A second locus for the 5S multigene family in Secale L.: sequence divergence in two lineages of the family

The 5S RNA genes in Secale sp. are arranged as tandem arrays of a 460- and 480-bp repeating sequence. These size classes were initially discovered by restriction endonuclease analysis using BamHI and subsequently by DNA sequencing of cloned units. The length variation between short and long units originated from major deletion-insertion events in the noncoding spacer region of the 5S DNA repeat units. In situ hybridization with [3H]cRNA and biotin-labelled probes synthesized from both the short and long 5S DNA units of S. cereale localized the sites on chromosome 1R and a new site on a chromosome identified as 5R. We propose that the chromosome 1R locus, which has been mapped previously, be named 5SDna-R1 and the second locus, reported in the present paper, be referred to as 5SDna-R2. A preferential hybridization of a probe from the long unit to the 5SDna-R2 locus and of a probe from the short unit to the 5SDna-R1 locus is reported. The clustering of long units in the 5SDna-R2 locus was confirmed by restriction endonuclease digestion of DNA from rye chromosome 5R additions to wheat. Nucleotide sequence alignment of 5S DNA repeat units from a number of Secale species, using both phenetic and cladistic computer programmes, demonstrated that two clear lineages corresponding to the long and short units existed in this genus. The different Secale species could not be unambiguously differentiated using the 5S DNA sequences.     

Evolutionary trends of different repetitive DNA sequences during speciation in the genus secale

The presence and distribution of two simple sequence repeats (SSRs), three highly repetitive sequences from rye, and the 5S rDNA have been investigated in 3 rye cultivars and 10 wild-related species of the genus SECALE: The following conclusions can be drawn in addition to detailed knowledge of the sequence content of chromatin in each accession studied: (1) Every species is unique in either or both the complement and chromosomal distribution of the six repeated sequences analyzed. (2) These sequences reveal multiple landmarks along all the rye chromosomes arms. (3) High polymorphism as well as heterozygosity between homologues in the distribution of the (AAG)(5) and (AAC)(5) was revealed in the outbreeding species of the Secale strictum complex. (4) It is possible to deduce trends in the complexity of repetitive DNA during the evolution of the genus. A possible evolutionary pathway that accounts for the present-day Secale species is presented.     

Evolutionary trends of microsatellites during the speciation process and phylogenetic relationships within the genus Secale

Eleven weedy or wild species or subspecies of the genus Secale L. were compared with a set of cultivated rye accessions, based on inter-simple sequence repeat (ISSR) markers to analyze their phylogenetic relationships. A total of 846 bands were amplified from reactions using 12 screening primers, including 79 loci with a mean of 10.1 alleles per locus. The number of amplified bands for each primer ranged from 12 to 134, with a mean of 70.5 amplified bands per primer. The presence and distribution of amplified bands in different accessions demonstrate that a rapid evolutionary trend of microsatellite repeats occurred during the speciation process from the perennial wild form to annual cultivated rye. In addition, variation, amplification, and deletion of microsatellites in genomes revealed phylogenetic relationships in the genus Secale. Analysis of the presence, number, and distribution of amplified bands in genomes, as well as the comparison with genetic similarity (GS) indices based on ISSR, indicate that Secale strictum subsp. africanum (Stapf) Hammer, Secale strictum anatolicum (Boiss.) Hammer, Secale sylvestre Host, and Secale strictum subsp. strictum (C. Presl) Hammer emerged in succession from a common ancestor of Secale following geographic separation and genetic differentiation. The annual weedy rye evolved from S. strictum subsp. strictum, which was domesticated as present-day cultivated rye. Data from ISSR analyses separated all investigated accessions of the genus Secale into three distinct groups. These results support the division of the genus Secale into three species: the annual wild species S. sylvestre; the perennial wild species S. strictum, including several differential subspecies forms such as strictum, africanum, and anatolicum; and S. cereale, including cultivated and weedy rye as subspecies forms.     

