Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18-24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18-24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12-13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12-13P was very similar to GISH results, suggesting that the 12-13P sequence constitutes a major part of the repetitive DNA of C. quinoa.     

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.     

Genetic control and distribution of leucine aminopeptidase in the cultivated chenopods (Chenopodium) and related weed taxa

Isozymes of leucine aminopeptidase (LAP) in leaf tissue of the cultivated chenopods (Chenopodium quinoa and C. nuttalliae) and their sympatric weedy relatives (C. hircinum and C. berlandieri) can be electrophoretically resolved into a sum total of five anodally migrating bands. These are the products of two unlinked gene loci which apparently reflect genetic duplication via allotetraploidy. Accessions from the Andean and Mexican areas of cultivation are characterized by a lack of electrophoretically detectable variation. Andean weed and cultigen accessions appear to be genetically identical at both Lap loci, as do weed and cultigen material from Mexico. The two cultigens, and their sympatric weeds, can be differentiated by variation at the Lap-B locus, whereas they are monomorphic at Lap-A. This locus is, however, highly polymorphic in weedy C. berlandieri populations of western North America.     

Intergenic spacer (IGS) polymorphism: a new genetic marker for differentiation of Toxoplasma gondii strains and Neospora caninum

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3' end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.     

基于荧光原位杂交的藜属植物核型分析

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

Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus.

We examined the sequence variability of the nontranscribed spacer (NTS) and internal-transcribed spacer (ITS1 and ITS2) domains of the rRNA locus of Perkinsus marinus from Maryland, Florida, and Louisiana. The sequence of P. marinus DNA including the 5S rRNA, NTS, small subunit (SSU) rRNA, ITSI, and ITS2 regions confirmed their contiguity in the rRNA locus and revealed differences at 28 positions with the SSU rRNA sequences published earlier. The 307-bp polymerase chain reaction (PCR)-amplified fragments from the NTS domain of the various P. marinus isolates revealed the presence of 2 distinct sequences, designated as types I and II, that differed at 6 defined nucleotide positions. Based on these differences, nested PCR and restriction enzyme digests were used to distinguish between the 2 types. Sequences of the ITS1 and ITS2 domains of samples from either NTS type I (n = 3) or type II (n = 3) showed no variation and were identical to published sequences. Frequencies of the P. marinus NTS sequence types I and II in infected oysters varied with the geographic origin of the samples. All Maryland samples examined (n = 19) corresponded to the NTS type I sequence, the type II was the most frequent in the Florida samples (n = 17), and both types were about equally represented in the Louisiana samples (n = 19), with both sequence types found in individual oyster specimens. Although it has been suggested that P. marinus is diploid, it remains to be determined if both NTS sequence types can be present in a single P. marinus trophozoite.     

Characterization of internal structure of the nucleolar organizing region in rice (Oryza sativa L.)

The genomic sequence of the nucleolar organizing region (NOR) in rice has not been characterized fully because of the difficulty in assembling repetitive sequences in silico. Here, we used a cytogenetic approach to elucidate the internal structure of the NOR. We detected one locus of the 18S rRNA genes on 'Nipponbare' chromosome. High-resolution fiber-fluorescence in situ hybridization made it possible to visualize each rRNA gene unit in the array of rRNA genes. Signals of pairs of alternating 18S and 25S rRNA genes were detected uniformly along the DNA fiber. Intergenic spacers were shorter than the transcribed region. The rRNA genes were infrequently interrupted. These and previous results based on the sequencing of genome fragments, PCR analysis and Southern blot hybridization suggest that the internal region of the NOR is filled with a uniform array of canonical rRNA genes separated by spacers carrying three 254-bp sub-repeats.     

Regular distribution of length heterogeneities within non-transcribed spacer regions of cloned and genomic rDNA of Saccharomyces cerevisiae.

A length difference of about 50 bp in the EcoRI fragment B of the rDNA from two different strains of Saccharomyces cerevisiae has been mapped in detail by sequencing of cloned fragments. This 2.4 kb EcoRI fragment contains the start of the 35S rRNA gene at one end and the 5S rRNA gene in the middle flanked by non-transcribed spacers, NTS1 and NTS2. The difference appeared as short deletions or insertions in five regularly spaced regions within the 1 kb NTS1, 3' to the 5S rRNA gene. The same regions of heterogeneities were displayed when all available sequence data of the NTS1 were compared. Four of the variable regions are located 160-170 bp apart, indicating that they might represent linker sequences between phased nucleosomes. Two variant clones, differing in the length of one subfragment of NTS1, were isolated for each strain. In both cases these represented the major variants among chromosomal NTS1 as revealed by sequencing of genomic fragments.     

