Study On Rapid Detection Of BMPR-IB Gene Polymorphism In Sheep By FRET Probe Genotyping Technology

Reproductive traits is one of the important economic traits of sheep, the level of repr- oductive performance is directly related to the production costs and production efficiency in the sheep industry , increasing the number of litter size of sheep is an effective measu- re for improving the production efficiency. BMPR-IB gene is one of the major gene for fecundity of sheep , point mutations (A746G)called FecB gene can be used as the molecular genetic marker for the selection of multiple births strain of sheep. The purpose of this study was to establish a genetyping method with automated, simple, rapid,accurate, and high throughput for BMPR-IB genetyping, providing a new method and technique for marker-assisted selection and breeding of sheep fecundity. Han sheep that reported with BMPR-IB gene polymorphism, Dorset that without BMPR-IB gene polymorphism and their F1 hybrid, F2 hybrid generation were selected as material for establishing the FRET probe genotyping technology,then BMPR-IB gene polymorphism in F2×F2 ram and twins sheep were rapidly detected and analyzed by FRET probe genotyping technology.At the beginning samples included three genotypes were found by PCR-RFLP, probes and different primer pairs were designed for establish FRET probe genotyping technology, then the results detected by two methods were verified by sequencing method, This study used FRET probe genotyping technology which was an improved FRET probe technology.The combination of fluorophore and quencher moiety insteaded of two commonly used fluorophore moiety, genotype were distinguished by differences of melting curve. The results were as follows:1. BMPR-IB gene intron 6 sequence were obtained, homology of the first 651bases with people was 84%, but the following with poor sequence homology.2. With 140 samples were verified, The experiment conditions were explored, indicat- ed that the accuracy of FRET probe genotyping technology for BMPR-IB gene(A746G) was 100% and was feasible.The method was rapid,accurate,simple, automate, resu- lts intuitive, easy to practice and applied to the detection of large-scale groups.3. Only two kinds of genotypes were detected in 158 F2×F2 ram, the B +genotype frequency was 0.03, the + +genotype frequency was 0.97; In 32 twins sheep also only two kinds of genotypes were detected, the B + genotype frequency was 0.22 , the + +genotype frequency was 0.78.4. With the increase of hybrid generation, B genotype frequency significantly decrea- sed.