Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information

Abstract

DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids.Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level.Chromosome mapping of these markers using a Lolium/Festuca substitution line 3 revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding. Keywordsintergeneric (interspecific) hybrid -intron polymorphismsinsertion-deletion (indel) -specificity to species -rice genome -comparative genomics

Targeted mapping of rice ESTs to the LmPi1 locus for grey leaf spot resistance in Italian ryegrass

“…Whole-genome sequencing of the model plants A. thaliana (The Arabidopsis Genome Initiative 2000) and rice (International Rice Genome Sequencing (2) intron-scanning primers are designed from conserved predicted exon sequences; and (3) polymorphisms in intron sequences, such as insertiondeletion (indel) and single nucleotide polymorphism (SNP), are detected by agarose gel electrophoresis, cleaved amplified polymorphic sequences (CAPS) analysis, or SSCP analysis (Bertin et al 2005;Feltus et al 2006;Tamura et al 2009). Thanks to a high frequency of polymorphism in the intron sequences and high levels of conservation of the exon sequences and exon/intron structures of genes among plant species, this strategy has proved successful and should be adaptable to many non-model plants.…”

The Perennial Ryegrass GenomeZipper: Targeted Use of Genome Resources for Comparative Grass Genomics

“…An excellent example of this is the rice genome, which has proven to be a valuable resource for comparative studies in many grass species at both the gene and genome levels (Sorrells et al, 2003;Stein et al, 2007;Hackauf et al, 2009;Tamura et al, 2009). More recently, the genome sequence of Brachypodium, a member of the Pooideae subfamily, has become available and provides great promise to become a powerful model system to study the genomes of economically more important pooid grasses, including wheat, barley, oat (Avena sativa), rye (Secale cereale), as well as forage and turf grass species (International Brachypodium Initiative, 2010).…”

A molecular marker approach using intron flanking EST-PCR to map candidate genes in peach (Prunus persica)

“…As previously has been described, the intron flanking approach showed to be a fast and efficient methodology to design polymorphic markers (Wei et al 2005;De Keyser et al 2009;Sargent et al 2009;Tamura et al 2009). This approach takes advantage of the Arabidopsis reference genome to predict potential polymorphic sites within candidate genes from different species.…”

“…These types of markers are based on the prediction of putative splicing sites and the amplification of them. Considering the assumption that intronic regions are richer in polymorphism than exonic ones, an increment in the use of these markers as fingerprinting tool has been reported for non-model species such as Rhododendron (Wei et al 2005;De Keyser et al 2009), Lolium, Festuca (Tamura et al 2009) and Rosaceae species (Sargent et al 2009). Taking in consideration the functional association of these type of markers, it could be a good approach in order to search for Quantitative Trait Loci (QTL), which in P. persica has been described already for commercially interesting traits such as soluble sugar and organic acids content (Dirlewanger et al 1999), fruit weight, skin colour, total soluble solids, juice acidity and juice pH (Eduardo et al 2011).…”

“…This approach consists in a local BLAST-N alignment of the candidate genes against genomic sequences that includes introns and untranslated regions (UTRs) of Arabidopsis thaliana (genome version TAIR10 plus introns and UTRs downloaded from www.arabidopsis.org). Taking in consideration that predictions of putative splicing sites through a comparative analysis have been demonstrate to be highly accurate between A. thaliana and non model species (Wei et al 2005;De Keyser et al 2009;Sargent et al 2009;Tamura et al 2009) we decide to take this approach for our analysis. In order to design intron flanking PCR molecular markers which will be representative of each candidate gene, the consensus sequence of the assembled contigs obtained from our local database were aligned using BLAST-N against the tenth version of the annotated A. thaliana genome which includes introns and UTRs (http://www.arabidopsis.org/wublast/index2.jsp).…”