【作者】 曾柏全；

【导师】 邓子牛；

【作者基本信息】 湖南农业大学 ， 果树学， 2009， 博士

【摘要】 柑橘在世界农业经济上占有很重要的地位,是我国第二大果树,湖南省柑橘面积和产量均占全国第一。宽皮柑橘是现代栽培柑橘的原始种,分析它们的遗传多样性对了解柑橘起源有重要意义,而湖南是我国拥有多样化野生柑橘类型的地理基因中心,由于柑橘与相近的属之间的杂交,加上高频率的芽变及栽培因素,柑橘分类与系统进化很复杂,存在很多分歧与争议,本文采用分子标记技术对宽皮柑橘进行系统的遗传分析,构建了遗传图谱,对于深入了解湖南省柑橘资源在全国原产中心的地位,在原基础上进一步整理与评价湖南柑橘资源,挖掘现有优异的种质资源,做好柑橘育种工作具有重要的理论和实践指导意义。本研究取得主要结果如下:1.优化并构建了适合宽皮柑橘的SRAP分子标记的反应体系。研究确定在25 uL的反应体系中,Mg²⁺浓度在1.5mmol/L,dNTP 0.3mmol/L,DNA模板50ng,上下引物各为0.2umol/L,Taq酶浓度为1.0U/25ul效果好,最佳PCR程序为94℃预变性5 min,然后94℃,1 min;50℃,1 min;72℃,1 min,35个循环,最后72℃延伸10 min。筛选了130对SRAP引物,其中31对引物扩增的带型清晰、丰富。2.优化并构建了适合宽皮柑橘的EST-SSR分子标记的反应体系。在25 uL的反应体系中,Mg²⁺浓度在2.5mmol/L,dNTP 0.2mmol/L,上下引物各为0.2umol/L,Taq酶浓度为1.0U/25ul效果好,最佳PCR程序为94℃预变性1 min,然后94℃,1 min;52℃,1 min;72℃,1 min,45个循环,最后72℃延伸3 min。3.从130对SRAP引物和23对EST-SSR引物中筛选获得了扩增多态性位点丰富的31对SRAP引物和11对EST-SSR引物,用11对SRAP引物和8对EST-SSR引物共扩增了128个位点,多态性位点达113个;UPGMA聚类分析,在遗传相似系数为0.795时,25份种质分成10组,揭示了金柑与宽皮柑橘、印度野橘亲缘关系较近。4.以9份野橘及近缘种为材料,采用了8对SRAP引物和9对EST-SSR引物,共扩增了115个位点,多态性位点101,PIC值为0.636。UPGMA聚类分析,在遗传相似系数为0.75时,9份种质可分为5类。推断野橘中存在圆型小叶莽山野橘、印度野橘→崇义野橘、立花橘→尖叶型莽山野橘、道县野橘的演化途径。南岭山脉是柑橘亚属的起源中心。5.13份温州蜜柑及本地广柑材料,采用了11对SRAP引物和11对EST-SSR引物,共扩增了108个基因位点,多态性位点40,PIC值为0.386。UPGMA聚类分析,在遗传相似系数为0.82时,可分为4类:宫川、尾张、本地广柑及9份特早熟蜜柑与早熟蜜柑,区分了由宫川变异而来的各品种。6.14份宜昌橙类与香橙类材料,采用了等11对SRAP引物和10对EST-SSR引物,共扩增了134个基因位点,多态性位点90,PIC值为0.596。三叶宜昌橙比怀化宜昌橙、贵州宜昌橙、宜昌橙6-3、公孙宜昌橙、宜昌橙25-86、宜昌橙2-3更为原始。7.采用了8对SRAP引物和10对EST-SSR引物扩增结果说明:长沙南橘与朱红橘的遗传相似系数为0.825,尖叶型莽山野橘与砂糖橘之间为0.82,茶籽柑与蓝柑和本地宽柑为0.81,粗皮狗屎柑与细皮狗屎柑和大丰黄皮桔为0.77。臭皮柑是原始的类型,区分了杂柑各品种。8、采用叶绿体SSR分析,并对扩增的PCR产物进行回收、克隆及测序,发现不仅存在单碱基的A与T微卫星序列,还发现存在AT、TC、CT、TA、GA的二碱基序列及ATT的三碱基序列。发现除枳壳外,金柑属及柑橘属样品均有“TAATAATAAAA”序列。DNAMAN软件的序列同源性分析发现道县粗皮小果野橘与立花橘叶绿体基因同源性很高,吉品与山下红、地中海橘与南柑20叶绿体基因序列的同源性高达99%。更多还原

