Molecular cytogenetic analysis of Aegilops cylindrica host.

The genomic constitution of Aegilops cylindrica Host (2n = 4x = 28, DcDcCcCc) was analyzed by C-banding, genomic in situ hybridization (GISH), and fluorescence in situ hybridization (FISH) using the DNA clones pSc119, pAs1, pTa71, and pTA794. The C-banding patterns of the Dc- and Cc-genome chromosomes of Ae. cylindrica are similar to those of D-and C-genome chromosomes of the diploid progenitor species Ae. tauschii Coss. and Ae. caudata L., respectively. These similarities permitted the genome allocation and identification of the homoeologous relationships of the Ae. cylindrica chromosomes. FISH analysis detected one major 18S-5.8S-25S rDNA locus in the short arm of chromosome 1Cc. Minor 18S-5.8S-25S rDNA loci were mapped in the short arms of 5Dc and 5Cc. 5S rDNA loci were identified in the short arm of chromosomes 1Cc, 5Dc, 5Cc, and 1Dc. GISH analysis detected intergenomic translocation in three of the five Ae. cylindrica accessions. The breakpoints in all translocations were non-centromeric with similar-sized segment exchanges.     

Molecular Cytogenetics ofMusaSpecies, Cultivars and Hybrids: Location of 18S-5.8S-25S and 5S rDNA and Telomere-like Sequences

The physical sites of 18S-5.8S-25S and 5S rRNA genes and telomericsequences in theMusaL. genome were localized by fluorescentinsituhybridization on mitotic chromosomes of selected lines.A single major intercalary site of the 18S-5.8S-25S rDNA wasobserved on the short arm of the nucleolar organizing chromosomein each genome. AA and BB genome diploids had a single pairof sites, triploids had three sites while a tetraploid hybridhad four sites. The probe is useful for quick determinationof ploidy, even using interphase nuclei from slowly growingtissue culture material. Variation in the intensity of signalswas observed among heterogeneousMusalines indicating variationin the number of copies of the 18S-5.8S-25S rRNA genes. Eightsubterminal sites of 5S rDNA were observed in Calcutta 4 (AA)while Butohan 2 (BB) had six sites; some were weaker in bothgenotypes. Triploid lines showed six to nine major sites of5S rDNA of widely varying intensity and near the limit of detection.The diploid hybrids had five to nine sites of 5S rDNA whilethe tetraploid hybrid had 11 sites. The telomeric sequence wasdetected as pairs of dots at the ends of all the chromosomesanalysed but no intercalary sequences were seen. The molecularcytogenetic studies ofMusausing repetitive and single copy DNAprobes should yield insight into the genome and its evolutionand provide data forMusabreeders, as well as generating geneticmarkers inMusa.Copyright 1998 Annals of Botany Company Genome evolution, nucleolar organizing regions, telomeres,in situhybridization, genetic markers, banana, plantain.     

A single species,two basic chromosomal numbers: case of Lygeum spartum (Poaceae)

Abstract

The existence of two chromosome numbers (2n?=?16 and 2n?=?40) in Lygeum spartum is confirmed in the Algerian steppe populations of Oran region. Chromosome counts were established for 11 Algerian populations and three supplementary populations from Italy, Spain and Greece. The karyotypes were characterized by chromosome markers obtained using fluorescence in situ hybridization and fluorochrome bandings. The organization of 5S and 18S-5.8S-26S (35S) rDNA was studied in both cytotypes. Six signals of 35S and 2 signals of 5S were observed in 2n?=?16 population, while 10 signals of 35S and 4 signals of 5S were detected in the population with 2n?=?40. All 35S loci were also strongly marked by chromomycin, but negatively stained by Hoechst, which indicates the presence of GC rich DNA in rDNA regions. The B chromosomes were found in both cytotypes, bearing a 35S locus in 2n?=?16 population. Genome size, determined by cytometry of 10 populations, ranged from 9.27?pg for 2n?=?16 to 26.63?pg for 2n?=?40 populations. The sequencing of plastid and nuclear DNA markers did not reveal major differences among 2n?=?16 and 2n?=?40 populations. However, given the differences between two cytotypes and based on their morphological and cytogenetic characteristics, the 2n?=?16 cytotype merits novel taxonomic treatment.     

