Chromosomal mapping of the major and minor ribosomal genes, (GATA)n and (TTAGGG)n by one-color and double-color FISH in the toadfish Halobatrachus didactylus (Teleostei: Batrachoididae)

The karyotype of Halobatrachus didactylus presents 46 chromosomes, composed of eight metacentric, 18 submetacentric, four subtelocentric, and 16 acrocentric chromosomes. The results of FISH showed that the major ribosomal genes were located in the terminal position of the short arm of a large submetacentric chromosome. They also showed a high variation in the hybridization signals. The products of amplification of 5S rDNA produced bands of about 420 pb. The PCR labeled products showed hybridization signals in the subcentromeric position of the long arm of a submetacentric chromosome of medium size. Double-color FISH indicated that the two ribosomal families are not co-located since they hybridizated in different chromosomal pairs. Telomeres of all the chromosomes hybridized with the (TTAGGG) _n probe. The GATA probe displayed a strong signal in the long arm of a submetacentric chromosome of medium size, in the subcentromeric position. The double-color FISH showed that the microsatellite GATA and the 5S rDNA gene are located in different chromosomal pairs. The majority presence of GATA probes in one pair of chromosomes is unusual and considering its distribution through different taxa it could be due to evolutionary mechanisms of heterochromatine accumulation, leading to the formation of differentiated sex chromosomes.     

Cytogenetic characterization of the sole Solea senegalensis (Teleostei: Pleuronectiformes: Soleidae): Ag-NOR, (GATA) n , (TTAGGG) n and ribosomal genes by one-color and two-color FISH

A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for the nucleolus organizer region (Ag-NOR) identification, one-color FISH for chromosomal mapping of 45S and 5S ribosomal DNAs (rDNAs), (GATA) _n , and (TTAGGG) _n , and two-color FISH for co-localization of both rDNAs. The Ag-NORs and the 45S rDNA were mapped to a medium-sized submetacentric chromosomal pair. Hybridization with the 5S rDNA showed a major signal on the short arm of a medium-sized submetacentric chromosome pair and a minor signal on a centromeric site of a small acrocentric chromosome pair. Differences in the Ag-NOR and 45S and 5S rDNAs FISH signal sizes were observed between homologous chromosomes and among individuals. A two-color FISH co-localized 45S and 5S rDNAs to a medium-sized submetacentric chromosomal pair. The hybridization with the telomeric (TTAGGG) _n repeat displayed small signals at all chromosomal telomeres. Finally, the (GATA) _n probe produced dispersed and small hybridization signals on all chromosome spreads, showing its ubiquitous existence in the genome. These results were compared with those from other Pleuronectiformes and discussed in terms of karyotype evolution.     

Chromosomal localization of 18S and 5S rDNA using FISH in the genus <Emphasis Type="Italic">Tor</Emphasis> (Pisces,Cyprinidae)

Dual color fluorescence in situ hybridization (FISH) was performed to study the simultaneous chromosomal localization of 18S and 5S ribosomal genes in the genus Tor for the first time. The 18S and 5S rDNAs in four Tor species were amplified, sequenced and mapped on the metaphase chromosomes. The number and distribution of 18S and 5S rDNA clusters were examined on metaphase chromosome spreads using FISH. The specimens of T. chelynoides, T. putitora and T. progeneius showed six bright fluorescent signals of 18S rDNA and T. tor exhibited ten such signals. The 5S rDNA signals were present only on one pair of chromosomes in all the four Tor species. Ag-NORs were observed on two pairs of chromosomes in T. chelynoides, T. putitora, T. progeneius and four pairs in T. tor. Comparison of the observed 18S rDNA FISH signals and Ag-NORs strongly suggested a possible inactivation of NORs localized at the telomeres of a subtelocentric and telocentric chromosome pairs in all four species. The 5S rDNA contained an identical 120 bp long coding region and 81 bp long highly divergent non-transcribed spacers in all species examined. 18S and 5S rDNA sequencing and chromosomal localization can be a useful genetic marker in species identification as well as phylogenetic and evolutionary studies.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

