Transcript JianYong Zhang

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.
Fenneropenaeus chinensis
15-20cm
delicious food
Larvae Rearing
Character Test
Family Conservation
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.
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.
3. Results
454 transcriptome pyrosequencing
Resistant shrimp-454 reads
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.
3. Results
Prawn-cDNA 454 sequencing
Resistant
Sensitive
Reads 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
Specific sequence of Resistant and Sensitive
Resistant
Seq number (contigs+singlets) 31,969
Specific sequence
18,331
Differential Expression
Sensitive
26,347
14,437
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
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
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 Genotyping
Genotype distributions of the twenty investigated SNPs in the 180 specimens
Genotype
SNP loci
Genotype
Type
MAF
AA
BB
AB
SNP loci
Type
MAF
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.