【作者】 王坤波；

【导师】 刘旭；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2009， 博士

【摘要】 恰当判别体细胞同源染色体进而获得更准确的基本数据,是核型分析最关键的环节。经典核型分析难以准确做到同源配对而往往失于偏颇,基于原位杂交的核型分析突破了这个瓶颈问题。澄清多倍体棉花的供体亲本,不仅可解决长期争论的理论问题,而且有助于棉花基因组测序首选棉种的决策,在进入全基因组测序时代,这一点非常重要。因此,利用我院现有材料优势,本人就较全面地开展了现有棉种基于原位杂交的核型分析。本研究收集到了21个棉种,涵盖了棉属除K基因组外的A~G和AD等8个基因组。用基因组DNA和rDNA作探针,获得了荧光原位杂交结果,并重点进行了核型分析,取得了较好的结果。现归纳如下:四倍体棉种的GISH,明显区分了两个亚组的染色体、其中一亚组染色体明显大于另外一亚组的,进而证实四倍体棉种的异源二倍体结构、棉属A基因组染色体大于D基因组;按照染色体相对长度由长到短编序时,发现亚组染色体的相对长度有交叉现象,即有的属于较小亚组的染色体比较大亚组的要大,这对于经典核型分析来说是无法回避的难题,说明基于原位杂交的核型分析更加准确可靠。核型基本参数比较和核型重合率聚类分析表明,阿非利加棉与亚洲棉及草棉都有很大的差异,可能有独特的进化途径,在棉属分类上应该给一个“种”的地位。澳洲棉和奈尔逊氏棉与C基因组斯特提棉有很大的差异,但与比克氏棉也有明显的差异,所以不支持将前两个棉种归到后者代表的G基因组或三个种归为一个基因组的观点。本研究用到的4个四倍体棉种,陆地棉与海岛棉亲缘关系最近,黄褐棉与二者及达尔文氏棉的亲缘关系很远。黄褐棉非常特殊,在四倍体棉种进化途径上是一个独立的分枝。进一步支持四倍体棉种两个亚组来源于A和D基因组的推论。四倍体棉种A亚组可能有共同的供体,即阿非利加棉;D亚组的供体分析不十分明朗,但肯定支持D亚组多系起源学说,且认为可能的供体种是雷蒙德氏棉和三裂棉,但雷蒙德氏棉可能不是陆地棉的父本供体。定位了二倍体棉种的45S rDNA,明显提高了对随体的鉴别能力。发现随体或45S rDNA位点在棉种的分布有“主区域”的特点,即集中于某些或某一序号染色体上,比如D基因组以第5、9和13号染色体为中心的3个区域。9个二倍体棉种定位了5S rDNA,其中7个棉种都与45S rDNA在同一染色体上,推测棉属两种rDNA可能有高度的共线性。核型资料显示棉属整体的进化程度不高。亚洲棉与绿顶棉为二倍体棉种进化程度最高与最低的两个典型。本研究还分析讨论了各个基因组的起源与演化的可能途径。更多还原

【Abstract】 It is the most critical technology link in the karyotype analysis to distinguish properly homologous chromosomes in mitotic cells and then obtain the accurate basic data about chromosomes. Non-FISH karyotype analysis is difficult to match accurately homologous chromosomes and often makes mistakes. FISH (fluorescent in situ hybridization) based karyotype analysis breaks through the bottleneck. Clarifying parental donors of polyploid cottons not only resolves the issues of long-term theoretical arguments in Gossypium and but also helps to select the prior cotton species for genome sequencing, which is very important in whole-genome sequencing era. Therefore, taking the advantage of materials in our institute, we carried out a comprehensive karyotype analysis of existing cotton species based on FISH. In this study we collected 21 Gossypium species including A, B, C, D, E, F, G genomes and AD genome except K genome. We used genomic DNA (gDNA) and rDNA as probes and obtained the FISH results. The results were as follows:GISHs of the tetraploid species showed clearly and directly the two subgenomes of chromosomes, and the chromosomes in one subgenome were generally bigger than those in another, which confirmed the theories that tetraploid cottons were disomic nature and chromosomes from A genome in Gossypium were bigger than the D genome chromosomes. We found that some chromosomes in smaller subgenome were bigger than some in larger one in all tetraploid GISHs, which was very difficult for non-FISH karyotype analysis method to properly match those chromosomes between the two subgenomes mainly according to the chromosome relative lengths from the longest to the shortest, which indicated FISH-based karyotype analysis was more accurate and reliable. Comparisons of the basic parameters of karyotype and cluster analysis of karyotype overlapping percentages showed that G. herbaceum var.africanum was rather different from G. arboreum and G. herbaceum. G. herbaceum var.africanum may have a unique evolution pathway and should be given a "species" status in genus Gossypium. G. australe and G. nelsonii were rather different from C genome species G. sturtianum, and also much different from G. bickii. Therefore the restults did not support the the suggestion that G. australe and G. nelsonii were classed into G. bickii involved G genome or included in the any other same genome with G. bickii.The specific relationships among the four tetraploid cottons tested here in this study showed that G. hirsutum was very closer to G. barbadense, whereas these two species including G. darwinii were very distinct from G. mustelinum. And G. mustelinum was very special as it should be an independent branch in the evolution of tetraploid cottons. Data from this study further supported the theory that the two subgenomes of the tetraploid cottons formed from A and D genome species. Probably, A–subgenome of the tetraploid cottons had a common donor, which was G. herbaceum var.africanum. And the detail donor species of D–subgenome for each tetraploid was not very clear, but it was certain that polyphyletic origination theory was supported. Also the possible donors were G. raimondii and G. trilobum. But G. raimondii may not be the donor species of G. hirsutum D-subgenome. The distinguishing ability of identification satellite on chromosome was significantly improved, after locating the 45S rDNAs on the diploid cotton chromosomes. The results showed that the distribution of the satellites and the 45S rDNA loci were characterized with the "cluster region". That is they focused on some or a certain number of chromosomes, such as the D genome species had 3 regions with chromosome 5, 9 and 13 as the region center. Moreover, the 5S rDNAs of 9 diploid cottons were located. The results showed that the loci of 5S and 45S rDNAs from seven species were in the same chromosome, speculating that 45S and 5S rDNAs may have a high degree of collinearity.The results about karyotype analysis showed the overall evolution level in Gossypium was low. G. arboreum and G. captis-viridis were the two typical species of the highest and lowest evolutional level among the diploid cottons, respectively. Moreover, the possible ways of origin and evolution of each genome were analyzed and discussed in the dissertation.更多还原