【作者】 张明科；

【导师】 巩振辉；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2008， 博士

【摘要】 大白菜（Brassica campestris L．ssp．Pekinensis（Lour．）Olsson）起源于中国，是具有中国传统特色又有较大影响的一种蔬菜作物，是我国蔬菜栽培中分布最广、种植面积最大的蔬菜作物之一。我国大白菜种质资源丰富，叶球颜色从白色到深绿色表现出很大的变异。近年来，不同球色育种已经成为新、奇、特大白菜品种选育的新方向。经初步研究，大白菜叶球紫色性状表现出数量性状的遗传特点。借助分子标记和QTL定位研究，可实现其分子标记辅助选择，极大地提高育种效率。本研究通过田间观察、统计紫色等性状，对大白菜紫色性状的遗传规律进行初步研究；利用改进的限制性位点扩增多态性（RSAP）标记技术，结合SRAP、SSR和RAPD等标记技术，以高代自交系紫菜薹和大白菜杂交产生的F₂代125个单株为材料，构建了一张大白菜分子连锁图谱。基于所构建的图谱与田间调查的紫色等9个性状的表型值，采用多重区间定位法，进行了紫色等性状的QTL定位研究。试验获得以下研究结果：1．对RSAP标记技术的引物进行了重新设计，以大白菜、紫菜薹自交系及其F₁、F₂的DNA为模板，对反应体系进行了优化，对其重复性进行了检验。技术要点为：通过在限制性位点序列的3’端增加3个选择性碱基，5’端设计为10～12个碱基的随机序列，设计了14条长度为19bp的（RSAP）新引物。PCR扩增的前5个循环退火温度为35℃，后35个循环为52℃。优化后的在25μL的反应体系中，最佳DNA用量为2．0μL(20．0ng·μL^-1)、Mg²⁺3．0μL(25mmol·L^-1)、Taq酶1．5U、dNTPs 2．0μL(2．5mmol·L^-1)、引物各0．6μL(10μmol·L^-1)。在此基础上，用小麦和荞麦自交系对其适用性进行了检验，结果良好。2．通过6个F₂和3个BC₁群体田间观察、统计及卡方（x²）适合性检验，研究了大白菜紫色性状的遗传规律。结果表明，叶柄色在4个F₂群体的符合3：1分离规律，2个BC₁群体符合1：1分离规律。说明叶柄紫色性状是由一对主基因控制的部分显性遗传，紫色深浅呈现剂量效应。1个分离异常的F₂群体的EST-SSR分析表明，5对共显性引物的带型均符合1：2：1分离比例，其中标记BC₂₁与控制果荚色的一个主效QTL连锁，表明果荚色也符合一对主效基因控制的理论。表型与基因型不完全相符，表明环境条件对紫色性状的表达影响很大，同时可能还有微效基因在发挥作用。3．采用BSA法，从640个RAPD引物中筛选出2个引物S₇₉和S₁₂₃，分别能扩增出与叶柄紫色性状连锁的条带S₇₉-934和S₁₂₃-750。连锁分析发现，标记S₇₉-934和S₁₂₃-750与紫色基因间的遗传距离分别为13．73cM和18．65cM，并且位于紫色基因的两边。回收S₇₉-934特异带，克隆并测序，比较分析表明，其与大白菜1号染色体上已知克隆KBrH077A05的全序列（113253bp）有99％的相似性，初步推断控制叶柄紫色性状的主基因位于大白菜1号染色体上。4．基于231个多态性标记，利用JoinMap 3．0软件，得到包含163个标记、11个连锁群和4个连锁片段的遗传图谱，其中包括117个RSAP标记、38个SRAP标记、5个SSR标记和3个RAPD标记。图谱覆盖总长度为821．3 cM，标记间平均图距为5．04 cM。并推断LG4与大白菜1号染色体对应。5．采用MIM法，结合性状田间调查数据，对大白菜紫色等9个农艺性状进行QTL定位及遗传效应研究，共检测出44个QTL，其中控制叶柄色的QTLs10个，控制花蕾颜色的QTLs3个，控制花薹颜色的QTLs6个，控制果荚颜色的QTLs4个，控制抽薹期的QTLs4个，控制初花期的QTLs5个，控制叶翅数目的QTLs5个，控制一级侧枝数目的QTLs3个，控制果喙长度的QTLs4个。各个性状都检测到了效应较大的QTL。针对叶柄紫色性状，只要对LG1上QTL cp1．2和LG4上的cp4．2进行标记辅助选择，就能达到较好的效果。更多还原

【Abstract】 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.更多还原