【Abstract】 Citrus is a very important fruit for agricultural economy in the world,and also is the second largest fruit in China.Mandarin（Citrus reticulata Blanco） is the original species of cultivated citrus.To understand the origin of citrus,it is important to analysis its genetic diversity.Mandarin is one of the most important citrus genotypes in China.Hunan,has abundant citrus native resources existing for over 2000 years.So Hunan is a geographic gene center of citrus.Citrus taxonomy and phylogency are very complicated,controversial and confusing,mainly due to sexual compatibility between citrus and related genera,highly frequency of bud mutation,long history of cultivation and wide dispersion.With the propose to clarify the relationships among the native citrus resources in Hunan,Molecular markers were developed.The main results as follows:1.A appropriate PCR reaction system for detecting gene in Mandarin with SRAP was developed.Each 25ul PCR reaction mixture consisted of 50ng of genemic DNA,1.5mmol/L of MgCl₂,0.3mmol/L of dNTP,0.2umol/L of enemprimer and 1.0 unit of Taq polymerase.The mixture was initially denatured at 94℃for 5min,then cycled 35 times at 94℃for 1min,50℃for 1min,and 72℃for 1min and finally extended at 72℃for 10min.2.A appropriate PCR reaction system for detecting gene in Mandarin with EST-SSR was developed.Each 25ul PCR reaction mixture consisted of 25ng of genemic DNA,2.5mmol/L of MgCl₂,0.2mmol/L of dNTP,0.2umol/L of enemprimer and 1.0 unit of Taq polymerase.The mixture was initially denatured at 94℃for 1min,then cycled 45 times at 94℃for 1min,52℃for 1min,and 72℃for 1min and finally extended at 72℃for 3min.3.Thirty-one pairs of SRAP primers exhibited the polymorphism among 130 SRAP primers combination and eleven pairs of EST-SSR primers exhibited the polymorphism among twenty-three EST-SSR primers combination.The dendrogram was obtained by UPGMA analysis.The 25 accessions were grouped into ten groups at the coefficient of 0.795.It is ascertained that C.ichangensis Swing,Citron, C.grandis Osbeck are among citrus genera.The results showed the closest relationship between C.reticulata Blanco and C.medica L,C.indica Tan.4.Nine pairs of SRAP primers and nine pairs of EST-SSR primers had been adopted in wild mandarin.115 clean and stable bands had been detected.The number of polymorphic bands were 101,and with polymorphic information content values of 0.636.The dendrogram was obtained by UPGMA analysis.The 9 accessions were grouped into five groups at the coefficient of 0.75.The relationships may be esisted that C.mangshanensis S.W.He et G.F.Liu and C.indica Tan→C.reticualta ’Congyi’,C.tachibana（Mak.） Tanaka→C. daoxianensis S.W.He et G.F.Liu→C.reticulate Blanco.Nanling Mountains is one of the native centers of the subgenus Eucitrus.5.Eleven pairs of SRAP primers and eleven pairs of EST-SSR primers had been adopted in the ’Guoqing’ Satsuma.108 clean and stable bands had been detected.The number of polymorphic bands were 40,and with polymorphic information content values of 0.386.The dendrogram was obtained by UPGMA analysis.The 14 accessions were grouped into four groups at the coefficient of 0.82.1) Gong chuan;2) Wei zhang;3) C.reticulata Blanco ’Bendiguanggan’;4) Others nine accessions of C.reticualta Blanco ’C.unshiu Marc’,and the various species mutated from C.unshiu Marc ’Gongchuan’ had been distinguished.6.Fourteen pairs of SRAP primers and ten pairs of EST-SSR primers had been adopted in the C.ichangensis Swingle and C.junos Sieb.ex Tan.134 clean and stable bands had been detected.The number of polymorphic bands were 90,and with polymorphic information content values of 0.596.Compared with othes C.ichangensis Swing（Such as C.ichangensis Swing ’Huaihua’,C.ichangensis Swing ’ Guizhou’,C.ichangensis Swing ’6-3’, C.ichangensis Swing ’Gongsun’,C.ichangensis Swing ’25-86’,C.ichangensis Swing ’2-3’）,C.ichangensis Swing ’Shanye’ was more primitive.It may be exsisted that C.ichangensis Swing ’Shanye’ is the ancestor of C.ichangensis Swing.7.Eight pairs of SRAP primers and ten pairs of EST-SSR primers had been adopted in the analysis.The results showed the closest relationship between C. reticulate Blanco ’Changsha’ and C.reticulate Blanco ’Zhuhong’ is 0.825,C. mangshanensis S.W.He et G.F.Liu and Shatangju is 0.82,C.reticulate Blanco ’Chazi’ and C.reticulate Blanco ’Bendiguanggan’ is 0.81,C.reticulata Blanco ’Xipi’ and C.reticulate Blanco ’Dafen’ is 0.92.Furthermore,Choupi Gan mandarin was the primary genotype among landraces and wild mandarins,and the various of Citrus Hybird had been distinguished.8.PCR amplified citrus chloroplast DNA was purified,cloned and sequenced in order to identify cpSSRs.These cpDNA microsatellites were usually single nucleotide repeats,mainly A·T repeats.Dinucleotide poly（AT,TC,CT,TA,GA） and A trinucleotide poly（TAA） motify were also found.The microsatellite sequence was "TAATAATAAAA".The chloroplast DNA homology between C.daoxianensis S.W.He et G.F.Liu and C.tachibana（Mak.） Tanaka was very high,between C.reticulate ’Sanxia hong’ and C.reticulate ’Ponkan’ was as high as 99%bv DNAMAN software.更多还原