RESUMO
BACKGROUND AND AIMS: Polyploidy is an important process that often generates genomic diversity within lineages, but it can also cause changes that result in loss of genomic material. Island lineages, while often polyploid, typically show chromosomal stasis but have not been investigated in detail regarding smaller-scale gene loss. Our aim was to investigate post-polyploidization genome dynamics in a chromosomally stable lineage of Malvaceae endemic to New Zealand. METHODS: We determined chromosome numbers and used fluorescence in situ hybridization to localize 18S and 5S rDNA. Gene sequencing of 18S rDNA, the internal transcribed spacers (ITS) with intervening 5.8S rDNA, and a low-copy nuclear gene, GBSSI-1, was undertaken to determine if gene loss occurred in the New Zealand lineage following polyploidy. KEY RESULTS: The chromosome number for all species investigated was 2nâ =â 42, with the first published report for the monotypic Australian genus Asterotrichion. The five species investigated all had two 5S rDNA signals localized interstitially on the long arm of one of the largest chromosome pairs. All species, except Plagianthus regius, had two 18S rDNA signals localized proximally on the short arm of one of the smallest chromosome pairs. Plagianthus regius had two additional 18S rDNA signals on a separate chromosome, giving a total of four. Sequencing of nuclear ribosomal 18S rDNA and the ITS cistron indicated loss of historical ribosomal repeats. Phylogenetic analysis of a low-copy nuclear gene, GBSSI-1, indicated that some lineages maintained three copies of the locus, while others have lost one or two copies. CONCLUSIONS: Although island endemic lineages show chromosomal stasis, with no additional changes in chromosome number, they may undergo smaller-scale processes of gene loss and concerted evolution ultimately leading to further genome restructuring and downsizing.
Assuntos
Cromossomos , Poliploidia , Filogenia , Hibridização in Situ Fluorescente , Austrália , DNA Ribossômico/genéticaRESUMO
The Schedonorus-Lolium complex of the subtribe Loliinae (Poaceae) includes several economically important forage and turf grasses. This complex encompasses Lolium Linnaeus, 1753, Festuca Linnaeus, 1753 subgenus Schedonorus (P. Beauvois, 1824) Petermann, 1849 and Micropyropsis Romero Zarco et Cabezudo, 1983. New FISH results of 5S and 18S-26S rDNA sequences are presented for three species and the results are interpreted in a review of distribution patterns of 5S and 18S-26S rDNA sequences among other species in the complex. Micropyropsistuberosa Romero Zarco et Cabezudo, 1983 (2n = 2x = 14) displayed a distribution pattern of rDNA sequences identical to that of F.pratensis Hudson, 1762, supporting a close phylogenetic relationship at the bottom of the phylogenetic tree. "Loliummultiflorum" Lamarck, 1779 accessions sourced from Morocco showed a different pattern from European L.multiflorum and could be a unique and previously uncharacterised taxon. North African Festucasimensis Hochstetter ex A. Richard, 1851 had a marker pattern consistent with allotetraploidy and uniparental loss of one 18S-26S rDNA locus. This allotetraploid has previously been suggested to have originated from a hybrid with Festucaglaucescens (Festucaarundinaceavar.glaucescens Boissier, 1844). However, the distribution patterns of the two rDNA sequences in this allotetraploid do not align with F.glaucescens, suggesting that its origin from this species is unlikely. Furthermore, comparisons with other higher alloploids in the complex indicate that F.simensis was a potential donor of two sub-genomes of allohexaploid Festucagigantea (Linnaeus) Villars, 1787. In the overall complex, the proximal locations of both rDNA markers were conserved among the diploid species. Two types of synteny of the two markers could, to a considerable extent, distinguish allo- and autogamous Lolium species. The ancestral parentage of the three Festuca allotetraploids has not yet been determined, but all three appear to have been sub-genome donors to the higher allopolypoids of sub-genus Schedonorus. Terminal locations of both the markers were absent from the diploids but were very frequently observed in the polyploids.
