The Study On Marking Techniques And Screening Of SSR Markers Associated With Cold Tolerance Of Large Yellow Croaker

Large yellow croaker [Pseudosciaena crocea (Richardson)] is an economicallyimportant marine species which is dying out as a result of overfish in China. The resourcesof large yellow croaker is severely depleted and faces many problems such as growthslows, poor immunity, weak tolerance, the individual becomes smaller,early sexualmaturity and so on, It is serious to P. crocea (Richardson) aquaculture that P. crocea(Richardson) can not survival in the winter in Zhejiang sea area. So how to improve coldtolerance of cultured large yellow croaker and breeding germplasm improvement is anextremely urgent. Through the group selection method, this study selected larvae of thelarge yellow croaker of Dai Qu Yang(cold tolerance F2generation reserve parent fish) asthe research samples, preliminary explored the two traditional sign technology’s(VIE andPIT)influence on the growth of large yellow croaker, finally got a better mark method.Onthe other hand, we had experiments to cold tolerance F2generation reserve parent fish withlow-temperature stress, the individuals associated with cold tolerance of large yellowcroaker were preliminary screened,then DNA microsatellite which is used widely onstudying biological diversity, classifying germplasm resource, identifying variety and soon,was employed to assess the genetic variations between the cultured populations of largeyellow croaker and screened a microsatellite mark locus associated with cold tolerance.Combined traditional tagging technology with modern gene marking technology,we offereda better tagging method and efficient molecule marking means for the study of the breedingand cultivating of follow-up large yellow croaker colony which associated with coldtolerance. The main results are as follows:1. The preliminary study of VIE and PIT on large yellow croakerGroup selection always have many groups, when the number of groups is more than2groups, using tagging technology can no only promise a unanimous breeding environmentand effective breeding result,but also reduce cost of aquaculture facilities and labor cost,makes the work of breeding easier and effectively. We marked the simples of large yellowcroaker with VIE and PIT in April2013,there were1200fishes divided into three groups.The results showed that the data of weight and body length growth were not significantlydifferent between VIE and PIT. But VIE was better than PIT in comparing the survival ratebetween which the data were significantly different (p<0.05). And the off-tag rates werelow and not significantly different (p＞0.05). The VIE had little influence on survival and growth than PIT,it was also simple to handle and lower cost(less than the12.5%ofPIT).so it turned out that VIE is more suitable for the next step of marking large yellowcroaker.2. Genetic diversity analysis and screening of microsatellite markers associated withcold tolerance of large yellow croaker (Pseudosciaena crocea R.)We had a experiment of artificielle hypothermie on3000large yellow croaker of DaiQu Yang which the body length were in9.2～11.5cm in January2013,then the selected100individuals of Dai Qu Yang F2offsprings of cold tolerance group and normal group of largeyellow croaker were applied to detect genetic differences.Each group contained50randomly and separately sampled individuals.The results showed that: there were total109alleles were detected by used13microsatellite marker loci in the two groups of largeyellow croaker of Dai Qu Yang, then observed85alleles,6.49average effectivealleles,observed heterozygosity was0.89, the average expected content heterozygosity was0.85.The average polymorphism information in2populations is0.81, all highlypolymorphic, indicating that13microsatellite loci in the breeding of Dai Qu Yang largeyellow croaker populations showed high polymorphism, genetic diversity is relativelyabundant. They could be good breeding materials.The research on the microsatellitemarkers associated with cold tolerance of large yellow croaker showed marked LYC0015were amplified in five alleles (fragment size was112,110,108,106and104bp) in twosamples, which the appear frequency of allele LYC0015112bpwas48%at the group of coldtolerance, while it was zero in the normal control group, indicating that the allele was moresensitive to temperature in large yellow croaker of Dai Qu Yang, there may be somelinkage relation with some kind of cold tolerance genes,this result can provide basicinformations for the study of breeding cold tolerance populations of Dai Qu Yang largeyellow croaker in the future. The SSR analysis of population genetic diversity betweencold tolerance populations and non-cold tolerance populations showed that showed ahigher overall level of polymorphism information content of the two large yellow croakerpopulations (PIC>0.5), there were rich polymorphism which had a good selection fertilitypotential. Through screening of the13microsatellite markers, the conclusions whichLYC0015sites was related to cold tolerance of large yellow croaker was further validate.