中国17种蔷薇属植物45S和5S的rDNA分布研究

为了探寻蔷薇属植物亲缘关系及系统发育研究的分子细胞遗传学证据,该研究采用双色FISH(荧光原位杂交)技术,对原产中国7个组的17种蔷薇属植物的45S和5S rDNA进行了定位分析。结果表明:(1)多数蔷薇属植物1组染色体对应1个45S rDNA位点和1个或2个5S rDNA位点,偶尔出现1~2个rDNA位点的丢失,但复伞房蔷薇(Rosa brunonii)的1组染色体对应了2个45S rDNA位点。(2)二倍体的蔷薇属植物至少有1对5S rDNA位点与45S rDNA位点共定位,而四倍体材料的5S rDNA位点与45S rDNA位点没有共定位,但所有四倍体材料均至少有1种rDNA信号纯合,表明它们应为二倍体直接加倍产生的同源四倍体。(3)绝大多数材料45S rDNA位于染色体短臂、5S rDNA位于染色体长臂,但缫丝花(R. roxburghii f. roxburghii)有1个5S rDNA信号位于染色体的短臂上,表明它与蔷薇属其他种的亲缘关系较远。(4)阿克苏地区和伊犁地区的疏花蔷薇的核型不同,且45S和5S rDNA的数量和位置不同,分子细胞遗传学证据也支持阿克苏地区的疏花蔷薇应为疏花蔷薇的新变种。(5)该研究中共有8个二倍体和6个四倍体蔷薇属植物的双色FISH为首次报道。研究认为,无论二倍体还是四倍体蔷薇属植物中出现的异形同源染色体、rDNA信号位置在同源染色体上的差异以及rDNA信号的增加和丢失,可能都与染色体结构变异和染色体重组有关,在分子细胞遗传学水平上证明染色体结构变异和染色体重组在蔷薇属植物演化过程中具有重要的作用。     

Karyotyping of three Pinaceae species via fluorescent in situ hybridization and computer-aided chromosome analysis

The positions of 18/25S rRNA genes, 5S RNA genes and of Arabidopsis-type telomeric repeats were localized by fluorescent in situ hybridization (FISH) on the chromosomes of three coniferous species; Picea abies, Larix decidua and Pinus sylvestris, each with 2n=24 chromosomes. Computer-aided chromosome analysis was performed on the basis of the chromosome length, the arm length ratio and the position of the hybridization signals. This enabled the chromosomes of the Norway spruce, 4 chromosomes of the European larch and 3 of the karyotype of the Scots pine to be individually distinguished. With respect to the chromosomal positions of rDNA and 5S rDNA loci, chromosome pair I of P. sylvestris is suggested to be homoeologous to pair II of P. abies, while another chromosome pair of P. sylvestris might be homoeologous to chromosome pair III of L. decidua.     

Molecular cytogenetic analysis and the establishment of a cell culture in the fish species Hollandichthys multifasciatus (Eigenmann & Norris, 1900) (Characiformes,Characidae)

Hollandichthys is a fish genus of the family Characidae that was until recently considered to be monotypic, with cytogenetic, morphological, and molecular data being restricted to a few local populations. In the present study, the karyotype of a population of Hollandichthys multifasciatus was analyzed using classical and molecular cytogenetic approaches for the investigation of potential markers that could provide new perspectives on the cytotaxonomy. H. multifasciatus presented a diploid number of 2n=50 chromosomes and a karyotype formula of 8m+10sm+32st. A single pair of chromosomes presented Ag-NORs signals, which coincided with the 18S rDNA sites visualized by FISH, whilst the 5S rDNA sequences were mapped in two chromosome pairs. The distribution of the U snRNA genes was mapped on the Hollandichthys chromosomes for the first time, with the probes revealing the presence of the U1 snDNA on the chromosomes of pair 20, U2 on pairs 6 and 19, U4 on pair 16, and U6 on the chromosomes of pair 11. The results of the present study indicated karyotypic differences in comparison with the other populations of H. multifasciatus studied previously, reinforcing the need for further research to identify isolated populations or the potential existence of cryptic Hollandichthys species.     

