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.     

Chromosomal Localization and Sequence Variation of 5S rRNA Gene in Five Capsicum Species

Chromosomal localization and sequence analysis of the 5S rRNA gene were carried out in five Capsicum species. Fluorescence in situ hybridization revealed that chromosomal location of the 5S rRNA gene was conserved in a single locus at a chromosome which was assigned to chromosome 1 by the synteny relationship with tomato. In sequence analysis, the repeating units of the 5S rRNA genes in the Capsicum species were variable in size from 278 bp to 300 bp. In sequence comparison of our results to the results with other Solanaceae plants as published by others, the coding region was highly conserved, but the spacer regions varied in size and sequence. T stretch regions, just after the end of the coding sequences, were more prominant in the Capsicum species than in two other plants. High G-C rich regions, which might have similar functions as that of the GC islands in the genes transcribed by RNA PolII, were observed after the T stretch region. Although we could not observe the TATA like sequences, an AT rich segment at -27 to -18 was detected in the 5S rRNA genes of the Capsicum species. Species relationship among the Capsicum species was also studied by the sequence comparison of the 5S rRNA genes. While C. chinense, C. frutescens, and C. annuum formed one lineage, C. baccatum was revealed to be an intermediate species between the former three species and C. pubescens.     

Phylogenetic and genomic relationships in Setaria italica and its close relatives based on the molecular diversity and chromosomal organization of 5S and 18S-5.8S-25S rDNA genes

We have analyzed the phylogenetic and genomic relationships in the genus Setaria Beauv. including diploid and tetraploid species, by means of the molecular diversity of the 5S rDNA spacer and chromosomal organization of the 5S and 18S-5.8S-25S rDNA genes. PCR amplification of the 5S rDNA sequences gave specific patterns. All the species studied here share a common band of about 340 bp. An additional band of an approximately 300-bp repeat unit was found for Setaria verticillata and the Chinese accessions of Setaria italica and Setaria viridis. An additional band of 450 bp was found in the sole species Setaria faberii. Fluorescent in situ hybridization was used for physical mapping of the 5S and 18S-5.8S-25S rDNA genes and showed that they are localized at two separate loci with no polymorphism of chromosome location among species. Two chromosome pairs carrying the 5S and 18S-5.8S-25S rDNA clusters can now be unambiguously identified using FISH. Phylogenetic trees based on the variation of the amplified 5S rDNA sequences showed a clear separation into four groups. The clustering was dependent on the genomic composition (genome A versus genome B) and confirmed the closest relationship of S. italica and S. viridis accessions from the same geographical region. Our results confirm previous hypotheses on the domestication centers of S. italica. They also show the wide difference between the A and B genomes, and even clarify the taxonomic position of S. verticillata. Received: 28 August 2000 / Accepted: 27 January 2001     

Mitochondrial DNA sequence variation of four <Emphasis Type="Italic">Saccharina</Emphasis> species (Laminariales,Phaeophyceae) growing in Japan

The complete mitochondrial DNA (mtDNA) was sequenced for four Saccharina species including three varieties of Saccharina japonica in Hokkaido: S. japonica; S. japonica var. religiosa; S. japonica var. ochotensis; S. japonica var. diabolica; Saccharina longipedalis; Saccharina angustata; and Saccharina coriacea. Furthermore, the structure and the sequence were compared among them. The total nucleotide length was 37,500–37,657 bp. All mtDNAs were mapped and no differences in the organization of the coding region were found. From the total alignment of S. japonica including the three varieties and S. longipedalis, nucleotide substitutions were detected at 68 sites, and a nucleotide insertion/deletion was detected at one site on rps19 for the whole genome. Variable regions useful for varieties distinction of S. japonica were trnI, trnM, rps19, ORF41, and seven spacers. The pairwise distance between S. japonica, S. angustata, and S. coraiacea was 0.000–0.116. Sequencing comparisons of rps7, rps11, rpl5, and tatC were thought to be useful tools for discrimination and phylogenetic analysis of Saccharina species having similar morphologies.     

