DHPLC for Genetic Marker Discovery and Screening in Oyster Genomics

Recipient Organization
UNIV OF DEL
700 PILOTTOWN ROAD
LEWES,DE 19958

Performing Department
COLLEGE OF MARINE STUDIES

Non Technical Summary
This grant will be used to purchase a high-throughput system for detecting genetic variation at the DNA level. The primary use of this apparatus will be to discover genetic markers for use in genetic mapping projects, with initial applications to oysters and poultry. Genetic mapping is used to locate genes affecting commercially important traits (growth rate, disease resistance, etc.), enabling more effective selective breeding programs and improving our understanding of the genetic basis of variation in performance. For many organisms such as oysters, genetic maps are only in the early stages of development, partly because of the lack of available markers. The new apparatus will make it possible to screen thousands of sites in any target genome in search of genetic variation, in a cost-effective manner. Our first application will be to develop several hundred markers for mapping the oyster genome, as part of the effort to develop lines that can resist the two diseases that are decimating the Atlantic coast oyster fishery; in addition, scientists in the University of Delaware College of Agriculture and Natural Resources will use the apparatus to develop markers for genetic improvement of broiler chickens.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
304	3723	1040	60%
311	3723	1080	40%

Knowledge Area
311 - Animal Diseases; 304 - Animal Genome;

Subject Of Investigation
3723 - Oysters;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;

Goals / Objectives
Using eastern oyster sequence databases (genomic DNA, >0.7 MB; >1000 ESTs), develop and test PCR primers to generate small (200-400 bp) amplicons. Screen amplicons for DNA sequence variation using high-throughput denaturing high performance liquid chromatography (DHPLC) with a Transgenomic Wave system, or a Spectrumedix temperature gradient capillary electrophoresis (TGCE) system. Characterize single nucleotide polymorphisms (SNPs) and insertions/deletions in polymorphic amplicons by direct sequencing. Provide screened markers and detection protocols to oyster genetics researchers for use in genetic mapping and analysis of germ plasm diversity.

Project Methods
PCR primers will be designed using primer design software (Oligo 6.0), with attention to primer location, in order to 1) reduce the frequency of null (non-amplifying) alleles caused by priming site polymorphism, and 2) produce amplicons with desirable DNA melting profiles, as determined by computer analysis (http://insertion.stanford.edu/melt.html). Amplicons will be screened for the presence of heteroduplex peaks indicative of sequence heterogeneity, using either DHPLC or TGCE. Identified polymorphisms will be characterized by direct sequencing to provide alternative detection methods (RFLP, various SNP detection techniques) for use by wider research community.

Progress 11/15/02 to 11/14/03

Outputs
The award was used to purchase a a high-throughput system for detecting genetic variation at the DNA level, a Spectrumedix SCE-2410 24-capillary electrophoresis unit, with Reveal Mutation Detection software. After an initial period of training and protocol development, we are using this system to detect sequence variation in several different farmed aquatic species. Current work includes discovery of DNA polymorphisms for genetic mapping in hard clams (subcontract to Virginia Institute of Marine Science, USDA-sponsored project), eastern oysters (research projects funded by NOAA Sea Grant) and Pacific oysters (USDA NRI award beginning in 2004). Preliminary work on marker development in farmed salmon species is in progress. Additional funds were leveraged from other sources to fit out the Spectrumedix SCE2410 system for automated dye terminator DNA sequencing, enabling the full process of single nucleotide polymorphism (SNP) discovery, characterization, and assay development for rapid genotyping.

Impacts
The acquisition of the instrument will provide the emerging aquacultural genetics/genomics research community with a powerful tool for rapid development of genetic markers for genetic mapping and analysis of germ plasm diversity. The instrumentation and technical expertise developed in the principal investigator's laboratory will be available to interested researchers at the University of Delaware and elsewhere.

Publications

No publications reported this period