Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root nodules and soil samples

Summary The isolation and pure culture of the symbiotic nitrogen-fixing frankiae has always been difficult. In the past the isolation of these actinomycetes directly from soil samples has proven impossible and isolations from root nodules of many genera has been only poorly successful. We report here a modified sucrose fractionation procedure which increased the success of isolations from root nodules and which permitted the isolation ofFrankia directly from soil samples. Crushed nodule suspensions or soil suspensions were incubated briefly in 0.7% phenol (carbolic acid) just before application to a sucrose density gradient. This phenol incubation decreased the number of contaminating eubacteria and fungi but more importantly increased the number ofFrankia developing on the isolation plates. If the phenol incubation was used solely without sucrose fractionation noFrankia were isolated, suggesting the death of the organisms due to phenol toxicity. The use of selective nitrogen-deficient media proved important for the isolation of frankiae from soils.     

A set of simple PCR markers converted from sequence specific RFLP markers on tomato chromosomes 9 to 12

A set of 24 simple PCR markers was generated for tomato chromosomes 9, 10, 11 and 12. Polymorphism was sought for between Lycopersicon esculentum and one of six other Lycopersicon species (L. parviflorum, L. cheesmanii, L. hirsutum, L. pennellii, L. peruvianum, and L. chilense). PCR primers, which were designed from mapped RFLP sequences, were used to amplify genomic DNA of the different species and the PCR amplification products were screened for polymorphism by testing restriction enzymes. With this approach, 24 (71%) of the 34 selected RFLPs were converted into simple PCR markers. By using a reference population, the map positions of these markers relative to the original RFLP markers were verified. These markers are locus specific and can be efficiently used for alignment of linkage maps, mapping target genes and marker assisted selection.     

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.     

Differentiation of pathogenic and nonpathogenic isolates of <Emphasis Type=Italic>Geotrichum candidum</Emphasis> sensu Suprapta et al. (1995) on citrus fruit based on PCR-RFLP analysis of rDNA ITS and PCR using specific primers designed in polygalacturonase genes

Pathogenic and nonpathogenic isolates of Geotrichum candidum sensu Suprapta et al. (1995) that affect citrus fruit are indistinguishable morphologically. In this work, differentiation of the two pathogenicity types based on the internal transcribed spacer regions of ribosomal DNA (rDNA-ITS) and polygalacturonase (PG) genes was attempted. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of rDNA ITS and PCR using specific primers to PG genes from each type could clearly differentiate the two pathogenicity types, and the profiles by PCR-RFLP of rDNA ITS genes corresponded with those by type-specific PCR of PG genes. These results indicate that the two pathogenic types can be differentiated at a molecular level and that PG genes are alternatively useful for distinguishing between the two types.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

Alder-Frankia interaction and alder-poplar association for biomass production

Summary Alders have an important role to play in biomass producing stands because of their N₂-fixing ability and their capacity to withstand soils having an excess of moisture. The objectives of preliminary trials were (1) to find if there is any alder-genotype xFrankia-strain interaction when the effect of inoculating the bacteria was compared to no inoculation in seed beds of different species and provenances of alder, (2) to measure the possible effect of black alders interplanted in poplars compared to pure poplar plots. Two trials were laid out to study the alder-Frankia interaction. Both produced interaction. In the first one the inoculation had a favorable effect onAlnus glutinosa at age 2 years andA. cordata at age 1 and 2 and no effect onA. rubra. In the second one the inoculation had a depressive effect at age 1 on 2 of 3 provenances ofA. rubra and no effect on 1A. rubra, 3A.glutinosa and 3A. cordata provenances.A closely spaced field trial associating one black alder provenance and the poplar clone UNAL gives no superiority of mixed plots compared to pure plots. The results suggest that the N₂-fixation of alders is not profitable to poplars at age 3 with a 1.5×2 m spacing.     

Identification of the DNAs of the ectomycorrhizal fungusTricholoma bakamatsutake using specific oligonucleotide probes and PCR primers

Partial nucleotides of the 18S rDNAs ofTricholoma bakamatsutake were sequenced and compared with those of six ectomycorrhizal fungi and a tree. Two probes, Probes 1 and 2, and a pair of primers were designed based on the variable positions in this region. The DNAs ofT. bakamatsutake were isolated from the colonized mycelia in the soil, field-collected fruit-bodies and artifically cultured mycelia. Hybridization with Probe 1 and PCR-amplification with the primers differentiated these DNAs of this fungus from those of eight ectomycorrhizal fungi and two tree species.     