Repetitive DNA and chromosome evolution in plants

Most higher plant genomes contain a high proportion of repeated sequences. Thus repetitive DNA is a major contributor to plant chromosome structure. The variation in total DNA content between species is due mostly to variation in repeated DNA content. Some repeats of the same family are arranged in tandem arrays, at the sites of heterochromatin. Examples from the Secale genus are described. Arrays of the same sequence are often present at many chromosomal sites. Heterochromatin often contains arrays of several unrelated sequences. The evolution of such arrays in populations is discussed. Other repeats are dispersed at many locations in the chromosomes. Many are likely to be or have evolved from transposable elements. The structures of some plant transposable elements, in particular the sequences of the terminal inverted repeats, are described. Some elements in soybean, antirrhinum and maize have the same inverted terminal repeat sequences. Other elements of maize and wheat share terminal homology with elements from yeast, Drosophila, man and mouse. The evolution of transposable elements in plant populations is discussed. The amplification, deletion and transposition of different repeated DNA sequences and the spread of the mutations in populations produces a turnover of repetitive DNA during evolution. This turnover process and the molecular mechanisms involved are discussed and shown to be responsible for divergence of chromosome structure between species. Turnover of repeated genes also occurs. The molecular processes affecting repeats imply that the older a repetitive DNA family the more likely it is to exist in different forms and in many locations within a species. Examples to support this hypothesis are provided from the Secale genus.     

Detection and mapping of SSRs in rye ESTs from aluminium-stressed roots

Aluminium toxicity is a major problem for crop production on acid soils. Rye (Secale cereale L.) has one of the most efficient group of genes for aluminium tolerance, at least, four independent and dominant loci, Alt1, Alt2, Alt3 and Alt4, located on chromosome arms 6RS, 3RS, 4RL and 7RS, have been described. The increasing availability of expressed sequence tags in rye and related cereals provides a valuable resource of non-anonymous DNA molecular markers. In order to obtain simple sequence repeat (SSR) markers related with Al tolerance more than 1,199 public accessible rye cDNA sequences from Al-stressed roots were exploited as a resource for SSR markers development. From a total of 21 S. cereale microsatellite (SCM) loci analysed, 12 were located on chromosomes 1R, 2R, 3R, 4R and 5R, using wheat–rye addition lines or mapped using a F₂ population segregating for Al tolerance. Seven SCM loci were included in a rye map with other SCIM and RAPD markers. Moreover, 14 SCM loci could be associated to proteins with known or unknown function. The possible implications of these sequences in aluminium tolerance mechanisms are discussed.     

The mapping of highly-repeated DNA families and their relationship to C-bands in chromosomes of Secale cereale

The relationship between the chromosomal location of heterochromatin C-bands and of four non-homologous repeated sequence families constituting 8 to 12% of total rye DNA has been investigated in chromosomes of rye (Secale cereale) by in situ hybridisation. Three rye varieties, a set of rye disomic additions to wheat and a triticale were studied. Only centromeric and nucleolar organizer region (NOR) associated C-bands failed to display hybridisation to at least one of the sequences and many telomeric blocks of heterochromatin contained all four repeated sequence families. Both between-variety differences in the chromosomal distribution of repeated sequences, and intravarietal heterozygosities were frequently noted and are probably widespread. — Previously reported deletions of heterochromatin from King II rye chromosomes added to the Holdfast wheat complement were correlated with deletions of some, but not all, of the highly repeated sequence families. A previously unreported loss of some families from King II rye chromosome 4R/7R in a Holdfast wheat genetic background was detected. This loss was not associated with complete deletion of a C-band. A deletion has also probably occurred from the short arm telomere of 4R/7R in the triticale variety Rosner. It is suggested that the families of repeats in rye telomeric heterochromatin which are absent from wheat are selected against in the wheat genetic background.     

Occurrence of rye (Secale cereale) 350-family DNA sequences inAgropyron and otherTriticeae

Evidence is presented that in the R and P genomes (Secale cereale andAgropyron cristatum, respectively) of theTriticeae there exist closely related 350-family DNA sequences in the terminal heterochromatin. This observation is compared to the relationships between these two genomes derived from a comparison of theNor and5 S DNA loci as well as the available data on morphological characters, chromosome pairing, and isozyme studies. It is concluded that the R and P genomes are not closely related and that the common presence of very similar 350-family DNA sequences reflects the parallel amplification of this family of DNA sequences.     