Localization of 5S and 25S rRNA genes on Somatic and meiotic chromosomes in <Emphasis Type="Italic">Capsicum</Emphasis> species of chili pepper

The loci of the 5S and 45S rRNA genes were localized on chromosomes in five species of Capsicum, namely, an-nuum, chacoense, frutescens, baccatum, and chinense by FISH. The 5S rDNA was localized to the distal region of one chromosome in all species observed. The number of 45S rDNA loci varied among species; one in annuum, two in chacoense, frutescens, and chinense, and four in baccatum, with the exceptions that ‘CM334’ of annuum had three loci and ‘tabasco’ of frutescens had one locus. ‘CM334’-derived BAC clones, 384B09 and 365P05, were screened with 5S rDNA as a probe, and BACs 278M03 and 262A23 were screened with 25S rDNA as a probe. Both ends of these BAC clones were sequenced. FISH with these BAC probes on pachytenes from ‘CM334’ plant showed one 5S rDNA locus and three 45S rDNA loci, consistent with the patterns on the somatic chromosomes. The 5S rDNA probe was also applied on extended DNA fibers to reveal that its coverage measured as long as 0.439 Mb in the pepper genome. FISH techniques applied on somatic and meiotic chromosomes and fibers have been established for chili to provide valuable information about the copy number variation of 45S rDNA and the actual physical size of the 5S rDNA in chili.     

Evolutionary dynamics of 5S rDNA location in acridid grasshoppers and its relationship with H3 histone gene and 45S rDNA location

We analyze the chromosomal location of 5S rDNA clusters in 29 species of grasshoppers belonging to the family Acrididae. There was extensive variation among species for the number and location of 5S rDNA sites. Out of 148 sites detected, 75% were proximally located, 21.6% were interstitial, and only 3.4% were distal. The number of 5S rDNA sites per species varied from a single chromosome pair (in six species) to all chromosome pairs (in five species), with a range of intermediate situations. Thirteen chromosomes from eight species carried two 5S rDNA clusters. At intraspecific level, differences among populations were detected in Eyprepocnemis plorans, and some heteromorphisms have also been observed in some species. Double FISH for 5S rDNA and H3 histone gene DNA, performed on 17 of these 29 species, revealed that both markers are sometimes placed in a same chromosome but at different location, whereas they appeared to co-localize in five species (Calliptamus barbarus, Heteracris adpersa, Aiolopus strepens, Oedipoda charpentieri and O. coerulescens). Double fiber-FISH in A. strepens and O. coerulescens showed that the two DNAs are closely interspersed with variable relative amounts of both classes of DNA. Finally, no correlation was observed between the number of 5S and 45S rDNA clusters in 23 species where this information was available. These results are discussed in the light of possible mechanisms of spread that led to the extensive variation in the number of clusters observed for both rDNA types in acridid grasshoppers.     

Marked Intragenomic Heterogeneity and Geographical Differentiation of nrDNA ITS in <Emphasis Type="BoldItalic">Larix</Emphasis><Emphasis Type="BoldItalic">potaninii</Emphasis> (Pinaceae)

Nuclear ribosomal DNA (nrDNA) of gymnosperms, especially Pinaceae, is characterized by slow concerted evolution and exhibits substantial ITS-region length variation (975–3663 bp), in sharp contrast to the narrow range (565–700 bp) in angiosperms. Here we examined intra- and intergenomic heterogeneity of the nrDNA internal transcribed spacer (ITS) region in four varieties of Larix potaninii, a species from the mountainous areas of western China. Two clones with more than a 100-bp deletion in ITS1 were detected in L. potaninii var. chinensis and L. potaninii var. australis, respectively. The deletion resulted in the loss of most part, including the motif sequence, of subrepeat 1 (SR1). Sequence divergence and phylogenetic analyses showed that some clones would be pseudogenes given their low GC content, high substitution rates, unique positions in the phylogenetic trees, or significant length variation. These clones might represent orphons or paralogues at minor loci resulting from large-scale gene or chromosome reorganization. Some recombinants characterized by chimeric structure and discordant phylogenetic positions in partitioned sequence analyses indicate that unequal crossing-over plays an important role in the process of nrDNA concerted evolution. In addition, some varieties of L. potaninii might have experienced an nrDNA founder effect parallel to their geographical isolation.     

