Isolation Of Microsatellite From Genome Of Leuciscus Waleckii Dybowski And Genetic Diversity Of The Wild Populations Of Five Leuciscus

Magnetic beads enriched method was used to isolate microsatellite DNA from the genome of Leuciscus waleckii. Firstly, the genomic DNA was extracted and digested with restriction enzyme Sau3AI, and targeted segments of 400-900 base pairs were collected by centrifugation of sucrose density gradient and ligated to adaptors.Then, the purified ligated–DNA was hybridized with biotin-labeled probes (CAG)8 , (TGA)8 ,(AGAT)6 and (GATT)6. After capture of target fragments through magnetic beads and enrichment by PCR amplification, the selected DNA fragments were cloned into the pMD18-T vector and transformed into competent E.coli DH5α. As a result, 1416 positive colonies were obtained through PCR screening and of 1047 colonies were sequenced successfully by the biological company. Sequencing of these positive colonies confirmed that 774 (73.94%) clones contained 1084 microsatellite loci. Among these sequences, 737 repeat motifs (67.99% ) were perfect, 166 repeat motifs (15.31%) were imperfect and 181 repeat motifs (16.70%) were compound. Approximately 105 primer pairs were designed successfully from 160 microsatellite loci with flanking sequences. Primer screening showed that 56 primer pairs can amplify target bands, and of 23 pairs are polymorphisms. 18 polymorphic microsatellite markers were selected to analyze the genetic structure of one population of L.waleckii sampled from Dali Lake. The result showed that the number of alleles ranged from 3 to 11, the average Ho and He were 0.6056 and 0.6288 separately, and the polymorphic information content (PIC) ranged from 0.2077 to 0.8820. The statistic results indicated that 67% loci (12/18) were in high polymorphic level (PIC>0.5).In this study,five populations of Leuciscus, totally 203 individuals, were screened for 24 microsatellite loci. Two Loci, HLJYL002 and HLJYL106, could amplified just in one population, and have alleles characterized by large deletions in other populations. And locus HLJYL007,HLJYL031,HLJYL042,HLJYL048,HLJYL052,HLJYL092,HLJYL100,HLJYL101,HLJC114�'�HLJC116 could amplified in four populations respectively. In these 5 wild populations, the average number of allele (A) was 7.78 to 11.65; the average number of effective allele (Ne) was 4.91 to 6.81; the value of average observed heterozygosity (Ho) in six populations was 0.6043 to 0.7077; the value of average excepted heterozygosity (He) was 0.7618 to 0.8313; and the value of average polymorphism information content (PIC) was 0.7174 to 0.7970.Hardy-weinberg equilibrium analysis revealed that at loci HLJYL035,HLJYL048 of Leuciscus baicalensis ; loci HLJYL035,HLJYL042,HLJYL046,HLJYL052,HLJYL101,HLJYL110 of Leuciscus waleckii from the Dali lake; loci HLJYL031,HLJYL046,HLJYL048,HLJYL110,HLJC57 of Leuciscus waleckii from the Songhua River showed equilibrium. All others loci showed disequilibrium.The genetic distance of five wild populations was calculated and cluster analysis was also carried out. The results showed that the populations of Leuciscus baicalensis and Leuciscus idus were similar, and Leuciscus brandti has close genetic distance to them. While the populations of Leuciscus waleckii from the Dali lake and Leuciscus waleckii from the Songhua River were similar.