Development and Application of SNP markers in Genome

Development and Application of SNP markers in Genome of shrimp (Fenneropenaeus chinensis) Jianyong Zhang Marine Biology 1

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. 2

Fenneropenaeus chinensis 15 -20 cm delicious food 3

Larvae Rearing Character Test Family Conservation 4 Varieties 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. 5

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. 6

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. 7

3. Results 454 transcriptome pyrosequencing Resistant shrimp-454 reads 8

Sensitive shrimp-454 reads 9

CAP 3 assembly default parameter： overlap 40 bp，identity 80% Match scores, mismatch scores, and gap penalties are all weighted by the quality values of the bases involved. 10

3. Results Prawn-c. DNA 454 sequencing Resistant Sensitive Reads number (ave len) 268, 511 (205 bp) 229, 335 (235 bp) Base number 48, 231, 158 bp 47, 352, 259 Number of assembled reads 220, 652 195, 637 Contig number 11, 750 11, 218 Max Contig len 3, 588 bp 3, 919 bp Contigs ave len 321 bp 355 bp Singlets number 20, 219 15, 129 11

Specific sequence of Resistant and Sensitive Resistant Sensitive Seq number (contigs+singlets) 31, 969 26, 347 Specific sequence 18, 331 Differential Expression 14, 437 12

Gene prediction base on sequencing R S Seq number (contigs+singlets) 31, 969 26, 347 Encode Protein 31, 836 26, 271 Protein annotate 5, 536 5, 443 Protein of GO Ontology 2, 773 2, 692 Gene prediction ：Get. ORF Gene Ontology analysis：gopipe 13

SNP calculation SNP loci 71, 724 Samesense mutation 17, 329 Non synonymous mutation 34, 642 Nonsense mutation 1, 478 Noncoding region 18, 275 Indel loci 31, 769 14

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): E 88 -8 15

SNP confirmation ARMA-PCR amplification The electrophoretogram was the genotyping by ARMS-PCR for SNP locus of contig 17838. 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, 18 and 21 indicated homozygous with genotype of TT and others were heterozygouse with genotype of CT 16

Family SNP Genotyping Genotype distributions of the twenty investigated SNPs in the 180 specimens Genotype SNP loci Ty pe A A B B A B Genotype MA F SNP loci Ty pe A A B B A B MA F C 3422 -126 T>C Ts 63 33 84 0. 4 17 C 9258 -329 C>G Tv 45 30 10 5 0. 4 58 C 4413 -277 T>C Ts 54 17 10 9 0. 3 97 C 14418 -530 C>A Tv 56 45 79 0. 4 69 C 9863 -273 G>C Tv 36 35 10 9 0. 4 97 C 17091 -559 A>C Tv 41 15 12 4 0. 4 28 C 11528 -234 A>C Tv 37 29 11 4 0. 4 78 C 4698 -355 C>T Ts 61 24 95 0. 3 97 C 14198 -323 A>C Tv 64 21 95 0. 3 78 C 5806 -373 C>A Tv 44 22 11 4 0. 4 39 C 18153 -299 C>T Ts 45 37 98 0. 4 78 C 12635 -182 T>A Tv 58 29 93 0. 4 19 C 18153 -524 T>C Ts 46 19 11 5 0. 4 25 C 17838 -344 T>C Ts 57 36 87 0. 4 42 Note: Transition: Ts; Transversion: 0. 4 Tv; AA is the wild genotype, BB is 10 the mutation genotype and AB is the C 244 -659 C 17838 -7370. 4 Tv 62 27 91 Ts 54 26 17 C>G 03 C>T 0 22 heterozygote; MAF: Minor allele frequency. C 6414 -458 - 0. 4 C 18477 -208 - 10 0. 4

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. 18

19