Study On Genetic Diversity Of Endangered Fish: Leuciscus Merzbacheri (Zugmayer1912) And Introgression Among The Three Leuciscus Subspecies In Sailimu Lake Of Xinjiang, China Using ISSR Markers

Leuciscus merzbacheri (Zugmayer) is a genus of fish belonging to Cypriniformes, Cyprinidae, Leuciscinae, Leuciscus cuvier. Zugmayer is an indigenous medium-sized fresh or brackish fish and other two Leuciscus species (L. baicalensis and L.idus), distributed over Junggar Basin of north-region of Xingjiang, Northwest China. Resently, most populations all over drainages suffered severe demographic decline and habitat fragmentation due to unreasonable development and utilization. Thus, early in 1998, the native fish species have been listed as vulnerable species in the China Red Date Book of Endangered Animals and it was listed as Xinjiang Aquatic wildlife protection list in 2004. However, limited genetic information is available for this rare dace, especially at molecule level. So it is imminent to launch this work. Here we describe the genetic diversity and introgression among Leuciscus Subspecies in Sailimu Lake by inter-simple sequence repeat markers (ISSR).In order to investigate the optimal ISSR-PCR system for Leuciscus merzabcheri for the ISSR-PCR approach defected by its unstable working condition. So, at first phage, genome DNA of L.merzbacheri were used as template DNA and L9 (34) orthogonal design was adopted to optimize ISSR amplification system, which involved four principal factors, Taq polymerase, Mg2+, dNTPs and Primers at three levels, respectively. Then, we gained the optimized ISSR-PCR system with 1×PCR buffer,0.5U Taq DNA polymerase,3.0 mM MgCl2,250μM dNTPs,0.75μM primer and 30 ng template DNA in total 25ul reaction volume. Furthermore, we screening amount of 9 primers from 32 primers and obtained their suitable anneal temperature observed this system. The results set a basic step to analysis the genetic diversity of the rarely fish species.Furthmore, the genetic diversity of 91 individuals from six wild populations (including three populations of L.merzbacheri, two populations of L.idus and one population of L.merzbacheri) in Junngr Basin was investigated. Using the nine primers,193 unambiguous DNA fragments were generated with 178 (91.75%) being polymorphic, indicating a notable genetic variation at the species level. However, at the population level, a low level polymorphism with the percentage of polymorphic bands ranging from 9.28 to 47.94%. Under the assumption of Hardy-Weinberg equilibrium, the Nei's gene diversity (Hpop) ranged from 0.0384(SG) to 0.1615(BG) with an average of 0.0994 at the population level, whereas Nei's diversity index (H1) value is 0.3347 at species level. Shannon index (Ipop) ranged from 0.0561(BG) to 0.2419 (SG), with an average of 0.1496 at the population level and at species level the Shannon index (I1) value is 0.4985.Thirdly, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations both within and between species. As the ISSR markers revealed,45.36% of the total genetic diversity resided at the intraspecies level among populations, whereas 30% and 24.64% were present at interspecies and within a population respectively. The Nei's Gst (0.7039) and gene flow among populations (Nm=0.2103) revealed seldom gene exchanges happens among populations.The dendrogram created from UPGMA analysis based on Nei and Li's coefficient showed that the L. baicalensis and Leuciscus merzbacheri aggregate at a cluster and other cluster gathered conforming significant genetic differentiation. This is the first report of the partitioning of genetic variability of Leuciscus at the DNA level. The ISSR results suggest there is a low level of genetic variation present at Leuciscus species. Cluster of UPGMA analysis indicate a slight cross breeding might occurs between the two species.In the present study, ISSR-PCR makers were used for investigation the genetic diversity of Leuciscus merzbacheri of Xinjiang. That's will helpful for us to understand the rare dace's evolution history and evolution capacity. In addition, we also studied whether there is an introgression among the three Leuciscus and analysis the level of hybridization among these dace. These valueable results not only offer the basic background knowledge for future Cytogenetics study and also protect the rare indigenous fish.