Physical mapping of rDNA sequences in four karyotypes of Ranunculus silerifolius (Ranunculaceae)

The chromosomal locations of the 18S-5.8S-26S rDNA and 5S rDNA sequences were examined in four cytotypes of Ranunculus silerifolius (the Matsuyama, Mugi, Otaru, and Karatsu types) using fluorescence in situ hybridization (FISH). Using the 18S-5.8S-26S rDNA probe, one pair of probe hybridization sites was detected by FISH in the interstitial region corresponding to the secondary constriction on the short arm of a satellite chromosome (chromosome pair 6) in all four karyotypes. FISH using 5S rDNA identified one pair of sites. The 5S rDNA locus was on different chromosomes in the four karyotypes: in the interstitial region of the short arm of the largest metacentric chromosome (chromosome pair 1) in the Matsuyama type, in the interstitial region of the short arm of the subtelocentric chromosome (pair 2) in the Mugi and Otaru types, and in the interstitial region of the short arm of the metacentric chromosome (pair 2) in the Karatsu type. This physical mapping of the 5S rDNA provides valuable information about karyotype evolution in R. silerifolius. Possible mechanisms of chromosome evolution are discussed.     

Parental origin and genome evolution in the allopolyploid Iris versicolor

BACKGROUND AIMS: One of the classic examples of an allopolyploid is Iris versicolor, 'Blue Flag' (2n = 108), first studied by Edgar Anderson and later popularized by George Ledyard Stebbins in cytogenetics and evolutionary text-books. It is revisited here using modern molecular and cytogenetic tools to investigate its putative allopolyploid origin involving progenitors of I. virginica (2n = 70) and I. setosa (2n = 38). METHODS: Genomic in situ hybridization (GISH), fluorescent in situ hybridization (FISH) and Southern hybridization with 5S and 18-26S ribosomal DNA (rDNA) probes were used to identify the parental origin of chromosomes, and to study the unit structure, relative abundance and chromosomal location of rDNA sequences. KEY RESULTS: GISH shows that I. versicolor has inherited the sum of the chromosome complement from the two progenitor species. In I. versicolor all the 18-26S rDNA units and loci are inherited from the progenitor of I. virginica, those loci from the I. setosa progenitor are absent. In contrast 5S rDNA loci and units from both progenitors are found, although one of the two 5S loci expected from the I. setosa progenitor is absent. CONCLUSIONS: These data confirm Anderson's hypothesis that I. versicolor is an allopolyploid involving progenitors of I. virginica and I. setosa. The number of 18-26S rDNA loci in I. versicolor is similar to that of progenitor I. virginica, suggestive of a first stage in genome diploidization. The locus loss is targeted at the I. setosa-origin subgenome, and this is discussed in relation to other polyploidy systems.     

The distribution of diploids and polyploids of theScilla scilloides complex in the northeastern district of China

A cytological analysis was made of 1504 individuals ofScilla scilloides Druce from the northeastern district of China. In terms of genome combination four cytotypes were found: AA (90.95%), AAAA (0.07%), AABB (8.92%) and AABB+2 (0.07%). In the cytotypes A and B denote genome A (x=8) and genome B (x=9), respectively. Most of 18 natural populations examined consist of one cytotype: indeed, all individuals examined of 15 of the populations are AA diploids, those of one population AABB allotetraploids, and individuals examined of two other populations have two cytotypes each, AA and AAAA, and AABB and AABB+2. In karyotype the genome A of the AA and AAAA was different from that of the AABB, and a direction of morphological change of chromosomes in the genome A is discussed in light of results of hybridization experiments reported elsewhere. Taken together with information available for the distribution of different cytotypes in China, Korea and Japan, the results of the present study support the view of Maekawa thatScilla scilloides was native to mainland China and lately introduced to Japan.     

