| Kumamoto oyster(Crassostrea sikamea)belongs to Mollusca,Lamellibranchia,Ostreidae,and Crassostrea,also call them “alias oysters”.It is a brackish water type with a preference for high-salinity Oyster group.Native to Southeast Asia,it is naturally distributed in China,Korea and Japan.Because of its good taste,rich nutrition,and high economic value,it was introduced to the east coast of the United States in 1943 and cultivated in a large area.In China,Kumamoto oyster is one of the important farming species in the coastal areas of East and South China,mainly distributed in some coastal areas such as Fujian,Guangdong and Zhejiang.However,due to breeding needs,the use of a small number of individuals for multi-generation breeding may result in a reduction in the genetic diversity of the breeding population and degradation of germplasm resources.Therefore,through the development of Kumamoto oyster microsatellite primers,the investigation of Kumamoto oyster germplasm resources and genetic diversity,the selection and breeding of Kumamoto oysters to promote the development of its breeding industry has very important practical significance.In this study,the magnetic bead enrichment method was used to develop highly polymorphic microsatellite molecular markers from the Kumamoto oyster genome,and 13 pairs of polymorphic primers were used to analyze the genetic diversity of 2 farmed populations and 7 wild populations(Every group were randomly sampled to collect 20 ～ 40 samples.After molecular identification,the number of experimental individuals was determined to be a total of 287),which provided a theoretical basis for the breeding of good varieties and the protection of germplasm resources.The main results of this study were as follows:(1)Development of Kumamoto oyster microsatellite by magnetic bead enrichment method.A total of 13 polymorphic microsatellite molecular markers were developed from the Kumamoto oyster genome,and the 13 sites were analyzed and evaluated for genetic diversity using the wild Kumamoto oyster population(26 individuals)in Zhuhai,Guangdong.A total of 133 alleles were amplified from all loci.The number of alleles(Na)varied from 2 to 22,the number of effective alleles(Ne)was 1.9538 ～ 16.4878,and the observed heterozygosity(Ho)was 0.0769 ～ 1.0000,the expected heterozygosity(He)was 0.0477 ～ 0.9578,Shannon-wiener index(I)was 0.6813 ～ 2.9285,the range of polymorphic information content(PIC)was 0.3690 ～ 0.9360,and the average PIC value was 0.7274.The results show that the 13 microsatellite markers developed have high polymorphism,and can be used for the analysis of Kumamoto oyster population genetic diversity,individual identification,breeding and breeding.(2)Genetic diversity analysis of Kumamoto oyster breeding and wild populations.Using the 13 Kumamoto oyster microsatellite molecular markers developed above to synthesize corresponding fluorescent primers,the genetic diversity of 2 Kumamoto oyster farming populations and 7 Kumamoto oyster wild populations were evaluated respectively.The results showed that a total of 1023 alleles were obtained from all loci after amplification.Among the two cultured populations: the Kumamoto oyster cultured population in American(The fifth generation of group breeding)(MG),the average number of alleles(Na),effective alleles(Ne),observed heterozygosity(Ho),expected heterozygosity(He),Shannon-Weiner index(I),and polymorphic information content(PIC)were: 9.0000,4.1342,0.7212,0.7158,1.5332,and 0.6630.(2)The average value of each genetic parameter(same as above)of the Kumamoto oyster cultured population in American(the second generation of population selection)(AF2)were: 6.9231,3.8111,0.6766,0.6750,1.4318,0.6294.Among the seven wild populations:(1)the wild population of Kumamoto oyster in Guangdong Zhanjiang(ZJ),the average number of alleles(Na),effective alleles(Ne),observed heterozygosity(Ho),expected heterozygosity(He),Shannon-Weiner index(I),and polymorphic information content(PIC)were: 8.6154,5.5450,0.7549,0.7801,1.7318,0.7205.(2)The average values of the genetic parameters(same as above)of the wild population(ZH)in Zhuhai,Guangdong were: 9.4615,5.9041,0.7200,0.7780,1.7789,0.7274.(3)The average value of each genetic parameter(same as above)of Shajing wild population(SJ)in Shenzhen,Guangdong were: 8.000,4.6810,0.8330,0.7564,1.6468,0.7122.(4)The average value of each genetic parameter(same as above)of the wild population(SZ)in the south of Shanzui Port,Taishan city,Guangdong were:9.3077,5.7513,0.7361,0.7690,1.7421,0.7203.(5)The average value of each genetic parameter(same as above)of the wild population(TS)in the north of Shanzui Port,Taishan city,Guangdong were: 10.9231,6.3702,0.7617,0.7595,1.7838,0.7128.(6)Theaverage value of each genetic parameter(same as above)of the wild population(TXX)in the southern part of Xiachuan island,Taishan city,Guangdong were: 8.8462,5.6870,0.7229,0.7528,1.6778,0.6971.(7)The average value of each genetic parameter(same as above)of the wild population(TSM)in northern Xiachuan island,Taishan city,Guangdong were: 8.6154,5.8357,0.8068,0.7920,1.7669,0.7349.The average polymorphic information content of Kumamoto oyster nine populations were all> 0.5,and all were highly polymorphic.Conclusions:(1)The 13 newly developed microsatellite markers showed moderate or high polymorphism,which could be used for population genetic analysis of Kumamoto Oyster.(2)The population genetic diversity index indicates that the population of nine Kumamoto oysters is at a highly polymorphic level and has great breeding potential.(3)The analysis of population genetic diversity found that the genetic diversity of two breeding populations was lower than that of the other seven wild populations,but remained at a high polymorphic level,indicating that the use of a large number of parents in the artificial breeding of Kumamoto oysters could effectively prevent the decline of genetic diversity of breeding populations,but artificial breeding also has a certain effect on genetic diversity of breeding population. |
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