Intraindividual and Interspecies Variation in the 5S rDNA of Coregonid Fish

This study was designed to characterize further the nontranscribed intergenic spacers (NTSs) of the 5S rRNA genes of fish and evaluate this marker as a tool for comparative studies. Two members of the closely related North American Great Lakes cisco species complex (Coregonus artedi and C. zenithicus) were chosen for comparison. Fluorescence in situ hybridization found the ciscoes to have a single multicopy 5S locus located in a C band-positive region of the largest submetacentric chromosome. The entire NTS was amplified from the two species by polymerase chain reaction with oligonucleotide primers anchored in the conserved 5S coding region. Complete sequences were determined for 25 clones from four individuals representing two discrete NTS length variants. Sequence analysis found the length variants to result from presence of a 130-bp direct repeat. No two sequences from a single fish were identical. Examination of sequence from the coding region revealed two types of 5S genes in addition to pseudogenes. This suggests the presence of both somatic and germline (oocyte) forms of the 5S gene in the genome of Coregonus. The amount of variation present among NTS sequences indicates that accumulation of variation (mutation) is greater in this multicopy gene than is gene conversion (homogenization). The high level of sequence variation makes the 5S NTS an inappropriate DNA sequence for comparisons of closely related taxa. Received: 22 August 1997 / Accepted: 31 October 1997     

Long-Term Evolution of 5S Ribosomal DNA Seems to Be Driven by Birth-and-Death Processes and Selection in Ensis Razor Shells (Mollusca: Bivalvia)

A study of nucleotide sequence variation of 5S ribosomal DNA from six Ensis species revealed that several 5S ribosomal DNA variants, based on differences in their nontranscribed spacers (NTS), occur in Ensis genomes. The 5S rRNA gene was not very polymorphic, compared with the NTS region. The phylogenetic analyses performed showed a between-species clustering of 5S ribosomal DNA variants. Sequence divergence levels between variants were very large, revealing a lack of sequence homogenization. These results strongly suggest that the long-term evolution of Ensis 5S ribosomal DNA is driven by birth-and-death processes and selection.     

Simple sequence repeat marker development and genetic mapping in quinoa (Chenopodium quinoa Willd.)

Quinoa is a regionally important grain crop in the Andean region of South America. Recently quinoa has gained international attention for its high nutritional value and tolerances of extreme abiotic stresses. DNA markers and linkage maps are important tools for germplasm conservation and crop improvement programmes. Here we report the development of 216 new polymorphic SSR (simple sequence repeats) markers from libraries enriched for GA, CAA and AAT repeats, as well as 6 SSR markers developed from bacterial artificial chromosome-end sequences (BES-SSRs). Heterozygosity (H) values of the SSR markers ranges from 0.12 to 0.90, with an average value of 0.57. A linkage map was constructed for a newly developed recombinant inbred lines (RIL) population using these SSR markers. Additional markers, including amplified fragment length polymorphisms (AFLPs), two 11S seed storage protein loci, and the nucleolar organizing region (NOR), were also placed on the linkage map. The linkage map presented here is the first SSR-based map in quinoa and contains 275 markers, including 200 SSR. The map consists of 38 linkage groups (LGs) covering 913 cM. Segregation distortion was observed in the mapping population for several marker loci, indicating possible chromosomal regions associated with selection or gametophytic lethality. As this map is based primarily on simple and easily-transferable SSR markers, it will be particularly valuable for research in laboratories in Andean regions of South America.     

Assessment of genome organization among diploid species (2n = 2x = 14) belonging to primary and tertiary gene pools of pearl millet using fluorescent in situ hybridization with rDNA probes.

Two contrasting forms of Pennisetum belonging to the primary and tertiary gene pools were assessed for genomic organization using in situ hybridization with rDNA probes (18S-5.8S-25S and 5S) on metaphase and interphase cell nuclei. The primary gene pool is represented by diploid (2n = 2x = 14) cultivated pearl millet (Pennisetum glaucum) and its close wild relatives (Pennisetum violaceum and Pennisetum mollissimum). Pennisetum schweinfurthii (2n = 2x = 14) was taken as representative of the tertiary gene pool, owing to its diploid status and its similarity to the accessions of the primary gene pool in chromosome number. Using the 18S-5.8S-25S probe, we observed two sites of distribution in the four species but at different locations. Within the primary gene pool, signals were detected on the telomeric part of the short arm of chromosome pair VI and at the nucleolar organizing region (NOR) of the satellited chromosome pair (VII). Signals were observed at the NOR of the two satellited chromosome pairs (I and IV) of P. schweinfurthii. The 5S probe was detected at the telomeric part of the short arm of metacentric chromosome pair IV of the three species of the primary gene pool, while it occured in an intercalary position on the short arm of chromosome pair II of P. schweinfurthii. These results showed a chromosomal similarity of rDNA sequence locations within the primary gene pool and are in agreement with the high genetic identity between wild and cultivated forms of pearl millet previously revealed by allozyme studies. Implications of genomic organization for genetic resource enhancement are discussed. Key words : Pennisetum, in situ hybridization, rDNA probes, genomic organization.     

