Diversity of culturable denitrifying bacteria

Sequence divergence in the ribosomal genes of known strains and isolates of aquatic denitrifying bacteria was investigated using restriction fragment length polymorphism (RFLP) analysis. The same cultures were characterized for their homology with antibody and gene probes for nitrite reductase (NiR), a key enzyme in the denitrification pathway, and for amplification with a set of polymerase chain reaction primers designed to amplify a portion of the NiR gene. The NiR probes were developed from Pseudomonas stutzeri (ATCC 14405) and several P. stutzeri strains were included in the analyses. The RFLP analysis clustered most of the P. stutzeri strains together, but detected considerable diversity within this group. Isolates from three aquatic environments exhibited within —and among — habitat diversity by RFLP. Hybridization with the NiR probes and amplification with the NiR primers were not correlated with the clustering of strains by rDNA RFLP analysis. The relationships among strains deduced from ribosomal DNA RFLP reflect heterogeneity within the P. stutzeri group and among other pseudomonads, and the patterns differ from those inferred from specificity of the NiR probes.Abbreviations NiR Nitrite reductase - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism     

Phylogenetic analysis of tnpR. genes in mercury resistant soil bacteria: the relationship between DNA sequencing and RFLP typing approaches

Abstract The diversity of resolvase ( tnpR ) genes carried by a number of mercury resistant soil bacteria has been investigated by DNA sequencing. The resulting DNA sequence information was compared to previously published tnp R. DNA sequences and to previously published restriction fragment length polymorphism (RFLP) data, permitting the relationships between DNA sequencing and RFLP approaches to be studied by the use of phylogenetic trees. DNA maximum likelihood and DNA parsimony were used to construct a variety of phylogenetic trees. DNA sequencing confirmed the validity of RFLP analysis and highlighted the importance of restriction endonuclease choice upon the resulting RFLP patterns and dendrogram topology. The tnp R genes of two previously uncharacterised mercury resistant bacteria, T2–7 and T2–12 were also studied. DNA sequence data placed T2–7 in a previously described gene class, tnp R-D and T2–12 in a new gene class, tnp R-F. The significance of this data with respect to the recombination and evolution events occurring within bacterial populations are discussed.     

Analysis of mitochondrial DNA restriction fragment length polymorphisms for examining genetic variability among isolates of Phellinus linteus

 Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNAs (mtDNAs) from nine Japanese wild isolates of Phellinus linteus was carried out to examine their genetic variability. BamHI and EcoRI digests of mtDNAs from these isolates produced four and five distinct RFLP patterns, respectively. By combining the RFLP patterns obtained with the two endonucleases, mtDNAs from the nine isolates could be assigned to five different genotypes, but no mtDNA variation was detected among the isolates collected from a small area. Distance values calculated among all pairs of mtDNA genotypes, based on the presence or absence of comigrating restriction fragments, were clearly smaller than those among the mtDNA genotypes of Lentinula edodes and Pleurotus ostreatus samples collected worldwide, suggesting the necessity of collecting P. linteus wild isolates for genetic resources from geographically wider areas. Received: June 27, 2002 / Accepted: August 19, 2002 Correspondence to:T. Nakamura     

Comparison of the genetic diversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O. sativa L.) using RFLP markers

Forty fourth single-copy RFLP markers were used to evaluate the genetic diversity of 122 accessions of common wild rice (CWR, Oryza rufipogon Griff.) and 75 entries of cultivated rice (Oryza sativa L. ) from more than ten Asian countries. A comparison of the parameters showing genetic diversity, including the percentage of polymorphic loci (P), the average number of alleles per locus (A), the number of genotypes (Ng), the average heterozygosity (Ho) and the average genetic multiplicity (Hs) of CWR and indica and japonica subspecies of cultivated rice from different countries and regions, indicated that CWR from China possesses the highest genetic diversity, followed by CWR from South Asia and Southeast Asia. The genetic diversity of CWR from India is the second highest. Although the average gene diversity (Hs)of the South Asian CWR is higher than that of the Southeast Asian CWR, its percentage of polymorphic loci (P), number of alleles (Na) and number of genotypes (Ng) are all smaller. It was also found that the genetic diversity of cultivated rice is obviously lower than that of CWR. At the 44 loci investigated, the number of polymorphic loci of cultivated rice is only 3/4 that of CWR, while the number of alleles, 60%, and the number of genotypes is about 1/2 that of CWR. Of the two subspecies studied, the genetic diversity of indica is higher than that of japonica. The average heterozygosity of the Chinese CWR is the highest among all the entries studied. The average heterozygosity of CWR is about two-times that of cultivated rice. It is suggested that during the course of evolution from wild rice to cultivated rice, many alleles were lost through natural and human selection, leading to the lower heterozygosity and genetic diversity of the cultivated rice. Received: 19 May 1999 / Accepted: 26 April 2000     

