Genetic diversity in watermelon (Citrullus lanatus) landraces from Zimbabwe revealed by RAPD and SSR markers.

Low polymorphism in cultivated watermelon has been reported in previous studies, based mainly on US Plant Introductions and watermelon cultivars, most of which were linked to breeding programmes associated with disease resistance. Since germplasm sampled in a putative centre of origin in southern Africa may harbour considerably higher variability, DNA marker-based diversity was estimated among 81 seedlings from eight accessions of watermelon collected in Zimbabwe; five accessions of cow-melons (Citrullus lanatus var. citroides) and three of sweet watermelons (C. lanatus var. lanatus). Two molecular marker methods were used, random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) also known as microsatellite DNA. Ten RAPD primers produced 138 markers of which 122 were polymorphic. Nine SSR primer pairs detected a total of 43 alleles with an average of 4.8 alleles per locus. The polymorphic information content (PIC) ranged from 0.47 to 0.77 for the RAPD primers and from 0.39 to 0.97 for the SSR loci. Similarity matrices obtained with SSR and RAPD, respectively, were highly correlated but only RAPD was able to provide each sample with an individual-specific DNA profile. Dendrograms and multidimensional scaling (MDS) produced two major clusters; one with the five cow-melon accessions and the other with the three sweet watermelon accessions. One of the most variable cow-melon accessions took an intermediate position in the MDS analysis, indicating the occurrence of gene flow between the two subspecies. Analysis of molecular variation (AMOVA) attributed most of the variability to within-accessions, and contrary to previous reports, sweet watermelon accessions apparently contain diversity of the same magnitude as the cow-melons.

Frequent silencing of rDNA loci on the univalent-forming genomes contrasts with their stable expression on the bivalent-forming genomes in polyploid dogroses (Rosa sect. Caninae).

The polyploid species in Rosa section Caninae (2n=21, 28 or 35) are characterized by an unusual reproductive system known as odd (or asymmetric) meiosis. Only two chromosome sets form bivalents in meiosis, whereas the remaining chromosomes are transmitted as univalents through the female germline. Evolution of ribosomal rRNA genes (rDNA) does not seem to be significantly affected by interlocus homogenization in dogroses. As a consequence, most species contain several rDNA families falling into two main clades (beta and gamma) thought to be differentially distributed between bivalent and univalent chromosomes, respectively. Here, we have investigated expression of rRNA gene families in five pentaploid species (R. canina, R. rubiginosa, R. dumalis, R. sherardii and R. caesia, 2n=35) and in one tetraploid (R. mollis, 2n=28). Using extensive sequencing of ITS clones and cleaved amplified polymorphism sequence (CAPS) analysis, we found that the beta-family was constitutively expressed in all species. However, there was large variation in the expression patterns of families constituting the gamma-clade. In addition, a single family can be active in one species, whereas silenced in another. The data show that the families on bivalent-forming chromosomes dominate rDNA expression in all dogrose species. We hypothesize that genes on bivalent genomes are stably expressed, whereas those on univalent genomes undergo variable levels of epigenetic silencing. Nonetheless, mosaic expression of univalent genomes could contribute to phenotypic variation between the species.

Molecular characterisation of indigenous Swedish apple cultivars based on SSR and S-allele analysis.

Trees of 68 apple cultivars, aimed for preservation by the 'National Program for diversity of cultivated plants' as mandate cultivars, were analysed using a set of 10 SSR (simple sequence repeat) primer pairs and the self-incompatibility (S-)locus to evaluate genetic diversity and reveal inter-cultivar relationships. The 12 polymorphic SSR loci exhibited 2 to 15 alleles, with expected heterozygozity (H(e)) ranging from 0.36 to 0.88 and a mean of 0.74. Numerous alleles were classified as rare or unique (35% and 18% respectively). For the S-locus, a total of 14 alleles were identified in this study. Five alleles, S1-S3, S5 and S7 had frequencies ranging from 11 to 18%, whereas the remaining 9 alleles were below 6%. All sexually obtained cultivars could be distinguished with the set of SSR loci. Sports were identical with their progenitors in two cases, but differed in one SSR allele in a third case. An SSR-based dendrogram, based on Roger's genetic distances, did not reveal any clear pattern of clustering. The genetic distances were, however, correlated with a corresponding matrix obtained in a previously conducted RAPD-based study of the same cultivars. Non-mandate parents of Swedish mandate cultivars together with some other reference cultivars were included in this study to check the accuracy of allele scoring, verify parentage and compare the results of this study with those presented in previously published studies. Some discrepancies in allele sizing were revealed and the possibilities of avoiding this problem are discussed.