Stability of RAPD fingerprints in potato: Effect of source tissue and primers

Variations in random amplified polymorphic DNA (RAPD) profiles from leaf, stem, root, and tuber tissues were observed in case of two glasshouse grown potato cultivars using 40 decamer primers suggesting possible danger of cultivar misidentification. Genomic DNA extracted from the above four tissues of four in vitro grown potato cultivars, however, produced more uniform RAPD fingerprints. A significant effect of random primers on fingerprint uniformity was observed in case of both glasshouse and in vitro grown samples. A new concept of stability index for random primers based on homogeneity of RAPD profiles obtained from different tissues of a single plant have been introduced. It is concluded that RAPD analysis of genomic DNA extracted from any tissue of in vitro grown potato plants using 14 selected decamer primers could be used to develop RAPD fingerprints for identification of Indian potato cultivars.     

Genetic relationship of Tricholoma matsutake and T. nauseosum from the Northern Hemisphere based on analyses of ribosomal DNA spacer regions

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.     

Phylogeny and biogeography of the flowering plant genus Styrax (Styracaceae) based on chloroplast DNA restriction sites and DNA sequences of the internal transcribed spacer region

Phylogenetic relationships within the flowering plant genus Styrax were investigated with DNA sequence data from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and with chloroplast DNA restriction site data from the genes trnK, rpoC1, and rpoC2. The data sets from each genome were analyzed separately and in combination with parsimony methods. The results strongly support the monophyly of each of the four series of the genus but provide little phylogenetic resolution among them. Reticulate evolution may at least partly explain discordance between the molecular phylogenetic estimates and a prior morphological estimate within series Cyrta. The historical biogeography of the genus was inferred with unweighted parsimony character optimization of trees recovered from a combined ITS and morphological data set, after a series of combinability tests for data set congruence was conducted. The results are consistent with the fossil record in supporting a Eurasian origin of Styrax. The nested phylogenetic position of the South American members of the genus within those from southern North America and Eurasia suggests that the boreotropics hypothesis best explains the amphi-Pacific tropical disjunct distribution occurring within section Valvatae. The pattern of relationship recovered among the species of section Styrax ((western North America + western Eurasia) (eastern North America + eastern Eurasia)) is rare among north-temperate Tertiary forest relicts. The monophyly of the group of species from western North America and western Eurasia provides qualified support for the Madrean-Tethyan hypothesis, which posits a Tertiary floristic connection among the semiarid regions in which these taxa occur. A single vicariance event between eastern Asia and eastern North America accounts for the pattern of relationship among intercontinental disjuncts in series Cyrta.     

PCR-RFLP of the ribosomal DNA internal transcribed spacers (ITS) provides markers for the A and B genomes in<Emphasis Type="Italic"> Musa</Emphasis> L.

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the diploid ancestors of modern bananas that are mostly diploid or triploid cultivars with various combinations of the A and B genomes, including AA, AAA, BB, AAB and ABB. The objective of this study was to identify molecular markers that will facilitate discrimination of the A and B genomes, based on restriction-site variations in the internal transcribed spacers (ITS) of the nuclear ribosomal RNA genes. The ITS regions of seven M. acuminata and five M. balbisiana accessions were each amplified by PCR using specific primers. All accessions produced a 700-bp fragment that is equivalent in size to the ITS of most plants. This fragment was then digested with ten restriction enzymes (AluI, CfoI, DdeI, HaeIII, HinfI, HpaII, MspI, RsaI, Sau3AI and TaqI) and fractionated in 2% agarose gels, stained with ethidium bromide and visualized under UV light. The RsaI digest revealed a single 530-bp fragment unique to the A genome and two fragments of 350-bp and 180-bp that were specific to the B genome. A further 56 accessions representing AA, AAA, AAB, AB and ABB cultivars, and synthetic hybrids, were amplified and screened with RsaI. All accessions with an exclusively A genome showed only the 530-bp fragment, while accessions having only the B-genome lacked the 530-bp fragment but had the 350-bp and 180-bp fragments. Interspecific cultivars possessed all three fragments. The staining intensity of the B-genome markers increased with the number of B-genome complements. These markers can be used to determine the genome constitution of Musa accessions and hybrids at the nursery stage, and, therefore, greatly facilitate genome classification in Musa breeding.Communicated by H.F. Linskens     

Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains

An Agrobacterium vitis-specific DNA fragment (pAVS3) was generated from PCR polymorphic bands amplified by primer URP 2R. A. vitis specificity of this fragment was confirmed by Southern hybridization with genomic DNA from different Agrobacterium species. Sequence-characterized amplified region (SCAR) markers were developed for A. vitis specific detection, using 24-mer oligonucleotide primers designed from the flanking ends of the 670 bp insert in pAVS3. The SCAR primers amplified target sequences only from A. vitis strains and not from other Agrobacterium species or other bacterial genera. First round PCR detected bacterial cells between 5×10² and 1×10³ cfu/ml and the detection sensitivity was increased to as few as 2 cfu/ml by nested PCR. This PCR protocol can be used to confirm the potential presence of infectious A. vitis strains in soil and furthermore, can identify A. vitis strains from naturally infected crown galls.     

Devising an <Emphasis Type="Italic">in vitro</Emphasis> nodulation system for two actinorhizal plants: <Emphasis Type="Italic">Allocasuarina verticillata</Emphasis> (Lam.) L. Johnson and <Emphasis Type="Italic">Casuarina glauca</Emphasis> sieb. ex spreng (Casuarinaceae)

Summary The purpose of this study was to establish an efficient in vitro nodulation device for producing actinorhizal root nodules on Allocasuarina verticillata and Casuarina glauca. Seeds from the two species were germinated aseptically and seedlings with at least two photosynthetic branchlets and a 3–5 cm long root system were transferred into Petri dishes containing a biphasic (solid/liquid) medium. To assess the nodulation capacity, four different culture media were tested. As soon as the root system developed and spread adequately on the surface of the medium, plants were deprived of nitrogen for at least 1 wk and inoculated with the Frankia strain. The time course nodulation for A. verticillata showed that the basal Hoagland medium supplemented with CaCO₃ and KNO₃ was most efficient, with 83% of plantlets forming nodules, while the medium supplemented with CaCO₃ reached 100% nodulation for C. glauca. This procedure can provide a valuable tool for the study of early events of actinorhizal nodulation and spatio-temporal expression of symbiotic genes in transgenic Casuarinaceae.     

Multiplex specific PCR for identification of the genera Actinopolyspora and Streptomonospora, two groups of strictly halophilic filamentous actinomycetes

The genera Actinopolyspora and Streptomonospora are two groups of extremely halophilic filamentous actinomycetes. Members of these two genera are isolated frequently, probably due to the high occurrence of these actinomycetes in the hypersaline soil environment. Although members of these genera can be identified by micromorphological criteria, the extensive chemotaxonomic characterization of each new isolates is a time-consuming task which cannot always be undertaken when handling large numbers of isolates as is the case in natural products screening programmes. In this work, the design of one set of genus-specific PCR primers which allows rapid detection of members of the genus Actinopolyspora by means of PCR amplification is presented. And we developed a multiplex PCR protocol for identification of the species of the genera Actinopolyspora and Streptomonospora, simultaneously. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic relationships among Mediterranean Pistacia species evaluated by RAPD and AFLP markers

Polymorphisms among Mediterranean basin Pistacia species and accessions within species were assessed by random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. Twenty-eight Pistacia accessions representing six species from geographically diverse locations in the Mediterranean area were analyzed. With RAPD, a total of 259 DNA fragments were amplified by 27 pre-selected primers, 254 were polymorphic fragments. AFLP analysis with 15 primer sets, produced 954 (93%) polymorphic bands out of a total of 1026. A Mantel test revealed an extremely high correlation (r=0.99) between similarity matrices generated from RAPD and AFLP data sets, indicating that similar results were obtained by the two techniques. Dendrograms constructed from the similarity matrices showed that Pistacia species could be clustered into two groups, one group containing all the #E5/E5#. lentiscus and the second group containing all other accessions. The latter group was divided into two subgroups, one consisting of #E5/E5#. palaestina and #E5/E5#. terebinthus; the other consisting of #E5/E5#. atlantica, #E5/E5#. khinjuk and #E5/E5#. vera. P. vera and P. khinjuk were highly similar, as were P. palaestina and P. terebinthus.     