Karyomorphological study of the Spiraea japonica complex (Rosaceae)

Reported in this paper is a karyomorphological study on one natural population of each of eight varieties in theSpiraea japonica complex. The interphase and mitotic prophase can be classified into the simple chromocenter type and the proximal type, respectively. The metaphase karyotypes of the eight varieties were formulated as follows:S. japonica var.japonica: 2n=18=14m+2sm+2st;S. japonica var.acuta: 2n=18=11m+4sm+3st;S. japonica var.incisa: 2n=18=12m+4sm+2st;S. japonica var.stellaris: 2n=18=15m+1sm+2st;S. japonica var.acuminata: 2n=18=14m+2sm+2st;S. japonica var.ovalifolia: 2n=18=10m+2sm+2st+4t;S. japonica var.glabra: 2n=18=10m+4sm+4st;S. japonica var.fortunei: 2n=36=17m+16sm+3st. Karyomorphological study reveals that the chromosome numbers within the complex are stable, and that karyomorphological divergence between the varieties lies mainly in chromosome size and organization. Based on the karyomorphological data and geographical distribution of the complex, the differentiation pattern as well as evolutionary mechanism of the complex is evaluated.     

Molecular characterization of the 5S ribosomal gene of the <Emphasis Type="Italic">Bradysia hygida</Emphasis>(Diptera:Sciaridae)

The rRNA genes are amongst the most extensively studied eukaryotic genes. They contain both highly conserved and rapidly evolving regions. The aim of this work was to clone and to sequence the Bradysia hygida 5S rDNA gene. A positive clone was sequenced and its 346 bp sequence was analyzed against the GenBank database. Sequence analysis revealed that the B. hygida 5S (Bh5S) rDNA gene is 120 bp long and is 87% identical to the aphid Acyrthosiphon magnoliae 5S rDNA gene. The Bh5S rDNA gene presents two unusual features: a GG pair at the 5' end of the gene sequence and the localization of the polyT signal immediately after the 3' end of the gene. In situ5S hybridization experiments revealed that the Bh5S rDNA gene is localized in the autosomal A chromosome     

Sequence polymorphism and chromosomal localization of 5S rDNA of three cultivated varieties of sweetpotato (Ipomoea batatas (L.) Lam.)

The 5S rDNA of plant is organized into clusters of tandem repeat units which include a coding region of 5S rRNA gene and variable sequences of nontranscribed spacer (NTS). In this study, we investigated sequence polymorphism and chromosomal localization of 5S rDNA in three cultivated varieties of sweet potato (Ipomoea batatas Lam.). Two different PCR products of 5S rDNA were amplified from all three varieties, as approximately 0.25 kb and 0.34 kb with multiples. In sequence analysis, the 5S rDNA ofI. batatas were discriminated from four consensus sequences by in reasonable sizes and molecular informative factors. Four consensus sequences were divided into three short sequences, including 263, 253, and 243 – 283 bp by sequence variation between 160 and 186 bp in NTS region, and one long sequence with 340 bp. To identify molecular relationship among varieties, phylogenetic analysis was applied. A total of 35 sequenced clones in this study were classified into four groups in phylogenetic tree. Interestingly, two varieties included all four groups, but one variety only two groups. To localize the physical map of 5S rDNA, fluorescencein situ hybridization (FISH) was performed in metaphase chromosomes of each varieties. In 90 chromosomes ofI. batatas, 6 loci of 5S rDNA were detected in chromosomes for all varieties. Our results will help to further more understand the genomic relationship inI. batatas, to investigate molecular relationship among varieties.     

BIOSYSTEMATIC STUDIES IN THE BOUTELOUA CURTIPENDULA COMPLEX. III. POLLEN SIZE AS RELATED TO CHROMOSOME NUMBERS

Pollen size statistics are presented for 10 closely related species of Bouteloua and relationships between pollen size and chromosome numbers are presented for 13 populations of 5 species and 3 varieties. With 1 exception, all populations of all taxa conformed to a general pattern of pollen size dependent upon chromosome number. Chromosome numbers varied from 2n = 20 to 2n = ca. 103, with several independent aneuploid series. Statistical analyses were made of pollen size as related to chromosome number in the 3 varieties of B. curtipendula. These data showed that tetraploids (2n = 40) of var. tenuis had significantly greater pollen size and coefficient of variation than diploids (2n = 20) of the same variety. Similarly, aneuploids of var. curtipendula with 2n = 45 to 2n = 64 chromosomes had significantly larger and more variable pollen than tetraploids (2n = 40) of the same variety. Highly significant positive regression coefficients were obtained from analyses of chromosome numbers and mean pollen size, and chromosome numbers and coefficient of variation, for var. curtipendula. Regression coefficients for var. caespitosa populations with chromosome numbers over the hexaploid (2n = 60) level were not significant.     

