Oysters represent an important fishery and aquaculture resource worldwide. In Brazil, the main native oyster species economically important for fisheries and mariculture are Crassostrea brasiliana (= C. gasar ) and C. rhizophorae . Currently, C. brasiliana is the one use in aquaculture operations due its higher size. However, the presence of exotic oysters in native oyster banks jeopardizes the native oyster genetic resources. This study aimed at identifying native and exotic specie s in the estuarine areas of Southeast coast of Brazil as well as to develop a panel of microsatellite loci of C. brasiliana by next generation sequencing (NGS) for further studies on genetic diversity and population structure of this native oyster.
The oysters were collected in mangrove roots and rocks in three estuaries of Southeast Coast of Brazil (Paraty , Bertioga and Cananeia) . Due the high plasticity phenotypic in oysters, we used 16S and COI sequences and PCR-RFLP to identify unambiguously the different oyster species. The DNA of C. brasiliana was employed to develop microsatellite markers by NGS. A genomic library was constructed following the manufacturer protocol of Illumina® Nextera DNA Library Preparation kit and the sequencing was conducted in a HiSeq2500 (Illumina, San Diego, USA).
Among 309 samples collected in the three estuaries , 72 were C. brasiliana, 110 C. rhizophorae and 127 were Saccostrea sp., an exotic species. C. brasiliana was not found in Paraty. The PCR-RFLP using the AluI enzyme was effective to discriminate C. brasiliana and C. rhizophorae , two cryptic species, while Saccostrea sp. was easily identified by its shell's morphological characteristics. The largest fragment generates by AluI have 410bp for C. rhizophorae and 205bp for C. brasiliana.
Microsatellite markers were isolated from a genomic library obtained by NGS. After a first selection, we identified 359 loci, of which 157 are dinucleotides, 45 trinucleotides and 157 tetranucleotides. A set of 30 primers were select ed, synthesized and the PCR conditions were standardized for each pair of primer. After polymorphism analysis and validation of selected primers, they will be used to evaluate the genetic diversity and structure of wild populations of C. brasiliana oysters of the Southeast coast of Brazil.
As verified in this work, C. brasiliana has been reduced in number and almost disappeared in places where it was abundant in the past (Bertioga and Paraty) . A rapid cutback of population size has serious consequences on population health and survival. The impact of the expansion of Saccostrea sp. in the long-term survival of the native oyster should be investigated. Against this background, this new novel of microsatellites markers will be useful to assess genetic variability in remaining populations as well as it will contribute for further studies on hatchery production associated with genetic improvement programs.
Research funded by the São Paulo State Research Support Foundation - FAPESP - Pr. 2016/16108-3