The Developement And Application Of Simple Sequence Repeat Markers From Dioscorea Oppostia Thunb. Based On Transcriptomic Sequencing

Huai yam(Dioscorea oppostia Thunb.)is one of the four famous Huaiqing Chinese medicines,Tiegun is one of the best Huaiyam cultivars,which has high nutritional values.However,there are many Huai yams of other cultivars sold on the market.Researches on molecular levels may help to distinguish diversity of yam germplasm resources and yam cultivars.There are a large number of simple sequence repeats(SSRs)distributed in the plant genome.The SSR markers are characterized by their high abundance,polymorphism and co-dominant inheritance.In this study,20 yam materials were used to develop SSR molecular markers based on the transcriptome sequencing data of Dioscorea oppostia Thunb transcriptome.The genetic diversity of 22 Chinese yam was analyzed using the 5 molecular markers developed and related bioinformatics software.The findings are as follows:1.Analysis of SSR distribution condition based on transcriptome sequencing data of Dioscorea oppostia Thunb.There are 71,739 Unigenes detected,totaling 59,233,267 bps,of which 17,405 Unigenes contain SSR loci.The frequency of occurrence(the ratio of Unigenes with SSR to total Unigenes)is 24.26%.The number of identified SSRs was 22425,and the frequency of occurrence(the ratio of the number of SSR to the total number of Unigenes)was 31.26%.The total number of SSR was 22,425,SSRs of mono repeats accounted for the largest proportion of 54.01%,SSRs of di-nucleotide repeats accounted for 23.04%,and SSRs of tri-nucleotide repeats accounted for 20.90%.More than four bases account for less.The number of SSRs with 10 repeats was the highest,which accounting for 26.82% of all the SSR.2.Development of SSR molecular markers based on Dioscorea oppostia Thunb.transcriptomePolymorphic bands were screened with 100 pairs of primers.The materials used were 20 kinds of yam,5 SSR molecular markers were screened finally.3.Cluster analysis of Dioscorea oppostia varietiesFive SSR primers were used to amplify 22 Dioscorea oppostia cultivars.According to the polymorphic loci the NTsys software to establish a cluster analysis tree,22 yam is divided into 8 categories.4.The analysis of genetic diversity in yam22 varieties of yam were amplified with 5 pairs of primers selected.The number of alleles(Na)observed,the number of effective alleles(Ne)in all varieties was 1.782,the varieties of different yam varieties were 1.2 ~ 2.0.Shannon's information index(I)was 0.542 in all varieties and 0.139 ~ 0.693 in different varieties of yam.The observed heterozygosity(Ho)was 0.782 in all varieties of yam,and the Ho value in different yam varieties was 0.2 ~ 1.0.The expected heterozygosity(He)was 0.391 in all varieties of yam and 0.1 ~ 0.5 in different varieties of yam.The unbiased expected heterozygosity(uHe)was 0.770 for all varieties of yam and 0.2-1.0 for different varieties of yam.5.The analysis of genetic differentiation in yam varieties.From the analysis of genetic differentiation among 22 yam varieties,two varieties of yam combinations,there are 231 ways in total.There were 69 pairs of Fst values between 0 and 0.05,indicating that the genetic differentiation among these 69 pairs was small and could not be considered.There were 36 pairs in the range of 0.05 ~ 0.15,indicating that there was a moderate degree of genetic differentiation between 36 pairs of yam,50 pairs between 0.15 and 0.25,and the degree of genetic differentiation among the groups was relatively large.The Fst values of the remaining 76 pairs of yam varieties were greater than 0.25,The degree of genetic differentiation is obvious.Among them,the genetic differentiation coefficient of Dioscorea zingiberensis C.H.Wright and the remaining 21 yam varieties were the largest,both greater than 0.25,indicating that the degree of differentiation between the groups is obvious.D.opposita cv.‘Dengxianfeng'in addition to D.opposita cv.‘Nancangcai',D.opposita cv.‘Songye No.1'genetic differentiation coefficient is less than 0.05,and the remaining 19 kinds of yam genetic differentiation coefficient is greater than 0.25.That is the degree of genetic differentiation among the groups was obvious.There was no significant difference in the degree of genetic differentiation between D.opposita cv.‘Tiegun'and D.opposita cv.‘Ribenyuan',D.opposita cv.‘Shennongshan No.4',D.opposita cv.‘Ribenqingsen',D.opposita cv.‘Ribenbai',D.opposita cv.‘Pingyao',D.opposita cv.‘Dahechangyu',D.opposita cv.‘Xiaoye No.2',D.opposita cv.‘Baiyu',D.opposita cv.‘Gaoyangxiaobaizui'and D.opposita cv.‘Lijiang No.2'.Most of the genetic differentiation coefficients between NC and other yams were between 0.05 and 0.15,and the degree of genetic differentiation among the populations was moderate.6.The analysis of genetic distance and genetic identity in yam varieties.Genetic distance and genetic identity were calculated for 22 different varieties of yam.The genetic distance between yam varieties was from 0 to 2.3864,and the genetic distance between Dioscorea zingiberensis C.H.Wright and the remaining yam varieties was larger with lower genetic agreement.Genetic difference is obvious.D.opposita cv.‘Dengxianfeng' with D.opposita cv.‘Nancangcai',D.opposita cv.‘Songye No.1'was the smaller genetic distance,between the remaining 19 kinds of yam genetic distance was greater than 1.Low genetic identity and genetic differentiation is obvious,the results are consistent with D.opposita cv.‘Dengxianfeng' population genetic differentiation between the value of Fst consistent.The genetic distance between NC and the other yam cultivars ranged from 0.1407 to 1.6181,the genetic distance and the genetic identity were moderate,and the degree of genetic differentiation was moderate,which was consistent with the genetic differentiation coefficient Fst of NC.The genetic distance is zero recently is the D.opposita cv.‘Tiegun' and D.opposita cv.‘Ribenqingsen',D.opposita cv.‘Ribenbai',D.opposita cv.‘Dahechangyu',D.opposita cv.‘Lijiang No.2' between two and two,D.opposita cv.‘Shennongshan No.4',D.opposita cv.‘Baiyu',D.opposita cv.‘Gaoyangxiaobaizui',D.opposita cv.‘Pingyao' between twenty-two,higher genetic agreement,that genetic differentiation is not obvious.The results are consistent with the results of the genetic differentiation coefficient Fst of each population.