construction of reproduction cdna microarray tiger shrimp (penaeus...
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Construction of reproduction cDNA microarray and identification of functionally important
genes in the reproductive system of the black tiger shrimp (Penaeus monodon)
Nitsara Karoonuthaisiri Kanchana Sittikankeaw
Umaporn UawisetwathanaSirawut Klinbunga
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Difficulties for maturation in captivity prevent the application of genetic improvement and selective breeding programs in this species.
• Outbreak of diseases• Reproductive maturation• Domestication• Shortage of wild-caught
broodstock
Motivation
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v Genomic health science
v Genotype-environment
interactions
v Domestication and
strain selection
v Marker assisted
selection (MAS)
v Heterosis
v Sex reversal and
breeding
v Monosex culture
vv Genomic health scienceGenomic health science
vv GenotypeGenotype--environment environment
interactionsinteractions
vv Domestication and Domestication and
strain selectionstrain selection
vv Marker assisted Marker assisted
selection (MAS)selection (MAS)
vv HeterosisHeterosis
vv Sex reversal and Sex reversal and
breedingbreeding
vv MonosexMonosex cultureculture
ESTs&
cDNA microarrays
ESTs&
cDNA microarrays
Geneticlinkage
maps
Geneticlinkage
maps
BioinformaticsBioinformatics
BACBAC
Genome sequencingGenome sequencing
SNPsSNPs
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Objectives
To construct Penaeus monodon
cDNA Microarrays
To use the arrays to identify genes
involved in ovarian development
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Reproduction Microarray: ReproArray
cDNAAmplification
Purification
Transfer
Fabrication
EST Library Construction
Testis Ovary
Colony PCR Plasmid Prep
Whatman Plate Purification
96-well à 384 well
SpotArray 72
PCR
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Colony PCR
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PCR ProductsBefore and After Purification
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Microarray Fabrication Summary
• Ovary– 1920 plasmids were amplified– 20 plates of 96-well PCR format– Sequenced and Blasted
• Testis– 3072 colony PCR reactions– 32 plates of 96-well PCR format– Sequenced and Blasted
• Overall– 4992 features were fabricated– 3 Print Runs (68 slides/print run)– 204 slides total
SpotArray 72 PerkinElmer
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Quality Check of Arrays
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Objectives
To construct Penaeus monodon
cDNA Microarrays
To use the arrays to identify genes involved in ovarian development
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Transcriptomic Studies
Reference Test Sample
RNA
cDNA
DNA Microarrays
Redà up-regulated
Green à down-regulated
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Anterior Lateral Posterior
Ovarian Developmental Stages
Genes involved in ovarian Genes involved in ovarian maturation of maturation of PP..monodonmonodon is yet to be revealed.is yet to be revealed.
Source: http://aims.gov.au/pages/research/mdef/mdef-02a-1.html
Stage 2
Stage 3
Stage 4Fixed in 70% alcohol causing a change of colors
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Examples of Result Image
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K Means Clustering
Gene expression patterns in female broodstockreproduction stages
A
B
C
D
Cy3 Stage I (GSI= 1.5)Cy5 Stage II III IV
Increase in GSI
GSI Values
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Acknowlegdements
• National Center for Genetic Engineering and Biotechnology (BIOTEC)
• Dr. Jingyue Ju and his research group (Columbia University, New York, USA)
• Members of Aquatic Molecular Genetics and Biology Lab (BIOTEC)
• Members of Microarray Laboratory (BIOTEC)
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