Genetic variation in cultivated strains of <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Genetic differences among Agaricus blazei strains were investigated using somatic incompatibility testing, isozyme analysis, restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD) analysis. Eight strains, one cultivated strain from Brazil and seven from Japan, were used in this study. Somatic incompatibility interactions were observed between the Brazilian cultivated strain and the Japanese strains. The Brazilian cultivated strain had its own distinct patterns of esterase isozyme and mtDNA RFLP, but all seven Japanese cultivated strains showed identical patterns. When the RAPD patterns, obtained using eight primers, were compared the eight strains had their own distinct RAPD profiles. Distance values were calculated between all pairs of the strains based on presence or absence of individual RAPD bands, and a dendrogram was constructed by unweighted pair-group method with arithmetic clustering (UPGMA) analysis. Seven Japanese cultivated strains were grouped to each other, and this group was finally linked to the Brazilian cultivated strain. Based on these results, the degree of genetic variation among the A. blazei strains used is discussed.     

Heterothallic life cycle in the white root rot fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

To prepare homologous DNA fragments as restriction fragment length polymorphism (RFLP) markers, the genes encoding phenol oxidase, chitinase, and xylanase were amplified from genomic DNA of Rosellinia necatrix strains. RFLP analysis using the amplified DNA fragments as probe was carried out, with segregation of the markers among two sets of F₁ progenies isolated from an independent perithecium. RFLP was frequently found using rpo1 as the RFLP marker among strains of R. necatrix, which was isolated from single ascospores and the circumference of the perithecium. In each set, RFLPs of some F₁ progenies were different from that of the parent strain. Random amplified polymorphic DNA (RAPD) also revealed that several strains, which were of different genotypes from the parent strain, were contained in the single ascospore culture isolated from the same perithecium. From these results, it is suggested that another strain, which was genetically different, was required for mating and development of the ascus in R. necatrix. Therefore, the life cycle in R. necatrix was presumed to be heterothallism. This is the first report about a heterothallic life cycle in R. necatrix.     

Non-random chloroplast segregation inNicotiana tabacum (+)N. rustica somatic hybrids selected by dual nuclear-encoded resistance

Nicotiana tabacum (+)N. rustica interspecific somatic hybrids were produced by fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistantNicotiana tabacum L. with leaf mesophyll protoplasts of transgenic kanamycin-resistantN. rustica L. Somatic hybrids were selected on the basis of resistance to both methotrexate and kanamycin. Evidence for nuclear hybridization was obtained for 21 hybrids by restriction-fragment-length-polymorphism (RFLP) analysis using a heterologous wheat nuclear ribosomal-DNA (rDNA) probe and by analysis of glutamate-oxaloacetate transaminase (GOT) isoenzymes. Chloroplasts segregated non-randomly as 20 of the somatic hybrids possessedN. rustica chloroplasts and only one hadN. tabacum chloroplasts. Patterns of mitochondrial inheritance were examined by hybridization of a heterologous wheat cytochrome oxidase subunit II (coxII) gene with genomic DNA of the somatic hybrids. Four somatic hybrids with hybridization patterns similar toN. rustica and 17 with hybridization patterns consistent with mitochondrial DNA (mtDNA) rearrangement or recombination were obtained. None of the somatic hybrids had patterns ofcoxll hybridization identical withN. tabacum. Male-fertility levels in the hybrids ranged from undetectable to 87% and only nine hybrids produced a limited amount of viable seed. There was no apparent correlation between the patterns of organelle inheritance in the somatic hybrids and the relative degree of fertility.Contribution No. 1439 Plant Research CentreCurrent address: Plant Biotechnology Institute, National Research Council, 110 Gymmasium Road, Saskatoon, Saskatchewan S7N OW9, Canada     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Genetic analysis of nuclear ribosomal DNA of Lentinula edodes

Genetic analysis of nuclear ribosomal DNA (rDNA) of Lentinula edodes was carried out using rDNA restriction fragment length polymorphisms (RFLPs) as genetic markers. Two compatible monokaryotic strains that differed in the endonuclease digestion patterns of their rDNA were used. The dikaryotic strain established by crossing them produced mixed RFLP patterns. Single-spore isolates derived from the dikaryotic strain showed three types of rDNA RFLP patterns: either one of the two parental types or a mixed type. From the frequency of the mixed type, the recombination value of rDNA tandem repeats was calculated to be 31.4%. Linkage analysis between rDNA and two incompatibility factors (A and B) revealed that rDNA was not linked to either factor. The rDNA genotypes did not affect mycelial growth among the single-spore isolates.     

