Showing papers on "RAPD published in 1994"

Analysis of population genetic structure with RAPD markers

Abstract: Recent advances in the application of the polymerase chain reaction make it possible to score individuals at a large number of loci The RAPD (random amplified polymorphic DNA) method is one such technique that has attracted widespread interest The analysis of population structure with RAPD data is hampered by the lack of complete genotypic information resulting from dominance, since this enhances the sampling variance associated with single loci as well as induces bias in parameter estimation We present estimators for several population-genetic parameters (gene and genotype frequencies, within- and between-population heterozygosities, degree of inbreeding and population subdivision, and degree of individual relatedness) along with expressions for their sampling variances Although completely unbiased estimators do not appear to be possible with RAPDs, several steps are suggested that will insure that the bias in parameter estimates is negligible To achieve the same degree of statistical power, on the order of 2 to 10 times more individuals need to be sampled per locus when dominant markers are relied upon, as compared to codominant (RFLP, isozyme) markers Moreover, to avoid bias in parameter estimation, the marker alleles for most of these loci should be in relatively low frequency Due to the need for pruning loci with low-frequency null alleles, more loci also need to be sampled with RAPDs than with more conventional markers, and some problems of bias cannot be completely eliminated

Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers

Abstract: We have used a "two-way pseudo-testcross" mapping strategy in combination with the random amplified polymorphic DNA (RAPD) assay to construct two moderate density genetic linkage maps for species of Eucalyptus. In the cross between two heterozygous individuals many single-dose RAPD markers will be heterozygous in one parent, null in the other and therefore segregate 1:1 in their F1 progeny following a testcross configuration. Meiosis and gametic segregation in each individual can be directly and efficiently analyzed using RAPD markers. We screened 305 primers of arbitrary sequence, and selected 151 to amplify a total of 558 markers. These markers were grouped at LOD 5.0, theta = 0.25, resulting in the maternal Eucalyptus grandis map having a total of 240 markers into 14 linkage groups (1552 cM) and the paternal Eucalyptus urophylla map with 251 markers in 11 linkage groups (1101 cM) (n = 11 in Eucalyptus). Framework maps ordered with a likelihood support > or = 1000:1 were assembled covering 95% of the estimated genome size in both individuals. Characterization of genome complexity of a sample of 48 mapped random amplified polymorphic DNA (RAPD) markers indicate that 53% amplify from low copy regions. These are the first reported high coverage linkage maps for any species of Eucalyptus and among the first for any hardwood tree species. We propose the combined use of RAPD markers and the pseudo-testcross configuration as a general strategy for the construction of single individual genetic linkage maps in outbred forest trees as well as in any highly heterozygous sexually reproducing living organisms. A survey of the occurrence of RAPD markers in different individuals suggests that the pseudo-testcross/RAPD mapping strategy should also be efficient at the intraspecific level and increasingly so with crosses of genetically divergent individuals. The ability to quickly construct single-tree genetic linkage maps in any forest species opens the way for a shift from the paradigm of a species index map to the heterodox proposal of constructing several maps for individual trees of a population, therefore mitigating the problem of linkage equilibrium between marker and trait loci for the application of marker assisted strategies in tree breeding.

A 300 kilobase interval genetic map of rice including 883 expressed sequences.

Abstract: We have constructed a high resolution rice genetic map containing 1,383 DNA markers at an average interval of 300 kilobases (kb). The markers, distributed along 1,575 cM on 12 linkage groups, comprise 883 cDNAs, 265 genomic DNAs, 147 randomly amplified polymorphic DNAs (RAPD) and 88 other DNAs. cDNAs were derived from rice root and callus, analysed by single-run sequencing and searched for similarities with known proteins. Nearly 260 rice genes are newly identified and mapped, and genomic DNA and cloned RAPD fragments were also sequenced to generate STSs. Our map is the first significant gene expression map in plants. It is also the densest genetic map available in plants and the first to be backed up comprehensively by clone sequence data.

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species.

Abstract: Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.

Application of the RAPD technique in tilapia fish: species and subspecies identification.

