| This experiment was done to explore the development of the ovary and follicles of Leizhou black ducks during pre-production(90 days),laying(120 days),and end of laying(500 days)period.Moreover,DNA methylation and laying traits relationship of in Leizhou black ducks were also analyzed.Morphological analysis of ovary and follicles of Leizhou black ducks during different production stages was carried out by HE staining.The DNA methylation degree of ovary tissue genome was detected by MSAP technique.Methylation polymorphisms were cloned and sequenced,which was then correlated with the laying traits of Leizhou black ducks.The expression of genes in high-yield and low-yield ovarian tissues was studied by RT-PCR:1.The morphological and histological observations of the ovarian follicles of Leizhou black duck showed that during different stages of production the volume and weight of ovary were significantly different from each other.The weight and volume of the ovary during the pre-production period was much lower compared to the production and end of production period.Leizhou black duck follicle diameter is SWF: <4mm,LWF:(4.49±0.43)mm,SYF:(5.81±0.37)mm,LYF:(6.94±0.29)mm,F6:(10.63±0.88)mm,F5:(14.11±0.61)mm,F4:(17.33± 1.13)mm,F3:(24.31±1.24)mm,F2:(30.70±1.42)mm,F1:(34.34±0.44)mm.The ovarian structure of Leizhou black duck can be divided into reproductive epithelium,tunica,ovarian cortex and ovarian medulla.During the development of follicles,the thickness of the granular layer and the membrane layer will also change.2.The methylation-sensitive polymorphism amplification technique(MSAP)was used to detect the genomic DNA methylation level of high-yield and low-yield Leizhou black duck ovary tissue.The results showed that there were three methylation patterns,permethylation and hemimethylation.And unmethylation patterns;total methylation levels in the high-yield group(49.74±16.94)%,permethylation levels(42.68±17.92)%,hemimethylation levels(7.07±3.78)%,non-methylation levels(50.27±16.94)%;total methylation level(45.47±8.58)%,permethylation level(38.29±13.17)%,hemimethylation level(7.18±4.67)%,non-methylation level in low-yield group(54.53 ± 8.58)%.The results showed that there was no significant difference in DNA methylation level between Leizhou black ducks in high-yield group and low-yield group(P>0.05).3.By cloning and sequencing,six ideal DNA methylation-specific fragment sequences were finally screened.By online Blast alignment,one fragment was found to be located in the non-coding region,and 100% homologous sequence gene was aligned with the aligned sequence.The gene annotation was LOC113844334.One other fragment was located in the coding region of the gene,with 99% homologous sequence to the duck genome,the gene annotation was LRRTM3(leucine rich repeat transmembrane neuronal.The other four fragments had no similar sequences.4.Quantitative analysis of the gene LRRTM3 obtained from the specific fragment recovery showed that the LRRTM3 gene was expressed in both high-yield and low-yield ovaries,and the low-yield group was significantly higher than the high-yield group,which was negatively correlated with methylation modification(P<0.05),presumed to be LRRTM3.Methylation of the gene sequence inhibits the expression of the gene in the ovary.The results of BSP methylation sequencing showed that the methylation degree of LRRTM3 gene in high-yield group was higher than that in low-yield group,but the difference was not significant.It is speculated that the regulation mechanism of LRRTM3 gene expression is not only regulated by epigenetics,but also likely to be influenced by molecular mechanisms such as population inheritance.Regulation,specific expression regulation pathways and mechanisms remain to be further studied. |
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