赖草(Leymus secalinus(Georgi)Tzvel.)中一个染色体标记的克隆与鉴定

通过构建Cot-1 DNA文库以及利用荧光原位杂交技术,从赖草(Leymus secalinus(Georgi)Tzvel.)中克隆获得一个在染色体多个部位具有点状杂交信号的重复序列。进一步对该序列在赖草基因组进行克隆和分析表明该序列为一个具有90 bp的重复单元(p Ls-90),并在基因组中呈串联状排列。利用p Ls-90对赖草进行分子核型分析,结果表明:p Ls-90在染色体的着丝粒、近着丝粒、臂间以及近端粒区均有分布,而且在每对染色体上信号分布模式不尽相同,结合染色体臂比可以清楚地对赖草的14对(28条)染色体进行识别。p Ls-90不仅可作为赖草种质鉴定和利用中染色体识别的理想标记,也可作为研究小麦族不同物种染色体组进化的有力工具。     

自身基因组原位杂交揭示植物基因组重复DNA沿染色体的分布

重复DNA沿染色体的分布是认识植物基因组的组织和进化的要素之一。本研究采用一种改良的基因组原位杂交程序,对基因组大小和重复DNA数量不同的6种植物进行了自身基因组原位杂交(self-genomic in situ hybridization,self-GISH)。在所有供试物种的染色体都观察到荧光标记探针DNA的不均匀分布。杂交信号图型在物种间有明显的差异,并与基因组的大小相关。小基因组拟南芥的染色体几乎只有近着丝粒区和核仁组织区被标记。基因组相对较小的水稻、高粱、甘蓝的杂交信号分散分布在染色体的全长,但在近着丝粒区或近端区以及某些异染色质臂的分布明显占优势。大基因组的玉米和大麦的所有染色体都被密集地标记,并在染色体全长显示出强标记区与弱标记或不标记区的交替排列。此外,甘蓝染色体的所有近着丝粒区和核仁组织区、大麦染色体的所有近着丝粒区和某些臂中间区还显示了增强的信号带。大麦增强的信号带带型与其N-带带型一致。水稻自身基因组原位杂交图型与水稻Cot-1DNA在水稻染色体上的荧光原位杂交图型基本一致。研究结果表明,自身基因组原位杂交信号实际上反映了基因组重复DNA序列对染色体的杂交,因而自身基因组原位杂交技术是显示植物基因组中重复DNA聚集区在染色体上的分布以及与重复DNA相关联的染色质分化的有效方法。     

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.     

垂穗披碱草与达乌里披碱草基因组细胞遗传学比较

采用顺序FISH-GISH技术,12个重复序列探针,包括9个三核苷酸简单重复序列、2个卫星DNA重复序列pSc119.2和pAs1以及5S rDNA,通过重复序列的物理定位对达乌里披碱草和垂穗披碱草基因组中部分重复序列的分布特征进行了比较分析,为进一步研究垂穗披碱草和达乌里披碱草的物种形成及演化提供新的分子细胞遗传学证据。结果表明:(1)所有的序列在这2个物种的染色体上都能产生可检测的杂交信号,且在2个物种中(AAC)_(10)、(ACT)_(10)、(CAT)_(10)都表现为共分布,(AAG)_(10)与(AGG)_(10)表现为近似共分布;2个物种的H基因组除5S rDNA序列外,其他序列都产生强烈且丰富的杂交位点,St与Y基因组不同重复序列探针的荧光位点数目有所差别,表现为5S rDNA、pSc119.2、(AAC)_(10)、(CAT)_(10)、(ACT)_(10)、(CAC)_(10)探针的信号位点较少或无信号,其余的探针信号位点稍多。(2)达乌里披碱草的第2对染色体上具有(AAC)_(10)、(CAT)_(10)、(ACT)_(10)的杂交位点、第6对染色体上具有(CAC)_(10)的杂交位点,而在垂穗披碱草的St基因组中未观察到上述序列杂交位点;达乌里披碱草St基因组仅有第4对染色体的端部具有pSc119.2杂交位点,而在垂穗披碱草St基因组中的pSc119.2杂交位点位于第5对染色体长臂的间隔区;相对于达乌里披碱草,垂穗披碱草St和Y基因组染色体含有更多的重复序列杂交位点。(3)达乌里披碱草的H/Y基因组间易位在不同材料间是稳定存在的,达乌里披碱草基因组相对稳定,不同材料间H基因组重复序列杂交信号多态性高于St和Y基因组;垂穗披碱草基因组的变异较大,不同材料间St和Y基因组重复序列杂交信号多态性高于H基因组。研究认为,垂穗披碱草和达乌里披碱草的H基因组均起源于布顿大麦,St基因组可能起源于不同的拟鹅观草属物种;与达乌里披碱草相比垂穗披碱草St与Y基因组可能具有更高的染色体结构变异性,而垂穗披碱草St与Y基因组变异较大的原因可能是与同区域分布的含StY基因组的物种发生了种间渗透杂交。     