Abstract: Random Amplified Polymorphic DNA (RAPD) analysis was applied to three species of the tilapia genus Oreochromis and four subspecies of O. niloticus. Thirteen random 10-mer primers were used to assay polymorphisms within and between populations. Different RAPD fragment patterns were observed for different species, although not always for different subspecies. Evidence is presented that RAPD markers might be useful for systematic investigation at the level of species and subspecies.

PCR technology: current innovations

Abstract: PCR as a Daily Used Technique in Molecular Biology (J.-P. Charlieu). The Design of Primers for PCR (A. Sharrocks). Purification of PCR Products (L. Mezei and D. Storts). Cloning PCR Products (L. Mezei and D. Storts). Amplifying of Unknown Flanking DNA by Single-Specific-Primer PCR (J. Stappert). Generation of Labeled DNA Probes by PCR (G. Konat). Nonisotopic Probe Generation by PCR (E.P.H. Yap, et al.). Direct PCR Screening of Lambda and Cosmid Libraries (H.G. Griffin). Methods for Generating Multiple Site Directed Mutations In Vitro (J. Stappert). Mutagenesis Using PCR (B. Tao). Direct Automated DNA Sequencing of ds and ss PCR Products (A. Lew). Distinction between Almost Identical DNA Sequences by Polymerase Chain Reaction (J.-P. Charlieu). Detection of Mutations by PCR (E.P.H. Yap, et al.). Mapped Restriction Site Polymorphisms (MRSPs) in PCR Products for Rapid Identification and Classification of Genetically Distinct Organisms (D. Ralph and M. McClelland). Detection of Polym orphic DNA Sequences at the 3' end of Alu Repeats by PCR (J.-P. Charlieu). Ligation-Anchored PCR (A. Troutt). PCR-Limiting Dilution Analysis (A. Troutt). Direct PCR from Whole Blood Using Formamide and Low Temperatures (A. Lew). Immuno-PCR: A Generic Method for Purifying Target for PCR (A. Lew). Nonisotopic Single Stranded Conformation Polymorphism (SSCP) Analysis of PCR Products (E.P.H. Yap, et al.). The Use of PCR-RAPD Analysis in Plant Taxonomy and Evolution (T. Demeke and R.P. Adams). Optimization of DNA Extraction and PCR Procedures for Random Amplified Polymorphic DNA (RAPD) Analysis in Plants (K. Yu and K.P. Pauls). The Use of RAPD Analysis to Tag Genes and Determine Relatedness in Heterologous Plant Populations Using Tetraploid Alfalfa as an Example (K. Yu and K.P. Pauls). DNA Recombination in the Course of PCR (A. Vartapetian). Thermostable DNA Polymerases for In Vitro DNA Amplifications (A. Bej and M. Mahbubani). PCR in Sequence-Tagged Site (STS) Content Genome Mapping (A. Srivastava). False Positives and Contamination in PCR (E.P.H. Yap, et al.). The Application of Polymerase Chain Reaction (PCR) Based Technologies to Forensic Analysis (L. Presley). Application of PCR Amplified DNA Markers in Identification of Individuals (L. Peltonen and A. Sajantila). PCR in Forensic Science (V. Pascali). Applications of Polymerase Chain Reaction Methodology in Clinical Diagnostics (A. Bej and M. Mahbubani). Applications of the Polymerase Chain Reaction (PCR) In Vitro DNA Amplification Method in Environmental Microbiology (A. Bej and M. Mahbubani). Detection of Foodborne Microbial Pathogens Using Polymerase Chain Reaction (D. Jones and A. Bej).

Molecular genetics of growth and development in Populus. III. A genetic linkage map of a hybrid poplar composed of RFLP, STS, and RAPD markers.

Abstract: We have evaluated three DNA-based marker types for linkage map construction in Populus: RFLPs detected by Southern blot hybridization, STSs detected by a combination of PCR and RFLP analysis, and RAPDs. The mapping pedigree consists of three generations, with the F1 produced by interspecific hybridization between a P. trichocarpa female and a P. deltoides male. The F2 generation was made by inbreeding to the maximum degree permitted by the dioecious mating system of Populus. The applicability of STSs and RAPDs outside the mapping pedigree has been investigated, showing that these PCR-based marker systems are well-suited to breeding designs involving interspecific hybridization. A Populus genome map (343 markers) has been constructed from a combination of all three types. The length of the Populus genome is estimated to be 2400–2800 cM.

Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis.

Abstract: The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in the dioecious species, Pistacia vera Progenies from two female parents pollinated by a common male parent were studied Two bulks of DNA were made in each cross, one from males and one from females, by pooling an equal weight of fresh leaves from each individual contributing to the bulk prior to DNA extraction DNA was extracted from each bulked sample and from each of the contributing individuals DNA was also extracted from 14 cultivars of P vera and from 94 open-pollinated, fewweeks-old P vera seedlings of unknown sex Seven hundred different decamer oligonucleotide primers were used to perform DNA amplification, with 1 of these (OPO08) producing a 945 bp amplification band that was present only in the bulked female samples and absent in the bulked male samples of the two crosses The relationship between band presence and female sex expression was conserved in every individual obtained from the two crosses and in the 14 cultivars unrelated to the crosses We propose that this band is tightly linked to the gene(s) that control sex determination in pistachio The OPO08945 RAPD marker could be used in a breeding program to screen the gender of pistachio plants long before they reach reproductive maturity, resulting in considerable savings of time and economic resources In order to verify that assumption we screened 94 additional seedlings with the OPO08 primer and obtained results consistent with a 1∶1 male:female ratio

Detection of genetic diversity and selective gene introgression in coffee using RAPD markers.

Abstract: RAPD (randomly amplified polymorphic DNA) markers generated by arbitary decamers have been successfully employed to detect genetic polymorphisms between coffee species and between Coffea arabica genotypes. The RAPD profiles were used to construct dendrograms and these were consistent with the known history and evolution of Coffea arabica. Material originating from Ethiopia and the arabica sub-groups — C. arabica var. typica and C. arabica var. bourbon — were clearly distinguished. RAPD analysis therefore reflects morphological differences between the sub-groups and the geographical origin of the coffee material. Species-specific amplification products were also identified, but, more importantly, amplification products specific to C. canephora were identified in two C. arabica genotypes, Rume Sudan and Catimor 5175. This diagnostic product is therefore indicative of interspecific gene flow in coffee and has biological implications for selective introgressive hybridisation in coffee. Our study demonstrates the power of the polymerase chain reaction technology for the generation of genetic markers for long-lived perennial tree and bush crops.

A detailed RFLP map of Sorghum bicolor x S. propinquum, suitable for high-density mapping, suggests ancestral duplication of Sorghum chromosomes or chromosomal segments.

Abstract: The first “complete” genetic linkage map of Sorghum section Sorghum is described, comprised of ten linkage groups putatively corresponding to the ten gametic chromosomes of S. bicolor and S. propinquum. The map includes 276 RFLP loci, predominately detected by PstI-digested S. bicolor genomic probes, segregating in 56 F2 progeny of a cross between S. bicolor and S. propinquum. Although prior cytological evidence suggests that the genomes of these species are largely homosequential, a high level of molecular divergence is evidenced by the abundant RFLP and RAPD polymorphisms, the marked deviations from Mendelian segregation in many regions of the genome, and several species-specific DNA probes. The remarkable level of DNA polymorphism between these species will facilitate development of a high-density genetic map. Further, the high level of DNA polymorphism permitted mapping of multiple loci for 21 (8.2%) DNA probes. Linkage relationships among eight (38%) of these probes suggest ancestral duplication of three genomic regions. Mapping of 13 maize genomic clones in this cross was consistent with prior results. Mapping of heterologous cDNAs from rice and oat suggests that it may be feasible to extend comparative mapping to these distantly-related species, and to ultimately generate a detailed description of chromosome rearrangements among cultivated Gramineae. Limited investigation of a small number of RFLPs showed several alleles common to S. bicolor and S. Halepense (“johnson-grass”), but few alleles common to S. propinquum and S. halepense, raising questions about the origin of S. halepense.

