Development Of SSR And SNP For Onchidiidae Based On Transcriptome Sequencing

Onchidiidae belongs to Mollusca, pulmonata. There are 5genus 6 species in our country. Among them 4 species contains Peronia verruculata, Paraoncidium reevesii, Onchidium struma, Platevindex mortoni received much attention. As transitional species between sea and land, the larvae of Onchidiidae live in water and the adult live in the intertidal area. With the similar characters like amphibians, Onchidiidae is good material as the study of marine invertebrates extending landward. In this paper, we developed microsatellite markers for P. reevesii and P. mortoni based on transcriptome sequencing Illumina HiseqTM 2000. In addition to this, 26 SNP markers for O. struma were isolated. The detailes is as follows:1, Development of microsatellite markers for Paraoncidium reevesii and Platevindex mortoni based on transcriptome sequencing Illumina HiseqTM 2000Repeats in the transcriptome sequences of Paraoncidium reevesii were searched using MISA and SSR Hunter1.3, 51 sequences with SSR were selected for primier designing. 24 primers were produced clearly defined bands after preparatory screening. The further test in Zhanjiang population indicated that 14 loci were showed polymorphism. The allele numbers per locus ranged from 2 to 4 and the observed and expected heterozygosity ranged from 0.0313 to 0.7143 and from 0.0313 to 0.7084, respectively. The polymorphic information contents（PIC） of each locus were between 0.0895 and 0.6405. Among 14 loci, 2 had a PIC>0.5, 10 had a 0.5>PIC>0.25, 2 had a PIC<0.25. 9 loci were detected not deviated from HWE（P>0.05）in 14 after Bonferroni correction. No pairs of loci showed significant linkage disequilibrium. 22 microsatellite loci were shown to be polymorphic in P. mortoni, with the expected and observed heterozygosity（HO and HE） ranged from 0.0278 to 0.9688 and from 0.0278 to 0.8370, respectively. Allele numbers per locus ranged from 2 to 5. 2 loci（PM46, PM60） were detected significant deviated from HWE（P<0.01）in 22 after Bonferroni correction. No pairs of loci showed significant linkage disequilibrium. These loci can be used for the research of population genetic and phylogenetic analysis of Onchidiidae. Furthermore, it will provide molecular data to conservation of P.reevesii resources.2, Isolation of EST-SNP markers for Onchidium struma based on transcriptome sequencing Illumina HiseqTM 2000Proposed site in the transcriptome sequences of Onchidium struma were searched using Samtools, 152212 SNP were detected after analysis. 57 sites were used to detect typing in the population collected from Chongming iland, Shanghai. 26 loci were successfully genotyped with polymorphism. The most type is conversion like A-G and T-C. The observed and expected heterozygosity ranged from 0.2553 to 1.0000 and from 0.0000 to 0.7447, respectively. The polymorphic information contents（PIC） of each locus were between 0.017 and 0.385. Among 26 loci, 7 had a 0.5>PIC>0.25, 19 had a PIC<0.25. 8 loci were detected not deviated from HWE（P>0.05）in 14 after Bonferroni correction. Shannon information index distribution ranged from 0.0503 to 0.6602. These loci will be beneficial to understand the breath way with skin and adaptation to life in terrestrial environment. Annotation for sequences with SNP were performed using blast X, GO and KEGG database. The result indicated that sequence S_Unigene1402_c0_seq1 has important function in synapse formation, Auditory receptor cells differentiation, enzyme localization in the respiratory chain, sequence S_Unigene508_c0_seq1 can regulate immune system, glandular secretion, DNA repair, cell apoptosis, sequence S_Unigene512_c0_seq1 involved in the formation of the nervous system, sequence S_Unigene685_c0_seq1 involved in cell proliferation, biological macromolecules metabolic.In the 26 SNP loci, 3 loci（S_Unigene508_c0_seq1₁42, S_Unigene685_c0_seq1₃534, S_Unigene508_c0_seq1₂83） were related to epidermis formation, one locus（S_Unigene3026_c0_seq1₃726） was related to epidermis formation and muscle formation, 3 loci（S_Unigene512_c0_seq1₉71, S_Unigene512_c0_seq1₅524, S_Unigene512_c0_seq1₅912） were related to angiogenesis associated and muscle formation simultaneously, one locus（S_Unigene11849_c0_seq1₈04） was related to formation of blood vessels and skin, the others loci were related to blood vessels development only.Onchidiidae has similar ‘amphibious’ feature with amphibian and different species have different characteristic. Development of SNP molecular markers in Onchidiidae will be benefit to further studying in ‘amphibious’ interrelated with gene and the protection of Onchidiidae.