Evolutionary differentiation of bimaculatus group anoles based on analyses of mtDNA and microsatellite data

The bimaculatus group of anoles inhabit the northern Lesser Antilles, as far south as Dominica. This study uses 1005 base pairs (bp) of mitochondrial DNA sequence data from two genes, cytochrome b (521 bp) and cytochrome oxidase subunit I (484 bp) to reconstruct phylogenetic relationships between species and populations of anoles from all islands banks. Allele frequency data from nuclear microsatellite loci are also analysed to assess their utility in uncovering historical relationships and provide independent corroboration for the mtDNA tree. Although the number of microsatellite loci used (six) was relatively small, some essential elements of the mitochondrial DNA phylogeny were recovered successfully. Anoles from Terre de Haut, Les Saintes, previously described as a subspecies of Anolis marmoratus, are shown to be more closely related to A. oculatus and their elevation to a full species, A. terraealtae, is supported. An island colonisation sequence inferred from the phylogeny shows a general pattern of North-to South colonisation. However, the Saban anole, A. sabanus, is shown to be derived from A. marmoratus populations from western Basse Terre following a longer-range, south to north translocation.     

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

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

The structure,amount and chromosomal localisation of defined repeated DNA sequences in species of the genus Secale

The structure, copy number and chromosomal location of arrays of four families of highly repeated sequences have been investigated in representative species of the genus Secale. The four unrelated families, previously characterised in Secale cereale, have repeating units of 480, 610, 630 and 120 base pairs respectively. The following general conclusions can be drawn in addition to detailed knowledge of the sequence content of heterochromatin in each accession studied: (1) Every species is unique in its complement or chromosomal distribution or both of the four highly repeated sequence families. S. montanum and S. cereale accessions studied here show the same complement of repeated sequences, but they differ substantially in the amounts they contain of the 610 and 630 base pair (bp) families, and in the distribution over the chromosomes of the 480 bp family. The structure of the repeating unit is also different in many members of the 480 bp family in S. montanum. — (2) The substantial differences between species in the amounts of the most highly repeated DNA sequences exist in the absence of any such conspicuous differences in most other repeated sequences which were detected as fluorescent bands after restriction enzyme digestion and gel electrophoresis. — (3) Each of the different highly repeated families can exist independently of the other families, though all the families have telomeric sites. Also, in the outbreeding species, heteromorphisms are frequent, and are particularly conspicuous in hybridisation detecting the 480 bp sequence family. — (4) The association of the highly repeated sequences with heterochromatin, discussed in the accompanying paper is generally true for other species in the genus, and the lower amounts of heterochromatin in other Secale species compared to S. cereale are associated with lower amounts of specific families of highly repeated DNA sequences. — (5) Analysis of highly repeated sequence families is likely to provide an easy method of identification of new accessions of Secale.     

Ribosomal RNA Multigene Loci: Nomads of the Triticeae Genomes

The nucleolus organizing regions (NORs) on the short arms of chromosomes 1A(m) and 5A(m) of diploid wheat, Triticum monococcum L., are at the most distal loci in the linkage maps of these two chromosome arms. This distal location differs from the interstitial location of the Nor loci on chromosome arms 1BS of tetraploid Triticum turgidum L. and hexaploid T. aestivum L., 5DS of T. aestivum and diploid Ae. tauschii Coss., and 5HS of barley. Moreover, the barley 5HS locus is at a different location than the 5DS locus. However, other markers, including the centromeres, are colinear. These findings showed that the major Nor loci have repeatedly changed position in the chromosome arms during the radiation of species in the tribe Triticeae without rearrangements of the linkage groups. It is suggested that Nor loci may change position via dispersion of minor loci, that are shown here to exist in the T. monococcum genome, magnification of gene copy numbers in these minor loci, and subsequent deletion of the original major loci. Implications of these findings for the use of rRNA nucleotide sequences in phylogenetic reconstructions are pointed out.     

