Bryophyte 5S rDNA was inserted into 45S rDNA repeat units after the divergence from higher land plants

The 5S ribosomal RNA genes (5S rDNA) are located independently from the 45S rDNA repeats containing 18S, 5.8S and 26S ribosomal RNA genes in higher eukaryotes. Southern blot and fluorescence in situ hybridization analyses demonstrated that the 5S rDNAs are encoded in the 45S rDNA repeat unit of a liverwort, Marchantia polymorpha, in contrast to higher plants. Sequencing analyses revealed that a single-repeat unit of the M. polymorpha nuclear rDNA, which is 16103 bp in length, contained a 5S rDNA downstream of 18S, 5.8S and 26S rDNA. To our knowledge, this is the first report on co-localization of the 5S and 45S rDNAs in the rDNA repeat of land plants. Furthermore, we detected a 5S rDNA in the rDNA repeat of a moss, Funaria hygrometrica, by a homology search in a database. These findings suggest that there has been structural re-organization of the rDNAs after divergence of the bryophytes from the other plant species in the course of evolution.     

Localization of 45S and 5S rDNA sites and karyotype of Chrysanthemum and its related genera by fluorescent in situ hybridization

The phylogenetic relationships among Chrysanthemum and its related genera (Anthemideae, Asteraceae) is poorly understood. In the present study, these relationships were investigated using 45S and 5S ribosomal DNA (rDNA)-targeted fluorescent in situ hybridization. The results showed that there were two 45S rDNA signals present in Crossostephium chinense, four 45S rDNA signals in Cercidiphyllum japonicum, Artemisia sieversiana, Artemisia annua and Artemisia absinthium, six 45S rDNA signals in Chrysanthemum boreale and Pyrethrum parthenium, eight 45S rDNA signals in Chrysanthemum nankingense, Chrysanthemum dichrum, Chrysanthemum lavandulifolium and Tanacetum vulgare, and ten 45S rDNA signals in Ajania przewalskii. For the 5S rDNA locus, two 5S rDNA signals were present in C. nankingense, C. dichrum, C. lavandulifolium, C. boreale, C. japonicum, C. chinense and P. parthenium, four in A. sieversiana, A. annua, A. absinthium and A. przewalskii, and six 5S in T. vulgare. In addition, karyotypes of the 12 species were investigated. From this study, we inferred that Chrysanthemum was closely related to Ajania, and that Chrysanthemum species originating from China and Japan may have evolved differently. These findings add a new level to the understanding of the phylogenetic relationships of Chrysanthemum and related genera.     

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.     

Physical mapping of 5S and 45S rDNA loci in pufferfishes (Tetraodontiformes)

Chromosomal features, location and variation of the major and minor rDNA genes cluster were studied in three pufferfish species: Sphoeroides greeleyi and Sphoeroides testudineus (Tetraodontidae) and Cyclichthys spinosus (Diodontidae). The location of the major rDNA was revealed with an 18S probe in two loci for all species. The minor rDNA loci (5S rDNA) was found in one chromosome pair in tetraodontid fishes and four sites located on two distinct chromosomal pairs in C. spinosus. A syntenical organization was not observed among the ribosomal genes. Signal homogeneity for GC/AT-DNA specific fluorochromes was observed in diodontid fish except in the NORs regions, which were CMA₃-positive. Giemsa karyotypes of tetraodontid species presents 2n = 46, having the same diploid value of other Sphoeroides species that have been investigated. On the other hand, the karyotype of C. spinosus, described for the first time, shows 2n = 50 chromosomes (4m + 18sm + 12st + 16a). The foreknowledge of the karyotypic structure of this group and also the physical mapping of certain genes could be very helpful for further DNA sequence analysis.     

