Diverged subpopulations in tropical Urochloa (Brachiaria) forage species indicate a role for facultative apomixis and varying ploidy in their population structure and evolution.

BACKGROUND: Urochloa (syn. Brachiaria) is a genus of tropical grasses sown as forage feedstock, particularly in marginal soils. Here we aimed to clarify the genetic diversity and population structure in Urochloa species to understand better how population evolution relates to ploidy level and occurrence of apomictic reproduction. METHODS: We explored the genetic diversity of 111 accessions from the five Urochloa species used to develop commercial cultivars. These accessions were conserved from wild materials collected at their centre of origin in Africa, and they tentatively represent the complete Urochloa gene pool used in breeding programmes. We used RNA-sequencing to generate 1.1 million single nucleotide polymorphism loci. We employed genetic admixture, principal component and phylogenetic analyses to define subpopulations. RESULTS: We observed three highly differentiated subpopulations in U. brizantha, which were unrelated to ploidy: one intermixed with U. decumbens, and two diverged from the former and the other species in the complex. We also observed two subpopulations in U. humidicola, unrelated to ploidy; one subpopulation had fewer accessions but included the only characterized sexual accession in the species. Our results also supported a division of U. decumbens between diploids and polyploids, and no subpopulations within U. ruziziensis and U. maxima. CONCLUSIONS: Polyploid U. decumbens are more closely related to polyploid U. brizantha than to diploid U. decumbens, which supports the divergence of both polyploid groups from a common tetraploid ancestor and provides evidence for the hybridization barrier of ploidy. The three differentiated subpopulations of apomictic polyploid U. brizantha accessions constitute diverged ecotypes, which can probably be utilized in hybrid breeding. Subpopulations were not observed in non-apomictic U. ruziziensis. Sexual Urochloa polyploids were not found (U. brizantha, U. decumbens) or were limited to small subpopulations (U. humidicola). The subpopulation structure observed in the Urochloa sexual-apomictic multiploidy complexes supports geographical parthenogenesis, where the polyploid genotypes exploit the evolutionary advantage of apomixis, i.e. uniparental reproduction and clonality, to occupy extensive geographical areas.

Introgression of chromosome segments from multiple alien species in wheat breeding lines with wheat streak mosaic virus resistance.

Pyramiding of alien-derived Wheat streak mosaic virus (WSMV) resistance and resistance enhancing genes in wheat is a cost-effective and environmentally safe strategy for disease control. PCR-based markers and cytogenetic analysis with genomic in situ hybridisation were applied to identify alien chromatin in four genetically diverse populations of wheat (Triticum aestivum) lines incorporating chromosome segments from Thinopyrum intermedium and Secale cereale (rye). Out of 20 experimental lines, 10 carried Th. intermedium chromatin as T4DL*4Ai#2S translocations, while, unexpectedly, 7 lines were positive for alien chromatin (Th. intermedium or rye) on chromosome 1B. The newly described rye 1RS chromatin, transmitted from early in the pedigree, was associated with enhanced WSMV resistance. Under field conditions, the 1RS chromatin alone showed some resistance, while together with the Th. intermedium 4Ai#2S offered superior resistance to that demonstrated by the known resistant cultivar Mace. Most alien wheat lines carry whole chromosome arms, and it is notable that these lines showed intra-arm recombination within the 1BS arm. The translocation breakpoints between 1BS and alien chromatin fell in three categories: (i) at or near to the centromere, (ii) intercalary between markers UL-Thin5 and Xgwm1130 and (iii) towards the telomere between Xgwm0911 and Xbarc194. Labelled genomic Th. intermedium DNA hybridised to the rye 1RS chromatin under high stringency conditions, indicating the presence of shared tandem repeats among the cereals. The novel small alien fragments may explain the difficulty in developing well-adapted lines carrying Wsm1 despite improved tolerance to the virus. The results will facilitate directed chromosome engineering producing agronomically desirable WSMV-resistant germplasm.

