Studies On Expression Characteristics Of Meiosis-related Genes (Ph1,Dmc1) In Autotriploid Carassius Auratus

Triploidy organisms are generally considered infertile because they cannot undergo normal meiosis.However,it is found that the natural autotriploid Carassius auratus can undergo normal meiosis,showing that both females and males are fertile.Artificial autotriploid Carassius auratus obtained by hybridization between red crucian carp Carassius auratus red var.(2n = 100,?)and autotetraploid fish(4n=200,?)in laboratory,the female is fertile while the male is sterile.In order to better understand the effect of autotriploid on the fertility of Carassius auratus,we used histology and molecular biology techniques to study gonad development,gene sequences,expression levels and methylation levels of meiosis related genes(Ph1,Dmc1)in natural and artificial autotriploid Carassius auratus.The main research contents and results are as follows:1.Gonadal structure and sperm DNA content of natural autotriploid Carassius auratus during breeding period were observed and detected by gonadal tissue section and flow cytometry,respectively.The results showed that females and males of natural autotriploid Carassius auratus had normal gonadal structure and could undergo normal meiosis to produce meiosis gametes.Gonadal structure and gamete morphology of artificial autotriploid Carassius auratus during reproduction wereobserved by gonadal tissue section and microscope respectively,and the results showed that females of artificial autotriploid Carassius auratus could produce normal egg cells,while males could not produce mature sperm.2.The cloning and analysis of CDS region sequences of Ph1 and Dmc1 genes showed that the sequence similarity of Ph1 and Dmc1 genes was 99.77% and 99.99% in artificial autotriploid Carassius auratus and its diploid ancestor,and 99.40% and 100% in natural autotriploid Carassius auratus and its diploid ancestor,respectively.The results showed that during the process of autotriploidy,meiosis related genes(Ph1 and Dmc1)were conserved,and no obvious mutation occurred in the gene sequence.3.Real-time PCR and promoter region methylation levels analysis of Ph1 and Dmc1 genes in the gonads of artificial and natural autotriploid Carassius auratus showed that compared with its diploid ancestors,Ph1 and Dmc1 genes had high expression levels and low methylation levels in the ovary and spermary of natural autotriploid Carassius auratus and in the ovary of artificial autotriploid Carassius auratus,while had low expression levels and high methylation levels in the spermary of artificial autotriploid Carassius auratus.This result indicated that the high expression of Ph1 and Dmc1 genes can promote meiosis recovery in the autotriploid Carassius auratus and then produce mature gametes,whilethe methylation levels of Ph1 and Dmc1 genes may be involved in the regulation of gene expression levels,thus affecting the meiosis process of the autotriploid Carassius auratus.By analyzing the gonadal development and meiosis of two different autotriploid Carassius auratus,this study provides new insights into the influence of autotriploid on vertebrate fertility,which is of great biological significance to fish evolution and genetic breeding.