Constructing The Molecular Linkage Map Of Pepper And The QTL Analysis Of Phytophthora Blight

Pepper (Capsicum annuum, L.) that Probably spread to Chinese in the end of theMing dynasty, originated from Central America and Latin America, is one of the mostwidely cultivated and consumed vegetables in China. Phytophthora blight is one ofthe most economically destructive diseases of pepper. The use of resistant cultivars isthe most important promising method to control the disease. Most scientists believethat the inheritance of the resistance is a quantitative trait which is very controlled bypolygenes. It has vital significance for molecular marker- assisted selection,thatconstructing the genetic linkage map and doing QTL analysis on the related resistantgene to Phytophthora capsici in pepper.83-60, a susceptible sweet pepper lines, and perennial, a resistant pungent line,and theirs'F2 generation were used as Mapping community in this study. Thegenomic DNA was extracted with N₂-CTAB method. Then constructed a geneticlinkage map by AFLP, RAPD and SSR markers. And through unified investigationresult of the experimental material's resistant Phytophthora blight. The main resultsare as follows:1. Obtained a system and procedure to suit the pepper RAPD analysisThrough the optimizated experiments to the system, obtained a system thatsuit the pepper RAPD analysis. The optinization of a 25μL RAPD-PCR system wasdetemined as followed: 2.5mmol·L^-1 Mg²⁺, 0.2 mmol·L^-1 dNTPs for each, 0.4μmol·L^-1 primer, 25～55ng template DNA, 1.0 U Taq DNA polymerase, and2.5μL PCR Buffer(10X). Unified the results of the annealing temperature's gradientexperiments, we obtained the amplification procedure conditions which werepredenature 95℃3 min followed by denature at 94℃30 s,anneaed primers at themost appropriate temperature for 60 s, extension 72℃for 90 s, cycling 45 times, lastextension 10 min then preserving an 4℃till to electrophoresis.2. Established a system and procedure to suit the pepper SSR analysisThrough the optimizated experiments to the system and unified the results of theannealing temperature's gradient experiments, We established a system andprocedure to suit the pepper SSR analysis. The condition was in a 20μL volume with1.875mmol·L^-1 Mg²⁺, 0.2 mmol·L^-1 dNTPs for each, 0.375μmol·L^-1 primer, 25～50 ng template DNA, 1.0 U Taq DNA polymerase, and 2.0μL PCR Buffer(10X).And amplification protocol was initial of 4min at 94℃, followed by denature at 94℃45 s, anneaed primers at the most appropriate temperature for for 45 s,extension 72℃for 90 s,cycling 35 times, last extension 8 min then preserving an 4℃till toelectrophoresis.3. A molecular linkage map of pepper, including 12 linkage groups and 70markers, was constructedBy analyzing the DNA of the parents and the F2 generation what using AFLP,RAPD and SSR method, we constructed a molecular linkage map by using JoinMap3.0 software, that included 12 linkage guoups, 429.03 cM, and 70 markers, making up of 54 AFLP markers, 15 RAPD markers, and 1 SSR marker. And the averageinterval of the map is about 6.13 cM between adjacent loci.4. 2 QTLs controlling pepper Phytophthora blight resistance was examinedinitiallyBsed on the map what we constructed, unified the Phytophthora blight resistanceappraisal result, 2 QTLs controlling pepper Phytophthora blight resistance wasexamined initially by using the QTL analysis with Windows QTL Cartographer V2.5software and Interval Mapping method. Both of the QTLs were located on linkagegroup LG4, with the explanation of 9.5% and 16.4ï¼….