Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resistant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866),DNA fingerprint technique, amplified ribosomal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes of Acinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high complementarity, and could be a useful tool in Acinetobacter genomic species identification. __________ Translated from Microbiology, 2007, 34(2): 303–306 [译自:微生物学通报]     

Relationships among <Emphasis Type="Italic">Leymus</Emphasis> species assessed by RAPD markers

The DNA genetic diversity of 40 accessions of genus Leymus was analyzed by random amplified polymorphic DNA (RAPD) markers. A total of 352 products were amplified by 34 10-mer arbitrary primers, among which 337 products (95.74 %) were found to be polymorphic. 5–14 polymorphic bands were amplified by each polymorphic primer, with an average of 9.91 bands. The data of 352 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Great genetic diversity in genus Leymus was observed, the genetic diversity among the different species more abundant than that of the different accessions, and the different accessions in a species or the species from the same areas were clustered together.     

Application of random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis for high-resolution identification of <Emphasis Type="Italic">Monascus</Emphasis> strains

Monascus fungi are commonly used for a variety of food products in Asia, and are also known to produce some biologically active compounds. Since the use of Monascus is expected to increase in food industries, strain-level identification and management of Monascus will be needed in the near future. In the present study, random amplified polymorphic DNA (RAPD) analysis coupled with microchip electrophoresis was applied for this purpose. Evaluations of the analysis stability revealed that reproducible results could be obtained, although template DNA fragmentation could influence the resulting RAPD pattern. RAPD analysis using 15 Monascus strains consisting of four species, M. ruber, M. pilosus, M. purpureus, and M. kaoliang showed that each strain generated a unique RAPD pattern, which allows strain-level identification of Monascus. In addition, the phylogenetic tree constructed from RAPD patterns reflected M. ruber–M. pilosus and M. purpureus–M. kaoliang clusters inferred from both ITS and β-tubulin gene sequences, which indicated that the RAPD pattern could reflect their phylogenetic traits to a certain extent. On the other hand, RAPD analysis did not support the monophyletic clustering of the four Monascus species used in this study, which suggests the necessity of reexamination of species boundaries in Monascus.     

Use of random amplified polymorphic DNA markers for the detection of Azospirillum strains in soil microcosms

Probes for the detection of Azospirillum strains were obtained from DNA fragments generated by random amplification of polymorphic DNA (RAPD) and tested to assess their specificity towards DNA extracted from pure cultures. The most specific probe, referred to as α4, produced a hybridization signal only with amplified DNA of A. lipoferum ATCC29731. This strain was inoculated, together with two other Azospirillum strains, in soil microcosms of different complexity and its presence tested with the probe α4. This probe confirmed its high specificity with amplified DNA extracted from the soil microcosm and in the presence of other A. lipoferum strains, indicating that the strategy for bacterial detection, based on RAPD markers, is useful for monitoring the presence of a particular strain under environment-like conditions. Other RAPD-derived probes, when tested on soil samples, did not show the same level of specificity as that shown on DNA from pure cultures. This result suggests that some precautions are necessary in the choice of a really specific RAPD marker. In a further development of this strategy, the α4 probe was sequenced and two pairs of “nested” primers were designed, which enabled a diagnostic polymerase chain reaction from soil samples that was specific for the A. lipoferum species. Received: 7 July 1997 / Accepted: 14 October 1997     

Genetic Relationship of <Emphasis Type="Italic">Curcuma</Emphasis> Species from Northeast India Using PCR-Based Markers

Molecular genetic fingerprints of nine Curcuma species from Northeast India were developed using PCR-based markers. The aim involves elucidating there intra- and inter-specific genetic diversity important for utilization, management, and conservation. Twelve random amplified polymorphic DNA (RAPD), 19 Inter simple sequence repeats (ISSRs), and four amplified fragment length polymorphism (AFLP) primers produced 266 polymorphic fragments. ISSR confirmed maximum polymorphism of 98.55% whereas RAPD and AFLP showed 93.22 and 97.27%, respectively. Marker index and polymorphic information content varied in the range of 8.64–48.1, 19.75–48.14, and 25–28 and 0.17–0.48, 0.19–0.48, and 0.25–0.29 for RAPD, ISSR, and AFLP markers, respectively. The average value of number of observed alleles, number of effective alleles, mean Nei’s gene diversity, and Shannon’s information index were 1.93–1.98, 1.37–1.62, 0.23–0.36, and 0.38–0.50, respectively, for three DNA markers used. Dendrograms based on three molecular data using unweighted pair group method with arithmetic mean (UPGMA) was congruent and classified the Curcuma species into two major clusters. Cophenetic correlation coefficient between dendrogram and original similarity matrix were significant for RAPD (r = 0.96), ISSR (r = 0.94), and AFLP (r = 0.97). Clustering was further supported by principle coordinate analysis. High genetic polymorphism documented is significant for conservation and further improvement of Curcuma species.     

