Molecular Breeding5: 11–20, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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Analysis of ribosomal RNA genes in somatic hybrids between wild andcultivated Solanumspecies

K. Harding∗ & S. MillamCrop Genetics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK(∗author for correspondence; e-mail khardi@scri.sari.ac.uk)

Received 16 March 1998; accepted in revised form 2 August 1998

Key words:plant breeding, protoplasts, somatic fusion, ribosomal RNA genes

Abstract

Ribosomal RNA genes were exploited as markers to identify somatic hybrids betweenSolanum tuberosumcv.Brodick and wild diploidSolanumspecies,S. megistacrolobum, S. sanctae-rosaeandS. sparsipilumand DNAmethylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probecombinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions ofthe ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas otherswere characteristic ofS. tuberosumcv. Brodick. This evidence is suggestive of somatic exchange/re-arrangementbetween the NOR region ofS. sanctae-rosaeandS. tuberosumcv. Brodick. Ribosomal RNA gene copy numberanalysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetrichybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation ofribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, othersshow an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.

Introduction

The production of novel plant germplasm via conven-tional plant breeding has long been recognized to bean exhaustive process requiring decades of sexual hy-bridization and selection. Since the development ofsomatic cell genetics and techniques in biotechnol-ogy, several approaches have been applied to reducebreeding time [15]. The genetic modification of plantgermplasm for crop improvement has progressed viatransformation and somatic hybridization techniquesrecently reviewed by Birch [5].

The genusSolanumcontains a diverse collection ofgermplasm and is an excellent source for the introduc-tion of new traits into potato (Solanum tuberosumL.)breeding programmes [11, 55]. Plant protoplasts andtissue culture techniques have been instrumental in theexploitation ofSolanumgermplasm [11, 14]. Therehave been many successful attempts and strategies toutilize Solanumgermplasm and produce somatic hy-brids (reviewed by [40]). Asymmetric fusions are a

potential alternative to reduce the transfer of unde-sirable traits [41, 50]; it is here, where the tools ofmolecular biology have an impact in allowing a morerapid and precise screening of somatic hybrids forselection in plant breeding programmes.

Wild Solanumgermplasm (Commonwealth PotatoCollection, SCRI) has been previously utilised in aprotoplast fusion programme to transfer the traits forpotato cyst nematode resistance (PCN) fromS. megis-tacrolobum, S. sanctae-rosaeand S. sparsipilumtoS. tuberosumcv. Brodick [4]. Advances in methodol-ogy for the identification of putative fusion productshave been recently reported. The analysis of puta-tive fusion products is essential to confirm hybridity[36, 38, 43] and expression of the desirable trait(s).This can also be done via morphological, biochemi-cal, cytological and molecular markers [23, 48], butother factors, such as agronomic performance, are alsoimportant, especially when the research is targetedtowards breeding objectives.

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Figure 1. Luminograph showing the detection of ribosomal RNAgenes after slot blotting isolated DNA (RNase treated) fromS. megistacrolobum(mga); S. sanctae-rosae(sct); S. sparsipilum(spl) andS. tuberosumcv. Brodick (brod). Calibration curve forrDNA signals expressed as integrated optical density units per unitarea (IOD/mm) plotted against rDNA copies/genome.

Ribosomal gene probes have been used in numer-ous studies of somatic hybrids (see [28, 58]). Thesesequences contain coding regions within the tran-scriptional (repeat) unit comprising the 25S, 5.8S and18S genes and are organized as a tandem array lo-cated in the nucleus organizer region (NOR). In thisstudy, the ribosomal RNA genes (rDNA) were ex-amined as markers for potential genetic changes inthe putative somatic hybrids and other attributes, suchas DNA methylation, acting as possible regulatoryfactors for gene expression [56]. In heterokaryons re-sulting from protoplast fusion, parental chromosomesare likely to exchange genetic information, inter-phase

nuclei are certain to interact and these mechanisms ofgene expression and regulation are fundamental in theformation of functional somatic hybrids. It is likelythat the rDNA is associated with DNA methylationchanges and gene expression [34, 56]. These RNAgene products play a role in the assembly of a func-tional ribosome [26, 33]. The impairment of rDNAexpression will reduce the pool of available RNA forthe maintenance of cytoplasmic ribosomes and thetranscription of essential mRNAs for desirable traits.This is likely to have consequences for protein syn-thesis, cellular growth, plant development [29] andoverall agronomic performance of somatic hybrids.