RESUMO
BACKGROUND: Unreduced gametes, a driving force in the widespread polyploidization and speciation of flowering plants, occur relatively frequently in interspecific or intergeneric hybrids. Studies of the mechanisms leading to 2n gamete formation, mainly in the wheat tribe Triticeae have shown that unreductional meiosis is often associated with chromosome asynapsis during the first meiotic division. The present study explored the mechanisms of meiotic nonreduction leading to functional unreduced gametes in an interspecific Trifolium (clover) hybrid with three sub-genomes from T. ambiguum and one sub-genome from T. occidentale. RESULTS: Unreductional meiosis leading to 2n gametes occurred when there was a high frequency of asynapsis during the first meiotic division. In this hybrid, approximately 39% of chromosomes were unpaired at metaphase I. Within the same cell at anaphase I, sister chromatids of univalents underwent precocious separation and formed laggard chromatids whereas paired chromosomes segregated without separation of sister chromatids as in normal meiosis. This asynchrony was frequently accompanied by incomplete or no movement of chromosomes toward the poles and restitution leading to unreduced chromosome constitutions. Reductional meiosis was restored in progeny where asynapsis frequencies were low. Two progeny plants with approximately 5 and 7% of unpaired chromosomes at metaphase I showed full restoration of reductional meiosis. CONCLUSIONS: The study revealed that formation of 2n gametes occurred when asynapsis (univalent) frequency at meiosis I was high, and that normal gamete production was restored in the next generation when asynapsis frequencies were low. Asynapsis-dependent 2n gamete formation, previously supported by evidence largely from wheat and its relatives and grasshopper, is also applicable to hybrids from the dicotyledonous plant genus Trifolium. The present results align well with those from these widely divergent organisms and strongly suggest common molecular mechanisms involved in unreduced gamete formation.
Assuntos
Células Germinativas Vegetais/crescimento & desenvolvimento , Meiose , Trifolium/crescimento & desenvolvimento , Células Germinativas Vegetais/metabolismo , Hibridização Genética , Trifolium/genéticaRESUMO
BACKGROUND: Trifolium ambiguum occurs as a 2x, 4x, 6x polyploid series in W Asia. The 6x form is the most agronomically desirable, having strong rhizomatous spread and drought tolerance. These traits would be potentially very valuable if they could be transferred to white clover (T. repens) which is the most important agronomic clover species. However, to-date, no fertile interspecific hybrids with 6x T. ambiguum are available. Previously, 2x T. occidentale from W Europe has produced synthetic fertile hybrids with both 2x and 4x T. ambiguum and these were inter-fertile with white clover. Here we ask whether 2x T. occidentale can form fertile hybrids with 6x T. ambiguum and act as a genetic bridge to white clover and bring these species together as part of a common gene pool. RESULTS: Ten verified F1 (6x T. ambiguum x 2x T. occidentale) hybrids were produced by embryo rescue and seven were studied further. All four investigated for chromosome number were 2n = 4x = 32 and FISH confirmed the expected 21 T. ambiguum and 8 T. occidentale chromosomes. Hybrid fertility was extremely low but 2n female gametes functioned with white clover pollen to produce seeds. Derived plants were confirmed using FISH and were successfully backcrossed to white clover to produce partially fertile breeding populations. CONCLUSIONS: Although T. occidentale and 6x T. ambiguum are widely separated by geography and ecological adaptation they have maintained enough genomic affinity to produce partially fertile hybrids. Inter-fertility of the hybrids with allotetraploid T. repens showed that T. occidentale can provide a genetic bridge between 6x T. ambiguum and white clover to produce plants with new phenotypes combining the traits of all three species. Use of this information should enable potentially valuable stress tolerance traits from 6x T. ambiguum to be used in white clover breeding for the first time.