Molecular genetics of rotifers: preliminary restriction napping of the mitochondrial genome of Brachionus plicatilis

Methods are presented to extract and purify mitochondrial DNA from the rotifer Brachionus plicatilis. The mtDNA obtained is of sufficient purity for digestion with restriction endonucleases. EcoR I restriction patterns are presented for 4 geographically separated clones. A restriction map based on digestion with 5 different restriction enzymes is included for one of these clones. Finally, use of mtDNA analysis for studies on the population structure and biogeography of rotifers is discussed.     

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F₁ hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F₁ hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.     

Somatic hybrids between Solanum etuberosum and diploid,tuber bearing Solanum clones

Electrofusion was used to obtain somatic hybrids between Solanum etuberosum (2n=2x=24) and two diploid potato lines. These hybridizations were conducted to determine if haploidxwild species hybrids are better fusion partners than conventional S. tuberosumGp. Tuberosum haploids. Restriction fragment length polymerase (RFLP) analyses of the putative somatic hybrids confirmed that each parental genome was present. The somatic hybrids between S. etuberosum and a haploid S. tuberosum clone, US-W730, were stunted and had curled, purple leaves. In contrast, somatic hybrids between S. etuberosum and a haploidxwild species hybrid (US-W 730 haploidx S. berthaultii), were vigorous and generally tuberized under field conditions. These hybrids were designated as E+BT somatic hybrids. Analyses of 23 E+BT somatic hybrids revealed a statistically significant bias towards the retention of S. etuberosum chloroplasts. Stylar incompatibilities were observed when the E+BT somatic hybrids were used as pollen donors in crosses with S. tuberosum cultivars. Reciprocal crosses did not show this incompatibility. The progeny were vigorous and had improved tuber traits when compared to the maternal E+BT parent. RFLP analyses of three sexual progeny lines confirmed the presence of all 12 S. etuberosum chromosomes. In two of these lines, RFLPs that marked each of the 24 chromosome arms of S. etuberosum were present. However, RFLP markers specific for regions on chromosomes 2, 7, and 11 were missing from the third clone. Because other markers for these chromosomes were present in the progeny line, these results indicated the likelihood of pairing and recombination between S. etuberosum and S. tuberosum chromosomes.     

Diploid Brassica napus somatic hybrids: characterization of nuclear and organellar DNA

Summary Five somatic hybrids between Brassica campestris and B. oleracea were obtained. Molecular, morphological and cytological information all suggest that the resynthesized B. napus plants were hybrids. All five plants were diploid (2n=38) and had mainly bivalents at meiosis. Seedset was low after selfing but normal after crossing with B. napus. Molecular proof of the hybrid nature of these plants was obtained by hybridization of a rDNA repeat to total DNA. Analysis of chloroplast DNA restriction patterns revealed that all hybrids had chloroplasts identical to the B. oleracea parent. The analysis of mitochondrial DNA indicated that three hybrids had restriction patterns identical to those of B. campestris, and the other two had restriction patterns similar to those of B. oleracea. The 11.3 kb plasmid present in mitochondria of the B. campestris parent was also found in mitochondria of all five hybrids. This suggests that the plasmid from a B. campestris type of mitochondria was transferred into mitochondria of a B. oleracea type.     

