Transcriptome Analysis And Study On Genetic Diversity Of Different Geographic Populations In Megalobrama Terminalis

Megalobrama terminalis, belonging to Family Cyprinidae, Genus Megalobrama, is the most widely distributed of bream genus which mainly distrubuted in the southeast coast area, Yangtze River, Yellow River, Heilongjiang River and the Russian Far East. However, due to the decreasing of the wild resource of M. terminalis, thus, in order to protect the wild resources and establish the breeding program of this species. In our laboratory,50full-sib families were bred using the broodstock original from Qiantang River. After,200five-month-old fishes from a family of F1generation were randomly selected for measuring body weight and length, of which,10(5%) fishes have highest growth rate (L) and10(5%) fishes have lowest growth rate (S) were sampled. The tissues of muscles, liver and brain were collected, extracted RNA and equally pooled into two separate samples of L and S, respectively. The Ion Torrent sequencing with the read length of200bp was used in this present study following to manufacture’s instruction. Briefly, after mRNA was isolated from total RNA of L and S, cDNA libraries of L and S were synthesized,318chip preparation, and sequencing using the Ion Torrent sequencer. Bioinformatics analysis was used to compare the differences between two transcriptomes of M. terminalis L and S in F1generation; using software for microsatellite searching to find out the SSRs in M. terminalis transcriptomes, discovery molecular markers in M. terminalis, study on genetic diversity of M. terminalis by using different geographic populations. The present study results were revealed following:(1) Based on the Ion Torrent sequencing with read length of200bp instruction and related kits, two cDNA libraries of L and S in F1generation of M. terminalis were constructed. After measuring, the concentration of cDNA library of L and S was5.06ng/μL and34.8ng/μL, respectively, which were higher than5ng/μL, accordance with the requirements of Ion Torrent RNA-Seq sequencing; and the length of the libraries was about200bp in average, which showed that the cDNA libraries of L and S in F1generation of M. terminalis were successfully constructed.(2) Using318chips, the transcriptomes of M. terminalis L and S in Fi generation were achieved from the Ion Torrent PGMTM sequencing. The L and S obtained2,163,601and3,893,877raw reads, respectively. After removal twice of the primer sequences, linkers and short sequences, the L obtained1,987,905cleaned reads and the S obtained3,795,177cleaned reads. The all cleaned reads of L were successfully assembled to27,673contigs, where N50was156bp and the average length was164(from101to2,824) bp. The all cleaned reads of S were successfully assembled to78,370contigs, where N50was152bp and the average length was162(101-3,376) bp. Compared with NR protein database, the L and S was hit to15,786and38,923contigs, and identification of10,800and21,718unique protein, respectively. In GO annotation, a number of3486contigs (in the L) and7134contigs (in the S) were annotated and divided into three biological categories. In KEGG analysis, the L had28growth-related transcripts, and the S had117growth-related transcripts. Through microsatellite searching software in the transcriptomes of M. terminalis L and S in F1generation,118and383microsatellite sequences were identified in the L and S, respectively. Among obtained microsatellites,46(in the L) and143(in the S) could be used to design microsatellite primer sequences. Using the M. terminalis SSR sequences,81microsatellite primer pairs were developed, and only two stable microsatellites were selected to amplify the target band of microsatellite loci.(3) Using of30previously developed high polymorphic microsatellite loci from blunt snout bream, a total of13microsatellite loci were amplified target band in M. terminalis by cross-species amplification. Of which these microsatellite loci,11were polymorphism. Based on two microsatellite loci from transcriptome profile and11microsatellite loci from cross-species amplification, the observed heterozygosity (Ho), expected heterozygosity (He) was calculated as in range of0.7000-0.9667and0.6480-0.8264, respectively; the polymorphism information content (PIC) was estimated in ranging of0.5710-0.7885(were higher than0.5). These13microsatellite loci may be used in studying on bream genetic diversity.(4) The13developed microsatellite loci were used to analyze the genetic diversity of four populations distributing in Qiantang River, the Beijiang River, Jinsha Reservoir and Heilongjiang. The average number of effective alleles of the four M. terminalis populations was in ranging of4.0196-4.4093. The observed heterozygosity and expected heterozygosity distributed between0.4-0.9667and0.8661-0.6073, respectively. The genetic differentiation fixation index Fst in the North River and Qiantang River population, the Beijiang River and Jinshahe Reservoir population was0.05<Fst<0.25. The UPGMA cluster results displayed that the Qiantang population and Heilongjiang population grouped together, and then with the Jinshahe Reservoir clustered together and finally with the Beijiang River populations clustered together. The results of AMOVA analysis showed that the most difference of genetic diversity was from different individuals rather than between populations or within population of M. terminalis.The paper provides basic information for further selection of large body and fast-growing of M. terminalis broodstock.