Structure and genomic organization of centromeric repeats in<Emphasis Type="Italic"> Arabidopsis</Emphasis> species

Centromeric repetitive sequences were isolated from Arabidopsis halleri ssp. gemmifera and A. lyrata ssp. kawasakiana. Two novel repeat families isolated from A. gemmifera were designated pAge1 and pAge2. These repeats are 180 bp in length and are organized in a head-to-tail manner. They are similar to the pAL1 repeats of A. thaliana and the pAa units of A. arenosa. Both A. gemmifera and A. kawasakiana possess the pAa, pAge1 and pAge2 repeat families. Sequence comparisons of different centromeric repeats revealed that these families share a highly conserved region of approximately 50 bp. Within each of the four repeat families, two or three regions showed low levels of sequence variation. The average difference in nucleotide sequence was approximately 10% within families and 30% between families, which resulted in clear distinctions between families upon phylogenetic analysis. FISH analysis revealed that the localization patterns for the pAa, pAge1 and pAge2 families were chromosome specific in A. gemmifera and A. kawasakiana. In one pair of chromosomes in A. gemmifera, and three pairs of chromosomes in A. kawasakiana, two repeat families were present. The presence of three families of centromeric repeats in A. gemmifera and A. kawasakiana indicates that the first step toward homogenization of centromeric repeats occurred at the chromosome level.Communicated by W. R. McCombie     

Patterns of DNA Variation Among Three Centromere Satellite Families in <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> and <Emphasis Type="Italic">A</Emphasis>. <Emphasis Type="Italic">lyrata</Emphasis>

We describe patterns of DNA variation among the three centromeric satellite families in Arabidopsis halleri and lyrata. The newly studied subspecies (A. halleri ssp. halleri and A. lyrata ssp. lyrata and petraea), like the previously studied A. halleri ssp. gemmifera and A. lyrata ssp. kawasakiana, have three different centromeric satellite families, the older pAa family (also present in A. arenosa) and two families, pAge1 and pAge2, that probably evolved more recently. Sequence variability is high in all three satellite families, and the pAa sequences do not cluster by their species of origin. Diversity in the pAge2 family is complex, and different from variation among copies of the other two families, showing clear evidence for exchange events among family members, especially in A. halleri ssp. halleri. In A. lyrata ssp. lyrata there is some evidence for recent rapid spread of pAge2 variants, suggesting selection favoring these sequences. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Brian Morton]     

High-resolution mapping of repetitive DNA by in situ hybridization: molecular and chromosomal features of prominent dispersed and discretely localized DNA families from the wild beet species Beta procumbens

Members of three prominent DNA families of Beta procumbens have been isolated as Sau3A repeats. Two families consisting of repeats of about 158 bp and 312 bp are organized as satellite DNAs (Sau3A satellites I and II), whereas the third family with a repeat length of 202 bp is interspersed throughout the genome. Multi-colour fluorescence in situ hybridization was used for physical mapping of the DNA families, and has shown that these tandemly organized families occur in large heterochromatic and DAPI positive blocks. The Sau3A satellite I hybridized exclusively around or near the centromeres of 10, 11 or 12 chromosomes. The Sau3A satellite family I showed high intraspecific variability and high-resolution physical mapping was performed on pachytene chromosomes using differentially labelled repeats. The physical order of satellite subfamily arrays along a chromosome was visualized and provided evidence that large arrays of plant satellite repeats are not contiguous and consist of distinct subfamily domains. Re-hybridization of a heterologous rRNA probe to mitotic metaphase chromosomes revealed that the 18S-5.8S-25S rRNA genes are located at subterminal position on one chromosome pair missing repeat clusters of the Sau3A satellite family I. It is known that arrays of Sau3A satellite I repeats are tightly linked to a nematode (Heterodera schachtii) resistance gene and our results show that the gene might be located close to the centromere. Large arrays of the Sau3A satellite II were found in centromeric regions of 16 chromosomes and, in addition, a considerable interspersion of repeats over all chromosomes was observed. The family of interspersed 202 bp repeats is uniformly distributed over all chromosomes and largely excluded from the rRNA gene cluster but shows local amplification in some regions. Southern hybridization has shown that all three families are specific for genomes of the section Procumbentes of the genus Beta.     

Physical mapping of rRNA genes by fluorescent in-situ hybridization and structural analysis of 5S rRNA genes and intergenic spacer sequences in sugar beet (Beta vulgaris)

A digoxigenin-labelled 5S rDNA probe (pTa-794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet,Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sizedB. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. TwoXbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.     