Assuntos
Genoma de Planta/genética , Trifolium/genética , Genótipo , Hibridização Genética , Fenótipo , Melhoramento Vegetal , Poliploidia , Sementes/genéticaRESUMO
BACKGROUND: The unambiguous identification of individual chromosomes is a key part of the genomic characterization of any species. In this respect, the development and application of chromosome banding techniques has revolutionised mammalian and especially, human genomics. However, partly because of the traditional use of chromosome squash preparations, consistent fluorescence banding has rarely been achieved in plants. Here, successful fluorescence chromosome banding has been achieved for the first time in perennial ryegrass (Lolium perenne), a forage and turf grass with a large genome and a symmetrical karyotype with chromosomes that are difficult to distinguish. RESULTS: Based on flame-dried chromosome preparations instead of squashes, a simple fluorescence Q-banding technique using quinacrine mustard, unambiguously identified each chromosome and enabled the development of a banded karyotype and ideogram of the species. This Q-banding technique was also shown to be compatible with sequential FISH mapping enabling labelled genes and molecular markers to be precisely assigned to specific cytogenetic bands. A technique for DAPI-banding, which gave a similar pattern to Q-banding, was also introduced. This was compatible with FISH mapping and was used to anchor a single copy gene from an earlier mapped linkage group of L. perenne, thus providing a step towards integration of the genetic and cytogenetic maps. CONCLUSIONS: By enabling the allocation of genes mapped by other methods to physically identified chromosome positions, this work will contribute to a better understanding of genomic structures and functions in grasses.
Assuntos
Bandeamento Cromossômico , Mapeamento Cromossômico , Hibridização in Situ Fluorescente , Lolium/genética , CariótipoRESUMO
BACKGROUND: Perennial ryegrass (Lolium perenne) has developed resistance to glyphosate within New Zealand vineyards following many years of herbicide application. The objectives of this work were to confirm resistance within two populations obtained from affected vineyards, and to determine the mechanism of resistance to glyphosate. RESULTS: Population O was confirmed to have a 25-fold resistance to glyphosate, whereas population J had a sevenfold resistance. Results of genotyping assays demonstrated a single nucleotide substitution at codon 106 of 5-enolpyruvylshikimate-3-phosphate synthase in population O but not population J. Glyphosate-resistant and glyphosate-susceptible populations did not differ in glyphosate absorption. However, in both resistant populations, much more of the absorbed (14) C-glyphosate remained in the treated leaf than occurred in the susceptible population. Significantly more glyphosate was found in the pseudostem region of susceptible plants compared with resistant plants. CONCLUSION: Both target-site and non-target-site mechanisms of glyphosate resistance were found in the perennial ryegrass population with 25-fold resistance, whereas only the non-target-site mechanism of resistance was found in the population with sevenfold resistance. This is the first study of the mechanism of glyphosate resistance in perennial ryegrass.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Glicina/análogos & derivados , Resistência a Herbicidas , Lolium/genética , Genótipo , Glicina/metabolismo , Herbicidas/metabolismo , Lolium/química , Lolium/enzimologia , Nova Zelândia , Folhas de Planta/química , Análise de Sequência de DNA , GlifosatoRESUMO
Land plant plastid genomes (plastomes) provide a tractable model for evolutionary study in that they are relatively compact and gene dense. Among the groups that display an appropriate level of variation for structural features, the inverted-repeat-lacking clade (IRLC) of papilionoid legumes presents the potential to advance general understanding of the mechanisms of genomic evolution. Here, are presented six complete plastome sequences from economically important species of the IRLC, a lineage previously represented by only five completed plastomes. A number of characters are compared across the IRLC including gene retention and divergence, synteny, repeat structure and functional gene transfer to the nucleus. The loss of clpP intron 2 was identified in one newly sequenced member of IRLC, Glycyrrhiza glabra. Using deeply sequenced nuclear transcriptomes from two species helped clarify the nature of the functional transfer of accD to the nucleus in Trifolium, which likely occurred in the lineage leading to subgenus Trifolium. Legumes are second only to cereal crops in agricultural importance based on area harvested and total production. Genetic improvement via plastid transformation of IRLC crop species is an appealing proposition. Comparative analyses of intergenic spacer regions emphasize the need for complete genome sequences for developing transformation vectors for plastid genetic engineering of legume crops.