The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S-5.8S-26S rDNA and a C genome specific DNA sequence in the genus Avena.

A physical map of the locations of the 5S rDNA genes and their relative positions with respect to 18S-5.8S-26S rDNA genes and a C genome specific repetitive DNA sequence was produced for the chromosomes of diploid, tetraploid, and hexaploid oat species using in situ hybridization. The A genome diploid species showed two pairs of rDNA loci and two pairs of 5S loci located on both arms of one pair of satellited chromosomes. The C genome diploid species showed two major pairs and one minor pair of rDNA loci. One pair of subtelocentric chromosomes carried rDNA and 5S loci physically separated on the long arm. The tetraploid species (AACC genomes) arising from these diploid ancestors showed two pairs of rDNA loci and three pairs of 5S loci. Two pairs of rDNA loci and 2 pairs of 5S loci were arranged as in the A genome diploid species. The third pair of 5S loci was located on one pair of A-C translocated chromosomes using simultaneous in situ hybridization with 5S rDNA genes and a C genome specific repetitive DNA sequence. The hexaploid species (AACCDD genomes) showed three pairs of rDNA loci and six pairs of 5S loci. One pair of 5S loci was located on each of two pairs of C-A/D translocated chromosomes. Comparative studies of the physical arrangement of rDNA and 5S loci in polyploid oats and the putative A and C genome progenitor species suggests that A genome diploid species could be the donor of both A and D genomes of polyploid oats. Key words : oats, 5S rDNA genes, 18S-5.8S-26S rDNA genes, C genome specific repetitive DNA sequence, in situ hybridization, genome evolution.     

Geographical distribution of cytotypes and genomic structures in natural populations of the<Emphasis Type="Italic">Scilla scilloides</Emphasis> complex in Korea

Cytotype distribution, B-chromosome frequency, and genomic constitution in the natural populations of theScilla scilloides complex in Korea were analyzed. Plants with various cytotypes were found: AA (2n = 16), BB (18), AAB (25), ABB (26), ABB (34), ABBB (35), BBBB (36), AABBB (43), AAABBB (51) and AAAABBBB (68). Allotetraploid AABB plants predominated with a frequency of 68.3%, and were found to distribute all over the Korean peninsula and Cheju-do. In diploids, the type AA plants distributed throughout the Korean midwest, while the type BB plants were limited to Cheju-do. Two other cytotypes, ABBB and AABBB, were found only in the southern part of the Korean peninsula including Cheju-do. Chromosomal variations, aneuploidy, and centromeric shifts were also found in the natural populations. The cytotypes AAB and AAAABBBB are reported here for the first time. B-chromosomes were found in 149 (85.6%) of 174 populations, the highest frequency being 81.8% in BB populations. The number of B-chromosomes per plant ranged from 1 to 31, and IB plants predominated (21.0%). Subtypes, with respect to the number and composition of B-chromosomes, indicated that sexual reproduction is still prevalent in AABB populations.     

Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species

Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus. Received: 26 February 1999 / Accepted: 16 March 1999     

Physical Mapping of rRNA Gene Loci and Inter-specific Relationships in Wild <Emphasis Type="Italic">Lilium</Emphasis> Distributed in Korea

Molecular cytogenetic analyses using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were carried out to elucidate inter-specific relationships among wild Lilium species distributed in Korea. FISH revealed four to eight 45S rRNA gene loci, which are located on chromosomes 1–7, 10, and 11 among the different species. In contrast, the 5S rRNA gene locus was conserved on the long arm of chromosome 3, occasionally with two adjacent sites on the same chromosome arm in a few species. The 5S rDNA site was located adjacent to the 45S rDNA site in only three species, Lilium distichum, Lilium hansonii, and Lilium tsingtauense. GISH analysis using genomic DNA probes detected strong hybridization of genomes between diploid and triploid Lilium lancifolium species, demonstrating that triploid plants were derived from diploid L. lancifolium and not from Lilium maximowiczii. Phylogenetic analysis of the ITS and NTS sequences supported the cytogenetic data as well as Comber’s classification of the genus Lilium.     