Differentiation of Fusarium oxysporum f. sp. vasinfectum races on cotton by random amplified polymorphic DNA (RAPD) analysis

Abstract: Assigbetse, K. B., Fernandez, D., Dubois, M. P., and Geiger, J.-P. 1994. Differentiation of Fusarium oxysporum f. sp. vasinfectum races on cotton by random amplified polymorphic DNA (RAPD) analysis. Phytopathology 84:622-626. We used pathogenicity and random amplified polymorphic DNA (RAPD) markers to assess genetic diversity among 46 isolates of Fusarium ox~~sporum f. sp. vasinfectuni of worldwide origin. Based on pathogenicity tests on five differential cotton cultivars and species, isolates were differentiated into three races (A, 3, and 4), restricted to defined geographic areas. The amount of genetic variation was evaluated by polymerase chain reaction amplification with a set of 11 random IO-mer primers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 83 band positions was scored (1/0) for the 11 primers tested. Genetic distances between each of the isolates were calculated, and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates clustered into three groups corresponding to their pathological reactions. We suggest that RAPD markers can be a quick and reliable alternative for differentiating isolates of F. o. vasinfectum into their respective pathogenicity group.

Analysis of a detailed genetic linkage map of Lactuca sativa (lettuce) constructed from RFLP and RAPD markers.

Abstract: A detailed genetic map has been constructed from the F2 population of a single intraspecific cross of Lactuca sativa (n = 9). It comprises 319 loci, including 152 restriction fragment length polymorphism (RFLP), 130 random amplified polymorphic DNA (RAPD), 7 isozyme, 19 disease resistance, and 11 morphological markers. Thirteen major, four minor linkage groups and several unlinked markers are identified for this genome which is estimated to be approximately 1950 cM. RFLP and RAPD markers show similar distributions throughout the genome and identified similar levels of polymorphism. RAPD loci were much quicker to identify but more difficult to order. Procedures for generating accurate genetic maps and their limitations are described.

Targeted mapping and linkage analysis of morphological isozyme, and RAPD markers in peach.

Abstract: Nine different F2 families of peach [Prunus persica (L.) Batsch] were analyzed for linkage relationships between 14 morphological and two isozyme loci. Linkage was detected between weeping (We) and white flower (W), 33 cM; double flower (Dl) and pillar (Br), 10 cM; and flesh color (Y) and malate dehydrogenase (Mdh1), 26 cM. A leaf variant phenotypically distinct from the previously reported wavy-leaf (Wa) mutant in peach was found in progeny of 'Davie II'. The new willow-leaf character (designated Wa2) was closely linked (0.4 cM) to a new dwarf phenotype (designated Dw3). Two families derived from the pollen-fertile cultivar 'White Glory' segregated for pollen sterility, but segregation did not follow a 3∶1 ratio. Evidence is presented suggesting that 'White Glory' possesses a pollen-sterility gene (designated Ps2) that is non-allelic to the previously reported pollen-sterility gene (Ps) in peach. Ps2 was linked to both weeping (We-Ps2, 15.5 cM) and white flower (Ps2-W, 25.3 cM). A genomic map of peach containing 83 RAPD, one isozyme, and four morphological markers was generated using an F2 family obtained by selfing an NC174RL x 'Pillar' F1. A total of 83 RAPD markers were assigned to 15 linkage groups. Various RAPD markers were linked to morphological traits. Bulked segregant analysis was used to identify RAPD markers flanking the red-leaf (Gr) and Mdh1 loci in the NC174RL x 'Pillar' and 'Marsun' x 'White Glory' F2 families, respectively. Three markers flanking Mdh1 and ten markers flanking Gr were identified. The combination of RAPD markers and bulked segregant analysis provides an efficient method of identifying markers flanking traits of interest. Markers linked to traits that can only be scored late in development are potentially useful for marker-aided selection in trees. Alternatives for obtaining additional map order information for repulsion-phase markers in large F2 populations are proposed.

