Development Of Microsatellite Mark And Construction Linkage Map For Pinctada Martensii (Dunker)

The pearl oyster, Pinetada martensii Dunker, is the primary economic shellfish used for marine pearls production in China. Early on1965artificial breeding of this species has been successfully carried out, since then the scale of its farming expand fastly and continually, and now it becomes the pillar industry in coastal areas of Guangdong, Guangxi and Hainan province. However, the long-term artificial breeding has lead to many serious problems, e.g. germplasm degradation, significantly lower genetic diversity and the decline of pearl yield and quality. Therefore, there is an urgent need for improving its varieties in production. Construction a linkage map is the most effective way to study genetic characteristics and could be used to facilitate its marker-assisted breeding.Microsatellite marker has the characteristics of high polymorphism, stable amplification, codominance and it is easy to automated testing. Those advantages made microsatellite become an important tool for the study of population genetic structure, molecular marker assisted breeding and protection of germplasm resources in economic shellfish. In this study, we isolated microsatellite loci from an enriched genomic library, and developed373microsatellites markers (171EST-SSR markers and202genomic). A male and a female, who are significantly different in phenotype, were selected as parents to construct a full-sibs family. With12microsatellite markers heterozygous in parents for paternity test in whole family,94offsprings and parents were selected to construct the genetic linkage map.We characterized sixteen polymorphic markers, including8genomic SSR and8EST-SSR, in a wild population and a full-sib family. In the wild population, all16markers were polymorphic, The number of allele in genomic SSR marker and EST-SSR marker are2±13and3～6, respectively. Expected (He) and observed (Ho) heterozugosity is0.78±0.13(0.54-0.90) and0.71±0.16(0.47-0.97) for genomic SSR marker, and0.65±0.03(0.60-0.69) and0.67±0.23(0.41-0.94) for EST-SSR marker, respectively. Four (50%) genomic-SSR and two (25%) EST-SSR deviated from Hardy-Weinberg equilibrium (P<0.05after Bonferroni correction). In the full-sib family, null allele is present at two loci, one genomic-SSR and one EST-SSR. Seven genomic-SSR and five EST-SSR are heterozygous segregating in at least one parent. Five out of seven genomic-SSR markers and four out of five EST-SSR markers segregations departed from the Mendelian segregation. The separation ratio of six genomic-SSR and three EST-SSR are1:1:1:1.Sex-specific linkage maps were constructed for Pinctada martensii using174microsatellite markers. Linkage mapping was performed on the full-sibs family. The female linkage map contained56markers in13linkage groups while the male linkage map had66markers in12linkage groups. The female linkage map spanning a total genetic distance of579.1cM, with an average interval of15.22cM; the male linkage map covered651.9cM, with an average interval of13.05cM. The genome coverages are59.4%and67.6%, respectively. An integrated map was constructed by incorporating the homologous parental linkage maps, producing12coupled linkage groups. Among all the coupled and uncoupled groups there are14groups having more than4markers, which is equal to the result of karyotype analysis. The integrated map spans972cM, with an average interval of12.15cM. The genome coverages of the integrated map was approximately70.56%. Theses maps are the first microsatellite linkage maps for Pincatada martensii and should be useful for further genetic analyses such as QTL mapping and MAS.