胡颓子科叶绿体基因组系统发育分析与演化趋势推断

【目的】为探索胡颓子科叶绿体基因组演化趋势,从而为胡颓子科植物物种鉴定以及资源开发利用提供理论依据。【方法】研究从头组装并注释了沙棘属（Hippophae）和野牛果属（Shepherdia）共4个类群的叶绿体基因组,结合已发表的叶绿体基因组序列,比较了胡颓子科各类群叶绿体基因组的基因构成、重复序列和结构特征,建立了系统发育树,并通过高分化区定位了该科叶绿体基因组的潜在DNA条形码区域。【结果】胡颓子科各属叶绿体基因组在四分体结构、基因数量和排列上高度相似;沙棘属和野牛果属的反向重复区（IR）和整个基因组重复序列数目较胡颓子属有扩张和增加的趋势。基于胡颓子科18个类群的叶绿体全基因组序列的系统发育树中,胡颓子属、沙棘属和野牛果属各自聚为一支,前者先分化出来,沙棘属和野牛果属有最近共同祖先;从长单拷贝区（LSC）和短单拷贝区（SSC）筛选出3个DNA条形码候选区,其中ycf1基因的鉴定效果最佳,基于此构建的各类群系统发育关系与基于全基因组序列的结果一致。【结论】胡颓子科的叶绿体基因组结构保守,但其非编码区序列在各属间存在明显差异,且IR区序列与重复序列在演化过程中分别有扩张和增多的趋势。研究选定的DNA条形码序列能很好区分胡颓子科各属之间以及胡颓子属内物种间关系。     

利用栽培稻C0t-1 DNA和基因组DNA比较分析宽叶野生稻基因组

利用AA染色体组栽培稻的中高度重复序列C0t-1 DNA和基因组DNA作为探针,通过荧光原位杂交技术对宽叶野生稻（Oryza latifolia）（CCDD染色体组）进行了比较基因组分析。结果显示,在宽叶野生稻染色体上,C0t-1 DNA的杂交信号没有基因组DNA的杂交信号明显;杂交信号主要分布在着丝粒、近着丝粒及端粒区域;随着洗脱严谨度的不同,杂交信号呈现出较高的种特异性。本研究以不同洗脱严谨度下的荧光原位杂交结果为依据,对宽叶野生稻进行的核型分析,可进一步提高稻属染色体识别的准确性。     

大赖草及近缘种染色体C—分带的研究

对Ｔｈｉｎｏｐｙｒｕｍｎｂｅｓａｒａｂｉｃｕｍ（Ｓａｖｕｌ．＆Ｒａｙｓ）Ａ．Ｌｖｅ、新麦草（Ｐｓａｔｈｙｒｏｓｔａｃｈｙｓｊｕｎｃｅａ（Ｆｉｓｃｈ．）Ｎｅｖｓｋｉ）和大赖草（Ｌｅｙｍｕｓｒａｃｅｍｏｓｕｓ（Ｌａｍ．）Ｔｚｖｅｌ．）染色体Ｃ分带的核型进行了比较研究。Ｔｈ．ｂｅｓａｒａｂｉｃｕｍ和新麦草的染色体在Ｃ分带带型上有明显的差异,显示了物种的特异性。３个物种的核型表明,Ｃ带带纹主要分布在染色体的末端,大部分染色体不显着丝粒带和中间带。在大赖草染色体上的末端带很明显。一些大赖草的染色体具有与Ｔｈ．ｂｅｓａｒａｂｉｃｕｍ和新麦草某些染色体相似的Ｃ带带型。对大赖草染色体组与Ｔｈ．ｂｅｓａｒａｂｉｃｕｍ和新麦草染色体组的关系进行了讨论。     

