拟南芥DNA探针的比较基因组原位杂交揭示的拟南芥与远缘植物基因组间的同源性

采用生物素标记的拟南芥基因组DNA探针在75%杂交严谨度下对双子叶植物番茄、蚕豆和单子叶植物水稻、玉米、大麦的染色体进行了比较基因组荧光原位杂交（comparative genomic in situ hybridization,cGISH）分析,以揭示拟南芥与远缘植物基因组间的同源性.cGISH信号代表了拟南芥基因组DNA中的重复DNA与靶物种染色体上同源序列的杂交.探针DNA在所有靶物种的全部染色体上都产生了杂交信号.杂交信号为散在分布,并呈现随基因组增大,杂交信号增多,且分布更加分散的趋势.所有靶物种的核仁组织区（NOR）都显示了明显强于其他区域的杂交信号,表明拟南芥基因组DNA探针可用于植物NOR的物理定位.在所有的靶物种中,信号主要分布在染色体的臂中间区和末端,着丝粒或近着丝粒区有少数信号分布.大麦染色体显示了与C-和N-带不同的独特的cGISH信号带型,表明此探针可用于不同植物染色体的识别.这些结果表明,拟南芥基因组与远缘植物基因组之间,除rDNA和端粒重复序列外,还存在其它同源的重复DNA;一些重复DNA序列在被子植物分歧进化为单子叶和双子叶植物之前就已存在,虽经历了长期的进化过程,至今在远缘物种之间仍保持了较高的同源性.结果还提示,大基因组中古老而保守的重复DNA在进化过程中发生了明显的扩增.     

基因组原位杂交鉴定玉米的外源染色体片段

利用基因组原位杂交技术,在稳定遗传的纯系５４０及其与玉米（ＺｅａｍａｙｓＬ．）自交系杂交后代Ｆ１即遗单６号中,成功地鉴定了渗入其中的二倍体多年生类玉米（ＺｅａｄｉｐｌｏｐｅｒｅｎｎｉｓＤｏｅｂｌｅｙ,ＤＰ）的染色体片段,采用ＤＡＢ（Ｄｉａｍｉｎｏｂｅｎｚｉｄｉｎｅｔｅｔｒａｈｙｄｒｏｃｈｌｏｒｉｄｅ）和荧光检出系统,二者获得了一致的结果,在纯系５４０的第１,２,５和８号两条同源染色体长臂上均检出杂     

普通小麦三个基因组之间的遗传关系及原位杂交分析

以普通小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍＬ．）的３个可能的二倍体供体种（乌拉尔图小麦（Ｔ．ｕｒａｒｔｕＴｈｕｍ．）拟斯卑尔脱山羊草（ＡｅｇｉｌｏｐｓｓｐｅｌｔｏｉｄｅｓＴａｕｓｃｈ）和粗山羊草（Ａｅ．ｔａｕｓｃｈｉｉＣｏｓｓ．）的基因组ＤＮＡ为研究对象,通过它们之间的相互杂交,比较杂交强度以及泳道中带纹的不同,并结合部分ＤＮＡ重复序列在基因组间含量差异的数据,得出结论：Ａ＾ｕ和Ｄ基因组的关系     

基因组中的重复DNA序列

综述了基因组中常见的重复DNA序列,介绍了其可能的产生机理、分布情况和生物学功能。     

双探针原位杂交揭示稻属BB,CC和EE基因组之间的分化

利用双探针原位要交技术,揭示了稻属Ｏｒｙｚａ ｏｆｆｉｃｉｎａｌｉｓ复合体中ＢＢ、ＣＣ和ＥＥ基因组之间的分化。以标记的ＢＢ基因组（来自二倍体的Ｏ．ｐｕｎｃｔａｔａＫｏｔｅｃｈｙ ｅｘ Ｓｔｅｕｄ．）的总ＤＮＡ为探针,同ＢＢＣＣ基因组（Ｏ．ｍｉｎｕｔａＪ．Ｓ．Ｐｒｅｓｌ．ｅｔ．Ｃ．Ｂ．Ｐｒｅｓｅｌ）的中期染色体杂交,结果表明,ＢＢ基因组的ＤＮＡ探针同与四倍体Ｏ．ｍｉｎｕｔａ中的ＢＢ基因组的染色体之间     

在植物粗线期染色体和DNA纤维上的荧光原位杂交技术

介绍了两种荧光原位杂交技术的详细实验步骤。第一种技术是在减数分裂粗线期染色体上的荧光原位杂交,包括从花粉母细胞中制备粗线期染色体和在这种染色体上定位ＤＮＡ序列,其分辨率水平能够达到１００ｋｂ。第二种技术是从植物细胞核中制备ＤＮＡ纤维,并在上面进行原位杂交,能够直接分析ＤＮＡ序列的分子排列关系,其分辨率水平能达到几个ｋｂ。为了说明这两种原位杂交技术在研究基因组和染色体结构、构建高分辨率的ＤＮＡ物理图谱上的能力,将展示用该技术直接分析番茄染色体端粒重复序列和端粒联接重复序列的染色体定位和ＤＮＡ分子排列。     

