Introduction

In plants, sexual polyploidization by the formation of unreduced (2n) gametes is an important feature in both nature and breeding programs. In potato, FDR and SDR mechanisms have been shown to give rise to 2n-gametes (Veilleux, 1985; Peloquin et al., 1989; Bretagnolle & Thompson, 1995). In both cases, the 2n-gametes are derived from two of the four strands of each bivalent of a pair of homologous chromosomes (Fig. 1.) The FDR gametes originate through an equational division of all chromosomes, as a result of which the nonsister chromatids are included in one and the same gamete. In this case, the 2n-gametes are highly heterozygous because homozygosity can occur only in the regions distal to chiasma formation. On the other hand, the SDR gametes originate from the restitution of chromosomes in the products of the first meiotic division, as a result of which the sister chromatids are included in one and the same 2n-gamete. For SDR, homozygosity occurs in regions proximal to chiasma formation (Fig. 1) Thus, depending on the modes of origin, 2n-gametes with quite different genetic compositions can be generated.

Fig. 1
figure 1

The genetic consequences of first division restitution and second division restitution for a proximal marker locus (P/p) and a distal marker locus (D/d), assuming one crossover between these markers. The maternal chromosomes and alleles are indicated in light grey and upper case, whereas the paternal chromosomes and alleles are indicated in dark grey and lower case (after Mendiburu & Peloquin, 1979).

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Through the use of appropriate meiotic mutants in potato, it has been possible to select genotypes that produce highly heterozygous FDR to relatively homozygous SDR populations of 2n-gametes (Ramanna, 1983; Werner & Peloquin, 1987; Werner et al., 1992; Douches & Quiros, 1988; Jongedijk et al., 1991; Bastiaanssen et al., 1996). It is believed that different meiotic aberrations can occur in one plant resulting in a mixture of FDR and SDR 2n-eggs (Conicella et al., 1991; Werner & Peloquin, 1991). However, in the ovules of potato, it is impossible to demonstrate cytological events that elucidate unequivocally the mode(s) of origin of 2n-eggs. In the absence of cytological methods for determining the mode(s) of origin of 2n-eggs in potato, genetic methods are employed (Mendiburu & Peloquin, 1979). In these methods it is assumed that homozygosity increases with genetic distances from the centromere in FDR, whereas it decreases in SDR (Fig. 1).

Genetic analysis of the tetraploid progeny from 2x×4x crosses can be used to deduce the mode of origin of 2n-eggs. Previous investigations (Douches & Quiros, 1987, 1988; Jongedijk et al., 1991; Werner et al., 1992; Barone et al., 1995) lacked segregation data for several loci on a single chromosome, preventing a detailed examination of the degree of heterozygosity, the restitution mechanism and the number of crossovers per chromosome in each individual 2n-egg. This also prevented firm conclusions on the exclusive occurrence of one restitution mechanism or a mixture of several restitution mechanisms in a genotype. In a previous study, RFLP markers of chromosomes 6 and 8 of potato were identified that are informative for multilocus analysis of tetraploid progenies that are derived from 2x×4x crosses (Bastiaanssen, 1997).

In the present investigation, the distal biochemical marker amylose-free starch (amf) was used to demonstrate differences in the mode(s) of origin of 2n-eggs in four diploid clones of potato on the basis of the segregation ratios in their tetraploid progenies. Four RFLP loci on chromosome 8 were then used in a tetraploid progeny of one 2n-egg producing genotype to elucidate the restitution mechanism(s) of 2n-egg formation, and to determine the extent of crossing-over.

Materials and methods

Plant materials

Four diploid clones that were derived from crosses between the diploid species S. tuberosum (tbr) and S. phureja (phu), S. chacoense (chc), S. microdontum (mcd) or S. spegazzinii (spg) were used for the analysis of 2n-egg formation (Table 1). These four interspecific hybrids were heterozygous (Amf/amf) for the amylose-free starch marker and showed normal chromosome pairing during meiosis. The hybrids produced 2n-eggs in variable frequencies, as was evident from their seed sets following 2x×4x crosses (Bastiaanssen, 1997).

