Detection of an efficient restriction enzyme combination for cDNA-AFLP analysis in Festuca mairei and evaluation of the identity of transcript-derived fragments.

In cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis, it is critical to choose a suitable pair of restriction enzymes for tagging sites in cDNA for amplification. Possibility of production of chimeric fragments from cDNA-AFLP analysis remains to be researched. The objectives of this study were to detect an efficient restriction enzyme combination for cDNA-AFLP analysis when Festuca species was used as template, and to evaluate the identity of transcript-derived fragments (TDFs) from cDNA-AFLP analysis. We found that NspI coupled TaqI was a pair of highly efficient enzymes by generating a much higher number of TDFs than the commonly used EcoRI and TaqI. This was the first study to apply NspI for AFLP analysis, prompting that this enzyme may have valuable application potential for other species. The identity of TDF was evaluated by sequencing a TDF and comparing it with the sequence of the template cDNA. The result showed that the chimeric fragments derived from ligation between digested fragments was generated and could not be eliminated by increasing adapter concentration. Although the existence of chimeric fragments should be carefully considered, the unexpected sequence in the chimeric TDF may not seriously influence the sequencing and BLAST searching analyses.

Comparison of tall fescue (Cyperales: Gramineae) to other cool-season turfgrasses for tolerance to European chafer (Coleoptera: Scarabaeidae).

Three cultivars of tall fescue, Festuca arundinacea Schreb., were compared with three cultivars each of fine fescue (Festuca spp.), Kentucky bluegrass (Poa pratensis L.), and perennial ryegrass (Lolium perenne L.) to evaluate tolerance to root-feeding by European chafer grubs, Rhizotrogus majalis (Razoumowsky). Potted turfgrasses were infested with initial densities equivalent to 33 or 66 grubs per 0.1 m2 on 19 August 2000. More grubs were added in late September and October, bringing the total to 66 or 143 grubs per 0.1 m2. Plant growth, root loss, weight gain, and survival of grubs were measured. The experiment was repeated in fall of 2001 with an initial density of 66 grubs per 0.1 m2. The proportion of root mass lost as a result of grub feeding was a function of turf species, root growth, grub survival, and grub growth during the test. Grubs gained the most weight and consumed the most roots when feeding on fine fescue. Fine fescue suffered the greatest percentage of root loss in 2000, despite having the most rapid root growth and largest mass in control pots. Cultivars of tall fescue appeared to be the most tolerant of grub feeding, having the smallest reduction in root mass in both years. Data from fine fescue, Kentucky bluegrass, and perennial ryegrass cultivars were not as consistent as tall fescue, because for some cultivars root growth and grub survival were different between years. We also found that grubs increased in mass by 20% when the mass of available roots was doubled.

Isolation and characterization of cold-regulated transcriptional activator LpCBF3 gene from perennial ryegrass (Lolium perenne L.).

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.

Monitoring of gene expression profiles and identification of candidate genes involved in drought responses in Festuca mairei.

To understand the molecular genetic basis underlying drought tolerance in grasses, the cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique was applied for identification of genes responding to drought stress in a xerophytic adapted plant, Festuca mairei. A total of 11,346 transcript derived fragments (TDFs) were detected, and 464 (4.1%) TDFs were identified as differentially expressed fragments (DEFs) during the drought treatment of the plant. The expression patterns of these DEFs included up-regulated ( approximately 30%), down-regulated ( approximately 54.3%), and the remainder ( approximately 16.7%) showing transient changes. The differential expression patterns of 171 DEFs were further confirmed by macroarray hybridization analysis. Sequences had been obtained for 163 DEFs, and 62 sequences had no significant hits to sequences currently in public databases. Predicted functions of remaining 101 sequences were subdivided into 17 categories. Down-regulated genes were highly represented by metabolism and cellular biogenesis. Up-regulated DEFs were enriched in genes involved in transcription, defense, cell cycle and DNA processing. Analysis of the 163 DEFs provides a first glimpse into the transcripts of F. mairei during drought stress treatment. The combination of data from studies on genetic model plants and on diverse plant species will enhance understanding of the drought tolerance mechanisms in plants.

Genetic diversity in colonial bentgrass (Agrostis capillaris L.) revealed by EcoRI-MseI and PstI-MseI AFLP markers.

Colonial bentgrass (Agrostis capillaris L.) is a potential source for genetic improvement of resistance to environmental stress and disease for other bentgrass species (Agrostis spp.). To conserve and study the existing genetic resources of colonial bentgrass for use in breeding, genetic diversity was investigated using amplified fragment length polymorphism (AFLP) markers. Included in this study were 22 accessions from US Department of Agriculture germplasm collected from 11 countries, in conjunction with 14 accessions from northern Spain and 3 commercial cultivars. Ten EcoRI-MseI and 6 PstI-MseI AFLP primer combinations produced 181 and 128 informative polymorphic bands, respectively. Cluster analysis of genetic similarity estimates revealed a high level of diversity in colonial bentgrass species with averages of 0.51 (EcoRI-MseI) and 0.63 (PstI-MseI). Greater genetic diversity was detected by the EcoRI-MseI AFLP primer combinations. A low but significant positive correlation (r = 0.44, p = 0.0099) between the 2 Jaccard similarity matrices was obtained by the Mantel test. Commercial cultivars of bentgrass showed a narrow genetic background. The assessment of genetic diversity among colonial bentgrass accessions suggested the potential value of the colonial bentgrass germplasm in turfgrass cultivar improvement.

Genetic variability of grey snow mould (Typhula incarnata).

Randomly amplified polymorphic DNA (RAPD) markers were used to assess the genetic diversity of isolates of grey snow mould ('gray snow mold'), Typhula incarnata, taken from infected turfgrasses from 40 different locations in the northern USA. Data from 115 markers using 37 RAPD primers showed 48 % polymorphism. The distance coefficients between isolates indicate the wide genetic diversity of T. incarnata across the sample area. Dendrograms generated using neighbour-joining (NJ) bootstrap analyses showed three clades and suggest possible recent colonization from common founder groups. Partitioning of the genetic variance using analysis of molecular variance (AMOVA) of four groups based on geographic locations (Michigan, lower and upper peninsula; Minnesota; Wisconsin) showed that genetic variation attributable among groups and within groups was 12.67 and 87.33 %, respectively. No correlation was found between geographic distance and pairwise genetic distance of the groups. High outcrossing and sexual recombination of T. incarnata may well be key factors explaining the genetic variability as shown with the low Fixation index (FST) and high average of genetic diversity per locus within groups.