Revealed Restriction Fragment Length Polymorphisms Research Articles

Mitochondrial SCAR and SSR Markers for distinguishing cytoplasmic male sterile lines from their isogenic maintainer lines in cotton

With 8 figures and 3 tablesAbstractCytoplasmic male sterility (CMS) line P30A of Upland cotton has been used in commercial production of F1 seeds. However, cytoplasm‐specific molecular markers in this ‘three‐line’ (CMS line P30A, maintainer line P30B and restorer line Y18R) system are mostly unknown. Twenty mitochondrial gene probes were used to identify the differences between P30B fertile (N‐) and P30A sterile (S‐) cytoplasm. Among six genes that revealed restriction fragment length polymorphisms (RFLPs), the gene for α‐subunit of F1ATPase (atpA) revealed significant differences between N‐ and S‐cytoplasm. All of the EcoRI restriction bands of atpA were amplified by inverse PCR (iPCR) technique, indicating that both N‐ and S‐cytoplasm contained an intact and a 3′ truncated atpA copy, but the truncating site (breakpoint) was different. According to the chimerical sequences following the breakpoints, three cytoplasm‐specific sequence characterized amplified region (SCAR) markers were developed. In addition, a simple sequence repeat (SSR) locus in the 3′ flanking sequences of the intact atpA gene was found between N‐ and S‐cytoplasm. The four PCR markers were used to distinguish CMS lines from maintainer lines.

Molecular characterization of cytoplasmic male sterility conditioned by Gossypium harknessii cytoplasm (CMS-D2) in upland cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait that fails to produce functional pollen grains. The CMS system is widely employed to facilitate the utilization of heterosis in major crops. However, little is known about the CMS associated genes in Upland cotton (Gossypium hirsutum). The objective of this study was to compare CMS cotton (CMS-D2) with the cytoplasm from G. harknessii and its isogenic maintainer line with the normal fertile Upland cotton cytoplasm to identify CMS-D2 specific gene(s) and to develop CMS-specific sequence characterized amplified region (SCAR) markers. Based on Southern blot analysis using 10 mitochondrial gene-specific probes (cob, cox2, atp6, atp9, nad3, cox3, atpA, cox1, nad6 and nad9), three probes (cox3, atpA, and nad6) revealed restriction fragment length polymorphisms (RFLP) between the CMS-D2 and its isogenic maintainer line. RT-PCR confirmed that the three genes were differentially expressed between the two lines. These results indicated that there existed structural and expression variations in the three genes when the mitochondrial D2 genome was transferred into Upland cotton. Genome walking and rapid amplification of cDNA ends (RACE) were further performed to analyze the sequences of these genes and their flanking regions. For cox3 and nad6, there was only one different nucleotide each in the gene regions between the two lines. Also some nucleotides upstream of the ATG codon were different. For atpA, the sequences downstream the atpA were significantly different between the two cytoplasmic lines. Furthermore, two nucleotides at the -4 and -5 position from ATG codon were also changed between the two cytoplasms (i.e., CG→AA), and this mutation also exists in RNA sequences. Interestingly, nine nucleotides (ATGCAACTA) were also inserted at the location of 899 bp upstream of ATG codon in the CMS line. The results suggest that the abnormal sequence and expression of atpA gene is associated with CMS expression in Upland cotton. According to the significant different sequences downstream the atpA gene, a CMS-D2 specific SCAR marker was developed. The CMS-specific PCR bands were verified for 10 cultivars containing either normal- or CMS-D2cytoplasm. This will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F1 seed lots.

A single-base deletion in soybean flavonoid 3'-hydroxylase gene is associated with gray pubescence color.