Assuntos
Biotecnologia , Evolução Molecular , Fabaceae/genética , Variação Genética , Genomas de Plastídeos/genética , Sequências Repetidas Invertidas/genética , Sequência de Aminoácidos , Técnicas de Transferência de Genes , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Trifolium/genéticaRESUMO
The discovery of CXCR7 as a new receptor for SDF-1 places many previously described SDF-1 functions attributed to CXCR4 in question, though whether CXCR7 acts as a signaling or "decoy" receptor has been in debate. It is known that CXCR7 is not expressed in normal blood leukocytes; however, the potential role of leukocyte CXCR7 in disease states has not been addressed. The aim of this study was to determine the expression and function of macrophage CXCR7 linked to atherosclerosis. Here, we show that CXCR7 was detected in macrophage-positive area of aortic atheroma of ApoE-null mice, but not in healthy aorta. During monocyte differentiation to macrophages, CXCR7 was up-regulated at mRNA and protein levels, with more expression in M1 than in M2 phenotype. In addition, CXCR7 induction was associated with a SDF-1 signaling switch from the pro-survival ERK and AKT pathways in monocytes to the pro-inflammatory JNK and p38 pathways in macrophages. The latter effect was mimicked by a CXCR7-selective agonist TC14012 and abolished by siRNA knockdown of CXCR7. Furthermore, CXCR7 activation increased macrophage phagocytic activity, which was suppressed by CXCR7 siRNA silencing or by inhibiting either the JNK or p38 pathways, but was not affected by blocking CXCR4. Finally, activation of CXCR7 by I-TAC showed a similar signaling and phagocytic activity in macrophages with no detectable CXCR3. We conclude that CXCR7 is induced during monocyte-to-macrophage differentiation, which is required for SDF-1 and I-TAC signaling to JNK and p38 pathways, leading to enhanced macrophage phagocytosis, thus possibly contributing to atherogenesis.
Assuntos
Aterosclerose/metabolismo , Quimiocina CXCL12/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Fagocitose , Receptores CXCR/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Quimiocina CXCL11/genética , Quimiocina CXCL11/metabolismo , Quimiocina CXCL12/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Mutantes , Monócitos/metabolismo , Monócitos/patologia , Oligopeptídeos/farmacologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/mortalidade , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR/genética , Receptores CXCR3/genética , Receptores CXCR3/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismoRESUMO
BACKGROUND: White clover (Trifolium repens) is a ubiquitous weed of the temperate world that through use of improved cultivars has also become the most important legume of grazed pastures world-wide. It has long been suspected to be allotetraploid, but the diploid ancestral species have remained elusive. Putative diploid ancestors were indicated by DNA sequence phylogeny to be T. pallescens and T. occidentale. Here, we use further DNA evidence as well as a combination of molecular cytogenetics (FISH and GISH) and experimental hybridization to test the hypothesis that white clover originated as a hybrid between T. pallescens and T. occidentale. RESULTS: T. pallescens plants were identified with chloroplast trnL intron DNA sequences identical to those of white clover. Similarly, T. occidentale plants with nuclear ITS sequences identical to white clover were also identified. Reciprocal GISH experiments, alternately using labeled genomic DNA probes from each of the putative ancestral species on the same white clover cells, showed that half of the chromosomes hybridized with each probe. F1 hybrids were generated by embryo rescue and these showed strong interspecific chromosome pairing and produced a significant frequency of unreduced gametes, indicating the likely mode of polyploidization. The F1 hybrids are inter-fertile with white clover and function as synthetic white clovers, a valuable new resource for the re-incorporation of ancestral genomes into modern white clover for future plant breeding. CONCLUSIONS: Evidence from DNA sequence analyses, molecular cytogenetics, interspecific hybridization and breeding experiments supports the hypothesis that a diploid alpine species (T. pallescens) hybridized with a diploid coastal species (T. occidentale) to generate tetraploid T. repens. The coming together of these two narrowly adapted species (one alpine and the other maritime), along with allotetraploidy, has led to a transgressive hybrid with a broad adaptive range.
