Showing papers on "RAPD published in 1991"

Identification of broccoli and cauliflower cultivars with RAPD markers.

Abstract: RAPD (Random Amplified Polymorphic DNA) markers generated by 4 arbitrary 10-mer primers, discriminated 14 broccoli and 12 cauliflower cultivars (Brassica oleracea L.) by banding profiles. The size of the amplified DNA fragments ranged from 300 to 2600 base pairs. Twenty-eight percent of the markers were fixed in both broccoli and cauliflower, whereas 12.5% were specific to either crop. The rest were polymorphic in either or both crops. The markers generated by two and three primers were sufficient to distinguish each of the broccoli and cauliflower cultivars, respectively. The average difference in markers was 14.5 between broccoli and cauliflower markers, 5.8 between two broccoli cultivars and 7.9 between two cauliflower cultivars. Larger differences for each crop were found between cultivars from different seed companies than within the same company. RAPD markers provide a quick and reliable alternative to identify broccoli and cauliflower cultivars.

Segregation of random amplified DNA markers in F1 progeny of conifers

Abstract: The recently developed approach to deriving genetic markers via amplification of random DNA segments with single primers of arbitrary nucleotide sequence was tested for its utility in genetic linkage mapping studies with conifers. Reaction conditions were optimized to reproducibly yield clean and specific amplification products. Template DNA from several genotypes of Douglas-fir (Pseudotsuga menziesii) and white spruce (Picea glauca) were tested against eight ten-base oligonucleotide primers. Most of the tested primer/parent tree combinations yielded polymorphic PCR products ("RAPD" markers). Selected primers were then used in PCR reactions with template DNA isolated from offspring in Douglas-fir and black spruce diallel crosses among the same parental lines. The diallel study confirmed the appropriate inheritance of RAPD markers in the F1 generation. The value of these dominant RAPD markers for genetic linkage mapping in trees was established from both theoretical and applied perspectives.

Identification of restriction fragment length polymorphism and random amplified polymorphic DNA markers linked to downy mildew resistance genes in lettuce, using near-isogenic lines.

Abstract: Near-isogenic lines were used to identify restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers linked to genes for resistance to downy mildew (Dm) in lettuce. Two pairs of near-isogenic lines that differed for Dm1 plus Dm3 and one pair of near-isogenic lines that differed for Dm11 were used as sources of DNA. Over 500 cDNAs and 212 arbitrary 10-mer oligonucleotide primers were screened for their ability to detect polymorphism between the near-isogenic lines. Four RFLP markers and four RAPD markers were identified as linked to the Dm1 and Dm3 region. Dm1 and Dm3 are members of a cluster of seven Dm genes. Marker CL922 was absolutely linked to Dm15 and Dm16, which are part of this cluster. Six RAPD markers were identified as linked to the Dm11 region. The use of RAPD markers allowed us to increase the density of markers in the two Dm regions in a short time. These regions were previously only sparsely populated with RFLP markers. The rapid screening and identification of tightly linked markers to the target genes demonstrated the potential of RAPD markers for saturating genetic maps.

Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA.

Abstract: We have used a PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), to assess genome variability between 21 isolates from F. solani f. sp. cucurbitae races 1 and 2. Based on RAPD marker patterns the isolates fell into two distinct groups corresponding to mating populations MPI and MPV. Four isolates that could not be assigned to one or other mating population by traditional means were distinguished by RAPD patterns. Seven polymorphic RAPD products were used to probe Southern blots of MPI and MPV genomic DNA. Six of the seven probes hybridized to single-copy sequences and five of the seven probes showed specificity for one or other mating population. We suggest that not only is the technique a rapid and reliable tool for isolate-typing of fungi but it also provides a rapid method for obtaining species- or race-specific hybridization probes.

Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD)

Abstract: A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps (“RAPD mapping”). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific “fingerprint” of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing “fingerprints” of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.

Development and chromosomal localization of genome-specific markers by polymerase chain reaction in Brassica.

Abstract: This paper reports the application of the RAPD (random amplification of polymorphic DNA sequence) markers in Brassica genetics. Forty-seven arbitrary decamer oligonucletides were used as primers to amplify genomic DNA by polymerase chain reaction. Some of the amplified products were genome specific and could be found in both diploid and derived amphidiploid species. Of a total of 65 such markers, 16 were A genome, 37 B genome, and 12 C genome specific. Of the 37 B-genome-specific markers, 11 were mapped on four independent chromosomes of B. nigra with the aid of existing B. napus-nigra disomic alien addition lines.