Nucleotide variation and physical mapping of ribosomal genes using FISH in genus <Emphasis Type="Italic">Tor</Emphasis> (Pisces,Cyprinidae)

Molecular cytogenetic studies were carried out for localization of 18S and 5S ribosomal DNAs on chromosomes of three cyprinid fish species viz., T. khudree, T. mussullah and T. mosal mahanadicus using two color fluorescence in situ hybridization (FISH). All the species typically possessed 100 diploid chromosomes with minor variation in karyo-morphology. The 18S rDNA signals were observed on two pair of chromosomes in T. khudree and T. mussullah, and three pairs in T. mosal mahanadicus. The location of 18S signals also showed affinity to silver nitrate and chromomycin A₃ staining. Similarly, variation in localization of 5S rDNA among the three species has been detected with the presence of FISH signals on one pair of chromosome in T. khudree and T. mussullah, and on two pairs in T. mosal mahanadicus. These molecular markers could be used as species specific markers for taxonomic identification and can further add in understanding the dynamics of genome organization and karyotypic evolution of these species. The 18S rDNA region was sequenced that generated 1811, 1810 and 1776 bp long 18S sequence in T. khudree, T. mussullah and T. mosal mahanadicus, respectively. The 18S rDNA sequence showed 95–98% identity among the subject species. Similarly, 5S sequencing generated 203 bp long fragments in these species with 100% identity in coding and 9.63% variability in non-transcribed spacer regions. The nucleotide sequence variations could be used for understanding the genetic diversity and will add new informative characters in comparative genomics. These results, in general, would enhance the value and interpretation of ecological assessment data for conservation of Tor species.     

FISH mapping of 18S-28S and 5S ribosomal DNA, (GATA)n and (TTAGGG)n telomeric repeats in the periwinkle Melarhaphe neritoides (Prosobranchia, Gastropoda, Caenogastropoda)

Spermatocyte chromosomes of Melarhaphe neritoides (Mollusca, Prosobranchia, Caenogastropoda) were studied using fluorescent in situ hybridization (FISH) with four repetitive DNA probes (18S rDNA, 5S rDNA, (TTAGGG)n and (GATA)n). Single-colour FISH consistently mapped one chromosome pair per spread using either 18S or 5S rDNA as probes. The telomeric sequence (TTAGGG)n hybridized with termini of all chromosomes whereas the (GATA)n probe did not label any areas. Simultaneous 18S-5S rDNA and 18S-(TTAGGG)n FISH demonstrated that repeated units of the three multicopy families are closely associated on the same chromosome pair.     

基于荧光原位杂交的藜属植物核型分析

为精确地识别藜属植物染色体组的核型特征,该文研究了4种来自青海高原的野生藜属植物(灰绿藜、藜、菊叶香藜及杂配藜)和1种从美国引进的栽培藜麦品种PI614932-HX(3)基于染色体荧光原位杂交(rDNA FISH)的核型。利用5S rDNA和45S rDNA对5种藜属植物有丝分裂中期的染色体进行FISH研究。藜属植物的核型分析结果表明:(1)藜属植物中存在二倍体(2n=2x=18)和四倍体(2n=4x=36)两种倍性,藜麦和灰绿藜为四倍体,其余3种为二倍体。(2)藜麦、灰绿藜、藜、菊叶香藜及杂配藜的核型公式分别为2n=4x=36=34m(2AST)+2sm,2n=4x=36=32m(4AST)+4sm,2n=2x=18=16m(4AST)+2sm,2n=2x=18=18m及2n=2x=18=16m+2sm。(3)染色体由大部分的中部着丝粒染色体(m)和少部分近中部着丝粒染色体(sm)组成。(4)核型类型除了菊叶香藜为1B以外,其余均属于2B类型。(5)在藜麦、灰绿藜及藜中具有分布位置不同、数量不等的双随体。5S rDNA、45S rDNA FISH结果表明:(1)藜麦和灰绿藜的染色体上存在2对5S rDNA位点和1对45S rDNA位点,藜、杂配藜的染色体上存在1对5S rDNA位点和1对45S rDNA位点,菊叶香藜的染色体上只存在1对5S rDNA位点。(2)5S rDNA和45S rDNA位点均位于染色体的短臂上。该研究首次获得了藜属植物基于5S rDNA和45S rDNA荧光原位杂交核型,为藜属植物亲缘关系研究和细胞生物学研究提供了分子细胞遗传学依据。     

