Rapid amplification and fixation of new restriction sites in the ribosomal dna repeats in the derivatives of a cross between maize and Tripsacum dactyloides

Some of the derivatives of a cross of maize (Zea mays L.) × Tripsacum dactyloides (L) L (2n = 72) have abnormal development leading to strange and striking morphologies. The Tripsacum chromosomes in these “tripsacoid” maize plants (with Tripsacum-like characteristics) were eliminated and the maize chromosomes were recovered through repeated backcrossing to maize. As an initial attempt to analyze the DNA alterations in tripsacoid maize, we have detected a few restriction site changes in the ribosomal DNA repeat of these plants (Hpa II, Bal I, Sst I, Mbo II, and Sph I) and a new Sph I site was mapped to the spacer region between the 26S and 17S genes. Several possible mechanisms for the generation of a new restriction site are discussed, and we propose that the transient presence of Tripsacum genome during the backcrossing in some way induced a rapid amplification and fixation of new restriction sites in a relatively short period of time.     

Characterization of pathogenic and nonpathogenic strains of Xanthomonas axonopodis pv. manihotis by PCR-based DNA fingerprinting techniques

Strains of Xanthomonas axonopodis pv. manihotis (Xam) were characterized for pathogenicity and for DNA polymorphism using different PCR-based techniques. Using amplified restriction fragment length polymorphism (AFLP), strains were distinguished from each other and also from other Xanthomonas strains. Cluster analysis showed a high correlation between DNA polymorphism and pathogenicity. Four Xam strains were further analyzed using three PCR-based techniques, AFLP, AFLP-pthB and RAPD-pthB. Various primer combinations were used including primers specific to a Xam pathogenicity gene (pthB) along with RAPD or AFLP primers. The AFLP primer combinations EcoRI+T/MseI+A and EcoRI+T/MseI+T were the most efficient to discriminate among pathogenic and nonpathogenic Xam strains. Polymorphic bands were excised from the gel, amplified and cloned. Sequences analysis showed significant homology with bacterial pathogenicity island, genes involved in pathogenic fitness and regulators of virulence. Three cloned AFLP fragments were used as probes in DNA blot experiments and two of them showed significant polymorphism.     

Phylogeny of mitochondrial DNA clones in tassel-eared squirrels Sciurus aberti

The tassel-eared squirrel, Sciurus aberti , includes six subspecies which occupy restrictive and apparently identical habitats in Ponderosa pine forests in the south-western United States and Mexico; the strict habitat requirement of this species is based on dietary requirements which are only fulfilled in these forests. To examine evolutionary relationships among certain subspecies of S. aberti , we obtained estimates of nucleotide diversity within subspecies as well as nucleotide divergence between subspecies using mitochondrial DNA (mtDNA) analysis. Restriction site polymorphisms were identified in samples of the four US subspecies: S. a. aberti (Abert), S. a. kaibabensis (Kaibab), S. a. ferreus (Ferreus), and S. a. chuscensis (Chuska) Fourteen mtDNA clones were resolved that were, with one exception, uniquely subspecific. Dendrograms constructed by neighbour-joining and maximum parsimony methods revealed two major assemblages: (1) an Abert/Kaibab group; and (2) a Ferreus/Chuska group. The Abert vs. Ferreus clones exhibited the greatest net nucleotide divergence, with a lineage separation estimate approximating 572 000 years ago assuming a nucleotide substitution rate of 7.15 × 10^-9/year/site. Five out of ten Chuska squirrels shared a clone with one Abert sample; the relative sizes of these two populations and their respective ranges as well as their close proximity support the proposal for relatively recent intermixing of Abert and Chuska populations resulting in what appears to be Abert → Chuska migration. Nucleotide diversity within subspecies ranked as Kaibab < Ferreus < Abert < Chuska; the relatively high diversity for the Chuska sample is based on the apparent introgression of Abert mtDNA. The relative diversity exhibited by Kaibab, Ferreus and Aberti samples corresponds to the range size of the respective subspecies.     