Development of Molecular Cytogenetics and Physical Mapping of Ribosomal RNA Genes in Lupinus

Identification of individual chromosomes in Lupinus is not possible due to gradient in size and similar morphology. To overcome this problem, molecular cytogenetics was developed for Lupinus. As an initial step in karyotype analysis, fluorescent in situ hybridization (FISH) was performed to determine genomic distribution of rRNA genes in L. hispanicus, L. luteus and L. × hispanicoluteus. It was found that all three diploid species posses two chromosome pairs carrying 18S-5.8S-25S rDNA and one chromosome pair carrying 5S rDNA. The use of probes for rDNA permitted unambiguous identification of three different pairs of chromosomes and revealed conservation of the number of rDNA loci among the three species. The study represents the first step in physical mapping of Lupinus genome through FISH by providing distinct chromosome landmarks. This revised version was published online in July 2006 with corrections to the Cover Date.     

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]     

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.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Karyotype and Chromosomal Location of 18S–28S and 5S Ribosomal DNA in the Scallops Pecten maximus and Mimachlamys varia (Bivalvia: Pectinidae)

This work describes the karyotype and chromosomal location of the ribosomal DNA (rDNA) of Pecten maximus and Mimachlamys varia, two commercial scallop species from Europe. According to the chromosome centromeric index values found, the karyotype of P. maximus is composed of 1 metacentric, 2 metacentric–submetacentric, 1 telocentric–subtelocentric and 15 telocentric pairs, and that of M. varia of 4 metacentric, 2 subtelocentric–submetacentric, 9 subtelocentric, 3 subtelocentric–telocentric and 1 telocentric–subtelocentric pairs. In P. maximus, 18S-28S rDNA was located by FISH on a metacentric–submetacentric pair, and in M. varia on a subtelocentric–submetacentric pair using both silver staining and FISH. PCR amplification of the 5S rDNA unit yielded a single product of about 460 bp (P. maximus) and 450 bp (M. varia), that used as probe revealed a 5S rDNA site on a telocentric pair in P. maximus and a subtelocentric pair in M. varia. Two-color FISH or sequential silver staining of 5S rDNA-FISH-metaphases corroborated that the two gene families are located on different chromosomes in both species. A comparative analysis of the data allowed the inference of karyotypic relationships within scallops.     

Characterization of Charr Chromosomes Using Fluorescence in Situ Hybridization

The chromosomal locations of several families of tandem repetitive DNA sequences and the 5S rDNA were determined using fluorescence in situ hybridization (FISH) in the five North American charr species: Salvelinus namaycush, S. fontinalis, S. alpinus, S. malma, and S. confluentus. The pattern of hybridization of three centromeric repetitive sequences previously isolated from S. namaycush and S. alpinus was unique in each species. Dual-color FISH experiments showed that in several species many of the centromeres had the EcoRI-DraI family in addition to either the AluI-RsaI type A or type B families. The EcoRI-DraI family which was found only at the centromeres of acrocentric chromosomes in S. namaycush, S. fontinalis and S. malma was also found at centromeres of selected metacentrics in S. alpinus (one pair) and S. confluentus (four pairs) whose chromosomes have undergone additional centric fusions compared to the other species. The locations of 5S rDNA sequences were different in each species except for the two most closely related (S. alpinus and S. malma). Two whole-arm chromosome paint probes, one specific for the short and the other for the long arm of the lake charr sex chromosomes, hybridize to the same chromosome pair in all species. Results with other paint probes suggest that independent centric fusions have occurred in S. alpinus and S. confluentus which is consistent with the phylogenetic tree obtained previously for Salvelinus with cytogenetic and DNA data.     

Chromosomal location and reorganization of the 45S and 5S rDNA in theBrachyscome lineariloba complex (Asteraceae)

The chromosomal locations of the 45S (18S-5.8S-26S) and 5S ribosomal DNA in theBrachyscome lineariloba complex and two related species have been determined by the use of multicolor fluorescencein situ hybridization (McFISH). TheBrachyscome lineariloba complex includes five cytodemes with 2n=4, 8, 10, 12 and 16. Each of the 5S and 45S rDNA loci occurs at two sites on chromosomes in cytodemes with 2n=4. While in cytodemes with 2n=8, 10, 12 and 16, the number of 5S rDNA sites increases from four to eight paralleled to the genomic addition of diploid (4 chromosomes) or haploid (2 chromosomes) dosage. Of the 5S rDNA sites, only one pair is major, except for the cytodeme with 2n=10. The remaining 5S rDNA sites are minor and seem to have reduced the unit number of the 5S rDNA during the successive genomic additions. The 45S rDNA site is detected only at two nucleolar organizing regions in all cytodemes regardless of successive genomic addition. The loss or diminution of 45S rDNA sequences seem to have proceeded more rapidly than 5S rDNA sequences in theB. lineariloba complex.     

