Chromosome pairing in Solanum commersonii–S. tuberosum sexual hybrids detected bycommersonii-specific RAPDs andcytological analysis
Amalia Barone, Angela Sebastiano, and Domenico Carputo
Abstract: BC1 pentaploid to near-pentaploidSolanum commersonii–S. tuberosumgenotypes obtained from 3x × 4xcrosses have been used for cytological and molecular studies on chromosome pairing and meiotic recombination.Microsporogenesis analysis at diakinesis revealed that chromosomes had a tendency to pair as bivalents, thoughmultivalents were also observed in many cells. Meiosis resulted in a high production of tetrads and pollen stainabilitywas high, ranging from 34.5% to 92.1%. DNA fromS. commersoniiand S. tuberosumwas amplified with 87decameric primers, resulting in the identification of 26S. commersonii-specific RAPDs. The analysis of these RAPDsin BC1 genotypes showed that a number ofcommersonii-specific markers was present in all BC1 genotypes. On theother hand, markers which were missing in one or more of the BC1 genotypes suggested that in some caseshomoeologous pairing occurred. Two linked markers were transmitted together in all of the BC1 genotypes except two.This provided evidence that a recombination event between these markers occurred during megasporogenesis of theirtriploid parents. The possibility of demonstrating other recombination events is discussed.
Résumé: Des génotypes BC1 Solanum commersonii–S. tuberosumpentaploïdes ou quasi-pentaploïdes obtenus suite àdes croisements 3x × 4x ont été utilisés pour examiner l’appariement chromosomique et la recombinaison méiotique àl’aide d’analyses cytologiques et moléculaires. L’analyse de la microsporogénèse en diacinèse a révélé que leschromosomes avaient tendance à former des bivalents bien que des multivalents aient également été observés chez denombreuses cellules. La méiose a produit de nombreuses tétrades et la fréquence de coloration du pollen était élevéepuisqu’elle variait entre 34,5% et 92,1%. L’ADN duS. commersoniiet du S. tuberosuma été amplifié avec 87 amorcesdécanucléotidiques ce qui a permis d’identifier 26 marqueurs RAPD spécifiques duS. commersonii. L’analyse de cesmarqueurs chez les génotypes BC1 a montré que plusieurs marqueurs spécifiques duS. commersoniiétaient présentschez tous les génotypes BC1. Par contre, l’absence de certains marqueurs chez un ou plusieurs génotypes BC1 suggéraitque des appariements homéologues avaient eu lieu. Deux marqueurs liés ont été transmis ensemble chez tous lesgénotypes BC1 sauf deux. Cela indique qu’une recombinaison s’est produite entre ces marqueurs durant lamégasporogénèse chez les parents triploïdes. La possibilité de démontrer l’existence de d’autres événements derecombinaison est discutée.
Mots clés: triploïde, pentaploïde, microsporogénèse, flux génique, recombinaison.
[Traduit par la Rédaction] Barone et al. 224
The wild tuber and non-tuber bearing relatives of the cul-tivated potatoSolanum tuberosumgroup Tuberosum (tbr)possess all the desirable traits lacking in the standard variet-
ies (Peloquin and Ortiz 1992). Among these wild species,S. commersonii(2n = 2x = 24) (cmm) has several valuabletraits, including resistance to pathogens and pests, and ahigh dry matter content in tubers (Hanneman and Bamberg1986). This species is also noteworthy for its resistance tolow temperatures. Palta (1994) reported a freezing toleranceof –4°C and the ability to cold acclimate down to –9°C.
Cmm is sexually isolated from both tbr haploids and sev-eral other 24-chromosome species. Johnston and Hanneman(1982) assigned to this species an endosperm balance num-ber (EBN) of 1. According to the EBN hypothesis formu-lated by Johnston et al. (1980), it cannot be crossed with tbrhaploids (2EBN) or other 2EBN diploid species due to a de-viation from the 2:1 maternal–paternal EBN ratio in the hy-brid endosperm. This 2:1 EBN ratio is a necessary conditionfor the normal development of the endosperm in the hybrid.Different strategies have been employed to overcome the
Received April 1, 1998. Accepted September 8, 1998.