Characterization of RUSI, a telomere-associated satellite DNA, in the genus Rumex (Polygonaceae).

A satellite-DNA family (RUSI) has been isolated and characterized in Rumexinduratus Boiss and Reuter (Polygonaceae), an Iberian endemic polygamous sorrel. The RUSI repeats are 170 bp in length and approximately 68% AT-rich containing different variants of degenerate telomere motifs--(TT)(n)AN(GG)(n) -, a typical feature of subtelomeric DNA repeats adjacent to telomeres, which have been referred to as telomere-associated sequences or TASs. In fact, fluorescent in situhybridization showed that this satellite DNA is located in subtelomeric positions of most of the chromosomes of R. induratus, with some centromeric loci. PCR and Southern-blot hybridization assays for sequence conservation in the genus Rumex, indicated that the RUSI sequences are restricted to the genomes of R. induratus and R. scutatus, both species of the section Scutati, suggesting that they are recently evolved. Sequence variation within the two species is high (mean value of sequence differences between repeats of 15% for R. induratus and 7.5% for R. scutatus) and the degree of sequence differentiation between species is low with no species-specific variants, postulated to be due to slowed rates of spreading of sequence variants by molecular homogenizing mechanisms. Characteristics of RUSI sequences are discussed in the light of their chromosomal location and analyzed for their evolutionary and phylogenetic implications.

Evolutionary dynamics and sites of illegitimate recombination revealed in the interspersion and sequence junctions of two nonhomologous satellite DNAs in cactophilic Drosophila species.

Satellite DNA (satDNA) is a major component of genomes but relatively little is known about the fine-scale organization of unrelated satDNAs residing at the same chromosome location, and the sequence structure and dynamics of satDNA junctions. We studied the organization and sequence junctions of two nonhomologous satDNAs, pBuM and DBC-150, in three species from the neotropical Drosophila buzzatii cluster (repleta group). In situ hybridization to microchromosomes, interphase nuclei and extended DNA fibers showed frequent interspersion of the two satellites in D. gouveai, D. antonietae and, to a lesser extent, D. seriema. We isolated by PCR six pBuM x DBC-150 junctions: four are exclusive to D. gouveai and two are exclusive to D. antonietae. The six junction breakpoints occur at different positions within monomers, suggesting independent origin. Four junctions showed abrupt transitions between the two satellites, whereas two junctions showed a distinct 10 bp tandem duplication before the junction. Unlike pBuM, DBC-150 junction repeats are more variable than randomly cloned monomers and showed diagnostic features in common to a 3-monomer higher-order repeat seen in the sister species D. serido. The high levels of interspersion between pBuM and DBC-150 repeats suggest extensive rearrangements between the two satellites, maybe favored by specific features of the microchromosomes. Our interpretation is that the junctions evolved by multiples events of illegitimate recombination between nonhomologous satDNA repeats, with subsequent rounds of unequal crossing-over expanding the copy number of some of the junctions.

Mapping in plants: progress and prospects.

Comprehensive genetic maps are now available for all of the world's important crop species. Data show a remarkable conservation of order of markers over family-wide taxonomic groupings and illuminate species relationships and mechanisms of genome evolution. Comparison of genetic and physical maps has revealed differences in genetic distance throughout genomes with implications for genome organization, gene isolation and transformation.

DNA aptamers against the MUC1 tumour marker: design of aptamer-antibody sandwich ELISA for the early diagnosis of epithelial tumours.

