Screening New Microsatellite Markers Of Milk Production Traits On BTA6

Many literatures have shown that there was a QTL (Quantitative Trait Loci) affecting milk production traits near marker BM143 on BTA6, but because of no enough makers, it can't be located precisely. The goal of our study was to find more new microsatellites near BM143 and make the QTL could be located precisely. The primers for the 7 microsatellites located in 10 cM near BM143 were designed in terms of sequences came from the Roslin Institute and NCBI. The targeted DNA fragments were got by PCR amplification, and then aligned the targeted DNA sequence by cloning and sequencing with the DNA sequence from NCBI. It showed that the two sequences are the same. Using the optimized PCR amplification conditions, we screened the BAC library by the method of 4D-two steps rapid PCR, then obtained the contigs that lie in between marker BMS2508 and BL1099 (29 positive clones). Digested all the DNA of positive clones by Rsal and Haelll, then extracted 200~800bp enzyme-digested fragments and ligated into fragments and adaptors. The adaptors-ligted fragments were heat denatured and hybridized with biotinylated probe (CA)12. Using streptavdin-coated magnetic beads to affinity capture hybridized mixture. Eliminated the fragments that not contain (TG) repeats after several eluted, then obtained single-stranded targeted fragments , which contain (TG) repeats and can became double-stranded by PCR amplification .ligated the double-stranded targeted fragments into pMDT-18 vector, then transformed into E.coli DH5 a competent cells to produced a microsatellite-enriched library. We got 60 clones that contain (CA/GT) with 5 or more repeats from sequenced 95 clones of the library, and the proportion of clones containing (CA/GT) repeats is 63.2%. After aligned the sequence by DNAMAN software, we obtained 21 new microsatellites, while among which there were 6 new microsatellites that (CA/GT) repeats located at 5' end or 3' end of insert fragments .which made us could not design primers to analysis their characters. Designed the primers of the other 15 new microsatellites, and 10 primers could obtain targeted fragments by PCR amplification. Using single BAC clone's DNA as template to PCR amplification and primary locate the new microsatellites near the marker BM 143.These new microsatellites will help to precisely locate the QTL affecting milk traits on BTA6, and provide the theory base to dairy molecular breeding.