Development of SSR marker by RNA-seq and its application in genotyping pearl sac in pearl oyster Pinctada fucata martensii

Abstract

Background: Pearl oyster Pinctada fucata martensii is cultured for producing round nucleated pearls. Pearl production involves a surgical operation, where a mantle tissue graft from a donor oyster and a round nucleus are implanted in the gonad of a host oyster. Whether the mantle graft implanted in the gonad of a host oyster contribute to the formation of a pearl sac that secretes pearl nacre to form pearl should be determined. In April 2012, two full-sib families were used separately for donor and host oysters for a nucleus insertion operation. The pearl sac was sampled from the host oysters at day 60 after nucleus operation. A large number of simple sequence repeat (SSR) markers were developed by using Illumina HiSeq^TM 2000 platform. The two full-sib families were also used to mine diagnostic SSR markers for genotyping donor oyster, host oyster and pearl sac.

Results: A total of 3,168 microsatellite loci were identified in 39,078unigenes, and 1,977 SSR primers were designed by Primer 3.0. Forty-seven SSR primers were validated, and the rate of successful amplification was 72.3%. Two diagnostic SSR primers could successfully genotyped pearl sac, donor oyster and host oyster. Donor and host oysters were both homogenous and the alleles in pearl sac were identical to those in donor and host oysters.

Conclusions: The present results confirmed that the mantle graft implanted in the gonad of host oyster contributed to the formation of the pearl sac in pearl oyster P. fucata martensii.