Aptamers are functional molecules able to bind tightly and selectively to disease markers, offering great potential for applications in disease diagnosis and therapy. MUC1 is a well-known tumour marker present in epithelial malignancies and is used in immunotherapeutic and diagnostic approaches. We report the selection of DNA aptamers that bind with high affinity and selectivity an MUC1 recombinant protein containing five repeats of the variable tandem repeat region. Aptamers were selected using the SELEX methodology from an initial library containing a 25-base-long variable region for their ability to bind to the unglycosylated form of the MUC1 protein. After ten rounds of in vitro selection and amplification, more than 90% of the pool of sequences consisted of target-binding molecules, which were cloned, sequenced and found to share no sequence consensus. The binding properties of these aptamers were quantified using ELISA and surface plasmon resonance. The lead aptamer sequence was subsequently used in the design of an aptamer-antibody hybrid sandwich ELISA for the identification and quantification of MUC1 in buffered solutions. Following optimisation of the operating conditions, the resulting enzyme immunoassay displayed an EC50 value of 25 microg/ml, a detection limit of 1 microg/ml and a linear range between 8 and 100 microg/ml for the MUC1 five tandem repeat analyte. In addition, recovery studies performed in buffer conditions resulted in averaged recoveries between 98.2 and 101.7% for all spiked samples, demonstrating the usability of the aptamer as a receptor in microtitre-based assays. Our results aim towards the formation of new diagnostic assays against this tumour marker for the early diagnosis of primary or metastatic disease in breast, bladder and other epithelial tumours.

Diversity of a major repetitive DNA sequence in diploid and polyploid Triticeae.

About 90 members of a major tandemly repeated DNA sequence family originally described in rye as pSc119.2 have been isolated from 11 diploid and polyploid Triticeae species using primers from along the length of the sequence for PCR amplification. Alignment and similarity analysis showed that the 120-bp repeat unit family is diverse with single nucleotide changes and few insertions and deletions occurring throughout the sequence, with no characteristic genome or species-specific variants having developed during evolution of the extant genomes. Fluorescent in situ hybridization showed that each of the large blocks of the repeat at chromosomal sites harboured many variants of the 120-bp repeat. There were substantial copy number differences between genomes, with abundant sub-terminal sites in rye, interstitial sites in the B genome of wheat, and relatively few sites in the A and D genome. We conclude that sequence homogenization events have not been operative in this repeat and that the common ancestor of the Triticeae tribe had multiple sequences of the 120-bp repeat with a range of variation not unlike that seen within and between species today. This diversity has been maintained when sites are moved within the genome and in all species since their divergence within the Triticeae.

Characterization of progenies of Triticum aestivum-Psathyrostachys juncea derivatives by using genomic in-situ hybridization.

Using genomic in-situ hybridization (GISH) technique, 7 translocation-addition lines, 6 translocation and translocation-addition lines, 2 ditelosomic addition lines and 1 translocation line were identified from Triticum aestivum L. -Psathyrostachys juncea (Fisch.) Nevski intergeneric hybrids, of which translocation-addition and translocation and translocation-addition lines were not found in other reports. No substitutions and disomic additions were detected in the, hybrids and breakages occurred in all P. juncea chromosomes studied. Results have shown that the improved GISH technique is a rapid and economical method for use in this field.

The physical organization of Triticeae chromosomes.

Molecular cytogenetics combines molecular information of DNA sequences with their chromosomal organization. Genomic in situ hybridization using total genomic DNA as a probe is proving particularly useful to paint chromosomes originating from different genomes in hybrids, alloploid species and alien plant breeding lines. Both the numbers and morphologies of alien chromosomes or chromosome segments can be detected at metaphase and interphase. The method also gives considerable information about species relationships and the distribution of common or diverse DNA sequences between closely related species. Painted chromosomes can be followed through all stages of the cell cycle of somatic and meiotic division, providing new information about chromosome behaviour and pairing at meiosis. In situ hybridization with defined probes enables the physical location of particular DNA sequences to be examined along chromosomes and the analysis of the long range organization of specific chromosome regions. The generation of an integrated genetical, physical and functional map will be useful for the understanding of the organization and structure of the cereal genome.

The chromosomal organization of simple sequence repeats in wheat and rye genomes.