Reproducible DNA fingerprinting with the random amplified polymorphic DNA (RAPD) method

Abstract: Much interest has recently arisen in methods for DNA fingerprinting based on the polymerase chain reaction (PCR). Among these, the Random Amplified Polymorphic DNA (RAPD) method, developed by Williams etal. (1), is currently receiving particular attention (2) because of its extreme simplicity and requirement for minimal amounts of genomic DNA. The basic strategy involves the PCR amplification of random fragments of genomic DNA with single or multiple (3) primers of arbitrary sequence. Polymorphism between individuals (or strains) is detected as differences between the pattern of DNA fragments amplified from the different DNAs using a given primer(s). Although RAPD PCR is an extremely powerful tool for such tasks as gene mapping, population and pedigree analysis, phylogenetic studies and bacterial strain identification, the reproducibility of RAPD fingerprints can be quite problematic (4-6). Since the pattern of fragments amplified is, in large part, a function of the sites on the template to which productive annealing of the oligonucleotide primer can occur, differences between DNA preparations that affect primer annealing could be one major source of irreproducibility of RAPD patterns. Here we show directly that ethanol precipitable contaminants in DNA are a major cause of irreproducibility. In preparing DNA for RAPD analysis using standard extraction techniques we found a perfect correlation between the procedure used to collect ethanol precipitated DNA and the reproducibility of the RAPD pattern. We always obtained highly reproducible patterns from DNAs that were wound on a glass rod, while patterns from DNAs collected by centrifugation displayed different degrees of variability. Since centrifugation of an ethanol precipitate of a previously wound DNA sample did not introduce variability, we concluded that winding the DNA must free it of the material that causes the variability exhibited by the centrifuged samples. The results in the figure demonstrate that the RAPD pattern of centrifuged DNA differs from that of wound DNA (lanes Al and Bl, respectively), that the pattern produced by wound DNA can be produced from centrifuged DNA if the latter is reprecipitated from ethanol but collected by winding on a glass rod (lanes A2), and that an altered pattern is obtained using wound DNA to which its supernatant material has been added back (lanes B2). Although RNase A digestion modified the RAPD pattern produced from centrifuged DNA, this treatment did not result in the same RAPD pattern produced with wound DNA (results not shown). Therefore, contaminating RNA may only be partly responsible for the variability observed with centrifuged samples. On the other hand, DNase treatment modified the RAPD pattern produced from wound DNA (results not shown), suggesting that either the presence of very short DNA fragments or shortened templates, or both, may lead to RAPD variability. These findings support the hypothesis that the ethanol precipitable contaminants include very low molecular weight DNA and/or RNA which, in some way, alter the formation of productive template/primer complexes. Whatever their nature, our results unequivocally show that ethanol precipitable contaminants in DNA extracted by standard techniques are a major, if not sole, source of variability in RAPD

Identification and localization of molecular markers linked to the Lr9 leaf rust resistance gene of wheat.

Abstract: Near-isogenic lines (NILs) for the leaf rust resistance gene Lr9 were screened for polymorphisms at the molecular level. RAPD (random amplified polymorphic DNA) primers as well as RFLP (restriction fragment length polymorphism) markers were used. Out of 395 RAPD primers tested, three showed polymorphisms between NILs, i.e., an additional band was found in resistant lines. One of these polymorphic bands was cloned and sequenced. Specific primers were synthesized, and after amplification only resistant lines showed an amplified product. Thus, these primers define a sequence-tagged site that is specific for the translocated fragment carrying the Lr9 gene. A cross between a resistant NIL and the spelt (Triticum spelta) variety 'Oberkulmer' was made, and F2 plants were analyzed for genetic linkage. All three polymorphisms detected by the PCR (polymerase chain reaction) and one RFLP marker (cMWG684) showed complete linkage to the Lr9 gene in 156 and 133 plants analyzed, respectively. A second RFLP marker (PSR546) was closely linked (8±2.4 cM) to the Lr9 gene and the other four DNA markers. As this marker maps to the distal part of the long arm of chromosome 6B of wheat, Lr9 and the other DNA markers also map to the distal region of 6BL. All three PCR markers detected the Lr9 gene in independently derived breeding lines and varieties, thus proving their general applicability in wheat breeding programs.

Differentiation of nearly identical germplasm accessions by a combination of molecular and morphologic analyses.

