RAPD

About: RAPD is a research topic. Over the lifetime, 15960 publications have been published within this topic receiving 360391 citations.

Molecular typing of Cryptococcus neoformans : Taxonomic and epidemiological aspects

Abstract: Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, serotype, and killer toxin sensitivity patterns of a wide range of saprobic, clinical, and veterinary isolates of both varieties of Cryptococcus neoformans were examined. C. neoformans var. neoformans and C. neoformans var. gattii differed in chromosomal makeup, W D pa tterns, and killer sensitivity patterns. These results suggest that there are two separate species rather than two varieties. No clear genetic or phenotypic differences were observed among the clinical, saprobic, and veterinary isolates within each taxon. The serotypes differed substantially in their RAPD characteristics. Geographical clustering was observed among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans. The isolates of each taxon that originated from restricted geographical areas often had identical or similar karyotypes and RAPD patterns, suggesting that clonal reproduction had occurred. The combination of PFGE and RAPD analysis allowed us to distinguish almost all isolates. This combination of techniques is recommended for further research on epidemiological, ecological, and population issues.

Mapping and Progress toward Map-Based Cloning of Brown Planthopper Biotype-4 Resistance Gene Introgressed from Oryza officinalis into Cultivated Rice, O. sativa

Abstract: Brown plant hopper (BPH), Nilaparvata lugens (Stal.), is a serious insect pest of rice in Asia, causing direct losses and vectoring Rice grassy stunt virus (RGSV) and Rice ragged stunt virus (RRSV). Recombinant inbred lines (RILs) developed from a cross between 'IR50' and 'IR54745-2-21-12-17-6' were used to identify random amplified polymorphic DNA (RAPD) markers closely linked to a BPH Biotype-4 resistance gene [Bphl3 (t)] derived from Oryza officinalis Wall. Bulked segregant analysis (BSA) using RAPD primers identified 11 polymorphic fragments. Six fragments, AJ09 260 a, AL05 220 a, AK10 690 a, AK10 430 c, AK10 380 d, and AJ01 200 a, were linked in coupling phase to the Bphl3 (t) locus. The remaining five fragments, AJ09 230 b, AJ09 180 c, AJ09 100 d, AL05 400 b, and AK10 340 e, were linked in repulsion. The most closely linked RAPD marker, AJ09 230 b, was converted to a codominant linked sequence tagged sites (STS) marker. This marker mapped 1.3 centimorgans (cM) from the resistance gene and was placed on rice chromosome 3 by means of 'IR64' × 'Azucena' doubled haploid (DH) population. The tightly linked STS marker could be used for marker-assisted selection (MAS). In addition, these markers will be useful for a positional cloning strategy to isolate the resistance gene.

Effects of Plant Tissue and DNA Purification Method on Randomly Amplified Polymorphic DNA-based Genetic Fingerprinting Analysis in Carrot

Abstract: ADDITIONAL INDEX WORDS. marker-assisted selection, polymerase chain reaction, PCR, RAPD ABSTRACT. Seven plant genomic DNA purification protocols were evaluated for genetic fingerprinting analysis using six tissues obtained from inbred carrot (Daucus carota L.) lines. Evaluations included 1) DNA yield, 2) DNA purity, 3) DNA cleavage with HindIII, 4) DNA integrity, and 5) DNA suitability for amplification in a random amplified polymorphic DNA (RAPD) system. Significant differences were observed among tissues and purification methods for the total amount of DNA. An extraction method using CTAB buffer + organic solvents gave the best results in DNA yield, purity, and HindIII cleavage when compared with the other six nonorganic extraction methods. Of the tissues examined, flowers yielded the most DNA (average value = 115 ng of DNA/mg of fresh tissue); followed by seeds (54 ng·mg -1 ), fresh leaves (48 ng·mg -1 ), lyophilized leaves (40 ng·mg -1 ), calli (22 ng·mg -1 ), and tap roots (4 ng·mg -1 ). For most of the preparations, the DNA showed no traces of degradation. However, DNA preparations were not consistently accessible to HindIII cleavage in all tissue-extraction method combina- tions. Uncut DNA was observed chiefly in extractions from flowers and fresh leaves suggesting a tissue-specific adverse effect on restriction endonuclease activity. Differences in RAPD band (amplicon) intensity and number were observed across tissues and DNA extraction methods using identical PCR conditions for RAPD. Callus was the best type of tissue for RAPD-based fingerprinting yielding a consistently higher number of more intense amplicons when compared to the other tissues. In flowers and seeds, only DNA obtained with the CTAB extraction method could be amplified. Polymorphisms deviating from genetic expectations were mainly observed in root and fresh leaf DNA, indicating that some RAPD markers may not present satisfactory levels of reproducibility. Judicious and uniform selection of DNA purification method as well as tissue source for DNA extraction are, therefore, important considerations for reliable RAPD-based DNA fingerprinting analysis in carrot. In addition, our studies allowed the identification of a better combination of procedures for use in routine manipulations of carr ot DNA such as RFLP-RAPD-based cultivar fingerprinting, molecular mapping, screening of transgenic plants, construction of genomic libraries, and gene cloning.

Identification of RAPD markers linked to a fertility restorer gene for the Ogura radish cytoplasmic male sterility of rapeseed (Brassica napus L.).

Abstract: Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.