The T locus of soybean (Glycine max (L.) Merr.) controls pubescence and seed coat color and is presumed to encode flavonoid 3'-hydroxylase (F3'H). The dominant T and the recessive t allele of the locus produce brown and gray pubescence, respectively. PCR primers were constructed based on the sequence of a soybean EST clone homologous to the F3'H gene. A putative full-length cDNA, sf3'h1 was isolated by 3' and 5' RACE. Sequence analysis revealed that sf3'h1 consists of 1690 nucleotides encoding 513 amino acids. It had 68% and 66% homology with corresponding F3'H protein sequences of petunia and Arabidopsis, respectively. A conserved amino acid sequence of F3'H proteins, GGEK, was found in the deduced polypeptide. Sequence analysis of the gene from a pair of near-isogenic lines for T, To7B (TT, brown) and To7G (tt, gray) revealed that they differed by a single C deletion in the coding region of To7G. The deletion changed the subsequent reading frame resulting in a truncated polypeptide lacking the GGEK consensus sequence and the heme-binding domain. Genomic Southern analysis probed by sf3'h1 revealed restriction fragment length polymorphisms between cultivars with different pubescence color. Further, sf3'h1 was mapped at the same position with T locus on LG3(c2). PCR-RFLP analysis was performed to detect the single-base deletion. To7B and three cultivars with brown pubescence exhibited shorter fragments, while To7G and three cultivars with gray pubescence had longer fragments due to the single-base deletion. The PCR-RFLP marker co-segregated with genotypes at the T locus in a F2 population segregating for the T locus. The above results strongly suggest that sJ3'h1 represents the T gene of soybean responsible for pubescence color and that the single-base deletion may be responsible for gray pubescence color.

Molecular tracking of infections by Leishmania infantum

Leishmania infantum is a major opportunistic parasite in patients with acquired immune deficiency syndrome and is very variable in these subjects. Isoenzyme characterization is not able to explain this variability, since half of the stocks isolated from patients co-infected with human immunodeficiency virus and Leishmania belong to zymodeme MON-1. Amplification of L. infantum minicircles by the polymerase chain reaction (PCR) and digestion of the amplified product to reveal restriction fragment length polymorphisms (RFLP) has proved very useful in distinguishing between relapses and reinfections in co-infected, treated patients. We have confirmed the existence of a leishmaniasis outbreak among intravenous drug users in north-east Spain, previously detected by isoenzymatic analysis. We have documented persistence of the same strain of Leishmania in 2 treated co-infected patients throughout several years, regardless of the theoretical rapid evolution ascribed to kinetoplast deoxyribonucleic acid minicircle sequences. We suggest using this PCR-RFLP technique to detect reinfections in treated co-infected subjects.

Spontaneous duplication of a 661 bp element within a two-component sensor regulator gene causes phenotypic switching in colonies of Pseudomonas tolaasii, cause of brown blotch disease of mushrooms.

Spontaneous sectoring of Pseudomonas tolaasii colonies results in a phenotypic switch from the smooth, pathogenic form (designated 1116S) to the rough non-pathogenic form (designated 1116R). This phenotypic switch can also be induced by mutation of the pheN master regulatory locus, which encodes a 99 kDa protein with homology to the conserved family of sensor regulator proteins. Southern blot analysis of genomic DNA from 1116S and 1116R probed with a 3.4 kb Xhol-BamHI fragment containing the pheN gene has revealed restriction fragment length polymorphisms in the pheN locus of 1116R. In order to characterize the genetic basis of this variation, the pheN locus (designated pheN') was cloned from 1116R and its nucleotide sequence determined. A 661 bp duplication was identified within pheN' introducing a frameshift mutation in the predicted pheN open reading frame (ORF). A resulting predicted ORF of pheN' designated ORF2 encodes a polypeptide of 706 amino acid residues, with a predicted molecular weight of 77 kDa, and which lacks part of the PheN sensor domain. Southern blot analysis of genomic DNA using a probe within the duplicated sequence revealed the presence of two bands in 1116R but only one band in the 1116S form. Polymerase chain reaction (PCR) analysis of 25 independently isolated 1116R sectors using primers flanking the duplication site in pheN confirmed the presence of the duplicated 661 bp sequence within this region in all of the sectors and the absence of the duplicated sequence in spontaneous revertants from 1116R to 1116S. Northern blot analysis of RNA from 1116S and 1116R using a pheN probe showed that ORF2 was transcribed in the 1116R form. The presence of a truncated PheN protein in 1116R was verified by Western blot analysis of total cell protein using a LemA antiserum, which revealed the presence of 99kDa and 77kDa cross-reactive bands in 1116S and 1116R respectively. It is concluded that the spontaneous colony-sectoring event that results in the 1116R phenotypic variant form of P. tolaasii arises owing to a 661 bp DNA duplication within the 5' end of the pheN gene, which results in loss of the periplasmic sensor domain of PheN and elimination of normal PheN function.