Physical mapping of rDNA loci in Brassica species.

The number of major rDNA loci (the genes coding for 18S-5.8S-26S rRNA) was investigated in the economically important Brassica species and their wild relatives by in situ hybridization of an rDNA probe to metaphase chromosomes and interphase nuclei. The diploid species B. nigra (B genome) has two major pairs of rDNA loci, B. oleracea (C genome) has two major pairs and one minor pair of loci, while B. campestris (A genome) has five pairs of loci. Among the three tetraploid species arising from these three diploid ancestors, B. carinata (BBCC genomes) has four loci, B. juncea (AABB genomes) has five major pairs and one minor pair of loci, and B. napus (AACC genomes) has six pairs of loci, indicating that the number of loci has been reduced during evolution. The complexity of the known rDNA restriction fragment length polymorphism patterns gave little indication of number of rDNA loci. It is probable that chromosome rearrangements have occurred during evolution of the amphidiploid species. The data will be useful for physical mapping of genes relative to rDNA loci, micro- and macro-evolutionary studies and analysis of aneuploids including addition and substitution lines used in Brassica breeding programs.     

Ribosomal DNA evolution and phylogeny in Aloe (Asphodelaceae)

All Aloe taxa (～400 species) share a conserved bimodal karyotype with a basic genome of four large and three small submetacentric/acrocentric chromosomes. We investigated the physical organization of 18S-5.8S-26S and 5S ribosomal DNA (rDNA) using fluorescent in situ hybridization (FISH) to 13 Aloe species. The organization was compared with a phylogenetic tree of 28 species (including the 13 used for FISH) constructed by sequence analysis of the internal transcribed spacer (ITS) of 18S-5.8S-26S rDNA. The phylogeny showed little divergence within Aloe, although distinct, well-supported clades were found. FISH analysis of 5S rDNA distribution showed a similar interstitial location on a large chromosome in all species examined. In contrast, the distribution of 18S-5.8S-26S rDNA was variable, with differences in number, location, and size of loci found between species. Nevertheless, within well-supported clades, all species had the same organizational patterns. Thus, despite the striking stability of karyotype structure and location of 5S rDNA, the distribution of 18S-5.8S-26S rDNA is not so constrained and has clearly changed during Aloe speciation.     

Nuclear DNA content,base composition,heterochromatin and rDNA in Picea omorika and Picea abies

Two closely related spruces, Picea abies and Picea omorika, a Balkan paleoendemic species, often share habitats, yet never hybridize in nature. The present study adresses their characteristics such as nuclear DNA content, base composition, heterochromatin and rDNA pattern. The genome size of P. abies was 10% larger than that of P. omorika when assessed by flow cytometry, respectively 2C=37.2 pg and 33.8 pg; although when estimated as total chromosome length it was virtually the same. The heterochromatin Chromomycin-A (CMA)/ DAPI fluorochrome banding patterns of both P. abies and P. omorika are given here for the first time. Simultaneous FISH (fluorescent in situ hybridization) using 18S-26S and 5S rDNA probes revealed 16 18S rDNA sites in P. omorika, 12 18S rDNA sites in P. abies, and a single 5S rDNA locus in both species. The genomes have about 41% GC. The number and position of CMA/DAPI bands and rDNA loci provide good chromosome markers to clarify the karyotypes of the two species. Received: 18 October 2000 / 14 June 2001     

Comparative physical mapping of the 18S-5.8S-26S rDNA in three sorghum species.

