【作者】 谭美莲；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2007， 博士

【摘要】 柑橘在世界农业经济中占有十分重要的地位。多胚性、雌／雄性败育以及遗传上高度杂合影响了柑橘杂交育种工作的开展和遗传图谱的构建。针对以上问题，首先，本研究配置了14个不同的杂交组合，采用常规杂交结合胚抢救和分子标记等技术，获取杂种后代，并对胚抢救过程中获得的枳杂种愈伤组织进行了再生能力的测定；然后利用分子标记技术，对红橘（C．reticulata Blanco）×枳（Poncirus trifoliata（L．）Raf）的F₁群体进行遗传分析，构建柑橘的遗传连锁框架图。本研究主要取得了以下结果：1、2003年和2004年连续两年，以HB柚（C．grandis（L．）Osbeck‘Hirado Buntan’）、华农本地早橘（C．reticulata‘Huanong’）、温州蜜柑（C．unshiu Marc．）、葡萄柚（C．paradisi Macq．）和红橘为母本，以枳、诺瓦橘柚、佩奇橘柚、费尔切尔德橘和默科特橘橙作父本，配置了14个不同的杂交组合进行杂交授粉，结果共收获果实1087个，种子4846粒。通过胚培养，共获得了杂交后代2029个单株，移栽成活1186株。为区分杂种苗和珠心苗，用形态识别和SSR分析，鉴定了990株枳的杂交后代，得到了423株三叶杂种（HB柚×枳262株，温州蜜柑×枳49株，红橘×枳111株，华农本地早橘×枳1株）；SSR分析鉴定了其它杂交后代的756个单株，结果共获得了614株杂种，其中HB柚×诺瓦244株，HB柚×佩奇179株，HB柚×费尔切尔德172株，华农本地早橘×诺瓦1株，华农本地早橘×佩奇7株，华农本地早橘×默科特3株，温州蜜柑×诺瓦8株。这些杂种资源为柑橘的品种选育、砧木育种及图谱构建提供了材料。2、以红橘、枳及它们的10个杂种单株为材料，共筛选了84对核SSR引物、68对EST-SSR引物、45条RAPD引物和1个CAPs标记（Lcyb基因），结果发现45对（53.6％）核SSR引物、30对（44.1％）EST-SSR引物、15条（33.3％）RAPD引物和Lcyb-CAPs标记具有多态性，并在子代个体中存在分离。用筛选出来的多态性引物对红橘×枳的F₁群体（107个单株）进行扩增，共得到了114个分离位点（53个核SSR位点，30个EST-SSR位点，30个RAPD位点，1个CAPs位点），其中有20.2％的位点（23个）表现了不同类型的偏分离，其余91个位点表现出符合1：1或1：1：1：1或1：2：1或3：1的孟德尔分离方式。核SSR、EST-SSR和RAPD位点分别表现出28.3％、6.7％和20.0％的偏分离比例。3、用Joinmap3.0软件，对91个符合孟德尔分离规律的位点，进行遗传连锁分析，构建了一张含有36个位点（15个SSR、19个EST-SSR、1个RAPD和1个Lcyb-CAPs）、9个连锁群、总长为329 cM的红橘×枳的图谱，并找到了与Lcyb基因相连锁的标记。7个新标记（其中5个为轻度偏分离标记）添加到图谱后，不但没有严重影响图谱原有标记的连锁关系，而且将总图距增加到了417 cM，说明轻度偏分离标记也可用于连锁图的构建。此外，用母本的81个位点、父本的57个位点、114个分离位点分别构建了含有11个连锁群和49个标记的母本连锁图、含有8个连锁群和20个标记的父本连锁图、含有14个连锁群和56个标记的双亲共有连锁图。母本、父本及双亲共有连锁图分别覆盖基因组总长的477 cM、202 cM和546cM。通过比较分析母本连锁图、父本连锁图及它们共有连锁图上的共有标记，发现不同连锁群之间存在共线性。此图谱主要由共显性标记组成，有利于各图谱之间的比较分析，并为图谱的进一步填充、抗性基因的定位打下基础。4、将温州蜜柑×枳授粉后80、85、90天及红橘×枳授粉后80、85天的种胚接种到MG1.0(MT+GA₃ 1.0 mg·L^-1)培养基中进行胚抢救，结果证明授粉后80天适合于这两个杂交组合的胚抢救；在所测的八种培养基中，MG0.5(MT+GA₃ 0.5mg·L^-1)培养基最适合于温州蜜柑×枳，MG1.0培养基最适合于红橘×枳。对胚抢救获得的两个枳杂种愈伤组织进行胚性能力测定，结果表明红橘×枳的杂种愈伤组织已丧失了胚性能力，在所用的七种培养基中都不能再生胚状体；温州蜜柑×枳的杂种愈伤组织还具有一定的再生能力，在M2（MT+5％乳糖）、M6(MT+500 mgl^-1ME+5％蔗糖)和M7(MT+500 mg l^-1 ME+3.0 mg l^-1 BA+5％蔗糖)三种培养基都能再生胚状体。更多还原

【Abstract】 Citrus is a very important fruit for agricultural economy in the world. Citrusbreeding and construction of linkage map has been hindered by several factors includingpolyembryony, female/male abortion and high hetetozygosis. In an effort to resolveabove difficults, at first, 14 different cross combinations of citrus were carried out toobtain the hybrid plants via embro rescue and molecular marker verification; Then, thelinkage framework map of citrus was constructed with molecular markers by the geneticanalysis of F₁ population from the red tangerine （C. reticulata Blanco）×trifoliate orange（Poncirus trifoliata （L.） Raf） cross and embryogenesis capacity of trifoliate orange （T）hybrid calli from embryo rescue was tested. The main results of this research are asfollows:1、A total of 1,087 fruits and 4,846 seeds were obtained from 14 different crosscombinations which was conducted in 2003 and 2004, with HB pummelo （C. grandis （L.）Osbeck ’Hirado Buntan’）, ’Bendizao’tangerine （C. reticulata ’Huanong’）, ’Guoqing’Satsuma （C. unshiu Marc.）, Grapefruit （C. paradisi Macq.） and Red tangerine （C.reticulata Blanco） as female, and trifoliate orange, ’Nova’ tangelo, ’Page’ tangelo,’Fairchild’ tangelo and ’Murcott’ tangor as pollen parents. A total of 2,029 progenies wasobtained from those crosses via embryo culture, 1,187 seedlings successfully survivedafter transplantation. Four hundred and twenty three （ 262 for HB×T, 49 for S×T, 111for R×T and 1 for B×T） out of 990 individuals exhibiting the trifoliate phenotype oftrifoliate orange were