High-throughout Screening Of SNP And Its Association With WSSV-resistance Traits In Fenneropenaeus Chinensis "Huanghai NO.2"

Fenneropenaeus chinensis is a commercially important farmed species in China,however, WSSV disease restrict the development of shrimp framing industry. Since thedevelopment of molecular biology techniques in the early1990s, DNA markers iswidely applied to the structural and functional analysis of important genes, the analysisof population genetic structure, development of genetic linkage map, marker-assistedselection and QTL analysis. The high density and mutational stability of SNP makethem particularly useful DNA markers for population genetics and for mappingsusceptibility genes for complex diseases. The purposes of this study were to identifythe facticity of putative SNPs with unlabeled probe high resolution melting curveanalysis method (HRM). Candidate SNP loci come from large-scale high-throughputsequencing results of Fenneropenaeus chinensis transcriptome454system. Accordingto the candidate SNP loci, primers and unlabled probes were designed to test and verify.132SNP loci were validated, and were used to analyze correlation with WSSVresistibility. Besides,132SNP loci were also used to estimate genetic diversity of theWSSV-resistant populations and WSSV-susceptible populations, which were artificiallyinfected with WSSV from2008to2012. The main results are as follows:1. Using the high resolution melting (HRM) genotyping approach,93SNP lociwere validated and were used to investigate the genetic structure of a breedingpopulation of F. chinensis in2012. The effective number of alleles ranged from1.0105to2.000. The average values of observed and expected heterozygosity of the SNP lociranged from0.0000to0.9792and from0.0104to0.5026, respectively. Polymorphism information content (PIC) values ranged from0.0103to0.6250, with an average valueof0.2182. Experimental results showed that the unlabeled probe high resolution meltingcurve analysis could be applied to screening SNP of Chinese shrimp. It is a simple,efficient, low-cost advantage of the new molecular genetic analysis. The results of thisstudy laid the technical foundation for the construction of the F. chinensis genetichigh-density SNP mapping and molecular marker-assisted breeding.2. F. chinensis “Huanghai No.2” which were obtained by selective breeding wereartificially infected with WSSV.144samples with the longest survival time weredefined as the WSSV-resistant group and144samples with the shortest survival timewere defined as the WSSV-susceptible group.132SNP loci were used to analyze thecorrelation between different genotypes and WSSV-resistibility by chi-square test.10genotypes in9SNP loci had statistical significant differences frequencies between thetwo populations. A/A of C1401-67TA, C/T of C6625-56CT, A/A of C364-89AT and C/Tof C12355-592CT were WSSV-resistant markers, A/G of C7695-204AG、T/T ofC7911-209CT, T/A of C9733-231TA, A/A of C12698-488CA, G/G of C283-145AG andT/T of C12355-592CT were WSSV-susceptible markers.3. More than70,000possible SNP loci based on Chinese shrimp transcriptome454high-throughput sequencing were analyzed,301candidate SNP loci were chosen forvalidation, of which93loci proved to be polymorphism. We investigated the mutationtypes of polymorphic loci and their correlation with the surrounding bases. The resultsshow that the propotion of mutation type of C/T was the highest, G/C was the lowest,and there was the transition/transversion bias2.0>0.5. With the number of A&Tnucleotide immediately neighbouring to the mutation point increases, thetransition/transversion bias decreases. There was a (G+C)/(A+T) bias of the wholesequence composition before and after mutation.4. Based on the sequence of transcriptome, the aldolase of Fenneropenaeuschinensis was cloned by RT-PCR and RACE. The aldolase cDNA was2127bp in length,which contains an ORF of1098bp, encoding365amino acids. The putative amino acid sequence was highly similar to aldolase from Arthropods, such as Bombyx mori andAntheraea yamamai. Sequence analysis indicated that aldolase of Fenneropenaeuschinensis belongs to the fructose-1,6-bisphosphate aldolase. The transcription ofaldolase gene in hepatopancreas of shrimps showed up-regulation first thendown-regulation，which indicates aldolase was involved in shrimp immue response.