Identification and classification of celery cultivars with RAPD markers

Summary Twenty-one celery (Apium graveolens L. var. dulce) cultivars, one celeriac (var. rapaceum) and one annual smallage (var. secalinum) cultivar were screened for polymorphic RAPD (Random Amplified Polymorphic DNA) markers with 28 arbitrary 10-mer primers. Among a total of 309 bands observed, 29 (9.3%) were polymorphic in the 23 cultivars screened, but only 19 (6.1%) markers were polymorphic within the 21 type dulce cultivars. These markers were sufficient to distinguish each of the cultivars used. The average marker difference was 6.4 between two celery cultivars, 16.7 between celery and annual smallage, 14.7 between celery and celeriac, and 12.0 between annual smallage and celeriac. The celery cultivars surveyed were classified into three groups based on the marker differences. The relationship among the dulce-type cultivars concluded from this research is basically consistent with the known lineage of the cultivars and the previous study using stem protein and isozyme markers. RAPD technology provides a new alternative for cultivar identification and classification in celery.     

Pre-germination genotypic screening using PCR amplification of half-seeds

A simple and rapid PCR-based method has been developed for determining the genotype of seeds before germination. Single half-seeds of rice (Oryza sativa L.) and wheat (Triticum aestivum L. em. Thell.) were preincubated, without grinding, in an aqueous extraction buffer. The resulting supernatants were then used in polymerase chain reaction (PCR) with oligonucleotide primers corresponding to rice single-copy sequences or a wheat microsatellite repeat. PCR products of identical size were amplified using either the half-seed extract or DNA isolated from leaf tissue. The remnant half-seeds can be maintained in ordered arrays using microtiter plates allowing the recovery of selected genotypes. Pre-germination genotypic screening of seed populations as described in this report should be useful for a variety of applications in plant breeding and genetics studies.     

Potential taxonomic use of random amplified polymorphic DNA (RAPD): a case study in Brassica

Summary The potential use of RAPDs for taxonomic studies were investigated using Brassica, Sinapis and Raphanus taxa. Principal coordinate analysis of 284 RAPD bands revealed the classical U triangle relationship between diploid and amphidiploid Brassica taxa. Raphanus sativus and S. alba were distinct from the Brassica taxa. It appears that at least ten primers with approximately 100 total bands are needed to adequately portray these relationships. Cultivars of cabbage and cauliflower were separated by RAPDs. Analysis of RAPDs from individual plants of B. carinata cv. dodola resulted in 69 RAPDs, with 91.7% monomorphic and 8.3% polymorphic bands. RAPDs appear to be useful for taxonomic studies at levels ranging from populations to species and perhaps genera.     

Rapid mapping of two genes for resistance to downy mildew from Lactuca serriola to existing clusters of resistance genes

Two resistances to downy mildew derived from Lactuca serriola were characterized genetically and mapped using molecular markers. Classical genetic analysis suggested monogenic inheritance; however, the presence of multiple, tightly-linked genes in each case could not be eliminated. Therefore, they were designated resistance factors R17 and R18. Analysis with molecular markers known to be linked to clusters of resistance genes quickly revealed linkage of R18 to the major cluster of resistance genes and provided six linked markers, three RAPD (Random Amplified Polymorphic DNA) markers and three codominant SCAR (Sequence Characterized Amplified Region) markers. The mapping of R17 required the screening of arbitrary RAPD markers using bulked segregant analysis; this provided five linked markers, three of which segregated in the basic mapping population. This demonstrated loose linkage to a second cluster of resistance genes and provided additional linked markers. Two RAPD markers linked to R17 were converted into SCARs. The identification of reliable PCR-based markers flanking each gene will aid in selection and in combining these resistance genes with others.     

Assessment of DNA pooling strategies for mapping of QTLs

The synthesis of DNA pools from segregating populations is an efficient strategy for identifying DNA markers closely linked to genes or genomic regions of interest. To-date, DNA pooling based solely upon phenotypic information, or bulked segregant analysis, has been employed only in the analysis of simply-inherited traits. We have assessed the utility of phenotype-based DNA pools for tagging (e.g., identifying DNA markers closely-linked to) quantitative trait loci (QTLs), segregating in the presence of other such loci, and expressing phenotypes which are influenced by the environment. Theoretical estimates suggest that QTL alleles with phenotypic effects of 0.75–1.0 standard deviations (SD), or larger, should be detectable in back-cross (BC), F₂ and recombinant inbred (RI) or doubled haploid (DH) populations of manageable size (100–200 plants/lines). However, post hoc analysis of three data sets, used in QTL mapping of tomato and rice, indicate that the majority of QTLs identified had allele effects of less than 0.75 SD, and thus could not be easily tagged in DNA pools. Segregation distortion can have a large effect on the allelic composition of DNA pools, necessitating the use of more individuals in the pools to minimize false positive and false negative results. In general, we suggest that use of phenotype-based DNA pools might be successful in tagging QTLs of very large effect, but is unlikely to permit comprehensive identification of the majority of QTLs affecting a complex trait. DNA pools constructed from a priori information should, however, be useful in identifying new DNA markers for regions of the genome known to contain QTLs.     

