【作者】 赵志刚；

【导师】 李再云；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 通过远缘杂交可以研究植物间亲缘及进化关系,同时还可以创建大量的异染色体系,其中异附加系在遗传和育种研究中具有广泛的用途。甘蓝型油菜（Brassicanapus,2n=38,AACC）和诸葛菜（Orychophragmus violaceus（L.）O.E.Schulz,2n=24）属间杂种及其后代的遗传学研究已经较为深入,在其有性杂种中发现的亲本基因组分开现象是继假配生殖（Pseudogamy）、半配生殖（Semigamy）和染色体消除（Chromosome elimination）之后,植物远缘杂交中观察到的新细胞学现象。尽管如此,在甘蓝型油菜和诸葛菜有性杂种后代并没有选择到稳定的附加系材料。本文对甘蓝型油菜和诸葛菜有性杂种高世代(F₈-F₁₀)材料进行形态学、细胞学、分子生物学多个方面的研究,进一步揭示有性远缘杂种后代的遗传规律;对甘蓝型油菜和诸葛菜属间体细胞杂种及与甘蓝型油菜回交后代进行了分子细胞遗传学研究并培育出附加系。主要结果如下:1.在甘蓝型油菜和诸葛菜属间杂种F₅群体中出现一株淡黄花的植株,其花粉母细胞具有31条染色体,在减数分裂后期Ⅰ呈15:16和12:19两种分离。该植株自交产生的18个植株具有广泛的形态学、染色体数目和育性的变化。在其产生的18株F₆植株中,第1-3株表型偏白菜型油菜,在第2株的F₇后代选择两株表型最偏白菜型油菜的植株分别连续自交到F₁₀形成Ⅰ、Ⅱ两个家系。1.1.形态学上这两个家系间、家系内世代间差异不大。在叶型、花期等性状上更接近白菜型油菜,而角果长度和千粒重等性状则更接近于甘蓝型油菜。1.2.两个家系都普遍具有自交不亲和性,其中Ⅱ类家系表现更为明显。1.3.两个家系随着世代的增加,体细胞染色体数目都有向甘蓝型油菜的2n=38升高和回归的趋势。与白菜型油菜杂种的减数分裂配对结果显示,这些偏白菜型油菜后代中丢失的可能是来自甘蓝的染色体。基因组原位杂交（GISH）分析未能在后代植株中检测出整条诸葛菜染色体或其片段。1.4.对两个F₁₀家系进行了AFLP分析,用16对引物只扩增出少量诸葛菜特异带,而新增带和缺失带所占比例较高。AFLP聚类分析将家系Ⅰ的大部分植株和家系Ⅱ的所有植株在相关系数约为0.92时分为明显的两组,体现出两者较近的遗传背景,又反映出两者不同的变化方向。两个家系和白菜型油菜、甘蓝各自的混合样都具有较小的相关系数,和白菜型油菜的相关系数大约0.68,小于和甘蓝的0.72。2.甘蓝型油菜华双三号和诸葛菜属间体细胞杂种及其后代的遗传学研究2.1.来自不同愈伤组织的三个体细胞杂种植株（Nos.98,100,101）均雄性不育,个别雌性部分可育。杂种98和101具有相似的植株形态,表现诸葛菜的花叶、多分枝和基部分枝特性,黄色花瓣间红色条纹。杂种100表型偏甘蓝型油菜,花瓣红条纹稀少。杂种的叶绿体DNA与甘蓝型油菜相同。AFLP分析表明所有体细胞杂种和诸葛菜有更近的遗传组成,101在苗期和花期具有不完全相同的DNA带型,田间腋芽培养植株和培养基上保存的芽长成的植株间也具有一定的DNA带型差异。三个杂种都是混倍体（2n=51-67）,包含19-28条诸葛菜染色体。减数分裂终变期诸葛菜染色体主要形成二价体,后期Ⅰ表现二极与多极分离。2.2.用华双三号为101授粉后获得20个BC₁后代,其中11株叶型偏甘蓝型油菜,9株偏花叶。这些BC₁后代都是雄性部分可育,雌性完全败育,胚珠畸形。所有BC₁植株都是混倍体（2n=41-54）,包含9-16条诸葛菜染色体。减数分裂终变期有大约一半的诸葛菜染色体参与了部分同源配对,有大量花粉母细胞发生了多极分现象,但所占比例明显低于杂种一代。2.3.用花粉育性较好的几个BC₁植株为华双三号授粉,获得大量的BC₂种子,BC₂种子具有较低的发芽率和较慢的发芽速度。花叶、雌不育和开花期等几个性状在不同组合的BC₂群体中发生了分离。细胞学检测的8株BC₂植株具有39-43条染色体,其中包含2-5条诸葛菜染色体。有接近一半的诸葛菜染色体进行了部分同源配对。后期Ⅰ诸葛菜染色体落后较为明显,但依然有相当多的后期Ⅱ、末期Ⅱ子核包含诸葛菜染色体。在大部分花药壁细胞有丝分裂过程中诸葛菜染色体均等分裂,而少部分不等分裂。2.4.在几个BC₂群体及自交后代中,选择到5个含一条诸葛菜染色体的甘蓝型油菜-诸葛菜单体附加系,分别表现花叶（O₁）、甘蓝型油菜（O₂）、花瓣褪色（O₃）、多分枝（O₄）和雌性不育（O₅）等形态特征。前4个附加系自交与自由授粉后均结实很好。2.5.在杂种及后代的体细胞及PMCs中,诸葛菜染色体的体积较大、染色较深,易与甘蓝型油菜的区分。更多还原

【Abstract】 Wide hybridization can be used to study the evolution of plants and establish alien chromosome lines.Alien addition lines are widely utilized for plant genetics and breeding. Cytogenetics of intergeneric hybrids between Brassica napus L.（2n=38） and Orychophragmus violaceus（L.） O.E.Schulz（2n=24） and their progenies have been extensively investigated.The separation of parental genomes during the mitotic and meiotic divisions of the hybrid cells is proposed,following pseudogamy,semigamy and chromosome elimination in plant wide crosses.However,no stable B.napus-O,violaceus addition lines have been obtained in the progenies of sexual hybrids.In this study,inbred lines(F₈-F₁₀) derived from one hybrid of these two species are