The development of two novel species-specific primers for identifying genus Crassostrea, with focus on C. sikamea and C. ariakensis

Abstract

Oyster reefs have important ecological functions and environmental service values, such as filtering water, providing fish habitats, maintaining biodiversity, and carbon sequestration. Oyster reef recovery has become one of the most important means for fishery resource conservation and ecosystems restoration of coastal regions . C. sikamea and C. ariakensis are two important reef oysters along the coast of China. However, it is very difficult to identify the larvae of these two oysters. To solve this problem, we developed two pairs of novel species-specific PCR primers for identifying these two oysters. The forward C. sikamea -specific primer is Cs-F: 5′-CGAAGAGGGGCATGATAAATGAGG-3′, and the reverse primer is Cs-R: 5′-ATATGAACTTCTCCAACCTCCCC-3′, both of them within the scope of mitochondrial ND5 gene. The forward C. ariakensis -specific primer is Ca-F: 5′-GGGCAAATAAAAGGCAAAACCC-3′, located between tRNA ser and tRNA pro , and the reverse primer is Ca-R: 5′-CATAAACTTCTGCAAGACTCCC-3′, lies between tRNA pro and small subunit ribosomal RNA (ssrRNA). These two primers can identify the larvae of C. ariakensis and C. sikamea from other oysters rapidly and effectively, with extremely high accurate rate (=100%). This molecular identification method does not require prior sorting of larvae. These two specific primers are good contribution to molecular identification of C. sikamea , C. ariakensis and other oysters, and could be a useful tool for oyster larvae identification and ecological restoration.

The development of multiplex PCR assays for the rapid identification of multiple Saccostrea species, and their practical applications in restoration and aquaculture

“…The development of species-specific primers can also allow for the analysis of larger sample sizes for ecological studies. Primer sets using the mitochondrial ND5 gene were developed for the identification of M. sikamea and M. ariakensis which are known to create mixed species oyster reefs in China [ 26 ]. The development of these primer sets facilitated research that explored settlement preferences between the two species and highlighted potential reasons for limited restoration success [ 27 ].…”