Analysis Of Physiological And Molecular Mechanisms Of Cold-resistance In Bermudagrass

Cold stress is a major environmental factor that limits plant growth, development, reproduction and distribution. It is important to find out the efifective methods to improve cold resistance of the plant, especially the industrial crop through analyzing the mechanism of cold resistance in multiple aspects such as ecological, physiological and molecular levels. Recently, along with the advance of experimental technology, the research on cold resistance of plant has increased rapidly. Researchers analyzed the mechanisms of cold resistance in different levels, including ecological, physiological, proteomic, metabolomic and transcriptomic levels. Moreover, Many cold-responsive genes were identified, and some of them were identified to improve the cold resistance of the transgenic plant.Bermudagrass(Cynodon dactylon) is a perennial and typical warm-season grass species in Poaceae, and it is distribute in tropical, subtropical and warm temperate zone. Bermudagrass is used in sport field, golf courses, park and homeland as turfgrass. It is also used as forage grass due to the rapid growth, high production and good palatability. In addition, the viability and reproduction speed of bermudagrass are relatively high. Bermudagrass can also be used as slope-protecting plant. Therefore, bermudagrass plays essential roles in economics, agriculture and forestry, environment industry. However, the widespread use of bermudagrass is limited because of its weak cold resistance.In this study, one cold-resistant and one cold-sensitive genotypes were screened from 106 wild bermudagrass acessions that collected from different areas across China. Genotype calibration and population structure analysis of bermudagrass germplasm were conducted with 144 pairs of SSR markers. Molecular markers related to cold resistance of bermudagrass were identified based on association analysis of genotype, phenotype and population structure, this results will contribute to cold-tolerance breeding of bermudagrass. Cold resistance genes in bermudagrass were identified use RNA-Seq technology. Effective and rapid methods to increase cold resistance of bermudagrass were explored through exogenous chemical substance application. The results are listed as follows:1. Investigation of cold resistance and construction of cold resistance phenome database of bermudagrassThe functional traits of bermudagrass germplasm were investigated after cold treatment. Cold resistance was evaluated by membership function method in fuzzy mathematics, and the cold resistance phenome database of bermudagrass was constructed. The results showed that, there were significant variations in functional trait values among the accessions under cold conditions. Positive correlation was detected between cold resistance capabilities and latitudes. Germplasms collected from higher latitudes had stronger cold resistance. On the contrary, germplasms collected from lower latitudes had weaker resistance. Results of discriminant and cluster analysis showed that, the accessions that were collected from adjacent latitudes were mostly clustered together. Cold resistant and cold sensitive varieties were selected based on the comprehensive evaluation.2. Association analysis of cold resistance of bermudagrass.Association analysis between SSR markers and cold resistance phenome data was conducted. According to the results of population atructure analysis, the accessions were identified into 2 structure groups, accessions collected from northwest of China (mostly from Xinjiang) were identified in the same group. The results of association analysis showed that, when the P value was set at P<0.01,285 SSR markers were identified to be associated with the identified functional traits, and 15 markers were identified significantly to be associated with the functional traits after the P value was corrected to P<3.5×10-4, the highest resolution was 57%. Among the 15 markers, CDGA2-1011-1012, CDCA1-27-28 and CDCA2-203-204 associated with different traits.3. Identification of cold resistance relative genes in bermudagrass and analysis of molecular mechanisms of cold responseCold responsive genes were comprehensively investigated by RNA-Seq. Multiple differtial expression genes including genes related to Ca2+ pathway, and transcription factors such as CBF, NAC, WRKY and bHLH were identified. Results of comparison of numbers of the differtial expression genes after different treatments showed that, global gene expressions were initiated more quickly and more effectively in resistance genotype than those in sensitive genotype, when they were exposed to cold stress. It is showed that, photosynthesis pathway was related to cold resistance of bermudagrass according to the results of KEGG pathway enrichment analysis.In addition, the full length of a transcription factor gene of WRKY family was cloned by RACE. The gene was named CdWRKY1. According to the results of VIGS, this gene was involved in cold resistance regulation of bermudagrass.4. Chemical molecular involved in cold resistance regulation of bermudagrass.Cold resistance of bermudagrass was investigated afterapplied of different chemical molecular. The results showed that, exogenous nitric oxide (NO) and melatonin (MLT) could improve the cold resistance of bermudagrass through affected the cell membrane stability, antioxidase activities, photosynthesis and metabolic process.In coclusion, positive correlation was detected between cold resistance capabilities and latitudes depending on the comprehensive evaluation. Fifteen markers were identified significantly to be associated with the functional traits according to the results of association analysis. Multiple differtial expression genes were identified by RNA-seq. Moreover, we found that exogenous NO and MLT could improve the cold resistance of bermudagrass. This study analyzed the mechanisms of cold resistance of bermudagrass, and contributed to the cold resistance breeding of bermudagrass in the future.