The asymmetric meiosis in pentaploid dogroses (Rosa sect. Caninae) is associated with a skewed distribution of rRNA gene families in the gametes.

In pentaploid dogroses, Rosa section Caninae (2n=5x=35), the pollen transmits one basic genome (x=7) derived from the seven segregating bivalents, whereas the egg transmits four basic genomes (4x=28) one set derived from the segregation of seven bivalents and three sets of univalent-forming chromosomes. Chromosomes from all five genomes carry 18-5.8-26S nuclear ribosomal DNA (rDNA) sites. This mode of sexual reproduction, known as permanent odd polyploidy, can potentially lead to the independent evolution of rDNA on bivalent- and univalent-forming chromosomes. To test this hypothesis, we analyzed rRNA gene families in pollen and somatic leaf tissue of R. canina, R. rubiginosa and R. dumalis. Six major rRNA gene families (alpha, beta, beta' gamma, delta and epsilon) were identified based on several highly polymorphic sites in the internal transcribed spacers (ITSs). At least two of the major rRNA gene families were found in each species indicating that rDNAs have not been homogenized across subgenomes. A comparison of ITS1 sequences from leaf and pollen showed differences: the shared beta rRNA gene family was more abundant among pollen clones compared to leaf clones and must constitute a major part of the rDNA loci on bivalent-forming chromosomes. The gamma and delta families were underrepresented in pollen genomes and are probably located predominantly (or solely) on the univalents. The results support the hypothesis that pentaploid dogroses inherited a bivalent-forming genome from a common proto-canina ancestor, a likely donor of the beta rDNA family. Allopolyploidy with distantly related species is likely to have driven evolution of Rosa section Caninae.

Unique genomic configuration revealed by microsatellite DNA in polyploid dogroses, Rosa sect. Caninae.

An allopolyploid complex with high genomic integrity has been studied. Dogroses transmit only seven chromosomes (from seven bivalents) through the pollen, whereas 21, 28 or 35 chromosomes (from seven bivalents and 14, 21 or 28 univalents) come from the egg cells. Seedlings derived from two interspecific crosses were analysed with flow cytometry and molecular markers to determine ploidy level, mode of reproduction and genomic constitution. Evidence was obtained for the formation of unreduced male and female gametes, which can take part in fertilization (producing seedlings with higher ploidy than the parental plants) or in apomictic reproduction. Random amplified polymorphic DNA (RAPD) and microsatellite analyses indicated that three seedlings (5%) were derived through apomixis, whereas the other 49 were hybrids. Bivalent formation appears to involve chromosomes that consistently share the same microsatellite alleles. Allele-sharing between the maternally transmitted and highly conserved univalent-forming chromosomes reflected the taxonomic distance between different genotypes. The frequently recombining bivalent-forming chromosomes were taxonomically less informative.

Assignment of allelic configuration in polyploids using the MAC-PR (microsatellite DNA allele counting-peak ratios) method.

Polysomic inheritance frequently results in the simultaneous occurrence of several microsatellite DNA alleles on a single locus. The MAC-PR (microsatellite DNA allele counting-peak ratios) method was recently developed for the analysis of polyploid plants and makes use of the quantitative values for microsatellite allele peak areas. To date, this approach has only been used in plants with known genetic relationships. We report here the application of MAC-PR for the first time to random samples of unknown pedigrees. We analysed six microsatellite loci using a set of tetraploid ornamental rose ( Rosa x hybrida L.) varieties. For each locus, all alleles were analysed in pairwise combinations in order to determine their copy number in the individual samples. This was accomplished by calculating the ratios between the peak areas for two alleles in all of the samples where these two alleles occurred together. The allele peak ratios observed were plotted in a histogram, and those histograms that produced at least two well-separated groups were selected for further analysis. Mean allelic peak ratio values for these groups were compared to the relationships expected between alleles in hypothetical configurations of the locus investigated. Using this approach, we were able to assign precise allelic configurations (the actual genotype) to almost all of the varieties analysed for five of the six loci investigated. MAC-PR also appears to be a very effective tool for detecting 'null' alleles in polyploid species.

