Random Genomic Probes Research Articles

Multiple and Simultaneous Detection of Specific Bacteria in Enriched Bacterial Communities Using a DNA Microarray Chip with Randomly Generated Genomic DNA Probes

A DNA microarray chip for detecting the presence of specific bacterial strains was developed using random genomic probes derived from genomic DNA, i.e., without any sequence information. Thirteen bacteria from different genuses were selected as targets. For the fabrication of the random genomic probes, genomic DNA from pure cultures of each bacterium was fractionated using several pairs of restriction endonucleases. After size fractionation of the genomic DNA fragments, random genomic libraries for each bacterium were constructed. From the library, specific probes were amplified by PCR and the probes were affixed to a slide glass to fabricate the DNA microarray chip. The results from tests with pure and mixed cultures of the bacteria used in the fabrication of the chips showed specific responses and only a small portion of cross-hybridization. This DNA microarray chip was also tested to detect the presence of specific bacteria in mixed populations. In these tests, it was demonstrated that this system provided a fast and specific response to the presence of bacterial species in mixed samples, even in activated sludge samples. This indicates that any DNA microarray chip for the detection of specific bacteria can be fabricated using the same protocols as presented in this study without requiring any genus level sequence information from pure isolates.

Genetic linkage mapping in Acacia mangium 1. Evaluation of restriction endonucleases, inheritance of RFLP loci and their conservation across species

Random genomic probes were used to assess levels of restriction fragment length polymorphism (RFLP) in two 2-generation outbred pedigrees of Acacia mangium Willd. Probes were evaluated for their ability to detect polymorphic loci in each pedigree and to determine the relative efficiency of different restriction enzymes in revealing polymorphisms. Sixty two percent of the probes which detected single- or low-copy number sequences revealed polymorphisms with at least one restriction enyzme. HpaII was the most efficient in detecting polymorphism among first-generation individuals. The recognition sequence of HpaII contains a CpG dimer, suggesting that cytosines in the CpG sequence may be hotspots for mutation in plant genomes, as previously reported in bacterial and mammalian genomes. Mendelian inheritance of 230 loci was demonstrated based on single-locus segregation in second-generation individuals. Less than 5% of loci showed evidence of segregation distortion. The proportion of fully informative loci (15%) was lower than previously reported in eucalypts reflecting the lower level of genetic diversity in A. mangium. The RFLP probes are suitable for the construction of a high-density genetic linkage map in A. mangium. Cross-hybridisation of the A.mangium RFLPs to DNA from species representing the three subgenera of the genus Acacia indicates that these markers could be used in breeding programs of other diploid acacias, for comparative studies of genome organisation, and for phylogenetic studies.

Genetic diversity among Paspalum spp. as determined by RFLPs

The genus Paspalum is characterized by over 400 species that are indigenous to a wide range of stressful habitats and marginal environments. Fifty-one accessions representing 29 Paspalum species were analyzed for DNA restriction fragment length polymorphisms (RFLPs). Fifteen random genomic probes were used in combination with restriction enzyme EcoRI to detect RFLPs, and data were analyzed phenetically. Hybridization with the 15 selected clones resulted in the detection of 261 RFLPs. Among the 261 restriction fragments scored, 204 (78.2%) were phenetically informative. Extensive RFLP variation was found between the species studied. Species affinities based on RFLP data were found to be in close agreement with previously determined relationships based on both morphological and cytological characteristics.

A Restriction Fragment Length Polymorphism Probe for Early Diagnosis of Gender in Asparagus officinalis L.

The use of a sex-linked molecular marker for early sex diagnosis in the dioecious species Asparagus officinalis L. was evaluated. Screening of random genomic probes as a part of a restriction fragment length polymorphism mapping project resulted in the identification of a sex-linked (6.9 cM) marker. The usefulness of this molecular tool was compared to morphological markers for prediction of gender in several genotypes. The level of polymorphism detected by this probe was high, and the level of incorrect sex attribution, as determined by this method, was low (≈7%).

Phylogenetic relationships of the pigeonpea (Cajanus cajan) based on nuclear restriction fragment length polymorphisms

Nuclear restriction fragment length polymorphisms (RFLPs) were used to determine phylogenetic relationships in the genus Cajanus using 15 random genomic probes and six restriction enzymes. Twenty-four accessions representing 12 species of four genera (Cajanus, Dunbaria, Eriosema, and Rhynchosia) were examined to determine phylogenetic relationships in the genus Cajanus. Eriosema parviflorum was selected as the out-group. Sufficient RFLP polymorphisms were detected among species to resolve in-group taxa into distinct clusters. Topologies of trees from parsimony and similarity matrix analyses were similar but not identical, and clustering patterns agreed broadly with published phylogenies based on seed protein data and, to a lesser extent, data from cytology and breeding experiments. Accessions of cultivated C. cajan shared more DNA fragments with C. scarabaeoides than with C. cajanifolia. Inconsistencies in taxonomic relationships based on data from morphology, cytology, crossability, and RFLPs are discussed.

