Research Note
DNA fingerprinting demonstrates extremely low
levels of genetic variation among blackberry cultivars grown in Finland
Kristiina Antonius
DepartmentofPlantBiology, UniversityofHelsinki,Finland. Current address:DepartmentofBiosciences, Division of Genetics, PO Box56,FIN-00014 UniversityofHelsinki, Finland,
email:kristiina.antonius@helsinki.fi GunWerlemark, Hilde Nybom
Balsgård-DepartmentofHorticultural PlantBreeding, SwedishUniversity ofAgriculturalSciences, Fjälkestadsvägen 123-1,S-29194 Kristianstad,Sweden
Mostblackberry plants cultivated inFinland closelyresemble the American speciesRubus allegh- eniensis. Thirty ninesuch blackberry accessionsintheUniversityof Helsinki clone collection were studiedby hybridization-based DNA fingerprintingandcomparedwithsomeknown cultivars ofR.
allegheniensis derivation. ‘lmperial’appears to be identical to the old cultivar ‘Majestät’,but ‘Earli- estofAll’differsconsiderably. In addition, 37of the accessions analysedalso haveDNA fingerprints that appear to becompletelyidentical to that of‘Majestät’!Theremaining twoaccessions,although identical to eachother,exhibitoneband not foundin‘Majestät’that isprobably causedbyasomatic
mutation.
Keywords',apomixis, clone,molecularmarker,Ml 3 probe, RFLP, Rosaceae,Rubus allegheniensis, Rosaceae
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 itsex- treme dailytemperature fluctuations,is particu- larly damagingasitcauses frost dehydration of otherwise healthy tissue when the ground is still frozen.
Nevertheless, blackberries have long been grownsuccessfully in shelteredgardens (Immo-
nen 1986, Selonen and Tigerstedt 1989). Dissem- ination and variation within the blackberry group were studied by Viksten (1984) witha view to broadening the selection of suitable plant mate- rial. Information on blackberry plants in culti- vation was requested from the public through mediaannouncements.
Of the more than 200 blackberry plants re- ported, 100were chosen to establisha collec- tionrepresenting different species, cultivars and provenances. Onlyafew contributors provided informationoncultivar identity;mostaccessions were identified through field observations and
©Agricultural and Food ScienceinFinland Manuscriptreceived April 1997
by comparison with herbarium specimensatthe University of Helsinkito species level only.
No fewer than74 of the 100 accessions close- ly resembled R. allegheniensis, awidely distrib- uted North American species. Partly dueto its outstanding winter hardiness, this species has been important in plant breeding, andnumerous successful cultivars have arisen from its tetra- ploid hybrids with other species (Jennings 1988).
One such is probably ‘Majestät’, apopular cul- tivar reported tobe of American origin(Reimer
1948) although itsexact derivation is unknown.
Morphologically it is similartowild material of R. allegheniensis.
The present study was undertaken in an ef- fort toprovide the unidentified R. alleghenien- sis-typeaccessions with cultivarnamesby com- paring their DNA fingerprints with those of known cultivars.
plants on the day before DNA extraction, and kept overnightat+4 °C. DNAwas isolatedac- cordingtoNybom and Schaal (1990), withsome minor modifications and an additional salt pre- cipitation step (Graham et al. 1996). Separate aliquots of 8 pg DNA were digested with the restriction enzyme Hae111 and electrophoresed on 0.7% agarose gels in TPE buffer according to Nybom and Schaal(1990).After Southern blot- ting toMagnagraph(MSI) nylonmembrane,the samples were hybridized witha32P hexamer-la- belled minisatellite DNA probe derived from the bacteriophage
Ml 3
(Nybom and Schaal 1990, Antonius and Nybom 1994). Autoradiograhps were developed for2 weeksat-80 °C using in- tensifyingscreens. Membranesweresubsequent- ly stripped and rehybridized with a synthetic (AC)/(TG) polydinucleotide probe asprevious- ly described (Antonius and Nybom 1994).Material and methods Results and discussion
Leaf samples were taken from all of the 39 R.
