Genetic Diversity Of Ten Species Of Wild Shallot And Garlic Based On SRAP And TRAP Markers In Xinjiang,China

The shallot and garlic is an important food,medicinal,vegetable,rearing and ornamental plant resource in the world.Onion,shallot and garlic are important economic crops widely cultivated in China.Xinjiang is an important distribution area of wild shallot and garlic in China and Central Asia,and its special geographical environment provides valuable genetic materials for the resistance breeding of Allium species.In this study,Ten species of wild shallot and garlic in Xinjiang were used as research materials.Through the extraction of genomic DNA from different organs,the effecs of genomic DNA on amplification results were compared.At the same time,the genetic diversity of ten species of wild shallot and garlic in Xinjiang based on SRAP and TRAP markers.In order to provide methods for the identification and kinship analysis of wild shallot and garlic in Xinjiang,and provide technical guidance for the identification of shallot and garlic vegetables in production.Below are key research findings:（1）Genomic DNA was extracted from different organs at different stages by modified SDS method.It research found that the purity of genomic DNA(OD₂₆₀/OD₂₈₀)obtained was between 1.714 and 1.968,with little change.The concentration of genomic DNA varied from 150 to 305μg/m L,with great change.For different stages of the same organ,the highest concentration of genomic DNA was in the young leaf,and the lowest was in the yellow leaf.The concentration of genomic DNA was highest inflorescence before the anthers scattered,and the inflorescence buds were the lowest.The genomic DNA concentration of seeds was highest at the fully matured seed and lowest in the green ripening stage.The detection of genomic DNA and the detection of PCR products showed that high quality genomic DNA can be obtained in these different organs,and the PCR product bands are clear.（2）15 pairs of forward and reverse primers were used for the SRAP markers.Forward and reverse primers were randomly assembled into 225 primer combinations.15 pairs of primer were screened for genetic diversity of ten species of wild shallot and garlic in Xinjiang in the later period.（3）Using the screened 15 pairs of primer combinations to screen for the final determination of the reaction program of SRAP by peogram and annealing temperature:the first 5 cycles of annealing temperature of 35℃and 35 cycles of annealing temperature at 52℃.2μL of 10×PCR Buffer,d NTPs concentration 0.225 mmol/L,primer concentration 0.250μmol/L,Taq enzyme dosage 1.000 U,and template DNA dosage 75.000 ng were the best SRAP-PCR reaction systems.The total of 263 loci were detected by SRAP-PCR amplification,with 259 polymorphic loci,polymorphic loci rate（PPB）of 98.45%,Nei’s genetic diversity index（H）of 0.4106,and Shannon genetic diversity index（I）of 0.5937.It shows that the genetic diversity of ten species wild shallot and garlic in Xinjiang is abundant.The genetic diversity（H_s）and total population genetic diversity（H_t）in the ten populations were 0.2099 and 0.4101,the gene differentiation coefficient(G_st)was 0.4882,and the gene flow（N_m）was 0.5241,It shows that the genetic variation mainly exists in the population and the gene mobility between populations is small.Based on the SM coefficient,the genetic similarity coefficient is 0.59～0.95,the test materials are divided into six major groups,indicating that the kinship is closer.（4）Two fixed primers and seven random primers were selected,and three fixed primers and three random primers designed based on the EST sequence of Allium plants searched by NCBI database were used to randomly select 50 pairs of TRAP primer combinations for primer selection.The results showed that the selected 11 pairs of TRAP primer combinations have certain versatility among different populations and their related species.（5）The selected 11 pairs of primer combinations were used for annealing temperature screening.The first annealing temperature of TRAP-PCR was 37℃,and the second annealing temperature was 53.2℃.After optimization,the optimal reaction system for TRAP-PCR was determined to be 2μL 10×PCR Buffer,0.250 mmol/L d NTPs,0.250μmol/L primer,0.750 U Taq enzyme,and 75.000 ng template DNA.TRAP markers detected the total of 202 loci with 188 polymorphic loci and the ratio of polymorphic sites was93.11%.The Nei’s genetic diversity index populations（H）was 0.1704 and the Shannon information index（I）was 0.2449.The diversity degree（H_s）was 0.1894;the total population genetic diversity（H_t）was 0.3939,the genetic differentiation coefficient(G_st)populations was 0.5192,and the gene flow（N_m）populations was0.4630,It shows that the genetic diversity of ten species of wild shallot and garlic in Xinjiang is relatively abundant,and the genetic variation mainly exists among populations,and the gene mobility between populations is small.Based on the SM coefficient,the similarity coefficient is 0.60～0.95,and the test materials are divided into four major groups,indicating that the kinship is closer.（6）A comparative study of SRAP and TRAP markers showed that the genetic diversity of ten species of wild shallot and garlic in Xinjiang is abundant,and there is no correlation between genetic distance and geographic distance.