Development Of SNP Markers In Psetta Maxima By High-resolution Melting(HRM) Analysis

1. The new way of the development of SNP marks established for our laboratoryMolecular marker has wide application prospects in high density genetic mapconstruction, character drawing and gene precise orientation, analysis of group geneticstructure and System growth etc. Single nucleotide polymorphisms (SNPs) molecularmarker is the third generation of molecular markers after the restriction fragmentlength polymorphism (RFLP) and the microsatellites (SSRs). It has vast quantities ofSites, Covers density and spreads over almost the entire genome. It is easy to amplifyfor short fragment, and easy to high throughput automatic analysis and to research ofgenetic mechanism. The new way of the development of SNP mark is established bycombining the High-resolution melting technology with bioinformatics analysis forlaboratory in this paper. The technical route is: Apply Megablast-VectorNTI softwareto cluster homologous sequences of the same species，and to join them together，thenscreen the putative SNP from the assembled contigs；And then design primers andprobes for Asymmetric PCR to amplify out products of target fragments containingsingle stranded ones；Put the probe join into the amplified product to make it hybridwith single stranded target fragment. Finally, put the hybrid products into the LightScanner analyzer for data acquisition, and apply related software for genotyped SNPsite to confirm it. The software used in the method is simple to operation, and theinstrument realizes closed tube operation with low cost, high precision, highthroughput. And it greatly reduces possible error from contamination in thisexperimental process. This method does not need the premise of the known mutations.It can intuitively and fast detect individual genetic variation of SNP sites and obtainindividual genotype in the site. It is good to lay the foundation for breeding workcombined with the traditional method for the future.2. Development of SNP markers in Psetta maximaThe experimental mines all Psetta maxima12428EST sequence in NCBI database. Putative SNPs were detected from assembled contigs using Vector NTIAdvance11. On the basis of the analysis of the contigs, strictly abide by the putativeSNP screening standards to select, then according to the screening of the putative SNPsite design primer and probe. SNP genotyping was performed using high-resolutionmelting (HRM) method. Primers and probes were designed using Primer5and Oligo7,respectively. Through steps of the asymmetric PCR and the molecules hybrid,Forty-two individuals from10character populations from8familys were used forvalidation of marker polymorphism. Data were retrieved and analyzed using LightScanner software. The results show that, from the analysis of the sequence obtain1457contigs and2738single sequences. In1457Contigs, the maximum of Contig size is411, average of6.65,522contigs Contain four and above EST sequence. More than160putative SNPs were found from522contigs,56of which were used in this studyfor marker development.37pairs of primers were successfully amplified, including45putative SNP site. The success rate is66.1%. After asymmetric PCR, probes which canhybrid with putative SNP sites have13. Hybridization Rate is28.9%.8sites ofhybridization displayed polymorphisms in different character populations, accountingfor61.5%of the hybrid sites. There are6transition sites, namely the G-A and C-T are3of each. There are2transversion sites, namely the T-G and A-T are1of each. SNPmark was developed in this paper based on the public database of EST sequenceinformation. It may be closely related with expressed genes or located genetic codingzone directly. It will lay the foundation for the genome compare, the gene location andcloning research, and it lay the foundation for genetic improvement, cultivating fineproperties such as good quality，high yield, Strong resistance of new varieties of studyfor the future. The other work finished during the master’s stage:Gynogenesis familys construction of Psetta maximaHeterologous sperm meiosis gynogenesis condition has been found out in thispaper in a brief. Fixed cold shock water temperature0±0.2℃, the cold shock startingtime6min30s, the cold shock duration25min, set gradient of ultraviolet radiation dose:250μj/cm2×1min、250μj/cm2×0.9min、250μj/cm2×0.8min to grope for best uv radiation dose. Fixed the sperm illuminate dose of250μj/cm2×0.9min and cold shocktreatment duration25min and treatment the water temperature0±0.2℃, set gradient ofcold shock start time after fertilization5min30s、6min30s、7min30to grope for bestcold shock starting time. On the basis of experiment result for conditions construct fivePsetta maxima heterologous sperm meiosis gynogenesis familys, and observe processof embryonic development and larva cultivation of gynogenesis family, and do growcontrast experiment between gynogenesis familys inside and the normal developmentdiploid filial generation. The results show that, in cold shock water temperature is0±0.2℃, the cold shock lasts for25min conditions, using pagrosomus major frozensperm induction Psetta maxima meiosis gynogenesis for the best processing conditionsis uv radiation dose250μ j/cm2×0.9min, cold shock starting after fertilization for6min30s. On the basis of experiment result for conditions construct five Psetta maximaheterologous sperm meiosis gynogenesis familys, Treatment of eggs differ from200mlto400ml. Get more than5000gynogenesis larvas then get more than40017monthsgynogenesis diploid filial generation after cultivation. The experiment has provedgynogenesis the stability of the condition. It provides a theory basis for the research ofgynogenesis application. The gynogenesis is a very ideal genetic gender controltechnology, using in production practice it can greatly enhance the breeding productionand increase economic benefits. At the same time, the gynogenesis technology canquickly build some pure familys of species of fishes. It has a broad prospect ofapplication to fish breeding job.