云实的45S rDNA检测和核型分析

该研究对药用植物云实的有丝分裂早中期、中期染色体进行了CPD(PI和DAPI组合)染色和相继的45S rDNA荧光原位杂交分析,并结合染色体测量和CPD带、45S rDNA杂交信号建立了其核型。结果显示:(1)云实的单倍基因组总长度为(30.38±1.58)μm;云实的核型公式为2n=24=14m+10sm (2SAT),染色体相对长度范围为11.22～7.12;核型不对称性参数CI、A1、A2、As K (%)、TF%、AI分别为41.63±6.70、0.27、0.16、58.18、41.82、2.57,核型属于2A类型。(2)云实的二倍体基因组具有9个45S rDNA位点,其中第3、8、9和12号染色体的短臂末端的位点成对存在,第10号染色体仅1个成员的短臂末端具有45S位点,呈现杂合性。该研究首次建立了云实的分子细胞遗传学核型,为该物种的基因组研究提供了基础资料。     

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。     

A tandemly repetitive centromeric DNA sequence of the fish Hoplias malabaricus (Characiformes: Erythrinidae) is derived from 5S rDNA

A substantial fraction of the eukaryotic genome consists of repetitive DNA sequences that include satellites, minisatellites, microsatellites, and transposable elements. Although extensively studied for the past three decades, the molecular forces that generate, propagate and maintain repetitive DNAs in the genomes are still discussed. To further understand the dynamics and the mechanisms of evolution of repetitive DNAs in vertebrate genome, we searched for repetitive sequences in the genome of the fish species Hoplias malabaricus. A satellite sequence, named 5SHindIII-DNA, which has a conspicuous similarity with 5S rRNA genes and spacers was identified. FISH experiments showed that the 5S rRNA bona fide gene repeats were clustered in the interstitial position of two chromosome pairs of H. malabaricus, while the satellite 5SHindIII-DNA sequences were clustered in the centromeric position in nine chromosome pairs of the species. The presence of the 5SHindIII-DNA sequences in the centromeres of several chromosomes indicates that this satellite family probably escaped from the selective pressure that maintains the structure and organization of the 5S rDNA repeats and become disperse into the genome. Although it is not feasible to explain how this sequence has been maintained in the centromeric regions, it is possible to hypothesize that it may be involved in some structural or functional role of the centromere organization.     

Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes,Prochilodontidae)

A single NOR-bearing chromosome pair was identified by silver nitrate staining in a previous study of the fish Prochilodus argenteus from the S ã o Francisco River (MG, Brazil), with a third metacentric chromosome sporadically bearing active NOR. The present study focused on an analysis of the chromosomal localization of both the major (45S) and the minor (5S) rRNA genes using FISH. The use of the 18S rDNA probe confirmed the previous Ag-NOR sites interstitially located in a large metacentric pair and also identified up to three other sites located in the telomeric regions of distinct chromosomes, characterizing an interindividual variation of these sites. In addition, the 5S rDNA site was revealed adjacent to the major NOR site, identified at the end of the large Ag-NOR bearing metacentric chromosome. In a few metaphases, an additional weak hybridization signal was observed in a third chromosome, possibly indicating the presence of another 5S rDNA cluster. Despite a lower karyotype diversification (2n=54 and FN=108) often observed among species of Prochilodontidae, variations involving both 45S and 5S rRNA genes could play an important role in their chromosome diversification.     

基于荧光原位杂交技术的紫薇属植物核型分析

以紫薇(Lagerstroemia indica)、尾叶紫薇(L.caudata)、屋久岛紫薇(L.fauriei)和福建紫薇(L.limii)4种紫薇属植物为材料,利用染色体荧光原位杂交技术(FISH)获得了4种紫薇属植物的有丝分裂中期染色体FISH图及核型参数,分析了45SrDNA在紫薇属植物染色体上的数量和分布特点。结果表明,4种紫薇属植物染色体上均具有1对45SrDNA杂交位点,位于较长染色体短臂的近端部,紫薇、尾叶紫薇、屋久岛紫薇和福建紫薇的核型公式分别为2n=48=2M+24m+22sm、2n=48=30m+18sm、2n=48=2M+20m+26sm和2n=48=2M+32m+14sm,均为2A型。该研究首次获得了紫薇属植物45SrDNA荧光原位杂交核型,为紫薇属植物亲缘关系研究和细胞生物学研究提供了分子细胞学依据。     