A. Barone.1 C.N.R.–I.M.O.F., Research Institute forVegetable and Ornamental Plant Breeding, Via Università133, 80055, Portici, (Na) Italy.A. Sebastiano and D. Carputo.Department of Agronomyand Plant Genetics, University of Naples, via Università 100,80055, Portici, (Na) Italy.
1Author to whom all correspondence should be addressed(e-mail: [email protected]).
sexual isolation of this interesting species. Somatic hybrid-ization between cmm (+) haploid tbr was obtained by Cardiet al. (1993), and resulted in tetraploid, hexaploid, andaneuploid hybrids with different levels of fertility. Sexualhybridization through ploidy and EBN manipulations hasalso been used to generate hybrids between cmm and tbr. In-direct transfer of the cmm genome through the bridge spe-cies S. chacoenseand S. gourlayi has been reported byElhenfeldt and Hanneman (1984) and Masuelli et al. (1992),respectively. Recently, direct transfer to tbr has beenachieved by crossing a tetraploid (2n = 4x = 48,2EBN) cmm clone with diploid Phureja-Tuberosum haploidhybrids (2n = 2x = 48, 2EBN) (Carputo et al. 1995).Through the function of 2n eggs, the triploid F1 hybrids ob-tained were used in 3x × 4x crosses with tbr varieties to gen-erate the first backcross (BC1). Two BC1 genotypes provedto be male and female fertile in crosses with cultivated vari-eties (Carputo et al. 1997). The fertility of these two geno-types is extremely interesting considering that odd ploidy ofBC1 genotypes can be the bottleneck in making thebackcross program successful.
A number of 3x × 4x cmm–tbr hybrids from differentcross combinations are now available. They represent usefuland unique materials as a link between the 1EBN diploidlevel of cmm and the 4EBN tetraploid level of tbr. Further-more, they represent elite genotypes for genetic studies,since they originated through the function of 2n eggs froman odd ploidy parent and thus may have unique chromosomenumbers. In this paper, various 3x × 4x cmm–tbr hybridshave been characterized through cytological and molecularanalysis in order to (i) determine their chromosomal consti-tution and (ii ) study the potential for homoeologous pairingand genetic recombination between cmm and tbr genomes.
Plant materialThe eleven BC1 genotypes analyzed were produced by Carputo
et al. (1997) from 3x × 4x crosses between four cmm–tbr triploidhybrids (A1, B3, B10, C1) and four tbr tetraploid genotypes(‘Blondie’, ‘Carmine’, ‘Tollocan’ varieties, and Wis 482 advancedselection). Seeds obtained from 3x × 4x crosses were treated withgibberellic acid (1500 ppm) for 24 h to break dormancy, sterilizedwith sodium hypochloride (10% for 20 min), rinsed several timeswith sterilized water, then sown in Petri dishes with Murashige andSkoog salts (1962), 1% sucrose, and 0.8% agar. From the obtainedseedlings, nodes with axillary buds were excised and cultured inMagenta GA7 vessels supplemented with the same medium de-scribed above, at 50 mmol–2s–1, with 16 h light, at 24°C. In vitroplants were used as a source of root tips for mitotic analysis andthen transplanted into pots in a temperature-controlled greenhouseto collect buds for meiotic studies and leaves for molecular analy-sis. Tubers of cmm (PI 243503), of the triploid hybrids and of thefour tbr tetraploids were planted in the same greenhouse to collectleaves.
Cytological analysisRoot tips were treated with 0.29 g/L 8-hydroxyquinoline for 5 h
and fixed in a 3:1 ethanol–acetic acid solution for 48 h. Followinghydrolysis for 55 min in 5N HCl at room temperature, the root tipswere stained for at least 2 h with Schiff’s reagent and squashed in45% acetic acid. For meiotic analyses, flower buds were fixed in a3:1 ethanol–acetic acid solution saturated with ferric chloride as amordent. Anthers were dissected and squashed in 1% acetocarmine
solution. Diakinesis, anaphase I, and anaphase II of pollen mothercells (PMCs) were examined. Pollen viability of the 3x × 4x hy-brids was estimated by staining fresh pollen of flowers collectedfrom greenhouse grown plants with 1% acetocarmine.