Behavioural Reproductive Isolation in a Rotifer Hybrid Zone

A hybrid zone between two Brachionus plicatilis rotifer mitochondrial DNA (mtDNA) lineages was recently described in the Iberian Peninsula between a pond (Santed 2) and a lake (Gallocanta). The patterns of mitochondrial and nuclear genetic variation observed suggested that gene flow is mainly male-mediated from the lake to the pond. Here we test two hypotheses: (a) that male-mediated gene flow occurs through assortative mating between individuals from these ponds, (b) that behavioural isolation occurs between the two mtDNA lineages. We isolated, reared and genotyped rotifer clones from resting eggs collected in the sediments of these and two other distant ponds. We devised a quick, inexpensive RFLP method to discriminate between B. plicatilis and its sibling species B. ‘Manjavaeas’ and between both mtDNA B. plicatilis lineages. Behavioural no-choice tests using new-born, virgin males and females were performed between five clones. B. ‘Manjavacas’ and B. plicatilis were reproductively isolated. B. plicatilis clones did not show evidence of reproductive isolation, regardless of their mtDNA lineage, except Santed 2 males, which discriminated strongly against Gallocanta females. These results could help to explain the discrepancies between mitochondrial and nuclear genetic variation reported in the two populations.     

Random chloroplast segregation and frequent mtDNA rearrangements in fertile somatic hybrids between Nicotiana tabacum L. and N. glutinosa L.

Patterns of organelle inheritance were examined among fertile somatic hybrids between allotetraploid Nicotiana tabacum L. (2n=4x=48) and a diploid wild relative N. glutinosa L. (2n=2x=24). Seventy somatic hybrids resistant to methotrexate and kanamycin were recovered following fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistant N. tabacum and kanamycin-resistant N. glutinosa. Evidence for hybridization of nuclear genomes was obtained by analysis of glutamate oxaloacetate transaminase and peroxidase isoenzymes and by restriction fragment length polymorphism (RFLP) analysis using a heterologous nuclear ribosomal DNA probe. Analysis of chloroplast genomes in a population of 41 hybrids revealed a random segregation of chloroplasts since 25 possessed N. glutinosa chloroplasts and 16 possessed N. tabacum chloroplasts. This contrasts with the markedly non-random segregation of plastids in N. tabacum (+)N. rustica and N. tabacum (+) N. debneyi somatic hybrids which we described previously and which were recovered using the same conditions for fusion and selection. The organization of the mitochondrial DNA (mtDNA) in 40 individuals was examined by RFLP analysis with a heterologous cytochrome B gene. Thirty-eight somatic hybrids possessed mitochondrial genomes which were rearranged with respect to the parental genomes, two carried mtDNA similar to N. tabacum, while none had mtDNA identical to N. glutinosa. The somatic hybrids were self-fertile and fertile in backcrosses with the tobacco parent.Contribution No. 1487 Plant Research Centre     

Natural Diversity of Nodular Microsymbionts of Alnus glutinosa in the Tormes River Basin

The genetic diversity of Frankia strains nodulating Alnus glutinosa along the basin of the Tormes River was studied on DNA extracted directly from nodules. Frankia strains inhabiting root nodules at 12 different locations, ranging in altitude from 409 to 1181 m, were characterized. For that, we amplified the whole IGS region between 16S–23S rDNA and performed a restriction fragment length polymorphism (RFLP) analysis with four restriction enzymes. Two different RFLP patterns (termed A and B) were obtained with HaeIII, indicating the existence of two different groups of Frankia strains. Three different nodule extracts from each of the two RFLP groups were selected for further analyses. Sequencing of the 16S–23S rDNA IGS showed a 100% of intragroup homology and also confirmed the difference (98.4% level of similarity) between the Frankia strains in the two nodule extract groups. The phylogenetic analyses based on the two 16S–23S rDNA IGS sequences obtained in this study and other previously published sequences indicated that Frankia strains TFAg5 and TFAg23 (chosen as representative of HaeIII–RFLP group A and B, respectively) are quite similar to other strains nodulating plants of A. rhombifolia and A. viridis in California (pairwise levels of similarity including gaps ranged from 97.8% to 98.6%), together with which they form a single group. To put the Frankia strains representative of each HaeIII–RFLP group in the context of overall Frankia diversity we amplified and sequenced the 16S rDNA and glnII gene from nodular DNA. An also remarkable fact found in this study was that Frankia strains belonging to the HaeIII–RFLP group A were distributed all along the river course, from the lowest site sampled to the highest, while Frankia strains placed into RFLP group B were restricted to the upper Tormes River, being exclusively found at altitudes of 946 m or higher.     

