Identification Of Cold Tolerance And Screening Of Cold Tolerant Germplasm Of Alfalfa

Alfalfa(Medicago sativa L.)is known as the "King of Forage",due to its high yield,rich nutritional value,good palatability and high economic value.In terms of wide ecological adaptability and strong stress resistance,it has been cultivated in large areas in Northwest,North and Northeast China.In this study,10 alfalfa varieties were used as experimental materials,the morphological characteristics,biological characteristics,agronomic traits,physical and chemical characteristics of the materials were systematically identified and evaluated from seeds,seedlings,field plants and roots by field observation and laboratory analysis.The differentially expressed genes related to cold resistance of alfalfa were screened by transcriptome sequencing analysis,aiming at exploring the response mechanism of cold resistance of alfalfa and providing basis for breeding cold-resistant varieties,germplasm innovation and molecular breeding of alfalfa.The main results are as follows:(1)Under low temperature,the germination rate,germination index,simplified vigor index,root length and bud length of all alfalfa materials showed a downward trend,and the peak period of germination delayed with the decrease of temperature.The temperature condition of 6℃ is the critical temperature for alfalfa germination,and the germination rate of all varieties above this temperature is higher than 50%;below this temperature,the germination rate is less than or partially reaches 50%.There are significant differences in simplified vigor index,root length and germination rate among varieties,which are related to the temperature conditions at germination,and can be used as evaluation indexes to identify the cold resistance of alfalfa during germination stage.According to the performance of these indexes,it was preliminarily determined that Longmu 806,Longmu801,Caoyuan No.3,Zhaodong and Gongnong No.2 alfalfa had strong cold tolerance by membership function screening analysis.(2)The contents of proline(Pro),soluble protein(SP),soluble sugar(SS)and the activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)in alfalfa materials increased first and then decreased with the extension of low temperature treatment time.The content of malondialdehyde(MDA)and electrical conductivity(EC)showed an upward trend,but there were obvious differences among different varieties.After 6 hours of low temperature treatment,the contents of Pro,SP and SS of Longmu 801 were the highest,while the activities of SOD and POD of Caoyuan No.3 and Gongnong No.2 were the highest.After 8h low-temperature stress,the MDA content and EC of Longmu 801 and Longmu 806 were the lowest,the SP and SS content of Longmu 801 was the highest,and the CAT and POD activities of Longmu 806 and Zhaodong alfalfa were the highest.Using the membership function,all materials can be classified according to the cold resistance grade.Longmu 801,Longmu 806 and Zhaodong alfalfa belong to strong cold resistance varieties,Caoyuan No.3 and Gongnong No.2 are strong resistant varieties,Aohan,Zhongmu No.1 and Queen alfalfa have weak cold resistance,and 420 and Sardi alfalfa have poor cold resistance.(3)The winter survival rate of varieties was positively correlated with neutral detergent fiber and yield(P<0.01),and was positively correlated with photosynthetic rate,leaf water vapor pressure,variable fluorescence(Fv),photochemical quenching coefficient(q P),photochemical efficiency(Fv/Fm),and potential photochemical efficiency(Fv/Fo)(P<0.05);There was a significant negative correlation between winter survival rate and plant height,internode length and relative feeding value(P<0.05).The forage yield of alfalfa varieties Caoyuan No.3,Zhaodong,Longmu 806,Longmu 801 and Zhongmu No.1is higher,and the forage quality of alfalfa varieties Longmu 806 and Zhongmu No.1 is excellent,which is suitable for planting in Hohhot.(4)Although alfalfa stopped growing in winter,the physiological and biochemical activities in the root system continued.The contents of Pro,SS,SP,MDA,abscisic acid and the activities of SOD,POD and CAT in the root system generally increased when the temperature decreased,and reached the peak in the coldest January(air temperature and ground temperature were the lowest),and then decreased with the temperature rising;However,EC also showed a trend of rising first and then falling.The varieties with lower fall dormancy rate and higher winter survival rate have more obvious change trend,and the content and activity of various contents in their roots are significantly higher than those of other varieties in the coldest period.In short,the determination and analysis of the content and activity of root contents in the coldest period is an effective means to identify the cold resistance of alfalfa varieties.The order of cold resistance of alfalfa varieties is: Longmu801 > Longmu 806 > Zhaodong > Caoyuan No.3 > Gongnong No.2 > Zhongmu No.1 >Aohan > Queen > 420 > Sardi.The winter survival rate of alfalfa is closely related to the size and position of root crown.The larger the root crown and the deeper the soil,the higher the winter survival rate of alfalfa in the following year.(5)A total of 4442 differentially expressed genes(DEGs)were obtained from alfalfa root crown,7 low temperature responsive transcription genes and key signal transduction pathways DEGs were identified,which belong to plant hormone signal transduction pathway,peroxisome pathway and transcription factor family(MYB,B3,AP2/ERF,WRKY).They mainly enriched in cell physiology and metabolic processes such as cell part,membrane part,response to stimulus and catalytic activity.The results of 7 DEGs by quantitative real-time PCR(q RT-PCR)are consistent with those by transcriptome sequencing(RNA-seq).The critical temperature of alfalfa seed germination was explored in this thesis,and the correlation between root crown size and soil location and alfalfa overwintering was made clear.Moreover,it also suggested that the content and activity of various physiological indexes in leaves and roots under the seedling stage and overwintering stage could be used as an important index to identify the alfalfa cold resistance.In the future,the identification and evaluation system of alfalfa cold resistance will be further improved and optimized,and the response mechanism of alfalfa cold resistance will be deeply revealed by combining metabonomics or proteomics.