| The sex determination mechanism has been a hot spot in life science research.Mollusca is the first major category of marine organisms,with rich species,diverse habitats,and complex sex determination mechanisms,but the research on the genetic mechanism of sex determination is obviously lagging behind.Haliotis discus hannai Ino is an important mariculture shellfish in our country,and H.sieboldii Reeve is a related species.This study intends to use them as the research object,identify their sex chromosomes through genome-wide association analysis and the distribution of sexually dimorphic SNPs,and develop molecular markers identification for sexdetermining.The main findings are as follows:(1)Combining the two methods of genome-wide association analysis and the distribution analysis of sexually dimorphic SNPs,the sex chromosomes were identified by locating the sexlinked genomic segments of H.discus hannai and H.sieboldii.Among them,the H.discus hannai has two SLSs,located at Chr.9(approximately 2.59 Mb)and Chr.2(approximately 50.02 Kb);the H.sieboldii also has two SLSs,located at Chr.4(about 23.20 Mb)and Chr.5(about 4.95 Mb).The result of chromosome collinearity analysis showed that Chr.2 and Chr.9 of H.discus hannai and Chr.4 and Chr.5 of H.sieboldii were not homologous chromosomes.In addition,28 genes containing sexually dimorphic SNP/In Del were annotated in H.discus hannai,and 189 genes were annotated in H.sieboldii,and the only intersection gene was the xyn B gene(involved in xylan degradation).(2)We used the sexually dimorphic In Del and SNP to develop sex molecular markers of H.discus hannai and H.sieboldii.In H.discus hannai,two co-dominant markers(MSP-2 and MSP-7)were developed based on the two sexually dimorphic In Del on Chr.9.At the same time,based on the three continuous sexually dimorphic SNPs on Chr.2,a dominant marker(2MSP-FX1)was developed.The verification results showed that the detection rates for both sexes of MSP-2 and2MSP-FX1 were 100%.In H.sieboldii,based on In Del and SNP of Chr.4,a total of 7 pairs of primers were designed,of which 3 pairs of primers in the verification experiment had a sex coincidence rate of over 95%,but no molecular markers completely linked to sex were found.The male and female individuals of the hybrids between H.discus hannai and H.sieboldii were examined by MSP-2 and X14-FR.The results showed that the sex molecular markers of H.discus hannai can be applied to the hybrids,but the markers of H.sieboldii were not applicable.(3)The sex chromosomes of H.discus hannai were identified by BAC-FISH.Using bioinformatics methods,the 2,411 BAC end sequences were compared with the reference genome of H.discus hannai.Among them,there were 3 BAC clones located on Chr.2,and there were 6BAC clones located on Chr.9.According to the results of the comparison and BAC interval repetitive sequence content statistics,clones 31-K11 and 37-A13 were selected for BAC-FISH to identify Chr.2 and Chr.9.The above results show that both H.discus hannai and H.sieboldii may have two pairs of sex chromosomes,and the sex chromosomes and sex determination system may have turnover,and there are also differences in the range of non-recombination regions and the degree of differentiation between the two abalones.The results of this thesis can lay a foundation for further research on the molecular mechanisms of sex determination of H.discus hannai and H.sieboldii. |
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