Cytological and molecular characterization of a novel monogenic dominant GMS in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

A novel genic male sterile (GMS) line in Brassica napus L., which was identified in 1999, was found to be controlled by a monogenic dominant gene, which we have designated as MDGMS. The microspores of the MDGMS abort before the degradation of the tapetal cell layer. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Among 880 random 10-mer oligonucleotide primers screened against the bulk DNA of sterile and fertile, one primer S243 (5′-CTATGCCGAC-3′) gave a repeatable 1500-bp DNA polymorphic segment S243₁₅₀₀ between the two bulks. Analysis of individual plants of each bulks and other types of GMS and cytoplasmic male sterility (CMS) lines suggest that the RAPD marker S243₁₅₀₀ is closely linked to the MDGMS locus in rapeseed. This RAPD marker has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs.     

Intra-specific relationships among Tibetan Eared-pheasants based on randomly amplified polymorphic DNA (RAPD) analysis

The Tibetan Eared-pheasant Crossoptilon harmani is a rare species native to China. A captive population has been established in the Beijing Zoo since 1999. In order to determine the kinship of the offsprings in 2001, randomly amplified polymorphic DNA (RAPD) was used to examine the parenthood of seven Tibetan Eared-pheasants in the Beijing Zoo. To amplify the genomic DNA of each individual, 53 arbitrary primers were selected. The results of amplifications showed that 14 primers had clear and distinct RAPD patterns. Totally, 226 amplified fragments were generated by RAPD in this study. Cluster analysis of the seven Tibetan Eared-pheasants indicated that all the four young birds had the same father (No.5 male). This study provides a practical method to determine the relationship of offsprings whose parents are unknown in birds. __________ Translated from Journal of Beijing Normal University (Natural Science), 2005, 41(5): 507–509 [译自: 北京师范大学学报 (自然科学版), 2005, 41(5): 507–509]     

Phylogenetic analyses within three sections of the genus Vicia

The averaged genomic similarities based on multilocus randomly amplified polymorphic DNA (RAPD) were calculated for eight species representing three sections of the genus Vicia: faba, bithynica and narbonensis. The frequency of appearance of the sequences corresponding to 25 decamers selected at random from genomes of different Fabace species was checked, and a high correlation with the frequency observed for Vicia allowed us to assume their similar weight in typing Vicia species. The RAPD-based similarity coefficients compared with those related to whole genome hybridization with barley rDNA and those based on restriction fragment length polymorphism (RFLP) revealed similar interspecies relationships. The averaged RAPD-based similarity coefficient (Pearson’s) was 0.68 for all the species, and was sectionspecific: 0.43 (bithynica), 0.50 (faba) and 0.73 (narbonensis). The averaged similarity coefficient for V. serratifolia (0.63) placed it apart from the rest (0.75) of its section. The results correspond to the interspecies relationships built upon non-genetic data. The averaged similarity coefficient for particular RAPD was related to the presence and type of tandemly repeated motif in a primer: 0.7–0.8 for heterodimers (GC, AG, CA, GT, CT), 0.5–0.6 for homodimers (CC, GG) and 0.6 for no repeat, indicating the sensitivity of diversity range to the type of target sequences.     

A Comparative Analysis of ISSR and RAPD Markers for Study of Genetic Diversity in Shisham (<Emphasis Type="Italic">Dalbergia sissoo</Emphasis>)

Shisham (Dalbergia sissoo) is one of the most preferred timber tree species of South Asia. Two DNA-based molecular marker techniques, intersimple sequence repeat (ISSR) and random amplified polymorphism DNA (RAPD), were compared to study the genetic diversity in this species. A total of 30 polymorphic primers (15 ISSR and 15 random) were used. Amplification of genomic DNA of 22 genotypes, using ISSR analysis, yielded 117 fragments, of which 64 were polymorphic. Number of amplified fragments with ISSR primers ranged from five to ten and varied in size from 180 to 1,900 bp. Percentage polymorphism ranged from 0 to 87.5. The 15 RAPD primers produced 144 bands across 22 genotypes, of which 84 were polymorphic. The number of amplified bands varied from five to 13, with size range from 180 to 2,400 bp. Percentage polymorphism ranged from 0 to 100, with an average of 58.3 across. RAPD markers were relatively more efficient than the ISSR assay. The mental test between two Jaccard’s similarity matrices gave r ≥ 0.90, showing very good fit correlation in between ISSR- and RAPD-based similarities. Clustering of isolates remained more or less the same in RAPD and combined data of RAPD and ISSR. The similarity coefficient ranged from 0.734 to 0.939, 0.563 to 0.946, and 0.648 to 0.920 with ISSR, RAPD, and combined dendrogram, respectively.     