A panel of probes detects DNA polymorphisms in human and non-human primate isolates of a periodontal pathogen, Actinobacillus actinomycetemcomitans

A panel of five DNA probes has been developed for use in genetic epidemiologic analysis of Actinobacillus actinomycetemcomitans, a bacterium associated with periodontal disease. Restriction fragment length polymorphism profiles were assessed using random genomic probes as well as probes for genes encoding potential virulence factors. These genotypes were compared with serotype and other phenotypic markers. Genotype was shown to be more stable than the phenotypic markers and should prove useful in epidemiological studies of periodontal disease. In addition, the use of a panel of probes has the advantage of being able to distinguish between the occurrence of a new isolate as opposed to a mutation in a strain already characterized. The panel of probes was tested on 35 isolates from humans, 18 isolates from monkeys, and two isolates from a baboon. The genetic probes revealed significant genetic diversity among the A. actinomycetemcomitans isolates. Importantly, most human isolates from an individual genotyped identically, whereas most isolates cultured from a given monkey were different, suggesting that one should exercise caution in comparison of A. actinomycetemcomitans infections in human and non-human primates.

RFLP-based phylogeny of Musa species in Papua New Guinea

Random genomic probes were used to detect restriction fragment length polymorphisms (RFLPs) in 26 accessions of Musa representing eight species from Papua New Guinea (PNG), M. textilis, M. jackeyi and one accession of Ensete. Ninety-eight phylogenetically informative characters were scored and analyzed cladistically and phenetically. Results generally agreed with previous morphology-based phylogenetic analyses. However, the closest wild relative of the edible M. fehi (fe'i banana) appears to be M. lolodensis. Musa angustigemma is sister species with M. boman and M. jackeyi and is distinct from M. peekelii, with which it is often united. Musa boman is unambiguously placed in section Australimusa. The diploid parthenocarpic landraces of section Musa unique to PNG are closely related to, but apparently distinct from, M. acuminata ssp. banksii. The evolution of the fe'i bananas and the M. acuminata-derived diploid landraces of PNG are discussed.

Restriction fragment length polymorphism (RFLP)-based phylogenetic analysis of Musa

Random genomic probes were used to detect RFLPs in 19 Musa species and subspecies. A total of 89 phylogenetically informative alleles were scored and analyzed cladistically and phenetically. Results were in general agreement with morphology-based phylogenetic analyses, with the following exceptions: our data unambiguously places M. boman in section Australimusa, and indicates M. beccarii is very closely related to M. acuminata. Additionally, no support was found for the separation of section Rhodochlamys from section Musa. A comparison of morphology-based and RFLP-based phylogenetic analyses is presented.

Restriction fragment length polymorphism evaluation of six peanut species within the Avachis section

Restriction fragment length polymorphisms (RFLP) were assessed among accessions within six peanut species of the Arachis section: tetraploid cultivated species, A. hypogaea; tetraploid wild species, A. monticola; and four diploid wild species, A. batizocoi,A. cardenasii, A. duranensis and A. glandulifera. While the two tetraploid species did not show polymorphism with 16 PstI-generated random genomic probes, two of seven seed cDNA probes detected polymorphisms. The RFLP variation detected by two seed cDNA probes appeared to be related to structural changes occurring within tetraploid species. The botanical var. 'fastigiata' (Valencia market type) of A. hypogaea subspecies fastigiata was shown to be the most variable. Arachis monticola was found to be more closely related to A. hypogaea subspecies hypogaea than to subspecies fastigiata. Diploid species A. cardenasii, A. duranensis, and A. glandulifera showed considerable intraspecific genetic diversity, but A. batizocoi showed little polymorphism. The genetic distance between the cultivated peanut and wild diploid species was found to be closest for A. duranensis.

Bamboo germplasm screening with nuclear restriction fragment length polymorphisms

Bamboo species are difficult to identify because flowering material is seldom available and taxonomy is of necessity based on vegetative characters. To evaluate the utility of restriction fragment length polymorphism (RFLP) analysis in bamboo systematics and germplasm screening, a library of random genomic probes from a Phyllostachys nigra PstI library was constructed. Probes from the library were used to screen bamboo germplasm consisting mostly of temperate bamboos of the genus Phyllostachys. RFLP variation was abundant, and species-specific patterns were readily obtained. Chloroplast DNA showed little variation among the bamboo accessions analyzed.

A Molecular Approach to Distinguish the Bean Rust and Siratro Rust Pathogens

Rust disease on the tropical pasture legume Macroptilium atropurpureum (siratro) is caused by Uromyces appendiculatus var. crassitunicatus. This pathogen was believed to be closely related to the bean (Phaseolus vulgaris) rust pathogen Uromyces appendiculatus var. appendiculatus. The genetic relationship between these two fungi was investigated. Total DNA hybridisations indicated that little homology exists between the high copy genomic DNA of these two rust fungi. Random genomic probes cloned from the bean rust fungus detected extensive Polymorphisms between the two, with only one probe from 17 being monomorphic. The ribosomal DNA repeat unit was also distinguished by RFLPs. It was calculated from the RFLP data that the bean rust fungus and the siratro rust fungus share only 8-14% sequence homology. The results indicate that the two fungi, although morphologically very similar, are not closely related genetically.

