Use of genome sequence data in the design and testing of SSR markers for Phytophthora species

<p>Abstract</p> <p>Background</p> <p>Microsatellites or single sequence repeats (SSRs) are a powerful choice of marker in the study of <it>Phytophthora </it>population biology, epidemiology, ecology, genetics and evolution. A strategy was tested in which the publicly available unigene datasets extracted from genome sequences of <it>P. infestans</it>, <it>P. sojae </it>and <it>P. ramorum </it>were mined for candidate SSR markers that could be applied to a wide range of <it>Phytophthora </it>species.</p> <p>Results</p> <p>A first approach, aimed at the identification of polymorphic SSR loci common to many <it>Phytophthora </it>species, yielded 171 reliable sequences containing 211 SSRs. Microsatellites were identified from 16 target species representing the breadth of diversity across the genus. Repeat number ranged from 3 to 16 with most having seven repeats or less and four being the most commonly found. Trinucleotide repeats such as (AAG)n, (AGG)n and (AGC)n were the most common followed by pentanucleotide, tetranucleotide and dinucleotide repeats. A second approach was aimed at the identification of useful loci common to a restricted number of species more closely related to <it>P. sojae </it>(<it>P. alni, P. cambivora, P. europaea </it>and <it>P. fragariae</it>). This analysis yielded 10 trinucleotide and 2 tetranucleotide SSRs which were repeated 4, 5 or 6 times.</p> <p>Conclusion</p> <p>Key studies on inter- and intra-specific variation of selected microsatellites remain. Despite the screening of conserved gene coding regions, the sequence diversity between species was high and the identification of useful SSR loci applicable to anything other than the most closely related pairs of <it>Phytophthora </it>species was challenging. That said, many novel SSR loci for species other than the three 'source species' (<it>P. infestans</it>, <it>P. sojae </it>and <it>P. ramorum</it>) are reported, offering great potential for the investigation of <it>Phytophthora </it>populations. In addition to the presence of microsatellites, many of the amplified regions may represent useful molecular marker regions for other studies as they are highly variable and easily amplifiable from different <it>Phytophthora </it>species.</p

Single-feature polymorphism discovery in the barley transcriptome

A probe-level model for analysis of GeneChip gene-expression data is presented which identified more than 10,000 single-feature polymorphisms (SFP) between two barley genotypes. The method has good sensitivity, as 67% of known single-nucleotide polymorphisms (SNP) were called as SFPs. This method is applicable to all oligonucleotide microarray data, accounts for SNP effects in gene-expression data and represents an efficient and versatile approach for highly parallel marker identification in large genomes

Identification, utilisation and mapping of novel transcriptome-based markers from blackcurrant (Ribes nigrum)

<p>Abstract</p> <p>Background</p> <p>Deep-level second generation sequencing (2GS) technologies are now being applied to non-model species as a viable and favourable alternative to Sanger sequencing. Large-scale SNP discovery was undertaken in blackcurrant (<it>Ribes nigrum </it>L.) using transcriptome-based 2GS 454 sequencing on the parental genotypes of a reference mapping population, to generate large numbers of novel markers for the construction of a high-density linkage map.</p> <p>Results</p> <p>Over 700,000 reads were produced, from which a total of 7,000 SNPs were found. A subset of polymorphic SNPs was selected to develop a 384-SNP OPA assay using the Illumina BeadXpress platform. Additionally, the data enabled identification of 3,000 novel EST-SSRs. The selected SNPs and SSRs were validated across diverse <it>Ribes </it>germplasm, including mapping populations and other selected <it>Ribes </it>species.</p> <p>SNP-based maps were developed from two blackcurrant mapping populations, incorporating 48% and 27% of assayed SNPs respectively. A relatively high proportion of visually monomorphic SNPs were investigated further by quantitative trait mapping of theta score outputs from BeadStudio analysis, and this enabled additional SNPs to be placed on the two maps.</p> <p>Conclusions</p> <p>The use of 2GS technology for the development of markers is superior to previously described methods, in both numbers of markers and biological informativeness of those markers. Whilst the numbers of reads and assembled contigs were comparable to similar sized studies of other non-model species, here a high proportion of novel genes were discovered across a wide range of putative function and localisation. The potential utility of markers developed using the 2GS approach in downstream breeding applications is discussed.</p

tropiTree:an NGS-based EST-SSR resource for 24 tropical tree species

The development of genetic tools for non-model organisms has been hampered by cost, but advances in next-generation sequencing (NGS) have created new opportunities. In ecological research, this raises the prospect for developing molecular markers to simultaneously study important genetic processes such as gene flow in multiple non-model plant species within complex natural and anthropogenic landscapes. Here, we report the use of bar-coded multiplexed paired-end Illumina NGS for the de novo development of expressed sequence tag-derived simple sequence repeat (EST-SSR) markers at low cost for a range of 24 tree species. Each chosen tree species is important in complex tropical agroforestry systems where little is currently known about many genetic processes. An average of more than 5,000 EST-SSRs was identified for each of the 24 sequenced species, whereas prior to analysis 20 of the species had fewer than 100 nucleotide sequence citations. To make results available to potential users in a suitable format, we have developed an open-access, interactive online database, tropiTree (http://bioinf.hutton.ac.uk/tropiTree), which has a range of visualisation and search facilities, and which is a model for the efficient presentation and application of NGS data

An eQTL Analysis of Partial Resistance to Puccinia hordei in Barley

Background - Genetic resistance to barley leaf rust caused by Puccinia hordei involves both R genes and quantitative trait loci. The R genes provide higher but less durable resistance than the quantitative trait loci. Consequently, exploring quantitative or partial resistance has become a favorable alternative for controlling disease. Four quantitative trait loci for partial resistance to leaf rust have been identified in the doubled haploid Steptoe (St)/Morex (Mx) mapping population. Further investigations are required to study the molecular mechanisms underpinning partial resistance and ultimately identify the causal genes.Methodology/Principal Findings - We explored partial resistance to barley leaf rust using a genetical genomics approach. We recorded RNA transcript abundance corresponding to each probe on a 15K Agilent custom barley microarray in seedlings from St and Mx and 144 doubled haploid lines of the St/Mx population. A total of 1154 and 1037 genes were, respectively, identified as being P. hordei-responsive among the St and Mx and differentially expressed between P. hordei-infected St and Mx. Normalized ratios from 72 distant-pair hybridisations were used to map the genetic determinants of variation in transcript abundance by expression quantitative trait locus (eQTL) mapping generating 15685 eQTL from 9557 genes. Correlation analysis identified 128 genes that were correlated with resistance, of which 89 had eQTL co-locating with the phenotypic quantitative trait loci (pQTL). Transcript abundance in the parents and conservation of synteny with rice allowed us to prioritise six genes as candidates for Rphq11, the pQTL of largest effect, and highlight one, a phospholipid hydroperoxide glutathione peroxidase (HvPHGPx) for detailed analysis.Conclusions/Significance - The eQTL approach yielded information that led to the identification of strong candidate genes underlying pQTL for resistance to leaf rust in barley and on the general pathogen response pathway. The dataset will facilitate a systems appraisal of this host-pathogen interaction and, potentially, for other traits measured in this populatio