Transcriptome Analysis And Development Of Microsatellite Markers In Paphia Undulata

Paphia undulata is one of the important farmed bivalve species in China and South East Asia,mainly living in tropic sea area.The research of P.undulata was mainly focused on artificial breeding and nutrition analysis,the study about transcriptome and genome of P.undulata were rarely reported.In this work,transcriptome of P.undulata foot tissue was sequenced using the high-throughput Illumina HiSeq^TM 2000 platform and immunity related gene were discovered and microsatellite markers were also developed.The key results as follows:1.In this study,more than 26 million sequencing reads of foot tissues were generated using Illumina Hiseq^TM 2000 sequencing platform.De novo assembly yielded 60,201 unigenes with a mean length of 875 bp and the N50 length of 1478 bp,among which 10,434 unigenes were longer than 1 kb and 23296 longer than 0.5 kb.In total,22,260 unigenes were successfully annotated in the Nr,Nt,Swiss-Prot,TrEMBL,COG,and KEGG databases.Among all the annotated unigenes,there were 6,718 unigenes were mapped to 237 KEGG pathways,15017were assigned with one or more GO terms and 5,786 unigenes could be assigned to the COG classifications.2.The transcriptome analysis of Paphia undulata indicated that a total of 34 immune genes on pattern recognition receptors,including 12 lectin genes,8 Toll-like receptor genes,2peptidoglycan recognition protein genes,5 scavenger receptor genes,5 thioester and C1q protein family related genes,and 2 others pattern recognition receptor genes.The Signal transduction related genes was 67,which included 24 Complememt pathway genes,14 NF-κB signaling pathway genes,10 MAPK signaling pathway genes,8 JAK/STAT signaling pathway genes and11 Toll-like receptor signaling pathway genes.There were 32 immune effector genes which included an antimicrobial peptide gene,7 heat shock protein genes,7 antioxidant enzymes genes,6 protease inhibitor genes,4 lysozyme genes,2 mucin genes and 5 other immune molecules.3.Using MISA software,7378 microsatellite markers were developed from the transcriptome data of Paphia undulata,in which the number of mono-nucleotide repeat is the most,tri-nucletide repeat is the second,penta-nucleotide repeat is the least and hexa-nucleotide repeat isn’t found.In which,95 loci were chosen to design the primers and the PCR products were verified in a wild group of Paphia undulata.The results showed that a total of 30 loci were successfully amplified,of which 15 loci were monomorphic and 15 loci were polymorphic.The allelic number（Na）and the efficient allelic number（Ne）in these 15 polymorphic loci were 4.733and 2.438,respectively.The observed heterozygosity（Ho）and the expected heterozygosity（He）were 0.270 and 0.516,respectively.The average of polymorphism information content（PIC）was0.457,in which 4 loci（PU29,PU32,PU49,PU70）showed highly polymorphic（0.500<PIC<1.000）.