Chromosome numbers of European blackberries (Rubus subg.Rubus,Rosaceae)

Chromosome numbers are presented for 32 collections of 29 European blackberry species (Rubus subg.Rubus) from Germany. One species is triploid (2n = 21), 27 species are tetraploid, (2n = 28), and one species is pentaploid (2n = 35). Chromosome numbers are reported for the first time ofR. adspersus, R. amisiensis, R. calvus, R. conothyrsoides, R. contractipes, R. demissus, R. elegantispinosus, R. ferocior, R. foliosus, R. hypomalacus, R. leucandrus, R. nemorosus, R. platyacanthus, R. praecox, R. rhombifolius, andR. rhytidophyllus. Chromosome numbers forR. dasyphyllus, R. gelertii, R. glandithyrsos, R. lamprocaulos, R. lindebergii, R. macrophyllus, R. montanus, R. muenteri, R. pedemontanus, R. polyanthemus, R. senticosus, R. silvaticus, andR. vigorosus are confirmed.     

Establishment and characterization of Rubus tissue culture systems for in vitro bioassays against phytotoxins from Rubus fungal pathogens

The Rubus species R. parviflorus, R. spectabilis and R. strigosus interfere with conifer seedling establishment on forest regeneration sites in Canada and the United States. As a first step towards microbial metabolite-based control, callus and cell suspension cultures of the Rubus species were developed as a bioassay system to detect phytotoxic compounds that may have relevance in a vegetation control context. Rapidly growing friable callus and suspension cultures were obtained from leaf disks of the three weedy Rubus species using similar culture media conditions (modified Murashige and Skoog) but required different plant growth regulators (R. parviflorus, 4.5 M 2,4-D; R. spectabilis, 26.9 M NAA/0.5 M zeatin; and R. strigosus, 12.4 M picloram). Cell growth and health attributes including callus circumference, degree of browning and suspension culture cell viability as measured by the TTC vital stain assay were developed and were rapid and convenient to use. We have established Rubus tissue culture systems that will make it possible for large scale screening of phytotoxic metabolites.     

Characterisation of Rubus niveus: a prerequisite to its biological control in oceanic islands

This study is intended to inform the search for a biocontrol solution to stem the spread of Rubus niveus, a morphologically-diverse and invasive Rubus species which has become an aggressive invader in areas of the world outside its native range. The fragile biodiversity of islands can be especially threatened by this plant – exemplars of this are the Galápagos archipelago and Hawaii. This report focuses on the morphological and molecular work carried out to establish the homogeneity or intra-specific variation of R. niveus populations within the Galápagos Islands, as well as their kinship with plants of the same name found in Asia. The premise of this study is that genetic closeness between these populations offers increased probability that pathogens of R. niveus in its native range will also provide effective control in the target environment. Increased knowledge of the genetic make-up of R. niveus in both its native and naturalised locations will also help to assess the feasibility of using organisms which have been found to be effective against other Rubus species, but not hitherto considered for R. niveus itself.     

Unusual filamentous virus-like particles in symptomless blackberry associated with severe leaf curling in the virus indicator Rubus macraei, and anomalous data using a degenerate badnavirus primer in PCR of Rubus species

Electron microscopy of ultrathin sections of leaves of symptomless Himalaya Giant blackberry and of the virus indicator species, Rubus macraei, showing severe leaf curl symptoms following graft inoculation with scions from this blackberry, detected highly flexuous virus‐like particles with an unusual ‘beaded’ structure. Such particles were restricted to a few vascular cells and were distinct from P‐protein common in some such cells. This virus, provisionally named Hawaiian rubus leaf curl virus (HRLCV), symptomlessly infected a wide range of Rubus species and cultivars. Badnavirus‐like bacilliform particles were observed in some cells of a single R. macraei plant showing leaf curl symptoms following graft inoculation with the causal agent of this disease symptom from Himalaya Giant blackberry after passage through red raspberry, but not in any other material. PCR with primer sets for the badnaviruses Rubus yellow net virus and Gooseberry veinbanding associated virus, showed that no Rubus sources studied contained these viruses. However, using a sequence‐specific primer set designed from the sequence of the product generated with a badnavirus degenerate primer set, a specific product was amplified from healthy plants of all of 16 raspberry cultivars and two Rubus species, but not from 16 blackberry cultivars (including cv. Himalaya Giant). All of these sources were free from viruses known to occur in Rubus. Sequence analysis of this product showed no homology with any known badnavirus, or with any other published sequences. It seems most likely therefore that a region of the raspberry genome has been amplified using the degenerate badnavirus primer set and that it is absent from the blackberry genome.     

