Cytoplasmic male sterility in sunflower: comparison of molecular biology and genetic studies

The genetics of male fertility restoration and the RFLP of mitochondrial DNA were studied for 16 sunflower cytoplasms (15 male-sterile and a male-fertile).Male fertility restoration/male sterility maintenance patterns distinguished 12 cytotypes. Four cytoplasms were completely unrestored so they were not distinguished genetically. The sunflower lines, tested for their restorer/maintenance reaction, showed that there was a continuous range between 0% and 100% of restorer genotypes according to the CMS considered. Restoration/maintenance patterns indicated that at least some restorer genes are specific to certain CMS.RFLP of mitochondrial DNA revealed specific differences between the cytotypes studied. Three restriction enzymes and 12 probes permitted distinction of 13 cytotypes. No relationship exists between CMS cytotypes and the species from which they originated.For genetical and mitochondrial RFLP studies, phenograms were constructed according to the similarity indexes between cytotypes. Most of the CMS defined by restoration patterns correspond with a restriction fragment pattern of mitochondrial DNA.     

现代生物技术在环境微生物学中的应用:Ⅲ.限制性片段长度多态性分析、变性/温度梯度凝胶电泳和报道基因

这是现代生物技术在环境微生物学中的应用系列综述文章的第三篇 ,讨论限制性片段长度多态性 (RFLP)分析、变性梯度凝胶电泳 (DGGE)和温度梯度凝胶电泳 (TGGE)以及报道基因。     

Applications of DNA genetic markers to the study of plant growth and development

DNA genetic markers, such as restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA markers (RAPDs), are powerful tools for studying the genetics of plant growth and development. DNA markers are defined sequences of DNA that can be used in traditional linkage mapping. Using DNA marker technology, scientists can uncover relationships between cloned cDNA sequences and classically characterized genes. DNA markers make it possible to dissect the contributions of multiple genetic loci underlying complex developmental processes. Moreover, changes in genome organization that occur during development or in response to environmental signals can be monitored using RFLP technology. In the future, it may be possible to clone any gene based solely on its map position. This will involve the use of tightly linked DNA markers as entry points for chromosome walking, in which a series of overlapping genomic clones reaching from the tightly linked DNA marker to the gene of interest are identified.     

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.     

Multiple gene analyses reveal extensive genetic diversity among ‘Candidatus Phytoplasma mali’ populations

This study focused on evaluating the genetic diversity among ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) populations in orchards of north‐western Italy, where apple proliferation (AP) disease is widespread and induces severe economic losses. ‘Ca. P. mali’ was detected through restriction fragment length polymorphism (RFLP) analysis of PCR‐amplified 16S rDNA in 101 of 114 samples examined. Collective RFLP patterns, obtained by restriction analyses of four amplified genomic segments (16S/23S rDNA, PR‐1, PR‐2 and PR‐3 non‐ribosomal region, ribosomal protein genes rplV‐rpsC and secY gene), revealed the presence of 12 distinct genetic lineages among 60 selected representative ‘Ca. P. mali’ isolates, underscoring an unexpected high degree of genetic heterogeneity among AP phytoplasma populations in north‐western Italy. Prevalence of distinct genetic lineages in diverse geographic regions opens new interesting avenues for studying the epidemiology of AP disease. Furthermore, lineage‐specific molecular markers identified in this work could be useful for investigating the biological life cycle of ‘Ca. P. mali’.     

A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses

We previously developed a PCR-based DNA fingerprinting technique named the Methylation Sensitive (MS)-AFLP method, which permits comparative genome-wide scanning of methylation status with a manageable number of fingerprinting experiments. The technique uses the methylation sensitive restriction enzyme Not I in the context of the existing Amplified Fragment Length Polymorphism (AFLP) method. Here we report the successful conversion of this gel electrophoresis-based DNA fingerprinting technique into a DNA microarray hybridization technique (DNA Microarray MS-AFLP). By performing a total of 30 (15×2 reciprocal labeling) DNA Microarray MS-AFLP hybridization experiments on genomic DNA from two breast and three prostate cancer cell lines in all pairwise combinations, and Southern hybridization experiments using more than 100 different probes, we have demonstrated that the DNA Microarray MS-AFLP is a reliable method for genetic and epigenetic analyses. No statistically significant differences were observed in the number of differences between the breast-prostate hybridization experiments and the breast-breast or prostate-prostate comparisons.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M. Johnston     