RAPD analysisThe PCR protocol reported by Williams et al. (1990) was
adapted with minor modifications on DNA extracted from leaf tis-sue following the procedure of Dellaporta et al. (1983). Each reac-tion consisted of 1× buffer (50 mM KCl, 10 mM Tris–HCl, pH 8.3,1.5 mM MgCl2, 0.001% gelatin), 0.1 mM of each dNTP, 0.2 mMprimer, 1.0 unit Taq DNA polymerase, and 20 ng genomic DNA.The thermocycler (Perkin Elmer Cetus DNA Thermocycler 480)was programmed for 45 cycles of 1 min at 94°C, 1 min at 35°C,2 min at 72°C, and a 7 minextension at 72°C. Amplification prod-ucts were separated by electrophoresis on 1.5% agarose gel in 1×TAE buffer. The primers employed, each 10 deoxyribonucleotidesin length were: (i) commercial 10-mers Operon Technologies(Alameda, California) from kits AN and H; (ii ) commercial 10-mers from the University of British Columbia (UBC set 100/1);and (iii ) 10-mer randomly designed and commercially synthesizedby PRIMM s.r.l. (Italy).
Southern analysis of PCR gelsPCR gels were denatured, neutralized, and Southern blotted
onto a nylon membrane (Amersham, N+) as described bySambrook et al. (1989). Cmm-specific RAPD fragments were ex-cised from the gel as a thin gel slice and purified using the QiagenGel Extraction Kit. The reamplified DNA was then labelled with a32P[dCTP] using the random priming reaction. DNA hybridizationand washings were performed as described by Gebhardt et al.(1989).
Table 1 reports chromosome number of BC1 genotypescoming from 3x × 4x crosses. Four hybrids were pentaploid(2n = 5x = 60) whereas all the others were aneuploid–pentaploids, with chromosome numbers ranging from 58(P11 and P15) to 67 (P6) (Fig. 1). Meiotic analysis showedthat metaphase I chromosomes had the tendency to clumptogether in a narrow spindle, making it difficult to observechromosome configurations at this meiotic stage. However,
at diakinesis spreading was sufficient to distinguish chromo-some associations. Chromosomes had a tendency to pair asbivalents but multivalents, either in chains or in rings, werealso observed in most PMC (Fig. 2). Anaphase I and II weretypical of odd ploidy genotypes with various chromosomedistribution at the poles (Fig. 2). Lagging chromosomeswere observed at anaphase I and II, but most of them wereincluded in the chromosome complement, since at telophaseI and II most PMC did not contain micronuclei (Fig. 2).
Meiosis resulted in a high production of tetrads with orwithout micronuclei and stainable pollen. Pollen stainabilitywas high (Table 1) ranging from 34.5% (hybrid P13) to92.1% (hybrid P3). All plants were vigorous and floweredprofusely, except two hybrids (P1 and P11) which did notflower at all.
DNA from PI 243503 of cmm and four tetraploid tbr wasused as a template for PCR amplification with 87 decamericprimers. The amplification product sizes ranged from 300 to
Fig. 2. Pollen mother cells ofS. commersonii–S. tuberosumsexual hybrids. (A and B) Diakinesis with a ring of four (r) and chains (c)of multivalents in hybrids P15 (2n = 58). (C) Anaphase I cell with 30–32 chromosome distribution in hybrid P1 (2n = 62).(D) Telophase II cell with no micronuclei in hybrid P5 (2n = 60).
3000 base pairs (bp), most of them falling in the 500–2000bp range. Eleven primers (12.6%) did not yield any product;23 primers (26.4%) were polymorphic and generated 28RAPDs present in cmm but not in tbr (Fig. 3). Southernanalysis of PCR gels was performed to confirm the specific-ity of these primers: it showed that two RAPDs selected onparental genotypes as cmm-specific markers hybridized withtbr even though no DNA of the corresponding size was visi-ble on PCR ethidium bromide stained gels. Only 26 RAPDswere actually present in cmm but not in tbr and thereforewere considered cmm-specific markers useful for our analy-sis.