土壤细菌16SrRNA基因变异型及其与植被的相关研究

绕过细菌的分离培养,直接提取土壤DNA,扩谱,克隆土壤细菌群体的16S核糖体RNA基因（16S,rDNA),根据该基因各种变异类型的限制性片段长度多型性,分析土壤细菌分子遗传多样性及其与植被的相关关系,植被的改变影响土壤养分,进而改变土壤细菌群落结构,土壤细菌遗传多样性和分化能反映植被的变化。     

鲫鱼遗传多样性的初步研究

用１７种限制性内切酶对鲫属普通鲫鱼低背型,高背型,异育银鲫,日本白鲫及华南鲤的变种红鲤共１２４个个体的线粒体ＤＮＡ进行了ＲＦＬＰ分析,１４种酶具多态,共计４３种限制性态型,１１种单倍型。     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

In vivo genetic systems in lactic acid bacteria

Abstract A review of in vivo genetic systems covers the key features of transduction and conjugation but emphasises the intramolecular and intermolecular DNA interactions that are often associated with these processes. As well as the transfer of many lactose plasmids, conjugal transfer of nisin genes and the use of conjugation to construct bacteriophage-resistant dairy starter cultures are discussed. The discovery and characterization of insertion sequences in Lactobacillus and Lactococcus and the exploitation of heterologous conjugation and transposition systems in the lactic acid bacteria are described.     

Mitochondrial DNA polymorphism in male-sterile cytoplasm of rice

Summary Mitochondrial DNAs (mtDNAs) were isolated and purified from ten strains of rice plants with male-sterile cytoplasm. The mtDNAs were digested with the restriction endonuclease PstI and the fragment patterns produced were analysed by 0.7% agarose gel electrophoresis. Restriction fragment length polymorphism was observed among the mtDNAs analysed; there were seven different patterns for the ten examined. Our results indicate that there are a variety of mtDNAs in cytoplasmically male-sterile rice.     

Genetic diversity of oilseedBrassica napus germ plasm based on restriction fragment length polymorphisms

Oilseed rape (Brassica napus) is an important oilseed crop worldwide. Cultivars have been developed for many growing regions, however little is known about genetic diversity inB. napus germ plasm. The purpose of the research presented here was to study the genetic diversity and relationships ofB. napus accessions using restriction fragment length polymorphisms (RFLPs). Eighty threeB. napus accessions were screened using 43 genomic DNA clones which revealed 161 polymorphic fragments. Each accession was uniquely identified by the markers with the exception of the near-isogenic cvs Triton and Tower. The RFLP data were analyzed by cluster analysis of similarity coefficients and by principal component analysis. Overall, there were three major groups of cultivars. The first group included only spring accessions, the second mostly winter accessions and the third, rutabagas and oilseed rape accessions from China and Japan. These results indicate that withinB. napus, winter and spring cultivars represent genetically distinct groups. The grouping of accessions by cluster analysis was generally consistent with known pedigrees. This consistency included the grouping of lines derived both by backcrossing or self-pollination with their parents.     

