Expressed sequence tags (ESTs) are important resources for gene discovery and molecular marker development. From over 147,000 ESTs of Medicago truncatula, we have identified 4,384 ESTs containing perfect simple sequence repeats (EST-SSR) of di-, tri-, tetra- or pentanucleotides. Six hundred sixteen primer pairs (PPs) were designed and screened over a panel of eight genotypes representing six Medicago spp. and subspecies. Nearly, 74% (455) of the PPs produced characteristic SSR bands of expected size length in at least one Medicago species. Four hundred six (89%) of these 455 PPs produced SSR bands in all eight genotypes tested. Only 17 PPs were M. truncatula -specific. High levels of polymorphism (>70%) were detected for these markers in alfalfa, M. truncatula, and other annual medics. About 48% of the reported markers are part of gene transcripts linked to putative functions. Our results indicate that the SSR markers developed from M. truncatula ESTs are valuable genetic markers for the Medicago genus. These markers will be useful in establishing the genomic relationships of M. truncatula to important forage legume crops such as alfalfa and other annual medics.
Simple sequence repeat (SSR) markers are highly informative and widely used for genetic and breeding studies. Currently, a very limited number of SSR markers are available for tall fescue (Festuca arundinacea Schreb.) and other forage grass species. A tall fescue genomic library enriched in (GA/CT)( n ) repeats was used to develop primer pairs (PPs) flanking SSRs and assess PP functionality across different forage, cereal, and turf grass species. A total of 511 PPs were developed and assessed for their utility in six different grass species. The parents and a subset of a tall fescue mapping population were used to select PPs for mapping in tall fescue. Survey results revealed that 48% (in rice) to 66% (in tall fescue) of the PPs produced clean SSR-type amplification products in different grass species. Polymorphism rates were higher in tall fescue (68%) compared to other species (46% ryegrass, 39% wheat, and 34% rice). A set of 194 SSR loci (38%) were identified which amplified across all six species. Loci segregating in the tall fescue mapping population were grouped as loci segregating from the female parent (HD28-56, 37%), the male parent (R43-64, 37%), and both parents (26%). Three percent of the loci that were polymorphic between parents were monomorphic in the pseudo F1 mapping population and the remaining loci segregated. Sequencing of amplified products obtained from PP NFFAG428 revealed a very high level of sequence similarity among the grass species under study. Our results are the first report of genomic SSR marker development from tall fescue and they demonstrate the usefulness of these SSRs for genetic linkage mapping in tall fescue and cross-species amplification.
Fescues (Festuca sp.) are major cool-season forage and turf grass species around the world. Heat stress is one of the limiting factors in the production of fescues as forage in the southern Great Plains of the US. Heat responsive gene transcripts were cloned by using suppression subtractive hybridization between a heat-tolerant and a heat-sensitive fescue genotype subjected to a slowly increased temperature mimicking the natural conditions. The temperature in the growth chamber containing the plants was gradually increased from 24 degrees C to 44 degrees C over a period of 2 weeks. Three subtractions were conducted between samples of the two genotypes collected after 12 h of exposure to 39, 42, and 44 degrees C. A total of 2495 ESTs were generated, of which 1800 clustered into 434 contigs and 656 were singlets. The putative functions of ESTs were predicted by BLASTX. Nearly 30% of the contigs and 39% of the singlets had no similarity to GenBank sequences. Differentially expressed genes selected by subtractions were classified into 10 broad categories according to their putative functions generated by BLAST analysis. Under heat-stress conditions, cell maintenance, chloroplast associated and photosynthesis-, protein synthesis-, signalling-, and transcription factor-related genes had higher expression levels in the heat-tolerant genotype. Genes related to metabolism and stress had higher expression in the heat-sensitive genotype. The expression of 17 selected gene transcripts were examined by RT-PCR using plant tissues of the two genotypes grown under heat stress and under optimal temperature conditions (24 degrees C) for fescue. Results from RT-PCR confirmed the differential expressions of these transcripts. The differential expressions of at least 11 of these genes were attributable to heat stress rather than to differences in the genetic backgrounds of the genotypes.
Tall fescue (Festuca arundinacea Schreb.) is commonly grown as forage and turf grass in the temperate regions of the world. Here, we report the first genetic map of tall fescue constructed with PCR-based markers. A combination of amplified fragment length polymorphisms (AFLPs) and expressed sequence tag-simple sequence repeats (EST-SSRs) of both tall fescue and those conserved in grass species was used for map construction. Genomic SSRs developed from Festuca x Lolium hybrids were also mapped. Two parental maps were initially constructed using a two-way pseudo-testcross mapping strategy. The female (HD28-56) map included 558 loci placed in 22 linkage groups (LGs) and covered 2,013 cM of the genome. In the male (R43-64) map, 579 loci were grouped in 22 LGs with a total map length of 1,722 cM. The marker density in the two maps varied from 3.61 cM (female parent) to 2.97 (male parent) cM per marker. These differences in map length indicated a reduced level of recombination in the male parent. Markers that revealed polymorphism within both parents and showed 3:1 segregation ratios were used as bridging loci to integrate the two parental maps as a bi-parental consensus. The integrated map covers 1,841 cM on 17 LGs, with an average of 54 loci per LG, and has an average marker density of 2.0 cM per marker. Homoeologous relationships among linkage groups of six of the seven predicted homeologous groups were identified. Three small groups from the HD28-56 map and four from the R43-64 map are yet to be integrated. Homoeologues of four of those groups were detected. Except for a few gaps, markers are well distributed throughout the genome. Clustering of those markers showing significant segregation distortion (23% of total) was observed in four of the LGs of the integrated map.
Background: Tall fescue (Festuca arundinacea Schreb) is a major cool season forage and turf grass species grown in the temperate regions of the world. In this paper we report the generation of a tall fescue expressed sequence tag (EST) database developed from nine cDNA libraries representing tissues from different plant organs, developmental stages, and abiotic stress factors. The results of inter-library and library-specific in silico expression analyses of these ESTs are also reported.
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