松科植物基因组总DNA提取方法的比较

目的:研究对比了用不同方法提取红松、樟子松和红皮云杉等3种松科植物叶片基因组DNA的效果,以寻找适合提取松科植物叶片基因组DNA的方法。方法:分别比较了用传统的CTAB法、SDS法以及3种改良的CTAB法提取的上述3种松科植物叶片基因组DNA的电泳、纯度和酶切效果。结果:采用不同方法提取的3种松科植物叶片基因组DNA的质量差异较大。结论:常规CTAB法和SDS法无法提取出高质量松科植物基因组DNA,而改良后的CTAB法的提取效果较好。     

基因组原位杂交鉴定牛筋草AA基因组在穇子染色体上的分布及探针长度优化方法

采用基因组原位杂交(Genomic in situ hybridization,GISH)方法研究了牛筋草(Eleusine indica)AA基因组在穇子(E.coracana)染色体上的分布,并探讨了AA、BB基因组的同源关系。用超声波破碎法进行预剪切,以缺口平移法标记的牛筋草总DNA为探针,BB基因组的E.floccifolia(Forssk.)Spreng.总DNA为封阻,与AABB基因组穇子的中期染色体进行杂交。结果表明,牛筋草AA基因组分布在穇子的18条染色体上。不加封阻或加过量封阻均不能鉴别AA基因组,说明AA和BB基因组间的分化程度不大,双方共享的重复序列较多。牛筋草与E.floccifolia总DNA分别用超声波破碎2 min和3 min后,可得到峰值为300-750 bp的DNA片段,这说明不同物种的超声波破碎时间需要调整,以获得合适长度的探针。     

小麦族植物DNA重复序列研究

近些年来从小麦族植物中分离到了许多DNA重复序列,并对其组织结构特点,物种分布特异性和在染色体上的分布特征做了广泛的研究,其中一些重复序列已被成功地用于检测遐入小麦的外源染色质和小麦族有关种属的进化研究,本文就以上诸方面进行了简要介绍。     

植物荧光原位杂交技术的发展及其在植物基因组分析中的应用

荧光原位杂交(FISH)是在染色体、间期核和DNA纤维上定位特定DNA序列的一种有效而精确的分子细胞遗传学方法。20年来,植物荧光原位杂交技术发展迅速:以增加检测的靶位数为目的,发展了双色FISH、多色FISH和多探针FISH鸡尾酒技术;为增加很小染色体目标的检测灵敏度,发展了BAC-FISH和酪胺信号放大FISH(TSA-FISH)等技术;以提高相邻杂交信号的空间分辨力为主要目的,发展了高分辨的粗线期染色体FISH、间期核FISH、DNA纤维FISH和超伸展的流式分拣植物染色体FISH技术。在植物基因组分析中,FISH技术发挥了不可替代的重要作用,它可用于:物理定位DNA序列,并为染色体的识别提供有效的标记;对相同DNA序列进行比较物理定位,探讨植物基因组的进化;构建植物基因组的物理图谱;揭示特定染色体区域的DNA分子组织;分析间期核中染色质的组织和细胞周期中染色体的动态变化;鉴定植物转基因。     

Genetic Relationship and Genomic in situ Hybridization Analysis of the Three Genomes in Triticum aestivum

Common wheat ( Triticum　aestivum L.) is an allohexaploid, consisting of three different genomes (Au, B and D ) which are genetically closely related. Genomic DNA of the three possible genome donors, T. urartu Thum., Aegilops speltoides Tausch and Ae. tauschii Coss.,were employed as probes to hybridize with the diploid genomic DNA digested by Eco RⅠand Hin dⅢ respectively. Both the hybridization strength and band patterns among the genomes would be good indicators of genome relationships. Combining distr ibution data of some repetitive DNA sequences cloned from T. urartu in the three genomes, the authors draw a conclusion that Au and D are more closely related to each other than either one to the B genome. Genomic in situ hybridization (GISH) of T. aestivum cv. Chinese Spring with genomic DNA probes of the three diploid progenitors respectively indicated that the three genomes could be discriminated clearly via GISH. The signals on the chromosomes of Au and D genomes were even. However, when Ae. speltoides DNA was used as probe, there were very strong cross hybridization and the signals condensed on some areas of the metaphasic chromosomes. In the interphase nucleus, the chromatin of B genome dispersed on the same region and the signals on the homologous chromosomes distributed symmetrically. Rich repetitive DNA sequences in B genome, especially the tandem repetitives, perhaps take an important role for the formation of the special hybridization pattern. The main difference between B and the other two genomes probably is in the repetitive DNA sequences.     

Genetic Relationships Among Five Basic Genomes St,E,A,B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

The St and E are two important basic genomes in the perennial tribe Triticeae (Poaceae). They exist in many perennialspecies and are very closely related to the A, B and D genomes of bread wheat (Triticum aestivum L.). Genomic Southernhybridization and genomic in situ hybridization (GISH) were used to analyze the genomic relationships between the twogenomes (St and E) and the three basic genomes (A, B and D) of T. aestivum. The semi-quantitative analysis of the Southernhybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, andrelatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion.St and E are the two basic genomes of Thinopyrum ponticum (StStE~eE~bE~x) and Th. intermedium (StE~eE~b), two perennialspecies successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of thespontaneous wheat-Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genomeand rarely in the B genome. This would develop further use of alien species for wheat improvement, especially thosecontaining St or E in their genome components.     

