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.     

Physical mapping of the 5S and 45S rDNA in teosintes

The physical locations of the 5S and 45S rDNA sequences were examined in three types of teosinte, Zea mays ssp. mexicana (2n = 20), Zea diploperennis (2n = 20) and Zea perennis (2n = 40) by biotinylated fluorescence in situ hybridization (FISH). The tested materials only showed one hybridization site of 5S rDNA on their genomes, but they were different in the position of the signals. The hybridization site of Zea mays ssp. mexicana was located on the long arm of chromosome 2, indicating that it is the same as the cultivated maize in the position of 5S rDNA, while the sites of Zea diploperennis and Zea perennis were on the short arms of other chromosomes. For 45S rDNA, one hybridization site was detected at secondary constriction region of the satellite chromosomes in Zea mays ssp. mexicana and Zea diploperennis, while in Zea perennis, besides the site located at the secondary constriction region, a second site on the short arm of another chromosome pair was observed. Our results provide additional evidence for Zea mays ssp. mexicana being a subspecies of Zea mays.     

Chromosome identification and karyotype analysis of Podophyllum hexandrum Roxb. ex Kunth using FISH

Podophyllum hexandrum is an important high altitude medicinal plant from Himalaya. Somatic chromosomes of this species were studied to delineate and physical mapping of repetitive rDNA sites to provide landmarks in chromosome identification. The karyotype formula of this species was found to be 6m + 2sm + 2st + 2t with secondary constriction in the chromosome 1 and 7. The FISH analysis of rDNA sites showed 4 sites for 18S rDNA and 2 sites for 5S rDNA. The chromosome number 1, 2, 5 and 6 can be identified based on 18S rDNA sites in their short arm and chromosome 1 and 2 can be identified by 5S rDNA site in the centromere region. The estimated genome size of this plant is 16.07 pg (1C).     

45S rDNA在小麦及其近缘物种染色体上的分布

将染色体C-分带和原位杂交技术相结合,系统研究了45S rDNA在栽培一粒小麦、野生二粒小麦、普通小麦、大麦、簇毛麦、硬簇麦、六倍体燕麦及鹅观草等物种染色体上的分布情况。这些物种染色体的次缢痕区都有45S rDNA位点, 某些非随体染色体上也有45S rDNA位点分布。以小麦—鹅观草1Rk^#1二体附加系为材料,通过顺序C分带-FISH技术首次将一个45S rDNA定位到1Rk^#1染色体短臂末端。     

水稻45S rDNA和5S rDNA的染色体定位研究

45SrDNA和5SrDNA是水稻中与核糖体RNA合成有关的2个功能片段,有关这2个序列在水稻染色体上的位置,不同研究者的研究结果不尽相同,在获得水稻染色体清晰制片的基础上,通过FISH确定了45SrDNA序列位于水稻的第9号和第10号染色体的短臂末端,并且第9号染色体上的拷贝数多于第10号染色体,5SrDNA序列位于第11号染色体短臂靠近着丝点处。     

Karyotype analysis of Lilium longiflorum and Lilium rubellum by chromosome banding and fluorescence in situ hybridisation.

Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red- and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.     

Production of wheat-Psathyrostachys huashanica chromosome addition lines

Psathyrostachys huashanica Keng (2n = 14; N(h)N(h)) is an endangered wheat-related species, with a distribution in the Huashan region of central China. It has many agronomically promising characters including resistance to disease and drought and winter hardiness. We produced hybrids between common wheat as the female parent and P. huashanica as the male parent. From the offspring, we selected chromosome addition lines of common wheat carrying each of all seven chromosomes of P. huashanica. Four chromosomes (B, D, E and F) were recovered in disomic lines and three (A, C and G) in monosomic addition lines. These alien chromosomes were distinguished from each other by cytological analyses. Chromosome A was characterized by a 45S rDNA site in the subtelomeric region of the short arm. Chromosome B carried one 5S and one 45S rDNA sites co-localized in an interstitial region of the short arm, and the expression of the alien high-molecular-weight glutenin was observed in the endosperm of line B. Chromosome D had a 45S rDNA signal in the interstitial region of the long arm. Chromosomes C, E, and F were distinguished by the EST-SSR markers Ltc0464, Ltc0096, and Xcfe175, respectively. The homoeologous group of each alien chromosome was implied from the results above, and the utilization of these addition lines for wheat breeding was discussed.     

