GENETIC ANALYSIS OF ISOZYME LOCI IN TETRAPLOIDPOTATOES (SOLANUM TUBEROSUM L.)

J. M. MARTINEZ-ZAPATER AND JOSE L. OLIVER

Departamento de Genética C-XV Facultad de Ciencias, Universidad Autónoma de Madrid,Madrid-34, Spain

Manuscript received February 27, 1984Revised copy accepted June 6. 1964

The genetic control of eight isozyme loci revealed by starch gel electropho-resis was studied through the analysis of three progenies derived from fourtetraploid cultivars of Solanum tuberosum (groups Andigena and Tuberosum).Duplicate gene expression was found in seven (Got-A, Got-B, Pgd-C, Pgi-B, Pgm-A, Pgm-B and Pa-C) isozymee loci. In another isozyme gene (Adh-A), the paren-tal genotypes were not adequate to diiinguish between monogenic or adigenic model of genetic control. Tetrasomic inheritance was demonstrated infour (Got-A, Got-B, Pgd-C and Pgi-B) isozyme loci. In the remaining duplicategenes, the parental genotypes precluded discrimination between disomic orttetrasomic models. Tetrasomic segregations of the chromosomal type weregenerally found. however, the isozyme phenotypes shown by three descendantsfrom selfing cv. Katahdin indicate the occurrence of chromatid sqregations.Although aneuploidy cannot be ruled out. Either autoploidy or amphidiploidywith lack of chromosome differentiation between the two diploid ancestors canaccount for the existence of traasomic inheritance in the common potato.

ISOZYME techniques have proved to be a very useful tool in biochemicalgenetic studies of both alloploid (ROOSE and GOTTLIEB~ 1976) and autoploid

(QUIRÓS 1982, 1983) plant species; they allow the analysis of many geneticmarkers of codominant expression. Allelic isozymes (allozymes) can be used asmarkers of chromosomes or chromosome regions for the design of selectionand breeding experiments. The use of allozymes as markers of loci in closeassociation with genes for given traits will be crucial to the future success ofplant breeding and genetic engineering (WHITT 1983). The elucidation of thegenetic control of isozyme systems is also necessary for their use as markersfor both varietal identification and phylogenetic analysis.

Cultivated potatoes belong to the series Tuberosa Rydb. of the genus So-lanum. Differences exist in the number of species considered by distinct sys-tematic classifications. Although HAWKES (1956a, 1978) recognizes eight spe-cies of cultivated potatoes, DODDS and PAXMAN (1962) consider only one species, S. tuberosum L., with five main groups: Stenotomum ( 2 x = 24). Phureja(2x = 24), Chaucha (3x = 36). Andigena (4x = 489 and Tuberosum (4x = 48).The derivation of the potato cultivated in the Northern Hemisphere (S. tub-erosum group Tubemsum) from the Sudamerican tetraploid potatoes is a well-

670 J. M. MARTINEZ-WPATER AND J. L. OLIVER

established fact, although some dispute remains about the group from whichit vtas derived (Andigena from Peru and Northern Bolivia vs. Tuberosum fromChile) (see SWAMINA~AN and MAG~ON 1961; H OWARD 1970; UCEKT 1970;HAWKEZS 1978; GRUN 1979 for revisions), Different polyploidization mecha-nisms have been implicated for the origin of cultivated tetraploid potatoes:autoploidy from the diploid group Stenotomum (HAWKES 1956b). intervarietalautoploidy (ST~BSNS 1957), segmental alloploidy (MATSUBAYASHI 1960) andamphidiploidy from Stenotomum and S. vernei (BROCHER 1964) or from Sten-otomum and S. sporsi@um (HAWKES 1967; HOWARD 1973; but see WOODCOCK

and HOWARD 1975). Because the frquency of multivalent associations atmeiosis varies among different potato cultivan, cytogenctic data have nat pro-vided sufficient evidence to decide which of these mechanisms is involved(SWAMINATHAN and MACOON 1961; HOWARD 1970). Tetrasomic ratios of in-heritance have been established only in a few genes controlling both morpho-logical characters and diseax resistances; furthermore, some of the reportedsegregations can be also explained by monogenic models (SWAMINATHAN andMACXK~N 1961; HOWARD 1370). More genes must k analyzed before anystrong inference on the nature of ploidy can be drawn.