characterized for morphology,cytology and genomic compositions.In addition,backcross progenies of two generations derived from the somatic hybrids between B.napus and O.violaceus after pollinated by B.napus parent are produced and characterized at morphological, cytogenetical and molecular levels,and several B.napus - O.violaceus monosomic addition lines with obvious phenotypic traits are established.The results are described as follows:1.One F₅ plant（2n=31） of one B.napus cv.Oro×O.violaceus hybrid with slightly yellow petals and the 12:19 and 15:16 segregations in its pollen mother cells（PMCs） produced 18 F₆ plants with distinct morphological characters and wide variations in fertility and chromosome numbers（2n=25-38）.Three plants（Nos.1-3） out of the 18 F₆ plants were morphologically B.rapa-like by having oval and less waxy but greener leaves and two of them（No.1 and 3） were totally sterile.Two most B.rapa-like F₇ plants from the plant No.2 were chosen through morphology for successive selfcrossing to F₁₀ generation and two populationsⅠ,Ⅱwere produced.1.1.A few morphological variations were observed within or between the two populations.They were more similar to B.rapa on leaves shape and flowering time etc, but more similar to B.napus on some botanical characters such as length of siliques and 1000 seed weight.1.2.Two populations both displayed self-incompatibility at some degrees withⅡbeing more evident.1.3.With progression of generations,chromosomes number of plants from two populations gradually increased and centred at the number of B.napus（2n=38）. Chromosome pairins at meiotic diakinesis of hybrids between B.rapa-like plants and B. rapa revealed that chromosomes lost in B.rapa-like plants possibly belonged to C genome of B.oleracea.No intact O.violaceus chromosomes or chromosome fragments were founded by genomic in situ hybridization（GISH） observations in these B. rapa-like progenies.1,4.AFLP analysis showed that B.rapa-like progenies(F₁₀) had more DNA bands novel for two parents and deleted from B.napus parent than specific bands for O. violaceus.The AFLP clustering analysis distinguished populationsⅠandⅡat 0.92 correlation coefficient,showing that they had very similar genomic compositions with a degree of genetic diversity.In addition,they had more distant genetic distances from B. rapa and B.oleracea.Genetic distance between the two populations and B.rapa seemed to be wider than that with B.oleracea.2 Genetic analysis of intergeneric somatic hybrids between B.napus cv. Huashuang 3 and O.violaceus and progenies2.1.Three hybrids（Nos.98,100,101） derived from three different calli were sterile for male and partially fertile for female in individual.Hybrids Nos.98,101 both showed the similar morphology