Genetic linkage map of creeping bentgrass and characterization of LpCBF3 gene for cold tolerance in perennial ryegrass

The colonial bentgrass (Agrostis capillaris L.) is a potential source of genetic improvement of resistance to environmental stress and disease for other bentgrass species (Agrostis spp.). Genetic diversity in the colonial bentgrass species was investigated using amplified fragment length polymorphism (AFLP) markers. Ten EcoRI/ MseI and six 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 (EcoR I/MseI) and 0.63 (PstI/ MseI). Greater genetic diversity was detected by the EcoR I/MseI AFLP primer combinations. A low but significant positive correlation (r=0.44, p=0.0099) between the two Jaccard similarity matrices was obtained by the Mantel test.; To improve creeping bentgrass disease resistance, interspecific hybrids between creeping bentgrass and colonial bentgrass were produced for introgression of resistance genes. The hybrids (2n=28) were confirmed by a species-specific sequence characterized amplified region (SCAR) marker of the colonial bentgrass genome. In addition, average seven ring bivalents, several univalents and multivalents were observed at meiosis of hybrids. Pollen fertility of the hybrids ranged from 4% to 34.5%. The hybrids were evaluated for gray snow mold disease resistance in the cold room and dollar spot disease resistance in the greenhouse. Some of hybrids had moderate snow mold and dollar spot resistance, exhibiting partially diminished disease symptoms.; A segregating progeny set of 188 F1 full-sib population was developed by two-way pseudo-testcross mapping strategy for linkage construction and QTL detection in creeping bentgrass. In the male parent map, 93 markers could be grouped in 14 linkages covering a total length of 793cM with an average interval of is 8.2cM, while in the female parent map 139 markers were clustered in other 14 linkages spanning 805cM with an average distance of 5.9cM between adjacent markers. Five putative QTLs were found in this segregation population, three QTLs for leaf width, one QTL for snow mold disease resistance, and one QTL for recovery.; A CBF homologous gene, LpCBF3, was isolated and characterized from a cold-tolerant perennial ryegrass accession (Lolium perenne L.). LpCBF3 encodes a protein of 237 amino acids with a molecular mass of 25.5 kDa. It carries sequences of the typical AP2 DNA-binding domain and an acidic activation which are present in the majority of the plant CBF protein family. Northern blotting and RT-PCR analysis found LpCBF3 reached the highest expression after 90 minutes of cold-treatment (4°C). In Arabidopsis, the over-expression of LpCBF3 under 35S promoter was resulting in plant with dwarfism, later flowering and freezing tolerance. These results lead us to propose potential implications and applications of LpCBF3 gene in turfgrass cold-tolerance and quality breeding program.