Melting pot of biodiversity: first insights into the evolutionary patterns of the Colchic bramble flora (Rubus subgenus Rubus,Rosaceae)

The Caucasus is a biodiversity hotspot of global significance, containing a number of highly diverse and species‐rich plant taxa. The region is also thought to be an important evolutionary hotspot for Rubus subgenus Rubus (brambles). However, Caucasian brambles have only been poorly studied to date and our knowledge of their evolutionary mechanisms, systematics and taxonomic variability remains rudimentary. Therefore, the objectives of this study were to shed light on the evolution, diversity and reproduction modes of Rubus in one of the two Caucasian glacial refugia, Colchis. Flow cytometry measurements were used to estimate DNA ploidy, a flow cytometric seed screen was conducted to determine reproduction mode and Sanger sequencing of two non‐coding plastid regions was used to reveal phylogenetic patterns. The most common ploidy level was tetraploid, followed by diploid and (rarely) triploid. Intra‐individual variation in reproduction mode was low, as the morphoseries Glandulosi and Radula exhibited strict sexuality and other taxa were mostly apomictic. A few exceptions were observed that deserve further attention, e.g. sexuality induced hypothetically by haploid pollen or by environmental conditions, a high proportion of triploid embryos or polyspermy. Plastid haplotype variability revealed specific, ancient evolutionary patterns with limited involvement of extant diploid taxa and recent isolation from European brambles. We provide the first insight into the variability and evolution of Colchic brambles, which serves as a starting point for further systematic and evolutionary studies.     

Foliar indumentum in central‐European Rubus species (Rosaceae) and its contribution to the systematics of the group

The foliar indumentum of 94 taxa of Rubus has been surveyed by scanning electron microscopy. The analysed species belonged to five subgenera. Rubus subgen. Rubus with its three sections and 21 series was the most numerously represented. In all studied species, indumentum was present, but the species differed more or less from each other with respect to the density of the indumentum on the abaxial leaf surface. Three main hair types were present: simple eglandular (unbranched) trichomes, branched eglandular trichomes, and very short secretory uni‐seriate trichomes; subtypes were distinguished according to trichome size. Apart from investigating separate traits, we described character patterns for the best represented and homogeneous series: Discolores, Subthyrsoidei, Rhamnifolii and Rubus. Other large groups (Sylvatici, Glandulosi and Micantes) were more diverse.     

Spontaneous hybrids between native and exotic Rubus in the Western United States produce offspring both by apomixis and by sexual recombination

Facultative asexual reproduction is a trait commonly found in invasive species. With a combination of sexual and asexual reproductive modes, such species may adapt to new environments via sexual recombination during range expansion, while at the same time having the benefits of asexuality such as the maintenance of fitness effects that depend upon heterozygosity. In the Western United States, native species of Rubus (Rosaceae) reproduce sexually whereas exotic naturalized Rubus species reproduce by pseudogamous apomixis. We hypothesized that new asexual lineages of Rubus could arise from hybridization in this range. To detect hybridization between native and exotic Rubus, we genotyped 579 individuals collected across California, Oregon and Washington with eight nuclear microsatellites and two chloroplast markers. Principal Coordinate Analysis and Bayesian clustering revealed a limited amount of hybridization of the native R. ursinus with the exotic R. armeniacus and R. pensilvanicus, as well as cultivated varieties. Genetic distances between these hybrids and their offspring indicated that both R. ursinus × R. armeniacus and R. ursinus × R. pensilvanicus produced a mix of apomictic and sexual seeds, with sexual seeds being more viable. Although neither of these hybrid types is currently considered invasive, they model the early stages of evolution of new invasive lineages, given the potential for fixed heterosis and the generation of novel genotypes. The hybrids also retain the ability to increase their fitness via sexual recombination and natural selection. Mixed reproductive systems such as those described here may be an important step in the evolution of asexual invasive species.     

