SSR Batches Developments And Their Application In Genetic Diversity Of Selective Strain And Pearl Sac Of Pearl Oyster Pinctada Martensii

The pearl oyster Pinctada martensii is one of the main oysters to produce seawaterpearl in China.We obtained102,762Unigene sequences by transcription library analysis of pearlsac.99,127expressed sequence tag (EST) without redundancy was developed. Thoroughlyscreened with the software of Misa, a total number of6034sequences containing SSRwere found and the ratio was6.1%to EST group library. Using Primer3software designEST-SSR,6595microsatellite loci were found but only4487loci could be used.6368wereperfect microsatellite loci and227loci belong to compound type.96.6%and3.4%of totalsite respectively. In perfect type, the trinucleotide and tetranucleotide repeats were mostcommon type in SSRs, accounting for30.3%and40%of the total site respectively. Themean ratio of amplification and the mean percentage of polymorphism site accounted for82.3%and60.6%of total sites respectively. Use EST sequence data, the synthesis ofpreliminary and screening for1400, of which the effect is good324. Genetic diversity ofyellow shell colored line, base population and fasting-growth line were detected by50polymorphism primers. A total of200alleles were detected across the50loci assayed. Allthe loci were polymorphic and the number of alleles ranged from2to8, with a meannumber of4alleles per locus and PIC values was0.413. A large number of polymorphismmicrosatellite site exploitation could provide effective tool for molecular genetics, pedigreeanalysis and wild resources protection research of the species.In April of2003, the base stock was developed by selecting yellow coloredindividuals as parental breeders in the Liushagang stocks. During the period of2004-2007,a successive three generation selection for yellow color in the base stock was carried out toproduce the third generation yellow colored line. In May of2010, the genetic variation ofthe third generation yellow colored line and base stock were evaluated by ten SSR primers.A total of40alleles were detected across the10loci assayed. All the loci werepolymorphic and the number of alleles ranged from2to6, with a mean number of4allelesper locus. The average effective allele numbers, observed heterozygosity, expectedheterozygosit and PIC values of the third generation yellow colored line and base stock were2.2862and2.2646,0.4119and0.4403,0.5333and0.5464,0.4722and0.4655,respectively. The genetic differentiation index was0.0389. This indicated that there existeda high level of genetic variation in the third generation yellow colored line after threegeneration selection for yellow shell color and SSR marker was suitable for investigatinggenetic variation of pearl oyster stocks.Thirty-six families were established by selecting mature breeders in the thirdgeneration selected line. Two families randomly sampled from the families were subjectedto nucleus-inserting operation. At days60after nucleus operation, pearl sac, muscle andmantle were sampled from the host oysters. The origin of pearl sac was detected by10SSRprimers. The results showed that the primer2-PM162、4-PM358and1-PM38weredetected in host oysters. Mixed cells in the connective tissue around the pearl oyster wereobserved by light microscope. The results showed that the cells of pearl sac originatedfrom the donor and host oysters.