Project Number: 1930-31000-008-03
Project Type: Reimbursable

Start Date: Aug 01, 2002
End Date: Jul 31, 2005

Objective:
The objective of this cooperative research project is to develop microsatellite genetic markers for use in selective breeding programs for striped bass.

Approach:
The National Center for Cool and Cold Water Aquaculture will contribute to the development of microsatellite markers for striped bass. This work will contribute towards the eventual realization of genetic mapping, quantitative trait loci identification, and marker assisted selection for striped bass. It has been estimated that approximately 50% of striped bass microsatellite markers are polymorphic (unpublished). To reach a goal of 100 useful markers, we must plan on successfully amplifying ~200 unique microsatellite marker sequences. In similar projects for rainbow trout, our success of PCR optimization was ~90%, so we must plan to design primers for ~225 microsatellites. Also, prior studies indicate that ~20% of clones will contain a microsatellite for which PCR primer design, PCR primer optimization, and polymorphism identification are successful. For this reason we will sequence 5 96-well plates of clones from microsatellite enriched libraries obtained as frozen glycerol stocks from Kent SeaTech (Westerman lab). The clones will be copied and sent to the USDA Nucleic Acid Core Facility (NAF) for DNA isolation and sequencing using robots and an ABI 3700. Sequences will be downloaded by FTP to the Rexroad laboratory and analyzed using Vector NTI Suite 7.0. Analysis will include removal of vector contamination and grouping of sequences according to microsatellite repeat. Sequence groups will then be aligned using Contig Express to identify unique microsatellite markers. Unique sequences of good quality will be used for PCR primer design using Oligo 6.0. Oligos will be ordered for primer optimization. PCR primer pairs which are successfully optimized for amplification of microsatellites from striped bass genomic DNA and miniprep DNA from the clone of origin will be sent to North Carolina State University for genotyping.