Genome discrimination and chromosome pairing in the Hordeum chilense × Aegilops tauschii amphiploid

Tritordeum (X Tritordeum Ascherson et Graebner) is a synthetic amphiploid belonging to the Triticeae tribe, which resulted from crosses between Hordeum chilense and wheat. It presents useful agronomic traits that could be transferred to wheat, widening its genetic basis. In situ hybridisation with total genomic DNA from H. chilense and cloned, repetitive DNA sequences (pTa71 and pAs1) probes were used to discriminate the parental origin of all chromosomes, to analyse the chromosome pairing and to identify the chromosomes in pollen mother cells (PMCs) at metaphase I of the tritordeum line HT251 (H^chH^chDD, 2n = 4x = 28). The H. chilense total genomic DNA and the ribosomal sequence pTa71 probes, allowed the unequivocal discrimination of the 14 chromosomes of H^ch genome-origin and the 14 chromosomes of D genome-origin. Chromosome pairing analysis revealed meiotic irregularities such as reduced percentage of PMCs with complete homologous pairing, high frequency of univalents, most of H. chilense-origin and a reduced frequency of intragenomic multivalents from both genomes. The H. chilense genome revealed high meiotic instability. After individual chromosome identification at metaphase I with the pAs1 probe, we found the occurrence of pairing between chromosomes of different homoeology groups. The possible interest of the tetraploid tritordeum in the improvement of other Triticeae species is also discussed.     

Development of synthetic Brassica napus lines for the analysis of “fixed heterosis” in allopolyploid plants

Summary Allopolyploids are widely spread in the plant kingdom. Their success might be explained by positive interactions between homoeologous genes on their different genomes, similar to the positive interactions between different alleles of one gene causing heterosis in heterozygous diploid genotypes. In allopolyploids, such interactions can also occur in homozygous genotypes, and may therefore be called “fixed heterosis”. As to our knowledge, no experimental data are available to support this hypothesis. We propose an experimental approach to quantify “fixed heterosis” in resynthesised Brassica napus and the detection of loci contributing to “fixed heterosis” via comparative QTL mapping in B. napus and its parental species B. rapa and B. oleracea. In order to develop a genetically balanced material, interspecific crosses between 21 Brassica rapa and 16 Brassica oleracea doubled haploid or inbred lines were performed. In total 3485 vital embryos have been obtained from 9514 pollinated buds. The success of interspecific hybridisation was highly depending on the maternal genotype (B. rapa) and ranged from 0 to 1.18 embryos per pollinated bud. For the genetic characterisation of the B. rapa and B. oleracea lines, a dendrogram was constructed based on 273 RAPD markers. Thus a well-characterised material is now available, which is suitable to analyse the effects of “fixed heterosis” and the interactions between homoeologous genes in allopolyploid species.     

Isolation and characterisation of somatic hybrids of diploid Solanum tuberosum and Solanum brevidens and the use of amylose-free starch mutation for detection of introgression

Summary Somatic hybrids of diploid amylose-free (amf) Solanum tuberosum and diploid S. brevidens were made by Poly-Ethylene-Glycol (PEG) or electrofusion methods. For the isolation of interspecific hybrids the use of selection markers (kanamycin and hygromycin resistance) was useful but not essential. In this 2x+2x interspecific combination 4x and 6x somatic hybrids were obtained. Seed set was the best in 4x×4x (S. tuberosum) backcrosses, but seed germination was the best in 6x×4x combinations, using in vitro germination of unripe seeds harvested 25 days after pollination. A high degree of pollen stainability (30–40%) was observed in 7 tetraploid hybrids and very low in all hexaploids. After iodine staining, the recessive amf marker was expressed by a red colour instead of blue, visible in starch-containing cells like columella cells of root tips, (micro)tubers or microspores. As expected, complementation was observed in starch-containing cells of the fusion hybrids. Segregation of the amf marker was clearly observed in microspores of 4x and 6x hybrids. Segregation ratios in the 4x hybrids showed variable recombination frequencies. In the backcross progeny of hexaploid F12-5 with a tetraploid amf mutant one amylose-free recombinant among 67 plants was found, indicating the occurrence of meiotic recombination in the megaspore mother cells.     

Root exudate-stimulated RNA accumulation in the arbuscular mycorrhizal fungus Gigaspora rosea

Plant root exudates induce the transition from asymbiosis to presymbiosis in arbuscular mycorrhizal fungi. In order to get an insight into this developmental switch, two libraries of Gigaspora rosea and one library of Gigaspora gigantea were screened for fragments of genes that show enhanced RNA accumulation 1 h after addition of a semi-purified exudate fraction of carrot roots. Among 150 clones, 40 seemed to contain inserts of root exudate-induced genes. One of the genes, GrosRbp1, putatively encoding an RNA binding protein involved in developmental control showed RNA accumulation which correlates to the extent of stimulation of presymbiotic hyphal branching.     

Sources of resistance to bacterial blight of stock (Matthiola incana R. Br.)

Summary Accessions ofM. tricuspidata (L.) R. Br.,M. aspera Boiss. andMatthiola longipetala (Vent.) DC. were tested for their reaction to bacterial blight of stock (M. incana R. Br.), caused byXanthomonas campestris pv.incanae. Potted plants were sprayed with bacterial cell suspension, adjusted to 10⁸ cells/ml. The tested accessions of the three species were found highly resistant, while control plants ofM. incana were severely infected. InterspecificF ₁ hybrid plants betweenM. incana andM. tricuspidata also exhibited high resistance to the disease. The implications and the expected difficulties in breeding stock for bacterial blight resistance are discussed. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No 1235-E, 1993 series.     

