Elimination Of The N + 1 Peak In Genotyping Of SSR And InDel Molecμlar Marker In Maize And Sorghum

Maize DNA fingerprint database with SSR markers provides a strong technical support for variety identification and breeding.InDel mμltiple complex system highlights its huge potential in variety identification because of fast and efficient feature.In the construction of maize DNA fingerprint database,when we analysis the fluorescence capillary electrophoresis data,some primers produce N + 1 peak that limits the automation of data analysis,reduces the data accuracy,affects the quality of the fingerprint and the collection standardization of fingerprint data.In this study,40 core primers of maize and sorghum and 20 primers of InDel mμltiple complex system were assessed,we used five kinds of N + 1 peak elimination methods to eliminate N + 1 peak produced by the primers in order to screen the good elimination method,including increasing the extension time of the last step in PCR procedure,adding a G(guanine base)in the reverse primer 5’ end,decreasing the amounts of DNA template,the increase of Taq concentration and the use of alternative polymerases.Finally,we use these methods to build optimized programs of N+1 peak elimination that is suitable for the SSR and InDel markers,achieving the optimization of maize DNA fingerprint database management system and InDel multiple complex system.The main resμlts are as follows:1.Through the statistics and analysis for a large number of fingerprint data of samples in maize DNA fingerprint database in the past two years,there are 13 primers produce the N+1 peak of 40 core primers of maize.We use five kinds of N+l peak elimination method to test 13 primers and compare the resμlt of their elimination of the N+1 peak,finally select three methods,then other primers are assessed used three methods and the fingerprint datas are normal,three optimized programs are put forward.2.24 sorghum materials are tested in the study,there are 15 primers produced N+1 peak of 40 core sorghum primers.For the N+1 peak of these primers,we eliminate them through two methods screened in last experiment,the res μ It shows that two methods can also eliminate the N+l peak,finally we establish two optimized programs.3.36 maize varieties and 20 InDel primers are assessed respectively used single and mμltiple PCR to screen primers produced N+1 peak,method one and method two eliminate N+l peak of maize S SR markers in the single PCR in the first experiment,proving that the two methods is also suitable for the N+1 elimination of corn InDel markers in single PCR,however,for the N+l elimination of corn InDel markers in mμltiplex PCR in complex system,when we use the two methods separately,the N+1 peak is not be eliminated,then two methods have been shown to promote fμll adenylation,therefore combine two methods,namely,increasing the extension of PCR reaction procedure in the last step of time of method two.The resμlt shows that this optimized method is applicable to the elimination of the N+1 peak in mμltiplex PCR of maize InDel markers,finally we set up the optimized program.