Genetic Relationships Among Five Basic Genomes St, E, A, B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

The St and E are two important basic genomes in the perennial tribe Triticeae （Poaceae）. They exist in many perennial species and are very closely related to the A, B and D genomes of bread wheat （Triticum aestivum L.）. Genomic Southern hybridization and genomic in situ hybridization （GISH） were used to analyze the genomic relationships between the two genomes （St and E） and the three basic genomes （A, B and D） of T. aestivum. The semi-quantitative analysis of the Southern hybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, and relatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion. St and E are the two basic genomes of Thinopyrum ponticum （StStE^eE^bE^x） and Th. intermedium （StE^eE^b）, two perennial species successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of the spontaneous wheat-Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genome and rarely in the B genome. This would develop further use of alien species for wheat improvement, especially those containing St or E in their genome components.     

Detection of Differentiation Among BB,CC and EE Genomes in the Genus Oryza by Two-probe Genomic in situ Hybridization (GISH)

双探针原位杂交揭示稻属BB、cc 和EE基因组之间的分化 李常宝1 张大明1* 葛颂1 卢宝荣2 洪德元1     

Chromosomal domains of chimpanzee are diverged from human as revealed by in situ hybridization using human genomic probe

Utilization of repetitive DNA probes to assess the taxonomic affinity between related species has become the most powerful tool in evolutionary biology today. Consequently, tremendous strides have recently been made towards establishing the phylogenetic relationship of humans with chimpanzee. We employed human genomic proe (P5080 B.5) to identify the degree of divergence of chimpanzee genome from humans. A small protion of structurally distinct genomic areas in chimpanzee could be identified by fluorescencein situ hybridization (FISH) technique when compared to human DNA. The genomic divergence is confined mainly to the chromosomal ends in chimpanzee and may be an important phylogenetic characteristic in human evolution.     

应用荧光原位杂交技术检测人类APPSWE基因在转基因小鼠染色体上的定位及位置效应

应用荧光原位杂交技术研究了人类淀粉样前体蛋白基因瑞典型突变（APPSWE）在转基因小鼠首建、F1及F2代小鼠染色体上的整合及定位,结果在2只首建转基因小鼠中,分别观察80个分裂相,出现杂交信号的核型分别为34及36个,检出率为42.5%和45%;1只F1及1只F2代转基因小鼠中,分别观察100个分裂相,出现杂交信号的核型分别为33及30个,检出率为33%和30%。转基因分别整合在8号、1号、17号和2号染色体上,提示转基因APPSWE已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给予子代,证实转基因在小鼠染色上的整合可能是随机的多点整合,同时,对不同整合位点的转基因小鼠进行了表型研究,结果发现不同整合位点对表型具明显影响。     

Macromolecular organization and genetic mapping of a rapidly evolving chromosome-specific tandem repeat family (B77) in cotton (Gossypium)

Isolation and characterization of the most prominent repetitive element families in the genome of tetraploid cotton (Gossypium barbadense L; [39]) revealed a small subset of families that showed very different properties in tetraploids than in their diploid progenitors, separated by 1-2 million years. One element, B77, was characterized in detail, and compared to the well-conserved 5S and 45S rRNA genes. The 572 bp B77 repeat was found to be concentrated in several discontinuous tandem arrays confined to a single 550 kb SalI fragment in tetraploid cotton. Genetic mapping based on the absence of the pentameric rung in the G. barbadense ladder showed that B77 maps to a D-subgenome chromosome. In situ hybridization supports the contention that the array is confined largely to a single chromosomal site in the D-subgenome. The B77 repeat has undergone a substantial increase in copy number since formation of tetraploid cotton from its diploid relatives. RFLPs observed among tetraploid cotton species suggest that amplification and/or rearrangement of the repeat may have continued after divergence of the five tetraploid cotton species. B77 contains many short direct repeats and shares significant DNA sequence homology with a Nicotiana alata retrotransposon Tna1-2 integrase motif. The recent amplification of B77 on linkage group D04 suggests that the D-subgenome of tetraploid cotton may be subject to different evolutionary constraints than the D-genome diploid chromosomes, which exhibit few genome-specific elements. Further, the abundance of B77 in G. gossypioides supports independent evidence that it may be the closest extant relative of the D-genome ancestor of cotton.     

植物染色体原位杂交技术的发展与现状

本文主要介绍植物染色体原位杂交技术的发展历史,评述适用于不同研究目的的各种主要原位杂交技术的基本原理和方法,并介绍该技术在植物细胞遗传学领域的应用和发展。 Abstract：In this paper,we briefly introduce the development of in situ hybridization of plant chromosome. The fundamental principle and method of many main kinds of in situ hybridization have been reviewed for different research purposes,and their application and development on plant cytogenetics also been recommended.