Occurrence of insertion sequence (IS) regions on plasmid deoxyribonucleic acid as direct and inverted nucleotide sequence duplications.

Insertion sequence (IS) regions have been identified previously as a cause of strongly polar mutations in Escherichia coli and several bacteriophages. The present experiments indicate that genetically characterized IS regions occur on bacterial plasmid deoxyribonucleic acid (DNA) as both direct and inverted DNA sequence duplications. The DNA insertion which has been shown previously (Sharp et al., 1973) to control expression of tetracycline resistance in the R6-5 plasmid, and which occurs as directly and inversely repeated DNA sequences adjacent to the region believed to contain the tetracycline resistance gene, has been identified as IS3. A second genetically characterized insertion sequence (IS1) has been identified as a direct DNA duplication occurring at both junctions of the resistance transfer factor and R-determinant components of R6-5 and related plasmids. A model is presented for the reversible dissociation of resistance transfer factor and R-determinant components of co-integrate R plasmids at the sites of DNA sequence homology provided by the repeated IS regions.     

Genomic organization of human 5 S rDNA and sequence of one tandem repeat

An organization of human 5 S rDNA repeats is inferred from Southern analyses of restriction digests of genomic DNA fractionated by pulsed-field and conventional gel electrophoreses. A single unit of 2.2 kb is repeated approximately 90 times within a 200-kb fragment (defined by enzymes that do not cleave within individual units, i.e., EcoR1, BglII, HindIII, and PvuII); a comparable number of 5 S sequences are scattered elsewhere in the genome. A lambda clone containing six complete 5 S repeats was obtained from a human placental DNA library. One repeat contains 2231 bp and includes poly(dG-dT).(dC-dA), tracts of polypyrimidine, and an Alu sequence in the spacer region. Also, 5-S-hybridizing clones, containing DNA inserts with an average size of 250 kb, have been obtained as yeast artificial chromosomes. Thus far, four clones have been partially characterized and shown to be 5 S sequences from loci separate from the tandem repeat units.     

Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.     

5S rDNA genome regions of Lens species.

The length variability of the nontranscribed spacer (NTS) of the 5S rDNA repeats was analyzed in species of the genus Lens by means of PCR amplification. The NTS ranged from approximately 227 to approximately 952 bp. The polymorphism detected was higher than previous NTS polymorphisms described in this genus. Three NTS length variants from Lens culinaris subsp. culinaris and 2 from Lens culinaris subsp. orientalis were sequenced. The culinaris NTS fragment lengths were 239, 371, and 838 bp, whereas the orientalis ones were 472 bp and 506 bp, respectively. As a result of sequence similarities, 2 families of sequences were distinguished, 1 including the sequences of 838 and 506 bp, and others with the sequences of 239, 371, and 472 bp. The 1st family was characterized by the presence of a repeated sequence designated A, whereas the 2nd family showed a single A sequence and other repeated sequences designated B, C, and D. The presence of an (AT)n microsatellite was also observed in the 2nd family of sequences. The fragments, which included the 239-bp and 838-bp NTS sequences, as well as the intergenic spacer (IGS) of the 18S-5.8S-26S ribosomal DNA also from L. culinaris subsp. culinaris, were used to localize the nucleolar organizer region (NOR) and the 5S rDNA loci in the chromosomes of several species of the genus Lens by means of fluorescence in situ hybridization (FISH). The selective hybridization of the 2 NTS probes allowed us to distinguish between different 5S rDNA chromosomal loci.     

Differential core histone binding behavior: RNA polymerase I promoter region vs 5S rDNA positioning DNA sequences

In order to further characterize the previously observed disruptive effect of the RNA polymerase I promoter sequence (Pol I) from Acanthamoeba castellanii on tandemly repeated 5S rDNA positioning sequences from sea urchin (Lytechinus variegatus), we compared the histone-binding ability of the isolated 199-bp Pol I promoter region to that of the 208-bp 5S rDNA and that of nucleosome core particle sequences isolated from chicken erythocytes. We found the 5S rDNA positioning sequence to be more efficient at forming nucleosomes than the RNA polymerase I promoter sequence. Nevertheless, examination of the free-DNA half-depletion points during the titrations suggested that twice as much histone had bound to RNA polymerase I promoter sequence as to the 5S nucleosome-positioning or core particle sequences. DNA bending analysis suggested two potential DNA bending loci in the RNA polymerase I promoter, whereas only one such locus was predicted for the 5S positioning sequence. Such mixed bending signals on the RNA polymerase I promoter could favor non-nucleosomal deposition of histones on these sequences.     

