RAPD And ISSR Analysis Of Genetic Diversity Of Seven Thymus Linn. Species

Plants of genus thymus linn. are to be generally called thyme,which belongs to Labiatae.With its use of edibility,medicine,aromatizetion,virescen- ce,environment protection and ornament and so on,thyme is a kind of aroma plant which has a great development potential.Presently,there is no report on the study of thymus linn. from the aspect of molecule in China, and especially the research of genetic diversity on DNA level is very inadequate,which restricts the development and the use of the genetic resource of thymus linn.This paper studys on the material of seven Plants of thymus linn. in Britain and Mongolian of china,using RAPD and ISSR-two kinds of molecular markers to compare their genetic diversities,and integrating the material of morphological and geographical distribution to do complete study and evaluation in the genetic diversity and relation of these plants of genus thymus linn..The main results are as follows:1.The method of improved CTAB was used to extract the DNA of species in Thymus Linn.,because the extracted DNA by the method was suitable for RAPD and ISSR analysis.2.Single factor design combining with orthogonal Design was used to obtain optimum scheme on reaction system for RAPD and ISSR of species in Thymus Linn..RAPD-PCR was performed in a 20μL reaction mixture with 2μL 10×Buffer Mg²⁺ free,80ng/20μl DNA,1.5U Taq polymerase,1.5mmol/L Mg²⁺,250μmol/L dNTP,0.8μmol/L random primer.The temperature profile used for RAPD-PCR was 94℃for 5 min;followed by 40 cycles of 94℃for l min,37℃for l min and 72℃for 1 min;and was terminated with a 10 min DNA extension step at 72℃;stored at 4℃.ISSR-PCR was performed in a 20μL reaction mixture with 2μL 10×Buffer Mg²⁺ free,20ng/20μl DNA,1U Taq polymerase,2mmol/L Mg²⁺,250μmol/L dNTP,0.8μmol/L primer.The temperature profile used for ISSR-PCR was 94℃for 5 min;followed by 35 cycles of 94℃for 0.5 min,50-56℃for l.5 min and 72℃for 1 min;and was terminated with a 7 min DNA extension step at 72℃;stored at 4℃. 3.The genetic diversity of Thymus Linn. revealed by RAPD and ISSR on 7 species level:Eighteen RAPD random primers amplified a total of 293 DNA loci,percentage of polymorphic loci is 93.5%,Shannon's information index,Nei's gene diversity and effective number of alleles were 0.4962,0.3278 and 1.5403,respectively.Thirteen ISSR primers amplified a total of 280 DNA loci,percentage of polymorphic loci is 95.7%,Shannon's information index,Nei's gene diversity and effective number of alleles were 0.5083,0.3376 and 1.5610,respectively.All of these two molecular markers revealed a highly genetic diversity in 7 species of Thymus Linn..However,all of these genetic diversity's parameters(PPB,I,H and Ne)of ISSR is much higher than that of RAPD.Plants of genus thymus linn. have richer genetic diversities and there are greater genetic variations among species, they are provided with the potential significances for further introduction and cultivation of new species with the use of germplasm resources, ornamental, ground cover plants, fixing sand and other aspects that adapt to the ecological needs of our country.4. The estimated genetic distance result got through the comparison of RAPD and ISSR,the two kinds of molecular markers shows that:the variation range of Nei's genetic distance value marked by ISSR is larger than the one marked by RAPD,and its genetic distance mean value is also larger than the one marked by RAPD,which further illuminates that ISSR marker can examine larger inter-species heritable difference than RAPD marker does.5.In the regard of the plant morphology,the most obvious characteristic of these seven species is the plant phenotype,that is creeping type or vertical type.In terms of the appearance,Thymus vulgaris, Thymus serpyllum and Thymus x citriodorus which are all vertical types,and all come from the Britain,show better consistency on the clustering results.Another main category is clustered of four species, namely,Thymus mongolicus,Thymus longicaulis,Thymus ciliatus and Thymus pannonicus are clustered into one category,these four species of patterns are all creeping type,among them Thymus mongolicus comes from Inner Mongolia in China,and the others are from the Britain.Molecular data and morphological characters revealed that:the species that their morphological characters were similar was tended to cluster together.At the same time,in terms of the information of geographical distribution,the six Thymus Linn. Species introduced from the Britain and one Thymus Linn. Species in Chinese Inner Mongolia are not completely separately clustered into one main category,which indicates that it lacks the inevitable contact between genetic relationship and geographical origin of molecular level.6. The results of UPGMA cluster analysis based on RAPD and ISSR fingerprint had the approximate trend,but not the same.The reasons may be as follows:1)The genome regions which are covered by two molecular markers are different,primers used by RAPD molecular markers are random primers, while those used by ISSR molecular markers are specific primers,the characteristics of the genome reflected by the two markers are different; 2)the numbers of loci of the genome reflected by the two markers are different.