Abstract: Polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPD) markers were used to study the extent of redundancy (duplication of genetic materials) within a genetic resources collection. Nine nearly phenotypically and identical accessions of butterhead lettuce (Lactuca sativa L.) were assayed for their genetic identities. A nonuniform, heterogeneous butterhead line and a crisphead cultivar were added for population comparison. PCR amplification using 13 oligonucleotide primers generated 93 polymorphic bands. The percentage of segregating bands was used to determine within-line variation; values ranged from 0.0 to 12.0%, except for the nonuniform line at 22.6%. Between-line similarity was measured using similarity coefficients and ranged from 0.919 to 0.985. The relationship between the crisphead accession and a composite of all butterhead accessions was 0.84. Selfed progeny of each line were measured for morphological uniformity. The variation obtained from these biological data was compa...

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Abstract: Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.

Potential use of random amplified polymorphic DNA (RAPD) technique to study the genetic diversity in Indian mustard (Brassica juncea) and its relationship to heterosis

Abstract: RAPD assays were performed, using 34 arbitrary decamer oligonucleotide primers and six combinations of two primers, to detect inherent variations and genetic relationships among 12 Indian and 11 exotic B. juncea genotypes. Of 595 amplification products identified, 500 of them were polymorphic across all genotypes. A low level of genetic variability was detected among the Indian genotypes, while considerable polymorphism was present among the exotic ones. Based on the pair-wise comparisons of amplification products the genetic similarity was calculated using Jaccard's similarity coefficients and a dendrogram was constructed using an unweighted pair group method was arithmetical averages (UPGMA). On the basis of this analysis the genotypes were clustered into two groups, A and B. Group A comprised only exotic genotypes, whereas all the Indian genotypes and four of the exotic genotypes were clustered in group B. Almost similar genotypic rankings could also be established by computing as few as 200 amplification products. In general, a high per cent of heterosis was recorded in crosses involving Indian x exotic genotypes. On the other hand, when crosses were made amongst Indian or exotic genotypes, about 80% of them exhibited negative heterosis. Results from this study indicate that, despite the lack of direct correlation between the genetic distance and the degree of heterosis, genetic diversity forms a very useful guide not only for investigating the relationships among Brassica genotypes but also in the selection of parents for heterotic hybrid combinations.

Differentiation of species and strains of entomopathogenic fungi by random amplification of polymorphic DNA (RAPD)

Abstract: Polymerase chain reaction (PCR)-based technology, involving random amplification of polymorphic DNA (RAPD), was used to assess the genomic variability between 24 isolates of deuteromycetous fungi (Metarhizium anisopliae, Metarhizium flavoviride, unidentified strains of Metarhizium and Beauveria bassiana) which were found to infect grasshoppers or locusts. M. flavoviride showed little intraspecific variability in PCR-amplified fragments when compared to M. anisopliae. The high level of variability in PCR-amplified fragments contained within M. anisopliae was similar to the total variability between B. bassiana, M. anisopliae and M. flavoviride, and suggests that M. anisopliae may include a number of cryptic species. Four polymorphic RAPD fragments were used to probe the genomic DNA of the various species and strains. On the basis of these probes the fungi can be grouped into M. flavoviride, M. anisopliae, or B. bassiana. According to PCR-amplified fragments, previously-unidentified Metarhizium strains were characterized as M. flavoviride. There was little evidence that these fungi, all isolated from, or virulent towards, grasshoppers or locusts, showed host-selection in PCR-amplified fragments. Nor was geographical origin a criterion for commonality based on PCR-amplified fragments. PCR-fragment-pattern polymorphisms and the construction of probes from one or more of these fragments may provide a useful and rapid tool for identifying species and strains of entomopathogenic fungi.

A PCR-based marker tightly linked to the nematode resistance gene, Mi, in tomato.

Abstract: A PCR-based codominant marker has been developed which is tightly linked to Mi, a dominant genetic locus in tomato that confers resistance to several species of root-knot nematode. DNA from tomato lines differing in nematode resistance was screened for random amplified polymorphic DNA markers linked to Mi using decamer primers. Several markers were identified. One amplified product, REX-1, obtained using a pair of decamer primers, was present as a dominant marker in all nematode-resistant tomato lines tested. REX-1 was cloned and the DNA sequences of its ends were determined and used to develop 20-mer primers. PCR amplification with the 20-mer primers produced a single amplified band in both susceptible and resistant tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq I. The linkage of REX-1 to Mi was verified in an F2 population. This marker is more tightly linked to Mi than is Aps-1, the currently-used isozyme marker, and allows screening of germplasm where the linkage between Mi and Aps-1 has been lost. Homozygous and heterozygous individuals can be distinguished and the procedure can be used for rapid, routine screening. The strategy used to obtain REX-1 is applicable to obtaining tightly-linked markers to other genetic loci. Such markers would allow rapid, concurrent screening for the segregation of several loci of interest.

