Euphytica 54 : 2 4 5-250, 1991 .©1991 Kluwer Academic Publishers . Printed in the Netherlands .

Restriction enzyme mapping the ribosomal RNA genes inSolanum tuberosum : Potato rDNA restriction enzyme map

K. Harding'Department of Genetics, School of Biological Sciences, Medical School, University of Nottingham,Nottingham, NG7 2 UH, UK; 'present address : Department of Biology, University of Crete, P . O. Box 1470,71110 Heraklion, Crete, Greece

Received 22 October 1990; accepted 4 April 1991

Key words: Solanum tuberosum, potato, rDNA, IGS variation

Summary

A restriction enzyme map of the ribosomal RNA genes (rDNA) in Solanum tuberosum cultivars GoldenWonder and Desiree has been constructed. An heterologous probe pTa 71 containing the rDNA derivedfrom wheat was used to detect and map the corresponding region in potato genomic DNA fragments . rDNArepeats of cultivars Desiree and Golden Wonder are similar with respect to their target sites for restrictionenzymes, however, these cultivars have diverged with respect to the length of their intergenic spacer (IGS)sequences. IGS length variants may reflect the different breeding systems for Golden Wonder and Desiree .Furthermore, some of the 25S genes of both cultivars appear to have a 100-150bp insertion/deletion neartheir 3' end . The presence of RFLPs (restriction fragment length polymorphisms) in the IGS region could beused as a DNA fingerprint for potato cultivar identification ; the ability to recognise IGS length variants maybe of significance to potato phylogenetics and breeding .

Introduction

The eukaryotic ribosome is composed of differenttypes of ribonucleic acid (RNA) . Ribosomal RNAis encoded by several genes situated at differentpositions in the nucleus . There are groups of genesclustered within a nuclear domain termed the 'nu-cleolar organiser region' (NOR) . The clustered ri-bosomal genes are organised into a transcriptionalunit comprising the 25-28S, 5 .8S and 18S genes .This structure can be repeated to several thousandcopies, organised in a long tandem array, and sep-arated by a variable intergenic spacer (IGS) region .

The molecular biology of the ribosomal RNAgenes has been studied in a limited number ofangiosperms . Detailed studies have been perform-ed in the tribe Triticeae (Appels & Dvorak, 1982 ;

McIntyre et al., 1988) . Variation in the IGS regionhas been used as a genetic marker for cultivar iden-tification, pedigree analysis (May & Appels, 1987)and evolutionary studies in populations of wildwheat (Flavell et al ., 1986) . In contrast to the varia-ble IGS region, the sequences encoding ribosomalRNA (rDNA) in eukaryotes are highly conserved(Messing et al ., 1984; Eckenrode et al., 1985) . Theconservation of rDNA sequences has enabled theuse of heterologous probes to examine the orga-nisation of rDNA in diverse species (Eckenrode etal., 1985 ; Rogers et al ., 1986) .The rDNA has been examined in a few members

of the family Solanaceae (Taliercio et al ., 1985 ;Borisjuk et al ., 1988) . In this paper, a comparativegenomic restriction enzyme map of the repeat unitsand their spacer lengths has been constructed for

246

two cultivars of potato . The use of rDNA sequenc-es as molecular markers for cultivar identificationin potato is discussed .

Materials and methods

Plant material and plasmid . S. tuberosum cultivarsGolden Wonder (GW) were obtained from DAFS,Edinburgh, Scotland and Desiree (Des) from IPSR(Cambridge). The plasmid pTa 71 (Gerlach & Bed-brook, 1979) was supplied as a gift from MikeO'Dell IPSR (Cambridge) .

DNA isolation and purification . DNA was extract-ed from freeze dried leaf material by the CTABprocedure (Rogers & Bendich, 1988), and purifiedby caesium chloride, ethidium bromide buoyantdensity centrifugation .

Restriction enzyme digestion, gel electrophoresis,and Southern blotting . Digestion of the DNA withrestriction enzymes (Bam HI = HI ; Eco RI = RIand Eco RV = RV) was performed according tothe manufacturers' instructions (BRL) . LambdaDNA digested with Eco RI and Hind III was usedas a molecular weight marker . Digested DNA(4 µg/track) was fractionated by agarose gel elec-trophoresis in TAE buffer, depurinated, dena-tured, neutralised and Southern blotted onto nitro-cellulose membrane filters as described by Maniatiset al., (1982) .

