【作者】 葛贤宏；

【导师】 李再云；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 在芸苔属与诸葛菜(Orychophragmus violaceus(L．)O．E．Schulz，2n=24)的属间杂交中发现亲本种染色体组分开的新细胞学现象及其遗传控制；芸苔属6个栽培种与诸葛菜属间杂种中的染色体行为受芸苔属亲本种含有的染色体组的影响；3个四倍体种与诸葛菜杂种中的不同染色体行为及差异，可由3个二倍体种与诸葛菜杂种中的不同染色体行为解释或预测。这是继假配生殖(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染色体行为，丰富了植物遗传学的内容。为了更深入研究染色体组分开现象的遗传和发生机制，本研究拟进行诸葛菜与人工合成的芸苔属异源六倍体(2n=54，AABBCC)的杂交。利用普通细胞学、基因组原位杂交(Genomic in situ hybridization，GISH)、扩增片段长度多态性(Amplified fragment length polymorphism，AFLP)及单链构象多态性分析(single-stranded DNA conformation polymorphism，SSCP)对杂种及其后代作系统的研究，探讨来源于三个不同基因组的芸薹属染色体在杂种细胞有丝分裂及减数分裂时的染色体行为。为此我们合成了三个不同来源的三基因组杂种，分别为埃塞俄比亚芥“G0-11”(B．carinata A．Braun，2n=34，BBCC)×白菜“上海青”(B．camprists，2n=20，AA)(组合BC．A)，甘蓝型油菜“中油821”(B．napus，2n=38，AACC)×黑芥(B．nigra(L．)Koch，2n=16)(组合AC．B)，人工合成的甘蓝型油菜×黑芥(组合A．C．B)的杂种一代。通过加倍获得三个不同来源的六倍体(分别表示为六倍体Ⅰ，Ⅱ，Ⅲ)，在细胞学观察的基础上以确认为六倍体的植株为母本与诸葛菜杂交。主要结果如下：1．普通细胞学分析表明三个三基因组杂种在减数分裂过程中均有较高数目的二价体形成。组合BC．A、AC．B、A．C．B分别有88.2％，96.8％和91.2％的细胞包含有6—10个二价体。同时包含有少于8个单价体的细胞比例在组合间有较大的差异，分别为14.7％，24.5％和29.8％。GISH分析表明三个组合的杂种在减数分裂过程中，均有相当比例的B基因组染色体参与了异源的染色体配对。三个组合中分别有31.9％，45.4％和50％的细胞包含有至少一条B基因组染色体参与了异源配对；在AC．B、A．C．B组合中同一个细胞中最多有三条B基因组染色体参与了异源的配对，而在组合BC．A最多只有两条。同时，分别有10.9％，16.6％和9.4％的花粉母细胞细胞至少形成一对B基因组染色体间的二价体。三个组合中，同一细胞中均出现了最多有两个二价体完全由B基因组染色体形成。上述结果进一步验证了芸薹属三个基因组在基因组间及基因组内存在的同源性。2．六倍体Ⅰ、Ⅱ、Ⅲ与诸葛菜杂交分别获得30，4及3株杂种单株，每个单株均来自不同的胚。杂种一代在表型上与母本的六倍体及六倍体自交后代有较大的差别，在苗期的叶形及成年植株茎杆颜色上表现有父本的特征。普通细胞学观察表明，所有单株的体细胞均由具不同数目染色体的细胞组成，即均为混倍体。体细胞最高的染色体数目为46，最低为20。杂种单株体细胞中出现频率最高的染色体数目有三种，一是44，有32株；二是42，有4株；三是36，有1株。可能的是杂种植株在合子细胞形成后的发育过程中经历了染色体组加倍和部分染色体消除的过程。3．GISH分析表明，所有杂种细胞中均未检测到诸葛菜的整条或者染色体的片段。同时根据B基因组DNA为探针的GISH结果，以细胞中B基因组染色体的数目及减数分裂时的配对及分离的方式，将所有杂种植株分为三大类：Ⅰ类具有16条B基因组染色体，以8Ⅱ的方式配对，以8:8分离，共32株；Ⅱ类具有14条B基因组染色体，以6Ⅱ+2Ⅰ方式配对，以7:7或者6:8的方式分离，共4株：Ⅲ类植株包含有上述两种细胞类型类型，共1株。GISH结果同时表明，六倍体Ⅰ中一对带有rDNA位点的A基因组染色体在所有杂种中均只有一条。GISH的结果表明所有的诸葛菜染色体在合子的发育过程中被全部消除。4．杂种一代的AFLP分析表明，六倍体Ⅰ与诸葛菜的杂种中具有较多诸葛菜的特异带，诸葛菜特异带占该单株的总带数的百分比在4.8％到7.1％之间。同时杂种单株中出现了母本的缺失带和双亲均无的新带，表明外源遗传物质的进入引起受体基因组结构的变化。六倍体Ⅱ、Ⅲ与诸葛菜的所有杂种中，25对引物中只有3对引物能够扩出诸葛菜的特异带，但是母本缺失带和新带的数量较多。5．六倍体与诸葛菜杂种及后代的AFLP，普通细胞学，表型及杂交分析表明，杂种及后代在基因组组成上更偏向于芥菜型油菜。由于B基因组染色体在绝大多数的杂种植株中均是完整的，说明所有杂种一代单株种中C基因组被消除或者说丢失的染色体要多于A基因组染色体，即在杂种细胞中三个基因组染色体表现出B＞A＞C的稳定性关系。6．SSCP分析发现在三种六倍体及六倍体与诸葛菜的杂种中，A／C来源的rRNA基因只在一个杂种植株中表达，而B基因组来源的rRNA基因在所有单株中均表达。A／C来源的rRNA基因表达的单株中B基因组染色体为14条，我们认为缺少的染色体上载有rDNA位点，该染色体的缺失导致核仁显性关系的变化。7．在六倍体Ⅰ和诸葛菜杂种一代的两个单株中，减数分裂四分体时期，当且仅当四分体形成时，小孢子中的染色质重新收缩呈染色体状，染色体随后随机的分到两极或者多极，同时在这些细胞中发现了两极和多极的纺锤体。随后细胞质分裂，单个的小孢子被分成了大小不等的几个微小孢子。原位杂交分析表明，染色体分离前没有经历复制的过程；染色体的两极或者多极分离是完全随机的，没有基因组的特异性。在另外一些细胞中，四分体形成后，四个小孢子逐渐融合成为一个大的细胞，染色体数目随之上升。这两种异常情况是相伴发生的，但是细胞额外分裂的频率要远高于融合的频率。这两种小孢子发育的异常只发生在两个植株的部分四分体中，甚至于同一个四分体的个别细胞中。8．六倍体Ⅲ和诸葛菜杂种的一个单株中，减数分裂粗线期之前未见异常，粗线期之后，染色体缺少配对的过程，单价体聚集在一起。聚在一起的染色体总是出现在细胞的边缘。随后这些染色体随机的分向两极，但是细胞质并不分裂，导致双核细胞的形成，双核细胞停止了进一步的发育。这些细胞在后期形成外壁，染色深，似成熟的花粉，但是细胞内空洞无物。在这些异常分裂的花粉母细胞中未发现纺锤体的出现。最后，我们认为芸薹属六倍体与诸葛菜杂种的染色体行为不同于二倍体或者四倍体与诸葛菜杂种的染色体行为。我们推测合子细胞在发育过程中经历了染色体组的加倍与双亲染色体组的部分分开。在染色体组部分分开的过程中，母本染色体的分开具有基因组的特异性，可能的是较多的C基因组染色体分到了诸葛菜染色体的一极，而包含有较多诸葛菜染色体的细胞最后被消除导致有较多C基因组染色体的消除。而B基因组染色体、较多的A基因组染色体、部分的C基因组及少量的诸葛菜染色体分到了同一极，但诸葛菜染色体在随后的细胞分裂过程中被消除。由于染色体的稳定性关系与核仁显性的一致性，我们认为基因组特异性rRNA基因的表达有助于该基因组染色体的稳定存在。诸葛菜染色体在消除的过程中亦可能与其他基因组的染色体发生交换和重组导致其遗传物质渗入。外源遗传物质的进入导致受体基因组结构及基因表达的改变，例如，杂种AFLP新带型的出现和小孢子发育的异常等。更多还原