Rapid molecular identification and characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains

Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S–23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)₅ primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2–3 d (in comparison to the standard methods).     

Detection of epigenetic variation in tissue-culture-derived plants of <Emphasis Type="Italic">Doritaenopsis</Emphasis> by methylation-sensitive amplification polymorphism (MSAP) analysis

Somaclones exhibiting variations with flower characteristics were recovered from the tissue-culture-derived plants of Doritaenopsis. Two molecular techniques, random amplified polymorphic DNA (RAPD) and methylation-sensitive amplification polymorphism (MSAP) analyses, were used to characterize the somaclones. RAPD analysis, using 100 randomly selected primers, failed to differentiate variants and normal plants, even though some primers (six out of 100 primers) exhibited 6–10 distinct banding patterns. However, MSAP analysis revealed the differences in the DNA methylation patterns in the normal and variant plants which were correlated with phenotypic variation. In all, 311, 337, 366, and 343 fragments were obtained with normal and V1, V2, and V3 variant plants, respectively; each representing recognition site cleaved by either or both of the isoshizomers were amplified using 12 combination of primers. A total of 36 (11.6%), 77 (22.9%), 73 (19.9%), and 47 (13.7%) sites were found to be methylated at cytosine in the genomes of normal and V1, V2, and V3 variant Doritaenopsis plants. This study demonstrates usefulness of MSAP to detect DNA methylation events in tissue cultured Doritaenopsis plants.     

Mating system and seed variation of <Emphasis Type="Italic">Acacia</Emphasis> hybrid (<Emphasis Type="Italic">A. mangium</Emphasis> × <Emphasis Type="Italic">A. auriculiformis</Emphasis>)

The mating system and seed variation of Acacia hybrid (A. mangium × A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean±s.e. t _m = 0.86±0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%–70%), followed by among pods within inflorescence (29%–37%), and the least variation among inflorescences within tree (<1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.     

Regeneration and analysis of interspecific asymmetric potato –Solanum ssp hybrid plants selected by micromanipulation or fluorescence-activated cell sorting (FACS)

 Recipient protoplasts from three Solanum tuberosum genotypes, cv ‘Folva’ (2n=4x=48), cv ‘Matilda’ (4n) and ‘161 : 14’ (2n), were electrofused with X-ray-irradiated donor protoplasts from two wild species S. spegazzinii (2n) or S. microdontum×S. vernei (2n). Prior to fusion, protoplasts were fluorescence-labelled with either fluorescein diacetate or scopoletin. Fusion products were identified by dual fluorescence and selected by micromanipulation or fluorescence-activated cell sorting (FACS). All putative hybrid plants were analysed by the random amplified polymorphic DNA (RAPD) technique. Our analysis demonstrates that each asymmetric hybrid plant has an individual and stable profile of donor-specific RAPD bands. The irradiation of donor protoplasts hampered the growth of selected heterofusion products in a dose-dependent way. Irradiation resulted in donor chromosome elimination, but not in a dose dependent way, in the tested interval. In asymmetric hybrids with the S. spegazzinii donor 33–68% of the donor-specific RAPD bands were missing, indicating a similar level of chromosome elimination. In asymmetric hybrid plants with the S. microdontum×S. vernei donor 74–95% of the donor RAPD bands were missing. Chromosome countings revealed that these hybrids had chromosome numbers equal to or below the chromosome numbers found in the tetraploid recipients. This is the first time that highly asymmetric hybrid plants between two tetraploid potato recipients and the donor S. microdontum×S. vernei have been obtained. Received : 16 December 1996 / Accepted: 21 February 1997     

RAPD fingerprints for identification and for taxonomic studies of elite poplar (Populus spp.) clones

RAPD (Random Amplified Polymorphic DNA) fingerprints have recently been used to estimate genetic and taxonomic relationships in plants. In this study RAPD analysis was performed on 32 clones belonging to different species of the genus Populus. Of these, 25 clones are registered in several countries for commercial use and, altogether, cover almost 50% of the worlds cultivated poplars. DNA was prepared from leaves and amplified by PCR using random oligonucleotide primers. Amplification products were separated by agarose-gel electrophoresis to reveal band polymorphisms. Four primers out of the 18 tested, were selected on the basis of the number and frequency of the polymorphisms produced. With these a total of 120 different DNA bands were reproducibly obtained, 92% of which were polymorphic. The polymorphisms were scored and used in band-sharing analyses to identify genetic relationships. With a few but interesting exceptions, these are consistent with the present taxonomy of the genus Populus and with the known predigrees of cultivated poplars. Moreover, the results show that RAPD analysis allows one to discriminate among all tested clones and can, therefore, be recommended as a convenient tool to defend plant breeders rights.     