5S rDNA variation and its phylogenetic inference in the genus <Emphasis Type="Italic">Leporinus</Emphasis> (Characiformes: Anostomidae)

5S rDNA sequences have proven to be valuable as genetic markers to distinguish closely related species and also in the understanding of the dynamic of repetitive sequences in the genomes. In the aim to contribute to the knowledge of the evolutionary history of Leporinus (Anostomidae) and also to contribute to the understanding of the 5S rDNA sequences organization in the fish genome, analyses of 5S rDNA sequences were conducted in seven species of this genus. The 5S rRNA gene sequence was highly conserved among Leporinus species, whereas NTS exhibit high levels of variations related to insertions, deletions, microrepeats, and base substitutions. The phylogenetic analysis of the 5S rDNA sequences clustered the species into two clades that are in agreement with cytogenetic and morphological data.     

Molecular cytogenetics studies in Reichardia tingetana: Physical mapping of heterochromatin,telomere repeats,and 5S and 45S rDNA by 4′, 6‐diamidino‐2‐phenylindole and fluorescence in situ hybridization

Abstract Molecular cytogenetics studies of A‐T‐rich regions, telomeres, and 5S and 45S rDNA sites on the chromosomes of Reichardia tingetana Roth (2n= 16; diploid) were done using 4′, 6‐diamidino‐2‐phenylindole (DAPI) and fluorescence in situ hybridization (FISH). The species were collected from three geographically isolated populations at Borg El Arab (salt marsh habitat), and Rashed and Shosha (sandy clay habitats) in Egypt. The three populations showed the chromosome number of all plants are diploid except for two tetraploid samples from Shosha. Plants from both Rashed and Shosha showed similarity in the distribution of six DAPI bands on six chromosomes, whereas those of Borg El Arab showed a distribution of 16 bands on 14 chromosomes. The FISH signals of the telomeres, and 5S and 45S rDNA, were at the telomeres of all chromosomes, two interstitial, and four terminal, respectively. The combination of DAPI and FISH showed colocalization of the DAPI bands with two 5S and two 45S rDNA loci. The increased number of DAPI bands in the cytotypes from the salt marsh habitat could indicate natural genetic adaptation through increasing the heterochromatin of A‐T‐rich regions.     

栽培玉米亚种5 S rDNA NTS序列分析及FISH定位方法比较

选取我国7个栽培玉米亚种材料,进行5 S rDNA的非转录间隔区（nontranscribed intergenic spacer,NTS）的序列分析,比较7个亚种材料NTS序列差异并进行聚类分析,探讨其亲缘关系。研究结果表明：7个材料的NTS区GC平均含量为45．67％,核苷酸位点变异位点个数1～15,转换／颠换率为0．83～2．0,特用玉米材料均存在不同程度的缺失;7个材料主要聚为两大类,第一类群中包括甜质、马齿、硬粒、爆裂和蜡质5个亚种材料,第二类群中包括粉质和甜粉2个亚种材料。同时利用荧光原位杂交技术（fluorescence in situ hybridization,FISH）对5 S rDNA进行定位,探针标记分别采用荧光素标记和生物素标记。结果表明：生物素标记检测系统灵敏度高、杂交信号强,更适合于5 S rDNA重复序列的定位检测。     

图像反卷积复原技术在玉米45S rDNA转录图式研究中的运用

由于成像焦平面外杂光的干扰,宽场荧光显微镜所得的图像往往较模糊,使其不适合用来观察植物细胞核内的精细结构。该实验讨论了反卷积软件中对图像复原结果有较大影响的几个重要参数的设置。经过合适的图像反卷积复原,由宽场荧光显微镜获取的玉米45S rRNA原位杂交信号图像得以清楚显示。对所得杂交图像的分析表明:玉米45S rDNA转录往往先发生在核仁外围,且随着核仁转录活性的提高逐渐向核仁内部扩散,并最终与5S rRNA一起,在核仁内部的空洞结构中形成成熟rRNA。研究结果显示,反卷积复原可有效提高宽场荧光显微镜所得二维图像的分辨率,从而使宽场荧光显微镜在植物细胞核内精细结构研究中发挥更多的作用。     