The basic problem with most of the traits genetically studied in the potatountil now is the lack of equivalence between phenotype and genotype due tocomplications introduced by varying he&abilities, dominance, epistasis andpleiotropy. The study of isoryme genes avoids all of these problems, allowingthe analysis of the complex segregations that can be expected in a polyploid.

We have undertaken an electrophoretic analysis of several enzymes in diploidand tetraploid groups of cultivated potatoes, as well as in some related wildspecies. The use of isozyme phenotypes for the identification of 67 Tuberosumvarieties, inciuding those of greatest agronomical interest in Europe and NorthAmerica, has been previously reported (MARTINEZ-ZAPATER and OLIVER1984). The phylogenetic relationships, as deduced from the analysis of genefrequencies, will be published elsewhere (OLIVES and MARTMEZ-ZAPAITLR1984). We report here the inheritance analysis that we have carried out infour tetraploid cultivars at eight variable isozyme loci.

MATERIALS A.!W METHODS

Three progenies from four tctnploid cultivars of S. hrbrrosum were analyzed: cv. Katahdinulfed, cv. Turia selfed and BUCGI 9 x DTCWM.

The number of plants arulyzcd at each isozymc @i is shown in Tables 2-4. Katahdin. Turkand Bucsa are typical Tuberosum cultivars, whereas the clone DTQSS is an Andigcna one. !ke&of the last two progenies and tukn of all these cultivan were provkkd by Estaci& de Mejorade la Patata, Vitoria, *in. To obtain berries, cut stems with flowers were taken from f-n-nplants and placed in jars of water. Plants were grwm from 8ccds under uniform condition5 in thegreenhouse.

Three organs of the plant were analyzed: young kavcs [&hoglucm isomcnsc VW. Wphoglucomutasc (PCH) and G-phosphogl uconate dchydrogcnau (PGD)]. tubtrs [akobol &hydra-gtnast (ADH) and glutamatt ouloacttatc transa&use (COT)]. and shoots [pmxida~ (POX)].In order to prrvcnt browning, the enzyme extra&on was accomplish4 by crushing the plantmaterial in a bufkrcd solution of vvtral reducing agents (VALIwDuI 1977). For PCD and PGMenzymes, gtyccrol (10%) was added to the exttactim buffer (ROOSE and GorrLm 19&o). Tht

ISOZYMES OF CULTIVATED POTATOES 671

TABLE 1

Atloz~ r&t& m&iliG~ d di&mt iwxpu s)ukm in the potato cultiwn cnalyud in tliisstudy

Allozymcslvuymc*yncm l b c d

ADH.A 0.51 0,47GOT-A 0.46 0.40COT-B 0.22 0.14PCDG 0.72 0.70PCI-B 0.37 0.33PGM-A 0.52 0.50PC&B 0.40 0.34POXC 0.54 0.50

enzyme extracts were &orbed directly onto paper wicka and subjected LO horizontal starch gelckctrophor&, with LiOH/bonte @H 8.1) electrode buffer and Tris/citmtc @H 6.3) gel buffer(&ANDER rl d. 1971). For PCD WC &ckd EDTA 0.4~ to gel and ekctrode buffers. SPmpla ofcv. Denim were included in all of the shb gels as internal marken to determine the elcct~rcticmobilities of the different &qmc bands.

Different g4 slicer ycrc assayed for six consistently rorabk enzymes: ADH [EC 1.1 .l.i (PA+ntmt 1973)). COT [EC 2.6.1.1 (Ganues 1973)], PC1 [EC 5.3.1.9 (Buxwut 1970)], PCM [EC2.7.5.1 (Bnmen 1970)); POX [EC 1.11.1.7 (SHAW and PRMAD 1970) with the pH modifKd at4.5 according to RJCK, ZOML and FOWS (1974)], PGD [EC 1.1.1.43 (BREW&R 1970)).