considered to be hybrids by morphology identification and SSRverification; Six hundred and fourteen of 756 plantlets derived from other crosses wereverified to be hybrids by SSR analysis. Of them, 244 for HB×’Nova’ tangelo, 179 forHB×’Page’ tangelo, 172 for HB×’Fairchild’ tangelo, 1 for ’Bendizao’tangerine×’Nova’tangelo, 7 for ’Bendizao’tangerine×’Page’ tangelo, 3 for ’Bendizao’tangerine×’Murcott’tangor and 8 for satsuma mandarin×’Nova’ tangelo. The hybrids provided the materialsfor citrus variety selection, rootstock breeding and construction of linkage map.2、Fouty five out of 84 genomic SSR primers, 30 out of 68 EST-SSR primers, 15 outof 45 RAPD primers and the Lcyb-CAPs marker exhibited the polymorphisms andsegregations among red tangerine, trifoliate orange and ten hybrids. Those polymorphic primers were screened out for amplification in the F₁ population （107 individuals） of redtangerine×trifoliate orange, and 114 segregated loci （53 SSR loci, 30 EST-SSR loci, 30RAPD loci and 1 Lcyb-CAPs loci） were obtained. Of which, 20.2% （23 loci） showed thedeviation of different Mendelian segregation type, and other loci （91 loci） segregated in1:1 or 1:1:1:1 or 1:2:1 or 3:1 ratio. The percentages of skewed loci for genomic SSR,EST-SSR and RAPD loci were 28.3%, 6.7% and 20% respectively.3、The consensus framework map of red tangerine×trifoliate orange was producedby linkage analysis using Joinmap3.0 software, 36 （15 SSR, 19 EST-SSR, 1 RAPD and 1Lcyb-CAPs） out of 91 examined loci were assigned to 9 linkage groups, with a totallength of 329 cM, and the loci linkage to the Lcyb gene were found. There was no severeeffects on the position of previous loci after adding 7 new loci（including 5 slightlyskewed loci） into the map, and the map length achieved 417 cM, which was suggestedthat the little distorted loci also could be use for map construction. Eighty one loci for redtangerine, 57 loci for trifoliate orange and 114 loci for red tangerine×trifoliate orangewere analyzed respectively, to construct the linkage maps. Eleven linkage groups with 49markers in red tangerine, 8 with 20 markers in trifoliate orange, and 14 with 56 markersin the cross pollinator （CP） consensus of both, were constructed. About 477 cM of redtangerine genome, 202 cM of trifoliate orange genome were covered. By comparison ofshared markers, a high level of colinearity existed among different linkage groups forthose three maps. This map was mainly composed of codominant markers; It will beessential for citrus comparison map, mapping further saturation and resistant genelocation.4、Immature embryos of 80, 85, 90 days after pollination （DAP） from the Satsumamandarin （S）×trifoliate orange （T） cross, 80 and 85 DAP from the Red tangerine （R）×trifoliate orange （T） cross, were cultured on MG1.0 medium consisting of MT basalmedium supplemented with 1.0 mgl^-1 GA₃ and 4% sucrose. The results showed that 80DAP was the optimal time for embryo rescue of the tested crosses. Among the eighttested media, MT medium supplemented with 0.5 mgl^-1 GA₃ was the best one forsatsuma mandarin, and MT plus 1.0 mgl^-1 GA₃ for red tangerine. By testing theembryogenesis capacity of hybrid calli derived from embryo rescue, it was observed thatno embryoids appeared on the tested seven media for hybrid calli of red tangerine×trifoliate orange, however, the capacity of embryogenesis still be possessed by hybridcalli of satsuma mandarin×trifoliate orange on M2 （MT+5% lactose）, M6 (MT+500 mgl^-1 ME+5% sucrose) and M7 (MT+500 mgl^-1 ME+3.0 mgl^-1BA+5% sucrose)midia.更多还原