Table 2 reports primer sequences and molecular weightsof 26 cmm-specific RAPDs tested on the four triploid cmm–tbr hybrids used to generate the BC1 genotypes. All themarkers tested were present in triploids B3, B10, and C1whereas triploid A1 lacked one cmm-specific marker (UBC-12), thus suggesting a heterozygous condition of this markerin the diploid parent cmm. The 25 cmm-specific markersoriginally present in all the triploid parents were also testedon eleven pentaploid to near-pentaploid BC1 genotypes. Outof 25 cmm-specific primers, 13 (52%) were present in allBC1 genotypes, whereas 12 sometimes were absent in somegenotypes (Fig. 4). In particular, seven markers (28% of the25 cmm-specific markers) were missing in one genotype,four (16%) were missing in 2 genotypes, and 1 (4%) wasmissing in 4 genotypes (Table 3). The presence of markersin the BC1 genotypes ranged from the P3 hybrid (2n = 60),
Fig. 3. Selection ofSolanum commersonii-specific RAPDs byPCR amplification of genomic DNA fromS. commersoniiandfour S. tuberosumtetraploids. The DNA was amplified withprimer UBC28 (CCGGCCTTAC). M indicates a molecularweight marker. Size markers in kilobases are shown at the right.
Table 2. Presence (+) ofSolanum commersonii-specific RAPDs in four triploid (3x) S. commersoni–S. tuberosumhybrids.
which contained all the markers, to the P15 hybrid (2n = 58)which lacked five markers.
The introgression of wild traits into cultivated speciesrequires meiotic recombination between homoeologouschro-mosomes. A prerequisite for recombination to occur is chro-mosome pairing and crossing over during meiosis, processeswhich are greatly determined by the relationship betweenthe genomes of the parental species. In fact, if the chromo-somes of the related species are not highly differentiated,chromosome pairing and recombination between homoeo-logous chromosomes may occur. The occurrence of homoeo-logous recombination between cmm and tbr is expected.Through RFLP analysis of chloroplast DNA Hosaka et al.(1984) reported that these two species are quite closely re-
lated. Matsubayashi (1991) further confirmed the relation-ship between these two species, and assigned the so-called“A genome” to both of them. Since chromosome pairinggreatly determines the extent of meiotic irregularities, andthus the fertility of the gametes, the pentaploid and near-pentaploid cmm–tbr hybrids obtained offer a number ofpractical applications. In fact, most of them are male and fe-male fertile (D. Carputo et al., in preparation), and thus canbe successfully used in backcross programs. Out of elevenBC1 genotypes analyzed, six were aneuploid pentaploid,with 58 to 67 chromosomes. The production of BC1 geno-types with such chromosome numbers provided evidencethat meiotic restitution involved poles with various chro-mosome numbers, as expected from chromosome distribu-tion at anaphase I of macrosporogenesis of a triploid. As aresult of this, 2n eggs with 34, 36, 38, 40 etc. chromosomeswere functional in the 3x × 4x crosses which generated our
Fig. 4. Screening of theSolanum commersonii-specific RAPD UBC28 (CCGGCCTTAC) on four triploids and on eleven pentaploid tonear-pentaploidS. commersonii–S. tuberosumhybrids. M indicates a molecular weight marker. Size markers in kilobases are shown atthe right.
BC1 progeny. The occurrence of genotypes with 63 and 67chromosomes is probably due to the occasional omission ofone chromosome from a restitution nucleus.