黑麂和费氏麂四种卫星DNA的克隆特征和染色体定位(英文）

近年来,分子细胞遗传学研究已基本证实了染色体的串联融合(端粒－着丝粒融合)是麂属动物核型演化的主要重排方式。尽管染色体串联融合的分子机制还不清楚,但通过染色体的非同源重组,着丝粒区域的卫星DNA被认为可能介导了染色体的融合。以前的研究发现在赤麂和小麂染色体的大部分假定的串联融合位点处存在着非随机分布的卫星DNA。然而在麂属的其他物种中,这些卫星DNA的组成以及在基因组中的分布情况尚未被研究。本研究从黑麂和费氏麂基因组中成功地克隆了4种卫星DNA（BMC5、BM700、BM1.1k和FM700）,并分析了这些卫星克隆的特征以及在小麂、黑麂、贡山麂和费氏麂染色体上的定位情况。结果表明,卫星I和II DNA (BMC5, BM700和FM700)的信号除了分布在这些麂属动物染色体的着丝粒区域外,也间隔地分布在这些物种的染色体臂上。其研究结果为黑麂、费氏麂和贡山麂的染色体核型也是从一个2n＝70的共同祖先核型通过一系列的串联融合进化而来的假说提供了直接的证据。     

慈姑基因组大小鉴定及核型分析

【目的】慈姑(Sagittaria sagittifolia L.)是中国重要的园艺经济作物,其基因组大小的鉴定和核型分析对慈姑基因组学研究及分子遗传学研究必不可少,现阶段慈姑的基因组大小、染色体形态的尚不明确,本研究以期为慈姑的育种和物种进化提供更多的细胞学参考。【方法】研究采用流式细胞技术、荧光原位杂交技术,并参考裂解液配方,探针配置,对慈姑品种‘紫金星’（ZJX）的根尖进行基因组大小进行鉴定,并对其染色体形态进行分析观察。【结果】（1）所测慈姑为二倍体植物,‘紫金星’慈姑基因组为16 Gb左右。（2）利用DAPI荧光染色,获得其染色体数目稳定,且与端粒荧光原位杂交的结果完全吻合,均为22条,染色体类型有中部着丝粒,近中部着丝粒以及近端部着丝,近端部着丝粒最多,且未发现随体;核型公式为：2n=2x=22=6m+4sm+12st,属于Stebbins类型中的3B型,核型不对称系数为99.13。【结论】慈姑基因组大小中等,核型极不对称,进化程度较高。     

利用荧光原位杂交技术检测导入普通小麦的大赖草染色质

利用以大赖草基因组ＤＮＡ为探针的荧光原位杂交技术对导入普通小麦的大赖草染色质进行检测。结果：９１）根尖体中花粉母细胞时期均可用于检测大赖草染色质。间期核中杂交信号点数与所含大赖草染色体数目之间的对应关系依染色体显强Ｃ－带末端数不同而不同;（２）利用荧光原位杂交,从普通小麦－大赖草单体异附加系的减数分裂前植株＾６０Ｃｏ－γ射线辐射后代中检测到一批大赖草端着丝粒染色体和小麦－大赖草染色体易位等结构变异     

Variations in abundance of 2 repetitive sequences in Leymus and Psathyrostachys species.

