Establishment Of Three Retrotransposon-Based Molecular Markers And Their Application In Identification Of Apple Bud Mutations

Retrotransposon are mobile genetic elements which transpose replicatively through RNA intermediatesThey are ubiquitous,present in high copy numbers as highly heterogeneous populations and show insertional polymorphism both with in and between species in plants and generate stable mutations.Compared with the conventional molecular markers Several molecular markers which were exploited Against these characteristics were frequently used to study genetic diversity,to establish phylogenies,to generate genetic linkage maps and to identify markers linked to agronomically importantly genes because of covering whole genomes and highlier level of polymorphism.Apple is one of the most vulnerable bud mutation in fruit crops.At present in production many excellent varieties and strains are obtained via bud sports selection.About half of the whole apple production come from the bud mutation in the world,bud mutation plays a very important pole in excellent Variety Breeding of apple.but the Conventional methods to identify apple bud mutation have some relatively difficult.In this paper,we establish and optimize IRAP and REMAP markers,which was used to identify apple bud mutations combined with SSAP markerThe main results were as follows:1 primers were designed based on Long Terminal Repeat(LTR) reserve region of retrotransposon in Apple.Several important reaction factors of IRAP and REMAP was studied in order to establish and optimize IRAP and REMAP systems in Apple.The final optimal IRAP system was as follows:in 25μL reaction system,template DNA 50ng,25mmol/L MgCl₂ 2.5μL,dNTP 2.0μL,10×Buffer 2.5μL,Taq DNA polymerase 1.0U,10μmol/LLTR primer 0.8μLThe final optimal REMAP system was as follows:in 25μL reaction system,template DNA 50ng,25mmol/L MgCl₂ 2.5μL,dNTP 2.0μL,10×Buffer 2.5μL,Taq DNApolymerase 1.0U,10μmol/L LTR primer 0.8μL,10μmol/L ISSR primer 0.8μL.2 Using the IRAP and REMAP markers identified the'Fuji'and' Delicious'bud mutations, Using the SSAP markers identified the'Fuji' bud mutations.Results showed the L7 primer revealed polymorphism in'Fuji'bud mutations in IRAP analysis;the L1 primer revealed polymorphism in 'Delicious'bud mutations in IRAP analysis.The primer component L1/P5 revealed polymorphism only in 'Fuji'in REMAP analysis.Fuji apple and its 14 bud mutations were subjected with Silver staining-SSAP marker.In six primer combinations selected from 60 primer combinations L12/E7 could discriminate all the bud mutations and maternal Fuji successfully and other 5 priner combinitiaons could discriminate Akifu 5,Yanful,Shengfangful,Nagafulfrom the mutation sports.The polymorphism in SSAP mainly comes from the transposition of retrotransposon.The results suggested that the bud mutations which have generated from Fuji appear to derive from retrotransposon insertion.It provided a early identification method of new variety breeding.