Identification of SCAR marker linking to longer frond length of <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Laminariales,Phaeophyta) using bulked-segregant analysis

To construct a molecular-marker-assisted selection (MAS) system, research was done on identifying molecular markers linking to longer frond length, a crucial selection index in the breeding of the commercially important seaweed Saccharina japonica. An F₂-segregant population of 92 individuals was obtained by crossing two prominent S. japonica strains. Genomic DNA from ten individuals with the longest frond and ten individuals with the shortest frond in the F₂-segregant population were mixed to create two DNA pools for screening polymorphic markers. In bulked-segregant analysis (BSA), out of 100 random amplified polymorphic DNA (RAPD) primers only two produced three polymorphic RAPD markers between the two DNA pools. In conversion of the three RAPD markers into sequence-characterized amplified region (SCAR) markers, only one was successfully converted into a SCAR marker FL-569 linking to the trait of longer frond. Test of the marker FL-569 showed that 80% of the individuals with longest fronds in a wild population and 87.5% of individuals with the longest fronds in an inbred line “Zhongke No. 2” could be detected by FL-569. Additionally, genetic linkage analysis showed that the SCAR marker could be integrated into the reported genetic map and QTL mapping showed that FL-569 linking to qL1-1. The obtained marker FL-569 will be beneficial to MAS in S. japonica breeding.     

Genetic variability of aflatoxin B<Subscript>1</Subscript> producing <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains isolated from discolored rice grains

Twenty-two aflatoxin B₁ (AFB1) producing Aspergillus flavus strains were isolated from 1,200 discolored rice grain samples collected from 20 states across India and tested their potential to produce AFB1 on different agar media. Further these isolates were characterized through randomly amplified polymorphic DNA method. All the strains of A. flavus were produced AFB1 on yeast extract sucrose agar media and none of the strains on A. flavus and A. parasiticus agar. Among the 22 strains, two strains from Tamil Nadu (DRAf 009) and Maharashtra (DRAf 015) produced high amount of AFB1 in all the media tested. To assess the genetic variability in A. flavus, the isolates were analyzed by using random amplified polymorphic DNA markers. Isolates showed 17–80% similarity with standard culture of A. flavus (MTCC 2799).     

The <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> species is abundant among hydrogen-producing facultative anaerobic bacteria in Lake Averno sediment

Lake Averno sediment was used to isolate the facultative anaerobic bacteria having the potential for H₂ production. Twenty-five out of 35 isolates recovered from the sediment sample produced hydrogen under anaerobic conditions from glucose with yields ranging from 0.1 to 0.49 mol H₂/mol glucose. Identification based on 16S rRNA gene sequence analysis revealed that most of them belong to the Firmicutes group, with a prevalence of the Paenibacillus polymyxa species. Seven distinct genomic fingerprints among the 11 P. polymyxa isolates were obtained using the random amplified polymorphic DNA (RAPD) technique. Glucose fermentation by P. polymyxa isolates was investigated. Glucose was totally consumed after 3 days of fermentation. The fermentation products were hydrogen (0.18–0.47 mol H₂/mol glucose), ethanol (0.1–0.5 mol ethanol/mol glucose), and 2,3-butanediol (0.1 mol 2,3-butanediol/mol glucose). Lower amounts of acetic, butyric, formic, lactic, and propionic acids were detected. All metabolic data concerning P. polymyxa isolates were analyzed by cluster analysis to reveal similarities and/or differences with clustering based on RAPD profiles. Despite the high metabolic similarity among almost all P. paenibacillus isolates, results of cluster analyses of metabolic and genetic data do not match completely.     

Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus,Lactobacillus crispatus,Lactobacillus amylovorus,Lactobacillus gallinarum,Lactobacillus gasseri,and Lactobacillus johnsonii

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.     

Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates

A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L.lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency. Received: 14 May 1998 / Accepted: 3 September 1998     

Development of SCAR markers linked to male sterility and very high linoleic acid content in safflower

Practically no molecular tools have been developed so far for safflower (Carthamus tinctorius L.) breeding. The objective of the present research was to develop molecular markers for the closely linked genes Li, controlling very high linoleic acid content, and Ms, controlling nuclear male sterility (NMS). A mapping population of 162 individuals was developed from the NMS line CL1 (64–79% linoleic acid) and the line CR-142 (84–90%), and phenotyped in the F₂ and F₃ generations. Bulked segregant analysis with random amplified polymorphic (RAPD) markers revealed linkage of five RAPD bands to the Li and Ms genes. RAPD fragments were converted into sequence-characterized amplified region (SCAR) markers. A linkage map including the five SCAR markers and the Li and Ms genes was constructed. SCAR markers flanked both loci at minimum distances of 15.7 cM from the Li locus and 3.7 cM from the Ms locus. These are the first molecular markers developed for trait selection in safflower.