Isozyme system, locus and alkk kttcr names were assigned following an ekctrophomic surveyof different dipioid and tctrapbid groups of S. tirosutn and of two diploid wild spcka, S.zpors@ilWm and S. pinnob’udw (OuvEm and MARR~NWZAP~TCR 1984). lrozymc system namc~begin with a apitaliud abbreviation for an already rccognued enzyme name (e.g., GOT); theisozymc ryunn with tk mat anodal migmtion was designated A, the next B and so forth (e.g.,GOT-A, GOT-B). In order to distinguish the isozyme locus from the protein it encoded, thecorraponding abbrcviatioa (#.g., Co&A, C&3) is italicized. For each isozymc locus, the alkle withthe grateat relative mobility was alkd (D, and then b, c, d, etc.

RESULTS

Electrophoretic patterns from 11 dinerent tissues and organs ( MARTINEZ-ZAPATER 1983; MAJ~TINEZ-ZAPATER and OLIVER 1984; OLIVER and MARTINEZ-ZAPATFJ~ 8984) provided an estimate of the number of isozyme systems foreach enzyme in Tuberosum cultivars, Eight isozyme systems were selected forstudy through progeny analysis. The relative mobilities of the alleles found inthe cultivars analyzed here are shown in Table 1. In addition, other electrophoretic bands, whose presence cannot be genetically explained, were detectedin PGD and PC1 enzymes. These were attributed to epigenetic modifications.Those detected at PC&B were similar to those reported by STAUB et al. (1982).

The four tetraploid cuitivars analyzed here were variable for some of theeight isozyme systems. Different classes of putative heterozygotes with reciprocal asymmetric banding intensities were observed. Let us consider, for ex-ample, a dimeric kqme system, ADH-B, for which five electrophoretic phe-notypes were found (Figure I). In addition ‘to the normal tribanded hetero-

672 J. M. MARTINEZ-ZAPATER AND J. L. OLIVER

RGVRE I .-Phenotypes for a dimeric potato isorymc (MH-B) in the twaploii group An&gemof S. Woncm. Note the change in the relative staining intcnsi~ of the hcterudimcric and ho-madimcric bands in symmetric tripk-bandcd phenotypes (lane 3) if compared with the asymmetricones .(hnn 5,6 and 7). Thus, in Lhc symmetric heterozygow the hcvrodimn (middle band) showsthe hi intensity. However, in the asymmetric hctcrtxygota one of the homodimcrr (stowband in lanes 5 and 6, fast bnd in lane 7) is the most sxaincd. Tbc f&lowing genotypes can kassigned: I and 2, cccc; 9, ootc; 4, ~oli; 5 and 6, octc; 7, mat.

zygote (lane S), two other tribanded phenoqpes with asymmetrical bandingintensities were observed: the first one has the slow bomodimeric band moreintensely stained (lanes 5 and 6). whereas the second one represents the reciprocal situation with the fast homodimeric band more intensely stained (lane 7).Slow (lanes 1 and 2) and fast (lane 4) homozygotes were also observed. Sincegroups Andigena and Tuberosum are recently originated tetraploids (UCENT,POZORSKY and POZORSKY 1983) duplicate gene expression must be expected(OLIVER cf al., 1983). In plants, gene dosage can result in an increase in theamount of gene product and, consequently, the relative intensity of individualelectrophoretic bands (CARLSON 1972; DeMaccro and LAMBRUKCS 1974;ROCBE and GWI-LIEB 1980). Thus, the best explanation for these phenotypesis that they are due to gene dosage effects (see, for example, ALLENDORF,UTTER and M AY 1975). That the relative band intensity is a result of genedosage in the potato is further supported by the obsenation that triploid plantsof varieties Negra and Chaucha Colorada always show an asymmetric pheno-type at at1 those isozyme loci for which they are heterozygous. In tetraploidpotatoes, reciprocal asymmetric banding intensities can be readily detected onthe gels, and they were observed at each one of tbe eight variable isozymesystems; thus, a genotype can be deduced for each electrophoretic phenotype(Figure 1).

