【作者】 陈海峰；

【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】 远缘杂交是作物遗传改良的重要途径,可以把野生种的优良性状向栽培种转移。十字花科中有很多优良的野生种质资源,可提供许多有用的细胞核和细胞质基因,用于油菜育种。我国现在大面积栽培的甘蓝型油菜（Brassic napus,2n=38,AACC）于二十世纪30与40年代从国外引进,遗传变异较为匮乏,故通过多种途径创造新的种质资源对其遗传改良具有重要意义。十字花科独行菜族的荠菜（Capsellabursa-pastoris（L.）Medic,2n=4x=32）广布全球,在中国和其它一些国家作蔬菜和药用。荠菜种子内的芥酸（0.68%）和硫甙（15.68μmol/g）符合双低（芥酸＜1%,硫甙＜30μmol/g）标准,为油菜遗传改良的天然双低种质资源,且高抗菌核病。本研究合成了白菜型油菜（B.rapa,2n=20）、甘蓝型油菜与荠菜的族间杂种,并对杂种及其后代进行了形态学、细胞学、分子生物学、脂肪酸和抗菌核病的分析。另外,对以前从甘蓝型油菜与诸葛菜的属间杂交中获得的一个缺体植株（2n=36）及后代进行了细胞遗传学研究。主要结果如下:1.油菜×荠菜。以白菜型和甘蓝型油菜为母本、荠菜为父本的有性族间杂交中,经大量人工授粉产生的大多数F₁植株在形态上和母本相似,只有少数表现为中间型。F₁植株具有父本荠菜的一些特征,如叶片小、深绿色、叶裂深、植株矮小、基部分枝、白色花瓣。根据体细胞（子房细胞）染色体数目,F₁植株分为五类:第一类2n=27—29,第二类2n=20,这两类植株来自白菜型油菜与荠菜的杂交;第三类为甘蓝型油菜单倍体,2n=19,第四类2n=29,第五类2n=38,这三类植株来自甘蓝型油菜与荠菜的杂交。基因组原位杂交（GISH）分析表明,第一类杂种植株的花粉母细胞（pollenmother cells,PMCs）中有1—2条荠菜染色体,而另在一些植株的子房细胞和PMCs中检测到荠菜的染色体片段。AFLP（amplified fragment length polymorphism）分析显示,除3个F₁植株（Nos.13,24,32）无荠菜特异带外,所有植株中都出现了荠菜特异带、油菜母本的缺失带和双亲均无的新带,而且各单株间多态性带的数目与组成都不一样,表明外源遗传物质的渗入是在不同水平上的,同时引起了受体基因组结构的变化。通过脂肪酸分析和菌核病鉴定,发现一些母本型植株后代的芥酸和硫甙含量降低、菌核病抗性显著提高。2.雄性不育材料。来自甘蓝型油菜奥罗与荠菜杂交的一个母本型植株（No.30）的雄蕊小、雄性完全不育。与奥罗回交一代的植株全可育,自交结实好。359个BC₁F₁植株中,276株可育,83株不育,可育株与不育株的比例为3:1(χ_c²=0.58＜χ_0.05²);在F₁×BC₁的109个植株中,58株可育,51株不育,可育株与不育株的比例为1:1(χ_c²=0.33＜χ_0.05²),说明该雄性不育性为一对隐性基因控制的核不育。花药切片观察表明不育株花药在造孢细胞时期开始出现异常,绒毡层多层、肥大,但是其减数分离是正常的。四分体时期绒毡层进一步恶化,多层、肥大、液泡化,开始降解,并挤压四分体,导致四分体不能形成单核花粉粒,小孢子开始退化。大多数后代植株的硫甙含量明显降低,一些植株达到双低标准,并且菌核病抗性显著提高。3.甘蓝型油菜缺体。对甘蓝型油菜奥罗与诸葛菜的一个混倍体杂种进行小孢子培养,获得的一个缺体（2n=36）植株,其株高只有70—80cm,比甘蓝型油菜开花早两个月左右。缺体植株的PMCs在终变期具有18个二价体,后期Ⅰ表现18:18的正常分离,但在14个自交一代植株中,6个植株31.2%—43.8%的PMCs在后期Ⅰ为17:19分离,这导致了后代中缺体—四体（2n=38）的形成。白菜型油菜×缺体的PMCs在终变期具有10Ⅱ+8Ⅰ,说明缺体缺失了一对来自甘蓝C基因组的染色体。奥罗×缺体后代的植株株高和开花时间与甘蓝型油菜接近,但在自交后代中没有发现缺体植株。缺体—四体的株高比奥罗矮5cm,开花时间比甘蓝型油菜奥罗早一个月左右,59%的PMCs在终变期具有17Ⅱ+1Ⅳ,其余的为19Ⅱ,自交结实较好。最后,我们提出了油菜与荠菜的杂种形成机制。我们认为在合子形成后或杂种胚发育的早期即发生了染色体的消除、加倍和外源渗入。通过杂交产生母本型植株,可快速向油菜转移荠菜优良性状（双低、抗菌核病）。同时,我们对缺体和缺体—四体在油菜育种、基因定位和功能分析中的运用进行了讨论。更多还原

【Abstract】 Wide hybridization plays an important role in crop improvement and has been used successfully to transfer desired traits from wild germplasm to large number of crop species. The Cruciferae family comprises a large number of wild species which provide rich germplasm with many useful nuclear and cytoplasmic genes for oilseed breeding. Brassica napus （2n=38, genomes AACC） was introduced into China for the first time from Korea in 1930s and subsequently from Europe in 1940s with limited genetic variability and thus the widening of its gene pool through suitable approaches including wide hybridizations is pivotal for further genetic improvement. The crucifer Capsella bursa-pastoris （L.） Medic （2n=4x=32） of tribe Lepidieae is a wild species distributed worldwide and has been used traditionally as vegetable and medicinal plants in China and some other countries for many centuries. It is a natural double-low （erucic acid <1%, glucosinolates <30μmol/g） germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Intertribal crosses were made between two Brassica species （B. rapa, 2n=20; B. napus, 2n=38） and C. bursa-pastoris with the latter as pollen parent, in order to introduce the desirable traits into the cultivated Brassica species. Progenies were investigated on morphology, cytology, molecular characteristics, fatty acid compositions and resistance to 5. sclerotiorum. On the other hand, one microspore-derived nullisomics （2n=36） without Orychophragmus violaceus （2n=24） chromosomes was obtained from the hybrid between B. napus and O.violaceus, and nulli-tetrasomics were obtained in the progenies of partial nullisomic plants. Cytogenetical analysis was applied to nullisomic and nulli-tetrasomics. The main results were as follows:1. Brassica species×C. bursa-pastoris. Majority of F₁ plants resembled female parents in morphology and only a few were morphologically intermediate between the parents. The F₁ plants expressed some characters of male parent, including small