Physical mapping of the 5S ribosomal RNA genes in Arabidopsis thaliana by multi-color fluorescence in situ hybridization with cosmid clones

The 5S ribosomal RNA genes were mapped to mitotic chromosomes of Arabidopsis thaliana by fluorescence in situ hybridization (FISH). In the ecotype Landsberg erecta, hybridization signals appeared on three pairs of chromosomes, two of which were metacentric and the other acrocentric. Hybridization signals on one pair of metacentric chromosomes were much stronger than those on the acrocentric and the other pair of metacentric chromosomes, probably reflecting the number of copies of the genes on the chromosomes. Other ecotypes, Columbia and Wassilewskija, had similar chromosomal distribution of the genes, but the hybridization signals on one pair of metacentric chromosomes were very weak, and detectable only in chromosomes prepared from young flower buds. The chromosomes and arms carrying the 5S rDNA were identified by multi-color FISH with cosmid clones and a centromeric 180 bp repeat as co-probes. The metacentric chromosome 5 and its L arm carries the largest cluster of the genes, and the short arm of acrocentric chromosome 4 carries a small cluster in all three ecotypes. Chromosome 3 had another small cluster of 5S rRNA genes on its L arm. Chromosomes 1 and 2 had no 5S rDNA cluster, but they are morphologically distinguishable; chromosome 1 is metacentric and 2 acrocentric. Using the 5S rDNA as a probe, therefore, all chromosomes of A. thaliana could be identified by FISH. Chromosome 1 is large and metacentric; chromosome 2 is acrocentric carrying 18S-5.8S-25S rDNA clusters on its short arm; chromosome 3 is metacentric carrying a small cluster of 5S rDNA genes on its L arm; chromosome 4 is acrocentric carrying both 18S-5.8S-25S and 5S rDNAs on its short (L) arm; and chromosome 5 is metacentric carrying a large cluster of 5S rDNA on its L arm.     

Chromosome analysis and FISH mapping of ribosomal DNA (rDNA), telomeric (TTAGGG)n and (GATA)n repeats in the leech Haemopis sanguisuga (L.) (Annelida: Hirudinea)

In the present paper the chromosome complement (n = 13; 2n = 26) of the common leech Haemopis sanguisuga (L.) (Annelida: Hirudinea: Hirudinidae) was analyzed using banding techniques and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG) _n and (GATA) _n ]. FISH with the rDNA probe consistently mapped major ribosomal clusters (18S–28S rDNA) in the pericentromeric region of one large metacentric chromosome pair; this region, which consisted of heterochromatin rich in GC base pairs, was preferentially stained by silver nitrate (Ag-NOR). The (TTAGGG) _n telomeric probe was hybridized with the termini of nearly all chromosomes, whereas the (GATA) _n probe did not label any chromosome areas.     

Molecular cytogenetics of <Emphasis Type="Italic">Oligosarcus hepsetus</Emphasis> (Teleostei,Characiformes) from two Brazilian locations

Karyotype and cytogenetic markers of Oligosarcus hepsetus from two Brazilian locations in the Paraíba do Sul River Basin (Brazil) were investigated using differential staining techniques (C-banding, silver (Ag)- and chromomycin A₃ (CMA₃)-staining) and fluorescent in situ hybridization (FISH) using 18 S rDNA and 5 S rDNA probes. The diploid chromosome number was invariably 2n = 50 with 3 pairs of metacentric, 5 pairs of submetacentric, 8 pairs of subtelocentric and 9 pairs of acrocentric chromosomes. No heteromorphic sex chromosomes were observed. The nucleolar organizer regions (NORs) were detected in the short arms of the largest acrocentric pair using Ag-, CMA₃- stainings and FISH with 18 S rDNA probe, the latter showing also positive labeling in the short arms of a small acrocentric pair, not visualized by the former methods. FISH with 5 S rDNA probe showed positive labeling in the two chromosome pairs. While the CMA₃-staining exhibited GC-rich heterochromatin segments in two pairs of chromosomes, including those coincided with Ag-NORs, the DAPI staining did not reveal any signal, indicating the absence of AT-rich heterochromatin. FISH with an As-51 satellite DNA probe derived from the closely related Astyanax scabripinnis did not reveal any positive signal, demonstrating the absence of this class of DNA in the genome of the specimens under study.     