DNA polymorphisms in the chicken growth hormone gene: response to selection for disease resistance and association with egg production

Analysis of the growth hormone (GH) gene in 12 strains of White Leghorn chickens revealed restriction fragment length polymorphisms (RFLPs) at three MspI sites and at a SacI site. Based on linkage disequilibrium analysis, they gave rise to eight different alleles (i.e. combinations of RFLPs), with five occurring at frequencies above 5% in at least one strain. Pairs of GH-RFLPs were at near maximal linkage disequilibrium, suggesting either a lack of recombination or the presence of selection pressure during evolution of the GH gene. Allele frequencies were determined in 12 non-inbred strains derived from three different genetic bases. These strains had been selected either for an array of egg production traits, resistance to Marek's disease or resistance to avian leukosis. Selection for disease resistance was consistently correlated with an increase in the frequency of one of the alleles. One strain segregated for only two alleles, which differed by three RFLPs. Analysis of variance in this strain indicated that the GH allele co-selected with resistance was associated with a delayed onset of ovulation but a higher persistency of ovulation as age progressed, resulting in an overall increase of egg production by 15% (age at first egg to 497 days). The resistance-associated GH allele was dominant for the onset of ovulation and recessive for the persistency of egg production. There was no significant effect of the GH genotype on juvenile body weight, egg weight or egg specific gravity.

The physical relationship of barley chromosome 5 (1H) to the linkage groups of rice chromosomes 5 and 10.

Using a recently developed polymerase chain reaction (PCR)-mediated approach for physical mapping of single-copy DNA sequences on microisolated chromosomes of barley, sequence-tagged sites of DNA probes that reveal restriction fragment length polymorphisms (RFLP) localized on the linkage maps of rice chromosomes 5 and 10 were allocated to cytologically defined regions of barley chromosome 5 (1H). The rice map of linkage group 5, of about 135 cM in size, falls into two separate parts, which are related to the distal portions of both the short and long arms of the barley chromosome. The markers on the rice map of chromosome 5 were found to be located within regions of the barley chromosome which show high recombination rates. The map of rice chromosome 10, of about 75 cM in size, on the other hand, is related to an interstitial segment of the long arm of chromosome 5 (1H) which is highly suppressed in recombination activity. For positional cloning of genes of this homoeologous region from the barley genome, the small rice genome will probably prove to be a useful tool. No markers located on rice chromosomes were detected within the pericentric Giemsa-positive heterochromatin of the barley chromosome, indicating that these barley-specific sequences form a block which separates the linkage segments conserved in rice. By our estimate approximately half of the barley-specific sequences of chromosome 5 (1H) show a dispersed distribution, while the other half separates the conserved linkage segments.

Decreased muscle cell proliferation in chicks with a deletion in the GH receptor gene

The increase in muscle weight in neonatal animals is a consequence of increased protein accretion and DNA content. GH increases protein accretion but direct effects of GH on myogenic cell proliferation have not been demonstrated. Sex-linked dwarfism in the chick is caused by mutation or deletion in the GH receptor gene and has provided a useful model to study the physiological consequences of GH insensitivity. This study determined the consequences of GH receptor gene mutation on muscle cell proliferation in vivo. Northern and Southern blotting and PCR analysis revealed restriction fragment length polymorphism patterns and a 1.7 kb deletion of the intracellular domain of the GH receptor gene in commercial dwarf broiler chicks, similar to the Connecticut strain in which there is a dysfunctional GH receptor. Cell proliferation was measured in muscle sections from normal and dwarf chicks after incorporation of 5-bromo-2'-deoxyuridine (BrdU; 25 mg/kg) in vivo at 2, 5 and 13 days of age. Incorporation of BrdU into nuclei was measured in frozen sections, counterstained with propidium iodide to estimate the total number of nuclei by quantitative image analysis, and the labelling index was calculated. Paraffin-embedded sections of breast muscle were stained using an anti-human IGF-I polyclonal antibody. Expression of IGF-I mRNA in muscle from each genotype at 5 days of age was measured by RNAse protection assay. The labelling index was similar in 2-day-old chicks from both genotypes (normal, 20.14 +/- 2.39%; dwarf, 19.79 +/- 5.83%). By day 5 the labelling index had decreased but was significantly higher (P < 0.02) in normal (12.53 +/- 3.36%) compared with the dwarf (6.25 +/- 1.39%). By 13 days of age, there was a further decrease in labelling index but no difference between the groups (normal, 4.92 +/- 1.28%; dwarf, 4.96 +/- 1.51%). IGF-I mRNA was expressed and IGF-I peptide was identified in muscle sections but there was no difference between genotypes. The results show that cell division in breast muscle in vivo is high in neonatal chicks but it declines with increasing age. The absence of a functional GH receptor in the dwarf is associated with a greater decline in DNA synthesis and suggests that GH may directly affect a proportion of cells, since there was no difference in IGF-I mRNA or peptide.

