| Marine organisms, the main souce of protein, palys an important role in our daily life. However, In recent decades, the quantity and quality of marine biological resources have been suffered a serious impact, and even some marine species are in danger of extinction by over-exploitation of marine resources like offshore operations and intensive ?shing. Unfortunately, the six species that Atrina vexillam, Lunella coronata granulata, Hemifusus termatamus, Megalonibea fusca, Siganus fuscescens and Sebastiscus marmoratus have not been paid too much attention by the aquaculture scholars. Due to these reasons, paying more attention in the aspects of consevertion and genetics is extremely urgent. For the purpose of protecting resources and rational exploitation, genetic diversity was studied using the microsatellite. The results of six species of microsatellite development and genetic diversity are as follows:1. 11 microsatellite makers of A. vexillam were developed using FIASCO. 236 fragments ranged from 500 bp to 1000 bp were selected to sequence, and 48 pairs of primers were designed. 30 individuals were brought from Hainan to evaluate the degree of polymorphism. The number of polymorphic alleles per locus ranged from 2 to 8. Polymorphism information content(PIC) ranged from 0.199-0.831. The observed and expected heterozygosities were 0.1000-0.8667 and 0.1244-0.8356, respectively. The PIC of 10 loci were high and mid, and 9 of the 11 polymorphic loci were in Hardy-Weinberg equilibrium(P>0.05).2. FIASCO method was used to screen microsatellite fragments from the digested product by restriction enzyme Fast Digest Tru1 I and construct genomic DNA library for L.coronata granulata. 310 clones in the size range of 500-1000 bp were selected for sequencing, and 60 pairs of primers were designed. Thirty wild individuals were used to estimate polymorphism. 10 loci were polymorphic, with the polymorphism information content ranging from 0.305-0.559. The observed and expected heterozygosity was from 0.0667-0.7333 and 0.0644-0.6628, respectively. The result of PIC detect showed 3 loci was high, 7 loci was mid, and 9 of the 10 polymorphic loci were in Hardy-Weinberg equilibrium(P>0.05).3. We used FIASCO method to isolate microsatellite fragments and construct genomic DNA library for H.termatamu. And then choosed 220 positive clones to sequence. Sequence analysis showed that among the microsatellite sequences, 171 repeating motifs were perfect, occupying 61.5%; 81 repeating motifs were imperfect, occupying 29.1%; 26 repeating motifs were conpound, occupying 9.4%. Besides the GT, CT repeats, several other types of repeats were also detected, such as GTT、TGG、GAA、CAA、TCTA、ACAG、TAGA、GTGA、GTCT、GATA and TTTTG. 58 primer pairs were designed. Thirty individuals of H.termatamus of artificial generation F1 were used to test genetic diversity for 58 primer pairs and finally 9 monomorphic microsatellite primer were screened. The result that all loci were monomorphic showed that genetic diversity of artificial generation F1 of H. termatamus was very low.4. FIASCO method was used to isolate microsatellite fragments and genomic DNA library was constructed for M.fusca. 242 fragments ranged from 500 bp to 1000 bp were selected to sequence, and 41 pairs of primers were designed. 30 individuals were collected from Xiamen to evaluate the degree of polymorphism. 10 loci were polymorphic, with the polymorphism information content(PIC) ranging from 0.000-0.624. The observed and expected heterozygosity was from 0.0667-0.7667 and 0.0644-0.5828, respectively. The result of PIC detect showed 2 loci was high, 6 loci was mid, and 2 was low, and 8 of the 10 polymorphic loci were in Hardy-Weinberg equilibrium(P>0.05).5. FIASCO method was used to obtain microsatellite sequences and 46 pairs of primers were designed. The result showed that the number of alleles per locus ranged from 2-12. The polymorphism information content varied from 0.210-0.849. The observed and expected heterozygosities ranged from 0.1429-0.8077 and 0.2246-8533, respectively. 13 loci were in Hardy–Weinberg equilibrium(HWE) while 2 loci(Sf1-37-2 and Sf1-47) deviated significantly from HWE.6. We use the same method to obtain microsatellite fragments and design 46 pairs of primers. According to the results, three loci were monomorphic and eleven loci were polymorphic. The results estimated by POPGEN 32 showed that the number of alleles per locus ranged from 2 to 8 and the observed and expected heterozygosities ranged from 0.186-0.969 and 0.170-0.782, respectively. The results also showed that only one loci deviated significantly from HWE while ten loci were in HWE. The polymorphism information content ranged from 0.155-0.752 by CERVUS3.0.In this study, FIASCO method was used to isolated the microsatellites for six marine organisms. As the result, microsatellite loci of five species were sucessfully screened except H. termatamus. This result suggested that microsatellites in many species could been isolated. But it also gave a reminder: the samples used to analysis the genetic diversity for species should be avoid collecting from F1 belonged to the same parent. Genetic evaluation of three marine fishes showed that the genetic diversity and PIC in M.fusca was low and was lower than S. fuscescens and S.marmoratus. Probably, this reflected the limited naturalre source distribution, the destruction of wildlife resources and the inbreeding depression in M.fusca. Thus, protecting resources for M.fusca is extremely urgent.In this research, we isolated 11 microsatellite loci in A. Vexillam, 10 microsatellite loci in L.coronata granulata, 10 microsatellite loci in M.fusca, 15 microsatellite loci in S. fuscescens and 11 microsatellite loci in S. marmoratus. These microsatellites chould be useful for the further study in the followed areas: population genetics, evolutionary genetics, traits analysis for QTL, germplasm identification, construction of genetic linkage map and genetic breeding. At last, we summarized main four main aspects involving microsatellite technology roadmap: the development of microsatellite loci, designing high-quality primer, testing PCR amplification and data analysis. |
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