Evaluation Of Genetic Diversity In Two Onchidiidae By ISSR And Development Of SSR

Onchidiidae mainly distributed in China’s southeast coastal areas. Inrecent years, coastal beaches had been vigorously developed and seriouspollution damaged to the environment, which caused a great threat to thesurvival of Onchidiidae. This study used methods of ISSR to investigatethe genetic diversity in the Onchidium struma and Platevindex mortonifrom different geographical populations in China. And we tried todesigned and selected microsatellite makers based on IlluminaHiSeq-2000paired-end sequencing. The main results are as follows.1.The genetic diversity of Onchidium struma collected fromYancheng(YC), Shanghai(SH), Wenzhou(WZ), Xiamen(XM),Shenzhen(SZ), Hainan(HN) in China, was studied by using the intersimple sequence repeat(ISRR) technique. The results indicated that thevalues of percentage of polymorphic band, Nei’s gene diversity andShannon’s information index were54.20%~70.99%,0.1564~0.2191and0.2421~0.3324,respectively for the six populations with the lowestvalues in SH population, the highest values in SZpopulation. The genetic distance between groups was0.0858~0.2596, andgenetic similarity coefficient was0.7573~0.9177. Genetic distanceand UPGMA tree showed that WZ was clustered with XMinto one clad firstly, and finally with HN. Mantel analysis indicatedno significant relationship between the genetic distance and the geneticdistance. According to AMOVA analysis, there were significant (P <0.001) genetic differences among the six populations,47.25%wasattributed to among populations and the rest (52.75%) to differenceswithin populations. The pairwise Fst=0.4725, gene differentiationcoefficient (Gst=0.3705) among populations and gene flow(Nm=0.8497)indicated that greater genetic differentiation occurred among thepopulations. Genetic drift was one of the main factors that affecting the population differentiation. Primer881was a maker that differ HN fromother groups.2.The genetic diversity of Platevindex mortoni collected fromCangnan(CN), Guangxi(GX)、Zhanjiang (ZJ)、Haikou (HN)in China, wasstudied by using the inter simple sequence repeat(ISRR) technique. Theresults indicated that the values of percentage of polymorphic band, Nei’sgene diversity and Shannon’s information index were50.00%～79.03%,0.1116~0.2201and0.1787~0.3392, respectively for the four populationswith the lowest values in GX population, the highestvalues in ZJ population. The genetic distance between groups was0.1081~0.2735, and genetic similarity coefficient was0.7607~0.8975.Genetic distance and UPGMA tree showed that CN wasclustered with ZJ into one clad, then with HN, and finallywith GX. Mantel analysis indicated no significant relationship betweenthe genetic distance and the genetic distance. According to AMOVAanalysis, there were significant (P <0.001) genetic differences among thefour populations,49.81%was attributed to among populations and the rest(50.19%) to differences within populations. The pairwise Fst=0.4981,gene differentiation coefficient (Gst=0.3828) among populations andgene flow(Nm=0.8063) indicated that greater genetic differentiationoccurred among the populations. Genetic drift was one of the mainfactors that affecting the population differentiation.3. In the present study, expressed sequence tagbased microsatellitemarkers were developed by Illumina paired-end Sequencing forOnchidium struma. A total number of19241SSRs were identified. Thedinucleotide repeat motif is the most abundant SSR, accounting for47.15%, followed by9.78%for trinucleotide repeats. Among theserepeated sequences,77microsatellites where repeat units were greaterthan4were randomly selected to test the variability in naturalpopulations.11polymorphic markers were identified and characterized.The number of alleles per locus varies from2to5, observed and expected heterozygosities ranged from0.1333to0.8462and from0.1282to0.6996,respectively. Four SSRs locus are significant departure from Hardy–Weinberg equilibrium after Bonferroni correction. High-throughputpyrosequencing allows fast and efficient isolation of microsatellitemarkers on a large scale, and can provide enough microsatellite markersfor population genetic studies, parentage and genetic mapping.Experimental results show that it is a fast and effective method ofdeveloping SSR based on Illumina paired-end sequencing results.