| Pigeons are typical monistic birds.It is difficult to distinguish between genders by simple appearance,which brings great difficulties to pigeon breeding and propagating.At present,the gender identification of pigeons is mainly distinguished by experienced breeding,this method has low accuracy,and it is not adapted to the gender judgment for early young birds.In this study,the genomic DNA of pigeon blood and feather samples was extracted,and PCR reaction,LAMP reaction and double LAMP reaction were used to determine the sex,and some pigeons were dissected to observe the accuracy of the gonad organ verification results,and the early pigeon sex identification system of PCR reaction,LAMP reaction and double LAMP reaction was established,which provided a method for solving the difficulty of sex identification of young pigeons.The results are as follows:1、A sex identification system of early young pigeon by PCR reaction was established.Test one,genomic DNA templates of blood samples of 65 adult pigeons were used as a control group,and genomic DNA extracted from feather samples were used as a test group,and a method for rapidly extracting feather DNA and gender identification was established through PCR gender identification according to the specificity of CHD1 gene.amplification results of the genomic DNA of the blood samples of the same individual were the same as those of the feather samples,one 446 bp band can be amplified from a male sample,and two 326 bp and 446 bp bands can be amplified from a female sample,and PCR gender identification results were consistent with observation results of gonadal organs;Test two,based on the optimized feather extraction scheme and PCR sex identification system,the sex of 100known-sex and 200 unknown-sex early pigeons was identified.the results showed that the detection rate of genomic DNA in the feather samples of 100 known-sex pigeons was 100%,which was 98%consistent with the observation results of gonad organs;the detection rate of genomic DNA in the feather samples of 200 unknown-sex pigeons was 90%;Test three,repeated verification was carried out,By extracting genomic DNA from feather samples,sex identification of 3000 pigeon by PCR reaction,and the detection rate was 90.4%.2、The LAMP response sex identification system of early young pigeon was established.Test one a blood genomic DNA of adult pigeons as a template,a conventional LAMP reactive primer was designed,and amplified by primers GAPDH,Sex,and optimize the reaction conditions,it was determined that the optimal annealing temperature of the primer was 62℃,the optimum reaction time was 45 min,the result of LAMP amplification reaction of that same individual was consistent with that of PCR reaction;Test two,the detection method was optimized by adding 1 μL 1000× SYBR Green Ⅰ nucleic acid dye,and a visual LAMP sex identification reaction system was established;Test three,the repeated verification was carried out,and the genomic DNA of 100 pigeon feather samples of known sex were subjected to visual LAMP reaction sex identification,and the detection rate was 97%,the coincidence rate of the visual LAMP reaction amplification result of the same individual with the PCR reaction result and the gonad organ observation result was 90.7%.3、A double LAMP response sex identification system for early young pigeons was established.Test one,the genomic DNA of adult pigeon blood was used as the template,and the fluorescence-labeled primers GAPDH and Sex were used for amplification,respectively to verify the effectiveness of the fluorescence-labeled primers;Test two,double LAMP reaction was performed using fluorescently labeled primers GAPDH and Sex,and the reaction conditions were optimized.The optimal concentration ratio of primers was 1:3 and the optimal concentration of PEI was 1 mol/L;Test three,repeated verification was performed.The genomic DNA of 100 pigeon feathers of known sex were sexed by double LAMP reaction,and the detection rate was 98%,the coincidence rate of the results of double LAMP reaction amplification and PCR of the same individual was 94.9%,and the coincidence rate with the observation results of gonadal organs was 96.9%.In summary,three methods for identifying the sex of young pigeons were established in this study,double LAMP reaction compared with LAMP reaction has a higher detection rate and accuracy,providing a method for rapid identification of the sex of young pigeons on the spot with good application prospect. |
PDF Full Text Request