Efficient DNA Fingerprinting Based on the Targeted Sequencing of Active Retrotransposon Insertion Sites Using a Bench-Top High-Throughput Sequencing Platform

In many crop species, DNA fingerprinting is required for the precise identification of cultivars to protect the rights of breeders. Many families of retrotransposons have multiple copies throughout the eukaryotic genome and their integrated copies are inherited genetically. Thus, their insertion polymorphisms among cultivars are useful for DNA fingerprinting. In this study, we conducted a DNA fingerprinting based on the insertion polymorphisms of active retrotransposon families (Rtsp-1 and LIb) in sweet potato. Using 38 cultivars, we identified 2,024 insertion sites in the two families with an Illumina MiSeq sequencing platform. Of these insertion sites, 91.4% appeared to be polymorphic among the cultivars and 376 cultivar-specific insertion sites were identified, which were converted directly into cultivar-specific sequence-characterized amplified region (SCAR) markers. A phylogenetic tree was constructed using these insertion sites, which corresponded well with known pedigree information, thereby indicating their suitability for genetic diversity studies. Thus, the genome-wide comparative analysis of active retrotransposon insertion sites using the bench-top MiSeq sequencing platform is highly effective for DNA fingerprinting without any requirement for whole genome sequence information. This approach may facilitate the development of practical polymerase chain reaction-based cultivar diagnostic system and could also be applied to the determination of genetic relationships.     

Pedigree assessment using RAPD-DGGE in cereal crop species

Summary The introduction of molecular biology methodologies to plant improvement programs offers an invaluable opportunity for extensive germplasm characterization. However, the detection of adequate DNA polymorphism in self-pollinating species remains on obstacle. We have optimized a denaturing-gradient-gel electrophoresis (DGGE) system which, when used in combination with random amplified polymorphic DNA (RAPD) analysis, greatly facilitates the detection of reproducible DNA polymorphism among closely related plant lines. We have used this approach to estimate pedigree relationships among a spectrum of plant materials in wheat, barley and oat. Based on analysis with one or two primers, we were able to distinguish soft from hard winter wheat, and 2-rowed from 6-rowed barley. Further analysis with additional primers allowed resolution of polymorpisms even among closely related lines in highly selected populations. We placed 17 cultivars of oat into two distinct clusters that differed significantly from previous oat pedigree assessments. We believe that DGGE-RAPD is a superior method for detecting DNA polymorphism when compared to RFLP, agarose-RAPD, or polyacrylamide-RAPD methods.     

MITOCHONDRIAL DNA VARIABILITY OF STRIPED DOLPHINS (STENELLA COERULEOALBA) IN THE SPANISH MEDITERRANEAN WATERS

Frozen muscle samples from 44 striped dolphins stranded on the Spanish Mediterranean coasts from 1990 to 1993 have been studied by means of mitochondrial DNA (mtDNA) restriction site analysis. Thirty-five of these dolphins were affected by a die-off occurring during this time in the western Mediterranean Sea. The mtDNA from each dolphin was digested with 15 restriction endonucleases that recognized 61 different restriction sites. The specific location of these sites on the mitochondrial gene map allowed us to determine the distribution of variability along this molecule. From the restriction analysis, a total of 15 different composite patterns or haplotypes was obtained and their phylogenetic relationships established both by cladistic and phenetic methods.
Estimates of sequence divergence among the 15 haplotypes ranged from 0.148% to 0.919%. Haplotype diversity (average heterozygosity) obtained was 0.789, a high value compared to other available data on cetaceans, especially considering that only a small area of the distribution of the species has been sampled. Neither population subdivision nor any evidence of correlation between haplotype frequencies and temporal or geographic distribution of dolphins has been detected.     

A modified AFLP with fluorescence-labelled primers and automated DNA sequencer detection for efficient fingerprinting analysis in plants

A modified AFLP (amplified fragment length polymorphism) technique is described. Fluorescence-labelled primers were used in the selective amplifications. The amplified fragments were detected on denaturing polyacrylamide gels using an automated ALF DNA sequencer with the fragment option. The modified AFLP technique avoids the use of isotopes or silver staining, but gives a much higher resolution than other AFLP detection systems.     

莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

Molecular analysis of the ADH1-C m allele of maize

The Adh1-C ^mallele and each gene in the Adh1-FC ^mduplication have been cloned and restriction-mapped. Of the C ^mallele 6 kb was sequenced. A single amino acid substitution of aspartate for tyrosine at residue 52 accounts for the altered enzymatic properties of the C ^mprotein. Comparison of the nucleotide sequence to that of Adh1-1F and Adh1-1S shows structural and restriction site polymorphisms in the 3 flanking DNA. C ^mlacks the insertion sequence present in 1F and 1S and contains a complex sequence composed of two direct repeats and an inverted repeat. The two genes of the duplication allele have similar restriction maps to C ^mand each other.     