Materials and methods

Plant materialWild Solanumgermplasm was obtained from theCommonwealth Potato Collection (CPC), ScottishCrop Research Institute,S. megistacrolobum(CPC3706, mga),S. sanctae-rosae(CPC 3779, sct),S. spar-sipilum(CPC 3533, spl) andS. tuberosumcv. Brodick(brod) from the departmental potato collection.

Protoplast isolation and fusionProcedures for protoplast isolation, fusion and tissueculture techniques for the production of somatic hy-brid plants are described elsewhere [4]. Protoplastsfor each cross were mixed (1:1 ratio) to give a finaldensity of 1× 106/ml, 200µl were placed in a sterileelectro-fusion chamber (Biojet Cell Fusion 50 powersupply, Kruss, Germany). Protoplasts were aligned inan alternating pulse of 120 V/cm at a frequency of1 MHz for 10 s. Fusion was induced by a 1200 V/cmpulse whereby protoplasts were recovered and resus-pended at a density of 1× 105/ml in culture medium[15]. From the population of some 300 putative hy-brids (150 mga× brod; 125 sct× brod, 50 spl× brod)somatic crosses were analysed for hybridity.

Preparation of DNALeaf material (1.0–5.0 g) was ground in liquid nitro-gen, the powder was transferred to extraction bufferand DNA was extracted as described [25].

Preparation of RNATotal RNA was extracted from fresh leaf material bystandard procedures [51].

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Figure 2. Luminograph of restriction enzyme rDNA profiles inS. megistacrolobum(mga), S. sanctae-rosae(sct), S. sparsipilum(spl) andS. tuberosumcv. Brodick (brod) after hybridization with aSolanum-specific intergenic spacer probe pS-6.

Preparation of rDNA standards

Estimates were based on genomic DNA reconstructionexperiments with a known rDNA probe (pTa 71, con-tains 9.0 kb rDNA wheat repeat and a 3.9 kb plasmid,pACYC184, see [17]) and tetraploidSolanum tubero-sum4C value of 3.6 pg [1] as follows. A single 9 kbcopy of pTa 71 is equivalent to 9× 10−6 pg (assume1 kb= 1×10−6 pg) and a single 4C genome is equiv-alent to 3.6× 10−12 g. Accordingly, in 10µg of brodgenomic DNA, there are 10×10−6 g/3.6×10−12 g=2.8× 10−6 4C genome copies. Therefore, the amountof 4C genome, in 10µg DNA, containing a singlerepeat unit of 9 kb, is [2.8 × 10−6] × [9 × 10−6]= 25.2 pg DNA. Assuming a 20µl slot blot loadingvolume, a× 10 copy number aliquot should contain252 pg of 4C genomic DNA (12.6 ng.ml). A correctionfor the insert is needed (pTa 71 contains 9.0 kb wheatrDNA and 3.9 kb is pACYC184) as ca. 30% of pTa 71is plasmid; therefore, a× 1000 rDNA copy number(1260 ng.ml, corrected to 1638 ng.ml) reconstructionsample was serially diluted (1000 to 100 copies) andapplied to the slot blot apparatus. Accordingly, 2.0µgDNA from the parents (mga, sct, spl and brod) and so-matic hybrids (83/1, 84/4, 90/4 and 90/6) were dilutedin a 2-fold series and applied to the slot blot apparatus.

Slot blot analysis

Genomic DNA and total RNA derived from the so-matic hybrids were denatured in a final concentrationof 0.4 M NaOH and 1 mM EDTA and slot-blottedonto a (Hoefer) apparatus containing a strip of ny-lon membrane (Tropilon or Hybond N) as described[25] prior to hybridization with plasmid pTa 71 andchemiluminescence detection.

Restriction enzyme digestion, gel electrophoresis andSouthern blotting

Digestion of DNA (5µg/track) was performed [26].Digested DNA was fractionated by agarose gel elec-trophoresis and Southern-blotted onto neutral nylonmembrane filters (Tropilon, Tropix) as described [51].

