Development Of Nuclear Microsatellis And SSR Analysis In An F₁ Hybrid Of Pyrus Pyrifolia Nakai

Nuclear microsatellite(nSSR) is very useful molecular marker,widely used in the study of plant genetic structure,phylogeny and relationship.But there are few nSSRs of Pyrus;this greatly limited the application of nSSR in Pyrus.Accurate forecast methods were used to isolate nSSR in Pyrus.These informational loci will be very useful in the study on the genetic structure,origin,evolution of Pyrus and its closely related speciesChina is one of Pyrus genus provenances.Pear plays an important role in fruitproduction in China.But it develops slowly in molecule.Constructing moleculegenetic map of pear using SSR markers will be more meaningful to Pyrus genus.In this paper, the map population was constructed using Xizilu×Kisui hybrid population.The parents have distinct difference incolor,it could be detected polymorphism locus.The result of experiment indicated that the population fitted to construct genetic map.The results were as follows.1 Genomic DNA was extracted from silica-gel-dried leaves of Pyrus with a modified CTAB method.A series of improved treatments were used in the progress.The DNA extracted with this protocol had high quality,less RNA,protein and other contamination,OD_260/280 ranging from 1.72 to 1.92.It could be suitable for a series of biotechnological researches.This protocol was also used to extract genomic DNA from cilica-gel-dried old or fresh leaves of mume,apple,peach and apricot and also gained high-quality DNA.So this proctocol is feasible and universal.2 Simple sequence repeat(SSR) markers are highly polymorphic,codominant,well-repeated and convenient.It has become a useful molecular marker technique in studying plant genetics and breeding,The theory and characteristics of SSR.But the isolation efficiency is still low,which greatly limited the application of SSR.We use modified affinity capture to enrich microsatellite and the model of accurate forecast(AFC) to isolation Microsatellite. While these primers were used to test 126 clones by PCR,38 clones screened from 60 positive clones were obtained specific sequences,The first ten anterior clones in the recommendation list created automatically by software were sequenced and all of them sequences were usable for SSR isolation.AFC pipeline has higher SSRs enrichment(63%), high sequence percentage(100%) and less cost per SSR locus.It is easy to be operated.It can be a new powerful SSR isolation approach.3 Citrus SSR reaction system was established by optimization of annealing temperature and the total volume for PCR reaction:the optimized total volume for citrus SSR reaction is a 25μl reaction mixture containing,10×Buffer 2.5μl,MgCl₂(25mM) 1.5μl,dNTP(2.5mM) 1.6μl.each Primer(15ng/μl) 1.0μl;Taq DNA polymerase(5U/μl)0.15μl and DNA(50ng/μl) 2.0μl;the proper annealing temperature cg the 28 pairs of SSR primers tested id form 45℃to 50℃.12 out of 40 pairs of SSR primers showed highly polymorphic loci between both parents,and thepolymorphic proportion is 30.7%.These primes are fit to construction citrus pyrus genetic map.4 Genetic linkage maps of Xizilu and Kisui Pear have been constructed using "double pseudotest-cross" strategy with SSR marker and linking JoinMap3.0software.There were 4 in this map,including 12 markers and the integrated map is130.0cM in length,the average distance was 10.8cM among markers.The largestgenetic distance among linkage groups was 26.0cM,the smallest was 6.0cM.Themost markers was Groupl and Group 3,they both have 4 markers,the length were 71.0cMand 50.0cM.The length group was Group 1 with 4 markers,the length was 71.0cM,the shortest group was Group2 with 2 markers,the length was 19.0cM.