Length and sequence heterogeneity in 5S rDNA of Populus deltoides.

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (pi) estimates that are less than 0.15% of those obtained for class 2 spacers (pi = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.     

Comparative analysis of intra-individual and inter-species DNA sequence variation in salmonid ribosomal DNA cistrons

This study examines sequence divergence in three spacer regions of the ribosomal DNA (rDNA) cistron, to test the hypothesis of unequal mutation rates. Portions of two transcribed spacers (ITS-1 and 5' ETS) and the non-transcribed spacer (NTS) or intergenic spacer (IGS) formed the basis of comparative analyses. Sequence divergence was measured both within an individual lake trout (Salvelinus namaycush) and among several related salmonid species (lake trout; brook trout, Salvelinus fontinalis; Arctic char, Salvelinus alpinus; Atlantic salmon, Salmo salar; and brown trout, Salmo trutta). Despite major differences in the length of the rDNA cistron within individual lake trout, minimal sequence difference was detected among cistrons. Interspecies comparisons found that molecular variation in the rDNA spacers did not conform to the predicted pattern of evolution (ITS spacers相似文献   

Physical mapping of 5S and 18S-26S ribosomal RNA gene families inAllium victorialis var.platyphyllum

A physical map of the 5S and 18S–26S rRNA genes was determined using bi-color fluorescencein situ hybridization technique inA. victorialis var.platyphyllum. 5S rRNA genes were positioned in the intercalary regions of the short arms in homologous chromosomes 6. Two major loci of the 18S-26S rRNA genes were detected in the secondary constrictions flanking with a pair of satellite and terminal region of short arm in chromosome 4. And two additional minor loci were heterotype, representing one signal on the terminal region of the short arm in one homolog of chromsome 2, and other on one homolog of chromosome 6 with linked 5S rRNA loci. In addition chromomycin A₃ (CMA,) fluorescent banding method was used to identify the relation between Nucleolus Organizer Region (NOR) sites and CMA, positive heterochromatin sites. In homologous chromosome 4 showing 18S–26S rDNA hybridization signals revealed also distinct CMA, positive band.     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

Satellite DNA landscapes after allotetraploidization of quinoa (Chenopodium quinoa) reveal unique A and B subgenomes

If two related plant species hybridize, their genomes may be combined and duplicated within a single nucleus, thereby forming an allotetraploid. How the emerging plant balances two co‐evolved genomes is still a matter of ongoing research. Here, we focus on satellite DNA (satDNA), the fastest turn‐over sequence class in eukaryotes, aiming to trace its emergence, amplification, and loss during plant speciation and allopolyploidization. As a model, we used Chenopodium quinoa Willd. (quinoa), an allopolyploid crop with 2n = 4x = 36 chromosomes. Quinoa originated by hybridization of an unknown female American Chenopodium diploid (AA genome) with an unknown male Old World diploid species (BB genome), dating back 3.3–6.3 million years. Applying short read clustering to quinoa (AABB), C. pallidicaule (AA), and C. suecicum (BB) whole genome shotgun sequences, we classified their repetitive fractions, and identified and characterized seven satDNA families, together with the 5S rDNA model repeat. We show unequal satDNA amplification (two families) and exclusive occurrence (four families) in the AA and BB diploids by read mapping as well as Southern, genomic, and fluorescent in situ hybridization. Whereas the satDNA distributions support C. suecicum as possible parental species, we were able to exclude C. pallidicaule as progenitor due to unique repeat profiles. Using quinoa long reads and scaffolds, we detected only limited evidence of intergenomic homogenization of satDNA after allopolyploidization, but were able to exclude dispersal of 5S rRNA genes between subgenomes. Our results exemplify the complex route of tandem repeat evolution through Chenopodium speciation and allopolyploidization, and may provide sequence targets for the identification of quinoa's progenitors.     

DNA sequence of the 16S rRNA/23S rRNA intercistronic spacer of two rDNA operons of the archaebacterium Methanococcus vannielii

The DNA sequence of the spacer (plus flanking) regions separating the 16S rRNA and 23S rRNA genes of two presumptive rDNA operons of the archaebacterium Methanococcus vannielii was determined. The spacers are 156 and 242 base pairs in size and they share a sequence homology of 49 base pairs following the 3' terminus of the 16S rRNA gene and of about 60 base pairs preceding the 5' end of the 23S rRNA gene. The 242 base pair spacer, in addition contains a sequence which can be transcribed into tRNAAla, whereas no tRNA-like secondary structure can be delineated from the 156 base pair spacer region. Almost complete sequence homology was detected between the end of the 16S rRNA gene and the 3' termini of either Escherichia coli or Halobacterium halobium 16S rRNA, whereas the putative 5' terminal 23S rRNA sequence shared partial homology with E. coli 23S rRNA and eukaryotic 5.8S rRNA.     

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.