Characterization of single nucleotide polymorphisms from expressed sequence tags of Chinese mitten crab Eriocheir sinensis

The Chinese mitten crab Eriocheir sinensis is an important aquaculture species in Asia and causes considerable economic and ecological damage as an invasive species in North America and Europe. Until recently, molecular markers available for genetic analysis in this species were limited to a few microsatellite sequences used in population studies. Here we describe the discovery of 3191 single nucleotide polymorphisms (SNPs) from the alignment of expressed sequence tags (ESTs) in E. sinensis. The observed frequency of SNPs was estimated at 0.78 per 100 bp of contig sequences. C/T substitutions were frequent and accounted for 32.2% of all SNPs. A subset of these SNPs (n = 38) was selected for validation by allele-specific PCR with melting temperature (T-m)-shift primers based on their observed frequency in sequence data; 12 (31.6%) of these were polymorphic in a panel of 40 wild-caught crabs. Eight (21.1%) did not amplify any product, and 18 (47.4%) failed due to amplification failure of 1 allele-specific primer. A table of optimal codons was deduced from the analysis of the E. sinensis EST dataset, and the results implied that the gene expression levels and GC-content might play important roles in codon usage bias. Sixteen codons ending with a G or C base were defined as 'optimal codons,' which may provide useful information for predicting gene expression in crabs. These are the first SNPs developed for the Chinese mitten crab and will provide a useful complement to currently available genetic markers.