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.

Bermudagrass is the most important warm-season pasture in the Southern USA with exceptional forage production potential and abiotic stress tolerance. However, it requires high nitrogen (N) supply to reach its full biomass and quality potential. Our objectives were to: (1) develop a nitrogen use efficiency (NUE) screening protocol for bermudagrass in controlled or semi-controlled conditions, (2) identify contrasting genotypes for NUE from natural variants and, (3) develop a knowledge base of NUE in bermudagrass. A collection consisting 290 Cynodon sp. genotypes was first pre-screened in the greenhouse. Thirty-nine genotypes with high NUE, five with low NUE were selected for further evaluations along with 5 checks in greenhouse and hoophouse under four N rates. Biomass, crude protein and N content were evaluated. N uptake efficiency (NUpE), N utilization efficiency (NUtE) and NUE were calculated based on biomass production. Genotypes showed significant influences (P \ 0.0001) in all of the response variables. The genotype 9 N rate interaction was not significant for NUE in both environments. NUE had strong correlation with biomass production and NUpE, which got stronger as N rates increased. In N limiting conditions, bermudagrass showed a trade-off between biomass maintenance and crude protein content. Lower N applications increased biomass production over crude protein. However, when N is abundant the crop has the ability to improve crude protein. Several genotypes presented high NUE due their high NUtE and NUpE. Genotypes with contrasting NUE were selected and subjected to further field evaluation. Superior genotypes for NUE will be used in the breeding program to enhance NUE in bermudagrass.

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.

Temperate grasses belonging to the Festuca-Lolium complex are important throughout the world in pasture and grassland agriculture. Tall fescue (Festuca arundinacea Schreb.) is the predominant species in the United States, covering approximately 15 million ha. Tall fescue has distinctive morphotypes, two of which are Continental (summer active) and Mediterranean (summer semidormant). This is the first report of a linkage map created for Mediterranean tall fescue, while updating the Continental map with additional simple sequence repeat and sequence-tagged site markers. Addition ally, this is the first time that diversity arrays technology (DArT) markers were used in the construction of a tall fescue map. The male parent (Continental), R43-64, map consisted of 594 markers arranged in 22 linkage groups (LGs) and covered a total of 1577 cM. The female parent (Mediterranean), 103-2, map was shorter (1258 cM) and consisted of only 208 markers arranged in 29 LGs. Marker densities for R43-64 and 103-2 were 2.65 and 6.08 cM per marker, respectively. When compared with the other Poaceae species, meadow fescue (F. pratensis Huds.), annual ryegrass (L. multiflorum Lam.), perennial ryegrass (L. perenne L.), Brachypodium distachyon (L.) Beauv., and barley (Hordeum vulgare L.), a total of 171 and 98 orthologous or homologous sequences, identified by DArT analysis, were identified in R43-64 and 103-2, respectively. By using genomic in situ hybridization, we aimed to identify potential progenitors of both morphotypes. However, no clear conclusion on genomic constitution was reached. These maps will aid in the search for quantitative trait loci of various traits as well as help define and distinguish genetic differences between the two morphotypes.T all fescue (Festuca arundinacea Schreb.) is a member of the Poaceae family and is closely related to the ryegrasses (Lolium spp.). It has been proposed that the Festuca subgenus Schedonorus should be merged with the Lolium genus (Darbyshire, 1993). However, it is clear that Lolium and Festuca fall into discrete taxa based on morphological traits as well as genetic differences (Köl-liker et al., 1999;Catalan et al., 2004;Mian et al., 2005;Kopecký et al., 2009a). Tall fescue is a cross-pollinated species possessing high levels of heterozygosity. Nearly all progeny are genetically unique, and substantial amounts of genetic variation occur both within (71%) and between (29%) populations (Cuyeu et al., 2013). Tall fescue, an allohexaploid (2n = 6x = 42), is part of a larger group of related plants that range from diploid (e.g., F. pratensis Huds.; 2n = 2x = 14) to decaploid (F. arundinacea subsp. cirtensis (St. Yves) J. Gamisans and var.

Jatropha curcas L. is cited as one of the best candidates for future oil and biodiesel production. It is widespread in many tropical and subtropical countries but has not yet received much genetic improvement. The objective of this study was to collect Jatropha germplasm and characterize it with molecular markers. A total of 64 genotypes, collected from seven geographic locations on two continents, were analyzed with 32 simple sequence repeat and two candidate gene-specific primers (ISPJ-1 gene and Curcin-P2 gene promoter). In general, markers were found to be highly conserved, and many (40%) were monomorphic in the studied populations. Polymorphic primers, which amplified population-specific fragments, were identified. The polymorphic information content of the polymorphic markers ranged from 0.03 to 0.47. Genetic similarity analysis identified two distinct groups at 0.73 DICE similarity coefficient. Group I included germplasm collected from the islands of Cuba and Cape Verde, and group II consisted of Brazil, Mozambique, and Senegal populations. Island genotypes were found to be very distinct compared to their mainland counterparts. Sequencing of monomorphic fragments identified single nucleotide polymorphism (SNP) between these two groups. Highresolution melting analysis of the SNP in the Jcps9 locus further confirmed the two gene pools. Sequencing of polymorphic fragments of the Jc03 locus identified a deletion in a (GT) 4 repeat motif in the genotypes in group II. Several population-specific microsatellites and SNP markers have been recognized. The distinct Jatropha genotypes and the population-specific molecular markers identified in this study will be valuable resources in breeding programs.