Isolation And Screening Of New Microsatellite DNA Markers And Genetic Diversity Analysis On A Familial F₂Population Of The Pearl Oyster Pinctada Fucata

The pearl oyster Pinctada fucata is an economically important species which is culturedfor pearl production in China. In the present study, new microsatellite DNA markersfrom P. fucata were isolated and screened by using FIASCO. Besides, genetic diversityof a radom population （Daya Bay） and genotype of a familial F₂population wereinvestigated using105microsatllite loci, including the new microsatellite markersrepectively screened from genomic DNA （this study）, primers designed by our own labbefore, EST sequences （from GenBank）, and published primers in literature. The mainresults are as follows：1. Screening of microsatellite DNA markers.（1） Microsatellite-enhanced genomicDNA library of the P. fucata oysters was constructed using repeat-enrichment methodwith biotin-labeled oligos （AC）15and streptavidin magnetic beads. Among randomlyselected2097clones,483clones （23.03%） contain microsatellite motifs after PCRscreening. Among others,135positive clones were sequenced and122microsatelliteloci （90.37%） were identified. By alignment,65microsatellite clones are unique.Sequence analysis of repeat motifs indicates that the microsatellites can be divided intothree types:70perfect types （82.36%）,7imperfect types （8.24%） and8compound（9.41%）. In addition, most microsatellite sequences contain5²0repeat units （95.74%）and the average is7.83. The proportion of （AC/GT）nrepeat is the highest （75.53%）.Sixty five pairs of primers were designed using Primer Premier5.0,20pairs can beeffectively amplified, and14loci showed polymorphism as tested on a randompopulation of Daya Bay in Guangdong. Ninteen out of20new loci can be effectivelyamplified as tested on parents and8individuals of F₂;（2） Nine polymorphic lociscreened by our own lab before was rescreened by using the parents and F₂. The resultshows that4loci can be effectively amplified;（3） Thirty five pairs of primers designed by our own lab can be effectively amplified as tested on the F₂family;（4） Thirty six locipublished in literatures can been effectively amplified;（5） Eighteen loci were designedbased on EST sequences downloaded from GeneBank, among which11can beeffectively amplified. Totally105loci are obtained. The loci above can be used ingenetic variation analysis and genetic mapping of pearl oysters in P. fucata.2. Genetic diversity analysis of the stock of the Daya Bay. A random populationfrom Daya Bay was characterized using five of the20loci. A total of29alleles werefound, and the DNA fragment lengths of these alleles ranged from122to402bp. Thenumber of alleles per locus was2⁹, with an average of5.8. The number of effectivealleles was1.5957⁵.6962, with an average of2.9554. The polymorphisminformation content （PIC） was0.3573⁰.8048, with an average of0.5204. Theobserved heterozygosity （Ho） ranged from0.3000to0.7000, with an average of0.4400. The expected heterozygosity （He） ranged from0.3797to0.8384, with an average of0.5737, showing rich genetic diversity in the pearl oyster stock of Daya Bay.3. Genetic diversity of a F₂population. Genotyping of the F₂and their parentswas investigated using105microsatllite loci. The PCR products were detected by8%non-denatured polyacrylamide gel electrophoresis. The results showed that the averageof Shannon index and Nei’s genetic diversity index of the F₂population were0.6433and0.4226respectively, meaning that the genetic diversity of the F₂was high.According to the PCR results,33loci （31.43%） were monomorphic,72werepolymorphic （68.57%）. The proportion （68.57%） of parents polymorphism loci in all theeffective amplified loci was slightly higher than the F₂generation （67.62%）, showingthat the genetic diversity of the F₂was reduced slightly. The genetic similarity analysisshowed that the average genetic identity between F₂and the parents is0.9722, geneticidentity between individuals is0.6280⁰.8836, and the average is0.7663. The resultshowed that germplasm resources of familial F₂was good, the genetic structure is stable,and the genetic consistency in the family was high. Genetic differentiation indexbetween the parents and F₂was low （FST=0.0110）. Clustering analysis showed that theparents and72individuals of F₂were clustered into one group, and the other22individuals were clustered into another. Fourteen （14.58%） individuals form parents andoffspring were clustered into7groups respectively, and the rest82individuals wereclustered alone. All these results indicated that there was little genetic differentiationbetween parents and offspring. 4. Segregation of microsatellite loci in the parents and F₂population. Chi-squaretests showed that49loci （68.06%） of72loci deviated from Hardy-Weinbergequilibrium （PHW<0.05）. The segregation of SSR markers in a F₂offspring and theparents of P. fucata were analyzed using72microsatellite polymorphic loci.2testshows that23loci （31.94%） were significant deviated from Mendelian first law （P <0.05）, with15distorted segregating loci with excess of heterozygotes, whereas7distorted segregating loci being excess of homozygotes, only one being inHardy-Weinberg equilibrium. There are25microsatellite segregating loci in male parentincluding four distorted segregating loci （16%）,11in female parent including threedistorted segregating loci （27.27%）. Obviously, the proportion of distorted segregatingloci in male is lower than in females, showing significant sex differences. Forty-nineloci （P>0.05,68.06%） were in accordance with Mendelian first law, among which20loci were polymorphic in both parents,21（42.86%） in male,8（16.33%） in female.These microsatellite loci can be used in construction of genetic map for P. fucata.