For each isozyme locus, we analyzed those progenies in which either one orboth parents were putative heterozygotes. Consideration of gene dosage effectswas not necessary to discriminate between the different genetic models. We

I!iOZYMES OF CULTIVATED POTATOES

TABLE 2

Segrqpiim of b and c alleles at A&-A isoryw &cut

6 7 3

Parenu

BUESA X DTO-53(bbbc x bbbb)

Phcnot~

Offspring X’ r

bbbb bbbcOhs.. no. 107 90EkR. ratio 1 1 1.47 0.23

tip., expected; ohs, observed.

tested first the hypothesis of a monogenic control, If this possibility was ruledout by x’ tests, the hypothesis of a digenic model with either disomic ortetrasomic inheritance was then tested. Once a particular mode of inheritancewas demonstrated, gene dosage effects were taken into account to again testthe particular model that follows each isozyme gene. For the sake of brevity,we only present here the tests that consider gene dosage effects (Tables 24).For the analysis of Pgd-C in the cross Buesa X DTO-33 (Table 4) all of thetribanded phenotypes were pooled together; the corresponding gels were notwell enough resolved to score gene dosage effects, and the analyses could notbe repeated due to the destruction of the plants for the first analysis.

The results obtained for Ad&A are shown in Table 2. Buesa shows a tri-banded electrophoretic phenotype with the fast homodimeric band more in-tensely stained, whereas DTO-33 shows only one band, corresponding to thefast homodimer. The progeny shows these two phenotypes in a 1:l ratio,indicating that variation observed for this isozyme system was under gene&zcontrol. However, a 1:l ratio would be expected whether control is monogenic(parental genotypes: b/c X b/b) or digenic (disomic: b/b b/c X b/b b/b; ortetrasomic: bbbc X bbbb), precluding discrimination between these geneticmodels.

For the remaining seven loci, evidence was obtained that supports duplicategene expression (Tables 3 and 4). The observed segregations for Pgm.4, Pgm-B and Pox-C fit well with expected ratios according to a digenic model (Table3). Because of the triplex constitution of one or both parents for these isozymeloci, we cannot distinguish between disomic or tetrasomic inheritance. How-ever, segregations for Got-A, Cot-B, PgdC and &i-B (Table 4) allowed us todiscriminate between both patterns of digenic inheritance, due to the dupk?cconstitutions of one or both parents in at least one of the progenies ataal~zed.The observed segregations fit well with a tetrasomic model in which chromesomal segregation occurs.

In the progeny from selfing cv. Katahdin we found three individuals withunexpected phenotypes, if tetrasomic inheritance with only chromosome rg-regation was assumed. For Got-A and Pgi-B we found individuals with asym-metrical banding intensities, the band corresponding to the homodim bbbeing most intensely stained; for Pgd-C another asymmetrical banding ap-peared, the band corresponding to the homodimer aa being most intenselystained.

674 J. M. MARTINEZ-WPATER AND J. L. OLlvER

TABLE 3

S8gregatiom analpdfor Pgm-A, Pgm-B and Pox-C isoryiw loci

-YP=

‘“-JJ

Pgl?l-A

RmLf

KATAHDlN S(abbb x abbb)

aabb92 (1)

abbb‘19 (2)

Pgm-B KATAWDlN S(bbbc x bbbc)

bbbb bbbc48 (1) 50 12)

bbCC

44 0) 0.37 0.83

BLJESA x DTO-33(bbbc x bbbb)

bbbb90 (1)

POX-C KATAHDIN S p’tT;‘I) 3;) 1;;) 4.33 0.35(aaac x aaac)

Expc~ted (Exp.) ratios were hose of a digenic model. S indicaws rlfing; w, obmcd.

DISCUSSION

in the analysis that WC have carried out on tetraploid potatoes, duplicategene expression has been demonstrated for seven of eight variable isozymeloci (Tables S mnd 4). Only for Adh-A were the parental genotypes not odequatcto distinguish between a monogenic or. a digenic model of genetic control(Table 2). However, the reciprocal asymmetric banding intensities observed inBuesa and other tetraploid cultivars for this locus (hMt~~-ZlrP~-rztt andOUVER 1984) indicate that it is duplicated as well. Thus, a digenic controlexists for all of the isoryme systems we have studied. These nsults suggestthat this would be the general pattern of inheritance in tetraploid potatoes.

Tetrasomic inheritance has been demonstrated in four of seven duplicateisotyme loci (Table 4). In three other duplicate genes, the parental genotypesprecluded discrimination between disomic or tetrasomic models (Table 9).Tetrasomic inheritance has been previously reported in both groups of tetra-ploid potatoes, Tuberosum and Andigena, for genes controlling morphologicalcharacters, as well as for those conferring disease resistances (Sw~aarrlrmmand MA-N 1961; HOWARD 1970).