非洲黑麦染色体特异性标记的建立与应用

以非洲黑麦、小麦-非洲黑麦双二倍体、安岳排灯麦等为材料筛选100条ISSR引物。其中, 引物UBC815可在非洲黑麦中扩增出1条长561 bp的特异性片段(命名为pSaUBC815₅₆₁), 而小麦对照均未扩出该片段。引物UBC815同样能在黑麦属的瓦维洛夫黑麦(Secale vavilovii Grossh.)、森林黑麦(Secale sylvestre Host.)等5个种扩增出pSaUBC815₅₆₁。根据pSaUBC815₅₆₁设计特异PCR引物U815-F、U815-R, 对小麦族多物种进行扩增, 表明pSaUBC815₅₆₁为黑麦属特有。进而利用一套中国春-Imperial黑麦二体附加系及小麦-黑麦异源材料进行扩增, 结果显示, pSaUBC815₅₆₁分布在黑麦整套染色体上, 并且所有后代材料都能扩增出pSaUBC815₅₆₁, 表明pSaUBC815₅₆₁可作为特异性标记用来检测小麦背景中的黑麦染色质。     

Discrimination of Repetitive Sequences Polymorphism in Secale cereale by Genomic In Situ Hybridization-Banding

Genomic in situ hybridization banding (GISH-banding), a technique slightly modified from conventional GISH, was used to probe the Chinese native rye (Secale cereale L.) DNA, and enabled us to visualize the Individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat (Triticum aeativum L.) background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S.cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattem of Secale vavilovli was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromatin is discussed.     

Rapid nested PCR-based detection of Ramularia collo-cygni direct from barley

Ramularia collo-cygni is a barley pathogen of increasing importance in Northern and Central Europe, New Zealand and South America. Accurate visual and microscopic identification of the pathogen from diseased tissue is difficult. A nested PCR-based diagnostic test has been developed as part of an initiative to map the distribution of the pathogen in Scotland. The entire nuclear ribosomal internal transcribed spacer and 5.8S rRNA gene regions from 14 isolates of diverse global origin exhibited complete homology following sequence characterization. Two pairs of species-specific primers, based on inter-specific sequence divergence with closely related species, were designed and empirically evaluated for diagnostic nested PCR. Nested primers Rcc3 and Rcc4 consistently amplified a single product of 256 bp from DNA of 24 R. collo-cygni isolates of diverse global provenance, but not from other Ramularia species, or other fungi commonly encountered in cereal pathosystems, as well as Hordeum or Secale DNA preparations. Using this approach, R. collo-cygni was successfully identified from naturally infected barley leaf, awn and grain samples of diverse geographical provenance, in particular from symptoms that lacked the presence of characteristic conidiophores. It is envisaged that this assay will become established as an important tool in continuing studies into the ecology, aetiology and epidemiology of this poorly understood yet economically damaging plant pathogen.     

微卫星位点筛选方法综述

微卫星标记因其丰富的多态性和共显性等特点,已得到了广泛的应用.应用微卫星标记首先需要获得微卫星位点的序列信息,用来设计引物.获得微卫星位点的方法有多种,本文综述了获得和富集微卫星位点的常用方法.最简便、最省时的方法是从公共数据库(如EMBL、Genbank、EST数据库等)或已发表的文献中查找到微卫星位点,但只限于已经有序列数据发布的物种.第二种方法是种间转移扩增,即从相近物种的数据库中查找微卫星位点,或使用已有数据发表的遗传距离相近物种的微卫星标记.第三种方法是从基因组DNA中筛选微卫星位点,其中用于富集微卫星的方法有引物法、磁珠杂交法、尼龙膜杂交法以及RAPD技术法.