Study On The Development Of Microsatellite Markers For Crassostrea Sikamea

Kumamotooyster(Crassostrea sikamea)is naturally distributed in Japan,China,Korea,and introduced to the United States for breeding.In China,Kumamoto oyster is the fourth economic species of oyster.With the expansion of the scale of breeding,the genetic diversity of the cultured population may be reduced.And problems such as the degradation of germplasm resources may arise.Therefore,the development of microsatellite primers for Kumamoto oysters,the investigation of the germplasm resources and genetic diversity of Kumamoto oysters,is of great practical significance for the breeding of Kumamoto oysters and the promotion of their breeding industry.(1)Using high-throughput sequencing,we characterized 20 pairs of polymorphic microsatellite primers from the Crassostrea sikamea genome,and we examined the markers in a wild population.A total of 330 alleles were found in 20 microsatellites.The observed number of alleles(Na)ranged 6~39 in average of 16.5,and the effective number of alleles(Ne)ranged 1.3529~33.3617.The observed and expected heterozygosity values range 0.2000~1.0000 and 0.2656~0.9877,respectively.The Shannon Wiener index ranged 0.6483~3.5858 and Polymorphic information content(PIC)ranged 0.2545~0.9692.16 microsatellite markers accord with Hardy-Weinberg equilibrium(HWE).Using these loci to analyze the genetic diversity of a cultured population,the average number of alleles was 10.25;the average number of effective alleles(Ne)was 5.8434.The observed heterozygosity(Ho)average was 0.6391;the expected heterozygosity(He)average was 0.7636;the Shannon-wiener index(I)average was 1.7914;and the polymorphic information content(PIC)average was0.7207.It was found that the genetic diversity of the C.sikamea cultured population was lower than that of the wild population,but it maintained a high-genetic diversity.The results of this study indicate that in the artificial breeding process of C.sikamea,the use of a large number of parents for breeding can effectively prevent the genetic diversity of the cultured population,but artificial breeding also has a certain impact on the genetic diversity of the cultured population.The 20 loci were surveyed the crossspecies proportions in Crassostrea angulate,Crassostrea gigas,Crassostrea hongkongensis,Crassostrea ariakensis,Saccostrea cucullata,Ostrea mordax,Hyotissa hyotis.Locis XB1-6,XB1-39,and XB1-45 can amplify the target band in 8 species,and the loci XB1-41 can only amplify the target band in C.sikamea.(2)Using a magnetic bead enrichment method,18 pairs of polymorphic microsatellite primers were developed from Kumamoto oyster.Primers were evaluated in 26 individuals from the wild population of Zhuhai,and 18 microsatellite markers showed high polymorphism in the wild population..A total of 266 alleles were amplified,and the observed number(Na)of alleles ranged from 2 to 33,and the average was 14.7778.The effective allele number ranged from 1.960 to 26.8889;the observed heterozygosity(Ho)ranged from 0.0714 to 0.9583;the expected heterozygosity(He)ranged from 0.5017 to 0.985;and the Shannon-wiener index(I)ranged from 0.6829 to 3.4061;The polymorphic information content(PIC)ranged from 0.3699 to 0.9615.Ten of these loci deviate from the 2.4 HardyWeinbergequilibrium,which may be related to factors such as homozygous excess,founder effect,and null allele.Because ofthe high polymorphism these primers and can be used for subsequent research on individual identification of Kumamoto oysters,population genetic diversity research,genetic structure analysis,and construction of genetic maps.