Individual chromosome assignment and chromosomal collinearity in Gossypium thurberi, G. trilobum and D subgenome of G. barbadense revealed by BAC-FISH

The experiment on individual chromosome assignments and chromosomal diversity was conducted using a multi-probe fluorescence in situ hybridization (FISH) system in D subgenome of tetraploid Gossypium barbadense (D(b)), G. thurberi (D(1)) and G. trilobum (D(8)), which the later two were the possible subgenome donors of tetraploid cottons. The FISH probes contained a set of bacterial artificial chromosome (BAC) clones specific to 13 individual chromosomes from D subgenome of G. hirsutum (D(h)), a D genome centromere-specific BAC clone 150D24, 45S and 5S ribosomal DNA (rDNA) clones, respectively. All tested chromosome orientations were confirmed by the centromere-specific BAC probe. In D(1) and D(8), four 45S rDNA loci were found assigning at the end of the short arm of chromosomes 03, 07, 09 and 11, while one 5S rDNA locus was successfully marked at pericentromeric region of the short arm of chromosome 09. In D(b), three 45S rDNA loci and two 5S rDNA loci were found out. Among them, two 45S rDNA loci were located at the terminal of the short arm of chromosomes D(b)07 and D(b)09, whilst one 5S rDNA locus was found situating near centromeric region of the short arm of chromosome D(b)09. The positions of the BAC clones specific to the 13 individual chromosomes from D(h) were compared between D(1), D(8) and D(b). The result showed the existence of chromosomal collinearity within D(1) and D(8), and as well between them and D(b). The results will serve as a base for understanding chromosome structure of cotton and polyploidy evolution of cotton genome and will provide bio-information for assembling the sequences of finished and the on-going cotton whole genome sequencing projects.     

Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species

Dual-color fluorescence in situ hybridization (FISH) analysis of three Cucurbitaceae species from different genera was conducted using 5S and 45S rDNA probes. In Benincasa hispida (Thunb.) Cogn. (2n=24), the 45S rDNA probe hybridized on two chromosomes, one in the short arm of a medium-sized metacentric chromosome and another at the satellite of a chromosome. The 5S rDNA hybridized at a site proximal to the centromere of the same short arm of the 45S rRNA gene locus that occupied almost the entire short arm. For Citrullus lanatus (Thunb.) Matsum & Nakai (2n=22), the 45S rDNA probe hybridized at sites in the short arms of two chromosomes and the 5S rDNA probe was co-localized with the 45S rRNA locus at the region proximal to the centromere in one chromosome. The 45S rRNA loci occupied almost all of the short arms in both chromosomes. In Cucurbita moschata Duch. (2n=40), the 45S rDNA probe hybridized in five chromosomes in which the 45S rRNA genes occupied almost two-thirds of the chromosomes in two large chromosomes and the entire short arm of a medium-sized chromosome. Two other loci were present in two medium-sized chromosomes, one in the proximal region in the short arm of a chromosome and another at the tip of the long arm of a chromosome. Chromosomes of B. hispida were relatively larger than those of the other two species. The karyotype of B. hispida is composed of two metacentrics and 10 submetacentrics, while that of C. lanatus is composed of seven metacentrics and four submetacentrics and that of C. moschata is composed of 18 metacentrics and two submetacentrics. Comparative chromosome evolution among the three Cucurbitaceae species was attempted using the karyotypes and the chromosomal distribution patterns of the 5S and 45S rDNAs. The results presented herein will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species, and will provide basic data for their breeding programs.     

Schistosoma mansoni: chromosomal localization of DNA repeat elements by in situ hybridization using biotinylated DNA probes

Localization of the SM alpha family of repeated DNA and the rDNA repeat on the chromosomes of Schistosoma mansoni by in situ hybridization is presented. Biotinylated DNA was hybridized to target chromosomes and hybridization was detected using either alkaline phosphatase-labeled avidin or fluorescein-labeled avidin and biotinylated anti-avidin antibody. Hybridization detection using a fluorescein conjugate was more specific and sensitive with less background noise than detection with alkaline phosphatase conjugates. SM alpha hybridizing sequences were found dispersed throughout the genome, hybridizing to the sex chromosomes and autosomes. The SM alpha probe showed specific hybridization to the euchromatic gap region within the large heterochromatic block of the short arm of the W chromosome. This specific hybridization coupled with the lack of chiasma formation in this region of the ZW bivalent (presumably due to the heterochromatinization of this region) may explain the pattern of sex-specific hybridization reported for the SM alpha family. The rDNA repeat was localized to the secondary constriction of the short arm of chromosome 3. Specifically, the rDNA probe hybridized with the stalk of the secondary constriction and with parts of both side regions, the satellite and the short arm proper.     