GENETIC CHARACTERIZATION OF STRAINS OF CYANOBACTERIA USING PCR‐RFLP OF THE cpcBA INTERGENIC SPACER AND FLANKING REGIONS1

ABSTRACTOligonucleotide primers, specific for conserved regions of the genes encoding the β‐ and α‐phycocyanin subunits of phycobilisomes (cpcB and cpcA) of cyanobacteria, were used to amplify a DNA fragment containing the intervening intergenic spacer region (cpcBA‐IGS) of 19 strains of three morphospecies of cyanobacteria. Six Australian strains were identified as Anabaena circinalis Rabenhorst, six strains were identified as Microcystis aeruginosa Kützing, and seven strains were identified as Nodularia spumigena Mertens. Restriction enzyme digestion of the amplification products from the strains revealed restriction fragment length polymorphism (RFLP) within all three morphospecies. Strains corresponding to M. aeruginosa were highly polymorphic: 11 of the 14 restriction enzymes used displayed RFLPs. The A. circinalis and N. spumigena strains were less variable: three of 14 enzymes and seven of 14 enzymes, respectively, showed RFLPs. The presence of genetic variation between strains within these three divergent morphospecies, which span two orders of cyanobacteria (Chroococcales Wettstein and Nostocales (Borzi) Geitler), show that the cpcBA‐ IGS fragment has broad application as a molecular marker for intrageneric studies of cyanobacteria systematics and genetics.

Restriction fragment length polymorphism of the T-cell receptor beta-chain gene in dogs

Since T-cells and the T-cell receptor (TCR) play a pivotal role in the response of the immune system, they are a target for pathogenesis studies in immune mediated diseases and have been used to generate markers for T-cell dependent diseases in humans and dogs. TCR rearrangement is generated at the genomic DNA level and can be analyzed by Southern blotting techniques. In the present study this method to detect rearrangement of the TCR beta chain in the dog was critically examined. To search for restriction fragment length differences due to either inherited polymorphism or in diseases with suspected superantigen influence (X-linked severe combined immune deficiency and canine juvenile polyarteriitis syndrome) 13 dog families of three different breeds were examined. In addition primary spleen cell cultures, stimulated with either phytohemagglutinin A (PHA) or staphylococcus enterotoxin A (SEA) and B (SEB) were studied. The germline digest pattern of the enzymes Pst I, Sst I, Bgl II, Eco RI and Eco RV were identical in all dogs examined with the exception of one dog with canine juvenile polyarteriitis syndrome. In this dog an additional band was found in the Bgl II and Eco RV digestion suggestive of specific TCR rearrangement. Bam HI digestion revealed restriction fragment length polymorphisms (RFLPs) showing Mendelian inheritance. After digestion of the genomic DNA extracted from PHA, SEA or SEB stimulated spleen cells and Southern blot analysis, no differences in fragment patterns between the unstimulated cells and the stimulated cells could be detected. An important point to consider before a specific pattern variation between dogs is classified to be a marker for a specific disease or is used in pathogenesis studies, is the possibility of an inherited RFLP, especially after Bam HI digestion. In such studies the combined examination of the parents and the offspring must be recommended.

The follicle-stimulating hormone receptor gene is polymorphic in premature ovarian failure and normal controls