Generally, for tetrasomic isozyme loci we found segregations to be of thechromosomal type (Table 4). However, in the progeny resulting from selfingcv. Katahdin one individual for each one of three isozyme loci (W-A, Pgd-Cand P&B) showed clectrophoretic phenotypes that could be exphined by chro-matid segregations; the corresponding genotypes would be rrbbb for C&-A, 4arrbfor Pgd-C and bbbc for Pgi-B. Chromatid segregation has been previously observed in group Tuberosum for genes showing tetrasomic inheritance (SWA-HSNATHAN and MAWON 1961; HOWARD 1970). However, rimihr electropho-

Grt-A KATAHDIN S@arib x aaab)

BUESA x DTO-55(a&b x &tab)

G&B BUfSA x DTO-55(add x cddd)

KATAHDIN S(hbb X abbb)

BUESA x D-l-O-33(a&b x a&b)

an. no.Exp. ntio

Dkmii or tctrasomic

aaaa wb25 40

1 2

aaaa aaabuh. no. 9 84Exp. ntio

Dhnic (at&b X aa/ab) 1 3TctrrWniC 1 5

cccd ccddob. no. 16 86hp. ratio

Dkomk (cd/cd x cd,‘dd) 1 3TCtrnwtnriC 1 5

ohs. 110.hp. latio

Diumk or tcttasomic

I

aabb91

abbb14

O-42 0.8 1

s 1 19.46 4).0015 1

cddd dddd80 15

3 15 I

4.67

9.296.57

1.44

676 J. M. MARTINEZ-WPATER AND J. L. OLWER

ISOZYMES OF CULTIVATED POTATOES 6 7 7

retie phenotypes would also result if these individuals originated from aneu-ploid gametes (CATCHESIDE 1959); then, the corresponding genotypes wouldbe abb for Got-A, aab for PgdC and bbc for Pgi-B. Trivalents and univalents,which have been observed at meiosis of S. tubctosum, could lead to the for-mation of aneuploid gametes (HOWARD 1970).

We have observed tetrasomic inheritance at one or more isozyme loci in twoTuberosum cultivars (Buesa and Turia) as well as in an Andigena cultivar(DTO-33). The existence of tetrasomic inheritance in tetraploid potatoes re-veals that lack of preferential pairing of chromosomes exists, a conclusion thatcan be also proposed from the observation of multivalent configurations atmeiosis (SWAMINATHAN and MACOON 196 1; HOWARD 1970). This may be dueto an autoploid origin of S. tubcronrm (HAWKES 1956b) or, if an amphidiploidorigin is assumed (HAWKFS 1967), to little chromosome differentiation betweenboth diploid ancestors. In most isozyme loci, the alleles of group Andigenawere a combination of those found in both group Stenotomum and the diploidweed S. sparsi;pilum (OLIVER and MARTINEZ-ZAPATER 1984), suggesting an am-phidiploid origin of group Andigena from those two diploid taxa, However,since Andigena was more related to Stenotomum (NEI unbii genetic distance D = 0.052) than to S. spartipilum (D = 0.241), the autoploidization ofStenotomum individuals and the subsequent hybridization with group Andi-gena may also occur. Therefore, models based on amphidiploidy, autoploidyand/or hybridization of the resulting tetraploids seem to be compatible withmolecular data. Although thi; does not mean that all of these processes arenecessarily involved, it clearly agrees with the proposal of UGENT (1970) on amultiple origin for cultivated potatoes (OLIVER and MARTINEZ-ZAPATER 1984).Thus, either autoploidy or amphidiploidy with lack of chromosome differen-tiation between the putative ancestors (Stenotomum and S. spursipilum)(HAWK= 1978) can account for the existence of tettasomic inheritance in thecommon potato.

We are grateful to H. W. HOWARD md CARLOG F. Qtmtos for critical reading of the manuscript.Helpful comments of two anonymous referees arc greatly appreciated. We ttnnk ~CNCI&N SAN-QIEZ-MONCE for providing us the potato material analyzed here. This rexarch was supported inpart by a grant fimm Caja de Ahornn y Monte de Piedad de Madrid, Spain.

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Correqwnding editor: M. R. H&-N