Structure of amylase genes in populations of Pacific Cupped oyster ( Crassostrea gigas): tissue expression and allelic polymorphism.

ABSTRACT

Using the previously determined complementary DNA Sequence of Crassostrea gigas amylase (Y08370), we designed several oligonucleotide primers and used them with polymerase chain reaction (PCR) technology to characterize oyster amylase gene sequences. Two genes encoding 2 different amylases were characterized and sequenced. The 2 genes are similarly organized with 8 exons and 7 introns. Intron insertions are found at the same location in the 2 genes. Sizes and nucleotide sequences are different for the different introns inside each gene and different for the corresponding introns in the 2 genes. Comparing the 2 genes, around 10% of the nucleotides are different along the exons, and comparing the 2 deduced protein sequences, a mean value of 10.4% of amino acids are changed. Genes A and B encode mature proteins of, respectively, 500 and 499 amino acids, which present 94% similarity. A microsatellite (TC(37)) that constitutes the largest part of intron 4 of gene A has been used as a polymorphic marker. A method consisting of a PCR step followed by EcoRI digestion of the obtained fragments was used to observe polymorphism in these 2 genes. Six and 4 alleles for genes A and B, respectively, have been sequenced, leading to a maximum of 2.9% base change. The 2 genes are ubiquitously expressed in the different digestive tissues with quantitative differences. Gene A is strongly expressed in the digestive gland and at a lower level in stomach, while gene B is preferentially expressed in the labial palps. The microsatellite repeat was used in the analysis of 4 populations of Crassostrea gigas from the French Atlantic coast. A high level of polymorphism observed with 30 different alleles of gene A inside the populations should allow their characterization using the mean value of the microsatellite allelic distribution. These populations showed a low level of differentiation ( F(st) between 0 and 0.011); however, the population of Bonne Anse appeared to be distinguished from the other populations.