Simultaneous detection and identification of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> (PepMV) isolates by multiplex one-step RT-PCR

A RT-PCR was developed for the simultaneous detection and identification of three groups of Pepino mosaic virus (PepMV): European/Peruvian, Chilean 1/US1 and Chilean 2/US2 groups, followed by a restriction analysis that allowed the separation of the European, Peruvian, Chilean 2 and US2 isolates (patent pending). The multiplex RT-PCR reaction was performed by a mix of six primers that amplified a part of the RNA-dependent RNA polymerase gene of PepMV plus an internal control. Amplifications resulted in a 980 bp, 703 bp or 549 bp PCR product for European/Peruvian, Chilean 1/US1 or Chilean 2/US2 groups, respectively. For the identification of the isolates present within the European/Peruvian and Chilean 2/US2 groups, the amplified PCR fragments were directly digested with SacI enzyme. The multiplex RT-PCR method presented higher sensitivity to detect CH1/US1 isolates in field samples than the RFLP-PCR method described by Hanssen et al. (European Journal of Plant Pathology 121:131–146, 2008). The detection limit observed with the multiplex RT-PCR was equal to or 3,125 times higher when compared to single RT-PCR or ELISA-DAS and molecular hybridisation methods, respectively. The use of the multiplex RT-PCR method in routine analysis of field tomato samples allowed the detection of 36.2 and 33.4% more positives when compared to the serological and molecular hybridisation methods, respectively, and the identification of plants infected with one, two or three isolates of PepMV.     

Growth and reproductive characteristics in backcross hybrids derived from Trifolium repens L. × T. nigrescens Viv. interspecific crosses

Interspecific hybridisation with the close relative, Trifolium nigrescens (Ball clover) is a possible strategy to achieve increased reproductive potential of white clover (Trifolium repens). Fertile F₁ plants have been used as the basis for two generations of backcrossing to T. repens as the recurrent parent. F₁ and backcrossed plants were assessed in both glasshouse and field for a range of morphological traits, including inflorescence production, and the level of water soluble carbohydrates in the stolons. Plants resulting from two generations of backcrossing had an increased allocation of dry matter to inflorescence production in comparison with T. repens. Variation within these plants for agronomic traits (e.g. stolon length, dry weights etc.) suggests that selection for these traits is feasible and in combination with increased inflorescence production offers a potentially valuable approach to germplasm improvement in white clover. This revised version was published online in August 2006 with corrections to the Cover Date.     

Methods of detection of Chlamydia psittaci in domesticated and wild birds

OBJECTIVE: To study the occurrence of Chlamydia psittaci in domesticated and wild birds and compare the sensitivity of molecular detection with cell culture isolation. DESIGN: Study of cell culture isolation and PCR detection of C psittaci in avian samples. PROCEDURE: Samples were obtained from 485 birds. Domesticated birds were selected at random from pet shops, private aviaries and zoos, while wild birds were captured locally, sampled, and immediately released. Swabs were collected from choanal slit, conjunctiva and cloaca of each bird and pooled. Samples were divided into equal portions for use in PCR dot-blot and cell culture detection. PCR and dot-blot detection was based on the ompB gene. RESULTS: Prevalence of infection varied markedly between flocks of captive birds. It was highest where there were frequent changes in the flock members or where there were many birds confined in small areas. C psittaci was not detected in wild birds or water birds. The sensitivity of cell culture compared to PCR dot-blot detection was 68%. All samples positive by cell culture were also positive by PCR. CONCLUSIONS: PCR-dot blot detection of C psittaci in birds appears to be more sensitive than cell culture isolation in this study. C psittaci infection of birds may occur in clinically normal captive birds.     

Identification of novel Trichoderma hamatum genes expressed during mycoparasitism

Trichoderma species are currently used as biocontrol agents for crop diseases caused by a number of fungal plant pathogens. However, their biocontrol performance in the field can be unreliable and it is likely that more consistent performance could be achieved through knowledge and manipulation of the genes involved. For example, induction of the genes could be optimised for variable environmental and physiological conditions, superior strains could be selected more effectively and novel st…     

Patterns of co-existence and hybridisation between narrowly endemic (Fundulus euryzonus) and broadly distributed (F. olivaceus) topminnows in a riverine contact zone

Abstract –  Two ecologically similar topminnow species ( Fundulus olivaceus and F. euryzonus ) were studied in a contact zone in the West Fork of the Amite River of the Lake Pontchartrain drainage. We assessed whether relative abundance and measures of fitness of each species, and their hybrids, were correlated with predictable abiotic or biotic factors. We sampled the full fish assemblage and measured a suite of local and landscape level environmental characteristics at ten sites. Topminnows were genotyped to identify individuals of hybrid ancestry. We compared the abundance and distribution of parental species in the context of overall fish assemblage structure and environmental variables. Hybridisation was generally rare with only nine of 244 genotyped topminnows exhibiting evidence of hybrid ancestry. The relative abundance of the two topminnows was related to site assemblage structure, species diversity and a number of local but not landscape level environmental variables. Both species were in better condition and females were more fecund at sites with greater abundance of F. olivaceus . Hybrid individuals were not morphological or ecological intermediates.