The Molecular Markers And QTL Location Of Chinese Cabbage Purple Trait

Chinese cabbage(Brassica campestris L.ssp.Pekinensis(Lour.)Olsson)originated fromChina,is one of important vegetables with Chinese characteristic and significant effect,andits distribution and area of cultivation are wide.Its germplasm is abundance in China and thecolor of leaf head varies from white to deep green.Recently,the leaf heads with differentcolor has become to new breeding direction.Through pilot study,the result showed that thepurple trait is quantitative character.Based on quantitative trait loci(QTL)analysis,markerassisted selection for purple trait can be an efficient way to this aim.First,by observed and recorded carefully in the field,the heredity law of purple trait wasstudied in this paper.Then,based on ameliorated RSAP(restriction site amplifiedpolymorphism)technique,joined with SRAP,SSR and RAPD,a genetic map was constructedbased on 125 F2 plants from two elite inbred lines.Based on the linkage map and phenotypevalues of 9 agronomic traits,the QTLs of purple trait et al were analyzed using multipleinterval mapping method.The main results of this study were as follows:1.The primers of RSAP marker technique were redesigned and its reaction system wasoptimized,using DNA from inbreed lines' young leaves of Chinese cabbage,Purple-caitai(Brassica compestris L.var.purpurea Bailey)and their F₁,F₂ as trial materials.Then,thereproducibility and applicability of new primers were tested.The sequence of restriction site(4-6 bases)located in the middle,three selective nucleotides were added to its 3'end and 10 to12 bases long of random sequence lied in its 5'end.Fourteen new primers of RSAP wereredesigned 19 nucleotides long.PCR amplification of new primers was run for the first 5cycles with an annealing temperature of 35℃,followed by 35 cycles with an annealingtemperature of 52℃;the optimum PCR reaction system of 25μL included 2.0μL DNAtemplates(20.0ng/μL),3.0μL of Mg²⁺(25mmol/L),1.5 U of Taq DNA polymerase,2.0μL ofdNTPs(2.5mmol/L)and 0.6μL of each primers(10μmol/L).New primers could amplifymore strips and polymorphism was better than former primers.In the varieties of buckwheatand wheat,new primer could amplify vivid strips too,which showed the applicability andreproducibility of new primers were very good,its application would be broad.2.Six F₂ populations and three BC₁were used to research the heredity law of Chinesecabbage purple trait,by observed,recorded and fitness tested,the color of petiole in 4 F₂ and 2 BC₁ generations accorded with 3:1 and 1:1 separate proportion respectively.The resultshowed that the genes controlled petiole purple character were one pair main genes,partiallydominant,and took on dose effect.Then,one F₂ generation with exceptional proportion wasanalyzed by EST-SSR technique,the statistical effects of strip types amplified with every of 5EST-SSR primers fitted the proportion 1:2:1,in which the marker BC₂₁linked to a main QTLof fruit color.This showed that the heredity of fruit color accorded with the above theory.Thephenotype didn't match to genotype completely,which showed that the expression of purplecharacter may be impacted by tiny genes and surroundings together.3.Using bulked segregant analysis(BSA),640 random primers were analyzed withRAPD technique.S₇₉and S₁₂₃could generate differential profiles linking to petiole purplecharacter among F₁ and its parents.Primer S₇₉could generate characteristic band S₇₉-934 andS₁₂₃could generate S₁₂₃-750.The linkage analysis showed that the distances to purple gene are13.73cM(band S₇₉-934)and 18.65cM(band S₁₂₃-750)respectively,which are located inboth sides of purple gene.Furthermore,characteristic bands S₇₉-934 was reclaimed,clonedand sequenced,its sequence had 99% identity with whole sequence(113253bp)of the cloneKBrH077A05 from 1^stchromosome of Chinese cabbage by BLAST analysis.Therefore,aspeculation was that main gene controlled purple character was located in 1^stchromosome ofChinese cabbage.4.Based on 231 polymorphic markers,a Chinese cabbage linkage map was constructedwith JoinMap 3.0 software.The map composed of 11 linkage groups and 4 segments with 163markers,included 117 RSAPs,38 SRAPs,5 SSRs and 3 RAPDs,which covered genome821.3 cM.The average distance between markers was 5.04 cM.Based on the linked markers,a conclusion was that 4^thlinkage group(LG4)corresponded to 1^stchromosome of Chinesecabbage.5.Using multiple interval mapping method,united with phenotype values of 9agronomic traits,a total of 44 QTLs was detected in Chinese cabbage,in which 10 for petiolecolor,3 for bud color,6 for color of flowering stem,4 for fruit color,4 for days to bolting,5for days to flowering,5 for leaf wing,3 for number of first branches and 4 for fruit beak.Main QTLs for per trait were identified.If aimed at deep purple petiole trait,only under thecondition of QTLs of cp1.2 and cp4.2 which located on LG1 and LG4 were selected bylinked markers,the aim would come true.