Table 1 The clones that were used for 2x×4x crosses to generate tetraploid progenies for genetic analysis of 2n-eggs
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The four diploid 2n-egg producing genotypes were crossed with four tetraploid S. tuberosum clones (Table 1) to generate progenies for genetic analysis. The tetraploid chromosome constitution of the progeny was ascertained by flow cytometry (Bastiaanssen, 1997).

Classification of the tetraploid progenies based on the amf marker

The tetraploid progenies from the 2x (Amf/amf)×4x (amf/amf/amf/amf) crosses were classified into the classes of duplex (Amf/Amf/amf/amf), simplex (Amf/amf/amf/amf), and nulliplex (amf/amf/amf/amf) genotypes on the basis of the starch phenotypes of microspores after staining in Lugol/chloral hydrate (1 : 2) (Jacobsen et al., 1989, 1991; Bastiaanssen et al., 1996). The nulliplex genotypes in this tetraploid progeny were identified on the basis of only red (amf/amf) microspores. The microspores of the simplex genotypes were expected to segregate blue (Amf/amf) : red (amf/amf) in the ratio of 1 : 1. The microspores of the duplex genotypes were expected to comprise the genotypes Amf/Amf: Amf/amf: amf/amf in the ratio of 1 : 4 : 1, resulting in a segregation ratio of blue (Amf/−) : red (amf/amf)=5 : 1. To distinguish the simplex and duplex genotypes, 100–500 microspores were scored.

Multilocus RFLP-analysis of the tetraploid progenies

In the present investigation, 138 tetraploid plants from a cross between the diploid mcd×tbr hybrid and Tetra 4 were analysed in more detail. The hybrid was heterozygous for four RFLP loci (m=TG536, TG482, TG434, TG346) on chromosome 8, and for each marker the alleles were distinguishable from the one to four different alleles (m♂) of the tetraploid male (Bastiaanssen, 1997). The four selected markers covered a large part of chromosome 8. Both the diploid mcd and tbr parent of the 2n-egg-producing hybrid were included in the RFLP analysis to identify the parental origin of the alleles (mmcd or mtbr) at each of the four loci. This was essential for the classification of the mmcdmmcdm♂m♂, mmcdmtbrm♂m♂ and mtbrmtbrm♂m♂ genotypes in the tetraploid progeny, and for the detection of crossovers between the mcd and tbr genomes in the hybrid. A genetic map was constructed from the RFLP scores using the computer package JoinMap 2.0 (supplied by P. Stam, Wageningen Agricultural University, The Netherlands).

Results

Assessment of the restitution mechanism through monitoring the segregation for the amf marker

The four diploid Amf/amf genotypes were crossed with three nulliplex males (Table 1) to generate tetraploid progenies. Because all plants received two amf alleles from the nulliplex male parent, nulliplex progeny plants were derived from amf/amf 2n-eggs, whereas simplex plants originated from Amf/amf 2n-eggs, and duplex plants originated from Amf/Amf 2n-eggs. From the segregation ratios of starch phenotypes of the microspores in the tetraploid progeny plants, it was possible to classify them as nulliplex, simplex and duplex and thus establish the genotypes for the amf marker (Table 2). In the progenies from the tbr×phu, chc×tbr and tbr×spg hybrids, the simplex class far outnumbered the nulliplex and duplex classes (Table 2) showing a high frequency of Amf/amf 2n-eggs. In contrast, the nulliplex and duplex classes far outnumbered the simplex class in the progeny of the mcd×tbr hybrid. This clearly indicated a different mode of origin of the 2n-eggs in the mcd×tbr hybrid.

