular markers.

Background: White clover (Trifolium repensL.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumesLotus japonicusandMedicago truncatulaas well asArabidopsis thalianawere also characterised.Results: A total of four paired homoeologous BACs were selected and sequenced to generate 173 kb of overlapping sequence between the O and P' sub-genomes. Equivalent gene content was generally observed, apart from small-scale deletions, in contrast to conservation of intergenic sequences, which varied between the four selected regions. Measurement of the number of synonymous substitutions between homoeologous genes led to estimation of a 4.2 million year divergence time between the two sub-genomes. Microsynteny was observed between the genomes of white clover andL. japonicusfor all four targeted regions, but correspondingM. truncatulagenomic regions were only identified for two BAC pairs.Conclusions: This study describes the first analysis of sub-genome structural conservation across selected genomic regions in white clover. Although the high levels of sequence conservation between the O and P' sub-genomes would complicate efforts for whole genome sequence assembly, the conserved microsynteny with model legume genomes, especially that ofL. japonicus, will be highly valuable for the future of white clover genomics and molecular breeding.

"QTL analysis of mineral content in perennial ryegrass (Lolium perenne L.)" published on 30 Oct 2005 by Wageningen Academic.