sized dark-green, deeply divided leaves, nanism, basal clustering branches and white petals. Based on cytological observation of somatic cells, the F₁ plants were classified into five types: two types from the cross with B. rapa, typeⅠhad 2n=27-29; typeⅡhad 2n=20; three types from the crosses with B. napus, typeⅢwas haploids with 2n=19; typeⅣhad 2n=29; typeⅤhad 2n=38. One to two chromosomes of C.bursa-pastoris were detected in pollen mother cells （PMCs） of type I plant by genomic in situ hybridization （GISH）, together with chromosomal segments in ovary cells and PMCs of some F₁ plants. Amplified fragment length polymorphism （AFLP） bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in F₁ plants except for three plants （Nos. 13, 24, 32） which had no specific bands, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum.2. Male sterile hybird. The F₁ plant No. 30 from the cross between B. napus cv. Oro and C. bursa-pastoris which resembled female parent in morphology was male sterile with rudimentary stamens. The BC₁ plants of the F₁ plant pollinated by ’Oro’ had good seed-set after selfing. In the selfing population of BC₁ plants, the ratio of male fertile to sterile plants was 3:1 （276 fertile, 83 sterile among 359 plants） (x_c²=0.58 < x_0.05²). Of the 109 plants from sterile F₁ x BC₁, 58 ones were male fertile and 51 male sterile, displaying a 1:1 ratio (X_c²=0.33 < x_0.05²). The result indicated that the male sterility （MS） in the hybrid was controlled by a recessive gene. Cytological observations showed that the tapetum was abnormal with multiple layers and hypertrophy since the stage of sporogenic cell, however, the meiosis was normal with chromosome pairing （19 bivalents） and segregation （19:19）. At the tetrad stage, the tapetum of male sterility was vacuolated and disaggregating with multiple layers and hypertrophy, and the tetrads were extruded by tapetum. So they could not produce normal uninucleate pollen grains, the microspores began to degenerating since the stage of tetrad. In most progenies, the content of glucosinolates was reduced obviously, some reached double-low standard, together with the significantly improved resistance to S. sclerotiorum.3. B. napus nullisomics. One microspore-derived nullisomics （2n=36） was obtained before from the hybrids between B. napus and Orychophragmus violaceus （2n=24）, its plant height was only 70-80 cm and flowering time was about two months earlier than B. napus. PMCs of the nullisomics had 18 bivalents at diakinesis and 18:18 segregations at anaphase I （AI）; however, of 14 nullisomic plants in its selfing progenies, six plants had 31.2-43.8% PMCs with 17:19 segregations at AI, which resulted in the production of nulli-tetrasomics （2n=38）. PMCs of B. rapa×nullisomics at diakinesis had 10 bivalents and 8 univalents, which indicated that the nullisomics lost one pair chromosomes of C genome from B. oleracea. The progenies of B. napus×nullisomics had plant height and flowering time similar to B. napus, but produced no nullisomic plants. Nulli-tetrasomics were only 5 cm shorter than B. napus and flowered about one month earlier than B. napus. The nulli-tetrasomics produced 59% diakinesis PMCs with 17 bivalents and one quadrivalent and the remainings with 19 bivalents and 19:19 AI segregations, and had good seed-set after selfing.Finally, it was proposed that chromosome elimination / doubling, and introgression were involved in the formation of these hybrids between Brassica species and C. bursa-pastoris during mitotic divisions of the zygotes or hybrid embryos, the Brassica -type progenies provided an opportunity to rapidly and successfully introduce useful traits of C. bursa-pastoris into Brassica species and to produce lines with improved oil quality and resistance to S. sclerotiorum. The utilization of the nullisomics and nulli-tetrasomics in plant breeding, location and functional analysis of genes was discussed.更多还原