【作者】 周建民；

【导师】 顾铭洪；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2004， 硕士

【摘要】 稻属（Oryza L．）是禾本科中重要的植物类群，该属含有两个栽培种及20余个野生种，分布广泛，形态多样。对于水稻的研究有着悠久的历史，现在水稻已成为研究单子叶植物的模式植物。水稻是如何起源、演化、发展的?这是贯穿整个水稻研究的重要命题。在较早的时候，人们主要是根据水稻的出土遣存、史书记载和地理生态型的差异，来研究水稻的起源演化问题。但直到现在，人们对水稻起源演化的认识还是非常有限的，并且各有其说，观点不一。 随着水稻重复序列研究的突破，尤其是水稻全基因组测序的完成，人们发现许多重复序列在水稻种间（亚种间）的分布存在明显的差异（Mawal，1995；Ohmido，2000；Qi Feng et al，2002）。利用FISH研究发现，一些串联重复序列在水稻中呈现特异性的分布，并且在典型的籼粳品种之间表现出明显的差异。本实验以中籼南京11(N₁₁)、粳稻巴利拉（Balilla）以及它们的杂交F₁代的DH群体为研究材料，将串联重复序列Os48、45S rDNA、RCS₂和5S rDNA标记成探针，利用荧光原位杂交技术（FISH）结合程氏指数分类，分析它们在籼粳杂交后代的分布，及与籼粳分化之间的关系。本研究表明，以上重复序列的特异分布与栽培稻籼粳型的分化有密切关系。主要结果有： 1．以N₁₁与Balilla杂交构建的DH群体有明显的偏籼分离现象。程氏指数的六个性状在后代分离中有明显的差异，其中稃毛和酚反应的分化与籼粳的实际分化关系最密切，颖壳色和穗轴长次之，而籽粒长宽比和叶毛与籼粳分化的关系不明显。扬州大学硕士学位论文 2，将串联重复序列455 rDNA标记成探针，对45个DH株系的中期染色体（包括程氏指数为19²4的株系）上进行的FISH分析表明，显示的杂交信号对数与釉梗分化有一定联系表现，在指数积分为0一18的粕型或偏釉型DH株系中，杂交信号显示为2对的株系居多，且这两对信号的强弱差异不大;在指数为19²4的粳型或偏粳型DH株系中，有4个系的杂交信号为1对，5个系的杂交信号为2对，但显示2对信号的强弱差异非常明显。 3.利用串联重复序列0548作为探针，对134个DH株系进行杂交分析，结果表明，随着程氏指数积分的减少，0548的杂交信号对数有增加的趋势，显示有2对杂交信号的DH株系只出现在程氏指数积分为23²4的梗型株中。但是大部分株系的杂交信号表现为两亲本间的中间类型。 4.利用水稻减数分裂粗线期染色体进行分析，发现在Nll染色体的ZL，SL，SL，gL，10L，11L和12L末端显示有0548的杂交信号;在Balina染色体的SL和6L末端出现了0548的杂交信号。中期（或前中期）染色体上显示2对杂交信号的6个DH株系的杂交信号都在第5号和第6号染色体的长臂末端上，说明这两条染色体上的0548与粳型分化有密切的关系。 5.在细线期染色体上用55 rDNA和RCS:作为探针进行FISH分析，发现在亲本N，l和Balilla之间有明显的差异，在N，;第11号染色体着丝粒近短臂处有2个55 rDNA串联重复，而在亲本BaliUa中只有一个55 rDNA重复位点。在含有2对0548杂交信号的粳型DH株系中，有4个株系表现出和亲本Balina一样的杂交信号，但是另2个株系的杂交信号却和FI的杂交结果一样，详细的原因还有待进一步研究。更多还原

【Abstract】 The genus Oryza L., besides two cultivated species (O. saliva L. and O. glaberrima Sted.), has 20 more wild species, distributed through most of the tropic and sub-tropic and differing evidently in growth traits between species and subspecies. Rice has been studied for long times. Now, it has become one of mode plants in studying monocotyledonous plants. The origin and evolution of cultivated rice is a radical problem, which has been studied, from the beginning, based on rice archeological discoveries, contrived circulation, history records, geographic ecotype differences and so on. However, up to now, the knowledge of rice is still very limited, and the opinions are as more as the scholars.With the breathtaking developments in rice repetitive DNA sequences research, especially with the completion of rice genomic projects, the distribution and copy number of repeated sequences in rice have been studied. Recently, some researchers found that some tandemly repeated sequences were distributed at specific defined chromosomal locations rather than randomly at many chromosomal locations, what’s more, visibly different between typical indica and japonica cultivars, using the technique of fluorescence in situ DNA hybridization (FISH). In our studies, the tandem repeats, Os48, 45S rDNA, 5S rDNA and RCS2, have been studied to reveal therelationships between the distributing differences of them and the differentiation of cultivated rice into indica and japonica. Based on the foundations of our studies and the reports of other scholars, we presumed that these tandem repeats played some important roles during the period of genetic differentiation of cultivated rice, and they could be used as molecular markers in studying rice evolution. The main results of our studies are as follows:1. The DH groups, derived from the FI individuals of a cross between NH (subsp. indica) and balilla (subsp. Japonica) , was obviously partial to indica in growth traits, utilizing Cheng’s systematic index. The six characters in Cheng’s index, however, embodied inconsistent distribution among DH groups, when they were analyzed respectively. This result indicated that there would be some errors in classification, when the six characters were applied averagely.2. The 45 S rDNA repeat sequence was tagged and hybridized to metaphase chromosomes of 45 DH lines, involving 9 DH lines whose scores of Cheng’s index were ranged from 19 to 24 (japonica lines) . The results revealed that, on the chromosomes of indica or partial indica lines whose Cheng’s index score were 0-18, more than two of thirds lines appeared two pairs of hybridization signals, whose sensitivity were close to each other. While, in japonica DH lines, we observed one pair of signals in four lines and two of that’s in five lines, and the sensitivity of the two signals differed distinctly.3. The hybridization of Os48 on metaphase chromosomes disclosed that the number of hybridization signals increased continuously with the gradual changes of DH groups from japonica to indica, and the more the signals appeared, the more the DH lines assembled. On the other hand, looked into the analyzing data, the signals were distributed normally among DH groups, differing from the classifying results by using Cheng’s index. The paradox perhaps is ascribed to the differential distribution of Os48 and the errors in classification using Cheng’s index.4. To obtain more realization about the distributing rules of Os48 in DH lines, we used pachytene chromosomes to do FISH analysis. The results of this experiment showed that the signals of Os48 were located at the euchromatic ends of long arms of chromosomes 2, 5, 8, 9, 10, 11 and 12 of indica variety NH, while, in balilla, the signals were detected at the long arm ends of chromosomes 5 and 6. When it comes to the six DH lines, which has two pairs of Os48 hybridization signals on metaphase chromosomes, the results were same to balilla.5. Other two repetitive DNA sequences, 5S rDNA and RCS2, were also studied on leptotene c更多还原