intermediate between two parents,displaying serrated leaves,many basic branches from O.violaceus and yellow petals with red stripes. Hybrids No.100 resembled B.napus mainly in morphology with pale yellow petals with slightly purple stripes.The cpDNA of all somatic hybrids was of B.napus type.AFLP analysis indicated that these hybrids had closer genetic relationship with O.violaceus than with B.napus,and the young and flowering plants of hybrid No.101 had some difference in AFLP profiles,and the same for the plants from cultured buds of the plant in field and from subcultured buds on medium.These hybrids were mixoploids （2n=51-67） with 19-28 O.violaceus chromosomes.Chromosomes in pollen mothe rcells （PMCs） at meiotic diakinesis mainly formd bivalents and showed bi-/multipolar segregations at anaphaseⅠ.2.2.After pollinated by B.napus parent and with embryo rescue,hybrid 101 gave rise to 20 BC₁ plants,in which 9 plants shown the typical serrated leaves of O.violaceus, the others had B.napus-type leaves.All BC₁ plants were partially fertile for male but totally sterile for female by showing the abnormal structure of ovules.They were mixoploids（2n=41-54） with 9-16 chromosomes from O.violaceus.About one half of O. violaceus chromosome in PMCs at meiotic diakinesis underwent homoeologous pairings in BC₁ plants.Multipolar segregations occurred with a lower frequency in AⅠPMCs of BC₁ plants than in hybrids.2.3.After polinated with the pollen grains of several BC₁ plants,Huashuang 3 produced plentiful BC₂ seeds which generally had low germination rate and speed.BC₂ plants showed segregations for female fertility,leaves shape and flowering time in some BC₂ combinations.Eight BC₂ plants examined by GISH had still some chromosome variations（2n=39-43） with 2-5 chromosomes from O.violaceus.Still nearly one half of O.violaceus chromosomes underwent homoeologous pairings in BC₂ plants.O. violaceus chromosomes were frequently lagged in AⅠPMCs,but many daughter groups at AⅡor nuclei at TⅡcontained O.violaceus chromosomes.During mitotic divisions of majority of anther-wall cells,the sister ehromatids of O.violaceus chromosomes were equally distributed into daughter cells but unequally for some.2.4.In BC₂ and several BC₂F₁ populations,five B.napus - O.violaceus monosomie addition lines with one O.violaceus chromosome were obtained,which showed specific phenotypic traits,such as serrated leaves of O.violaceus（O₁）,B.napusphenotype （O₂）,pale petals（O₃）,basal clustering branches（O₄） and female sterility（O₅）. The first four lines showed high seed-sets by selfing or open- pollination.2.5.In these hybrids and progenies,the chromosomes of O.violaceus were easily distinguished from those of B.napus by their large size and dark staining.更多还原