To examine the FSH receptor gene for detectable abnormalities in women with premature ovarian failure. Study of genomic DNA from controls and from patients with 46,XX premature ovarian failure (POF). Clinics and laboratories of university gynecology and obstetrics departments. Twenty-one women with 46,XX POF and 40 normal fertile controls. Deoxyribonucleic acid was analyzed in patients and controls by Southern blot analysis, polymerase chain reaction (PCR), and denaturing gradient gel electrophoresis. Southern blots were hybridized with the FSH receptor complementary DNA and other smaller DNA probes. Exons 1, 5 to 6, and 10 were amplified by PCR and electrophoresed on agarose gels. Polymerase chain reaction products from exons 1 and 10 were electrophoresed on denaturing gradient gels. Fragments obtained by Southern blot analysis and PCR were compared in patients and controls. Polymerase chain reaction fragments electrophoresed on denaturing gels also were compared in patients and controls. No FSH receptor gene deletions or other mutations were identified in women with POF. Southern blots containing PstI- and HindIII-digested DNA revealed restriction fragment length polymorphisms in both patients and controls. Denaturing gradient gel electrophoresis analysis of PCR fragments of exon 10 also demonstrated DNA sequence polymorphisms in both patients and controls. Follicle-stimulating hormone receptor gene deletions are not common in women with POF, although the gene is polymorphic. We cannot exclude point mutations in other regions of the FSH receptor gene in some patients with POF.

Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO

A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle.

Analysis of Heat Shock Response in Perennial Ryegrass using Maize Heat Shock Protein Clones

Evidence suggests that heat shock proteins (HSP) may be involved in differences in thermal tolerance among perennial ryegrass (Lolium perenne L.) cultivars. It seems possible that HSP could be useful in developing laboratory assays for thermal tolerance in perennial ryegrass. To evaluate the possibility of using HSP genes or gene products to screen for thermal tolerance, thermal‐tolerant and thermal‐sensitive cultivars were germinated and heat treatments were conducted in a dry germinator in constant light. The two cultivars exhibited differences in root, shoot, and total seedling length when briefly subjected to high temperatures (55 °C) following an initial acclimation period (40 °C). In vitro translation products of total RNA indicated that different messenger RNA (mRNA) populations were present in seedlings exposed to stress temperatures. Hybridizations of two maize (Zea mays L.) heat shock clones, pMON9501 (HSP 70) and pZmHSP26 (HSP 26), to genomic DNA indicated a high degree of homology between the two perennial ryegrass cultivars and the HSP 70 clone and a moderate homology of the cultivars with HSP 26. The HSP 70 clone revealed restriction fragment length polymorphisms between the cultivars. Hybridizations of the two maize heat shock clones to Northern blots with perennial ryegrass total RNA showed that HSP 70 and HSP 26 mRNA were heat inducible. There were detectable differences in the levels of heat‐induced HSP 26 mRNA between the two cultivars. Our findings indicated that analysis of HSP could provide the basis of a rapid and reliable characterization of thermal tolerance in perennial ryegrass.

Cloning and characterisation of a repetitive DNA sequence from Theileria mutans: application as a species-specific probe.

Repetitive DNA sequences were isolated from a Theileria mutans genomic library by screening with T. mutans total DNA. DNA sequence analysis demonstrated that a section of one of the clones contained a complex series of overlapping perfect repeats ranging between 99 and 20 bp in size. The T. mutans repetitive sequence did not contain large open reading frames (ORFs), unlike T. parva Tpr repetitive DNA sequences. When used as a hybridisation probe the repetitive sequence revealed restriction-fragment-length polymorphisms (RFLPs) between the EcoRI-digested DNAs of two T. mutans stocks. The T. mutans repetitive probe gave a signal with 1 ng of purified T. mutans piroplasm DNA and detected T. mutans sequences in whole-blood DNA isolated from an experimentally infected animal when the piroplasm parasitaemia was equal to or above 0.4%. Oligonucleotide primers derived from the repetitive sequence allowed more sensitive detection of T. mutans DNA by polymerase chain reaction (PCR) amplification. Using the PCR, T. mutans DNA was amplified from an experimentally infected animal with a parasitaemia of < 0.1%.

Differentiation and Diagnosis of Pseudocercosporella herpotrichoides (Fron) Deighton with Genomic DNA Probes

AbstractRepetitive genomic clones were used to differentiate between varieties within the species Pseudocercosporella herpotrichoides. From 21 clones tested 13 revealed restriction fragment length polymorphisms among isolates. Cluster analysis was performed based on these data. Differentiation of isolates according to growth speed corresponds better to genetic information than differentiation on the basis of spore morphology. Pathogen‐specific probes can be used in early diagnosis of the pathogen in infected plant material. Furthermore polymorphic pathogen‐specific probes allow varieties to be differentiated directly in infected plant material without isolation and culture of the fungus.

