Influence Factors of in Situ Hybridization in Triticeae

In order to increase the efficiency, accuracy, fidelity and reliability of in situ hybridization to identify the alien chromosomes and chromosome fragments in triticeae, major steps including probe labelling, chromosome denaturation, DNA concentration for blocking and post-hybridization washing in in situ hybridization were optimized. The results are as fel-lows. (1) The cloned repetitive DNA sequence could be biotin labelled more efficiently by nick translation than by random oligonucleotide labelling method: whereas the random oligonucleotide labelling is more suitable for genomic DNA probe and the labelling efficiency could be increased by prolonging the labelling time appropriately. (2) Denaturation of the biotinylated probe and chromosomes together in oven at 75 ℃ showed the satisfactory results of in situ hybridization, but the contour of treated rye chromosomes often became blurred when the temperature of denaturation was higher than 85℃. When 70% formamide (in 2 × SSC) was used to denature the chromosome DNA, rye chromosomes often swelled although the biotinylated signals could be detected. (3) The unlabeled DNA concentrations for blocking were tested in genomic in situ hybridization to detect the Haynaldia villosa chromosomes with biotin labelled H. villosa genomic DNA as probe. The best contrast between H. villosa and wheat chromosomes was obtained without using the blocking DNA (unlabeled wheat genomic DNA). (4) Post-hybridization washes were carried out in 50% formamide (in 2 × SSC) or in 2 × SSC at different temperature. When the post-hybridization washing temperature were increased gradually from room temperature to 42℃ in 50% formamide (in 2 × SSC). specific in situ hybridization signals on chromosome in triticeae were observed using both biotinylated repetitive DNA and genomic DNA as probe. With the improved resolution of this protocol, in situ hybridization would be widely applied to wheat breeding and genetics researches.     

The physical location of fourteen RFLP markers in rice (Oryza sativa L.)

A biotin-labeled in situ hybridization technique was used in order to physically map RFLP markers to the chromosomes of rice (Oryza sativa L.). Fourteen RFLP markers, associated with the ends of the linkage groups on rice chromosomes 7, 8, 11, 12, were physically mapped onto specific regions of the chromosomes. The average detection rate of in situ hybridization was 5.91%. The markers were located on seven different chromosome arms. Ten of the fourteen markers were distributed near the chromosome ends. This demonstrated that the RFLP linkage groups involved covered a wide physical distance and that the centromeric region was bisected by all but one linkage group. Two markers covered a short genetic distance but were physically distant, while two covering a longer genetic distance were physically closer together. This indicates that considerable variation can, and does, exist between genetic and physical maps.This paper is a contribution of the U.S. Department of Agriculture, Agricultural Research Service, and Missouri Agricultural Experiment Station, Journal Series No. 11 882All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicapThis paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the U.S. Department of Agriculture or the University of Missouri     

Genome differentiation in Aegilops. 4. Evolution of the U-genome cluster

Phylogenetic relationships of polyploid Aegilops species sharing the U-genome were investigated by analyzing heterochromatin banding patterns of their somatic metaphase chromosomes as revealed by C-banding and fluorescence in situ hybridization (FISH) with the heterochromatin-limited repetitive DNA probes pSc119, pAs1, as well as the distribution of NOR and 5S DNA loci revealed by pTa71 (18S-26S rDNA), and pTa794 (5S rDNA) probes. Seven tetraploid (Ae. triuncialis, Ae. peregrina, Ae. kotschyi, Ae. geniculata, Ae. biuncialis, Ae. columnaris, and 4x Ae. neglecta) and one hexaploid (6x Ae. neglecta) Aegilops species of the U-genome cluster were studied. The U^t and C^t chromosomes of 4x Ae. triuncialis (U^tC^t) were similar to the diploid donors Ae. umbellulata (U) and Ae. caudata (C). However, the size of the NOR locus on chromosome 5U^t was reduced. Karyotypic analyses confirmed that 4x Ae. peregrina (S^pU^p) was derived from a hybridization of the diploid species Ae. umbellulata with Ae. longissima, whereas Ae. umbellulata and Ae. sharonensis (or an immediate precursor) were the diploid progenitor species of Ae. kotschyi (S^kU^k). In both 4x species, the NORs on S-genome chromosomes were inactivated and were accompanied with a decrease or loss of rDNA sequences. Karyotypes of the tetraploid species, Ae. geniculata (U^gM^g) and Ae. biuncialis (U^bM^b) differed from each other and from the putative diploid progenitors Ae. umbellulata and Ae. comosa indicating that various types of chromosomal alterations occurred during speciation. Inactivation of major NORs on the M-genome chromosomes, redistribution of 5S rDNA sites, and loss of some minor 18S-26S rDNA loci were observed in Ae. geniculata and Ae. biuncialis. Significant differences in the total amount and distribution of heterochromatin, the number and location of 5S and 18S-26S rDNA loci observed between Ae. columnaris (U^cX^c)/4x Ae. neglecta (UⁿXⁿ) and Ae. geniculata/Ae. biuncialis indicate that these species have different origins. Similarities in C-banding and FISH patterns of most Ae. columnaris and 4x Ae. neglecta chromosomes suggest that they were probably derived from a common ancestor, whereas distinct differences of three chromosome pairs may indicate that the divergence of these species was probably associated with chromosomal rearrangements and/or introgressive hybridization. Ae. umbellulata contributed the U genome, however, the source of their second genomes remains unknown. The formation of 6x Ae. neglecta (UⁿXⁿNⁿ) was not associated with large modifications of the parental genomes.     

