| Mesocentrotus nudus,also known as Dalian purple sea urchin,northern purple sea urchin,belongs to the family Echinodermata,Echinoidea,Camarodonta,and Strongylocentrotidae,and is widely found in China’s Shandong Province and along the Korean Peninsula,northern Japan,and Russia’s Far East coast.Sea urchin gonads are the only edible part,and the most economical part.Sea urchin gonads are rich in unsaturated fatty acids and flavor amino acids,taste delicious,gradually become a popular world cuisine.With the increasing demand of the market,the scale of its breeding is also expanding.Sea urchins are gonochorism,but most species of sea urchins have no obvious gender dimorphism and are unable to distinguish sex from appearance,which somewhat limits the process of artificial breeding of M.nudus.In addition,there are significant differences in immunity,growth rate and nutritional composition of urchins between different genders.Therefore,the research on the mechanism of urchin gonadal development and the identification of sex molecular markers has become one of the hot spots.In this study,we identified genome-specific DNA molecular markers in M.nudus by type IIB endonuclease restriction-site associated DNA sequencing(2b-RAD),genome survey and genome-wide association studies.A rapid and accurate PCR-based method for identifying the sex of M.nudus in vivo was developed by sequencing the gendered candidate DNA molecular markers and single nucleotide polymorphism(SNP)sites in different breeding and wild populations.The main findings of the study are as follows:1.Tube foot genomic DNA from 10 male and 10 female M.nudus was proposed for 2b-RADseq,sequenced to 137,684,505 raw reads and filtered to 129,116,728 clean reads.A total of2,384,989 tags and 43,922 polymorphic SNPs were obtained by comparing female and male derived clean reads.2.Genome survey sequencing was performed to obtain 684,230,824 raw reads in female urchins and 669,375,890 raw reads in male urchins,filtered and assembled to obtain female and male genomic sketches.After splicing and assembly,1,582,413 scaffolds were obtained in females,with the longest scaffold being 52,656 bp and the average length 1,579 bp.There were 5,953,609 scaffolds in males,with the longest scaffold at 10,758 bp and an average length of 1,579 bp.3.The tags obtained by 2b-RAD-seq were compared with the genome survey results,and a total of 13 female-specific candidate tags and 20,201 sex-related SNPs were obtained.Among the top 10 sex-related SNPs with confidence,all females are heterozygous and all males are homozygous,suggesting that a ZW/ZZ-type sex determination system may exist in M.nudus.4.The obtained 13 female-specific candidate tags and the top 10 sex-related SNPs were verified by PCR in 10 females and 9 male M.nudus,and finally confirmed that there are 3 femalespecific tags only in females The target band can be amplified in the individual,but there is no amplification product in the male individual.In all individuals analyzed,SNP1 was A/G heterozygous in females and A/A homozygous in males,SNP2 was A/G heterozygous in females,and SNP2 was T/T homozygous in males,SNP3 is C/T heterozygous in females and C/C homozygous in males.5.Three female-specific tags were verified by PCR amplification in 95 breeding population and 19 wild population,and it was found that the accuracy rate of using these three tags to identify the genetic sex of M.nudus was 100%.The results of this study have realized the rapid and accurate identification of the genetic sex of M.nudus,realized the precise collocation of male and female in the breeding process,improved the efficiency of its genetic breeding,and laid a foundation for revealing the sex determination mechanism of sea urchins,which provided support for the development of sea urchin sexcontrolled breeding. |
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