Screening Of Trap Markers And Analysis Of Genetic Diversity In Common Carp(Cyprinus Carpio)

The newly developed TRAP (Target Region Amplified Polymorphism) was a reliable, stable and rapid PCR-based technique. There are several advantages of TRAP over other DNA marker techniques. First, it combines the favorable features of the AFLP (highly informative) and the RAPD (easy to perform) methods. Second, TRAP uses bioinformatics tools and EST database information to generate polymorphic markers around targeted candidate gene sequences, while RAPD and AFLP are generated by random anonymous sequences. TRAP maker has been effectively used in plant genetic breeding research. At present, the major markers applied in common carp(Cyprinus carpio) genetic structure research include SSR marker, RAPD markers and so on. But the literature of TRAP mraker applied in common carp genetic research hasn’t been found. In this study, this novel molecular marker was applied in common carp genetics and breeding research.The reaction system for TRAP makers of common carp one of the major cultured fishes, was designed in this paper. The factors of reaction system including Mg2+, dNTPs, Taq DNA polymerase, DNA template and primer concentrations were optimized and a stable, repeatable TRAP-PCR system for common carp was established. The PCR was performed with a final reaction volume of15μl with the following components:1.5mmol/L of Mg2+,0.35mmol/L of dNTPs,6pmol/L of each unlabeled700-or800-arbitrary primers,10pmol/L of the fixed primer,60ng of DNA template and1.0U Taq DNA polymerase. Thermal cycling consisted of an initial denaturing of template DNA at94℃for4min, then5cycles of denaturing at94℃for45s, annealing at35℃for45s, prolonging at72℃for lmin, followed by35cycles of denaturing at94℃for45s, annealing at53℃for45s, prolonging at72℃for1min, and a final prolonging at72℃for10min. The optimized TRAP marker reaction system was used to assess genetic variability among five populations of C carpio. Of the168bands amplified by10primer combinations,134(80.41%) were polymorphic. The average of polymorphism information content (PIC) was0.29. AMOVA analysis indicated that the genetic variation mainly came from individuals within populations (96.97%), while the inter-population components account for3.03%of the total variation. The cluster analysis based on TRAP analysis could really reflect the variation degree of these populations based on TRAP polymorphism of the target gene.TRAP markers were used to amplify and analyze the genomic DNA of three common carp populations including Jian Carp (JL), Huanghe Carp (HL), and the improved common carp (XY). Of the409bands amplified by28primer combinations,312(75.51%) were polymorphic. The average of polymorphism information content (PIC) was0.27. In our study, the highest and lowest average heterozygosity was found in XY population (0.2533) and JL population (0.2236), respectively. Nei’s genetic diversity index (h) are also presented, the averages in each populations were0.2103,0.2423and0.2076. The proportion of polymorphic loci, the most diverse group of the populations examined in our work was the XY population (65.22%) and the least was HL population (61.37%). The genetic distance and genetic identity results also suggested that the improved common carp inherited more genetic material from Jian carp than from Huanghe carp. Two specific molecular bands were also found in XY (200bp) and HL (220bp).In addition, candidate genes that maybe relates to the trait of growth were used to design TRAP’s fixed primer. The genetic structure of the improved common carp and growth trait associated with body weight and body length were analyzed using twenty-five polymorphic TRAP markers. Of the353bands amplified by25primer combinations,230(65%) were polymorphic. The average of polymorphism information content (PIC), Nei’s gene diversity and Shannon’s Information index was0.28、0.2193and0.3289, respectively, all of which indicated that the population genetic diversity was medium. Three loci (ghlTrap04-140%4rTrap04-308、 igf4Ga5-135) maybe associate with growth trait were examined by the procedures of generalized linear modeling (GLM) using SAS program. The result indicated that only the loci (4rTrap04-308) probably associate with both body weight and body length significantly (P<0.01), and need to identify in the future. In our study, a possible functional gene locus related to growth was found in common carp by the application of TRAP markers for the first time. It provided basis for locolization of QTL concerning improtant traits and marker-assistant selection.