Cytogenetic characterization and mapping of rDNAs,core histone genes and telomeric sequences in <Emphasis Type="Italic">Venerupis aurea</Emphasis> and <Emphasis Type="Italic">Tapes rhomboides</Emphasis> (Bivalvia: Veneridae)

We describe the chromosomal location of GC-rich regions, 28S and 5S rDNA, core histone genes, and telomeric sequences in the veneroid bivalve species Venerupis aurea and Tapes (Venerupis) rhomboides, using fluorochrome staining with propidium iodide, DAPI and chromomycin A3 (CMA) and fluorescent in situ hybridization (FISH). DAPI dull/CMA bright bands were coincident with the chromosomal location of 28S rDNA in both species. The major rDNA was interstitially clustered at a single locus on the short arms of the metacentric chromosome pair 5 in V. aurea, whereas in T. rhomboides it was subtelomerically clustered on the long arms of the subtelocentric chromosome pair 17. 5S rDNA also was a single subtelomeric cluster on the long arms of subtelocentric pair 17 in V. aurea and on the short arms of the metacentric pair 9 in T. rhomboides. Furthermore, V. aurea showed four telomeric histone gene clusters on three metacentric pairs, at both ends of chromosome 2 and on the long arms of chromosomes 3 and 8, whereas histone genes in T. rhomboides clustered interstitially on the long arms of the metacentric pair 5 and proximally on the long arms of the subtelocentric pair 12. Double and triple FISH experiments demonstrated that rDNA and H3 histone genes localized on different chromosome pairs in the two clam species. Telomeric signals were found at both ends of every single chromosome in both species. Chromosomal location of these three gene families in two species of Veneridae provides a clue to karyotype evolution in this commercially important bivalve family.     

Chromosome Analysis and Molecular Characterization of Highly Repeated DNAs in the Aphid Acyrthosiphon Pisum (Aphididae, Hemiptera)

Despite the interest in aphid biology, information on chromatin organization of their holocentric chromosomes is still limited to few species. In order to fill this gap, we have performed an extensive survey on pea aphid mitotic chromosomes using both classical and molecular cytogenetic techniques. Our results after silver, CMA₃ and DAPI-staining, C-banding and fluorescent in situ hybridization (FISH) using 28S rDNA and 5S rDNA as probes evidenced a tendency of repetitive DNAs to be concentrated on the X chromosomes. FISH experiments with the telomeric probe (TTAGG) _n revealed bright hybridization signals on each telomere of all Acyrthosiphon pisum chromosomes. No interstitial signals were seen. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chromosome phylogeny of <Emphasis Type="Italic">Zamia</Emphasis> and <Emphasis Type="Italic">Ceratozamia</Emphasis> by means of Robertsonian changes detected by fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization (FISH) technique of rDNA

 Appearance and location of 45S rDNA and 5S rDNA signals were compared in chromosomes of nine species of the aneuploid Zamia and their taxonomically and phylogenetically closely related Ceratozamia mexicana. The 45S rDNA signal was detected in the proximal region of six chromosomes in Zamia angustifolia, Z. integrifolia, Z. pumila and Z. pygmaea (all 2n=16); in the proximal region of 6–14 chromosomes in Z. furfuracea, Z. loddigesii, Z. skinneri and Z. vazquezii (all 2n=18); and on the proximal region of 20 chromosomes in Z. muricata (2n=23). The 5S rDNA signals were commonly seen near the terminal region of the short arm of two metacentric chromosomes in the four species with 2n=16 and Z. furfuracea, Z. loddigesii and Z. vazquezii with 2n=18. Other 5S rDNA signals were seen near the terminal region of two terminal-centromeric chromosomes in Z. skinneri and near the terminal region of a metacentric and a telocentric chromosomes in Z. muricata. In contrast, those with 45S and 5S rDNA signals were exhibited in chromosomes of Ceratozamia mexicana in a different manner from those in the nine species of Zamia; the 45S rDNA signal in the terminal region of four metacentric and two submetacentric chromosomes and the 5S rDNA signal near the proximal region of two metacentric chromosomes. Received November 1, 1999 Accepted January 10, 2001     