黄褐棉45S rDNA的FISH定位及核型分析

黄褐棉是棉属5个四倍体棉种之一,利用荧光原位杂交技术将45S rDNA定位在黄褐棉2、4、9号染色体,2号染色体上的45S rDNA特别大,信号位于随体并覆盖了染色体的短臂,比二倍体和四倍体棉种的45SrDNA都要大得多;另外的2对信号很小,形状与陆地棉中的弱信号类似。黄褐棉的核型公式为：2n=4x=52=50m（2SAT）＋2sm,属于2B类型,第2对染色体为亚中着丝粒染色体,其余都为中部着丝粒染色体。黄褐棉的核型、随体数、45S rDNA与其他四倍体棉种区别很大,黄褐棉是一个非常特殊的四倍体棉种。     

Chromosomal Characteristics of rDNA in European Grayling <Emphasis Type="Italic">Thymallus thymallus</Emphasis> (Salmonidae)

The chromosomal characteristics, locations and variations of two classes of ribosomal DNA (5S and 18S) were studied in European grayling karyotype (Thymallus thymallus, Salmonidae). Major rDNA sites as revealed by sequential CMA₃/Ag staining and confirmed by in situ hybridization with a 18S rDNA probe were situated in two loci and were found to be polymorphic in size and displaying several distinct forms. The 5S rDNA was located by PRINS on three pairs of subtelocentric chromosomes, additional minor signal was present at the centromere of one metacentric element. 5S sites were not associated with NORs. The dosage compensation mechanism was proposed as an explanation of high frequency of lethal rDNA-deleted forms of the NOR-bearing chromosomes. Double variable pattern in the number and location of NORs supported the bi-directional evolution of salmonid rDNA loci.     

Internal transcribed spacer sequence analyses indicate cytoevolutionary patterns within Brachycome Cass. (Asteraceae)

The sequences of the Internal Transcribed Spacer regions (ITS1 and ITS2) within the genes coding for cytoplasmic ribosomal (r) RNAs on the A chromosome complement of 34 members of the higher plant genus Brachycome (synonym Brachyscome) have been compared. The ITS1 sequence of species within the B. lineariloba complex contains a 56 bp tract that is absent from at least 12 Brachycome species but is present in other species within Brachycome as well as other Asteraceae. Phylogenetic data support the suggestion that the number of chromosomes reduced in several independent Brachycome lineages during speciation. Comparisons with the B chromosome ITS2 of B. dichromosomatica cytodeme A1 suggests an origin of the B chromosome at a time prior to the divergence of the four cytodemes of B. dichromosomatica.     

Molecular organization of 5S rDNA in bitterlings (Cyprinidae)

Molecular organization and nucleotide sequences of the 5S rRNA gene and NTS were investigated in freshwater fish, bitterlings (Acheilognathinae), including 10 species/subspecies of four genera, Acheilognathus, Pseudoperilampus, Rhodeus, and Tanakia, to understand the evolutionary trait of 5S rDNA arrays. Southern hybridization analysis revealed a general trend with tandem repeats of 5S rDNA in all the examined bitterlings. Sequence analysis demonstrated a conserved 120 bp sequence of the 5S rRNA gene and a short NTS of 56–67 bp with two distinct portions, a conserved (5′-flanking portion; at positions −1 to −38) and a variable part (3′-flanking portion), in 6 of 10 species/subspecies examined. The conserved NTS region was most likely an external promoter so far observed in various vertebrates, whereas the variable NTS region could be divided into two types due to its nucleotide polymorphisms. Molecular phylogeny using the 5S rRNA gene and NTS sequences suggested the occurrence of 5S rDNA duplication before speciation and a concerted evolution for the gene and conserved NTS regions, but a birth-and-death process to maintain the variable NTS region. Thus, the 5S rDNA in the examined bitterlings might have evolved under a mixed process of evolution.     