Comparative karyotype analysis in Haplopappus Cass. and Grindelia Willd. (Asteraceae) by double FISH with rRNA specific genes

The genera Grindelia Willd. and Haplopappus Cass. belong to the family Asteraceae - Astereae and are distributed in America and South America, respectively. Previous cytotaxonomic studies showed for South American species of Grindelia 2n=12 and for Haplopappus 2n=10 and 2n=12. Both Grindelia species (G. anethifolia, G. prunelloides), newly analyzed with molecular-cytological methods, exhibited symmetric karyotypes (AsI %=55.46 and 55.95) with metacentric chromosome sets (5m + 1m-sat) and 2n=12 chromosomes. The NOR was detected after fluorescence in situ hybridization (FISH) with 18/25S rDNA in the satellite chromosome 2. In contrast H. Happlopappus glutinosus, H. grindeloides and H. stolpii showed exclusively a higher asymmetric index (66.83%, 67.01% and 68.87%, respectively) with submetacentric chromosome sets (4sm + 1sm–sat). The sat-chromosomes 3 of H. glutinosus and H. grindelioides were both significantly different in their length from chromosomes 2 and 4. Furthermore in Grindelia the FISH with 5S rDNA could estimate signals in the short arms of chromosomes 3 or 4, that were not significantly differentiated in their length. Contrary to these findings in Grindelia, the position of 5S rDNA in Haplopappus was detected in the long arms of chromosome 1 (H. grindelioides and H. stolpii) and chromosome 2 (with two different loci) and chromosome 4 of H. glutinosus. The lengths of all measured chromosome arms with 5S rDNA were significantly different to those of the neighbours in the karyotypes. The two-color FISH of 5S and 18/25S rDNA had provided clear karyotypic markers for three (Haplopappus glutinosus) and two (H. grindelioides and H. stolpii) chromosomes. The number and position of rDNA signals were relatively highly conserved in the investigated five species without the double marked chromosome 2 of H. glutinosus, which shows an evolutionary dynamic of this 5S rRNA specific gene cluster. This investigation supports the assumption that the evolution of New World members of Grindelia and Haplopappus has not been accompanied by large karyotypic changes, but small chromosomal rearrangements have undoubtedly occurred (e.g. 5S rDNA localizations).     

Physical mapping of rDNA sequences in four karyotypes of Ranunculus silerifolius (Ranunculaceae)

The chromosomal locations of the 18S-5.8S-26S rDNA and 5S rDNA sequences were examined in four cytotypes of Ranunculus silerifolius (the Matsuyama, Mugi, Otaru, and Karatsu types) using fluorescence in situ hybridization (FISH). Using the 18S-5.8S-26S rDNA probe, one pair of probe hybridization sites was detected by FISH in the interstitial region corresponding to the secondary constriction on the short arm of a satellite chromosome (chromosome pair 6) in all four karyotypes. FISH using 5S rDNA identified one pair of sites. The 5S rDNA locus was on different chromosomes in the four karyotypes: in the interstitial region of the short arm of the largest metacentric chromosome (chromosome pair 1) in the Matsuyama type, in the interstitial region of the short arm of the subtelocentric chromosome (pair 2) in the Mugi and Otaru types, and in the interstitial region of the short arm of the metacentric chromosome (pair 2) in the Karatsu type. This physical mapping of the 5S rDNA provides valuable information about karyotype evolution in R. silerifolius. Possible mechanisms of chromosome evolution are discussed.     

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.     

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

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

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     

Diversity of centromeric repeats in two closely related wild rice species, Oryza officinalis and Oryza rhizomatis

Oryza officinalis (CC, 2n=24) and Oryza rhizomatis (CC, 2n=24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.     