Evolutionary divergence of human chromosome 9 as revealed by the position of the ABL protooncogene in higher primates

Attempts to solve the fundamental questions regarding the descent of man are dogged by superstitions and unexamined orthodoxies. The origin of humans, established a decade ago based upon cytological analysis of ape chromosomes, continues to be called into question. Although molecular methods have provided a framework for tracing the paths of human evolution, conclusive evidence remains elusive. We have used a single ABL gene probe derived from human chromosome 9 to assess the direction of change in the equivalent ape chromosomes. This approach has resulted in a few surprises which again challenge the prevailing view of early primate evolution based solely on chromosome banding patterns. The ABL protooncogene is present on human chromosome 9 at band q34. Similar DNA sequences presumed to represent an ABL gene, are present on chromosome 11 in chimpanzee (Pan troglodytes) but at a different relative location, indicating that the mechanism of the origin of human chromosome 9 is far more complex than has previously been suggested. Nevertheless, in gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus), the equivalent to human chromosome band 9 q34 is apparently located on chromosome 13 at a putative telomeric position and no discernible differences could be established. Despite the presence of the ABL protooncogene on human equivalent ape chromosomes, molecular systematics will continue to generate enigmas in the evolutionary context until the entire genome is sequenced.     

用染色体原位抑制杂交法研究人和猕猴染色体同源性

本文用生物素标记的人类1号、2号、4号染色体DNA探针进行染色体原位抑制(chromosomal in situ suppression,简称CISS)杂交以研究人和猕猴染色体的同源性。结果表明:人1号染色体与猕猴1号染色体同源。其中与猕猴lpter→lq33的同源程度高,与猕猴lq33→lqter的同源程度相对较低;人2号染色体与猕猴13号染色体长臂、9号染色体长臂和部分短臂同源;人4号染色体与猕猴2号染色体同源。结合染色体带型比较分析,本文对人和猕猴染色体的演化关系进行了探讨,该研究进一步证明了染色体重排可能是灵长类染色体进化的主要机制。     

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside     

Molecular phylogeny of the genus Triticum L

The genus Triticum L. includes the major cereal crop, common or bread wheat (hexaploid Triticum aestivum L.), and other important cultivated species. Here, we conducted a phylogenetic analysis of all known wheat species and the closely related Aegilops species. This analysis was based on chloroplast matK gene comparison along with trnL intron sequences of some species. Polyploid wheat species are successfully divided only into two groups – Emmer (sections Dicoccoides and Triticum) and Timopheevii (section Timopheevii). Results reveal strictly maternal plastid inheritance of synthetic wheat amphiploids included in the study. A concordance of chloroplast origin with the definite nuclear genomes of polyploid species that were inherited at the last hybridization events was found. Our analysis suggests that there were two ancestral representatives of Aegilops speltoides Tausch that participated in the speciation of polyploid wheats with B and G genome in their genome composition. However, G genome species are younger in evolution than ones with B genome. B genome-specific PCR primers were developed for amplification of Acc-1 gene.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

麦类作物原位杂交影响因素的研究

为了更好地利用原位杂交技术进行麦类作物外源染色体的检测和基因定位,对麦类作物原位杂交的影响因素进行了研究。（１）利用缺口转译法对克隆的ＤＮＡ 片段进行生物素标记,即节省时间,又可以获得较高的标记效率;对麦类作物的基因组ＤＮＡ,宜采用随机寡核苷酸引物标记法进行生物素标记,适当延长标记时间可以提高标记效率。（２）共变性法比较适宜麦类作物的原位杂交,分别变性法如掌握不当易使染色体产生膨胀现象,变性温度过高也会使黑麦（Ｓｅｃａｌｅ ｃｅｒｅａｌ）染色体的轮廓模糊不清。（３）应根据麦类作物亲本之间的亲缘关系决定封阻ＤＮＡ 的使用浓度,并利用生物素标记的簇毛麦（Ｈａｙｎａｌｄｉａ ｖｉｌｌｏｓａ）基因组ＤＮＡ 为探针,从普通小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）－簇毛麦双二倍体的染色体中识别了簇毛麦的染色体。（４）麦类作物的原位杂交受洗脱强度的影响很大,利用甲酰胺进行洗脱可以获得背景清晰的原位杂交带型。随着原位杂交技术分辨率的不断提高,该技术还将用于单拷贝基因的染色体定位     

Conservatism of sites of tRNA loci among the linkage groups of severalDrosophila species

Summary The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization.¹²⁵I-labeled tRNA probes fromDrosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from fourDrosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, andDrosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA ₂ ^Arg and tRNA ₂ ^Lys hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution ofDrosophila species.     