Restriction fragment length polymorphisms in Colletotrichum gloeosporioides infecting Stylosanthes spp. in Australia

Restriction fragment length polymorphisms (RFLPs) have been used to estimate the relationship between four isolates of each of the Type A and B forms of Colletotrichum gloeosporioides which cause anthracnose on Stylosanthes spp. They were distinguished by polymorphisms in ribosomal DNA repeats and after hybridization with both low and high copy random genomic probes. The nucleotide sequence divergence estimate confirmed the existence of two distinct pathogen populations in Australia. Two high copy probes indicated that genetic diversity was low within the Type B population. One diagnostic high copy probe could be used to distinguish a teleomorphic isolate and isolates from the tropical legumes Centrosema pubescens and Aeschynomene falcata, from the Type A and B isolates. RFLPs are, therefore, extremely useful for determining genetic relationships within C. gloeosporioides.

Efficient linkage of 10 loci in the proximal region of the mouse X chromosome

Interspecific Mus species crosses were used to construct a multilocus genetic map of the mouse X chromosome that extends for more than 50 cM. In these studies, we established the segregation of eight loci in more than 200 backcross progeny from crosses of M. musculus and M. spretus with a common inbred strain ( C57 BL 6 JRos ). Genetic devergence at the level of the nucleotide sequences makes these crosses a useful cumulative genetic resource for mapping additional genes defined by genomic or cDNA probes in a highly efficient manner. We have therefore devised a mapping strategy that uses a subset of these backcrosses that are recombinant between successive anchor loci to both localize and order an additional set of six genes without necessarily resorting to an analysis of the entire backcross series. Using this approach, we have defined the linkage of cytochrome b 245 β-chain ( Cybb), synapsin ( Syn-1), and two members of the X-linked lymphocyte-regulated gene family ( Xlr-1, Xlr-2), as well as DXSmh141 and DXSmh172, two loci defined by random genomic probes. All six loci have been localized to the proximal portion of the mouse X chromosome and their order has been defined as Cybb, Otc, Syn-1 Timp , DXSmh141 Xlr-1 , DXSmh172, Hprt, Xlr-2, Cf-9. Gene order was established by minimizing multiple recombination events across the region spanning an estimated 20 cM of the proximal X chromosome. The possible significance of the Xlr loci is discussed with respect to other X-chromosome loci that regulate the immune response.

Comparison of restriction endonucleases and sources of probes for their efficiency in detecting restriction fragment length polymorphisms in lettuce (Lactuca sativa L.)

As a first step in developing a detailed genetic map of lettuce (Lactuca sativa L.), 156 cDNA and 123 genomic DNA clones of lettuce were compared for their efficiency to detect restriction fragment length polymorphism (RFLP) between four lines of lettuce. Polymorphism was detected 2.5 times more frequently with cDNA probes than random genomic probes. Less polymorphism was detected with cDNA clones homologous to single copy than with cDNA clones homologous to multiple copy DNA sequences. A lower percentage of polymorphism was detected with genomic DNA clones homologous to repetitive sequences than with other types of probes. Digests with each of nine restriction endonucleases were compared; increased polymorphism was not correlated with the presence of a CpG dimer in the recognition sequence of the restriction endonuclease. Digests with enzymes recognizing four base pairs, however, displayed RFLPs less frequently. The six pairwise comparisons of the four lettuce lines showed different frequencies of polymorphism which only approximately corresponded to genetic distances obtained from previous isozyme analyses.

Mapping of the mouse X chromosome using random genomic probes and an interspecific mouse cross.

Two libraries enriched in murine X chromosome material have been constructed in the lambda vector NM 1149 from flow-sorted chromosomes. Inserts of unique genomic sequence DNA were purified and their X chromosome specificity characterised by hybridisation to a panel of somatic cell hybrid lines. Of the first five such X chromosome-specific probes characterised, all detect restriction fragment length polymorphisms (RFLPs) between inbred mouse laboratory strains such as C57BL/6 and BALB/c and the SPE/Pas mouse strain established from a wild Mus spretus mouse, when their DNAs are digested with the restriction enzyme TaqI. Taking advantage of these RFLPs, all five probes have been localised on the X chromosome using an interspecific backcross between the B6CBARI and SPE/Pas mouse strains segregating the X chromosome markers hypoxanthine phosphoribosyl transferase (Hprt) and Tabby (Ta). Three of the probes map to the region between the centromere and Hprt, and two distal to Ta. Since such X-specific sequence probes detect RFLPs between M. spretus and M. musculus domesticus DNAs with high frequency, a large panel of well localised probes should soon be available for studies of biological problems associated with the X chromosome which can best be approached using the murine species.