大豆疫霉菌一个DNA指纹分析重复序列探针的鉴定

【目的】大豆疫霉菌指纹分析的建立和黑龙江与新疆大豆疫霉菌群体的群体遗传分析。【方法】利用生物信息学方法寻找大豆疫霉菌(Phytophthora sojae)的中度重复序列,并对黑龙江和新疆大豆疫霉菌进行DNA指纹分析。【结果】分析得到一个中度重复序列,定名为PS1227。Southern blot分析表明PS1227在大豆疫霉菌基因组中约有34条可辨的介于1.5-23kb之间的杂交条带,其中21个杂交条带在49个供试菌系中表现多态性。单游动孢子分析表明PS1227指纹特征在病菌无性生殖阶段表现稳定。利用PS1227标记,本实验发现采自黑龙江HP4002、SY6和GJ0105菌系分别与新疆的DW303、71228和71222菌系具有完全相同的指纹特征。【结论】获得一个可用于大豆疫霉菌流行学和群体生物学研究的指纹分析序列PS1227,在分子水平证实了新疆大豆疫霉菌可能由黑龙江传入。     

Heterogeneous aerobic benzene-degrading communities in oxygen-depleted groundwaters

A sandstone aquifer beneath a petrochemicals plant (SIReN site, UK) is heterogeneously contaminated with benzene and oxygen-depleted. Despite low redox potentials in three of the most contaminated groundwaters (benzene concentrations from 17.8 to 294 mg L(-1)), we observed aerobic benzene degradation in microcosms, indicating the presence in situ of a latent community of obligate aerobic microorganisms or an active community of facultative aerobes responding rapidly to oxygen ingress. Moreover, benzene degradation occurred at the ambient pH of 8.9 and 9.4, considerably more alkaline conditions than previously reported. 16S rRNA analyses showed that the groundwater microcosm communities were distinct from each other, despite sharing the function of aerobic benzene degradation. From DNA fingerprinting, one consortium was dominated by Acidovorax spp., another by Pseudomonas spp.; these benzene-degrading consortia were similar to the in situ communities, perhaps indicating that these organisms are active in situ and degrading benzene microaerophilically or by denitrification. Conversely, in the third sample, benzene degradation occurred only after the community changed from a Rhodoferax-dominated community to a mix of Rhodococcus and Hydrogenophaga spp. Four of the main benzene-degrading strains were brought into culture: Hydrogenophaga and Pseudomonas spp., and two strains of Rhodococcus erythropolis, a ubiquitous and metabolically versatile organism.     

Capturing of host DNA by a plant retroelement: Bs1 encodes plasma membrane H+-ATPase domains

The recently identified maize retroelement Bs1 encodes domains of the plasma membrane H⁺-ATPase. This is the first example of host DNA captured by a plant retroelement and resembles the acquisition of oncogenes by vertebrate retroviruses. The ability to capture sequences from its host provides plant retroelements with a mechanism to alter gene structure which could be important for evolutionary adaptive change.     

Restriction site heteroplasmy in the mitochondrial DNA of the marine fish Sciaenops ocellatus (L.)

Restriction site heteroplasmy involving the enzymes NcoI and XbaI was detected in the mitochondrial DNAs of two individuals of the marine fish Sciaenops ocellatus. This represents only the sixth documented example of mitochondrial DNA restriction site heteroplasmy in animals. Two heteroplasmic individuals were found in a survey of nearly 750 individuals, suggesting that in most studies the incidence of mitochondrial DNA site heteroplasmy may be too low to be routinely detected.     

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.     

A bacteriophage lambda vector for cloning with BamHI and Sau3A

A phage lambda cloning vector has been constructed which contains a single site for the restriction endonuclease BamHI. Since Sau3A and BglII produce the same cohesive ends as BamHI, this vector can also be used to clone DNA fragments generated with either of these enzymes. We have used this vector to construct an Escherichia coli library using partial digestion with Sau3A. This vector will be most useful for applications requiring genetic analysis of cloned E. coli genes.     

Cloning, quantification and characterization of a minisatellite DNA sequence from common bean Phaseolus vulgaris L.

 We describe the cloning and the characterization of a 130-bp DNA fragment, called OPG9-130, amplified from bean (Phaseolus vulgaris L.) genomic DNA. This fragment corresponds to a minisatellite DNA sequence containing seven repeats of 15 bp which differ slightly from each other in their sequence. Southern analysis showed that the core sequence of 15 bp is repeated in clusters dispersed throughout the genome. The use of this fragment as a probe allowed us to identify common bean lines by their DNA fingerprints. We suggest that OPG9-130 will be useful for line identification as well as for the analysis of genetic relatedness between bean species and lines. Received: 14 February 1998 / Accepted: 10 February 1998