RAPD And ISSR Analysis Of Genetic Diversity In Musard Germplasm Resourse

Mustard(Brassica juncea Czem et Coss.) is one of the important vegetables originated from China.Classification of mustard had been conducted for almost one century.But the views are different and controversial.The genetic diversity of 28 mustard accessions was investigated using RAPD and ISSR markers.The two assays differed in the amount of polymorphism detected.The study showed the results as follows:1.The technique of mustard DNA extraction was established:with fresh young leaves,using the developed CTAB method,some measures were taken to prevent oxidation of phenolic compounds by adding a proper amount of PVP during grinding,and DNA sample extracted by the methods had high purity.2.The RAPD and ISSR analysis system were optimized:the concentration of 40ng DNA templet, 10pmol/μl primer,10×PCR buffer,0.3mmol/L dNTPs,1UTaq polymerase,2.0mmol/L Mg²⁺ in the 20μL RAPD reaction system.The optional annealing temperature for RAPD-PCR reaction was proposed by gradient PCR.the concentration of 40ng DNA templet,10pmol primer,10×PCR buffer, 0.5mmol/L dNTPs,1Utaq polymerase,2.0mmol/L Mg²⁺ in the 20μL ISSR reaction system.The optional annealing temperature for ISSR-PCR reaction was proposed by 51℃gradient PCR.3.Twenty RAPD primers were screened out from 300 RAPD primers for PCR amplification of the 28 kinds of mustard DNA,generated a total of 162 clear and reproducible bands of which 140 were polymorphic,with an average of 7.0 polymorphic fragments per primer.Eighth ISSR primers were screened out from 50 ISSR primers for PCR amplification of the 28 kinds of mustard DNA, generated a totol of 143 clear and reproducible bands,of which 124 were polymorphic,with an average of 6.9 polymorphic fragments perprimer.4.The value of GS(genetic similarity indexes) was estimated between 0.55 and 0.96 of 28 mustard materials based on the RAPD analysis and the average was 0.72.The polygenetic tree of the genetic relationship in 28 individuals was drawn.It showed that when the value of GS was from 0.72 to 0.76,28 kinds of mustard materials could be divided into three categories.The value of GS was estimated between 0.57 and 0.96 of 28 materials based on the ISSR analysis,and the average was 0.74.The polygenetic tree of the genetic relationship in 28 individuals were drawn.It showed that if when the value of GS was 0.72 to 0.76,28 kinds of orna-mental gourds materials could be divided into three categories.5.The value of GS was estimated between 0.58 and 0.97 of 28 materials based on the RAPD and ISSR analysis,and the average was 0.76,which was between RAPD and ISSR,but it was of no significant difference。The polygenetic tree of the genetic relationship in 28 individuals was drawn. It showed that when the value of GS was from 0.70 to 0.74,28 kinds of mustard materials could be divided into three categories.It was closely similar with the RAPD and the ISSR,Comparatively,it was more similar to the latter one.The two methodologies gave different views of the amount of variation present,but both of them showed a high level of genetic variation in mustard.The following conclusions can be drawn from this study whether based on RAPD data and ISSR data:(1)the degree of genetic variation within the studied accessions was notably great.(2)The DNA-based markers would be more useful in detecting genetic diversity in closely related accessions but the capicity of different molecular markers for classifying the genetic relationship of closely related accessions were different In addition,a dendrogram that took into account all fragments produced by RAPD and ISSR reflected better the relationships than made dendrograms based on only one type of markers.