Soybean Genome Analysis: DNA Polymorphisms are Identified by Oligonucleotide Primers of Arbitrary Sequence

Abstract: We have recently constructed a complete linkage map of Glycine max using restriction fragment length polymorphism technology. The map contains more than 550 markers which describe 23 linkage groups and 2700 cM of DNA. Given the level of polymorphism between soybean cultivars, for a single genetic map to have general utility in a breeding program, the map must contain over 1300 markers. To reach this goal, we have developed new technology for genetic mapping that promises to automate genotype determination. The new assay is called a RAPD assay after Random Amplified Polymorphic DNA. It is based on the observation that single oligonucleotide primers of arbitrary sequence can be used to amplify discrete loci in a complex genome.

Molecular genetic studies on Brassica napus L.

Abstract: The feasibility of using two different methods of assaying for DNA polymer phisms has been assessed. They were Restriction fragment length polymorphisms (RFLPs) as revealed by a range of characterised Brassica cDNA sequences and Random amplified polymorphic DNA (RAPD). These techniques have been shown to reveal DNA polymorphisms between varieties of Brassica napus L.. Further more, the sequence and organisation of a Hind III family of highly repetitive DNA sequences were also studied on Brassica napus L. RFLPs associated with rape extensin, ext A, and Brassica oleraceae self- incompatibility genes were observed when DNA samples from 19 commercial varieties of B. napus were analysed using the cDNA probes pRR566 (coding for root-specific extensin) and pBOS2 (coding for S(_5) self-incompatibility allele in B. oleraceae). Both cDNA clones were able to reveal RFLP patterns with varying degrees of polymorphism depending on the restriction enzymes used in the digestion of genomic DNAs. Although both probes could generate complex RFLP band patterns, those revealed by pB0S2 were generally easier to analyse and more suitable for DNA fingerprinting while those revealed by pRR566 were less distinct as a result of extensive background hybridisations. The probe pRR566, with certain restriction enzymes generated simpler RFLP band patterns that were more suitable for segregation analyses. Segregation analysis of F(_1) individuals revealed additive RFLP band patterns of both parental varieties, while that of F(_2) individuals revealed RFLP band patterns of each parental varieties as well as the additive pattern. When analysed for possible association with varietal glucosinolate content, none of the RFLP band patterns showed such linkage. A cDNA library was constructed from pod material of a high glucosinolate variety in an attempt to obtain clones which could reveal RFLP patterns associated with glucosinolate content. Differential screening using total cDNAs from pod materials of high and low glucosinolate varieties failed to isolate any cDNA clones useful as RFLP markers. Another DNA polymorphism assay studied, RAPD, was able to detect inter- and intraspecific variation in Brassica sp.. Analysis of six phylogenetically-related but distinct Brassica sp. revealed extensive variation in the RAPD band patternsof amplification products; with some amphidiploid species sharing conserved band patterns with their ancestral species. RAPD analysis on 17 varieties of rape revealed polymorphic as well as highly conserved RAPD band patterns depending on the primer used. One of the primers was able to amplify a polymorphic band which could be associated with low glucosinolate varieties i.e. present almost exclusively in low glucosinolate varieties. Species-specific as well as variety-specific band patterns were also observed during the RAPD analysis. Finally, sequence and organisation of a Hind III family of repetitive sequences was studied. The monomeric and polymeric forms (trimer and tetramer) of the repetitive sequences were successfully cloned into pUCl8. Sequence analysis of the two clones containing the polymeric forms revealed that the monomers were arranged in tandem array and that all internal Hind III recognition sites were lost due to point mutation(s) which occurred within the six basepair recognition site. A consensus monomeric sequence was deduced from sequence comparison of the 8 copies of the monomeric sequences present in the 3 clones and the deviation from the consensus sequence of each of the eight monomers was less than 3%. No two monomeric sequences were identical. It was estimated that the number of copies of the monomeric sequences in a haploid genome was approximately 0.3 million copies. Estimates of the proportional representation of each of the polymeric sequences based on the number of copies of the monomers in each polymer were also calculated.