DNA fingerprinting demonstrates extremely low levels of genetic variation among blackberry cultivars grown in Finland

Kristiina Antonius Department of Plant Biology, University ofHelsinki, Finland. Current address: Department ofBiosciences, Division of Genetics, PO Box 56, FIN-00014 University ofHelsinki, Finland, email: kristiina.antonius@helsinki.fi Gun Werlemark, Hilde Nybom Balsgård-Department ofHorticultural Plant Breeding, Swedish University ofAgricultural Sciences, Fjälkestadsvägen 123-1, S-291 94 Kristianstad, Sweden

ntroduction Blackberry (Rubus spp.) cultivation in Finland is severely hindered by insufficient winter har- diness and late fruit ripening in the available plant material.The spring weather, with its ex- treme daily temperature fluctuations, is particularly damaging as it causes frost dehydration of otherwise healthy tissue when the ground is still frozen.
Nevertheless, blackberries have long been grown successfully in sheltered gardens (Immo-  nen 1986, Selonen and Tigerstedt 1989).Dissem- ination and variation within the blackberry group were studied by Viksten (1984) with a view to broadening the selection of suitable plant mate- rial.Information on blackberry plants in culti- vation was requested from the public through media announcements.Of the more than 200 blackberry plants re- ported, 100 were chosen to establish a collectionrepresenting different species, cultivars and provenances.Only a few contributors provided information on cultivar identity; most accessions were identified through field observations and by comparison with herbarium specimens at the University of Helsinki to species level only.
No fewer than 74 of the 100 accessions close- ly resembled R. allegheniensis, a widely distrib- uted North American species.Partly due to its outstanding winter hardiness, this species has been important in plant breeding, and numerous successful cultivars have arisen from its tetraploid hybrids with other species (Jennings 1988).One such is probably 'Majestät', a popular cul- tivar reported to be of American origin (Reimer  1948) although its exact derivation is unknown.Morphologically it is similar to wild material of R. allegheniensis.
The present study was undertaken in an ef- fort to provide the unidentified R. allegheniensis-type accessions with cultivar names by com- paring their DNA fingerprints with those of known cultivars.plants on the day before DNA extraction, and kept overnight at + 4 °C.DNA was isolated ac- cording to Nybom and Schaal (1990), with some minor modifications and an additional salt precipitation step (Graham et al. 1996).Separate aliquots of 8 pg DNA were digested with the restriction enzyme Hae 111 and electrophoresed on 0.7% agarose gels in TPE buffer according to Nybom and Schaal (1990).After Southern blot- ting to Magnagraph (MSI) nylon membrane, the samples were hybridized with a 32 P hexamer-la- belled minisatellite DNA probe derived from the bacteriophage Ml 3 (Nybom and Schaal 1990,   Antonius and Nybom 1994).Autoradiograhps were developed for 2 weeks at -80 °C using intensifying screens.Membranes were subsequently stripped and rehybridized with a synthetic (AC)/(TG) polydinucleotide probe as previously described (Antonius and Nybom 1994).