Mapping of ribosomal DNA and (TTAGGG)n telomeric sequence by FISH in the bivalve Patinopecten yessoensis (Jay, 1857)

The chromosome set of Patinopecten yessoensis (Jay, 1857) wascharacterized using Giemsa staining, DAPI staining and fluorescencein situ hybridization (FISH) with three repetitive DNA probes[18S–28S rDNA, 5S rDNA and telomeric (TTAGGG)_n]. DAPIstaining showed that AT-rich regions were located on the centromereof almost all chromosomes and interstitial banding was not observed.FISH showed that 18S–28S rDNA spread over the short armsof two subtelocentric chromosome pairs and 5S rDNA was locatedon the long arm of one subtelocentric chromosome pair. SequentialFISH demonstrated that 18S–28S and 5S rDNA were locatedon different chromosomes. FISH also showed that the vertebratetelomeric sequence (TTAGGG)_n was located on both ends of eachchromosome and no interstitial signals were detected. Sequential18S–28S rDNA and (TTAGGG)_n FISH indicated that repeatedunits of the two multicopy families were closely associatedon the same chromosome pair. (Received 4 January 2007; accepted 1 September 2007)     

Cytogenetic markers for the characterization of Capsicum annuum L. cultivars

Karyotypic studies with conventional staining have been unsuccessful due to the uniformity of Capsicum chromosomes. In this study, we found diagnostic chromosome characters that permit to characterize cultivars; this is the first cytological characterization of both rDNAs (18S and 5S) in a species of Capsicum using a genus-specific probe and the most exhaustive in C. annuum to date. The heterochromatic banding patterns enabled us to identify cultivars, and fluorescent in situ hybridization (FISH) showed one 5S rDNA locus largely conserved within the cultivars, whereas high variation in the number of 18S rDNA loci was observed. One of the most obvious differences is the presence of an additional active nucleolar organizer region in pair #12 and the dispersal of inactive 18S rDNA signals. These results indicate that fluorochrome banding together with silver impregnation and FISH procedures are very useful for the identification of chromosomes and the interpretation of chromosomal variation between cultivars. The functional role of this variation is still uncertain, but our results show that copy number variation of repetitive DNA during the course of evolution might provide an excellent experimental system for studying genome rearrangements accompanying functional divergence in domesticated C. annuum.     

Multicolor FISH mapping of the dioecious model plant,<Emphasis Type="Italic"> Silene latifolia</Emphasis>

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.Communicated by J.S. Heslop-Harrison     

Characterization of Japanese flounder karyotype by chromosome bandings and fluorescence in situ hybridization with DNA markers

The chromosomes of Japanese flounder, Paralichthys olivaceus, were examined by conventional differential staining methods including G-, Q-, C-, silver (Ag)-, fluorochrome, and replication R-bandings and by fluorescence in situ hybridization (FISH) with 5S and 18S rDNAs and telomeric DNA as probes. Replication R-banding substantially made it possible to identify 24 homologous pairs by their RBG-banding pattern and relative length. Both rDNA loci were mapped to chromosome 1, where 5S and 18S rDNA loci were located at the centromeric region and secondary constriction, respectively. C-banding revealed that both rDNA loci were heterochromatic, and 18S rDNA loci were positive for chromomycin A₃ but negative for 4′,6-diamidino-2-phenylindole (DAPI) staining. Telomeric FISH signals were observed at all chromosome ends and at the interstitial region of some chromosomes. The observed results were discussed in relation to the karyotype evolution in the order Pleuronectiformes.