Highly variable microsatellite markers for the fungal and algal symbionts of the lichen Lobaria pulmonaria and challenges in developing biont-specific molecular markers for fungal associations

The availability of highly variable markers for the partners of a fungal symbiosis enables the integrated investigation of ecological and evolutionary processes at the symbiotic level. In this article we analyze the specificity of the first and to date only microsatellite markers that had been developed for an epiphytic lichen (Lobaria pulmonaria). We used DNA extracts from cultures of the fungal and of the green algal symbionts of L. pulmonaria as well as total DNA extracts from related Lobaria species associated with the same algal partner, and got evidence that five of the previously described microsatellite markers, proposed to be fungus-specific, are indeed alga-specific. Hence, highly variable microsatellite primer sets available for both, the algal and the fungal symbionts of L. pulmonaria are now at our hands, which allow us to investigate so far unexplored biological processes of lichen symbionts, such as codispersal and coevolution. In a broader sense, our work evaluates and discusses the challenges in developing biont-specific molecular markers for fungi forming close associations with other organisms.     

Segregation and conditional probability association of molecular markers with traits in autotetraploid alfalfa

The complications introduced by the autotetraploid, outcrossing nature of alfalfa (Medicago sativa L.) as related to detecting associations of marker loci and traits of interest are discussed, and a new method of detecting marker-trait associations is suggested. This method utilizes plant populations that are likely to have been produced through the plant breeding process: populations selected for one trait, and the base, unselected population. Marker allele frequency shifts between the populations are indicative of genomic regions involved in trait expression, and may indicate alleles that have reached the triplex or homozygous state and do not segregate in S₁ or F₁ populations. However, because many, perhaps hundreds, of sequential frequency comparisons are needed to detect fragments in significantly different proportions in the two populations, the type I error rate is very high. A resampling-based analysis method is proposed to address the concern of the type I error rate, and identify marker alleles associated with this trait of interest. The utility of marker-trait associations thus defined for identifying individual plants from heterogeneous populations was investigated through model-building and conditional probability studies. Factors investigated that influenced the utility of the marker associations and (in the base population) the frequencies of the trait and marker, and the frequencies of the markers in plants exhibiting the trait and in the plants not exhibiting the trait. The frequency of occurrence of a marker in undesirable plants profoundly influenced the efficiency with which the marker could be used to select desirable plants, however, under some circumstances, markers or combinations of markers can be highly efficient for selecting rare, desirable plants from a heterogeneous base population.     

Development and application of microsatellite markers for genomic analysis of papaya

Papaya has a relatively small genome, displays high levels of phenotypic diversity, and is amenable to transformation, making it attractive as a fruit tree model system. The high level of phenotypic diversity seen among papaya cultivars in the field does not correlate with the low levels of genotypic polymorphism thus far elucidated. The highly mutable nature of microsatellites or simple sequence repeats (SSRs) make them potentially powerful markers for distinguishing deoxyribonucleic acid (DNA) polymorphisms between closely related genotypes. Genomic research for papaya has resulted in a significant quantity of sequence data. We mined 28.1 Mb of bacterial artificial chromosomes end sequences, 5.8 Mb of complementary DNA, and 1.6 Mb of random genomic sequences for SSRs. We generated 938 SSR markers and tested for polymorphism among seven varieties that had been used to produce five mapping populations. The level of polymorphism was highest for Kaek Dum × 2H94 with 210 markers, followed by UH928 × SunUp with 194, AU9 × SunUp with 189, UH918 × SunUp with 177, and Kapoho × SunUp displaying the lowest level with 97. Variation in levels of polymorphism, motif predominance, and motif length between the genomic and genic fractions indicated differential selection pressures acting on the microsatellites in these two fractions. The microsatellites developed in this study will greatly assist in the genetic and physical mapping of the papaya genome as well as enhance breeders’ ability to improve the crop. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.