The physical distribution of ten simple-sequence repeated DNA motifs (SSRs) was studied on chromosomes of bread wheat, rye and hexaploid triticale. Oligomers with repeated di-, tri- or tetra-nucleotide motifs were used as probes for fluorescence in situ hybridization to root-tip metaphase and anther pachytene chromosomes. All motifs showed dispersed hybridization signals of varying strengths on all chromosomes. In addition, the motifs (AG)12, (CAT)5, (AAG)5, (GCC)5 and, in particular, (GACA)4 hybridized strongly to pericentromeric and multiple intercalary sites on the B genome chromosomes and on chromosome 4A of wheat, giving diagnostic patterns that resembled N-banding. In rye, all chromosomes showed strong hybridization of (GACA)4 at many intercalary sites that did not correspond to any other known banding pattern, but allowed identification of all R genome chromosome arms. Overall, SSR hybridization signals were found in related chromosome positions independently of the motif used and showed remarkably similar distribution patterns in wheat and rye, indicating the special role of SSRs in chromosome organization as a possible ancient genomic component of the tribe Triticeae (Gramineae).

Heterochromatin and nucleolus-organizer-region behaviour at male pachytene of Sus scrofa domestica.

In the domestic pig (2n = 38) two types of constitutive heterochromatin can be differentiated by fluorescence counterstaining techniques. All 24 biarmed autosomes and the X chromosome have chromomycin A3-positive centromeric C-bands, whereas all 12 acrocentric chromosomes exhibit DA-DAPI-positive centromeric heterochromatin. Fluorescence analysis of male pachytene nuclei revealed that the DA-DAPI-positive C-bands form one or two large chromocentres per cell, while the chromomycin A3-bright C-material is well scattered. Hence, the bivalents formed by the acrocentric chromosome pairs are centromerically associated, whilst the submetacentric bivalents are not. Counce-Meyer spreading techniques were used to study the structure of synaptonemal complexes (SCs) both by light and electron microscopy. In general, the SCs of the domestic pig resemble those described for other mammals. The SC formed by the X and the Y may include up to 94.5% of the Y chromosome. In silver-stained microspreads each of the bivalents (nos. 8 and 10) bearing the nucleolus-organizer-regions (NORs) is connected to a pair of nucleoli, indicating that all four NORs are active during early meiotic stages. By contrast, in the majority of mitotic metaphases of phytohaemagglutinin-stimulated lymphocytes only one pair (no. 10) exhibited Ag-NOR staining. The significance of the chromosome disposition in the pachytene nucleus is discussed with regard to heterochromatin composition and karyotype evolution.

BIS 1, a major component of the cereal genome and a tool for studying genomic organization.

We report the cloning and characterization of an element (BIS 1) in barley. Related sequences were also found in wheat and rye genomes. BIS 1-related sequences may be some of the more frequent of the complex repeats in the barley genome, and their dispersion throughout the barley genome suggests that they are capable of both mobility and amplification. BIS 1 sequences have been used to study the gross structure of the barley genome. These studies indicate that the genome may be formed from larger genomic structures of tens of kilobases which may be repeated. Surprisingly, these studies also show that these large genomic structures also occur in similar proportions and at similar size distribution in the wheat genome.

The use of fluorochromes in the cytogenetics of the small-grained cereals (Triticeae).

This paper describes some of the major advances that have been made in the cytogenetics of the small-grained cereals (Triticeae) using fluorochromes to detect nucleic acids in situ. The method, widely known as fluorescence in situ hybridization, has made a contribution in several areas including (i) chromosome mapping programmes, and (ii) cereal breeding programmes. Flow cytometry of cereal chromosomes has now been developed for the generation of chromosome enriched libraries; these libraries will ultimately be of use in both the cereal mapping and breeding programmes. Fluorescence in situ hybridization has also made a major contribution to the understanding of cereal genome structure by elucidating the distribution of different classes of DNA sequence. By using suitable nucleic acid probes whole chromosomes can now be identified in interphase nuclei. The labelling patterns have revealed a structured arrangement of chromosomes at interphase. Not only are chromosomes organized but the ribosomal RNA genes also show structured patterns of condensation and expression. Progress in each of these areas has been rapid in recent years and this progress is described.