18-26S rDNA在4种重楼属植物中的定位

为探讨rDNA在重楼属Paris L.中的分布规律,利用荧光原位杂交（FISH）对4种重楼属植物 的18-26S rDNA进行了定位。所有植物均为二倍体,基因组由A、B、C、D和 E5条染色体构成。（1）滇重楼P.polyphylla var.yunnanensis:2n=10=6m+4t,C和D染色体的 短臂上各有1个18-26S rDNA位点;（2）长柱重楼P.forrestii:2n=10=6m+4t,B染色体的长臂 、C和D染色体的短臂上各有1个位点;（3）五指莲P.axialis:2n=10=6m（2sat）+4t（2sat） +1-2B,C和D染色体的短臂上各有1个位点;在有1个B染色体的细胞中,B染色体没有信号点, 而有2个B染色体的细胞中,只有1个B染色体上有信号点,表明B染色体上有基因存在且其分裂 不均等;（4）大理重楼P.daliensis:2n=10=4m+2sm+2st+2t,C染色体的短臂上有1个位点。1 8-26S rDNA位点不仅出现在染色体的次缢痕上,也出现在非次缢痕位点。另外,4个种中C染 色体短臂末端均有18-26S rDNA。     

High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice

Large-scale physical mapping has been a major challenge for plant geneticists due to the lack of techniques that are widely affordable and can be applied to different species. Here we present a physical map of rice chromosome 10 developed by fluorescence in situ hybridization (FISH) mapping of bacterial artificial chromosome (BAC) clones on meiotic pachytene chromosomes. This physical map is fully integrated with a genetic linkage map of rice chromosome 10 because each BAC clone is anchored by a genetically mapped restriction fragment length polymorphism marker. The pachytene chromosome-based FISH mapping shows a superior resolving power compared to the somatic metaphase chromosome-based methods. The telomere-centromere orientation of DNA clones separated by 40 kb can be resolved on early pachytene chromosomes. Genetic recombination is generally evenly distributed along rice chromosome 10. However, the highly heterochromatic short arm shows a lower recombination frequency than the largely euchromatic long arm. Suppression of recombination was found in the centromeric region, but the affected region is far smaller than those reported in wheat and barley. Our FISH mapping effort also revealed the precise genetic position of the centromere on chromosome 10.     

Mapping of18-26S rDNA loci in four species of the genus Paris by fluorescence in situ hybridization（FISH）

18-26S rDNA loci were mapped on chromosomes in four species of Par is,and the num-ber and position of rDNA sites in these species were compared f or analysis of the distribution of the sites. All the plants were diploids,and t he genome consisted of five chromosomes,A,B,C,D and E. （1）P. polyphylla var. yunnanensis,2n=10=6m+4t. Two18-26S rDNA loci were de-tected on the short arms o f C and D chromosomes;（2）P. forrestii,2n=10=6m+4t. One locus was detected on th e long arm of B chromosome,and also two loci on the short arms of C and D chromosomes;（3）P. axialis. 2n=10=6m（2sat）+4t（2sat）+1-2B. Two loci were detected o n the short arms of C and D chromosomes. One locus was detected in the cell with t wo B-chromosomes（B）,but none was detected in that with only one B chromosome, indicating that rRNA gene existed on B chromsome,and an unequal division occurr ed during mitotic cycle of B-chromosomes. （4）P. daliensis,2n=10=4m+2sm+2st+2t. O ne locus was detected on the short arm of D chromo-some. The signals of18-26S rD NA appeared not only in the second constriction but also in the other regions of chromosome. It is noteworthy that one locus was detected in the terminal region o n the short arm of C chromosome in all the four species studied.     

番茄的CPD带型和45S rDNA位点的鉴别

采用CPD（PI和DAPI组合）染色对番茄减数分裂粗线期和有丝分裂中期染色体进行了显带分析,随后用两种不同的45S rDNA克隆在相同的分裂相进行了荧光原位杂交定位分析。CPD染色在8条粗线期染色体上显示出了10条红色的CPD带纹,在6对有丝分裂中期染色体上显示出了12条CPD带纹。有丝分裂中期染色体上的CPD带纹与粗线期染色体上显著的带纹具有对应性。用改良的CPD染色程序清晰而稳定地显示出这些特征性的CPD带纹为番茄的染色体,特别是有丝分裂中期染色体提供了新的识别标记。用番茄的一个45S rDNA克隆进行的荧光原位杂交,不仅在位于2号染色体短臂的随体上显示了强的杂交信号,而且在粗线期染色体的5个CPD带区或有丝分裂中期染色体的4对CPD带区显示了弱的杂交信号。然而,用来自小麦的45S rDNA克隆pTa71进行的原位杂交却只在随体上显示了杂交信号。鉴于所用的两个45S rDNA克隆在序列上的差异,推断在番茄基因组中只有随体含有45S rDNA单位的编码区,即番茄只有一对45S rDNA位点。     

