Genetic Structure And Genetic Diversity In Chinese Populations Of Black-spotted Frog (Pelophylax Nigromaculata) Revealed By Inter Simple Sequence Repeats (ISSR)

Chapter 1:The purpose of this review is to introduce principles, characteristics and applications of a nuclear DNA marker, inter simple sequence repeats (ISSR), which is used widely in the genetic research.Chapter 2:Inter simple sequence repeats (ISSR) polymorphism was used to examine the genetic structure and genetic diversity of black-spotted frog, Pelophylax nigromaculata, in temperate and subtropical China. Twenty-five ISSR primers were screened and 10 primers were selected for their ability to produce clear and reproducible patterns of multiple bands. A total of 127 DNA bands were amplified, 103 of which were polymorphic and the percent of polymorphic bands (PPB) was 81.10%. A relatively high level of intraspecific genetic diversity was revealed: He = 0.1518, I = 0.2233 and PPB =38.67% at population level; He = 0.2664, / = 0.4000 and PPB = 81.10% at species level. This study demonstrated that the genetic polymorphism in the populations of the middle and east China was higher than that of the peripheral populations in north, south and southwest China.The twenty-eight populations were divided into two groups on the basis of unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis. The largest group which included most populations was defined as mainland lineage A. The other group was northeast lineage B which only included a few populations in northeast China. Further more, there were two sub-lineages within the mainland lineage A: lineage Al and lineage A2. Lineage Al covers most of the species' range in China, A2 occurred in southwest plateau.Genetic distance (Fst) between populations ranged from 0.15181 to 0.77390, with an average of 0.50086. The genetic distances between lineages A and B were generally higher than the average and that between populations within lineages A or B. The results indicate the deep genetic differentiation between lineages A and B. Inaddition, we also found that lineages A and B coexisted in DL area in northeast China, and there were gene flow between the two lineages. We concluded that this area is the second contact zone of lineages A and B.