Phenotypic And Genetic Diversities Of Cuticular Waxes On Medicago Ruthenica(L.)

Plant phenotypic diversity refers to the external results of plants in the process of evolution under the interactions between environment and genes.Understanding the plant phenotypic diversity can explain the genetic laws and variation degree of different plant populations.Medicago ruthenica(L.)is a perennial high-quality forage of Medicago in Leguminosae,showing multiple characteristics on high protein,low saponin,drought resistance,cold resistance,and barren resistance.Because of its wide ecological range,there are abundant phenotypic and genetic diversity among different populations and different individuals in the same population.Plant cuticular wax is a protective structure covering plant epidermis,and plays an important role in preventing non-stomatal water dispersion,preventing germs from invading,and reflecting ultraviolet light.To understand the phenotype and genetic diversity of cuticular wax in different Medicago ruthenica populations will help us to understand how environmental and genetic factors affect the deposition of cuticular wax on plant leaves and improve their adaptation.In this thesis,seed and leaf morphology and cuticle wax diversity of 32Medicago ruthenica populations from different habitats were analyzed in a common garden experiment;CDS sequences of four genes(Mr FAR3-1,Mr FAR3-2,Mr CER1and Mr KCS1)were obtained by PCR cloning technology,and the promoter sequences of these four genes were cloned by using the chromatid step-by-step method;the relationship between wax content and gene expression was analyzed.At the same time,combining with the stress experiment,the response mechanism of cuticular wax to enhanced UV radiation and drought stress was analyzed.The main results are as follows:1.There were significant differences in seed morphology and leaf morphology between different materials of Medicago ruthenica.The variation range of seed length was 2.47～3.04 mm,the variation range of seed width was 1.63～2.08 mm,the variation range of 1000 grain weight was 2.08～3.35 g,the variation range of leaf area was1.23～4.51 cm~2,the variation range of leaf length was 0.65～2.21 cm,the variation range of leaf width was 0.40～1.11 cm,the variation range of leaf length width ratio was1.08～3.40.According to principal component analysis and coefficient of variation analysis,it was determined that five characters,such as leaf area,leaf length,leaf length-width ratio,thousand-grain weight,and seed length were the main characters that caused phenotypic diversity of Medicago ruthenica.2.The cuticular wax of Medicago ruthenica mainly consists of primary alcohols,alkanes,aldehydes and unknown components.Among them,the primary alcohols accounted for the highest proportion(71.77%～88.67%)in various waxy components,while aldehydes(3.91～14.23%)and alkanes(4.48%～11.85%)accounted for a relatively low proportion.There were significant differences in wax components and contents among different populations,and the coefficient of variation was 33.25%～50.89%.The expression levels of four wax synthesis genes(Mr FAR3-1,Mr FAR3-2,Mr CER1and Mr KCS1)in different populations was also significantly different,in which the expression of Mr FAR3-2 and Mr CER1 genes was positively correlated with the primary alcohol content,and the expression of Mr CER1 genes was positively correlated with the total wax content.3.The similarity of the promoter regions of Mr FAR3-1,Mr FAR3-2,Mr CER1 and Mr KCS1 gene in 32 Medicago ruthenica materials was 94.8%,93.85%,92.34%and97.73%respectively;the HD index was 0.996,0.960,0.988 and 0.984 respectively;the PI index was 0.01144,0.00579,0.01490 and 0.00526 respectively;the number of polymorphic loci was 88,55,126 and 77 respectively.The Tajima’s D values and Fu and Li’s D*and F*values of the four waxy genes(except Mr CER1)were not significant,which was consistent with the neutral evolution model,indicating that they were not affected by natural selection,while Mr CER1,on the contrary,was affected by natural selection.4.Enhanced UV radiation changed the crystal structure of Medicago ruthenica,made it melt,At the same time,also promoted the accumulation of the total amount of cuticular wax in the leaves of Medicago ruthenica.Enhanced UV radiation resulted in a significant increase in the expression of four waxy genes in different Medicago ruthenica materials,Among them,the expression of Mr KCS1 gene increase by≥20times,and the expression of the other three genes increase at least≥3 times.It was suggested that the increase of expression level of waxy gene induced by enhanced UV-B radiation could change the crystal structure and wax deposition,and finally enhanced the stress resistance of plants.5.Drought stress promoted the synthesis of wax in the cuticle of Medicago ruthenica.The content of cuticular wax in 02912,02929,GS3734 and GS4193increased by 58.14%,15.22%,58.47%and 28.44%,respectively.Drought stress increased the content of primary alcohols and aldehydes,but had little effect on the crystal structure of wax.After drought stress,the relative expression of four waxy synthesis genes was significantly increased,among which Mr FAR3-1 was the most sensitive and the relative expression increased at least≥8 times.According to comprehensive analysis,the population of Medicago ruthenica distributed in different environments changed the promoter sequences of waxy genes involved in the cuticular wax of leaves during the process of adapting to their growth environment,and induced the expression of waxy genes,and finally made the cuticle wax deposition and components change,showing diverse characteristics.