Development and Application of SNP markers in Genome of shrimp (Fenneropenaeus chinensis) Jianyong Zhang Marine Biology

1 、 Introduction The Chinese shrimp, Fenneropenaeus chinensis, widely naturally distributed in the coastal waters of north China, has especially become an important economic mariculture species in this region The current studies on shrimp mainly concentrated on the research of molecular marker development and application, gene clone, disease resistance and high yield breeding, etc.

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Larvae RearingFamily Conservation Character TestVarieties Propagation

White Spot Syndrome Virus (WSSV) WSSV was first found in South Asia and then spread to America, Europe and Australia. The mortality rate of WSSV-infected shrimp was almost 100% in 3 to 10 days. Because of its rapid spread and high mortality rates, WSSV is an extremely virulent pathogen in shrimp culture.

Purposes 454 pyrosequencing based transcriptome analysis of shrimp was carried out to discover genes and single nucleotide polymorphism （ SNP ） loci involved in disease resistance to WSSV. Identifying the facticity of putative SNPs and analyzing genetic diversity of family or constructing genetic linkage map.

2. Materials and Method Resistant shrimp and Sensitive shrimp to WSSV were sequenced based transcriptome using Roche 454 GS FLX system by Chinese National Human Genome Center (Shanghai). Analyzing sequencing data with software. Thirty individuals from each of six shrimp families were sampled to identify putative SNP loci with amplification refractory mutation system (ARMS) PCR method.

Resistant shrimp-454 reads 3. Results 454 transcriptome pyrosequencing

Sensitive shrimp-454 reads

CAP3 assembly default parameter ： overlap 40bp ， identity 80% Match scores, mismatch scores, and gap penalties are all weighted by the quality values of the bases involved.

Prawn-cDNA 454 sequencing ResistantSensitive Reads number (ave len)268,511 (205bp)229,335 (235bp) Base number48,231,158bp47,352,259 Number of assembled reads220,652195,637 Contig number11,75011,218 Max Contig len3,588bp3,919bp Contigs ave len321bp355bp Singlets number20,21915, Results

ResistantSensitive Seq number (contigs+singlets)31,96926,347 Specific sequence18,33114,437 Specific sequence of Resistant and Sensitive Differential Expression

Gene prediction base on sequencing RS Seq number (contigs+singlets)31,96926,347 Encode Protein31,83626,271 Protein annotate 5,5365,443 Protein of GO Ontology2,7732,692 Gene prediction ： GetORF Gene Ontology analysis ： gopipe

SNP calculation SNP loci71,724 Samesense mutation17,329 Non synonymous mutation34,642 Nonsense mutation1,478 Noncoding region18,275 Indel loci31,769

SNP confirmation ARMA-PCR amplification Eighty putative SNPs loci were chosen and were validated by PCR-amplified from F. chinensis genomic DNA. Primers were designed using the primer design computer program made accessible by Ye et al. A total of 20 SNPs loci were validated within 80 putative loci, both the outer and the expected inner bands were amplified. Ye S, Dhillon S, Ke X, Andrew R C. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res, 2001, 29(17): E88-8

SNP confirmation ARMA-PCR amplification The electrophoretogram was the genotyping by ARMS-PCR for SNP locus of contig The lane marked M denoted molecular marker. The panel of 1, 6,10, 11, 16, 22, 23, 25 and 28 indicated that the SNP loci were homozygous with genotype of CC, the panel of 2, 4, 9, 12, 12, 18 and 21 indicated homozygous with genotype of TT and others were heterozygouse with genotype of CT

Family SNP Genotyping Genotype distributions of the twenty investigated SNPs in the 180 specimens SNP lociType Genotype MAFSNP lociType Genotype MAF AABBABAABBAB C T>CTs C C>GTv C T>CTs C C>ATv C G>CTv C A>CTv C A>CTv C C>TTs C A>CTv C C>ATv C C>TTs C T>ATv C T>CTs C T>CTs C C>GTv C C>TTs C G>TTv C C>ATv C A>GTs C A>GTs Note: Transition: Ts; Transversion: Tv; AA is the wild genotype, BB is the mutation genotype and AB is the heterozygote; MAF: Minor allele frequency.

Conclusion Pyrosequencing technology is a valuable method for SNP identification. Tetra-primer ARMS is a simple and effective method for SNP genotyping. A single Tetra-primer-ARMS PCR procedure was sufficient for the detection of two different mutations in a SNP locus. The SNPs study of F. chinensis family is suggesting that SNP markers have adequate levels of polymorphisms to make them useful for genetic and breeding studies in F. chinensis.