Molecular and physical organization of highly repetitive, undermethylated DNA from Pennisetum glaucum

A HaeIIl monomer of a repetitive DNA family from Pennisetum glaucum (L.) R. Br. cv. Massue has been cloned and characterized. The repeat is 137 bp long and is organized in head-to-tail orientation in tandem arrays. The HaeIII monomer contains 55% A+T residues. The distribution of this highly repetitive sequence in different Pennisetum species and in other cereals was investigated. The HaeIII satellite is present in all Pennisetum species investigated but absent from other genera examined. In situ hybridization revealed a centromeric localization of this sequence on all seven chromosome pairs and indicated chromosome-specific differences in copy number. Methylation was investigated by comparative restriction enzyme analysis (Msp/HpaII) which showed a greater extent of methylation of the internal C of the enzyme recognition site 5-CCGG. A South-Western analysis, using an anti-methylcytosine antibody to examine the methylation status in P. glaucum confirmed that the sequence is not highly methylated.     

额外小染色体的分子细胞遗传学研究

本文应用’H标记的7.3kb的rRNA基因探针进行染色体原位杂交技术,并结合多种细胞遗传学技术对一个额外小染色体的家族进行分析研究。在该家族调查的三代人中有8名成员带有相同的额外小染色体,携带者表型均正常。结果证实该额外小染色体是D组或G组染色体的短臂。并就其额外小染色体的起源,遗传效应及生育等问题进行了扼要的讨论。     

Molecular genetics of Rhabdomys pumilio subspecies boundaries: mtDNA phylogeography and karyotypic analysis by fluorescence in situ hybridization

The phylogeography of the African four-striped mouse, Rhabdomys pumilio, was investigated using complete sequences of the mtDNA cytochrome b gene (1140 bp) and a combination of fluorescence in situ hybridization (FISH) and conventional cytogenetic banding techniques (G- and C-banding). Two cytotypes (2n=46 and 2n=48) were identified by cytogenetic analysis. There is no evidence of diploid number variation within populations, difference in gross chromosome morphology or of subtle interchromosomal rearrangements at levels detected by ZOO-FISH. Analysis of the mtDNA cytochrome b resulted in two major lineages that correspond roughly to the xeric and mesic biotic zones of southern Africa. One mtDNA clade comprises specimens with 2n=48 and the other representatives of two cytotypes (2n=48 and 2n=46). The mean sequence divergence (12%, range 8.3–15.6%) separating the two mtDNA clades is comparable to among-species variation within murid genera suggesting their recognition as distinct species, the prior names for which would be R. dilectus and R. pumilio. Low sequence divergences and the diploid number dichotomy within the mesic lineage support the recognition of two subspecies corresponding to R. d. dilectus (2n=46) and R. d. chakae (2n=48). Our data do not support subspecific delimitation within the nominate, R. pumilio. Molecular dating places cladogenesis of the two putative species at less than five million years, a period characterised by extensive climatic oscillations which are thought to have resulted in habitat fragmentation throughout much of the species range.     

Genome analysis of species in the genus Hystrix (Triticeae; Poaceae)

Genomic in situ hybridisation (GISH) and Southern genomic hybridisation were applied in order to gain further knowledge regarding generic delimitation of the genus Hystrix as well as to clarify the genomes of the Hystrix species H. patula, H. longearistata, H. coreana, H. duthiei and H. komarovii. The hybridisation intensity of different genomic probes was compared among the Hystrix species and with other Triticeae species. The Southern- and GISH results confirm that H. patula contains the StH genome and show that H. komarovii most likely has a variant of this StH genome. The other Hystrix species under study, i.e. H. longearistata, H. coreana and H. duthiei, contain an Ns basic genome, and most probably two variants of this basic genome, Ns ¹ Ns ² . The genus Hystrix is thus not a monophyletic group of species.     

原位杂交技术在检测染色体畸变中的研究

实验运用原位杂交技术检测肿瘤组织的染色体畸变。用3、7、8、9、12号染色体着丝粒特异性DNA探针,分别对外周静脉血的淋巴细胞、新鲜实体瘤组织、瘤组织石蜡切片进行染色体检测。二个染色体杂交点代表一对正常的染色体、三个染色体杂交点显示该细胞有染色体三体。整个实验过程简单、快速、准确、易重复。此方法和传统的细胞遗传学方法相结合,对探讨肿瘤的发生、发展有很大的意义。     

植物染色体原位杂交技术及其在稻属研究中的应用

本文介绍了植物染色体原位杂交技术,以及该技术在稻属特定DNA序列定位、基因组间关系、外源染色体鉴定等研究中的应用.