DNA and RNA hybridization conditions

Plasmid pTa 71 [17] containing the ribosomal genesderived from wheat and pPS-6 [8] containing the inter-genic spacer region was biotin-labelled and hybridizedas described [25].

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Detection of DNA/RNA sequences bychemiluminescenceHybridized membranes were treated and, with chemi-luminescent detection, done in cassettes containingX-ray film (Fuji HR-G) without screens at room tem-perature [21].

DNA methylation assayThe methylation status of DNA sequences is evaluatedby comparing the activities of an isoschizomer pair ofrestriction enzymes according to Hardinget al. [26].

Results

rDNA slot-blot analysis

All slot blots (see Materials and methods) were hy-bridized to a biotin-labelled rDNA probe (pTa 71) andhybridization signals detected by chemiluminescence(Figure 1). These signals were scanned by an imageanalysis system (Visage, 4.6L, Millipore) and the in-tegrated optical density units per unit area (IOD/mm)plotted against rDNA copies/genome (Figure 1). Thiscalibration curve (including the correction factor of 5for the 2µg of loaded DNA) was used to estimatethe rDNA repeat unit inSolanumparents and somatichybrids. The results (mean of two independent experi-ments) showed the following rDNA copy numbers pergenome: brod, 1900; sct, 3500; somatic hybrids 83/1,3250; 84/4, 3000; 90/4, 3550; 90/6, 2000.

Total RNA isolated from brod and the somatichybrids (83/1, 90/4 and 90/6) was slot-blotted and hy-bridized to the probe pTa 71. The results (not shown)indicate that the levels of RNA (18S+ 25S) derivedfrom the ribosomal RNA genes were lower in some ofthese somatic hybrids relative to brod. In understand-ing the underlying mechanisms of gene regulation vizthe genotypic versus phenotypic interactions, thesedata indicate low levels of expression, for example forthe polygenic trait plant height: in field-grown sam-ples, there was a height reduction for the hybrids (50,35, 45 cm) relative to brod (67, 55 cm).

rDNA hybridization in RFLP analysis

DNA was extracted from severalSolanumplants (mga,sct, spl, brod) and digested withEcoRV prior toagarose electrophoresis and Southern blotting and hy-bridization with pTa 71 (results not shown). All theseSolanumrDNA repeat units co-migrated the same dis-tance (8.4–9.0 kb); notably, mga show 2 size classes

Figure 3. Luminograph (A) of rDNA profiles in somatic hybridsafter digestion withApaI and hybridization to pPS-6 and (B) thesame genomic DNA fragments in an ethidium bromide-stained gel.

of rDNA consistent with other findings [8, 19, 49,52]. There are differences in rDNA profiles betweenbrod and wild-typeSolanumspecies. The commonrDNA hybridization fragments reflect the highly con-served coding regions within rDNA tandem array.Figure 2 shows several rDNA polymorphisms in mga,sct, spl and brod after digestion withBamHI, EcoRIandBamHI/EcoRI after hybridization with aSolanumspecific intergenic spacer (IGS) probe (pPS-6).

Preliminary screening of the somatic hybrids wasdone with the restriction enzymes (BamHI, EcoRV),the pTa 71 probe after chemiluminescence. An rDNAanalysis was performed on somatic hybrids (35/9,38/1, 94/9 and 87/9) derived from protoplast fusionsbetween brod and sct. These four hybrids appear tohave an rDNA background of cv. Brodick, but hybrids35/9 and 38/1 apparently lack a 4.3 kb double rDNAfragment present in brod but not in sct (results notshown). A comparison between these rDNA fragmentpatterns with morphological data (see [4]) indicatesthe somatic hybrids 35/9 and 94/9 to be intermediatefor these parents based on morphological characters,

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Table 1. Methylation status of the rDNA in parental plants and somatic hybrids

