Study On Genetic Diversity And Genetic Linkage Map Construction In Medicago Polymorpha

Burr medic(Medicago polymorpha L)is one of the annual medics belonging to the genus Medicago.This genus consists of approximately 87 species,including the widely cultivated major forage crop species alfalfa(Medicago sativa)and the legume model species barrel medic(Medicago truncatula),which play important roles in agriculture and livestock.Because of its low sensitivity to photoperiod and vernalization,Medicago polymorpha has widely spread all over the world.In addition to nitrogen fixation and forage quality attributes common to legumes.Medicago polyymorpha is very useful for soil restoration,green manure,winter cover for erosion and so on,and is successfully used in green manure,cover crop,ley-farming,pasture improvement and so on.Besides that Medicago polymorpha also possesses some important agronomical features such as adaptability to difficult climates and soils,good winter growth,persistence,ability to be heavily grazed and so on,it has regarded as a useful genetic resource for alfalfa improvement.Recently,with the concerning about food safety,Medicago polymorpha becoming more and more popular for both consumers and government as a green organic food.A market-oriented Medicago polynmorpha industry comes into rapid development.The areas and produces of Medicago polymorpha are constantly increasing.Without saying that Medicago polymorpha is a multi-purpose legume forage with great potential.However,compared with the rapid development of industrialization,the development of genetic breeding of Medicago polymorpha still slowly,lacking of quality and high-yielded varieties and many problems in production remain to be solved.The study of genetic diversity and genetic linkage map construction in Medicago polymorpha will not only benefit for Medicago polymorpha breeding,but also can provide an important reference for annual medics,and even alfalfa.The main results are as follow.(1)In this paper,five resources from different areas were collected as the experimental materials,germination rates were counted at different temperature treats(5??10??15??20??25??3??35??40?).The results showed that hardseededness was the main factor for seed germination;hardseededness degrees were different in Medicago polymorpha from different areas,and break the coat could significantly improve the germination rate.The highest germination rate of Medicago polymorpha was at 20?,and 25? was the most suitable temperature for root growing,however the seeding will die at 40?.(2)The results showed that materials from different places were different in plant height,branch number,leaf length,leaf width and cutting yields.The second cutting was significant positive correlation to grass yield,and compensation existed in different cuttings.Among the main agronomic characters of Medicago polymorpha,second cutting was the most important factor on the yield,followed by plant height,third cutting,first cutting,branch number,leaf width,leaf length,fourth cutting and root length.(3)There are great genetic variations in the leaf length,leaf width,plant height,branch number,stem diameter,tem-leaf ratio,fresh weight,dry weight and fresh-dry ratio in different Medicago polymorpha accessions.Among those,the plant height and branch number have the largest variations than others,which can be used as morphological characteristics for distinguish different Medicago polymorpha accessions.Correlation analysis showed that the leaf length,leaf width and plant height were significantly positive correlation;the branch number,stem diameter and tem-leaf ratio have significant positive correlation;fresh weight and dry weight was significantly positive correlation;stem diameter with fresh weight and dry weight show a positive correlation.The results can give a benefit guide for variety breeding based on phenotypic traits.(4)In this study,6.26 Gb of data containing 24,831,153 reads were obtained from a diverse collection of 10 burr medic accessions by a recently developed specific length amplified fragment sequencing(SLAF-seq)method.The average sequencing depth was 11.37 and 52,237 high-quality SNPs were identified with minor allele frequencies(MAFs)>0.05 and integral>90%.Our analyses revealed that Medicago polymorpha harbored nucleotide diversity(??=0.0045 and,? = 0.0043).Three populations divided by phylogenetic relationship were consistent with phenotypic characteristic,and confirmed by population structure and principal components analysis(PCA).Wild population harbored a higher allelic variation compared to the cultivated population,which exhibited high levels of population differentiation.A low level of gene flow was found between different populations,although without associations with their geographical distributions.Results of this study allowed the discovery of a large number of SNP markers,which will help us to better understand the domestication history and intraspecific relationship of Medicago polymorpha.(5)In this study,124 F2 individuals from the cross between 'Wenling' and 'Haian' and their parents were used to construct a molecular genetic linkage map by using the specific length amplified fragment sequencing technique.DNA sequencing generated 85.18 Gb data consisting of 453,946,824 paired-end reads.In all,261,501 high-quality specific length amplified fragments were detected,of which 23,221 were polymorphic for a polymorphism rate of 8.88%.The average coverage of each SLAF marker was 32.39-fold for the parents compared with 8.27-fold for the F2 offspring.Among the 23,221 polymorphic SLAFs,17,345 were encoded(a ratio of 15.82%).Finally,2,408 SLAFs were used for map construction.2,408 SLAFs were assigned to 7 linkage groups.The total length of the map was 839.01 cM,with an average distance of 0.35 cM between each maker.The largest LG,LG6 harbored 324 markers covering a length of 195.28 cM,the largest average inter-marker distance was 0.60 cM;LGl was the most saturated,comprising 760 markers with an average marker density of 0.19 cM;and the smallest LG,LG4 contained 143 markers with a length of 65.68 cM and an average inter-marker distance of 0.35 cM.To our knowledge,this is the first genetic linkage map for Medicago polymorpha.This high-density genetic map will benefit for comparative mapping,quantitative trait loci mapping and molecular-assisted breeding of Medicago polymorpha.At the same time,according to constructed genetic linkage map;9 QTLs were detected for 7 yield-related traits,which were located on LG1?LG3 and LG6.Of them,two for the leaf length,one for the leaf width,one for the branch number,one for the plant height,one for the beginning of flowering time,one for the per plant dry weight,and two for the per plant pod weight.