| Fenneropenaeus penicillatus Alcock is of high commercial value because it was once one of the most important aquaculture shrimps distributing along the southeast coast of China. However, the wild resource of F. penicillatus Alcock decreased rapidly due to over-exploitation, environmental degradation of spawning ground and other factors. F. penicillatus Alcock was listed as an endangered species in "China Species Red List" in 2005. In order to broaden the knowledge of its germplasm resource, to conserve, and to provide theoretic basis for the establishment of its natural reserve, microsatellite markers and sequence analysis of mitochondrial DNA gene(16SrRNA and D-loop)were used, together with the theory of marine biotechnology, ecology, and conservation genetics, to analyze the genetic diversity and differentiation of F. penicillatus Alcock which are distributed along the southeast coast of China.We developed 20 pairs of polymorphic primers of F. penicillatus Alcock using FIASCO method, and then applied 10 of these 20 polymorphic primers to analyze the genetic diversity and differentiation of 12 wild F. penicillatus Alcock populations along the southeast coast of China. Besides, sequence analysis of mtDNA 16 S rRNA and D-loop genes were used to study the genetic diversity and differentiation, and molecular neutral test of F. penicillatus Alcock.(1)Microsatellites enriched genomic library was constructed by bio-(CT)15 and bio-(GT)15. A total of 184 fragments ranging from 400 bp to 1200 bp were selected to be sequenced, and 80 pairs of primers were designed. A total of 30 wild individuals from Dongshan population were used to analyze the polymorphism of these primers, and 20 of them were polymorphism and could be used for the further population genetic diversity and differentiation study. According to the results, the number of alleles per locus ranged from 2 to 7, polymorphism information content(PIC) ranged from 0.0078 to 0.9771, the observed and expected heterozygosities was 0.0357-0.9130 and 0.1308-0.7405, respectively. Finally, ten primer pairs with high polymorphism and clear bands were chosen in the further population genetic research.(2)Ten primer pairs were used to analyze the genetic diversity and differentiation of 12 wild F. penicillatus Alcock populations along the southeast coast of China. The mean number of alleles per locus(A) was from 2.9000 to 3.9000, with an average of 3.3916. The mean effective number of alleles per locus(Ae) was from 1.6913 to 2.2915, with an average of 1.9326. The average of the observed heterozygosities(Ho) was 0.2503, ranging from 0.2028 to 0.3092, and the average of the expected heterozygosities(He) was 0.3849, ranging from 0.2746 to 0.5216. Thus, the genetic diversity of 12 wild F. penicillatus Alcock populations was not high. The genetic distance among the 12 wild F. penicillatus Alcock populations ranged from 0.0127 to 0.3676. FST value(FST) was 0.0329, and the gene flow(Nm) was 7.3448. The result showed that certain level of genetic differentiation existed among 12 wild F. penicillatus Alcock populations, but the genetic differentiation was not high.(3)According to the results of mtDNA 16 S rRNA sequence analysis, 16 variable sites were founded in 85 F. penicillatus Alcock individuals from 12 wild populations along the southeast of China. These 16 variable sites were all polymorphic sites, and 14 haplotypes were defined. The average number of nucleotide differences was 1.400, and the nucleotide diversity was 0.00264. Among 12 populations, the average number of nucleotide differences was from 0.000 to 2.476, and the nucleotide diversity was from 000000 to 0.00466. The genetic distance among 12 wild F. penicillatus Alcock populations ranged from 0.000 to 0.009. AMOVA analysis showed that no significant genetic differentiation existed among 12 wild populations. The molecular neutral test(Tajima’s D and Fu’s Fs test) revealed that F. penicillatus Alcock populations departured from neutrality model significantly.(4)According to the results of mtDNA D-loop sequence analysis, 184 variable sites were found in 83 individuals in 12 F. penicillatus Alcock populations and 79 haplotypes were defined. The average number of nucleotide differences was 36.065, the nucleotide diversity was 0.06283, and haplotype diversity was 0.999. Among 12 F. penicillatus Alcock populations, the average number of nucleotide differences ranged from 8.036 to 54.900, and the nucleotide diversity ranged from 0.01378 to 0.09433. The genetic distance among 12 F. penicillatus Alcock populations ranged from 0.019 to 0.171. AMOVA analysis showed that there exists certain level of genetic differentiation among 12 wild F. penicillatus Alcock populations. The molecular neutral test(Tajima’s D and Fu’s Fs test) showed that 12 F. penicillatus Alcock populations conformed to neutrality model.The genetic diversity of 12 wild F. penicillatus Alcock populations which were distributed along the southeast of China was not high. And although there is certain level of genetic differentiation among these 12 populations, the genetic differentiation was not high. |
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