Plant Retriction Density per Percentage of Methylated

material enzymes unit area methylation sequence

brod EcoRV 470 0.0 –

brod EcoRV/HpaII 410 87.2 CmCpGG

brod EcoRV/MspI 265 56.4 mCmCpGG

sct EcoRV 737 0.0 –

sct EcoRV/HpaII 707 95.9 CmCpGG

sct EcoRV/MspI 450 61.0 mCmCpGG

83/1 EcoRV 578 0.0 –

83/1 EcoRV/HpaII 443 76.6 CmCpGG

83/1 EcoRV/MspI 558 96.5 mCmCpGG

84/4a EcoRV 639 – –

84/4a EcoRV/HpaII 479 74.9 CmCpGG

84/4a EcoRV/MspI 290 45.4 mCmCpGG

84/4b EcoRV 554 – –

84/4b EcoRV/HpaII 407 73.5 CmCpGG

84/4b EcoRV/MspI 175 31.5 mCmCpGG

90/4a EcoRV 871 – –

90/4a EcoRV/HpaII 741 85.1 CmCpGG

90/4a EcoRV/MspI 521 59.8 mCmCpGG

90/4b EcoRV 757 – –

90/4b EcoRV/HpaII 527 69.6 CmCpGG

90/4b EcoRV/MspI 536 70.8 mCmCpGG

90/6a EcoRV 797 – –

90/6a EcoRV/HpaII 612 76.8 CmCpGG

90/6a EcoRV/MspI 481 60.4 mCmCpGG

90/6b EcoRV 846 – –

90/6b EcoRV/HpaII 626 74.0 CmCpGG

90/6b EcoRV/MspI 686 81.1 mCmCpGG

whereas 38/1 and 87/9 are morphologically related tocv. Brodick.

Figure 3 shows a selection of putative somatic hy-brids (83/1, 84/4, 90/4 and 90/6) derived from the sct× brod cross digested withApaI and hybridized totheSolanumIGS probe pPS-6. Prior to Southern blot-ting, the ethidium bromide-stained gel revealed somegenomic fragment differences in the somatic hybridscompared to the parents (Figure 3B) whereas, afterrDNA hybridization, these ethidium bromide genomicfragments did not correspond to the rDNA fragmentprofiles (Figure 3A). Clearly, the intermediate profilesindicate possible rDNA hybridity. This may reflectdifferent repeat unit class sizes within the NOR re-

gion due to spacer length heterogeneity resulting fromsomatic rDNA re-arrangements.

rDNA methylation analysis

To examine some of the variable hybridization fea-tures attributable to DNA methylation and possi-ble consequences for gene expression, DNA fromthe parents (sct and brod) and the somatic hybrids(83/1, 84/4, 90/4 and 90/6) was digested withEcoRV,EcoRV/HpaII and EcoRV/MspI and hybridized withthe pTa 71 probe. The methylation-sensitive restric-tion enzymeHpaII and the insensitiveMspI recog-nize the same target sequence CCGG, butHpaIIactivity is inhibited by methylation of the internal

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Table 2. Methylation status of the rDNA in parental plants and somatic hybrids

Plant Retriction Density per Percentage of Methylated

material enzymes unit area methylation sequence

brod EcoRV 167 – –

brod EcoRV/EcoRII 127 76.0 CmCpGG

brod EcoRV/BstNI 0 0 mCmCpGG

sct EcoRV 290 – –

sct EcoRV/EcoRII 197 67.9 CmCpGG

sct EcoRV/BstNI 0 0 mCmCpGG

83/1 EcoRV 298 – –

83/1 EcoRV/EcoRII 258 86.6 CmCpGG

83/1 EcoRV/BstNI 0 0 mCmCpGG

84/4a EcoRV 365 – –

84/4a EcoRV/EcoRII 358 98.0 CmCpGG

84/4a EcoRV/BstNI 0 0 mCmCpGG

84/4b EcoRV 456 – –

84/4b EcoRV/EcoRII 353 77.4 CmCpGG

84/4b EcoRV/BstNI 0 0 mCmCpGG

90/4a EcoRV 377 – –

90/4a EcoRV/EcoRII 333 88.3 CmCpGG

90/4a EcoRV/BstNI 0 0 mCmCpGG

90/4b EcoRV 446 – –

90/4b EcoRV/EcoRII 446 100 CmCpGG

90/4b EcoRV/BstNI 0 0 mCmCpGG

90/6a EcoRV 338 – –

90/6a EcoRV/EcoRII 186 55.0 CmCpGG

90/6a EcoRV/BstNI 0 0 mCmCpGG

90/6b EcoRV 412 – –

90/6b EcoRV/EcoRII 305 74.0 CmCpGG

90/6b EcoRV/BstNI 0 0 mCmCpGG

cytosine (CmCGG), whereasMspI activity is inhib-ited by methylation of the external cytosine in thetarget sequence (mCCGG or mCmCGG). Also, theisoschizomersEcoRII and BstNI recognize the sametarget sequence CC(A/T)GG.EcoRII activity is inhib-ited when the internal cytosine (CmCAGG) is methy-lated; BstNI cleaves several methylated sequences(CmCAGG, mCCAGG and mCmCAGG), but is in-hibited if both external and internal cytosines arehemi-methylated (hmChmCGG).

