De Novo Assembly and Characterization of Tall Fescue Transcriptome under Water Stress.

Water stress is a fundamental problem for tall fescue [Lolium arundinaceum (Schreb.) Darbysh.] cultivation in the south-central United States. Genetic improvement of tall fescue for water-stress tolerance is the key strategy for improving its persistence in the region. Genotypes with contrasting characteristics for relative water content and osmotic potential were identified from a tall fescue population. Transcriptome profiling between water-stress-tolerant (B400) and water-stress-susceptible (W279) genotypes was performed to unravel the genetic regulatory mechanism of water-stress responses in tall fescue. RNA samples from leaf, shoot, root, and inflorescence were pooled and sequenced through Illumina paired-end sequencing. A total of 199,399 contigs were assembled with an average length of 585 bp. Between the two genotypes, 2986 reference transcripts (RTs) were significantly differentially expressed and 1048 of them could be annotated and found to associate with metabolic pathways and enzyme coding genes. In total, 175 differentially expressed RTs were reported for various stress-related functions. Among those, 65 encoded kinase proteins, 40 each encoded transposons, and transporter proteins were previously reported to be involved with abiotic stress responses. A total of 6348 simple sequence repeats and 6658 single-nucleotide polymorphisms were identified in the contig sequences. Primers were developed from the corresponding sequences, which might be used as candidate gene markers in tall fescue. This study will lead to identification of genes or transcription factors related to water-stress tolerance and development of a comprehensive molecular marker system to facilitate marker-assisted breeding in tall fescue.

Mapping with RAD (restriction-site associated DNA) markers to rapidly identify QTL for stem rust resistance in Lolium perenne.

A mapping population was created to detect quantitative trait loci (QTL) for resistance to stem rust caused by Puccinia graminis subsp. graminicola in Lolium perenne. A susceptible and a resistant plant were crossed to produce a pseudo-testcross population of 193 F(1) individuals. Markers were produced by the restriction-site associated DNA (RAD) process, which uses massively parallel and multiplexed sequencing of reduced-representation libraries. Additional simple sequence repeat (SSR) and sequence-tagged site (STS) markers were combined with the RAD markers to produce maps for the female (738 cM) and male (721 cM) parents. Stem rust phenotypes (number of pustules per plant) were determined in replicated greenhouse trials by inoculation with a field-collected, genetically heterogeneous population of urediniospores. The F(1) progeny displayed continuous distribution of phenotypes and transgressive segregation. We detected three resistance QTL. The most prominent QTL (qLpPg1) is located near 41 cM on linkage group (LG) 7 with a 2-LOD interval of 8 cM, and accounts for 30-38% of the stem rust phenotypic variance. QTL were detected also on LG1 (qLpPg2) and LG6 (qLpPg3), each accounting for approximately 10% of phenotypic variance. Alleles of loci closely linked to these QTL originated from the resistant parent for qLpPg1 and from both parents for qLpPg2 and qLpPg3. Observed quantitative nature of the resistance may be due to partial-resistance effects against all pathogen genotypes, or qualitative effects completely preventing infection by only some genotypes in the genetically mixed inoculum. RAD markers facilitated rapid construction of new genetic maps in this outcrossing species and will enable development of sequence-based markers linked to stem rust resistance in L. perenne.

Genetic linkage mapping of an annual x perennial ryegrass population.

Annual (Lolium multiflorum Lam.) and perennial ( L. perenne L.) ryegrass are two common forage and turfgrass species grown throughout the world. Perennial ryegrass is most commonly used for turfgrass purposes, and contamination by annual ryegrass, through physical seed mixing or gene flow, can result in a significant reduction in turfgrass quality. Seed certifying agencies in the United States currently use a test called seedling root fluorescence (SRF) to detect contamination between these species. The SRF test, however, can be inaccurate and therefore, the development of additional markers for species separation is needed. Male and female molecular-marker linkage maps of an interspecific annual x perennial ryegrass mapping population were developed to determine the map location of the SRF character and to identify additional genomic regions useful for species separation. A total of 235 AFLP markers, 81 RAPD markers, 16 comparative grass RFLPs, 106 SSR markers, 2 isozyme loci and 2 morphological characteristics, 8-h flowering, and SRF were used to construct the maps. RFLP markers from oat and barley and SSR markers from tall fescue and other grasses allowed the linkage groups to be numbered, relative to the Triticeae and the International Lolium Genome Initative reference population P150/112. The three-generation population structure allowed both male and female maps to be constructed. The male and female maps each have seven linkage groups, but differ in map length with the male map being 537 cm long and the female map 712 cm long. Regions of skewed segregation were identified in both maps with linkage groups 1, 3, and 6 of the male map showing the highest percentage of skewed markers. The (SRF) character mapped to linkage group 1 in both the male and female maps, and the 8-h flowering character was also localized to this linkage group on the female map. In addition, the Sod-1 isozyme marker, which can separate annual and perennial ryegrasses, mapped to linkage group 7. These results indicate that Lolium linkage groups 1 and 7 may provide additional markers and candidate genes for use in ryegrass species separation.