The physical locations of the 18S-5.8S-26S rDNA sequences were examined in three sorghum species by fluorescence in situ hybridization (FISH) using biotin-labeled heterologous 18S-5.8S-26S rDNA probe (pTa71). Each 18S-5.8S-26S rDNA locus occurred at two sites on the chromosomes in Sorghum bicolor (2n = 20) and S. versicolor (2n = 10), but at four sites on the chromosomes of S. halepense (2n = 40) and the tetraploid S. versicolor (2n = 20). Positions of the rDNA loci varied from the interstitial to terminal position among the four accessions of the three sorghum species. The rDNA data are useful for investigation of chromosome evolution and phylogeny. This study excluded S. versicolor as the possible progenitor of S. bicolor.     

中间偃麦草(Thinopyrum intermedium)18S-5.8S-26S rDNA位点在染色体上的分布及对其核ITS序列异质性的分析(英文)

中间偃麦草(Thinopyrum intermedium(Host)Barkworth et Dewey)是禾本科小麦族植物中的一个异源六倍体物种,是重要的牧草植物,在小麦的抗病育种中发挥了重要作用。利用荧光原位杂交(FISH)技术,在体细胞中期染色体上,对18S-5.8S-26S rDNA位点进行了物理定位,发现该物种有3～4对染色体携带18S-5.8S-26S rDNA主位点。结合基因组原位杂交(GISH)分析,证明中间偃麦草的St基因组中有一对同源染色体短臂末端携带一个主位点,其余2～3对主位点位于E基因组染色体上。对不同来源的材料研究表明:18S-5.8S-26S rDNA位点的数目(包括主位点和小位点)、位置、拷贝数在不同收集材料之间的差异较大,甚至在同一个体的不同细胞中也存在差异。讨论了rDNA物理作图数据在分析系统发育问题中的局限性。结合中间偃麦草的三个可能的二倍体基因组供体(Th.bessarabicum、Th. elongatum和Pseudoroegneria stipifolia)rDNA位点分析的结果,对中间偃麦草进化过程中rDNA位点的变化进行了分析,同时,对其中一份材料的核ITS序列进行了克隆、测序和系统发育分析,发现在中间偃麦草中,ITS序列具有很高的异质性。     

Development of Molecular Cytogenetics and Physical Mapping of Ribosomal RNA Genes in Lupinus

Identification of individual chromosomes in Lupinus is not possible due to gradient in size and similar morphology. To overcome this problem, molecular cytogenetics was developed for Lupinus. As an initial step in karyotype analysis, fluorescent in situ hybridization (FISH) was performed to determine genomic distribution of rRNA genes in L. hispanicus, L. luteus and L. × hispanicoluteus. It was found that all three diploid species posses two chromosome pairs carrying 18S-5.8S-25S rDNA and one chromosome pair carrying 5S rDNA. The use of probes for rDNA permitted unambiguous identification of three different pairs of chromosomes and revealed conservation of the number of rDNA loci among the three species. The study represents the first step in physical mapping of Lupinus genome through FISH by providing distinct chromosome landmarks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physical localization of the 18S-5.8S-26S rDNA and sequence analysis of ITS regions in Thinopyrum ponticum (Poaceae: Triticeae): implications for concerted evolution

