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

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.     

The comparison of RFLP,RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.Abbreviations RFLP restriction fragment length plymorphism - RAPD random-amplified polymorphic DNA - AFLP amplified fragment length polymorphism - SSR simple sequence repeat - PCR polymerase chain reaction - TBE Tris-borate-EDTA buffer - MI marker index - SENA sum of effective numbers of alleles     

Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.)

A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials.The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.Abbreviations PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism.     

Combined RAPD and RFLP molecular linkage map of asparagus.

Two linkage maps of asparagus (Asparagus officinalis L.) were constructed using a double pseudotestcross mapping strategy with restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and allozymes as markers in a population generated from crossing MW25 x A19, two heterozygous parents. All data were inverted and combined with the natural data to detect linkages in repulsion phase. Two sets of data, one for each parent, were formed according to the inheritance patterns of the markers. The maternal MW25 map has a total of 163 marker loci placed in 13 linkage groups covering 1281 cM, with an average and a maximum distance between adjacent loci of 7.9 and 29 cM, respectively. The paternal A19 map has 183 marker loci covering 1324 cM in 9 linkage groups, with an average and a maximum distance between two adjacent loci of 7.7 and 29 cM, respectively. Six multiallelic RFLPs segregating in the pattern a/c x b/c and eight heterozygous loci (four RAPDs, and four RFLPs segregating in the pattern a/b x a/b (HZ loci)) were common to both maps. These 14 loci were used as bridges to align homologous groups between the two maps. In this case, RFLPs were more frequent and informative than RAPDs. Nine linkage groups in the MW25 map were homologous to six groups in the A19 map. In two cases, two or more bridge loci were common to a group; thus, the orientation of homologous linkage groups was also determined. In four other cases, only one locus was common to the two homologous groups and the orientation was unknown. Mdh, four RFLPs, and 14 RAPDs were assigned to chromosome L5, which also has the sex locus M.     

Linkages between restriction fragment length,isozyme, and morphological markers in lentil

Summary A genetic linkage map of lentil comprising 333 centimorgans (cM) was constructed from 20 restriction fragment length, 8 isozyme, and 6 morphological markers segregating in a single interspecific cross (Lens culinaris × L. orientalis). Because the genotypes at marker loci were determined for about 66 F₂ plants, linkages are only reported for estimates of recombination less than 30 cM. Probes for identification of restriction fragment length polymorphisms (RFLPs) were isolated from a cDNA and EcoRI and PstI partial genomic libraries of lentil. The cDNA library gave the highest frequency of relatively low-copy-number probes. The cDNAs were about twice as efficient, relative to random genomic fragments, in RFLP detection per probe. Nine markers showed significant deviations from the expected F₂ ratios and tended to show a predominance of alleles from the cultigen. Assuming a genome size of 10 Morgans, 50% of the lentil genome could be linked within 10 cM of the 34 markers and the map is of sufficient size to attempt mapping of quantitative trait loci.     

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.     

Construction of genetic linkage maps in maize and tomato using restriction fragment length polymorphisms

Summary Genetic linkage maps were constructed for both maize and tomato, utilizing restriction fragment length polymorphisms (RFLPs) as the source of genetic markers. In order to detect these RFLPs, unique DNA sequence clones were prepared from either maize or tomato tissue and hybridized to Southern blots containing restriction enzyme-digested genomic DNA from different homozygous lines. A subsequent comparison of the RFLP inheritance patterns in F2 populations from tomato and maize permitted arrangement of the loci detected by these clones into genetic linkage groups for both species.     

Systematics and randomly amplified polymorphic DNA in the genusLeucaena (Leguminosae,Mimosoideae)

Randomly amplified polymorphic DNA (RAPD) was used to examine genomic diversity in taxa of the neotropical legume genusLeucaena. Data were analysed using both similarity- and parsimony-based approaches and the data compared to a parsimonybased analysis of chloroplast DNA restriction fragment length polymorphisms (RFLP). Distance-based methods of RAPD analysis produced groups inconsistent with those identified by RFLP analysis. Parsimony-based analysis of the data produced groupings largely consistent with those identified using RFLPs. The major differences were grouping of the two subspecies ofLeucaena diversifolia (subsp.diversifolia and subsp.stenocarpa) in the RAPD tree, but their separation in the RFLP tree. The value of RAPD data in systematics as a result of these data and our understanding of the molecular basis of RAPDs are discussed.     