It is expected that during backcrosses, portions ofthe cmm genome will be progressively lost, possibly com-bined with homoeologous recombination. For this reason,molecular markers that identify cmm genome would facili-tate the analysis and the selection of backcross progeny.DNA-based markers have already been used to verify theintrogression of wildSolanumspecies into the cultivated po-tato gene pool (McGrath et al. 1994; Masuelli et al. 1995).Among the various techniques, the PCR-based one, knownas randomly amplified polymorphic DNA (RAPD) (Wil-liams et al. 1990), has been used in the present study to fol-low the introgression of the cmm genome fromtriploid cmm–tbr hybrids to pentaploid or near pentaploidBC1 genotypes obtained through the function of 2n eggs ofthe triploid parent.
Molecular analysis by RAPDs allowed the selection of26 cmm-specific markers. Only bright bands were consid-ered during the evaluation and each PCR-amplification wasreplicated three times to verify band reproducibility.The cmm-specific RAPDs were all homozygous in the dip-loid parent cmm, except marker UBC-12, which was hetero-zygous, as suggested by its absence in the triploid A1. Thismarker was not analyzed on BC1 genotypes since its pres-ence or absence would be the result of segregation ratherthan chromosome pairing. The analysis of all other cmm-specific markers in BC1 genotypes showed that a numberof cmm-specific RAPDs was always present in all of theBC1 genotypes. This can be the consequence of homologouspairing between the two cmm chromosomes, but may wellbe the result of pairing between cmm and tbr chromosomesfollowed by crossing over between the centromere and themolecular marker. In the first case, all derived 2n eggs of thetriploid parent will show the cmm-specific marker. In thesecond case, 2n eggs will show the cmm-specific marker de-pending on the distribution of the cmm chromosome whichdid not take part to the homoeologous pairing.
The cmm-specific markers lacking in one or more of theBC1 genotypes suggested that in some cases ahomoeologous pairing occurs with the formation of biva-lents and (or) multivalents between cmm and tbr chromo-somes. Diakinesis analysis of triploid hybrids confirmedtrivalent formation, even though the tendency of chromo-somes to clump together in the equatorial plate made it diffi-cult to obtain detailed data on chromosomal configurations.However, Masuelli and Camadro (1992) found a mean num-ber of 4.8 trivalents/cell in triploids between cmm andS. gourlayi, belonging to seriesTuberosa, the same as in tbr.Based on cytological analysis, the authors suggested a highhomology between cmm andS. gourlayiand tbr. Cytologicalstudies on our pentaploids and near-pentaploid genotypesconfirmed the potential for homoeologous pairing be-tween cmm and tbr genomes. Multivalent associations of upto five chromosomes were observed even though chromo-some pairing was mainly between bivalents. The high fre-quency of bivalents was expected, since this is a commonfeature of most 24-chromosome species and hybrids(Swaminathan and Howard 1953). High frequency of biva-
lents does not exclude homoeologous pairing since short po-tato chromosomes may not allow multivalent association, asalso reported in alfalfa (Obajimi and Bingham 1973).
Molecular analysis suggested evidence not only forhomoeologous pairing but also for genetic recombinationbetween cmm and tbr genomes. In fact, a tight linkage be-tween markers OPH3 and UBC28 has been found in the BC25x × 4x progeny between pentaploid P5 and cultivar Blondie(data not shown). In the BC1 pentaploid to near-pentaploidpopulation studied here these two markers were transmittedtogether in all genotypes except P1 and P15. This providedevidence that a recombination event between these markersoccurred during megasporogenesis of P1 and P15’s triploidparents (B10 and A1, respectively). Furthermore, it might bepossible to demonstrate other recombination events. In fact,it is well known that theoretically molecular markers distrib-ute randomly on chromosomes, thus other cmm-specificmarkers which are lacking could be assigned to chromo-somes carrying one or more of the 13 markers which are al-ways present. Assignment of the 25 cmm-specific RAPDmarkers to association groups and their localization on thepotato map will determine the extent of recombination oc-curring between cmm and tbr, and will indicate which chro-mosomes are involved in pairing and recombination. RFLPmarkers already mapped on the RFLP potato map (Gebhardtet al. 1991) are currently being tested together withthese cmm-specific RAPDs in a tetraploid near tetraploidBC2 progeny produced from 5x × 4x crosses.
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