Cold Resistance Evaluation And Temperature Response Mechanism Of Bermudagrass At Different Latitudes

Bermudagrass(Cynodon dactylon(L.)Pers)is a perennial herbaceous warm season plant,and is widely distributed in tropical and subtropical areas.Bermudagrass is mainly distributed in the southern of Yellow River basin.With the increase of latitude gradient,the green period of bermudagrass lawn becomes shorter and suffered by cold damage easily.Bermudagrass cannot overwinter safely at high latitudes.Extreme temperature is one of primary abiotic factors determining the geographical distributions of bermudagrass.In this study,310 bermudagrass materials were collected from 16 regions along the latitude gradient from south to north(22.59°N to 36.31°N).Three tetraploid Bermudagrass materials were selected from each latitude,and the physiological indexes of Bermudagrass under different temperature were studied by artificial simulated temperature(5℃,20℃and 35℃).The comprehensive cold resistance of bermudagrass at different latitudes was evaluated by membership function method.Besides,the cold resistance of Bermudagrass was identified by semi-lethal temperature method,and the wild Bermudagrass germplasm with relatively strong and weak cold resistance was identified by combining the two methods.Based on the evaluation of cold resistance,water content and nutrient content,combined with the observation of root anatomical section and microscope,the changes of bermudagrass root system under freezing injury and the strategies to deal with freezing injury were analyzed.Transcriptome sequencing technology was used to explore the temperature response mechanism of bermudagrass related to physiological changes.Single nucleotide polymorphisms(SNPs)were used to analyze the genetic structure of Bermuda grass populations along the latitudinal gradient.What’s more,the response mechanism of ethylene to low temperature was investigated by applying ethylene treatment agent.The results are listed as follows:1.Comprehensive evaluation of physiological and biochemical responses and cold tolerance of bermudagrass at low temperature in different latitudes.After low temperature treatment,the EL,MDA content,ABA content,POD enzyme and SOD enzyme activities of bermudagrass in 16 regions increased with the decrease of temperature.CAT and APX activities varied in all materials.Pn,Gs and Tr of photosynthetic indexes decreased with temperature decreasing.After comprehensive analysis of physiological indexes by membership function method,the order of cold resistance of bermudagrass in 16 regions was as follows:Zhumadian>Yinde>Zhongshan>Xiantao>Youxian>Xinyang>Guidong>Renhua>Liuyang>Huixian>Guangzhou>LinXiang>Cixian>Xuchang>Zhengzhou>XiaoChang.In addition,the cold resistance of wild Bermuda grass in 16 regions at different latitudes was evaluated by using the conductivity method combined with logistic equation.The two methods were combined to identify the Bermuda grass in Xiantao and Zhumadian as the material with strong cold resistance.2.Evaluation of cold resistance and adaptation mechanism of bermudagrass roots under freezing injury in different longitude and latitude.Bermudagrass is sensitive to cold.In winter,the root system which is sensitive to low temperature will wither or die in winter,resulting in slow green recovery and less aboveground biomass in the next year.The changes of C,N content and C/N ratio of roots in two periods(normal growth period and winter low temperature period)were compared.Through the identification of cold resistance of roots under normal growth,the influence of root structure on cold resistance was explored combining with the microscopic structure of root paraffin sections.The results showed that free water content decreased,bound water content increased,and ratio of free water with bound water decreased in winter.The main strategy of root freezing injury was to avoid the formation of intercellular ice crystals and reduce the damage caused by the presence of ice crystals.The total C content of latitudinal gradient materials increased obviously in winter indicated that root material accumulated in winter.Based on the analysis of root cold tolerance and root structure,the root cold resistance was greatly affected by xylem and phloem.3.Genetic diversity patterns along latitudinal gradients of Bermudagrass at different temperatures137 Bermuda grass transcriptome libraries with 16 latitudinal gradients were selected to explore the differences of molecular markers and analyze the genetic diversity and structure of Bermuda grass along latitudinal gradients at different temperatures.More high-quality single nucleotide polymorphisms(SNPs)were obtained in populations at middle and low latitudes.Populations of bermudagrass at high latitudes differed from populations at middle and low latitudes,which was supported by a principal component analysis(PCA)and genetic structure analysis,performed at different temperatures.We observed more genetic variation for low-latitude populations at 5℃,according to an analysis of three phylogenetic trees at different temperature levels,suggesting that low temperatures affected samples with low cold resistance.Based on the results of phylogenetic analysis,we found that samples from high latitudes evolved earlier than most samples at low latitudes4.Transcriptome analysis and molecular mechanism of cold resistance in bermudagrass at low temperature.The transcriptome library of 16 bermudagrass leaves under temperature treatment was constructed.A total of 15,224 and 9230 differential expressed genes(DEGs)were identified in T5/T20 and T35/T20 combinations by comparative transcriptome analysis.Through the functional enrichment of DEGs,we found that differential gene enrichment is related to the photosystem,protein kinase,transporter transcription factor(TFs),MAPK signaling,pathway in plant hormone signal transduction and circadian rhythm pathway.Through WGCNA analysis in the correlation between DEGs and physiological indicators,results showed that differential gene expression profiles in networks are associated with the plant growth,photosystem,redox system,and transcriptional regulation to cold stress in bermudagrass.In particular,genes encoding HSP70/90 and HsfA3/A8 are not only regulated by temperature stress,but also directly or indirectly interplay with the processes of peroxide scavenging and chlorophyll synthesis under cold stress.5.Study on the mechanism of ethylene in response to low temperature in bermudagrassThe control group and low-temperature treatment group were set up,including three ethylene treatment groups:ethylene donor ACC,ethylene inhibitor AVG and growth regulator ethephon.The results shown,EL and MDA content decreased in low temperature treatment group,AVG treatment was the lowest in cold-tolerant materials,and ethephon treatment was the lowest in cold-sensitive materials.POD and SOD activities increased and CAT activities decreased.ACC treatment significantly increased the content of ABA in bermudagrass.The results of principal component analysis showed that the three treatments slightly reduced the cold resistance of cold-tolerant bermudagrass,while ACC treatment significantly improved the cold resistance of cold-sensitive bermudagrass.RT-PCR results showed that the expression levels of several ethylene related genes were the same as the physiological changes,suggesting that CdERS,CdEIN3 and CdEREBP may be involved in the positive regulation of bermudagrass cold response.In summary,the physiological changes of bermudagrass in 16 regions on the latitude gradient under low temperature were analyzed,and the cold-tolerant and low-temperature sensitive bermudagrass materials were selected through comprehensive analysis.Through the correlation analysis with latitude,it was found that there was a great difference between the cold resistance of bermudagrass at high latitude and that at middle and low latitudes.Transcriptome sequencing results were used to excavate the genes and related pathways of bermudagrass in response to cold stress,and SNP was used to analyze the genetic structure of bermudagrass.The results showed that there was genetic differentiation between bermudagrass at high latitudes and middle and low latitudes.The difference of cold resistance of bermudagrass was the result of long-term adaptation evolution,and the latitude distribution had a profound effect on the cold tolerance of bermudagrass.In addition,the effects of ethylene on different materials with low temperature tolerance were also found to be different.These results provide important clues and resources for further research on the response of bermudagrass to low temperature stress and breeding of bermudagrass for cold tolerance.