Fluorescence in situ hybridization was used in Thinopyrum ponticum, a decaploid species, and its related diploid species, to investigate the distribution of the 18S-5.8S-26S rDNA. The distribution of rDNA was similar in all three diploid species (Th. bessarabicum, Th. elongatum and Pseudoroegneria stipifolia). Two pairs of loci were observed in each somatic cell at metaphase and interphase. One pair was located near the terminal end and the other in the interstitial regions of the short arms of one pair of chromosomes. However, all of the major loci in Th. ponticum were located on the terminal end of the short arms of chromosomes, and one chromosome had only one major locus. The maximum number of major loci detected on metaphase spreads was 20, which was the sum of that of its progenitors. The interstitial loci that exist in the possible diploid genome donor species were probably 'lost' during the evolutionary process of the decaploid species. A number of minor loci were also detected on whole regions of two pairs of homologous chromosomes. These results suggested that the position of rDNA loci in the Triticeae might be changeable rather than fixed. Positional changes of 18S-5.8S-26S rDNA loci between Th. ponticum and its candidate genome donors indicate that it is almost impossible to find a genome in the polyploid species that is completely identical to that of its diploid donors. The possible evolutionary significance of the distribution of the rDNA is also discussed. Internal transcribed spacer (ITS) regions of nuclear DNA in Th. ponticum were investigated by PCR amplification and sequencing. The sequence data from five positive clones selected at random, together with restriction site analysis, indicated that the ITS repeated units are nearly homogeneous in this autoallodecapolypoid species. Combined with in situ hybridization results, the data led to the conclusion that the ITS region has experienced interlocus as well as intralocus concerted evolution. Phylogenetic analyses showed that the sequences from Th. ponticum have concerted to the E genome repeat type.     

Chromosomal location and reorganization of the 45S and 5S rDNA in theBrachyscome lineariloba complex (Asteraceae)

The chromosomal locations of the 45S (18S-5.8S-26S) and 5S ribosomal DNA in theBrachyscome lineariloba complex and two related species have been determined by the use of multicolor fluorescencein situ hybridization (McFISH). TheBrachyscome lineariloba complex includes five cytodemes with 2n=4, 8, 10, 12 and 16. Each of the 5S and 45S rDNA loci occurs at two sites on chromosomes in cytodemes with 2n=4. While in cytodemes with 2n=8, 10, 12 and 16, the number of 5S rDNA sites increases from four to eight paralleled to the genomic addition of diploid (4 chromosomes) or haploid (2 chromosomes) dosage. Of the 5S rDNA sites, only one pair is major, except for the cytodeme with 2n=10. The remaining 5S rDNA sites are minor and seem to have reduced the unit number of the 5S rDNA during the successive genomic additions. The 45S rDNA site is detected only at two nucleolar organizing regions in all cytodemes regardless of successive genomic addition. The loss or diminution of 45S rDNA sequences seem to have proceeded more rapidly than 5S rDNA sequences in theB. lineariloba complex.     

不同染色技术和双色荧光原位杂交技术对宽翅菘蓝（Isatis violascens Bunge）的细胞遗传学分析

为了解十字花科菘蓝属植物宽翅菘蓝（宽翅菘蓝,Isatis violascens Bunge）的染色体结构,本文利用45SrDNA和5SrDNA双色荧光原位杂交、银染技术和CMA3对宽翅菘蓝进行分子细胞遗传学研究。45SrDNA和5SrDNA荧光原位杂交结果显示宽翅菘蓝染色体上有1对45SrDNA信号和1对5SrDNA信号;银染结果显示其中期染色体有1对银染点;CMA3染色结果发现宽翅菘蓝中期染色体存在1对CMA3信号。     

基因组原位杂交技术及其在园艺植物基因组研究中的应用

基因组原位杂交(GISH)技术可以鉴定植物多倍体物种起源、杂种亲本染色体来源和组成,分析栽培种与其近缘野生种的亲缘关系,研究减数分裂染色体行为等。基因组原位杂交包括多色基因组原位杂交、比较基因组原位杂交和自身基因组原位杂交等。基因组原位杂交技术的关键步骤是染色体制片、探针制备及长度优化、探针与封阻的浓度比例和杂交后洗脱强度。该文对近年来国内外有关基因组原位杂交技术的发展及其在园艺植物基因组研究中的应用现状进行了综述,并指出随着多种园艺植物全基因组的测定,未来应从基因组信息中寻找更多的染色体特异性标记,结合荧光显带及荧光原位杂交技术,为深入研究园艺植物的起源以及遗传关系鉴定等提供技术支持。