Figure 4 shows the main rDNA repeat fragment intheEcoRV digests and similar residual low-molecular-weight rDNA fragments presenting a sub-set of rDNAunits within the tandem array. To quantify the de-gree of rDNA methylation, the density per unit area

(IOD/mm) was measured for each repeat fragment perDNA sample. The values for the residual rDNA frag-ments after double digestion were expressed as a per-centage of the total (EcoRV digestion) and indicatesthe degree of sequence-specific methylation withinthe rDNA tandem array. Table 1 shows the methy-lation status of the site-specific sequences within therDNA for the parents and somatic hybrids. These re-sults (mean of two independent experiments) indicatesite-specific sequence methylation of the CmCpGGand mCmCpGG positions for the parents. While thistrend is true for some somatic hybrids, others (84/4a,84/4b) notably display a reduction in the mCmCpGGcompared to those (83/1, 90/4b 90/6b) showing an in-crease in the mCmCpGG sequence. These trends are

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Figure 4. Luminograph showing rDNA profiles of parental plants (brod and sct) and somatic hybrids after digestion withEcoRV, EcoRV/HpaIIandEcoRV/MspI and hybridization to pTa 71.

reflected also in theEcoRII/BstNI profiles (Figure 3and Table 2). The rDNA repeat unit was digested tocompletion in all samples with theEcoRV/BstNI com-bination. TheseEcoRV/BstNI somatic hybrid rDNAprofiles were identical to brod and theEcoRV/EcoRIIprofiles indicate that these sequences were highlymethylated.

Discussion

The analysis of putative somatic hybrids is importantto confirm their hybrid nature. Some biochemical andmolecular markers have been examined forSolanumspecies which include the use of protein profiles [18],rDNA probes [9, 44], species-specific sequences [42]and randomly-amplified polymorphic DNA (RAPD)markers [2, 59]. The object of these procedures hasmainly been to establish a differential molecular pro-file in the parental plants and to compare those profileswith the putative somatic hybrid plants. Apart frommarking the ‘bands on a gel’ to confirm hybridity,studies focusing on the application of molecular tech-niques to examine functional attributes essential forplant growth and development are scanty [35, 37]. Inother reports, agronomic performance as shown in theplant characters, hybrid vigour, yield, maturity andheight [53], key features in plant breeding have beendescribed.

S. sanctae-rosaeis a source of resistance to bothGlobodera rostochiensisand G. pallida [55]. Thebreeding objective in the present study was to transferPCN resistance from wildSolanumspecies via proto-plast fusion intoS. tuberosumcv. Brodick plants. Thescreening of putative protoplast fusion products forthis single trait was an objective, as was developmentof procedures diagnostic of functional characters, asthe many nuclear/cytoplasmic hybrid combinationsmay well affect the plants ability to express theseresistance genes and other genes for essential devel-opmental functions. This was seen in the variabledegree of PCN resistance found in the range of somatichybrid crosses. Resistance values againstG. pallidarange from susceptible brod (mean 15/20 cysts/rootsystem) to a mean of 18 cysts/root system in hybrid83/1; 9 cysts/root system in 83/4; 8 cysts/root systemin 90/4 and 9 cysts/root system in 90/6 (F. Dale, pers.comm.). Equally so, the bringing together of two evo-lutionarily distant genomes is likely to be disruptive tothe cell cycle, the ordering of chromosomes, and thecell signal-trigger mechanisms essential for the timelyexpression of gene sequences. The function of thesecellular factors will most probably have an influenceon the genes and metabolite pathways determinativeof agronomic performance.