RAPDs identify varietal misclassification and regional divergence in cranberry [Vaccinium macrocarpon (Ait.) Pursh]

The majority of cultivated cranberry varieties were selected from native populations in the 1800s and early 1900s from sites in Massachusetts, New Jersey, and Wisconsin. Since their initial selections 100–150 years ago, varietal identities have become increasingly confused; primarily the result of there being a paucity of qualitative markers to effectively distinguish among varieties. Random amplified polymorphic DNA (RAPD) technology has the potential for allowing a more definitive classification of varieties and was used in this study to characterize 22 cranberry varieties. Twenty-two decamer primers amplified 162 scorable DNA fragments, of which 66 (41%) were polymorphic. On the basis of these 66 silver-stained RAPDs (ssRAPDs), 17 unique profiles were identified rather than the expected 22. Fourteen varieties had unique ssRAPD profiles, while the remaining 8 were represented by 3 ssRAPD profiles. Permuational analyses of the data suggest that the observed ssRAPD profile duplications are examples of varietal misclassification. Further analyses identified 2 ssRAPD markers that were found only in Eastern varieties (from Mass. and N. J.) and not in Wisconsin varieties. With varieties differing on average by 22 bands, ssRAPDs are shown to be effective in varietal identification and the assessment of genetic diversity in cranberry.New Jersey Agricultural Experiment Station, Publ No. D-12163-8-93, Supported by State funds.     

DNA fingerprinting of Peronospora parasitica,a biotrophic fungal pathogen of crucifers

The fungus Peronospora parasitica (Pers. ex Fr.) Fr. is an obligate biotroph infecting a wide range of host species in the family Cruciferae. Isolates from different hosts are morphologically similar, and pathotypes are usually distinguished on the basis of host range. Random Amplified Polymorphic DNA (RAPD) fingerprints were generated from a range of P. parasitica isolates from different Brassica species. Reaction conditions, in particular DNA template, primer and Mg²⁺ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by Southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus.     

Consistency of population genetics parameters estimated from isozyme and RAPDs dataset in species of genus <Emphasis Type="Italic">Prosopis</Emphasis> (Leguminosae,Mimosoideae)

Genetic variability, population structure and differentiation among 17 populations of 5 species and 2 natural interspecific hybrids of section Algarobia of genus Prosopis were analyzed from data of 23 isozyme and 28 RAPD loci. Both markers indicated that the studied populations are highly variable. P. alba populations in average showed lower values of genetic variability estimates from isozyme data, but this trend was not observed for RAPD markers. The hierarchical analyses of the distribution of genetic variability showed that the highest proportion of variation occurred within populations, the differentiation among species was intermediate and the lowest component was observed among populations within species. The consistency between results from both dataset implies that they are not biased and reflect the actual genetic structure of the populations analyzed. The matrices of Euclidean distances obtained from the two sets of markers were highly correlated according to Mantel test. In both cases the corresponding phenogram and MDS plot tended to cluster conspecific populations while hybrid populations were not intermediate between putative parents. Some disagreements between isozyme and RAPD phenograms were observed mainly in the affinities of hybrid populations. Such inconsistencies might result from reticular rather than dichotomic evolutionary relationships. The phenetic associations retrieved gave no support to the division of the section Algarobia into series.     

Spread of violets in polluted pine forests: morphological and molecular evidence for the ecological importance of interspecific hybridization

Hybridization between plant species occurs frequently but hybrids are often restricted to ecotones or disturbed habitats. In this study we show that introgressive hybrids between the tetraploid Viola riviniana and the diploid V. reichenbachiana invaded pine forests of the Dübener Heide (central Germany), an area affected by calcareous pollutants. The spread of these violet populations was correlated with the impact of pollution on habitat conditions. We compared morphology, cytology and random amplified polymorphic DNA (RAPD) bands among six Viola populations from the Dübener Heide and three populations of each pure species. RAPD analysis using 12 primers revealed 141 scorable bands. We considered bands as species specific if they occurred in at least 75% of the plants in one pure violet species but in none of the other. Seven bands were specific to V. riviniana and 11 bands were specific to V. reichenbachiana . Two plants of a V. reichenbachiana population were identified as hybrids. Of the Viola populations from the Dübener Heide, one was diploid and could be classified as V. reichenbachiana by morphology and RAPD markers. However, the majority of the Dübener Heide populations were tetraploid, and of a more variable morphology than V. riviniana and V. reichenbachiana , showing different combinations of intermediate characters, characters of the pure species and extreme characters. Despite their overall genetic similarity to V. riviniana , these plants could be identified as introgressive hybrids between V. riviniana and V. reichenbachiana by species-specific RAPD bands. Therefore, we propose that recurrent hybridization and backcrossing resulted in novel genotypes adapted to the changed environment of polluted pine forests.