Resistance to Botrytis cinerea in canes of Rubus idaeus and some related species

Mycelial inoculation of canes with Botrytis cinerea proved a useful method for assessing Rubus material for resistance. When canes were inoculated in summer resistant genotypes developed relatively small lesions which produced few small sclerotia in the following spring. The size of lesions in autumn generally provided the best discrimination between genotypes. Very strong resistance was found in several species, of which Rubus pileatus and R. occidentalis are the most useful for breeding; hybrids of raspberry with these species or with R. crataegifolius also had strong resistance. The resistance shown by a derivative of red raspberry cv. Chief was less strong, as was the resistance conferred by gene H, which determines cane pubescence.     

Pollen Morphology and Wall Stratification in Rubus L. (Rosaceae) in the Iberian Peninsula

A palynological study using LM and SEM was made of the morphology, ornamentation, apertural system and structure of the pollen wall, of the 18 species of the genus Rubus which are present in the Iberian Peninsula. We demonstrate: a) the stenopalynous character of the genus (isopolar, subprolate, 3-lobate, 3-zonocolporate and striate-perforate, except for R. genevierii whose ornamentation is striate-gemmate-perforate and with a striated margin); b) the lalongate shape of the endoaperture, due to the disappearance or thinning of the nexine; and c) the existence of two structural types, Type I granular and Type II columellar with clearly-defined columellar spaces or compact without intercolumellar spaces.     

Rubus canduliger, a new regional species from the Netherlands, with notes on the range structure and dynamics of brambles (Rubus, Rosaceae)

Rubus canduliger is described as a new regional bramble species belonging to the seriesDiscolores. It has been recognized in the Netherlands as a distinct species for more than 70 years but identified as the central EuropeanR. grabowskii. The latter, however, differs by its tapering prickles, regular leaves with longer petiolule of the terminal leaflet, broader, often pyramidal inflorescence, and white or pink flowers with carpels hairy at the top.R. canduliger has a well-defined range north of the Rhine river around Arnhem where it occurs in urban and other disturbed habitats. Historical events, e.g. transport of young trees in the 16th century may explain most records south of the Rhine river, 25–50 km outside the main range. The ecology and phytosociology of the new bramble are discussed. Its distribution is compared toR. trichanthus, another regional species with a similar range asR. canduliger, but occurring in more natural habitats. In pseudogamous European blackberries (Rubus sectionRubus =R. fruticosus agg.), range size is considered to be related to the age of the species. Several widespread species have disjunct distribution ranges that are suggested to be the result of long-distance dispersal by migratory birds but unintentional anthropogenic transport of seeds may be involved as well. We hypothesize that in northwestern Europe fragmentation of continuous ranges of widespread species was caused by ongoing deforestation and subsequent degradation (acidification) of originally base-rich sandy and loamy soils from the Iron Age onward. Many regional species, however, have originated and spread in the resulting man-made landscapes. A classification of range types of brambles differing in size and internal structure is presented.     

Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids

Rubus yellow net virus (RYNV) infects Rubus species and cultivars worldwide and is an essential component of raspberry veinbanding mosaic (RVBMD), a virus disease complex that causes serious decline in plant vigour and productivity. The virus is transmitted, probably in a semi‐persistent manner, by the large raspberry aphid, Amphorophora idaei in Europe, and A. agathonica in North America. The particles of RYNV are bacilliform in shape and measure 80–150 × 25–30 nm, similar to those of badnaviruses. A1.7 kb fragment of the viral DNA was amplified by PCR and then directly sequenced. Analysis of this sequence suggests that RYNV is possibly a distinct species in the genus Badnavirus and is most closely related to Gooseberry vein banding associated virus (GVBAV) and Spiraea yellow leaf spot virus, two other badnaviruses described recently. Using the sequence derived from the PCR‐amplified viral DNA fragment, RYNV‐specific primers were designed and used in PCR to assay for RYNV in a range of Rubus germplasm infected with RYNV, with other unrelated viruses and virus‐like diseases found in Rubus, and in healthy plants. RYNV was detected in all glasshouse cultures of RYNV‐infected plants, whether alone or in complex infections with other viruses, but not from healthy Rubus plants, nor from plants infected with other viruses. It was also detected in field‐grown raspberry plants with and without symptoms of RVBMD and in raspberry plants infected with RYNV by viruliferous A. idaei. RYNV was also detected by PCR in A. idaei following access feeds on RYNV‐infected plants of 1 h or more. PCR failed to amplify DNA from gooseberry infected with GVBAV confirming the specificity of the RYNV analysis. PCR detection of RYNV in dormant raspberry buds allows assays to be made outside the natural growing season, providing a useful application for plant introduction and quarantine programmes.     

Chloroplast DNA diversity in the genusRubus (Rosaceae) revealed by Southern hybridization

The variability in chloroplast DNA type of 20Rubus genotypes was examined by Southern hybridization. DNA extracted from theRubus accessions was digested with two restriction enzymes (EcoRI and EcoRV) and heterologous chloroplast DNA sequences from barley and pea were used as probes to detectRubus chloroplast DNA sequences on Southern blots ofRubus total DNA. Restriction fragment length polymorphism was detected and a total of 92 restriction fragments were generated by the probe/enzyme combinations examined. Cladistic principles based on the parsimony assumption were used to assemble a phylogenetic tree based on chloroplast restriction fragment length data. The phylogenetic tree grouped the taxonomically defined species and is in general agreement with information based on morphological criteria. However, the Japanese red raspberryR. illecebrosus was shown to have diverged considerably in terms of evolutionary time from other species in subg.Idaeobatus. Furthermore, the molecular approach provides a quantitative estimate of the relationship between species that is difficult to obtain from morphological data. In order to complement the chloroplast DNA information a ribosomal DNA probe was also included in the analysis and provided further information on the phylogenetic relationships withinRubus.     

The genome of black raspberry (Rubus occidentalis)

Black raspberry (Rubus occidentalis) is an important specialty fruit crop in the US Pacific Northwest that can hybridize with the globally commercialized red raspberry (R. idaeus). Here we report a 243 Mb draft genome of black raspberry that will serve as a useful reference for the Rosaceae and Rubus fruit crops (raspberry, blackberry, and their hybrids). The black raspberry genome is largely collinear to the diploid woodland strawberry (Fragaria vesca) with a conserved karyotype and few notable structural rearrangements. Centromeric satellite repeats are widely dispersed across the black raspberry genome, in contrast to the tight association with the centromere observed in most plants. Among the 28 005 predicted protein‐coding genes, we identified 290 very recent small‐scale gene duplicates enriched for sugar metabolism, fruit development, and anthocyanin related genes which may be related to key agronomic traits during black raspberry domestication. This contrasts patterns of recent duplications in the wild woodland strawberry F. vesca, which show no patterns of enrichment, suggesting gene duplications contributed to domestication traits. Expression profiles from a fruit ripening series and roots exposed to Verticillium dahliae shed insight into fruit development and disease response, respectively. The resources presented here will expedite the development of improved black and red raspberry, blackberry and other Rubus cultivars.     

