| Gene mutation is a widespread phenomenon during biological evolution,which includes both advantageous and deleterious mutations.Advantageous mutations increase the diversity of species to a certain extent and have a certain screening function for the generation of dominant species,while deleterious mutations can cause metabolic disorders under gene regulation in an organism state that has reached a high level of coordination with the external environment,thus exhibiting different degrees of physiological or pathological phenomena,such as dysplasia or malformation,reduced viability,infertility or even lethality.At present,the phenomenon of lethal and semi lethal gene mutations has been widely studied in animals and plants,but studies on the effects of lethal mutations on egg production performance of local chickens and their mechanisms of action have rarely been reported.In the early stage of this study,a mutation site n.100005430 T>G on chromosome 3 was identified in the Lushi green-shelled hen genome sequencing database with only two genotypes:wild-type homozygous(TT)and mutant heterozygous(TG),and the mutation was presumed to be homozygous lethal.On this basis,this study first used the restriction enzyme digestion fragment length polymorphism(RFLP)technology to analyze the local chicken Gushi chicken and Xichuan silky chicken,high-yielding layer Hailan Brown and Roman,and fast broiler Anka chicken.The individual genotypes of the natural populations of different breeds of chickens were analyzed to verify the distribution of the mutation sites in the natural populations.Then,the inheritance pattern of this mutation site and the time when its lethal effect occurred were investigated by hybridization and testcross tests as well as genotypic analysis of dead embryos at different embryonic ages during incubation.Third,the effect of different genotypes on the egg production performance of Lushi chickens was analyzed.Fourth,using histological analysis,we analyzed the development of follicles and their granulosa cell layer(GL)and membranous cell layer(TL)of Gushi hens of different genotypes.Finally,haplotype analysis using whole genome resequencing data from 1000 Lushi chickens was used to screen for candidate genes in linkage disequilibrium with this mutation locus,with the hope of elucidating the mechanism of lethality at this mutation locus through an in-depth study of the molecular mechanism of action of this gene.The main results are as follows:1.Through the genotypic analysis of Gushi Chicken,Xichuan Silky Chicken,Five Black Green Shell Layers,Lushi Green Shell Layers,Changshun Green Shell Layers,Silk Feather Silky Chickens,Hailan Brown Layers,Roman Layers,Anka chicken and F2 generation resource population of Gushi chicken × Anka chicken,only wild homozygous genotype TT exists in the genome of high-yielding layer chickens,and only two genotypes,wild homozygous genotype TT and mutant heterozygous genotype TG,were present in chickens and broiler breeds elsewhere.2.400 Gushi hens with TG genotype and 50 Gushi cocks with TG genotype were fertilized at a ratio of 8:1.The ratio of TT genotype to TG genotype individuals in the hybrid offspring was 118:181,which was significantly different(P<0.05)from the theoretical ratio TT:TG=1:2(in the absence of GG genotype).80 Gushi hens with TG genotype and 10 Gushi cocks with TT genotype were inseminated at an 8:1 ratio,and among the tested offspring,the ratio of TT genotype to TG genotype individuals was 116:64,which was very different from the theoretical value of TT:TG=1:1(P<0.01).80 TT genotype Gushi hens and 10 TG genotype Gushi cocks insemination was 8:1.The ratio of TT genotype to TG genotype in the offspring was 54:64,which was consistent with the theoretical value TT:TG=1:1.Only two genotypes,TT and TG,were also detected in the genomes of embryos(126 total)that died at different embryonic ages during hatching.Statistical analysis of the hatching situation of 3547 eggs produced by different cross combinations found that the fertilisation and hatching rates of eggs were better than those of other crosses when the hen and cock were both wild homozygous genotypes(TT).Taken together,it is concluded that some type G oocytes undergo apoptosis early in development.3.Statistical analysis of egg production records of 270 randomly offspring hens with different genotypes(78 with TT genotype and 191 with TG genotype)revealed no significant difference in starting day of life between individuals with TT genotype and TG genotype,but the mean cumulative number of eggs produced in TT genotype hens at different stages of egg production was always higher than that in TG genotype hens,indicating that TT genotype hens are superior to TG ones in overall egg production performance.Selected 9 Gushi hens each with 10 W,15 W,20 W,28 W healthy and stable laying performance under the same breeding environment to compare and analyze the development status of ovaries and follicles.The results showed that,there was no difference in body weight,ovarian weight and ovarian index between the two genotypes in the early stages of individual development,but after entering the peak period of egg production,the ovarian weight and ovarian index of the TT genotype hens were larger than those of the TG genotype hen.In the early stage of egg production(20 W),there was no significant difference in the number of pre-grade follicles and grade follicles,follicular axis length and quality between TT genotype and TG genotype individuals.At the peak of egg production(28 w),the number of follicles in individuals with the TT genotype was generally more than that with the TG genotype.A comparative analysis of the follicular cell membrane and granular cell layer of the two genotypes at the early stage of laying(20 W)and the peak period of laying(28 W)found that the thickness of the granular cell layer of the TT genotype was significantly greater than that of TG genotype Individual(P<0.01).Based on these results,it is inferred that the granulosa cell layer development of TT genotype individuals has an advantage over TG genotype during the pre-grading follicle stage.Compared with TG genotype individuals,they can secrete more steroid hormones to provide nutritional support for the development of later grade follicles and accelerate Follicle selection,development and ovulation.4.Through haplotype analysis,it was found that there is a haplotype in the 28 kb segment downstream of the lethal mutation site(Chr3:100005430 T>G)in Gushi chicken that is in linkage disequilibrium with the lethal site,which is located in the first exon of gene RAD51AP2.This mutation is homozygous and lethal like the lethal locus,so RAD51AP2 can be used as a lethal locus candidate gene for follow-up research.In summary,based on previous researches,this work further verified the existence of two genotypes,TT and TG,in local chickens and broiler chickens,while only TT genotype existed in high-yield laying hens,and there was no GG genotype in all living organisms.The results of the hybridization and testcross tests showed that type G spermatozoa had equivalent fertilization ability to type T spermatozoa,but some of the type G oocytes might be arrested early in development and undergo apoptosis or could not complete fertilization normally with type G spermatozoa.The mean cumulative number of eggs produced was higher in individuals with the TT genotype than in those with the TG genotype.At the peak of egg production,the ovarian weight and ovarian index of individuals with the TT genotype were greater than those with the TG genotype,individuals with the TT genotype generally had a greater number of pre grade follicles than those with the TG genotype,and the granulosa cell layer thickness of pre grade follicles was significantly thicker than that of individuals with the TG genotype.RAD51AP2 gene is a lethal candidate gene. |
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