| Babylonia lutosa, commonly named as yellow conch, is a kind of euryhaline and eurythermalconch which is distributed mainly over the Hainan, Guangxi, Guangdong and Fujian provinces in China. Because of its high nutrition value, B. Lutosa has become popular in global market in last few years. Advantage has been taken from its fast growth and high selling price to develop this novel species by marine aquaculture. However, the nature resources of B. lutosa has decline sharply, mainly attributed to environment deterioration and overfishing. The Ministry of Agriculture of China has set up a special public welfare project to effectively protect and sustainably develop this aquaculture resource.With the support of the project, in this work, the transcriptome data of the B. lutosa was obtained by High-throughput sequencing, from which growth related genes containing EST-SSR sequences were identified. The developed EST-SSR genetic markers were applied to analyze hereditary constitution and to study the correlation between growth characteristics in the first generation (F1) of B. lutosa artificial breeds. The mitochondrial genes of B. lutosawere also taken for genetic diversity study. This work will be beneficial for the B. lutosa germplasm resources protection, artificial breeding efficiency evaluation and molecular-marker-assisted selection (MAS).The main results are as follows:1.Transcriptomic analysis for B. Lutosa using High-throughput sequencingDe novo assembly and transcriptomic analysis on B. lutosa were conducted by High-throughput sequencing technology. A totla of 119,113 quality-filtered and trimmed reads were obtained from mixed RNA samples that were collected from pleopod and hepatopancreas of three B. lutosa individuals. Among which,109,359 unigenes were assigned to GO (gene ontology) classification and 13,962 EST-SSRs were screened. By KEGG analyzing, there were 8,272 isotigs annotated with Enzyme Commission (EC), which participated in 232 metabolic pathways.2.Exploration and cloning of EST-SSR containing growth-related genesThere were 7 EST-SSR containing genes were identified from B. lutosa transcriptome. One of the genes, TBC-1-A, were amplified by RACE technique, in the 3’-end non-coding region of which, however, the rcpeatitive sequence was not found. It is a deduction that the coloned gene could be a isoform in the protein family.3.Correlation analysis on growth characteristics in artificially bred B. lutosa F1Except for the hight of screw body and spiral,all the morphological characteristis are positively correlated with body weight.The influence on body weight from strong to weak in turn was shell width, aperture width, body whorl height and body spiral height, which could be used as reference index for selective breeding.4.Development of B. lutosa EST-SSR markersIn total,53 EST-SSRs with polymorphism were developed.The number of alleles per locus range from 2 to 7. The average observed heterozygosities, average expected heterozygosities and polymorphic information content of locus is 0.4813,0.4832 and 0.4682, respectively. It could be concluded that the genetic diversity in wild population was relatively low.5.Genetic diversity analysison wild populations by EST-SSR markersBy using the developed EST-SSR markers, three wild populations of B. lutosa were studied, which were collected in Bei hai(Guang xi province), Lian jiang(Fu jian province) and Chang le(Fujian province), respectively. It was exhibited that the genetic distance is 0.6132 between wild populations of Bei hai and Lian jiang,0.6465 between Bei hai and Chang le, and 0.0996 between Lian jiang and Chang le, which suggested that there was little gene flow between Fu jian province and Guang xi province.6.Correlation of EST-SSR markers with growth traits in B. lutosaOptimal scaling regression analysis on the correlation of loci genotype and characteristics was carried out in 206 individuals of Fi artificial breeds of B. lutosa.The results showed that eight locus had significant impacts on body weight, among which, genotype BC at BL-106 loci and AB at BL-107 loci had extreme significance and were considered as dominant genotype for the growth traits. BL-106 loci belonged toTBCl-2B gene that encoded growth-related protein.These two genetype can be used as growth markers for molecular marker assisted breeding.7.Mitochondrial genome cloning and genetic diversity analysis of B. lutosaBased on 14 primers amplification, the whole mitochondrial genome of B. Lutosa was cloned. By the homology analysis on mitochondrial genes sequences among 12 species of class Neogastopoda, it was suggestted that there were four highly variable and two conserved regions. Moreover, consensus primers of 16S rRNA and CO1 gene were used to study the genetic diversity of mitochondrial genome of different B. lutosa population. There was no mutation found in 16S rRNA and CO1 gene, however, some mutation loci were isolated in non-coding regions. |
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