Hybridisation conditions, radiolabelling and aut-oradiography . Prehybridisation was performed inplastic bags (Hybaid) containing 5 x Denhardt'ssolution (Denhardt, 1966), 0 .1% SDS and 4 x SSCat 65'C for 4h. Plasmid pTa 71 or isolated gelfragments containing the ribosomal genes derivedfrom wheat were radiolabelled by the method ofnicktranslation using a- 32P-dCTP described by Ma-niatis et al ., (1982), or by the method of randompriming described by Feinberg & Vogelstein(1984). Hybridisation was performed for 16-20hunder the same conditions as pre-hybridisation .Filters were washed after hybridisation in 4 x SSC,0.1 % SDS for 4 min at 65° C, and 2 x SSC at 20'C

for 2 min under rapid buffer flow using the Hybaidsystem . Autoradiography was performed in cas-settes with G3 screens (Fuji) and X-ray film (FujiHR-G) at - 80'C .

Preparation of restriction fragments for labellingand mapping procedures . The plasmid pTa 71 wasdigested with Eco RI and fractionated on a 0 .7%low melting point agarose (Seaplaque GTG) gel inTBE (89 mM Tris.HC1 pH 8.0, 2 mM EDTA and89 mM boric acid) buffer. The 9.05 kb fragmentwas removed, the gel slice melted at 65'C for10 min and maintained at 37'C, and further re-striction enzyme digestions were performed in thegel with Bam HI, Sal I, and Taq I (BRL) . Dena-tured DNA fragments were labelled and hybrid-ised to single (Eco RV, Bam HI, Eco RI, Bgl I, BglII, Sst I, and Xba I), double (Eco RV/Bam HI, EcoRV/Eco RI, Eco RV/Bgl I, Eco RV/Bgl II, EcoRV/Sst I, Eco RV/Xba I) and triple (Eco RV/EcoRI/Bam HI) digests of Golden Wonder and Desir-ee DNA. Mapping of the ribosomal genes in potatowas performed by taking different fragments de-rived from pTa 71 and sequentially hybridisingthem to unwashed filters . Additional fragmentswere detected after each successive hybridisationby autoradiography .

Results

The pTa 71 probe (containing a 9 .05 kb clonedinsert) was digested with Eco RI and Bam HI, andone of the resultant fragments (0 .9 kb containingpart of the wheat 25S gene) was radiolabelled andhybridised to potato DNA. In relation to the wheatrestriction enzyme map the Barn HI 5 .4 and 4 .25 kbfragments of Golden Wonder and the 5.9, 5 .3, 5 .1,4 .6, 4.1 and 3.9 kb fragments of Desiree span theIGS region represented diagramatically in Fig . lb .The length difference of the 5 .4 and 4.25 kb frag-ments suggests that there may be a Bam HI sitelocated within the IGS of Golden Wonder . Eco RIdigests of Golden Wonder and Desiree producedthe same 2.5, 2.6 and 3.85 kb fragments . Theseresults indicate that the Eco RI fragments werederived from the coding regions as shown in Fig .

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Fig. 1 . Autoradiograph of Southern blots showing the fragments detected by a 0 .9 kb probe derived from pTa 71 in digested DNAsamples of Golden Wonder and Desiree . Figures la and b : Construction of a restriction enzyme map of potato rDNA . Restrictionenzyme fragments are illustrated by arrowed lines and molecular weights . The boxes represent the 18S, 5 .8S (unlabelled small box) and25S ribosomal genes, the variable IGS region is shown by the dotted lines in Fig . 1b .

la. The Eco RI Eco RV digests of Golden Wonderand Desiree produced the same 3 .35 kb fragment(derived from the Eco RI 3 .85 kb fragment), sug-gesting that there are Eco RI polymorphic siteslocated within the coding regions for some of therepeats within the tandem array . The presence ofthe Eco RI 2.5 and 2.6 kb fragments may be indica-tive of two variants for the 25S gene, differing fromeach other by 100 bp. Triple Bam HI/Eco RI/EcoRV digests of Golden Wonder and Desiree pro-duced the same 2 .15,1 .0 and 0 .85 kb fragments andthis further supports the possibility of two 25S genevariants. The 2.15 fragment indicates there is aBam HI polymorphic site for some of the repeatswithin the tandem array .

Figure 2a shows the fragments detected by thepTa 71 probe after hybridisation to filters contain-

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247

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ing Eco RI digested potato DNA. There are 1major and 3 minor fragments in Golden Wonderand 6 major fragments in Desiree . In contrast, thefragments detected by the radiolabelled 0 .33 kbprobe show Eco RI digests of Golden Wonder toproduce a major 3 .45 and minor 3.65 and 3 .7 kbfragments, whereas, Desiree produced major 3 .2,3.4 and 3.9, and minor 3 .6 and 3.7 kb fragments .Triple Barn HI/Eco RI/Eco RV digests of GoldenWonder produced a single 3.15 kb fragment, Desir-ee produced the 2 .85, 3 .1, 3.3 (minor) and 3 .6 kbfragments . In both cultivars the 0 .33 kb probe didnot detect the 3 .85 kb fragment, as observed in Fig .2a; this fragment is part of the 18-25S coding regionand not the IGS region .