Assuntos
Quimera/genética , Evolução Molecular , Genoma de Planta/genética , Trifolium/genética , Adaptação Biológica/genética , Sequência de Bases , Cruzamento , Quimera/classificação , Pareamento Cromossômico , Cromossomos de Plantas/genética , Análise Citogenética , DNA de Cloroplastos/química , DNA de Cloroplastos/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Diploide , Genótipo , Hibridização Genética , Dados de Sequência Molecular , Filogenia , Folhas de Planta/genética , Pólen/genética , Sementes/genética , Análise de Sequência de DNA , Tetraploidia , Trifolium/classificaçãoRESUMO
BACKGROUND AND AIMS: DNA sequence similarities and hybridization patterns in Trifolium (clovers) section Trifoliastrum suggest that rapid radiation from a common ancestral source led to this complex of diverse species distributed across Europe, western Asia and North Africa. Two of the most geographically and ecologically divergent of these species are the rhizomatous T. ambiguum from high altitudes in eastern Europe and western Asia and the stoloniferous T. occidentale from sea level in western Europe. Attempts were made to hybridize these species to ascertain whether, despite this separation, gene flow could be achieved, indicating the retention of the genetic factors necessary for hybridization. METHODS: Three F(1) hybrids formed after embryo rescue were described, characterized by conventional and molecular cytogenetics, subjected to fertility tests and progeny generations were developed. RESULTS AND CONCLUSIONS: Partially fertile hybrids between Trifolium ambiguum and T. occidentale were obtained for the first time. The F(1) hybrids produced seeds after open-pollination, and also produced triploid progeny in backcrosses to T. occidentale from the functioning of unreduced gametes in the hybrids. These plants were fertile and produced progeny with T. occidentale and with T. repens. Meiotic chromosome pairing in the F(1) showed six to eight bivalents per pollen mother cell, indicating pairing between the parental genomes. A chromosome-doubled form of one hybrid, produced using colchicine, showed some multivalents, indicative of interspecific chromosome pairing. The hybrid plants were robust and combined phenotypic characteristics of both species, having stolons, thick roots and a few rhizomes. Results show that despite separation by the entire breadth of Europe, the speciation process is incomplete, and these taxa have partially retained most of the genetic compatibilities needed for hybridization (possibly except for endosperm development, which was not tested). The fertile progeny populations could lead to new clover breeding strategies based on new hybrid forms.
Assuntos
Quimera/genética , Diploide , Trifolium/genética , Pareamento Cromossômico , Europa (Continente) , Fluxo Gênico , Transferência Genética Horizontal , Especiação Genética , Genoma de Planta , Hibridização Genética , TriploidiaRESUMO
Suckling clover, Trifolium dubium Sibth., is a European grassland legume that has spread to many parts of the world. The present work shows that it is an allotetraploid (2n = 4x = 30) combining the genomes of T. campestre Schreb. (2n = 2x = 14) and T. micranthum Viv. (2n = 2x = 16), two diploid species of similar geographic distribution. T. dubium has two nuclear ITS sequences that closely match those of T. campestre and T. micranthum. Genomic in situ hybridisation using genomic DNA of T. campestre and T. micranthum as probes has differentiated the ancestral sets of chromosomes in T. dubium cells. Comparative fluorescence in situ hybridisation analyses of 5S and 18S-26S rDNA loci were also consistent with an allotetraploid structure of the T. dubium genome. A marked preponderance of ITS repeats from T. campestre over those from T. micranthum indicated that concerted evolution has resulted in partial homogenisation of these sequences by depletion of the T. micranthum-derived 18S-26S rDNA repeats. In parallel with this, the epigenetic phenomenon of nucleolar dominance has been observed in T. dubium such that the chromatin associated with the 18S-26S rDNA loci derived from T. campestre is decondensed (transcriptionally active), whilst that from T. micranthum remains highly condensed throughout the cell cycle. T. dubium, therefore, appears to have arisen by way of hybridisation between forms of the diploid species T. campestre and T. micranthum accompanied by chromosome doubling. The observed genomic changes in rDNA resulting from interspecific hybridisation provide evidence for the process of genome diploidisation in T. dubium.