【Abstract】 The phenomenon of complete and partial parent genome separation was found inhybrids between six Brassica species and Orychophragmus violaceus （L.） O.E.Schulz（2n=24）. Results from the hybridization also indicated that the different chromosomebehavior in different combinations might be arised by differetn maternal parent genome.The different chromosome behavior of hybrids with three Brassica diploids （B. rape, B.nigra and B. oleracea） might contribute to the different cytogenetics of hybrids with threetetraploids （B. napus, B. juncea and B. carinata）. Those findings are new chromosomebehaviors in plant wide hybridization beside pseudogamy, semigamy and chromosomeelimination and enriched the knowledge of plant genetics. To further study the mechanismbehind the phenomenon, we synthesized Brassica allohexaploid from differentcombinations and cross with O.violaceus as maternal parent. Cytology, genomic in situhybridization （GISH）, amplified fragment length polymorphisms （AFLP） andsingle-strand DNA conformation polymorphisms （SSCP） analyses were applied tohybrids. To obtain different allohexaploid, three different tri-genomic hybrids （ABC,2n=27） from crosses BC.A: B.carinata （G0-11, BBCC, 2n=34）×B.camprists （shanghaiqin,AA, 2n=20）, AC. B: B. napus （zhong you 821, AACC, 2n=38）×B. nigra （Koch, BB, 2n=16）,A. C. B: artificial B. napus×B. nigra were used for chromosome double. Three typeallohexaploids were named hexaploidⅠ,Ⅱ,Ⅲrespectively. Main results were describedas follows:1. A relatively high proportion of bivalents were formed in all tri-genomic hybridsfrom different combinations. Hybrids from combination of BC. A, AC. B, A. C. B had88. 2%, 96. 8% and 91. 2% PMCs （Pollen Mother Cells） with 6-10 bivalents, 14.7%, 24.5%and 9.8% PMCs with univalents less than 8 respectively. GISH analyses also revealed thateach type of hybrids had 31. 9%, 45. 4% and 50% PMCs contained at least one B-genomechromosome paired with A/C chromosomes and maximally three B-chromosomes inallosyndesis per cell observed in AC.B and A.C.B combinations. Each type of hybrids had10.9%, 16.6% and 9.4% PMCs with at least one bivalent formed by B chromosomes. Amaximum of two bivalents formed by autosyndesis within B genome at diakinesisappeared in all combinations. The accurate analyses of auto- and allo-syndetic pairing forB genome in trigenomic combinations provided further evidence for the hypothesis thatthree basic genomes of the cultivated Brassica species were secondary polyploids andderived from one common ancestral genome with a lower chromosome number. 2. 30, 4 and 3 hybrid plants were produced from crosses hexaploidⅠ,Ⅱ,Ⅲ×O.violaceus respectively. All hybrids were different from the hexaploid or self progeniesof hexaploid on phenotype. Young leaves and stem of adult plants showed some traits ofO.violaceus. Cytology observation revealed that all hybrids contained cells with differentchromosomes which indicated the mixploidy of the F₁ plants. The highest chromosomenumber was 46, and the lowest was 20. Cells with 44 chromosomes were found withhighest frequency in 32 hybrid plants, 42 in 4 hybrid plants and 36 in only hybrid plant.3. GISH analyses found no intact chromosome or chromosome fragments fromO.violaceus in all three type hybrids. According to the number and behavior of B genomechromosomes, all hybrid plants were classified into three types. All somatic cells of TypesⅠplants contained 16 B genome chromosomes which formed 8Ⅱat diakinesis and shown8:8 