Construction of reproduction cDNA microarray tiger shrimp (Penaeus...

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



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



Objectives

To construct Penaeus monodon

cDNA Microarrays

To use the arrays to identify genes

involved in ovarian development



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



Colony PCR



PCR ProductsBefore and After Purification



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



Quality Check of Arrays



Objectives

To construct Penaeus monodon

cDNA Microarrays

To use the arrays to identify genes involved in ovarian development



Transcriptomic Studies

Reference Test Sample

RNA

cDNA

DNA Microarrays

Redà up-regulated

Green à down-regulated



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



Examples of Result Image



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



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)



Construction of reproduction cDNA microarray tiger shrimp (Penaeus...

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