DNA methylation is known to play a role in theformation of heterochromatin know for its transcrip-tional inactivity [54, 56]. The findings reported hereindicate changes in DNA methylation pattern in the

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Figure 5. Luminograph showing rDNA profiles of parental plants(brod and sct) and rDNA profiles in somatic hybrids after digestionwith EcoRV, EcoRV/EcoRII andEcoRV/BstNI and hybridization topTa 71.

somatic hybrids (see Tables 1 and 2). The differ-ence between (a) and (b) somatic hybrids is the timeof leaf collection for glasshouse-grown plants (Fig-ures 4 and 5): (a) represents the start of the growingseason (May) whereas (b) is ca. 3–4 months later.Clearly, there appears to be a seasonal effect where theplants’ ‘methylation status’ increases towards matura-tion. DNA methylation has been negatively correlatedwith gene activity [39], and it may account for thisobservation as gene expression is known to diminishwith senescence. DNA methylation has been shown toinhibit gene expression in protoplasts [27], and thereis likely to be some residual effect of somaclonal vari-ation resulting from protoplast isolation and culture[46]. There were variable levels of DNA methylation

in the rDNA of the somatic hybrids relative to theparental plants, and this ‘methylation imprint’ may ex-ist in other regions of the genome influencing patternsof gene expression [39] and DNA repair processes[10] resulting from protoplast fusion and heterokaryonformation.

There are reports of potato genomic instabilityresulting from in vitro culture, for example, a re-duction in the number of rDNA copies [30], severalrDNA RFLPs [20] and variable hybridization rDNAfragments [22, 24]. Qualitative changes showing theappearance of ‘new’ rDNA repeats were detected inprotoplast-derived plants [45]. Similarly, the use ofunknown repetitive DNA sequence probes showedvariation in the intensity of the hybridization signal intwo potato somaclonal variants [3]. Studies here quan-tify the rDNA repeat (copy) number in the diploid wildspecies (mga, sct and spl) and cultivated tetraploidbrod. Where the euploid base numbern = x = 12,therefore, diploids (2n) have 24 chromosomes andtetraploids (4x, 4 genome copies) have 48 chromo-somes. The symmetric fusion products predictablyhave 24 plus 48 chromosomes equivalent to 6 genomecopies. This requirement also assumes a single NORregion per haploid set and each NOR region containsan equal number of rDNA repeat units. The rDNAcopy number values findings here are consistent withother reports for Solanaceous species [49, 52].

The rDNA is located in the NOR region at thedistal end of chromosome 2 [6, 7, 12] and this re-gion is proposed to be a ‘hot spot’ for genetic changevia recombinational mechanisms [49]. This may wellfacilitate chromosomal exchange during heterokaryonformation [57]. The precise location of the rDNA lociwithin the nucleolus is controversial and their pat-terns of gene expression remain unclear [47] but thepositions of actively transcribed genes within rDNAtandem arrays have been shown to be fundamen-tally different in wheat and rye [32]. Sexual hybridsbetween wheat and rye often show suppression of ac-tivity of the NOR region on chromosomes of rye whenin competition with major nucleolar organising chro-mosomes of wheat and this has lead to the model forrDNA expression within the nucleolus [31].

Plant protoplasts are suitable tools for physiologi-cal studies [16] and, regarding findings reported here,the formation of somatic hybrids is a highly appropri-ate model to study fundamental processes in geneticsand molecular biology. Also these results, with rDNAprobes, show the presence of RFLPs as a diagnostictool to aid species/cultivar identification. This ability

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to recognize RFLP and IGS rDNA length variants isof significance to phylogenetic studies [8] and plantbreeding and provides important analytical tools toexamine possible changes resulting fromin vitro cul-ture. This work further demonstrates the application ofrDNA probes to identify regions of chromosome 2 aspossible recombinational ‘hot spots’ upon somatic hy-bridization. This was a useful approach to gain insightinto the gene mechanism, where methylation profilesdiffering between somatic hybrids are indicative oftranscritional inactivity [56]. This has significance inunderstanding the factors contributing towards agro-nomic performance and residual tissue culture effectslikely to influence both single and polygenic traits.

Acknowledgements

The authors kindly acknowledge the assistance fromSCRI staff and the financial support (RO432) fromSOAFD and the Potato Marketing Board.

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