Indigestible carbohydrates alter the intestinal microbiota but do not influence the performance of broiler chickens

Aims:  Prebiotics are a potential alternative to in-feed antimicrobials to improve performance of chickens. We investigated the effects of mannanoligosaccharide (MOS) and fructooligosaccharide (FOS) on growth, performance and the intestinal microbiota.
Methods and Results:  Cobb 500 birds were fed either: Control, starter diet without antimicrobials; ZnB, Control + 50 ppm zinc bacitracin; MOS, Control + 5 g kg⁻¹ MOS; or FOS, Control + 5 g kg⁻¹ FOS. An energy metabolism study was conducted and intestinal microbial communities assessed by T-RFLP and Lac PCR-DGGE. Diet did not influence performance. Ileal microbial communities were significantly different in ZnB-fed birds compared to all diets, and FOS-fed chickens compared to Control. MOS-fed chickens had a different caecal profile to ZnB and FOS-fed birds. Consensus Lac PCR-DGGE profiles indicated Lactobacillus communities clustered according to diet with Lactobacillus johnsonii characteristic of ZnB diet. Control and MOS-fed chickens displayed significantly different jejunal Lactobacillus profiles to each other whilst ileal profiles were different between MOS and FOS-fed birds.
Conclusion:  Prebiotics influenced the intestinal microbiota, but did not affect performance.
Significance and Impact of the Study:  In light of pressure for in-feed antimicrobial withdrawal, the impact of alternative compounds on the intestinal microbiota and bird performance is critical to the poultry industry.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE)

Three molecular tools, amplified fragment length polymorphism (AFLP), denaturing gradient gel electrophoresis (DGGE) and random amplified polymorphic DNA (RAPD) analysis, were explored for their usefulness to identify isolates of Malassezia yeasts. All seven species could be separated by AFLP and DGGE. Using AFLP, four genotypes could be distinguished within M. furfur. AFLP genotype 4 contained only isolates from deep human sources, and ca. 80% of these isolates were from patients with systemic disease. Most of the systemic isolates belonged to a single RAPD genotype. This suggests that systemic conditions strongly select for a particular genotype. Although the clinical use of DGGE may be limited due to technical demands, it remains a powerful tool for the analysis of complex clinical samples.     

Denaturing gradient gel electrophoresis identifies genomic DNA polymorphism with high frequency in maize

Summary We have used denaturing gradient gel electrophoresis (DGGE) to identify genomic DNA polymorphism in maize (Zea mays L.). DGGE probes detect polymorphism in maize at a frequency comparable to the incidence of restriction fragment length polymorphism (RFLP). Probes identifying polymorphism were mapped to maize chromosome arms by utilizing DGGE and maize lines carrying B-A chromosomal translocations. The methods for library construction, probe screening, and genome analysis, described here for maize, can also be applied to the genomic analysis of other organisms.     

DNA fingerprints from blood mixes in chickens and in Turkeys

Abstract

Although DNA fingerprints are useful in individual identification and genetic linkage studies, expensive and time‐consuming laboratory procedures limit their practical application. By mixing blood from individuals within a population, a DNA fingerprint pattern representing the population can be obtained. The pattern was identical to that in which DNA from individuals was mixed, and was not improved by adjusting blood volumes according to hemoglobin levels.     

Polymorphism in mitochondrial DNA (mtDNA) of yak (Bos grunniens)

Mitochondrial DNAs (mtDNA) from 21 yaks (Bos grunniens) were assayed for restriction fragment length polymorphisms by using 20 restriction endonucleases, six of which (AvaI, AvaII, BglII, EcoRI, HindIII, and HpaI) detected polymorphism. Four different mtDNA haplotypes were identified. Combining this with previous reports about the mtDNA RFLPs of B. indicus and B. taurus, there are obvious differences in mtDNA polymorphism between the yak and other Bos species. We estimated that the divergence times between the ancestor of B. grunniens and the ancestor of B. taurus or B. indicus were about 1.2–2.2 and 1.01–2.02 million years ago, respectively.     

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri , Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii . Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.     

