Construction Of AFLP Analyse System And Application In China Holstein Dairy Cattle

AFLP (Amplified Fragment length Polymorphism) invented and developed by Zabeau and Vos is a new kind of molecular marker. Its principle is based on the polymorphism of DNA fragment which is producted by restricted double enzymes (freqent cutter and rare cutter) and selective amplification. AFLP technology integrate the RFLP technology with stabilization and credibility and PCR technology with simpleness and high efficiency, so it is suited for the analysis of heredity polymorphism, making heredity map, identfication of parentage, and research of molecular marker. Holstein is most breed in China , and dairy cattle herd improvement is very important assignment, AFLP technology as a molecular marker can provide help.Because AFLP technology is more complex than others and with high request, detective method about AFLP of the Holstein wasn't reported at home.(At abroad, isotope marker and fluorescence was mostly used, but isotope is expensive and not safty, fluorescence is more expensive and need special apparatus, silver staining result is not stable if it is not optimized.) First , the condition on construction of AFLP fingerprinting map is optimized, and then AFLP analysis system of silver staining with low cost was set up.Second, genome DNA was digested by EcoR I/Taq Iå’ŒEcoR I/ Mse I . 100 primer sets was analyzed by effect and polymorphism. To seek for AFLP primer sets which were suited for Holstein.At last, 60 Holstein dairy cattles (from more than 1200 Holstein dairy cattles with full DHI, 10 with very high milk yield and 10 with very low milk yield, 10 with high dairy ingredient and 10 with low dairy ingredient, 10 high somatic cells and 10 with low somatic cells) was analysised through AFLP technology. Discussed the feasibility about AFLP applied to breeding of Holstein economically important traits. The relevant results are as follows:1. AFLP analysis system was established in Chinese Holstein diary cattle .The results of several reactions during AFLP analysis had been detected such as DNA extraction ,double enzymes restriction, adapter-ligation pre-amplification selective amplification.2. Silver-stainning technology system also was established, temperature was very important for the whole process, especially the silver-stainning solution and apparenting solution. At last, the optimum temperature was found and nicer coloration effect was appeared. Low cost, short time for detecting, free of isotope contamination, long time of preserving, all proved that silver-stainning was a safe and effective detecting method.