Comparative analysis of genetic relationships in barley based on RFLP and RAPD markers

Genetic relationships have seldom been analyzed with different types of molecular markers in order to compare the information provided by each marker class. We investigated genetic relationships among nine barley cultivars using separate cluster analyses based on restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs). Genomic DNA restricted with three enzymes and hybridized with 68 probes revealed 415 RFLPs (74.2% of all bands). Among the 128 primers used for RAPD analysis, 100 provided a reproducible profile, 89 of which revealed 202 polymorphic and 561 monomorphic bands (26.5 and 73.5%, respectively). A nonrandom distribution of 62 RAPDs with a tendency to cluster near centromeric regions was produced when these RAPDs were mapped using 76 doubled-haploid lines derived from a cross between two of the nine cultivars. The correlation between the RFLP and RAPD similarity matrices computed for the 36 pairwise comparisons among the nine cultivars was equal to 0.83. The dendrograms obtained by cluster analyses of the RFLP and RAPD data differed. These results indicate that in barley the information provided by RFLPs and RAPDs is not equivalent, most likely as a consequence of the fact that the two marker classes explore, at least in part, different portions of the genome.     

RFLP and RAPD mapping in flax (Linum usitatissimum)

A map of flax (Linum usitatissimum) using restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs), and comprising 15 linkage groups containing 94 markers, has been developed covering about 1000 cM. The mapping populations were the F₂ populations from two crosses between diverse cultivars. From one cross, CI1303 and Stormont Cirrus, 20 RFLP and 520 RAPD markers were analyzed. Thirteen RFLP and 80 RAPD markers were on the 15 linkage groups, in addition to one sequence-tagged site (STS). Seven polymorphic RAPD markers were found to have unusual segregation patterns. RAPDs were expressed as dominant markers, but for these markers a prevalence of the progeny lacked a band rather than the expected one-fourth ratio. However, these exceptions may be related to the instability of the genome of Stormont Cirrus in which stable and heritable genomic changes can be induced by environmental factors. The current map could be used for the identification of markers linked to loci controlling the ability to generate heritable changes in response to environmental growth conditions, and to develop anchor loci with STSs for a more general application. Received: 20 March 1999 / Accepted: 16 December 1999     

Characterization of 215 simple sequence repeat markers in creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is a versatile, cross-pollinated, temperate and perennial turfgrass species. It occurs naturally in a wide variety of habitats and is also cultivated on golf courses, bowling greens and tennis courts worldwide. Isozymes and amplified fragment length polymorphisms (AFLPs) have been used to determine genetic diversity, and restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPDs) were used to construct a genetic linkage map of this species. In the current report, we developed and characterized 215 unique genomic simple sequence repeat (SSR) markers in creeping bentgrass. The SSRs reported here are the first available markers in creeping bentgrass to date. Eight hundred and eighteen alleles were amplified by 215 SSR loci, an average of 3.72 alleles per locus. Fifty-nine per cent of those alleles segregated in a 1:1 Mendelian fashion (P > 0.05). Twenty-two per cent had a distorted segregation ratio (P ≤ 0.05). These SSR markers will be useful for assessing genetic diversity in creeping bentgrass and will be important for the development of genetic linkage maps and identifying quantitative trait loci. These markers could enhance breeding programmes by improving the efficiency of selection techniques.     