Dna evolution and colonization sequence of island lizards in relation to geological history: mtdna rflp, cytochrome b, cytochrome oxidase, 12s rrna sequence, and nuclear rapd analysis

Abstract: A novel source of nuclear DNA information from random amplified polymorphisms (RAPD) and a wide-range mitochondrial DNA information (cytochrome b, cytochrome oxidase, and 12s rRNA sequence, RFLP from 4-base and 6-base recognition endonucleases) are used to reconstruct the population phylogeny of the western Canary Island lizard, Gallotia galloti, which, for geological reasons, has been subject to dispersal but not vicariance. Interpretation of DNA phylogenies in terms of colonization sequence indicates that G. galloti arose in Tenerife and dispersed westward in two independent pathways: north from north Tenerife to La Palma, and south from south Tenerife to Gomera to Hierro. The direction and timing of colonization by DNA divergence is entirely compatible with geological time and sequence of island origin.

SCAR, RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean.

Abstract: Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).

Restriction fragment length polymorphism analysis of loci associated with disease resistance genes and developmental traits in Pisum sativum L.

Abstract: An F2 population of pea (Pisum sativum L.) consisting of 174 plants was analysed by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) techniques. Ascochyta pisi race C resistance, plant height, flowering earliness and number of nodes were measured in order to map the genes responsible for their variation. We have constructed a partial linkage map including 3 morphological character genes, 4 disease resistance genes, 56 RFLP loci, 4 microsatellite loci and 2 RAPD loci. Molecular markers linked to each resistance gene were found: Fusarium wilt (6 cM from Fw), powdery mildew (11 cM from er) and pea common Mosaic virus (15 cM from mo). QTLs (quantitative traits loci) for Ascochyta pisi race C resistance were mapped, with most of the variation explained by only three chromosomal regions. The QTL with the largest effect, on chromosome 4, was also mapped using a qualitative, Mendelian approach. Another QTL displayed a transgressive segregation, i.e. the parental line that was susceptible to Ascochyta blight had a resistance allele at this QTL. Analysis of correlations between developmental traits in terms of QTL effects and positions suggested a common genetic control of the number of nodes and earliness, and a loose relationship between these traits and height.

Evaluation of RFLP and RAPD markers in a comparison of Brassica napus breeding lines.

Abstract: RFLP and RAPD markers were evaluated and compared for their ability to determine genetic relationships in a set of three B. napus breeding lines. Using a total of 50 RFLP and 92 RAPD markers, the relatedness between the lines was determined. In total, the RFLP and the RAPD analysis revealed more than 500 and 400 bands, respectively. The relative frequencies of loci with allele differences were estimated from the band data. The RFLP and RAPD marker sets detected very similar relationships among the three lines, consistent with known pedigree data. Bootstrap analyses showed that the use of approximately 30 probes or primers would have been sufficient to achieve these relationships. This indicates that RAPD markers have the same resolving power as RFLP markers when used on exactly the same set of B. napus genotypes. Since RAPD markers are easier and quicker to use, these markers may be preferred in applications where the relationships between closely-related breeding lines are of interest. The use of RAPD markers in fingerprinting applications may, however, not be warranted, and this is discussed in relation to the reliability of RAPD markers.

