A microsatellite panel for triploid verification in the Pacific oyster(Crassostrea gigas)

In the Pacific oyster(Crassostrea gigas),triploidy induction is the most common genetic method to enhance production yield through phenotypic improvement.Production of triploids necessarily requires a proper method to verify the ploidy level after induction.Various methods have been developed such as flow cytometry,chromosome counting by karyotype analysis,counting of nucleoli,particle size analysis and so on.However,each method has some drawbacks considering size of sample,accuracy,complexity in conduction and cost.This study aims to identify a microsatellite panel for triploid validation in the Pacific oyster and further explore the number of microsatellites required to successfully verify ploidy status.Triploid oysters are usually induced by suppressing the extrusion of the second polar body.If the distance between a microsatellite and centromere is great,homologous chromosomes might cross over at prophase I of most meiosis allowing the triploid progenies having both of the two maternal alleles.This study screened the 56 microsatellites located in the centromere mapping and obtained seven successfully amplified loci at which the female parent is heterozygous and does not share any allele with the male parent.Using these seven microsatellites,40 triploid oysters were verified from the 115 individuals treated by cytochalasin B(CB,0.5 mg/L)for 15 min starting from the time when about 50% of the eggs released the first polar body.Ploidy status was then verified by flow cytometry and the correspondence was 100%.The ability of a microsatellite in identifying triploids relies on its frequency of microsatellite-centromere recombination(y).To further explore the number of microsatellites required to successfully identify all the triploids,different combinations of microsatellites were analyzed.The results indicate approximate 100% accuracy when product of(1-y)of all microsatellites is no more than 0.005.This study shows that microsatellite markers can serve as an accurate,fast,cost-effective and technically simple method for triploid verification in the Pacific oyster.The protocol described in this work will help in the application of triploids verification using molecular markers in other species.