The Ns genome of the genus Psathyrostachys is a component of the polyploid genome in the genus Leymus. Using fluorescence in situ hybridization (FISH), the occurrence and abundance of 2 tandem repetitive sequences from Leymus racemosus (Lam.) Tzvelev, pLrTaiI-1 (TaiI family) and pLrPstI-1 (1 class of 350-bp family), were assayed in 4 species of the genera Psathyrostachys and Leymus. The pLrPstI-1 sequence was absent in all 4 Psathyrostachys species. While P. fragilis and P. huashanica did not have the pLrTaiI-1 sequence, 15 accessions of P. juncea and 2 accessions of P. lanuginosa had pLrTaiI-1 sites ranging in number from 7 to 16 and from 2 to 21, respectively. The numbers of pLrTaiI-1 and pLrPstI-1 sites were 1-24 and 0-30, respectively, in L. ramosus; 2-31 and 5-36 in L. racemosus; 0-4 and 0 in L. mollis; 2-9 and 24-27 in L. secalinus. The FISH assay on pLrTaiI-1 was successfully converted to a sequence-tagged-site polymerase chain reaction (STS-PCR) test using a primer pair designed from the sequence of this repetitive DNA. Seventy-three accessions representing 27 Leymus species were assayed for the abundance of pLrTaiI-1 by STS-PCR. With a few exceptions of uniformity in some accessions, nearly all Leymus species observed were heterogeneous for the abundance of pLrTaiI-1 sequence and no Leymus species was totally devoid of this repetitive sequence. These findings may have significance for the understanding of phylogeny, nature of polyploidy, adaptive ranges, and breeding potential of Leymus species.     

Highly repetitive sequences in B chromosomes of Secale cereale revealed by fluorescence in situ hybridization.

An analysis of the presence and distribution of the rye and wheat repeated sequences in rye B chromosomes was carried out by fluorescent in situ hybridization. Probes used consisted of three highly repetitive sequences from rye (pSc119.2, pSc74, and pSc34) and the multigene families for the 25S-5.8S-18S and 5S rDNA from wheat (pTa71 and pTa794, respectively). pSc74 and pSc119.2 showed hybridization signals in the telomeric regions of rye B chromosomes. The remaining DNA clones did not hybridize to the B chromosomes.     

Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis

Hybrids derived from wheat (Triticum aestivum L.) × rye (Secale cereale L.) have been widely studied because of their important roles in wheat cultivar improvement. Repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 are usually used as probes in fluorescence in situ hybridization (FISH) analysis of wheat, rye, and hybrids derived from wheat × rye. Usually, some of these repetitive sequences for FISH analysis were needed to be amplified from a bacterial plasmid, extracted from bacterial cells, and labeled by nick translation. Therefore, the conventional procedure of probe preparation using these repetitive sequences is time-consuming and labor-intensive. In this study, some appropriate oligonucleotide probes have been developed which can replace the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 in FISH analysis of wheat, rye, and hybrids derived from wheat × rye. These oligonucleotides can be synthesized easily and cheaply. Therefore, FISH analysis of wheat and hybrids derived from wheat × rye using these oligonucleotide probes becomes easier and more economical.     

Molecular cytogenetics of Leymus: Mapping the Ns genome-specific repetitive sequences

The objective of this paper is to summarize the work in my group on FISH (fluorescent in situ hybridization) mapping of Ns-specific repetitive DNA sequences fromLeymus and discuss the results in the context of classification based on the genome system currently used among Triticeae researchers. The key question here is whether the genome composition of a tetraploid Leymus species should be NsXm or NsNs (Ns₁Ns₂). Different types of Leymus-specific dispersed retroelement-like repeats have been isolated and characterized. Because the sequences occur in significantly high copy number in Leymus, based on strong hybridization signal in Southern blots, they are considered essentially specific to Leymus. They are also abundant in Psathyrostachys, the progenitor of Ns genome in Leymus. These dispersed repeats are found to distribute over the whole of all Leymus chromosomes, without any differentiation between chromosomes that have been suggested to be of different genomic origins, meaning that all genomes in Leymus are the same. GISH (genomic in situ hybridization) experiments on Leymus chromosomes using Psathyrostachys genomic DNA as probes further support the NsNs (Ns₁Ns₂) genome constitution for Leymus. The Xm genome of an unknown origin might have been there in the beginning of the allopolyploidization process, but the Ns genome-specific elements must have spread predominantly and rapidly across genomes, thus homogenizing the nuclear genomes of Leymus. I present here for the first time evidence that Ns-specific dispersed repeats can spread in a very short time, from Leymus over to wheat in Triticum × Leymus hybrids growing in artificial conditions.     

Variation in highly repetitive DNA composition of heterochromatin in rye studied by fluorescence in situ hybridization.

