Comparative Study On Physiological Characteristics And Transcriptome Of Different Alfalfa Varieties In Response To Low Temperature

In recent years,the global climate change has resulted in frequent occurrences of extreme weather events,including low temperatures,which pose a serious threat to the growth and survival of cultivated forage and crops,leading to significant losses in agricultural production.Alfalfa（Medicago stavia L.）,hailed as the“Queen of forage”,plays a pivotal role in animal husbandry and dairy production.However,alfalfa is mainly planted in the northern China,where cold winters and frequent“cold snaps”in spring cause difficulties in overwintering and low greening rates,thus affecting forage quality and yield of alfalfa.Therefore,it is of great significance to screen cold-resistant alfalfa varieties and identify key genes involved in low-temperature adaptation mechanisms for improving the productivity of alfalfa in northern China.In this study,therefore,ten alfalfa varieties from diverse regions were selected as experimental materials and a cold tolerance evaluation procedure for alfalfa seedlings was established based on four aspects:biomembrane stability,accumulation of osmoregulatory substances,antioxidant enzyme activity,and photosynthesis.The cold tolerance of the tested alfalfa varieties was classified and ranked through correlation analysis,principal component analysis,fuzzy function evaluation,and cluster analysis.A stepwise linear regression analysis was conducted to develop a cold tolerance evaluation equation for alfalfa seedlings.Finally,two alfalfa varieties with the highest resistance and sensitivity to cold were selected as materials for transcriptome comparison under low temperature stress in order to mine differential genes and identify key genes involved in cold response.The main findings of this study are presented as follows:1.The physiological response pattern of alfalfa seedlings under low-temperature conditions was determined.At 4°C,the relative electrical conductivity（ERC）,malondialdehyde（MDA）,proline（Pro）,soluble sugar（SS）,soluble protein（SP）content,and the activities of superoxide dismutase（SOD）and peroxidase（POD）in alfalfa leaves exhibited an increasing trend,while the net photosynthetic rate（Pn）,stomatal conductance（Gs）,transpiration rate（Tr）,and intercellular CO₂ concentration（Ci）showed a decreasing trend.There were significant variations in these physiological indicators among different varieties,with the most notable elevation in proline content and SOD and catalase（CAT）activities in’Longmu 801’.2.The cold tolerance of 10 alfalfa varieties was classified and ranked.The 10 alfalfa varieties were categorized into three groups based on their cold tolerance using correlation analysis,principal component analysis,and fuzzy function evaluation.The cold-resistant varieties included’Longmu 801’and’Zhaodong’,while the moderately cold-resistant ones were’Bara 520 YQ’,’Gongnong No.1’,’Baimu 401’,and’Aohan’.The cold-sensitive varieties comprised of’Challenger’,’Dongnong No.1’,’Knight’,and’Xinmu No.4’.By determining the leaf semi-lethal temperature(LT₅₀)of each variety,it was observed that among the 10 varieties,’Longmu 801’exhibited the lowest LT₅₀whereas’Xinmu No.4’had the highest LT₅₀,which is consistent with the results from membership function analysis.3.A cold tolerance evaluation equation was developed for alfalfa seedlings,which is expressed as D=0.138+0.178×SP+0.203×POD+0.17×Pro.Moreover,soluble protein,proline,malondialdehyde,soluble sugar,relative electrical conductivity and catalase activity were identified as key physiological indicators for distinguishing’Longmu 801’and’Xinmu No.4’under low-temperature stress conditions.4.Candidate genes regulating cold tolerance in alfalfa were identified via transcriptome comparison between two varieties of alfalfa.Through differential gene screening,weighted gene co-expression network analysis（WGCNA）,time series analysis（TSA）,least absolute shrinkage and selection operator（LASSO）regression model screening,and artificial neural network（ANN）verification,we successfully screened seven candidate genes closely related to cold tolerance of alfalfa,including MS.gene003665（XK1）,MS.gene65385（CLCe）,MS.gene99779（BRX1-2）,MS.gene045296（NTF2A）,MS.gene49520（ABI4）,MS.gene61387,and MS.gene36642.These results provided theoretical basis for revealing the physiological and molecular regulation mechanism of alfalfa seedling response to low temperature,and laid a foundation for the cultivation of cold-resistant alfalfa varieties.