Two classes of 5S rDNA unit arrays of the silver fir, Abies alba Mill.: structure, localization and evolution

The structure and organization of the 5S ribosomal DNA units of the silver fir, Abies alba Mill., as well as their position in the chromosome complement were investigated. PCR amplification of the gene and nontranscribed spacer region, sequence analysis and Southern hybridization, using a homologous probe, detected DNA sequences of approximately 550 bp and 700 bp. Sequence analysis of the spacers revealed that the difference in length between the sequences occurred in the middle spacer region as a result of the amplification of a 75-bp sequence of the short unit class, which is organized in four 54- to 68-bp tandem repeats in the long spacer unit. The 5S rDNA transcribed region is 120 bp long and shows high sequence similarity with other gymnosperm species. The comparative analysis of 5 and 3 flanking sequences of 5S rRNA genes of silver fir and other gymnosperms indicates that A. alba spacer units have the same rate of evolution and are more closely related to Larix and Pseudotsuga than to Pinus and Picea. Southern hybridization and fluorescence in situ hybridization of metaphase chromosomes of A. alba suggest that the short and long spacer units are organized as separate tandem arrays at two chromosomal loci on chromosomes V and XI.     

Identification of Mytilus spp. and Pecten maximus in Irish Waters by Standard PCR of the 18S rDNA Gene and Multiplex PCR of the 16S rDNA Gene

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.     

Origin and Inheritance of Group I Introns in 26S rRNA Genes of Gaeumannomyces graminis

Studies of the distribution of the three group I introns (intron A, intron T, and intron AT) in the 26S rDNA of Gaeumannomyces graminis had suggested that they were transferred to a common ancestor of G. graminis var. avenae and var. tritici after it had branched off from var. graminis. Intron AT and intron A exhibited vertical inheritance and coevolved in concert with their hosts. Intron loss could occur after its acquisition. Loss of any one of the three introns could occur in var. tritici whereas only loss of intron T had been found in the majority of var. avenae isolates. The existence of isolates of var. tritici and var. avenae with three introns suggested that intron loss could be reversed by intron acquisition and that the whole process is a dynamic one. This process of intron acquisition and intron loss reached different equilibrium points for different varieties and subgroups, which explained the irregular distribution of these introns in G. graminis. Each of the three group I introns was more closely related to other intron sequences that share the same insertion point in the 26S rDNA than to each other. These introns in distantly related organisms appeared to have a common ancestry. This system had provided a good model for studies on both the lateral transfer and common ancestry of group I introns in the 26S rRNA genes. Received: 17 May 1996 / Accepted: 14 January 1997     

Isolation and characterization of microsatellite markers in Perilla frutescens Brit

We isolated and characterized 13 polymorphic microsatellite primers from Perilla frutescens Brit. var. frutescens by using a modified method that involves one‐way PCR amplification with single primer prior to enrichment with an ‘oligo hook’. The efficiency of this procedure for isolating unique microsatellite sequences was approximately 77%. The number of alleles per microsatellite locus ranged from three to 10 with an average of 6.5 alleles per locus while fragment size varied from 156 to 298 bp. The observed and expected heterozygosities ranged from 0.52 to 0.86 and 0.52 to 0.89, respectively. These newly isolated microsatellite markers are expected to provide valuable resources for different genetic studies currently underway in our Perilla genome research program.     

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.     

凝胶电泳与共头霉分类

对于共头霉的唯一已知种总状共头霉Syncephalastrum racemosum Cohn ex Schroet.(S 1)和新发现的具单孢孢子囊的新种单孢共头霉下的三个变种,原变种S.monosporum Zheng et al. var. monosporum( S7, S8, S9),冠囊变种var. cristatum Zheng et al. (S10, S11)及多重生变种var. pluriproliferum Zheng et al.(S12) 6株菌的蛋白和酯酶进行了聚丙烯酰胺凝胶电泳的研究.这4个分类群的蛋白图谱和酯酶酶谱互不相同,而同一分类群内各个菌株之间则有很高的相似性.从计算出的相似性百分数来看,3个新变种相互之间有较为密切的关系,而和S. racemosum虽然较疏远,但也有相当程度的亲缘关系.这些结果被认为支持了根据形态鉴定而定这三个新变种属于同一新种内并将其和S. racemosum一并归属于一个属内.由此可见,聚丙烯酰胺凝胶电泳是共头霉分类的有用的辅助手段.     

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.