Cytogenetic characterization of the bay scallop, Argopecten irradians irradians, by multiple staining techniques and fluorescence in situ hybridization

The chromosomes of Argopecten irradians irradians were studied by various cytogenetic approaches. Conventional chromosome characterization built on C-banding, DAPI-staining, and silver staining was complemented by the physical mapping of ribosomal DNA and telomeric sequence (TTAGGG)n by FISH. Results showed that the constitutive heterochromatin revealed by C-banding was mainly distributed at telomeric and centromeric regions. However, interstitial C-bands were also observed. The pattern of DAPI banding was almost consistent with that of C-banding. Silver staining revealed that NORs were located on the short arms of chromosome 3 and 10, and this was further confirmed by FISH using 18S-28S rDNA. 5S rDNA was mapped as two distinguishable loci on the long arm of chromosome 11. 18S-28S and 5S rDNA were located on different chromosomes by sequential FISH. FISH also showed that the vertebrate telomeric sequence (TTAGGG)n was located on both ends of each chromosome and no interstitial signals were detected. Sequential 18S-28S rDNA and (TTAGGG)n FISH demonstrated that repeated units of the two multicopy families were closely associated on the same chromosome pair.     

Ribosomal DNA distribution in somatic chromosomes of Stangeria eriopus (Stangeriaceae, Cycadales) and molecular-cytotaxonomic relationships to some other cycad genera

Somatic chromosomes ofStangeria eriopus (Stangeriaceae, Cycadales) were investigated by fluorescentin situ hybridization (FISH) using an 18S ribosomal DNA (rDNA) probe.Stangeria eriopus showed a chromosome number of 2n=16 with a karyotype of 12 median-, 2 subterminal-, and 2 terminal-centromeric chromosomes. FISH study ofS. eriopus revealed 16 signals made up of rDNA sites located on the terminal regions of the long arms of the 7 median- and 2 subterminal-centromeric chromosomes, on terminal region of the short arm of the 1 median-centromeric chromosome, on the terminal regions of the long and the short arms of 1 median- and 2 terminal-centromeric chromosomes. This result suggests that, not only karyomorphologically but also molecular-cytologically, the genusStangeria may be more closely related to the genusCeratozamia than the genusBowenia or the genusMicrocycas previously hypothesized.     

Ribosomal DNA,tri- and bi-partite pericentromeres in the permanent translocation heterozygote <Emphasis Type="Italic">Rhoeo spathacea</Emphasis>

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A₃-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.     

Physical mapping of the 18S-25S and 5S ribosomal RNA genes in diploid bananas

Fluorescent in situ hybridisation (FISH) was used to determine the number and distribution of the 18S-25S and 5S rDNA sites on mitotic chromosomes of 6 wild and 2 edible diploid (2n=22) accessions belonging to the two banana species, Musa acuminata and M. balbisiana. FISH with the 18S-25S probe resulted in signals on one pair of chromosomes, the position of signals corresponded to the secondary constriction at the end of a short arm. The intensity of labelling was different between the homologues and the larger site corresponded to a larger secondary constriction. This labelling pattern was observed consistently in all genotypes. On the other hand, differences in the number of 5S sites were observed between the accessions. While in some of the wild seeded species, the 5S rDNA was localised on two pairs of chromosomes, hybridisation signals appeared on three pairs of chromosomes in other wild accessions. Quite unexpectedly, only five sites of 5S rDNA were reproducibly observed in the two vegetatively propagated diploid edible cultivars, Pisang Mas and Niyarma Yik, evidence for structural heterozygosity. A dual colour FISH showed that in all accessions, the satellite chromosomes carrying the 18S-25S loci did not carry the 5S loci. The results demonstrate that molecular cytogenetics can be applied to Musa and that physical cytogenetic maps can be generated. This revised version was published online in July 2006 with corrections to the Cover Date.     

植物45S rDNA的染色体位置的CPD染色和FISH分析

采用PI和DAPI组合(CPD)染色结合45SrDNA探针的荧光原位杂交(FISH)对分属6个科的16种植物的45S rDNA的染色体位置进行了分析。在所有供试植物中,共检测到53个45S rDNA位点。大多数45S rDNA位点分布在染色体的短臂;位于染色体臂内和染色体末端的位点的比例大体相当;多数位于染色体臂内的45S rDNA位点有次缢痕形成,但rDNA重复单位簇所处的位置存在差异。根据45S rDNA所处的染色体臂的不同、距着丝粒远近的差异、形成次缢痕与否以及rDNA重复单位簇相对于次缢痕的位置等特征,将植物的45S rDNA位点划分为12种染色体分布类型。基于我们的结果和其他的报道对45S rDNA位点、核仁组织区(NOR)、次缢痕和随体相互之间的关系进行了分析。