The Development Of CSNPs In Zhikong Scallop(Chlamys Farreri) Transcriptome And Construction Of Haplotype

1.SNPs identification for Zhikong scallopIn this research,five hundred contigs based on 454 sequencing of the transcriptome of Zhikong scallop(Chlamys farreri Jones et Preston) are selected randomly,then primers and probes are designed for candidate sites.Two hundred and fifty-nine pairs of primers are amplied successfully,which take account for 65.8% of all the designed.The sizes of products between 70 bps and 900 bps,and then primers of products between 70 bps and 150 bps are picked.Six individuals are used for testing validation of probes,getting 101 sites for genotyping later.Consequently,44 cSNPs(exclude indel) are identified following high resolution melting(HRM) by taking 48 representative unrelated individuals,which orignate from 5 populations. The 44 loci are bi-allelic loci including 34 transition and 10 transversion,which origin from 27646 bps of cDNA. They account for 17.4% of all the candidates,equivalent to 630 bps/SNP.Forty-eight individuals from Qingdao population are utilized for charactering.Over all,HE range from 0.100 to 0.505,HO distribute from 0.1042 to 0.875. Four of analyses exhibited non-Mendelian segregation ratios before Bonferroni correction, with 0.8% significant after correction, demonstrating that SNPs markers can be considered Mendelian traits. There are 40 loci conformed to Hardy-Weinberg equilibrium,which are useful for many studies. These results suggest that the transcriptome of Zhikong scallop has relatively high degree of variation. These markers are potentially useful for evaluation of genetic diversity and population structure of Zhikong scallop.2.Construction of Haplotype using SNPsIn this part,SNPs are used to haplotype construction.Firstly,contigs contained typing SNPs are clustered to get 8 blocks,which are about 376 bps to 2359 bps in length and 972 bps of average space,using 48 individuals from Qingdao population.Every block have three or four typing SNPs. Block C34869CHF and C5273CHF have a concordant sequence length to expected for PAGE testing. Then PHASE2.1 procedure are used for haplotype construction of eight blocks,and frequency of calculation of haplotypes.Consequently,8 to 16 alleles are inferred in all the blocks,which have 12 alleles in average.Particularly,block C16019CHF and C5471CHF contain three and two major forms respectively.Frequencies of other alleles are distribute from 0.000206 to 0.069202,displaying prominent differences.Moreover,there are 28 and 29 individuals could be sure in forms of haplotype completely for the two blocks.Distinctly,16 alleles of block C34869CHF exhabit similar frequencies,and just one individual could be make sure for haplotype.Other five blocks have parallel characters to block C16019CHF and C5471CHF.At last,the LD analysis between two loci are carried out.When P<0.05,the loci of 8 blocks have no LD relationship in Qingdao population.