Inheritance of isozyme and RFLP markers in Brassica campestris and comparison with B. oleracea

Summary Using primarily cDNA restriction fragment length polymorphism markers (RFLPs) previously located to Brassica oleracea (cabbage, 2n=18) chromosomes, we initiated a comparative RFLP map in an F₂ population of B. campestris (turnip x mock pak-choi, 2n=20). As with B. oleracea, the genome of B. campestris showed extensive gene duplication, and the majority of detected duplicated loci were unlinked. Only 6 of the 49 identified loci were represented as a single copy, and 3 of these 6 were clustered on a single linkage group showing a distorted segregation ratio. Comparison with B. Oleracea indicates this synteny is conserved between species. Two other linkage groups also appeared syntenic between B. oleracea and B. campestris. One single copy locus appears to have changed synteny between B. oleracea and B. campestris. These observations suggest that B. oleracea and B. campestris share a common ancestor, but that chromosome repatterning has occurred during or after speciation. Within B. campestris, 5 loci appeared duplicated in one parent or the other, and 2 of these were linked. Differentiation through subspecies-specific duplication or deletion events is suggested as one mechansim for the evolution of numerous morphotypes within each of these species.     

Genetic diversity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria

Individual merRTΔP regions were amplified from DNA directly isolated from soil and sediment samples using consensus primers derived from the conserved mer sequences of Tn501, Tn21 and pMER419. Soil and sediment samples were taken from four sites in the British Isles; one ‘pristine’ (SB) and three polluted (SO, SE, T2) with respect to mercury. The sizes of the PCR products amplified (= 1 kb) were consistent with their generation from mer determinants related to the archetypal elements found in Gram negative bacteria. Forty-five individual clones of sequences obtained from these four sites were isolated which hybridized (> 70% homology) to a merRTΔP probe from Tn501. The diversity of these amplified mer genes was analysed using Restriction Fragment Length Polymorphism (RFLP) profiling. Fourteen RFLP classes were distinguished, 12 of which proved to be novel and only two of which had been identified in an earlier study of 40 Gram negative mercury resistant bacteria cultured from the same four sites. UPGMA analysis was used to examine the relationships between the 22 classes of determinant identified. The T2 site, which has the longest history of mercury exposure, was found to have the greatest level of diversity in terms of numbers of classes of determinant, while the SO site, which had the highest mercury levels showed relatively low variation. Variation of mer genes within and between the sequences from cultivated bacteria and from total bacterial DNA shows clearly that analysing only sequences from cultivated organisms results in a gross underestimation of genetic variation.     

Evaluation of genetic diversity of Panicum turgidum Forssk from Saudi Arabia

The genetic diversity of 177 accessions of Panicum turgidum Forssk, representing ten populations collected from four geographical regions in Saudi Arabia, was analyzed using amplified fragment length polymorphism (AFLP) markers. A set of four primer-pairs with two/three selective nucleotides scored 836 AFLP amplified fragments (putative loci/genome landmarks), all of which were polymorphic. Populations collected from the southern region of the country showed the highest genetic diversity parameters, whereas those collected from the central regions showed the lowest values. Analysis of molecular variance (AMOVA) revealed that 78% of the genetic variability was attributable to differences within populations. Pairwise values for population differentiation and genetic structure were statistically significant for all variances. The UPGMA dendrogram, validated by principal coordinate analysis-grouped accessions, corresponded to the geographical origin of the accessions. Mantel’s test showed that there was a significant correlation between the genetic and geographical distances (r = 0.35, P < 0.04). In summary, the AFLP assay demonstrated the existence of substantial genetic variation in P. turgidum. The relationship between the genetic diversity and geographical source of P. turgidum populations of Saudi Arabia, as revealed through this comprehensive study, will enable effective resource management and restoration of new areas without compromising adaptation and genetic diversity.     

Genetic diversity among Synechococcus spp. (cyanobacteria) isolated from the pelagial of Lake Constance

Abstract: Seven phycoerythrin (PE)-rich and six phycocyanin (PC)-rich unicellular cyanobacteria of the Synechococcus type were isolated from the pelagial of Lake Constance. The genetic diversity among the isolates was evaluated using restriction fragment length polymorphism (RFLP) within the psbA gene family. Nine out of 13 isolates exhibit different DNA structures in the probed areas and, furthermore, they differ from morphologically similar strains collected from other lakes. The data set does not support a principal distinction between PC-rich and PE-rich strains but it reveals less heterogeneity in the coding region of the psbA genes among PE-rich isolates than among PC-rich isolates. The isolation of distinct strains in different seasons suggests species diversity and seasonal occurrence of Synechococcus spp. in Lake Constance.     

