| Amphioxus, a unique and endangered transition species between vertebrate andnon-vertebrate, had been considered as a new model animal. LiuWuDian, located on thenortheast of Xiamen Island, was the World-Famous habitat of B. belcheri for high commercialvalue, but in recent decades, the resource of B. belcheri has been diminishing as a result ofoverfishing, environmental degradation of the habitat and sand extraction from the sea bottom,and was listed as Class II protected species in China. In order to assist us with protecting thisspecial species, population genetics of B. belcheri Gray populations was studied using themicrosatellite and mtDNA. ESU and MU on the conservation of B. belcheri Gray were definedbased on the population genetics, and replenish conservation genetics of B. belcheri Gray forfurther. The aim of our study is to provide genetic information for reasonable conservationstrategies of B. belcheri Gray.We developed8pairs of polymorphic primers of B. belcheri Gray using FIASCO, and studythe genetic diversity and genetic differentiation of B. belcheri Gray with additional3primers ofB. japonicum. Besides, the sequence characteristic, genetic diversity, genetic variation andmolecular neutral test of B. belcheri Gray were analyzed using mtDNA12S rRNA, COII andCOIII.1. Microsatellits enriched genome library,(CA)nand (GA)n, was conducted by bio-(GT)15andbio-(CT)15using FIASCO.100fragments ranged from300bp to1200bp were selected tosequence, and41pairs of primers were designed. The polymorphism of these primers wasanalyzed in30individuals. The analysis of the genetic diversity by8primer pairs ofpolymorphic indicated that number of alleles per locus ranged from3to10, polymorphisminformation content ranged from0.279to0.847, the observed and expected heterozygositiesranged from0.1667to0.9333and from0.3100to0.8617, respectively. The study also indicatedthat the PIC of8primer pairs were high and these primer pairs can be used in the populationanalysis.2. The genetic diversity and differentiation of4populations of B. belcheri Gray were analyzedusing8pairs of microsatellite primers (above) and additional3pairs of primers of B. japonicum.The mean number of alleles per locus (A) ranged from4.6364to5.4545, with an average of5.0682. And the mean effective number of alleles per locus (Ae) ranged from2.3138to2.8661, with an average of2.5111. The average of the observed heterozygosities (Ho) was0.4497,ranging from0.3966to0.4839, and the average of the expected heterozygosities (He) was0.5315, ranging from0.5045to0.5699. All populations as a whole, the mean number of allelesper locus, the mean effective number of alleles per locus, the observed heterozygosity and theexpected heterozygosity is6.3636,2.6405,0.4494and0.5493, respectively. The genetic distanceamong the4populations ranged from0.0388to0.0709, the FSTvalues (FST) was0.0329, and thegene flow was7.3448. The UPGMA dendrogram shows that,4populations of B. belcheri Grayclustered into two major groups, the Dadeng Island and East of southern line to18populationsbelonged to one group, the Huangcuo and West of southern line to18populations belonged tothe other group.3. The study of mtDNA12S rRNA showed that,30variable sites were founded in23individuals within4populations of B. belcheri Gray, included3sites with gaps or missing and27polymorphic sites.23haplotypes were defined from these individuals. As a whole, theaverage number of differences was3.285, and the nucleotide diversity was0.00515. Withinpopulation, the average number of differences ranged from2.600to3.733, the nucleotidediversity ranged from0.00408to0.00585, and all of the haplotype diversities were1.000. Thegenetic distance among the4populations ranged from0.0042to0.0049, with the mean0.0046.AMOVA analysis revealed that no significant genetic differentiation among4populations. Themolecular neutral test (Tajima’s D and Fu’s Fs test) revealed that4populations of B. belcheriGray departured from neutrality model.4. The study of mtDNA COII showed that,39variable sites were founded in22individualswithin4populations of B. belcheri Gray,21haplotypes were defined from these individuals. Asa whole, the average number of differences was5.745, the nucleotide diversity was0.00831, andhaplotype diversity was0.996. Within population, the average number of differences rangedfrom4.133to7.500, the nucleotide diversity ranged from0.00598to0.01085, and all of thehaplotype diversities were1.000. The genetic distance among the4populations ranged from0.0067to0.0106, with the mean0.0084. AMOVA analysis revealed that no significant geneticdifferentiation among4populations. The molecular neutral test (Tajima’s D and Fu’s Fs test)revealed that4populations of B. belcheri Gray departured from neutrality model.5. The study of mtDNA COIII shows that,56variable sites were founded in23individualswithin4populations of B. belcheri Gray,22haplotypes were defined from these individuals. Asa whole, the average number of differences was6.656, the nucleotide diversity was0.00844, andhaplotype diversity was0.996. Within population, the average number of differences rangedfrom3.867to9.733, the nucleotide diversity ranged from0.00490to0.01234, and all of thehaplotype diversities were1.000. The genetic distance among the4populations ranged from 0.0062to0.0110, with the mean0.0085. AMOVA analysis revealed that no significant geneticdifferentiation among4populations. The molecular neutral test (Tajima’s D and Fu’s Fs test)revealed that4populations of B. belcheri Gray departured from neutrality model.The above four results revealed that the genetic diversity among the4populations ofB.belcheri Gray was moderate, and the genetic differentiation was no significant. The B.belcheriGray should be protected immediately. Based on analysis of genetic diversity, status of resourcesand ecological habitat, this study advanced six conservation strategies, and recommended to listDadeng Island to the Amphioxus Conservation Area. |
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