allegheniensis-type accessions hitherto uniden- tified bycultivar, aswellasfrom three cultivars on the same collection; ‘Majestät’, ‘lmperial’
and ‘Earliest of AH’. ‘lmperial’ was obtained from the Institute ofHorticulture, the Agricul- tural Research Centre ofFinland, atPiikkiö,and
‘Earliest of AH’ from Rastlösa atJorvas, Kirk- konummi, Finland. Three accessions of
‘Majestät’ were included in the analysis: one obtained from Turku HorticulturalSchool,Fin- land; onefrom Professor E. Kaukovirta of the Department ofHorticulture,University of Hel- sinki, Finland; andonefrom 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 andyoung, notyetfullyex- panded leaveswerecollected from field-grown
DNA fingerprinting, based onthe hybridization of restriction-enzyme digested DNA samplesto hypervariable minisatelliteDNAprobes, has pre- viously proved its abilitytodiscriminate between closely related Rubusgenotypes (Nybom etal.
1989, Nybom and Hall 1991).In this study, we obtained clear and informative DNA fingerprints after hybridization of the various Rubusacces- 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 havingsevenbands notpresent in ‘Majestät’. At least another four bands appeared in thesame position in allsam- ples. This level of inter-cultivar differentiation is of thesame magnitudeas that previously re- ported among blackberry cultivars (Nybom etal.
1989, Nybom and Hall 1991).
Our sample of ‘lmperial’ turned out to be completely identicaltothat of ‘Majestät’. How- ever, samples of ‘lmperial’ derived from other sources should also be analysed to rule outthe
possibility of misidentification of the plant ma- terial used in ourstudy.
Surprisingly, 37 of the 39previously uniden- tified accessions showed exactly thesameband pattern as ‘Majestät’. The two exceptions, A34 and A4O, had one additional DNA band in the same position, but were otherwise completely identicalto ‘Majestät’. Whereas pathogenic in- festation cannot be completely ruled out as the cause of thisextraband, asomatic mutation in- volving some chromosomal rearrangement is more likely. Somatic mutations have previously been showntocausevery little(if any) variation in the DNA fingerprints, e.g. among sports of apple cultivars (Nybom 1990). By contrast, ge- netic recombination due to sexual seedset in
blackberries generally gives riseto widely dif- ferent bandpatterns (Nybom 1995).
Hybridization with the (AC)/(TG) probe yielded DNA fingerprints ofsamecomplexityas with the
Ml 3
probe but, aspreviously reported in Rubus (Antonius and Nybom 1996),these bands wereless distinct and consequentlymore difficulttoscore. Onlytwopatters were found:‘Earliest of All’ showed three bandsnotpresent in the other samples, whereas these showed four bands not present in'Earliest of AH’. At least anotherten bands appeartobe in thesameposi- tion for all samples analysed.
Chromosome level is closely associated with the reproductive system in blackberry taxa,dip- loid species being sexual and polyploid species Fig. 1.Contact copy ofanautoradiopraphwithDNA fingerprintsfromhybridizationof//aelll-digested DNA samplestoan Ml 3minisatelliteDNAprobe; Ml=‘Majestat’fromTurku,M2=‘Majestät’formHelsinki,E=‘Earliest ofAH’,I=Tmperial’;
the remainder arepreviouslyunidentified accessions inthe Helsinki Rubus collection. A34and A4Oaretwo accessions, each withoneadditional band(position shownbyarrow),comparedwith the standardDNA fingerprintfor‘Majestät’.Size markersinthe right-hand margin (7.2,6.4, 4.8, 4.3, 3.7and2.3kb) wereobtained fromaXDNA-B.v/EII digest.
facultatively apomictic, i.e. abletoproduce seeds without prior fertilization (Nybom 1988). Al- though the wild species R. allegheniensis has been reported to be diploid (Jennings 1988), Finnish accessions of its putative derivative,
‘Majestät’, have previously proved tobe tetra-
ploid and apomictic as deduced from chromo- some counts and from evaluation of offspring derived from cross-pollination experiments (An- tonius and Nybom 1996). Consequently, seed collected from ‘Majestät’ would mainly produce plants identicaltothe seedparent.This could be one explanation for the identical DNA finger- printpatterns of ‘Majestät’ and ‘lmperial’.
