Development and Application of SNP markers in Genome of shrimp (Fenneropenaeus

chinensis)

Jianyong Zhang

Marine Biology

1 、 Introduction

• The Chinese shrimp, Fenneropenaeus chinensis, widely natur

ally distributed in the coastal waters of north China, has espe

cially become an important economic mariculture species in t

his region

• The current studies on shrimp mainly concentrated on the res

earch of molecular marker development and application, gene

clone, disease resistance and high yield breeding, etc.

Fenneropenaeus chinensis

15-20cm

delicious food

Larvae Rearing Family Conservation

Character Test Varieties Propagation

White Spot Syndrome Virus (WSSV)

• WSSV was first found in South Asia and then spread to Ame

rica, 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 nu

cleotide polymorphism （ SNP） loci involved in

disease resistance to WSSV.

• Identifying the facticity of putative SNPs and analyzin

g genetic diversity of family or constructing genetic li

nkage map.

2. Materials and Method

• Resistant shrimp and Sensitive shrimp to WSSV were sequ

enced based transcriptome using Roche 454 GS FLX syste

m by Chinese National Human Genome Center (Shanghai).

• Analyzing sequencing data with software.

• Thirty individuals from each of six shrimp families were s

ampled to identify putative SNP loci with amplification ref

ractory mutation system (ARMS) PCR method.

Resistant shrimp-454 reads

3. Results454 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

Resistant SensitiveReads number (ave len) 268,511 (205bp) 229,335 (235bp)

Base number 48,231,158bp 47,352,259

Number of assembled reads 220,652 195,637

Contig number 11,750 11,218

Max Contig len 3,588bp 3,919bp

Contigs ave len 321bp 355bp

Singlets number 20,219 15,129

3. Results

Resistant SensitiveSeq number (contigs+singlets)

31,969 26,347

Specific sequence 18,331 14,437

Specific sequence of Resistant and Sensitive

Differential Expression

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 ： GetORF

Gene Ontology analysis： gopipe

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

SNP confirmation

ARMA-PCR amplification

• Eighty putative SNPs loci were chosen and were validated by PC

R-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, bot

h 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 contig17838. 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 GenotypingGenotype distributions of the twenty investigated SNPs in the 180 specimens

SNP loci Type

Genotype

MAF SNP loci Type

Genotype

MAF

AA BB AB AA BB AB

C3422-126-T>C Ts 63 33 84 0.417 C9258-329-C>G Tv 45 30 105 0.458

C4413-277-T>C Ts 54 17 109 0.397 C14418-530-C>A Tv 56 45 79 0.469

C9863-273-G>C Tv 36 35 109 0.497 C17091-559-A>C Tv 41 15 124 0.428

C11528-234-A>C Tv 37 29 114 0.478 C4698-355-C>T Ts 61 24 95 0.397

C14198-323-A>C Tv 64 21 95 0.378 C5806-373-C>A Tv 44 22 114 0.439

C18153-299-C>T Ts 45 37 98 0.478 C12635-182-T>A Tv 58 29 93 0.419

C18153-524-T>C Ts 46 19 115 0.425 C17838-344-T>C Ts 57 36 87 0.442

C244-659-C>G Tv 62 27 91 0.403 C17838-737-C>T Ts 54 26 100 0.422

C6414-458-G>T Tv 64 36 80 0.422 C18477-208-C>A Tv 54 25 101 0.419

C6707-288-A>G Ts 60 27 93 0.408 C929-994-A>G Ts 38 23 119 0.458

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.