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PROJECTS: GENETICS OF THE PURPLE SEA URCHIN USING ARRAY TECHNOLOGY

S. purpuratusAdaptive evolution in a species distributed along an environmental gradient is a tug-of-war between the forces of natural selection and gene flow. Some alleles may freely move between populations via dispersal, while others are favored or removed by natural selection. The challenge is to identify which are the genes targeted by natural selection. Recent advances have allowed us to approach this task in a novel way.  Using the published genome sequence and the latest array technology, we have developed a technique to scan all 23,000 genes of the purple sea urchin, Strongylocentrotus purpuratus, genome for the signature of natural selection acting between populations.

The purple sea urchin is an excellent species for this work because its range spans a large environment gradient from the cold waters of Alaska to the warm waters of Mexico. In addition, population sizes are large, minimizing the effects of genetic drift and maximizing the effects of selection.  Finally, they have high dispersal potential because of their pelagically dispersing larvae. Previous studies have shown little to no population genetic structure. Thus, genes that have diverged between populations would have to have a major selective advantage to withstand the homogenizing effects of gene flow, making the purple sea urchin a robust system for the study of natural selection.

Our genome scan is based on detecting restriction cut site polymorphisms differing from the published genome sequence. Custom high-density oligonucleotide arrays are designed with 50 base pair probes centered on restriction cut sites. A genomic DNA sample from an individual is processed by random shearing, restriction digestion, and fluorescent labeling before hybridization to an array. Restriction digested fragments will not bind to the tile, while DNA that has a mutation in the restriction cut site will not cut and will bind to the tile. Fluorescent imaging will reveal single-nucleotide-polymorphisms (SNPs) by variations in signal intensity.

Results from 14 array hybridizations show a major effect of restriction digestion on the signal intensity of a given feature and very low variation among replicate features on an array, among replicate experiments, and among control features between individuals. We are currently in the process of generating a population data set of 40 individuals, 20 from the northern end of the species range and 20 from the south. The major effect of restriction digestion, low variability among replicates, and high repeatability, will allow the detection of SNPs and non-SNPs for each feature using this population data set. Candidate markers for adaptively evolved genes will be those where the presence and absence of SNPs perfectly segregates between populations. Genes identified as potentially adaptive will sequenced across the species range to look for clinal variation and genetic patterns will be compared to ecological and biogeographic patterns.

Genes identified in this study can be examined in other species that have similar or different life history characteristics or species ranges to investigate if other organisms have convergently evolved similar adaptive solutions to their local ecology. This technology can also be applied to study organisms that do not have a sequenced genome; one can screen a cDNA library sequence from any organism for restriction cut site polymorphisms comparing two groups, for example, diseased and not-diseased, or high and low intertidal zone. Finally, uncovering the genomic underpinnings of how populations adapt to local environmental conditions will help biologists understand local adaptation as a mechanism for speciation, help conservationists manage and protect populations, and help scientists predict how a species might respond to global climate change.

Hopkins Marine Station, Stanford University, 120 Ocean View Blvd., Pacific Grove, CA 93950