The molecular characterization of heterochromatin in six lines of rye has been performed using fluorescence in situ hybridization (FISH). The highly repetitive rye DNA sequences pSc 119.2, pSc74, and pSc34, and the probes pTa71 and pSc794 containing the 25S-5.8S-18S rDNA (NOR) and the 5S rDNA multigene families, respectively, were used. This allowed the individual identification of all seven rye chromosomes and most chromosome arms in all lines. All varieties showed similar but not identical patterns. A standard in situ hybridization map was constructed following the nomenclature system recommended for C-bands. All FISH sites observed appeared to correspond well with C-band locations, but not all C-banding sites coincided with hybridization sites of the repetitive DNA probes used. Quantitative and qualitative differences between different varieties were found for in situ hybridization response at corresponding sites. Variation between plants and even between homologous chromosomes of the same plant was found in open-pollinated lines. In inbred lines, the in situ pattern of the homologues was practically identical and no variation between plants was detected. The observed quantitative and qualitative differences are consistent with a corresponding variation for C-bands detected both within and between cultivars.     

Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21

We have isolated and characterized two human middle repetitive alphoid DNA fragments, L1.26 and L1.84, which localize to two different sets of chromosomes. In situ hybridization revealed both repeats to have major and minor binding sites on the pericentric regions of several chromosomes. Probe L1.26 maps predominantly to chromosomes 13 and 21. Probe L1.84 locates to chromosome 18. Minor hybridization sites for both probes include chromosomes 2, 8, 9, and 20; in addition, L1.26 revealed minor sites on chromosomes 18 and 22. The binding to these sites strongly depends on hybridization conditions. In Southern blot hybridizations to total human DNA, both L1.26 and L1.84 give the same ladder pattern, with a step size of 170 bp, indicating their presence as tandem repeats, but with different band intensities for each probe. The chromosome-specific nature of particular multimers was confirmed by Southern blot analyses of a human-rodent hybrid cell panel. We conclude that L1.26 and L1.84, with their related sequences, constitute subfamilies of alphoid DNA that are specific for subsets of chromosomes and, in some cases, possibly even for single chromosomes.     

The cotton centromere contains a Ty3-gypsy-like LTR retroelement

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species.     

Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography

The vast majority of probes used in fluorescence in situ hybridization (FISH) contain repetitive DNA. This DNA is usually competed out of a hybridization reaction by the addition of an unlabeled blocking agent, Cot-1 DNA. We have successfully removed repetitive DNA from two complex FISH probe sets: a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) single human chromosome library and genomic DNA. The procedure involved hybridizing in solution a DOP-PCR-amplifiable probe set with a 50-fold excess of biotin-labeled Cot-1 DNA, and capturing the Cot-1 DNA-containing hybrids using streptavidin magnetic particles, followed by purification and reamplification of the unbound fraction. Probes were checked for depletion of repeats by hybridization to chromosomes without Cot-1 DNA. Results showed hybridization patterns comparable to those achieved with untreated probes hybridized with Cot-1 DNA. Received: 21 January 1997 / Accepted: 2 April 1997     

Genus-specific localization of the TaiI family of tandem-repetitive sequences in either the centromeric or subtelomeric regions in Triticeae species (Poaceae) and its evolution in wheat.

The TaiI family sequences are classified as tandem repetitive DNA sequences present in the genome of tribe Triticeae, and are localized in the centromeric regions of common wheat, but in the subtelomeric heterochromatic regions of Leymus racemosus and related species. In this study, we investigated the chromosomal distribution of TaiI family sequences in other Triticeae species. The results demonstrated a centromeric localization in genera Triticum and Aegilops and subtelomeric localization in other genera, thus showing a genus-dependent localization of TaiI family sequences in one or the other region. The copy numbers of TaiI family sequences in species in the same genus varied greatly, whether in the centromeric or subtelomeric regions (depending on genus). We also examined the evolution of TaiI family sequences during polyploidization of hexaploid common wheat. A comparison of chromosomal locations of the major TaiI family signals in common wheat and in its ancestral species suggested that the centromeric TaiI family sequences in common wheat were inherited from its ancestors with little modification, whereas a mixed origin for the B genome of common wheat was indicated.