Immonen(1986) mentions another blackber- rycultivar, ‘Wilson’sFrtihe’, which has beenon
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. alleg- heniensis (Hämet-Ahti etal. 1992), and there- sults ofourstudy indicate that onlyonecultivar in this group, ‘Majestät’, has become reallysuc- cessful.
Acknowledgements. Financial support wasreceived from the FinnishMinistryofAgricultureandForestry, and from the Swedish Research Council for Agricultural Sciences.
References
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SELOSTUS
Suomessa viljeltävissä karhunvatukkalajikkeissa hyvin vähän geneettistä vaihtelua
KristiinaAntonius, GunWerlemark, HildeNybom Helsingin yliopisto ja Swedish UniversityofAgriculturalSciences
Saatavillaolevien taimien heikkotalvenkestävyys ja marjojen myöhäinen kypsyminen vaikeuttavat kar- hunvatukoiden (Ruhus spp.) viljelyä Suomessa.Vik- stenetsi tutkimuksessaan Suomessa menestyviäkar- hunvatukoita tiedotusvälineissä julkistettujen kysely- jenavulla. Vastausten perusteella Helsingin yliopis- tolle kerättiin n. 100 karhunvatukkanäytteen klooni- kokoelma. Näistä 74kloonia muistuttaa läheisesti Pohjois-Amerikassa laajalle levinnyttäRubusalleghe- niesis-lajia. Lajiketiedot puuttuivatsuurimmalta osal- takerätyistä näytteistäTämän tutkimuksen tarkoituk- sena on tunnistaa lajikkeita vertaamalla tuntematto- mien näytteiden ja tunnettujen lajikkeidenDNA-sor- menjälkiäkeskenään.
Kaikista 39 toistaiseksi tuntemattomastaR. al- legheniensis -tyyppisestä kloonista jakokoelmassa olevista‘Majestät’, Tmprial’ ja ‘Earliestiof AU’ -la- jikkeistakerättiin lehtinäytteet. DNA eristettiin hiu- kan muunnellun Nybomin ja Schaalin ohjeen mu- kaan.
DNA-sormenjälkitekniikka tuotti kaikille tutki- tuille Rubus-suvunnäytteilleselkeät kuviot. ‘Majes- tät’ja ‘EarliestofAli’-lajikkeiden DNA-sormenjäl- jeterosivat selkeästi toisistaan usean viivan osalta.
Tässä tutkimuksessa mukana ollut TmperiaT-lajik- keen näyte osoittautuipuolestaan täysin samanlaisek-
si ‘Majestät’-lajikkeenkanssa. Jotta taimimateriaalin sekaantuminen taivääränimeäminen voitaisiin var- mistaa,olisi analysoitava useampiaeri lähteistä pe- räisin oleviaTmperialMajikkeen näytteitä.
Aiemmin tuntemattomasta39näytteestä37osoit- tautuitäysinsamanlaiseksi ‘MajestätMajikkeenkans- sa.Kahden näytteen sormenjälkikuvioista löytyi sa- malta kohdalta yksi ylimääräinen viiva muutointäy- sin samanlaiseen ‘MajestätMajikkeenkuvioon verrat- tuna.Muutoksen aiheuttaa todennäköisesti yksittäi- nen somaattinen mutaatio,mikä yleensä tuottaahy- vin vähäistä vaihteluaDNA-sormenjälkikuvioihin, kuten ontodettu esim. omenoilla.
‘MajestätMajikkeen on aikaisemmin todettu li- sääntyvän apomiktisesti, eli suurin osa sen siemen- jälkeläisistä on täysin emokasvin kaltaisia. Tämä saattaa olla yksi selitys ‘Majestät’ ja TmperiaT-la- jikkeidenidenttisilleDNA-sormenjälkikuvioille. Im- monen mainitsee vieläyhden karhunvatukkalajik- keen, ‘Wilson’sFriihe’, jokaon ilmeisesti myös sa- manlainen kuin ‘Majestät’. Hämet-Ahdin ja muiden mukaan suurin osaSuomessaviljeltävistäkarhunva-
tukoista muistuttaaR. allegheniensis -lajia, ja tämän tutkimuksen tulosten mukaan vain ‘MajestätMajike tästäryhmästäontodella menestynyt.