segregation at anaphase; TypesⅡcontained 14 B genome chromosomes whichformed 6Ⅱ+2Ⅰat diakinesis and showed 6:8 or 7:7 segregation at anaphase. TypesⅢcontained above two chromosomes number and behavior. GISH analyses also found thatone chromosome from A genome with rDNA locus was eliminated in all hybrids betweenhexploidⅠand O.violaceus. Obviously, complete O.violaceus chromosomes and partialmaternal chromosomes were eliminated.4. AFLP analyses revealed that all hybrids contained bands specific for O.violaceus,especially for hybrids between hexaploid I and O.violaceus which contained 4.8%-7.1%bands specific for O.violaceus. Deleted bands for maternal parent and novel bands werealso found in all hybrids. Possilably, introgressinon of O.violaceus chromatin disturbedthe maternal genome.5. Cytology, morphology, AFLP and cross analyses indicated that the genome ofhybrids and their self progenies resembled B.juncea. For intact B-genome chromosomeswere found in most of the hybrids, more chromosomes from C genome were eliminatedthan from A genome which indicated that the stability of three genome showingrelationships with B＞A＞C.6. RT-PCR SSCP analyses revealed that rRNA genes from A/C genome had beentranscripted in only hybrid but rRNA genes from B genome were transcripted in allhybrids. The plant contained 14 chromosomes which indicated that the deleted twochromosomes might have rDNA locus.7. Here extra divisions and nuclei fusions were observed to occur in microsporenuclei of partial hybrids between synthetic Brassica hexaploid I and O.violaceus. Abnormal spindle were formed and chromosomes were separated into several nuclei ofvariable sizes after bi-, or multi-polar divisions in the four cells of tetrads. As aconsequence, more than eight mini-microspores of different sizes were produced by onetetrad. Genomic in situ hybridization results indicated that no chromosome replicationoccurred during such divisions. In some tetrads, the four nuclei were fused to form onelarge cell with increased chromosome number. The extra divisions or fusions appearedonly in some flower buds of one plant, some anthers in the same buds, or even inindividual cells of tetrads. The possible mechanisms behind these cytological phenomenawere discussed.8. In meiosis of one hybrid between hexaploidⅢand O.violaceus, no chromosomepairing was found at diakinesis. Univalents then congregated which were found always atedge of the cell. Congregated chromosomes then separated randomly to two poles.However, cytoplasm did not divide. Cells with two nuclei then stopped development butformed thick ektexine which were dyed darkly. No spindle was found when specific dyefor spindle was used for those cells.Finally, chromosome behavior of hybrids between Brassica hexaploid andO.violaceus was different from that of hybrids between diploid or tetraploid andO.violaceus. Probably, zygote cells experienced chromosome double and partial parentgenome separation during development. More C genome chromosomes were divided intocells with more O.violaceus chromosomes and those cells were eliminated later. Thespecific genome rRNA gene expression might help to stabilize its chromosomes.O.violaceus chromatin introgression lead to alternation of maternal genome structure anddisturbed gene expression, such as new AFLP bands emergence and abnormal meiosis.更多还原