Screening Of Trap Markers And Analysis Of Genetic Diversity In Red Tilapia(Tilapia Mossambica)

Tilapia (Oreochromis spp) is one of most important economic breeding fish in the world, which has been strongly recommended by the FAO as an important source of animal protein. Red tilapia were imported from Malaysia by Freshwater Fisheries Research Center of Chines Academy of Fishery Science,2009, they have been domesticated and began mixing breeding. TRAP (Target Region Amplified Polymorphism) which is a PCR-based molecular marker was developed about sunflower. It got the attention of the molecular breeding due to its simple, efficient, and reliable and easy to be sequenced. TRAP uses EST or cDNA sequence information and bioinformatics tools to design fixed primer polymorphic markers tend to produce tightly linked to the target gene sequences. TRAP has been used in the analysis of genetic diversity of germplasm resources, screen the important growth traits marker, construction genetic map and molecular marker-assisted breeding. At present, the main method of the analysis genetic structure in tilapia were with SSR and SNP markers. TRAP markers for analysis of the genetic structure of tilapia reported hasn’t been found. The TRAP markers were applied in the tilapia breeding research.First, this study conducted apreliminary analysis of basic morphological traits of red tilapia, morphometric measurement was tanken on1182-year-old tilapia sampled. And calculated the correlation coefficient between body weight,body length, body width and body height.The coefficients, determination coefficients and correlation index were calculated.The mulptiple regression equation about body weight was developed as Y=-474.877+1.813Χ1+5.874Χ2+2.709Χ3, while y is body weight,Χ1Χ2, Χ3is body length, body width, body height respectively.Secondly, in present study, we select the optimal the stable and repeatable reaction system of TRAP marker in tilapia using L16(45) orthogonal experiment and single-factor trail were designed to.The analytical results indicated that the dNTPs concentration, random primer concentration and DNA concentration were same, while the concentration of Mg2+and Taq polymerase dosage vary in the two approaches.We also observe the interaction between different factors. The orthogonal design is simpler, more scientific and more reasonable than single factor design. The optimal TRAP reaction system for tilapia contained60ng of DNA template,1.5mmol/L of Mg2+,0.3mmol/L of dNTPs,0.5U of Taq DNA polymerase,7pmol/L of random primers and10pmol/L of the fixed primer in a final volume of15μL. The amplification program was:predenaturing at94℃for2min; denaturing at94℃for45s, annealing at38℃for45s, and prolonging at72℃for lmin, the reaction ran5cycles followed by other35cycles of denaturing at94℃for45s, annealing at53℃for45s and prolonging at72℃for lmin, and finally prolonging at72℃for8min. The stability and repeatability of the reaction system were verified in the four populations of tilapia. The TRAP marker could be used in evaluation of genetic diversity, germplasm identification, molecular marker-assisted breeding and other related research in tilapia.Furthermore, in order to obtain TRAP markers about the growth traits in red tilapia, the optimal TRAP reaction system for tilapia were used to analyze the genetic diversity of red tilapia group and used of the t test to screen the molecular markers associated with the growth traits(body weight, body, caudal peduncle length).The TRAP amplified results showed that of the251bands amplified by15primer combinations,216were polymorphic loci, the average proportion of polymorphic loci was86%. The average polymorphism information content(PIC), Shannon’s information index, Nei’s genetic diversity index of the studied population was0.21,0.466,0.316, respectively. The result indicated that the loci ghl-Trap1380005maybe association with weight and body significant (P<0.01), and ghl-Em110with caudal peduncle length and weight by preliminary screen. The Path analysis results and growth associated molecular markers can be combined to provide a reference for assisted breeding of red tilapia.