Characterisation of mildew resistant wheat-rye substitution lines and identification of an inverted chromosome by fluorescent in situ hybridisation.

Seven different mildew resistant wheat lines derived from crosses between triticale and bread wheat were examined by molecular cytogenetics and chromosome C-banding in order to determine their chromosomal composition. Genomic in situ hybridisation (GISH) showed the presence of rye germplasm in all the lines and identified three substitution lines, three double substitution lines and one addition-substitution line. C-banding identified rye chromosomes 1R and 4R in the addition-substitution line, rye chromosomes 1R and 6R in two substitution lines and 1R and 2R in the third line, and rye chromosome 1R in the three substitution lines. Two of the latter lines (7-102 and 7-169) contained a modified form of the chromosome; fluorescent in situ hybridisation (FISH) using five different repetitive DNA-probes showed a pericentric inversion of 1R in both lines. The breakpoints of the 1R inversion were between (1) the 5S rDNA site and the NOR-region on the satellite of the short arm, and (2) between two AAC(5) sites close to the centromere on the long arm. The role of the rye chromosomes in the mildew resistance, the utilisation of the inverted 1R and the significance of the lines in wheat breeding are discussed.

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.

The large-scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes.

Repetitive DNA sequences in the terminal heterochromatin of rye (Secale cereale) chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandemly repeated, subtelomeric DNA families with complex but contrasting higher order structural organizations. Polymerase chain reaction analysis with a single primer showed a fraction of the repeat units of one family organized in a "head-to-head" orientation. Such structures suggest evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. In situ hybridization and pulse field gel electrophoresis showed the order of the repeats and the heterogeneity in the lengths of individual arrays. After Xbal digestion and pulse field gel electrophoresis, the telomeric and two subtelomeric clones showed strong hybridization signals from 40 to 100 kb, with a maximum at 50 to 60 kb. We suggest that these fragments define a basic higher order structure and DNA loop domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.

The chromosomes of Citrus and Poncirus species and hybrids: identification of characteristic chromosomes and physical mapping of rDNA loci using in situ hybridization and fluorochrome banding.

In situ hybridization of 18S-5.8S-25S rDNA probes labeled with biotin or rhodamine and 5S rDNA probes labeled with digoxigenin was used to locate rDNA sites on root-tip metaphase chromosomes of Citrus sinensis L. (2n = 2x = 18), Poncirus trifoliata L. Raf. (2n = 2x = 18), and Citrus x Poncirus hybrids (2n = 2x = 18). Counterstaining with the fluorochromes chromomycin A3 and DAPI uniquely identified many but not all chromosomes. C. sinensis had five 18S-25S rDNA sites, P. trifoliata had seven, and three different Citrus x Poncirus hybrids had five or six sites. Four 5S rDNA sites were detected, mostly linked to 18S-25S rDNA sites. Overall we observed high levels of chromosomal heterozygosity in all accessions examined.

Genomic in situ hybridization to sectioned nuclei shows chromosome domains in grass hybrids.

In situ hybridization using biotinylated total genomic DNA and avidin detection systems was adapted for examination of thin-sectioned plant material in the light and electron microscopes. Root tip material was preserved prior to sectioning, so that the in vivo disposition of the chromatin was maintained. Use of total genomic DNA from Secale africanum as a probe enabled the chromatin from the two parental genomes in the grass hybrid Hordeum chilense x S. africanum to be distinguished. The biotinylated probe preferentially labelled the chromosomes of S. africanum origin. DNA-DNA hybrids were visualized at the light-microscope level by Texas Red fluorescence and at the electron-microscope level by the enzymic precipitation of DAB (diaminobenzidine) or by colloidal gold particles. The use of thin sections allowed the location of probe hybridization to be established unequivocally in both metaphase and interphase nuclei. Analysis of interphase nuclei showed that chromatin originating from the two parental genomes did not intermix but occupied distinct domains.