Plant & Animal Genome IX Conference Plant & Animal Genome IX Conference
Workshop: Aquaculture
W10_07.html
Forty-nine simple sequence repeat (SSR) or microsatellite regions of the Crassostrea virginica genome were identified by screening DNA sequences of a small-insert genomic library . PCR primers have been designed to amplify more than 50 of these microsatellite regions. Amplification reactions have been optimized for 13 loci representing 7 di-, 3 tri- and 3 tetranucleotide repeat sequences. These markers were successfully used to screen 5 pedigreed reference families. Comparisons of parental and adult F1 progeny genotypes indicated non-Mendelian inheritance patterns. A protocol for isolation of DNA from individual larvae and embryos is being optimized to circumvent segregation distortion problems that would confound mapping. In addition to SSR regions identified from the small insert library, five microsatellite-enriched libraries were constructed for tri- and tetranucleotide repeat sequences. More than half of the sequences from the enriched libraries contain the targeted repeat motifs. Many SSRs from both the small insert and microsatellite enriched libraries exhibit imperfect repeat sequences. PCR primers have been designed and are being tested for 18 additional SSR loci. Of these, null alleles were identified at 3 loci following initial primer design. Redesigned primers have allowed amplification of previously undetected alleles. With three additional markers null allele difficulties are suspected, since several individuals cannot be successfully amplified at otherwise clearly amplifiable loci. Allelic profiles for 6 allozyme, 9 single-copy nuclear and the 13 microsatellite loci have been generated for 40-60 F1 individuals from the reference families for preliminary linkage analysis.