Microsatellite DNA marker inheritance indicates preferential pairing between two highly homologous genomes in polyploid and hemisexual dog-roses, Rosa L. Sect. Caninae DC.

According to previous cytological evidence, the hemisexual dog-rose species, Rosa sect. Caninae, transmit only seven chromosomes (derived from seven bivalents) through their pollen grains, whereas egg cells contain 21, 28 or 35 chromosomes (derived from seven bivalents and 14, 21 or 28 univalents) depending on ploidy level. Two sets of reciprocal pairwise interspecific crosses involving the pentaploid species pair R. dumalis and R. rubiginosa, and the pentaploid/tetraploid species pair R. sherardii and R. villosa, were analysed for 13 and 12 microsatellite DNA loci, respectively. Single loci were represented by a maximum of three simultaneously occurring alleles in R. villosa, and four alleles in the other three parental plants. In the experimentally derived offspring, the theoretical maximum of five alleles was found for only one locus in the pentaploid progenies. Microsatellite DNA allele composition was identical with that of the maternal parent in 10 offspring plants, which were probably derived through apomixis. Almost all microsatellite DNA alleles were shared with the maternal parent also in the remaining offspring, but 1-4 alleles shared only with the paternal parent, indicating sexual seed formation. Analysis of quantitative peak differences allowed a tentative estimation of allelic configuration in the individual plants, and suggested that bivalent formation preferentially takes place between chromosomes that consistently share the same microsatellite alleles and therefore appear to be highly homologous. Moreover, alleles that were shared between the species in each cross combination comparatively often appear to reside on the bivalent-forming chromosomes, whereas species-specific alleles instead occur comparatively often on the univalent-forming chromosomes and are therefore inherited through the maternal parent only. Recombination then takes place between very similar genomes also in interspecific crosses, resulting in a reproduction system that is essentially a mixture between apomixis and selfing.

Skewed distribution of morphological character scores and molecular markers in three interspecific crosses in Rosa section Caninae.

The dogroses, Rosa section Caninae, are all polyploid and characterised by their unbalanced meiosis; the pollen parent contributes one genome, whereas the seed parent contributes 3-5 genomes depending upon ploidy level of the species. As a result, genetically determined traits are expected to be matroclinally inherited. In the present study, the transmittal of genetic material was studied using manually scored reproductive characters (ovary and sepals), automated image analysis of leaflet shape (Fourier coefficients), and molecular markers (RAPD). The plant material consisted of a pair of reciprocal crosses between R. sherardii and R. villosa, a R. rubiginosa x R. sherardii cross and offspring obtained from selfing or within-population crosses of the parental species. All but one of the maternal markers were transmitted to all the offspring plants, whereas only 41% of the paternal markers were transmitted to all of them, 23% were never transmitted and 36% reached only one or two of the offspring plants. Canonical variates analyses (CVA) based on the vegetative characters could easily separate the offspring groups representing parental species and also all three hybridogenous offspring groups from each other, whereas CVA based on reproductive characters failed to separate R. sherardii x R. villosa from its seed parent, but otherwise distinguished all offspring groups. The study shows that the expression of characters as well as molecular marker inheritance is dependent upon the direction of the cross, and on the species involved. It also demonstrates the importance of employing several different types of character sets for an improved comprehension of the effects of the peculiar canina meiosis.

Combined analyses of RAPDs, cpDNA and morphology demonstrate spontaneous hybridization in the plant genus Chaenomeles.