Isolation of Region-Specific Cosmids by Hybridization with Microdissection Clones from Human Chromosome 10q11.1-q21.1

A region-specific plasmid library composed of 20,000 recombinants was constructed by microdissection of human chromosome 10 (10q11.2-q21.1) and subsequent amplification with the primer-linker method of polymerase chain reaction (PCR). Hybridization with total human DNA showed that 32 of 217 microclones studied contained highly repetitive sequences. Further analysis of the remaining 185 microclones proved that 43 microclones, each having an insert longer than 200 bp, contained unique sequences of human chromosome 10 origin. Twenty-five microclones randomly selected from the 43 were used directly as probes to isolate corresponding cosmid clones, resulting in 32 cosmids corresponding to 14 microclones. Of the 25 cosmids that could be mapped by fluorescence in situ hybridization, 24 proved to originate from the microdissected or adjacent region (10p11.2-q22.3) and 1 from a rather distal region (10q24.3-q25.1). In addition, 15 of the 32 cosmids revealed restriction fragment length polymorphisms, including 1 with a variable number of tandem repeats marker. The microdissection library and the obtained cosmids are valuable resources for constructing high-resolution physical and linkage maps of the pericentromeric region of chromosome 10, where the gene predisposing to multiple endocrine neoplasia type 2A (MEN2A) has been mapped.

Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression

A tomato phytoene synthase gene, Psy1, has recently been isolated as the clone GTOM5 and shown by sequence identity to be the gene from which the major fruit-ripening cDNA clone TOM5 was derived. Sequence analysis of transcripts from two allelic yellow-fruited tomato mutants, mapped to chromosome 3, has shown the lack of carotenoids in fruit of these mutants to be due to the production of aberrant TOM5 transcripts which are unlikely to encode a functional phytoene synthase enzyme. In one mutant (yellow flesh) the aberrant transcript contained a sequence that, by its strong hybridization to a wide size range of genomic fragments, appeared to be repeated many times within the genome. Southern and PCR analysis of the phytoene synthase genes in the mutant revealed restriction fragment length polymorphisms, suggesting that the production of altered mRNAs was associated with specific genomic rearrangements. Constitutive over-expression of a TOM5 cDNA clone in transgenic mutant plants restored synthesis of the carotenoid lycopene in ripening fruit and also led to unscheduled pigment production in other cell types. In some mutant plants transformed with the TOM5 cDNA construct, inhibition of carotenoid production in immature green fruit, leaves and flowers was observed, due to the phenomenon of co-suppression, indicating that different insertion events with the same gene construct can lead to overexpression or co-suppression in transgenic plants. Green organs of these plants were susceptible to photobleaching, due to the lack of carotenoids. These results suggest the existence of separate Psy genes for carotenoid synthesis in green organs.

Heat shock protein synthesis and restriction fragment length polymorphism in a virulent and a weakly virulent strain of Leptosphaeria maculans

Two strains of Leptosphaeria maculans, differing in virulence on Brassica napus and with distinct cultural characteristics, were tested for their heat shock response. Following exposure to elevated temperatures, both the strains responded by rapidly synthesizing a set of heat shock proteins (HSPs). Protein synthesis profiles, visualized by labelling with [35S]methionine and resolution on one-dimensional Laemmli gels, revealed a classical heat shock specific pattern: three major thermally induced polypeptides corresponding to the 90-, 80-, and 70-kDa HSPs were observed in both strains. At the optimum heat shock temperature of 40 °C, 11 and 5 new polypeptides appeared labelled in the virulent and weakly virulent strains, respectively. At 45 °C only the three prominent high molecular mass polypeptides were synthesized by the virulent strain, while the weakly virulent strain synthesized these and also showed sustained synthesis of several other normal cellular proteins. Protein synthesis was completely abolished on transfer to 48 °C. Qualitative and quantitative differences were detected in the overall polypeptide profiles of the two strains. Southern blots of genomic DNA of the two strains, digested with three restriction endonucleases and probed with a fragment of the Neurospora crassa hsp70 gene, revealed restriction fragment length polymorphisms with all the three enzymes. Key words: Leptosphaeria maculans, virulent–avirulent pathotypes, heat shock proteins, RFLP, hsp70.

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases EcoRI or HaeIII. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus. Digestion with EcoRI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae. Digestion of genomic and mitochondrial DNA with HaeIII showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium.

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genusVerticillium

Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases EcoRI or HaeIII. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus. Digestion with EcoRI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae. Digestion of genomic and mitochondrial DNA with HaeIII showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium.