Parentage Determination Based On Microsatellite And Its Application Of Hatchery-releasing For Orange-spotted Grouper(Epinephelus Coioides)

The orange-spotted grouper,Epinephelus coioides,is a valuable and popular seafood fish in the Indian Ocean,the Red Sea,the Mediterranean Sea,the Northern Pacific Ocean,the Western Pacific Ocean and the Southeast Asia,and is one of the major mariculture species in China.Due to the overfishing,water pollution and other human factors,the population of wild orange-spotted grouper decreased sharply.We always use culture population to increase the amount of resources,but failed to screen the germplasm resources with genetic assessment.In this paper,microsatellite or simple sequence repeats(SSRs)was used as genetic markers to identify the paternity and identify the hatchery-released population and the wild population accurately.The technique of the mixed population of the grouper was established to evaluate population genetic to provide relevant theoretical basis and guidance for hatchery release.In this study,we constructed two sets of paternity testing systems based on77 polymorphic SSR markers developed by genome-wide sequencing and analyzed their genetic diversity.The results showed that:(1)Based on 77 2-4 base repeat microsatellite sequences obtained from the Illumina II second genome sequencing in our laboratory,we screened seven three-base SSRs and five four-base SSRs.These microsatellite markers are stable,highly polymorphic,highly heterozygous and with the same annealing temperature of 58℃.Through optimization of conditions,we established two sets of multiplex fluorescence PCR system,and verified by a whole sib family,with the identification rate of100% and can stable amplification.(2)To build a crossed family with 27 parents(15 females and 12 males)and produce 226 F1 offspring.In addition,two completely different wild populations from Hainan and Daya Bay were selected,which were 38 and 41,respectively.Then they were mixed with progeny F1 of breeding population and used to simulate the population of hatchery-released and further verify the accuracy of the patertage assignment.The results showed that only 4 individuals were misidentified with an accuracy rate of 98.68%,which could be used for screening grouper grouper.(3)Using the established two sets of paternity testing system,12 SSR markers were used to analyze the genetic diversity of two populations in Hainan,Guangdong and the populations to be released,and found obvious genetic differentiation.In this study,we established two sets of high-resolution parentage assignments for orange-spotted grouper.The technology can be applied to the hatchery-released populations,and provide theoretical guidance for the genetic breeding of orange-spotted grouper.