Genome differentiation in Aegilops. 4. Evolution of the U-genome cluster

Phylogenetic relationships of polyploid Aegilops species sharing the U-genome were investigated by analyzing heterochromatin banding patterns of their somatic metaphase chromosomes as revealed by C-banding and fluorescence in situ hybridization (FISH) with the heterochromatin-limited repetitive DNA probes pSc119, pAs1, as well as the distribution of NOR and 5S DNA loci revealed by pTa71 (18S-26S rDNA), and pTa794 (5S rDNA) probes. Seven tetraploid (Ae. triuncialis, Ae. peregrina, Ae. kotschyi, Ae. geniculata, Ae. biuncialis, Ae. columnaris, and 4x Ae. neglecta) and one hexaploid (6x Ae. neglecta) Aegilops species of the U-genome cluster were studied. The U^t and C^t chromosomes of 4x Ae. triuncialis (U^tC^t) were similar to the diploid donors Ae. umbellulata (U) and Ae. caudata (C). However, the size of the NOR locus on chromosome 5U^t was reduced. Karyotypic analyses confirmed that 4x Ae. peregrina (S^pU^p) was derived from a hybridization of the diploid species Ae. umbellulata with Ae. longissima, whereas Ae. umbellulata and Ae. sharonensis (or an immediate precursor) were the diploid progenitor species of Ae. kotschyi (S^kU^k). In both 4x species, the NORs on S-genome chromosomes were inactivated and were accompanied with a decrease or loss of rDNA sequences. Karyotypes of the tetraploid species, Ae. geniculata (U^gM^g) and Ae. biuncialis (U^bM^b) differed from each other and from the putative diploid progenitors Ae. umbellulata and Ae. comosa indicating that various types of chromosomal alterations occurred during speciation. Inactivation of major NORs on the M-genome chromosomes, redistribution of 5S rDNA sites, and loss of some minor 18S-26S rDNA loci were observed in Ae. geniculata and Ae. biuncialis. Significant differences in the total amount and distribution of heterochromatin, the number and location of 5S and 18S-26S rDNA loci observed between Ae. columnaris (U^cX^c)/4x Ae. neglecta (UⁿXⁿ) and Ae. geniculata/Ae. biuncialis indicate that these species have different origins. Similarities in C-banding and FISH patterns of most Ae. columnaris and 4x Ae. neglecta chromosomes suggest that they were probably derived from a common ancestor, whereas distinct differences of three chromosome pairs may indicate that the divergence of these species was probably associated with chromosomal rearrangements and/or introgressive hybridization. Ae. umbellulata contributed the U genome, however, the source of their second genomes remains unknown. The formation of 6x Ae. neglecta (UⁿXⁿNⁿ) was not associated with large modifications of the parental genomes.     

Physical mapping of 5S rDNA reveals a new locus on 3R and unexpected complexity in a rye translocation used in chromosome mapping

Using fluorescence in situ hybridization (FISH) with probe pScT7, three different 5S rDNA loci were detected in the satellite of rye chromosome 1R (5SDna-R1) and in the short arms of chromosomes 3R (5SDna-R3) and 5R (5SDna-R2) respectively. All three loci showed polymorphism for the hybridization signal intensity. In order to determine the localization of these rye 5S rDNA multigene loci with higher precision within the corresponding chromosome arms, the probe pScT7 was physically mapped by FISH in relation to the following five translocations (Wageningen Tester Set): T850W (1RS/4RL), T248W (1RS/6RS), T273W (1RS/5RL), T305W (2RS/5RS) and T240W (3RS/5RL). Accurate physical maps of the translocation breakpoints had previously been made using electron microscope analysis of spread pachytene synaptonemal complexes of heterozygotes for the different translocations. The results indicate that locus 5SDna-R3 is located between the breakpoint of translocation T240W and the telomere, whereas locus 5SDna-R2 is located between the breakpoint of translocation T305W and the centromere, the hybridization of probe pScT7 on T305W translocated chromosomes demonstrating the complex nature of this translocation. On the other hand, the simultaneous detection of probes pScT7 and pTA71 (18S-5.8S-26S rDNA) with two different fluorochromes, indicated that the breakpoints of translocations T850W and T248W are located between loci Nor-R1 and 5SDna-R1.     

Mapping the nucleolus organizer region,seed protein loci and isozyme loci on chromosome 1R in rye

Summary The nucleolus organizer region located on the short arm of chromosome 1R of rye consists of a large cluster of genes that code for ribosomal RNA (designated the Nor-R1 locus). The genes in the cluster are separated by spacer regions which can vary in length in different rye lines. Differences in the spacer regions were scored in two families of F₂ progeny. Segregation also occurred, in one or both of the families, at two seed protein loci and at two isozyme loci also located on chromosome 1R. The seed protein loci were identified as the Sec 1 locus controlling -secalins located on the short arm of chromosome 1R and the Sec 3 locus controlling high-molecular-weight secalins located on the long arm of 1R. The two isozyme loci were the Gpi-R1 locus controlling glucose-phosphate isomerase isozymes and the Pgd 2 locus controlling phosphogluconate dehydrogenase isozymes. The data indicated linkage between all five loci and map distances were calculated. The results indicate a gene order: Pgd 2 ... Sec 3 ... [centromere] ... Nor-R1 ... Gpi-R1 ... Sec 1. Evidence was obtained that rye possesses a minor 5S RNA locus (chromosome location unknown) in addition to the major 5S RNA locus previously shown to be located on the short arm of chromosome 1R.