Use of genetic fingerprinting and random amplified polymorphic DNA to characterize pathotypes of Fusarium oxysporum f. sp. ciceris infecting chickpea

Abstract: Isolates of Fusarium oxysporum f. sp. ciceris induce either yellowing or wilt syndromes in chickpea and can be characterized into seven races by means of biological typing. DNA from 63 isolates of F. o. ciceris and from 11 isolates of other fungi was amplified by the random amplified polymorphic DNA (RAPD) technique by using the polymerase chain reaction with single primers. The primers used were based either on known ribosomal DNA sequences from Penicillium hordei or on sequencing primers. The amplification products were analyzed for polymorphisms by gel electrophoresis to determine whether pathotypes and/or races could be distinguished at the molecular level

Genetic structure of Pyrenophora teres populations determined with random amplified polymorphic DNA markers

Abstract: The genetic structure of Pyrenophora teres, an ascomycete fungus that causes net blotch of barley, was examined using random amplified polymorphic DNA (RAPD) markers. Twenty-seven random oligonucleotide primers were screened against DNA from 16 isolates of P. teres of diverse geographic origin. Five primers gave scorable, reproducible DNA products (bands) suitable for population genetic studies. Genetic analyses of bands produced by two of the primers revealed single locus segregation in three of four crosses, indicating that these RAPDs can be interpreted as alleles at genetic loci. Allele frequencies were determined for 10 putative RAPD loci from five primers in 22–35 isolates of P. teres sampled from each of five geographically separated populations in Canada, Germany, and the U.S.A. Eight RAPD loci were polymorphic in at least one population and two loci were monomorphic in all five populations. Variation in allele frequencies (allelic diversity) among the five populations was partitioned into within-...

Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation-responsive loci.

Abstract: Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments was obtained with approximately half of the 140 random primers tested, revealing 266 segregating loci. These were tested for linkage using 60 BC1 progeny from an intergeneric cross of Citrus grandis (L.) Osb. x [Citrus grandis (L.) Osb. x Poncirus trifoliata (L.) Raf.]. A core linkage map was constructed that consists of nine linkage groups containing 109 RAPD markers and 51 previously-mapped RFLP and isozyme markers. A further 79 markers that could not be ordered unambiguously because of their allelic constitution were associated with individual linkage groups and are shown in relation to the core map. The core map has a total length of 1192 cM with an average distance of 7.5 cM between loci and is estimated to cover 70–80% of the genome. Loci with distorted segregation patterns clustered on several linkage groups. Individual clusters of loci were skewed in allelic composition toward one or the other parent, usually C. grandis. This relatively-saturated linkage map will eventually be used to identify quantitative trait loci for cold and salt-tolerance in Citrus. As a beginning we have mapped three loci detected by a cold-acclimation-responsive cDNA.

Evaluation of "sequence-tagged-site" PCR products as molecular markers in wheat.

Abstract: The polymerase chain reaction (PCR) is an attractive technique for many genome mapping and characterization projects. One PCR approach which has been evaluated involves the use of randomly amplified polymorphic DNA (RAPD). An alternative to RAPDs is the sequence-tagged-site (STS) approach, whereby PCR primers are designed from mapped low-copy-number sequences. In this study, we sequenced and designed primers from 22 wheat RFLP clones in addition to testing 15 primer sets that had been previously used to amplify DNA sequences in the barley genome. Our results indicated that most of the primers amplified sequences that mapped to the expected chromosomes in wheat. Additionally, 9 of 16 primer sets tested revealed polymorphisms among 20 hexaploid wheat genotypes when PCR products were digested with restriction enzymes. These results suggest that the STS-based PCR analysis will be useful for generation of informative molecular markers in hexaploid wheat.

Intraspecific molecular variation among Trichoderma harzianum isolates colonizing mushroom compost in the British Isles.

Abstract: The genetic diversity in Trichoderma harzianum isolates from mushroom compost was assessed using various molecular techniques. Restriction fragment length polymorphism (RFLP) analysis of ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) divided the 81 isolates into three major groups, 1, 2 and 3. There was no variation within a group in rDNA, while a low degree of polymorphism was detected in mtDNA. Random amplified polymorphic DNA (RAPD) analysis of 30 randomly chosen isolates, with six primers, in general confirmed the RFLP groups. Nucleotide sequence determination of rDNA internal transcribed spacer (ITS) 1 revealed three distinct ITS types, 1, 2 and 3, possessed by isolates from the respective groups 1, 2 and 3. Based on these molecular data, group 2 isolates, which are aggressive colonizers of mushroom compost, could be clearly distinguished from the isolates belonging to the other two groups.