Evaluation of the impact of DNA extraction methods on BAC bacterial community composition measured by denaturing gradient gel electrophoresis

Aims: The impact of DNA extraction methods on biological activated carbon (BAC) DNA yield and bacterial community was evaluated. Methods and Results: Three different DNA extraction methods were compared: method a, method b and method c. Method c with ultrasonic pretreatment improved cell lysis efficiency (from 34% to 87%) and DNA yield [from 10·58 μg g^?1 (dry wt) of carbon to 21·42 μg g^?1 (dry wt) of carbon]. denaturing gradient gel electrophoresis profiles obtained by method c recovered the five seeded bacteria (Bacillus subtilis Strain WSO 6, Pseudomonas putida Strain WSO 7, Acinetobacter lwoffii WSO 10, Pseudomonas pertucinogena WSO 11 and Brevibacterium mcbrellneri WSO 13). Conclusions: The results showed method c with ultrasonic pretreatment was the most successful for the analysis of BAC bacterial community because it was effective in the detachment of bacteria and cell lysis, thereby resulting in good yields. Significance and Impact of Study: These results must be taken into consideration when extracting DNA for analysing BAC bacterial community.     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

Mitochondrial DNA analysis of Sporothrix schenckii in North and South America

Mitochondrial DNA (mtDNA) types based on restriction fragment length polymorphism (RFLP) patterns with HaeIII were investigated in clinical isolates of Sporothrix schenckii in North and South America. In addition to 14 mtDNA types (Types 1–14) so far reported, six new mtDNA types, Types 15–20 were found in this study. Type 3 was divided into two subtypes, Subtype 3A and Subtype 3B based on RFLP with Msp1. Type 14 was also divided into three subtypes, Subtype 14A, Subtype 14B and Subtype 14C based on RFLP with Hha1. Nineteen isolates in the United States consisted of 1 isolate of Type 1, 12 of Type 2, 2 of Type 4, 3 of Type 14 (1 of Subtype 14B and 2 of Subtype 14C) and 1 of Type 15. Twenty nine isolates in Venezuela consisted of 13 of Type 3 (Subtype 3B), 6 of Type 4, 1 of Type 18, 3 of Type 19 and 6 of Type 20. Thirteen isolates in Argentina consisted of 2 of Type 3 (Subtype 3A), 4 of Type 4, 4 of Type 16 and 3 of Type 17. One isolate in Brazil was Type 3 (Subtype 3A). Based on the phylogeny of 20 mtDNA types (Types 1–20) constructed by estimating sequence divergences of mtDNA, mtDNA types were clustered into two groups: Group A (Types 1–3, Type 11 and Types 14–19) and Group B (Types 4–10, Types 12–13 and Type 20). These results suggest that S. schenckiiisolates in North and South America mainly belong to Group A. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transposon signatures: species-specific molecular markers that utilize a class of multiple-copy nuclear DNA

Transposable elements are mobile sequences found in nuclear genomes and can potentially serve as molecular markers in various phylogenetic and population genetic investigations. A PCR-based method that utilizes restriction site variation of element copies within a genome is developed. These patterns of site variation, referred to as transposon signatures, are useful in differentiating between closely related groups. Signature data using the magellan retrotransposon, for example, is useful in examining relationships within the genus Zea and Tripsacum. This method allows transposable elements, or even other multiple-copy nuclear DNA sequences, to be generally utilized as molecular markers in discriminating between other closely related species and subspecies.     

Comparison of 23S polymerase chain reaction-restriction fragment length polymorphism and amplified fragment length polymorphism techniques as typing systems for thermophilic campylobacters

In this study, we evaluated the combination of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP) molecular typing techniques for the analysis of thermophilic campylobacter species isolated from clinical and poultry samples. 23S PCR-RFLP analysis performed to fingerprint 69 strains exhibited an excellent level of typability. Eleven different types were defined at 100% linkage level following numerical analysis of band patterns. Differentiation of Campylobacter jejuni and Campylobacter coli at species level was achieved although no significant relationship could be observed between the profiles and the origin of the strains. Simplified AFLP analysis of the isolates disclosed the presence of 66 different banding patterns. The resulting dendrogram showed a high diversity among the strains studied. All the isolates were grouped within eight main types with a 69% homology degree among them. Differentiation at subspecies level was possible but no significant relationship could be observed between the AFLP profiles and the origin of the strains. When used in combination, 23S PCR-RFLP and single-enzyme AFLP methods can be applied to determine taxonomic and epidemiological relationships among thermophilic campylobacters.