Targeted isolation of simple sequence repeat markers through the use of bacterial artificial chromosomes

 Simple sequence repeats (SSRs) are versatile DNA markers that are readily assayed and highly informative. Unfortunately, non-targeted approaches to SSR development often leave large genomic regions without SSR markers. In some cases these same genomic regions are already populated by other types of DNA markers, especially restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and amplified fragment length polymorphisms (AFLPs). To identify SSR markers in such regions, bacterial artificial chromosome (BAC) clones can be used as intermediaries. First, one or more BAC clones in a region of interest are identified through the use of an existing DNA marker. BAC clones uncovered in this initial step are then used to create a small insert DNA library that can be screened for the presence of SSR-containing clones. Because BAC inserts are often 100-kb pairs or more in size, most contain one or more SSRs. This strategy was applied to two regions of the soybean genome near genes that condition resistance to the soybean cyst nematode on molecular linkage groups G and A2. This targeted approach to identifying new DNA markers can readily be extended to other types of DNA markers, including single nucleotide polymorphisms. Received: 13 August 1998 / Accepted: 13 October 1998     

Development of a second generation linkage map for almond using RAPD and SSR markers.

Fifty-four RAPD (random amplified polymorphic DNA) markers and 6 SSRs (simple sequence repeats) were included in a molecular marker map with 120 RFLPs (restriction fragment length polymorphisms) and 7 isozyme genes previously constructed using the offspring of a cross between the almond (Prunus amygdalus) cultivars 'Ferragnès' and 'Tuono'. Only highly reproducible RAPDs segregating 1:1 were used. To identify these markers, a total of 325 primers were screened, from which 41 produced RAPDs useful for mapping. Polymorphism was detected in six of the eight Prunus SSRs (simple sequence repeats) studied, thus enabling these to be mapped. All markers were placed on the 8 linkage groups previously identified. The number of new markers included in the map of 'Ferragnès' was 33 for a total of 126, and 30 in the map of 'Tuono' for a total of 99. The sizes of the maps of 'Ferragnès' (415 cM) and 'Tuono' (416 cM) were similar, representing a 5% increase over the maps constructed solely with isozymes and RFLPs. The estimated total size of the almond map was of 457 cM. Some markers were placed in zones with low density of markers and others in the extreme of linkage groups. The use of RAPD markers to complete genetic maps constructed with transferable markers is discussed.     

Comparison of allozyme,RFLP, and RAPD markers for revealing genetic variation within and between trembling aspen and bigtooth aspen

We examined genetic variation in allozyme loci, nuclear DNA restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs) in 130 trembling aspen (Populus tremuloides) and 105 bigtooth aspen (P. grandidentata) trees. In trembling aspen 10 out of 13 allozyme loci assayed (77%) were polymorphic (P), with 2.8 alleles per locus (A) and an expected heterozygosity (H_e) of 0.25. In contrast, bigtooth aspen had a much lower allozyme genetic variability (P=29%; A=1.4; H_e=0.08). The two species could be distinguished by mutually exclusive alleles at Idh-1, and bigtooth aspen has what appears to be a duplicate 6PG locus not present in trembling aspen. We used 138 random aspen genomic probes to reveal RFLPs in HindIII digests of aspen DNA. The majority of the probes were from sequences of low copy number. RFLP results were consistent with those of the allozyme analyses, with trembling aspen displaying higher genetic variation than bigtooth aspen (P=71%, A=2.7, and H_e=0.25 for trembling aspen; P=65%, A=1.8, and H_e=0.13 for bigtooth aspen). The two species could be distinguished by RFLPs revealed by 21 probes (15% of total probes assayed). RAPD patterns in both species were studied using four arbitrary decamer primers that revealed a total of 61 different amplified DNA fragments in trembling aspen and 56 in bigtooth aspen. Assuming a Hardy-Weinberg equilibrium, estimates of P=100%, A=2, and H_e=0.30 in trembling aspen and P=88%, A=1.9, and H_e=0.31 in bigtooth aspen were obtained from the RAPD data. Five amplified DNA fragments were species diagnostic. All individuals within both species, except for 2 that likely belong to the same clone, could be distinguished by comparing their RAPD patterns. These results indicate that (1) RFLPs and allozymes reveal comparable patterns of genetic variation in the two species, (2) trembling aspen is more genetically variable than bigtooth aspen at both the allozyme and DNA levels, (3) one can generate more polymorphic and species-specific loci with DNA markers than with allozymes in aspen, and (4) RAPDs provide a very powerful tool for fingerprinting aspen individuals.     

