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.

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.

Evolutionary implications of permanent odd polyploidy in the stable sexual, pentaploid of Rosa canina L.

In Rosa canina (2n = 5x = 35), the pollen and ovular parents contribute, respectively, seven and 28 chromosomes to the zygote. At meiosis I, 14 chromosomes form seven bivalents and 21 chromosomes remain as univalents. Fluorescent in situ hybridization to mitotic and pollen mother cells (PMC) of R. canina showed that 10 chromosomes (two per genome) carry ribosomal DNA (rDNA) loci. Five chromosomes carry terminal 18S-5.8S-26S rDNA loci; three of these also carry paracentric 5S rDNA loci and were designated as marker chromosomes 1. Five chromosomes carry only 5S rDNA loci and three of these were designated as marker chromosomes 2. The remaining four of the 10 chromosomes with rDNA loci were individually identifiable by the type and relative sizes of their rDNA loci and were numbered separately. At PMC meiosis, two marker chromosomes 1 and two marker chromosomes 2 formed bivalents, whereas the others were unpaired. In a gynogenetic haploid of R. canina (n = 4x = 28), obtained after pollination with gamma-irradiated pollen, chromosomes at meiosis I in PMC remained predominantly unpaired. The data indicate only one pair of truly homologous genomes in R. canina. The 21 unpaired chromosomes probably remain as univalents through multiple generations and do not recombine. The long-term evolutionary consequence for the univalents is likely to be genetic degradation through accumulated mutational change as in the mammalian Y chromosome and chromosomes of asexual species. But there is no indication that univalents carry degenerate 5S rDNA families. This may point to a recent evolution of the R. canina meiotic system.

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.