Phylogeny of korean rubus (rosaceae) based on its (nrDNA) and trnL/F intergenic region (cpdna)

We examined the phylogeny of the genusRubus in Korea using an internal transcribed spacer (ITS) of the nuclear ribosomal DNA and a trnL-trnF (trnL/F) intergenic region of the chloroplast DNA. In all, 21 ingroup species (1.2 kb for each species) were analyzed using parsimony, resulting in 672 aligned sequences from ITS, and 502 bases with trnL/F. Individual and combined analysis of ITS and trnL/F data proved that the genusRubus is a monophyletic group. This phylogeny also substantiated a previous sectional classification scheme rather than a subgerius classification scheme. However, our results did not support the earlier sectional classification by Focke (sect.Corchorifolii), but did support the sectional classification of Nakai: sect.Crataegifolii (R. crataegifolius, R. takesimensis andR. trifidus), and sect.Villosii (R. corchorifolius). Most of these species, which are found in Korea and belong to subg.Idaeobatus, appeared in two different groups in all data sets. This suggests that this subgenus is a polyphyletic group that has gone through at least two independent evolutionary processes. The taxa, when mapped onto the combined tree, showed that the occurrence of their morphological characters of simple and compound leaves was concurrent in KoreanRubus. ITS sequence data were consistent overall with the geographical distribution of each species. Furthermore, the trnL/F sequence data provided phylogenetic information within closely related species.     

Apomixis versus sexuality in blackberries (Rubus subgen.Rubus,Rosaceae)

InRubus L. a connection seems to exist between the degree of meiotic disturbances on the one hand, and the production of unreduced embryo sacs, pollen fertility and relative seed set on the other hand. Severe meiotic disturbances commonly encountered in apomictic taxa decrease pollen fertility and thereby seed set since pollen is necessary for endosperm development. By contrast interspecific hybrids between apomictic taxa appear to be sexual and exhibit high pollen fertilities, probably due to an improved meiosis. Thus, apomixis leads to a decreased fertility inRubus, not the opposite, as often discussed.     

Isolation and characterization of simple sequence repeat loci in Rubus hochstetterorum and their use in other species from the Rosaceae family

The identification of microsatellite loci in Rubus hochstetterorum provides an important tool for the characterization and conservation of wild populations of this species. Cross‐species amplification of markers may be of particular interest for the study of other Rubus species. In this study, 41 simple sequence repeat markers were identified in a genomic library of R. hochstetterorum. Fifteen of the identified microsatellite loci were characterized in a set of 30 samples and revealed to be polymorphic with three to 19 alleles per locus. All the identified markers allowed cross‐species amplification in at least one of the other three tested species from the Rosaceae family.     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

Genetic analysis of natural hybrids between endemic and alien Rubus (Rosaceae) species in Hawai`i

A population of putative hybrids between theendemic Rubus hawaiensis and naturalizedR. rosifolius was discovered inKpahulu Valley, on the island of Maui inthe Hawaiian archipelago. The goal of thisstudy was to molecularly characterize thisnatural hybridization event, investigate themode of hybridization, and determine the malefertility of the hybrid individuals. Bothmorphological and RAPD marker data indicatethat the putative hybrid individuals are theprogeny of R. rosifolius and R.hawaiensis. All 39 hybrid individuals sampledhad the chloroplast DNA haplotype of R.rosifolius. Thus hybridization appears to beasymmetric, with R. rosifolius acting asthe maternal parent. All hybrid individualsassessed for pollen stainability were sterile,and there was no evidence of backcrossing toeither parent. This result suggests thathybrids are of the first filial generation andthat variation among hybrids reflectsdifferences within the parental populations.Sympatric populations of R. hawaiensisand R. rosifolius occur on four islandsand six additional alien species of Rubusare naturalized and sympatric with R. hawaiensisin Hawai`i. Further investigationis merited to assess whether hybridization maypose a threat to the long term viability ofR. hawaiensis. This study highlights theincreasing frequency and negative consequencesof native-alien hybridization and theimportance of maintaining active alien speciescontrol programs in the Hawaiian Islands.