Material and methods
Results and discussion Leaf samples were taken from all of the 39 R. allegheniensistype accessions hitherto uniden- tified by cultivar, as well as from three cultivars on the same collection; 'Majestät', 'lmperial' and 'Earliest of AH'.'lmperial' was obtained from the Institute of Horticulture, the Agricultural Research Centre of Finland, at Piikkiö, and 'Earliest of AH' from Rastlösa at Jorvas, Kirkkonummi, Finland.Three accessions of 'Majestät' were included in the analysis: one obtained from Turku Horticultural School, Finland; one from Professor E. Kaukovirta of the Department of Horticulture, University of Hel- sinki, Finland; and one from Poland through Paul Olsson's plant nursery in Finland.'lmperial' and 'Majestät' are strikingly similar, but 'Earliest of All' deviates in having darker and more reddish foliage and thornfree canes.The latter also appears to be more susceptible to winter injury.
Healthy-looking and young, not yet fully ex- panded leaves were collected from field-grown DNA fingerprinting, based on the hybridization of restriction-enzyme digested DNA samples to hypervariable minisatelliteDNA probes, has previously proved its ability to discriminate between closely related Rubus genotypes (Nybom et al.  1989, Nybom and Hall 1991).In this study, we obtained clear and informative DNA fingerprints after hybridization of the various Rubus acces- sions (Fig. 1).The cultivars 'Majestät' and 'Ear- liest of All' had different DNA band patterns, 'Majestät' having six bands not present in 'Ear- liest ofAH', and this cultivar having seven bands not present in 'Majestät'.At least another four bands appeared in the same position in all sam- ples.This level of inter-cultivar differentiation is of the same magnitude as that previously re- ported among blackberry cultivars (Nybom et al.  1989, Nybom and Hall 1991).
Our sample of 'lmperial' turned out to be completely identical to that of 'Majestät'.How- ever, samples of 'lmperial' derived from other sources should also be analysed to rule out the Vol.6 (1997): 241-245.possibility of misidentification of the plant ma- terial used in our study.
Surprisingly, 37 of the 39 previously uniden- tified accessions showed exactly the same band pattern as 'Majestät'.The two exceptions, A34 and A4O, had one additional DNA band in the same position, but were otherwise completely identical to 'Majestät'.Whereas pathogenic infestation cannot be completely ruled out as the cause of this extra band, a somatic mutation involving some chromosomal rearrangement is more likely.Somatic mutations have previously been shown to cause very little (if any) variation in the DNA fingerprints, e.g.among sports of apple cultivars (Nybom 1990).By contrast, genetic recombination due to sexual seed set in blackberries generally gives rise to widely dif- ferent band patterns (Nybom 1995).
Hybridization with the (AC)/(TG) probe yielded DNA fingerprints of same complexity as with the Ml 3 probe but, as previously reported in Rubus (Antonius and Nybom 1996), these bands were less distinct and consequently more difficult to score.Only two patters were found: 'Earliest of All' showed three bands not present in the other samples, whereas these showed four bands not present in'Earliest of AH'.At least another ten bands appear to be in the same position for all samples analysed.
Chromosome level is closely associated with the reproductive system in blackberry taxa, diploid species being sexual and polyploid species Fig. 1.Contact copy of an autoradiopraph with DNA fingerprints from hybridization of //aelll-digested DNA samples to an Ml 3 minisatellite DNA probe; Ml='Majestat' from Turku, M2='Majestät' form Helsinki, E='Earliest of AH', I=Tmperial'; the remainder are previously unidentified accessions in the Helsinki Rubus collection.A34 and A4O are two accessions, each with one additional band (position shown by arrow), compared with the standard DNA fingerprint for 'Majestät' .Size markers in the right-hand margin (7.2, 6.4, 4.8, 4.3, 3.7 and 2.3 kb) were obtained from a X DNA-B.v/EIIdigest.facultatively apomictic, i.e. able to produce seeds without prior fertilization (Nybom 1988).Although the wild species R. allegheniensis has been reported to be diploid (Jennings 1988), Finnish accessions of its putative derivative, 'Majestät', have previously proved to be tetra- ploid and apomictic as deduced from chromo- some counts and from evaluation of offspring derived from cross-pollination experiments (Antonius and Nybom 1996).Consequently, seed collected from 'Majestät' would mainly produce plants identical to the seed parent.This could be one explanation for the identical DNA fingerprint patterns of 'Majestät' and 'lmperial'.Immonen(1986) mentions another blackberry cultivar, 'Wilson's Frtihe', which has been on sale in Finland and is apparently identical to 'Majestät'.Obvioulsy this single cultivar has been renamed several times.Most blackberries grown in Finland closely resemble R. allegheniensis (Hämet-Ahti et al. 1992), and the re- sults of our study indicate that only one cultivar in this group, 'Majestät', has become really suc- cessful.