The restriction enzyme map for Bgl I, Bgl II,Barn HI, Eco RI, Eco RV, Sst I, and Xba I of

248

b~--3.85W.

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Fig. 2a. Autoradiograph of Southern blots showing the fragments detected by the radiolabelled pTa 71 probe after hybridisation tofilters containing Eco RI digested potato DNA . The IGS fragments 3 .45, 3 .65, 3 .7 and 3 .85 kb in Golden Wonder and the 3 .2, 3 .4, 3 .6,3 .7, 3.85 and 3 .9 kb fragments were detected in Desiree . Fig . 2b: Autoradiograph of Southern blots showing the fragments detected bythe radiolabelled 0.33 kb wheat probe after hybridisation to digested DNA samples from Golden Wonder and Desiree . The major IGSEco RI fragments are represented by the unbroken arrowed lines in the restriction enzyme map and larger numbers in Fig . 2b . Whereasthe minor IGS Eco RI fragments are represented by the broken arrowed lines and smaller numbers in Fig . 2b : The `spurious' IGS 3 .85 kbEco RI fragment is illustrated by the bold unbroken arrowed line .

Golden Wonder and Desiree is shown in Fig . 3 . ty. Desiree was shown to have several IGS frag-The 25S gene variant II has a 100-150 by deletion ments compared to Golden Wonder . This featurebetween the Sst I and Eco RI site at the 3' end .

was further elucidated (Fig . 2) demonstrating thatthese cultivars have diverged with respect to thelength of their IGS . Variation in the length of the

Discussion

IGS is a feature of many plant species . A variationof 300 by resulted in three size classes of wheat

The differences between the potato cultivars (Fig . rDNA (Gerlach & Bedbrook, 1979), and in Vicia1) may be explained by spacer length heterogenei-

faba, at least 20 different size classes have been

RI

RI

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Fig. 3 . A restriction enzyme map of the ribosomal genes of Golden Wonder and Desiree . The distances between restriction enzyme sitesare ± 50 bp .

Xbal

RI

HI SstIHI

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reported (Rogers et al ., 1986) . The rDNA repeatsof the cultivars Desiree and Golden Wonder havesimilar restriction enzyme sites, as well as somepolymorphic sites . The 25S genes appear to have a100-150 by insertion/deletion in order to producethe variant shown in Fig . 3. The region of thisinsertion/deletion was located between the Sst Iand terminal Eco RI site . Furthermore, the repro-ducible presence of identical partial digestion prod-ucts in both cultivars may be due to (1) site muta-tion or (2) methylated DNA sequences . WheatrDNA has been shown to contain methylated se-quences (Flavell et al ., 1983), and potato rDNAmay be methylated and therefore refractory tocomplete digestion by some restriction enzymes(McClelland, 1983) .

In an early study of the inheritance of rDNA inXenopus (Reeder et al ., 1976), spacer length varia-nts were observed in 2 out of 50 individuals of theF1 progeny . Therefore, it is possible to speculatethat species entering into sexual reproduction mayshow a greater degree of IGS variation comparedto asexual species . This may be relevant to thecultivars of S . tuberosum, as the number of IGSvariant fragments may possibly reflect the differentbreeding systems for Golden Wonder and Desiree .Desiree is produced from a sexual cross, whereas

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249

Golden Wonder is a periclinal chimeric sport de-rived from the cultivar Langworthy (Cassells et al .,1986). Although Desiree can be propagated throughtrue potato seed (TPS) and tubers, Golden Wonderis pollen sterile (Howard, 1970) and therefore clonal-ly propagated.

The use of an heterologous probe (derived fromwheat) to restriction enzyme map the ribosomalgenes of potato is possible due to the highly con-served nature of the coding regions . The degree ofrDNA conservation between monocots and dicotsis shown by the 93 .5% homology in the 18S gene ofsoybean and maize (Eckenrode et al., 1985) . In-deed, the use of pTa 71 and other wheat rDNAprobes for similar studies has been reported forbarley (Molnar et al., 1989 ; Saghai-Maroof et al .,1984), Lilium henryi (von Kalm et al., 1986) andrice (Barnes & Pental, 1986) . The procedure de-scribed in this paper may be applied to other plantspecies, especially where the cloning of rDNA frag-ments may be problematic due to methylated se-quences (Woodcock et al ., 1989). The ability torecognise IGS length variants may be of signif-icance to phylogenetic studies and plant breeding,particularly as RFLPs in the IGS could be used as aDNA fingerprint for cultivar identification . Theuse of rDNA sequences as molecular markers for

250

cultivar identification in potato may provide anadditional approach to the tuber protein profileanalysis used to assess European potato varieties .The recognition of quantitative traits, especiallythose linked to NOR loci may be of benefit to plantbreeding .

Acknowledgements

The author would like to thank Ms . Helen Smithfor technical assistance, and the InternationalBoard for Plant Genetic Resources (Rome) forfunding the project .

References

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