Restriction fragment length polymorphisms as genetic markers in soybean,Glycine max (L.) merrill

Summary Restriction Fragment Length Polymorphisms (RFLP) have been identified between widely distant cultivars (Minsoy and Noir 1 ) of soybean Glycine max (L.) Merrill. Using as probes randomly chosen clones of DNA, one in five probes revealed a polymorphism. More than half of these polymorphisms appear to result from rearrangements of the genomic DNA. Twenty seven markers were analyzed for linkage in F₂ plants. Eleven of these markers were contained in four linkage groups. Five cultivars were compared in a search for new alleles. When RFLP markers corresponding to low copy DNA were used to analyze three other cultivars — Sooty, Forrest and Mandarin (Ottawa) — few new alleles were found. Using these probes, five different markers could be used to differentiate the five cultivars. Complex probes, which correspond to repeated DNA, revealed different polymorphisms in different cultivars and a single such probe could be used to distinguish the five cultivars from each other.     

Clonal differences within M-types of the group A streptococcus revealed by pulsed field gel electrophoresis

Digestion of chromosomal DNA with the rare cutting restriction enzyme SfiI in association with pulsed field gel electrophoresis was used to observe restriction fragment length polymorphisms (RFLP) among isolates of group A Streptococcus. Streptococci examined included isolates belonging to the same M-type (epidemiologically related and unrelated), and isolates from other M-types. RFLP patterns were quite distinct between all serotypes tested. More importantly, isolates from within a serotype could be differentiated by this technique.     

Molecular-marker analysis of seed-weight: genomic locations,gene action,and evidence for orthologous evolution among three legume species

The objectives of this study were to use molecular markers to: (1) identify quantitative trait loci (QTL) controlling seed-weight in soybean, (2) characterize the genetic basis of seed-weight expression, and (3) determine whether soybean shares orthologous seed-weight genes with cowpea and/or mung bean. An F₂ population was developed between a large-seeded Glycine max breeding line and a small-seeded G. soja plant introduction. DNA samples from 150 F₂ individuals were analyzed with 91 polymorphic genetic markers, including RFLPs, RAPDs and SSRs. Seed-weight was analyzed by randomly sampling 100 seeds from each of 150 greenhouse-grown F₂ individuals, and their 150 F₂₃ lines, from a replicated field trial. Markers associated with seed-weight were identified using the computer program MapMaker-QTL and a one-way analysis of variance. Three and five markers were significantly associated with seed-weight variation (P<0.01) in the F₂ and F₂₃ generations, respectively. Tests for digenic epistasis revealed three significant interactions in both generations. In a combined analysis, these markers and interactions explained 50 and 60% of the phenotypic variation for seed-weight in the F₂ and F₂₃ generations, respectively. Comparison of our results in soybean (Glycine) with those previously reported in cowpea and mung bean (Vigna) indicated that soybean and cowpea share an orthologous seed-weight gene. In both species, a genomic region significantly associated with seed-weight spanned the same RFLP markers in the same linkage order. A significant digenic interaction involving this genomic region was conserved in all three species. These results suggest that the exploitation of comparative QTL mapping is an invaluable tool for quantitative geneticists working with poorly characterized plant systems.     

Variability in nuclear DNA among nonhuman primates: Application of molecular genetic techniques to intra- and inter-species genetic analyses

Nuclear DNA clones and sequence information derived from human genetic analyses were used to detect and characterize intra- and inter-species DNA variation at several nuclear loci in hominoids and cercopithecoids. Restriction fragment length polymorphisms were found at five loci among captive rhesus monkeys. Cross-species polymerase chain reaction (PCR) amplification detected an insertion within the beta-globin gene cluster in hylobatids. The combined use of cross-species PCR and denaturing gradient gel electrophoresis detected both species differences and intra-species polymorphism in the homeobox cluster 2 of hominoids. These results a) demonstrate that DNA clones and nucleotide sequence information from human molecular genetics can be used to facilitate studies of the molecular genetics of nonhuman primates, and b) document specific examples of intra- and inter-species molecular variability at several loci. © 1992 Wiley-Liss, Inc.