Analysis Of Genetic Structure Of The Comb Pen Shell Atrina Pectinata And Development Of Microsatellite Loci In Acrossocheilus Labiatus

The comb pen shell, Atrina pectinata Linnaeus, is a large economic shellfish with highvalue of utilization. In recent decades, the natural resource of A. pectinata has been declineddue to habitats destruction and overfishing. This study analysised the genetic diversity anddifferentiation of A. pectinata by AFLP and28S rDNA, in order to provide importantknowledge for further research in germplasm evaluation, resource protection and selectivebreeding of A. pectinata.1. Ten pairs of selective primers were found and used to analyze the five wild A.pectinata populations which collected from Lianyungang Jiangsu (JS), Dongshan Fujian (DS),Zhanjiang Guangdong (ZJ), Beihai Guangxi (BH) and Lingao Hainan (HN) by amplifiedfragment length polymorphism technology (AFLP).984loci were amplified in the fivepopulations.The genetic diversity results showed that the mean number of alleles per locus (A) variedfrom1.4715to1.7998, with an average of1.5632; the mean effective number of alleles perlocus (Ae) ranged from1.1956to1.4151, with an average of1.2732; the percentage ofpolymorphic loci (P) was from47.15%to79.98%, with an average of56.32%; the mean ofNei’s gene diversity (H) and Shannon’s information index (I) was0.1664and0.2564, rangingfrom0.1227to0.2506and0.1933to0.3834, respectively. Especially, DS population had richgenetic diversity with highest level indexes, but HN stock had the lowest one.As a whole, the genetic differentiation of the five populations was large. The total geneticvariation (Ht), the genetic variation among populations (HS), the coefficient of genedifferentiation among populations (GST) and the number of migrants per generation (Nm) was0.2914,0.1665,0.4288and0.6662, respectively. The genetic distance (D) of the fivepopulations ranged from0.0276to0.2829; the genetic identity (I) varied from0.7536to0.9728; the GSTvalue was between0.0734and0.4182, the Nmvalue was from0.6956to6.3134. There were significant correlations (P<0.05) between geographical distance andgenetic distance, GSTvalue of the five populations, with the correlations were0.733and0.636.The UPGMA dendrogram displays that, ZJ population had closest relationship with BHpopulation. All the five stocks clustered into two major groups. The ZJ, BH, HN and JSpopulations belonged to one group, and DS population belonged to the other group. 2. The study of28S rDNA showed that there was a significant difference between twokinds of haplotypes respectively named with JY-28S-1and JY-28S-2in50samples from5populations of A. pectinata. The consistency of the two haplotypes was about fourty percent.In JY-28S-1haplotype,26variable sites were found in26individuals within5populations of A. pectinata, including7sites with gaps or missing and19sites with basesubstitution (10bps being transition and9bps being transversion).7haplotypes were definedfrom the samples. The haplotype diversity (Hd), the average number of nucleotide differences(k) and the nucleotide diversity (Pi) was0.465,2.492and0.00251, respectively. Within5populations, the Hd value ranged from0.000to1.000; the k value varied from0.000to23.000;the Pi value ranged from0.000000to0.023069. In JY-28S-2haplotype,24variable sites werefound in24individuals within5populations of A. pectinata, including3sites with gaps ormissing and21sites with base substitution (11bps being transition and10bps beingtransversion).6haplotypes were defined. The Hd value, the k value and the Pi value was0.562,7.591and0.00769, respectively. Within5populations, the Hd value ranged from0.000to0.600; the k value varied from0.000to0.667; the Pi value ranged from0.000000to0.000674.Based on JY-28S-1and JY-28S-1haplotype, the genetic distances ranged from0.000to0.010and from0.000to0.019; the FSTvalues were0.67187and0.97363(P<0.01) suggestingthat there was much genetic variation among the populations. Cluster analysis by NJ treeshowed that all the JY-28S-1individuals were divided into two groups (ZJ, BH and HNpopulations belonged to one small group first, then clustered with JS and DS populations asone large group, and the28SDS17individual from DS population belonged to the other group),and the JY-28S-2samples were also clustered into two groups (JS, ZJ, BH and HNpopulations belonged to one group, and DS population clustered as the other group).Tajima’s D test displayed that the five stocks had significant deviated from neutralevolution based on JY-28S-2haplotype, but did not based on JY-28S-1haplotype. Fu’s Fs testshowed that the five populations had significant deviated from neutral evolution both based onJY-28S-1haplotype and JY-28S-2haplotype. It is generally acknowledged that Fu’s Fs test ismore responsive than Tajima’s D test.The above analysis by AFLP and28S rDNA revealed that the genetic diversity among thefive populations of A. pectinata was low, and the genetic differentiation was significant,maybe due to geographic isolation, genetic flow restriction, overfishing or habitat destruction.These imply that JS population, DS population and ZJ-BH-HN populations may belong tothree different subspecieses. The resource of A. pectinata has been damaged. So it is necessaryto make a series of effective protection strategies and work for A. pectinata.28S rDNA in A. pectinata is conservative with slow evolutionary rate. But in this study, two haplotypes withsignificant difference were found in28S rDNA of A. pectinata. The relevant reasons need thefurther research.Acrossocheilus labiatus Regan is a species of Acrossocheilus belonging to Cypriniformes,which occurs in the east of Asia. At present, the study in breeding, resource investigation,biochemistry and molecular biology is very poor. This study used magnetic bead hybridizationmethod to isolate microsatellite fragments from the digested product by restriction enzymeMse I and construct genomic DNA library for A. labiatus.262clones in the size range of400~800bp were selected for sequencing, and78primer pairs were designed from163microsatellites. Thirty wild individuals of A. labiatus were used to estimate polymorphism.25loci were polymorphic, with the number of alleles per locus (Na) varing from2to8, thepolymorphism information content (PIC) ranging from0.090to0.825. The observed andexpected heterozygosity (Hoand He) was from0.000to0.900and0.067to0.844, respectively.The PIC of21loci was high, and12of the25polymorphic loci were in Hardy-Weinbergequilibrium (P>0.05). Besides,8monomorphism loci were found.25polymorphism loci willbe useful for the further study of genetics and molecular ecology for A. labiatus.