Localization of 5S rRNA Loci in Three Coregonid Species (Salmonidae)

In the present study the chromosome distribution of the 5S rDNA loci and its relation to the major rDNA genes were investigated in three Coregonid species (Salmonidae): Coregonus lavaretus, Coregonus peled and Coregonus albula, a family which has experienced large karyotype rearrangements along its evolution starting from a tetraploid ancestor. 5S PRINS/CMA₃ sequential staining together with previous data enabled us to locate 5S rRNA genes and nucleolar organizer regions (NORs) in the three species analyzed. PRINS revealed the 5S rDNA cluster at the distal part of the long arm of a similar submetacentric chromosome pair in the three species. Our data indicate that 5S rDNA clusters have probably conserved chromosomal location in the genus Coregonus, whereas 45S rDNA (NOR) sites are clearly differentiated, from a single locus in C. peled, to multiple loci in C. lavaretus and highly polymorphic multichromosomal location in C. albula.     

Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus

BACKGROUND AND AIMS: Studying the genome structure of pines has been hindered by their large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes in different groups of pines is extremely limited. However, techniques are now available that can surmount these difficulties. The purpose of this study was to exploit some of these techniques to characterize the genome differentiation between the two subgenera of Pinus: Pinus and Strobus. METHODS: Double-probe fluorescence in-situ hybridization (FISH) was used to localize the 5S and 18S rDNA loci on chromosomes of five species from the subgenus Strobus: P. bungeana, P. koraiensis, P. armandii, P. wallichiana and P. strobus. * KEY RESULTS: The rDNA FISH pattern varied considerably among the five species, with P. bungeana being the most distinct. By comparing the results obtained with those of previous rDNA FISH studies of members of the subgenus Pinus, several general features of rDNA loci distribution in the genus Pinus can be discerned: (a) species of subgenus Strobus generally have more rDNA loci than species of subgenus Pinus, correlating with their larger genomes in the subgenus Strobus; (b) there is a clear differentiation in 5S and 18S rDNA loci linkage patterns between the two subgenera; (c) variations in the rDNA FISH pattern correlate with phylogenetic relationships among species within the subgenus; (d) P. bungeana has fewer 18S rDNA sites than other pines investigated to date, but they give intense signals, and may reflect the primary distribution of the 18S-25S rDNA loci in the genus. CONCLUSIONS: The stable differentiation in rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in the splitting of the two subgenera, and transpositional events rather than major structural changes are likely responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes.     

Karyotype analysis and physical mapping of 45S rDNA in eight species of Sophora, Robinia , and Amorpha

The karyotype analysis and physical locations of 45S rDNA were carried out by means of fluorescence in situ hybridization in three species, and two forms of Sophora, two species of Robina, and one species of Amorpha. S. japonica L., S. japonica L. f. oligophylla Franch., S. japonica L. f. pendula Loud., and S. xanthantha C. Y. Ma. are all tetraploids with 2n = 28. There were four 45S rDNA sites in pericentromeric regions of two pairs of chromosomes in each of them. S. rubriflora Tsoong. is a triploid with 2n = 21, and three sites were located in each satellite of group 5 chromosomes. In R. pseudoacacia L. (2n = 2x = 22), we examined four intensive signals in telomeric regions of two pairs of satellite chromosomes. In R. hispida L. (2n = 2x = 30), there were four other signals in centromeric regions besides those like in R. pseudoacacia. Amorpha fruticosa L. has most chromosomes (2n = 40) among the eight materials, however, there were only six 45S rDNA loci and they laid in centromeric regions, and satellites of three pairs of chromosomes. 45S rDNA is a valuable chromosomal landmark in karyotype analysis. The distribution and genomic organization of rDNA in the three genera were also discussed. __________ Translated from Acta Botanica Yunnanica, 2005, 27(3): 261–268 [译自: 云南植物研究, 2005, 27(3): 261–268]