Evidence of spontaneous hybridization between two partially sympatric species of Chaenomeles, C. cathayensis and C. speciosa, has been obtained through analysis of offspring families from these two species, as well as from two presumed interspecific hybrid populations. A combination of different methods was applied. Analysis of diagnostic RAPD markers and of chloroplast DNA haplotypes supported the notion of spontaneous hybridization, and suggested that there has been symmetrical, rather than unidirectional, introgression between C. cathayensis and C. speciosa. RAPDs and morphological characters revealed concordant patterns of genetic relatedness among the studied offspring families. Some putative hybrid families had mainly intermediate characters, whereas others appeared to be later generation hybrids as they were genetically and phenotypically rather similar to families that appeared to represent pure species. The RAPD-based proportion of between-family variability was considerably higher in the putatively hybridogenous populations than in populations of the pure species. Within-family gene diversity estimates ranged from C. speciosa (max. Hj = 0.235) to C. cathayensis (min. Hj = 0.094) with the presumed hybrid families taking intermediate values.

Application of DNA fingerprinting to the taxonomy of European blackberry species.

Thousands of blackberry species (Rubus subgenus Rubus) have been described from Central and Northern Europe. These species are usually polyploid and pseudogamous. Since apomixis is only facultative, the species have retained the possibility to produce some offspring by sexual recombination. Hybridization of restriction-enzyme-digested DNA samples to the M13, (AC)/(TG) and 33.15 probes has yield valuable information on the occurrence and distribution of genetic variation in Rubus. Intraspecific variation proved to be very restricted, with identical DNA fingerprints sometimes being exhibited by Swedish, Danish and German populations of the same species. By comparison, interspecific variation was usually pronounced, suggesting that most species have been derived through genetic recombination. Speciation through interspecific hybridization was demonstrated in one case. Several critical taxa were investigated; some of these proved to have unique DNA fingerprints, whereas others appear to be identical with previously described species. Evaluation of raspberry cultivars, obtained through several generations of vegetative propagation or with various in vitro techniques, failed to detect any DNA fingerprint variation, suggesting that the fingerprints are somatically stable. Blackberry seedlings, obtained by pollinating various species as well as interspecific hybrids with pollen from yet other species, were successfully evaluated with DNA fingerprinting to determine the relative proportions of the apomictic and sexual seed set.

Genetic variation detected by use of the M13 "DNA fingerprint" probe in Malus, Prunus, and Rubus (Rosaceae).

Recently, "DNA fingerprints" have been reported in a wide array of organisms. We used the M13 repeat probe on several genera and species in the angiosperm family Rosaceae. Four apple cultivars could be differentiated when any one of five restriction enzymes was used to analyze minisatellite DNA. Similarly, four individual trees of Prunus serotina (black cherry) exhibited different "fingerprints" with each of four enyzmes. A total of 14 Rubus (blackberries and raspberries) plants representing four species were investigated with two enzymes. Extensive inter-and intraspecific variation was found. However, some closely growing plants had identical "fingerprints", probably due to their being derived through vegetative propagation.

DNA "fingerprints" applied to paternity analysis in apples (Malus x domestica).

Analysis of minisatellite DNA sequences, yielding so called DNA "fingerprints", has proven useful in paternity analysis for several different organisms. Here 64 apple seedlings, grown from seeds collected in an orchard with three cultivars, were analyzed using the M13 "fingerprint" probe. Paternity could be determined for 56 of the seedlings, 2 of which were derived through selfing. The analysis was facilitated by the occurrence of a multiallelic locus. The five different fragments determined by this locus migrated to similar positions, whether digesting the DNA with restriction enzymes TaqI or RsaI.

Pollen-limited seed set in pseudogamous blackberries (Rubus L. subgen. Rubus).

Pseudogamous blackberry species are polyploid and usually exhibit meiotic irregularities causing severe reduction in pollen viability. When species means were compared, relative seed set was strongly correlated with the number of good pollen grains produced per flower divided by the number of ovules (good-pollen/ovule ratio).Partial correlation analysis for percentage viable pollen and relative seed set, controlling for good-pollen/ovule ratio, revealed an almost significant relationship, presumably because meiotic irregularities have a similar effect on both pollen viability and the viability of meiotically derived embryo sacs. Seed germination, on the other hand, showed no relationship with relative seed set or with pollen viability.The automatic selfing capacity exhibited by most of these blackberry species does not appear to be related to either pollen production or relative seed set.