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

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.Abbreviations RFLP restriction fragment length polymorphism - STS sequence-tagged site - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA     

Identification of microsatellite loci in apricot

The sequences of 21 primer pairs of microsatellite loci screened from a genomic library of apricot (Prunus armeniaca L.) are reported in this study. All the identified microsatellite loci were characterized in a set of 25 apricot cultivars and revealed to be polymorphic with 3–12 alleles per locus. These markers showed to be more informative than isozymes and restriction fragment length polymorphisms (RFLPs) reported in the literature.     

Character compatibility of molecular markers to distinguish asexual and sexual reproduction

Particularly in polyploids, the potential of the high variability of dominant markers such as random amplified polymorphic DNA fragments (RAPDs) and amplified fragment length polymorphisms (AFLPs) in population genetic studies and analysis of breeding systems is reduced due to their dominant nature. In contrast, the criterion of character compatibility is hindered neither by dominance nor by polyploidy as allelic interpretation is not necessary. Character compatibility, which can be used to detect events of genetic exchange (or recombination), is particularly informative if these events are expected to be rare such as in taxa with extensive vegetative reproduction or apomixis. Binary unordered characters such as presence and absence of anonymous DNA markers are incompatible if all four pairwise combinations of character states are present among the individuals studied. Because incompatible character state distributions defy any progenitor–derivative relationship among individuals, they provide strong evidence for genetic exchange. Both the absolute number of incompatible character combinations and the probability of compatibility can be used as a measure of incompatibility. Although these measures may not directly relate to the frequency of genetic exchange, they provide a useful tool to heuristically explore data sets. The most commonly used input for multivariate analyses and analysis of molecular variance in population genetic studies of (dis)similarity of marker distributions are amalgamates of mutation and recombination. Character compatibility can be used to complement these traditional methods of analysis. Advantages and disadvantages of character incompatibility relative to multilocus analysis of modes of reproduction and population genetics are demonstrated with data from RAPDs, isozymes, and restriction fragment length polymorphisms (RFLPs) of the nuclear ribosomal and chloroplast genome.     

DNA polymorphisms in grain sorghum (Sorghum bicolor (L.) Moench)

Molecular markers [random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)] were used to determine the frequency of DNA polymorphism in grain sorghum (Sorghum bicolor (L.) Moench). Twenty-nine oligonucleotide primers were employed for RAPDs, generating a total of 262 DNA fragments, of which 145 were polymorphic in at least one pairwise comparison between 36 genotypes. Individual primers differed significantly in their ability to detect genetic polymorphism in the species. The overall frequency of polymorphisms was low with a mean frequency of 0.117 polymorphisms per RAPD band being obtained from all pairwise comparisons between genotypes, with maximum and minimum values of 0.212 and 0.039, respectively. Results from phenetic analysis of bandsharing data were consistent with current sub-specific groupings of the species, with clusters of Durra, Zerazera, Caud-Nig, Caud-Kaura and Caffrorum being discernible. The results also indicated that individuals of a similar taxonomic grouping but different geographic origin may be genetically less identical than previously considered. Similar frequencies of polymorphism to that obtained with RAPDs were obtained with RFLPs. Results from these experiments indicated that a high level of genetic uniformity exists within S. bicolor.     

Genetic Mapping and Non-Mendelian Segregation of Mating Type Loci in the Oomycete, Phytophthora Infestans

DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in this normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses as RAPD markers, restriction fragment length polymorphisms (RFLPs), single-strand conformational polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balanced lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude from progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.     

Identification of chrysanthemum cultivars and stability of DNA fingerprint patterns

Several techniques of DNA analysis were applied to identify chrysanthemum cultivars. Unrelated cultivars could be distinguished by using RAPDs (random amplified polymorphic DNAs), inter-SSR (simple sequence repeat) PCR (polymerase chain reaction), hybridization-based DNA fingerprinting, as well as RFLPs (restriction fragment length polymorphisms). Cultivars with different flower colours and belonging to one family, i.e. vegetatively derived from 1 cultivar, appeared to have the same DNA fragment patterns, whichever technique was applied. The absence of polymorphisms between different accessions of the same cultivar indicated a high stability of the observed patterns.