Establishment Of Recessive Hereditary Disease Of Hesitant And Detection Of Molecular Marker Assotionated With Milk Traits

Cows recessive genetic diseases (such as CVM, DUMPS, BLAD, etc.) will cause cows miscarriage, stillbirth, premature delivery, high estrus returning rate, low milk yield which cause huge losses to the dairy production. At the same time, lactation trait is typical quantity economic trait, which is determined by multiple genes and affected by environment. The progress of cow breeding is very slow only using traditional breeding techniques. MAS can shorten the generation interval and improve the accuracy of selection. Therefore, traditional breeding techniques combined with the MAS can improve breeding efficiency and accelerate genetic progress. Prerequisite for application of MAS technology is to obtain molecular markers which have the significant correlation with economic traits. Candidate gene approach is an effective method of screening of molecular markers. Seed selection of cattle using preference of superordinary genes combined with elimination of deleterious genes can effectively improve the performance of dairy cow.In this study, Molecular biology detection technology of CVM、DUMPS、BLAD which affected milk yield and cow health was established. We also use PCR-RFLP and gene sequencing to validate the AS-PCR technology. The samples were tested in order to carry out the harmful recessive diseases gene. And leptin gene associated with breast development, K-Cn gene, ABCG2 gene with the formation of milk protein, related with cell proliferation and differentiation respectively. These genes were selected as candidate genes using candidate gene approach. Five polymorphic sites of these genes were detected by PCR-RFLP and sequencing and and also analyzd the association between polymorphism and milk traits was analyzed. The main results of this study are as follows:1. Established allele specific PCR for detection of recessive genetic diseases-DUMPS. Using PCR-RFLP and gene sequencing to validate the AS-PCR technology and the accuracy of AS-PCR was 100%. The AS-PCR technology had many advantages, for example fast speed (3 hours each time), lower cost (reduced costs for enzyme, being applicable to large-scale testing (at least 96 each time).2. Established allele specific PCR for detection of recessive genetic diseases-BLAD. Using PCR-RFLP and gene sequencing to validate the AS-PCR technology and the accuracy of AS-PCR was 100%. The AS-PCR technology had many advantages, for example fast speed (3 hours each time), lower cost (reduced costs for enzyme, being applicable to large-scale testing (at least 96 each time). 3. DUMPS and BLAD recessive genetic disease of the 417 Holstein cows and 132 semen samples were detected using the AS-PCR established in this study. One DUMPS carrier was found. The detection rate of DUMPS carriers was 0.18% and harmful gene frequency was 0.09%. There was no harmful gene of BLAD found in Experimental cattle. The cow numbers of DUMPS carriers were provided to the production for providing an important scientific basis of the selection of breeding programs.4. CVM recessive genetic disease of the 217 Holstein cows and 125 semen samples were detected using the CRS-PCR established early in our Laboratory. Ten CVM carriers were found. The detection rate of CVM carriers was 2.92% and harmful gene frequency was 1.5%. The cow numbers of CVM carriers were provided to the production for providing an important scientific basis of the selection of breeding programs.5. The T/C mutation of the leptin exon 3 was detected using PCR-RFLP and sequencing and there were TT, TC, CC genotypes. Milk yield of TC genotype was significantly higher than CC genotype milk yield (P<0.01). However, milk fat percentage and milk protein percentage between the different genotypes, were not significantly different between the different genotypes (P> 0.05).6. The C/A and G/A mutation of theκ-Cn gene exon 4 were detected using PCR-RFLP and sequencing and there were two genotypes. Milk yield of AA genotype was extremely significant higher than AC (AG) genotype milk yield (P<0.01). Milk protein of AC (AG) genotype was significantly higher than AA genotype milk protein (P<0.05). However, milk fat percentage a between the different genotypes, were not significantly different (P> 0.05);7. The A/C mutation of the ABCG2 gene exon 14 was detected using PCR-RFLP and sequencing. But this mutation was not detected in this experiment.8. The A/T mutation of the leptin exon 2 was detected using PCR-RFLP and sequencing and there were AA, AT genotypes. But milk yield, milk fat percentage and milk protein percentage between the different genotypes, were not significantly different (P> 0.05).