DNA-fingerprinting (AFLP and RFLP) for genotypic identification in species of the <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> complex

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man’s selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods—amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of laccase and manganese peroxidase genes—to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.     

Natural nodulation of <Emphasis Type="Italic">Acacia mangium</Emphasis>–<Emphasis Type="Italic">Acacia auriculiformis</Emphasis> hybrids: distribution of the indigenous strains in the nodules

Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP) of the InterGenic Spacer (IGS) between rDNA 16S and 23S was used to identify indigenous strains nodulating four clones of Acacia mangium–Acacia auriculiformis hybrids cultivated in non-sterilized sandy soil from Sangalkam (Senegal) under greenhouse conditions. The experiment was for 4 months. The analysis of restriction fragment length polymorphism obtained with MspI and HaeIII restriction enzymes allowed the identification of 15 different IGS Groups with a distribution which significantly differed according to the clone of the hybrid (strains of one clone can belong to three and five different IGS Groups). Three large multi-lobed nodules were obtained on the root system of clone 3.26 within 5 months. Also, the nature of the rhizobia contained in each lobe was determined. The results showed that the lobes of large nodules can be occupied by one or two strains and the nodules analysed were mainly occupied by those belonging to IGS Group 12.     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.     

Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method

The restriction patterns of two chloroplast fragments and one mitochondrial DNA fragment, amplified by PCR with universal primers, were studied to determine the mode of inheritance of these organelles in 143 progeny of five intraspecific crosses in pedunculate oak (Quercus robur L.). The results indicate that both genomes are maternally inherited, an observation which agrees with the commonly observed pattern of inheritance in angiosperms. They confirm that both chloroplast DNA and mitochondrial DNA can be used as a source of seed-specific markers for the study of the geographic structure of oaks. This is the first report of organelle inheritance within the Fagaceae, an important and widespread tree family.     

Genotyping of Trichophyton rubrum by analysis of ribosomal-DNA intergenic spacer regions

An attempt was made to explore the genotyping of Trichophyton rubrum (T. rubrum) and the relationship between genotype and geographical origin using ribosomal restriction endonuclease polymorphic analysis. The total DNA was extracted by cetyltrimethyl ammonium bromide (CTAB). The probe was amplified from part of the 18S, ITSI, 5.8S, and ITSII region of T. rubrum standard strain with the universal fungal primers NS5 [5′-AACTT AAAGG AATTG ACGGA AG-3′] and ITS4 [5′-TCCTC CGCTT ATTGA TATGC-3′]. The genomic DNA of 49 clinical T. rubrum isolates digested by EcoR1 were hybridized with this probe, and the hybridization patterns were used as the basis of genotyping. Of the data from 49 strains of T. rubrum studied (21 from Nanjing, 26 from Dalian, and two from Beijing), 20 individual patterns (DNA Type A–T) were identified, among which Type A–C accounted for 48.98% of all the strains. The DNA patterns of Nanjing strains were represented by three bands, those of Dalian strains were represented by four bands. The DNA typing of T. rubrum by Southern blotting was highly sensitive and highly distinguishable. The DNA patterns of Nanjing strains were obviously different from those of Dalian strains.     

Rearrangements in the mitochondrial genome of somatic hybrid cell lines of Pennisetum americanum (L.) K. Schum. + Panicum maximum Jacq.

Summary Mitochondrial DNA from three somatic hybrid cell lines of Pennisetum americanum + Panicum maximum was compared with mitochondrial DNA of the parents. Gel electrophoresis of BamHI-restricted mitochondrial DNA indicated that extensive rearrangements had occurred in each of the three hybrid lines. The hybrid restriction patterns showed a combination of some bands from each parent plus novel fragments not present in either parent. Additional evidence for rearrangements was obtained by hybridization of eight DNA probes, carrying sequences of known coding regions, to Southern blots. Each of the somatic hybrids exhibited a partial combination of the parental mitochondrial genomes. These data suggest recombination or amplification of the mitochondrial genomes in the somatic hybrids.