RESUMO
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: 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
The predominant root-knot nematode in New Zealand pastures is Meloidogyne trifoliophila, and a recurrent selection program in Trifolium repens has developed resistance to this species. No data are available, however, on the mechanisms of resistance in T. repens or resistant genotypes of T. semipilosum. The development of M. trifoliophila in roots of T. repens and T. semipilosum was examined weekly after a 2-day inoculation with eggs. More second-stage juveniles (J2) were found in two resistant genotypes of T. repens than in two susceptible ones 1 week after inoculation. J2 did not develop further in resistant genotypes, but in susceptible plants development proceeded to the adult stage, visible at 4 weeks after inoculation. The mode of action of resistance to M. trifoliophila in T. repens and in T. semipilosum was compared after a 24-hour inoculation with J2. Numbers of J2 per root tip ranged from 0 to 12 with a median of one for each species. At 24 hours after inoculation (HAI), similar numbers of J2 were seen in the cortex oriented toward the root tip in both resistant and susceptible genotypes of both plant species. At 48 HAI, accumulations of J2 were seen in the meristem in both resistant and susceptible genotypes of both plant species. At 72 HAI, differences in nematode responses were evident between resistant and susceptible genotypes of both plant species; in susceptible roots, J2 heads were embedded in the developing stele. At this time, a browning reaction in resistant genotypes of both plant species indicated a hypersensitive response, and differences in the reaction were recorded between T. repens and T. semipilosum. More study is needed to determine whether the resistance reaction in T. semipilosum is suitable for introgression or insertion into T. repens.