Table 2 Segregation of the amf marker in progenies from Amf/amf×amf/amf/amf/amf crosses and its application to test the expected second division restitution (SDR) mode of 2n-egg formation in the four diploid clones of potato
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The amf locus was previously shown to be the most distal marker on chromosome 8 of potato, and it was suggested that in normal synaptic plants a single crossover occurred between this locus and the centromere (Bastiaanssen et al., 1996). Heterozygous 2n-eggs (Amf/amf) could result from either FDR or SDR, as indicated for the distal locus in Fig. 1. Assuming normal frequency of crossovers, FDR would give rise to no more than 50% of heterozygous (Amf/amf) 2n-eggs. Thus, the most likely explanation for the high frequency of heterozygous (Amf/amf) 2n-eggs in the tbr×phu, chc×tbr and tbr×spg hybrids (Table 2) was SDR following the frequent occurrence of one crossover between the centromere and the amf locus. The suggested SDR 2n-egg formation was confirmed through RFLP analysis of a tetraploid progeny of the tbr×spg hybrid, in which homozygosity decreased with genetic distance from the centromere (Bastiaanssen, 1997).

The mcd×tbr hybrid behaved differently, producing a high frequency of homozygous (Amf/Amf and amf/amf) 2n-eggs. In the case of FDR 2n-egg formation, the frequency of these Amf/Amf and amf/amf 2n-eggs would not exceed 50%, whereas in the case of SDR 2n-egg formation, the observed high frequency could only occur with severely reduced crossing-over between the centromere and the amf locus. Another explanation might be that the 2n-eggs of the mcd×tbr hybrid resulted from the doubling of chromosomes in the haploid products of normal meiosis, a previously undescribed restitution mechanism in potato. If postmeiotic doubling of the chromosomes had occurred in the mcd×tbr hybrid, the 2n-eggs should be completely homozygous and show crossover events between the mcd and tbr genomes. In order to test this, the tetraploid progeny derived from the cross mcd×tbr hybrid× Tetra 4 was used for RFLP analysis.

RFLP analysis of the tetraploid progeny derived from the mcd×tbr hybrid and Tetra 4

The 138 tetraploid plants from a mcd×tbr hybrid× Tetra 4 cross were genotyped at each of four fully informative marker loci (TG536, TG481, TG434 and TG346) on chromosome 8. An example of a Southern blot showing polymorphisms for the probe TG346 is shown in Fig. 2.

Fig. 2
figure 2

Southern blots showing the absence of tetraploid progeny plants with both alleles of the mcd×tbr hybrid potato. The parental clones are shown in lanes 1–5; mcd parent (lane 1) and tbr parent (lane 2) of the diploid mcd×tbr hybrid (lane 3), the male parent Tetra 4 (lane 4), a mixture of mcd×tbr hybrid and Tetra 4 (lane 5). Sixteen plants of the tetraploid progeny of the cross mcd×tbr hybrid×Tetra 4 are shown in lanes 6–21. DNA samples were digested with EcoRV and hybridized to probe TG346. λ-DNA was used as a marker for the size of DNA-fragments (lane λ).

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The marker loci TG536, TG481, TG434 and TG346 cover 89% of the genetic length of the linkage map of tomato chromosome 8 (Tanksley et al., 1992), and can be expected to give similar coverage in potato. The markers gave 10 distinct classes of genotypes in the tetraploid progeny with two remarkable features (Fig. 3). First, all plants had either the mmcd allele or the allele mtbr at all four loci. This indicated that all progeny plants were mmcdmmcdm♂m♂ or mtbrmtbrm♂m♂ and consequently originated from 2n-eggs in which chromosome 8 was completely homozygous. This homozygosity closely corresponded to the segregation pattern for the amf marker of the same chromosome (Table 2). Secondly, most genotypes (80) had mmcd alleles and mtbr alleles in homozygous condition for different markers of chromosome 8 (columns 3–10 in Fig. 3), showing frequent crossing-over between the two genomes in the mcd×tbr hybrid.

Fig. 3
figure 3

Genotype frequency distribution of the tetraploid progeny derived from the potato cross mcd×tbr hybrid×Tetra 4 to show the allelic composition of the 138 plants at four RFLP loci of chromosome 8. The order of the loci is the same as that on the tomato map of Tanksley et al. (1992). The bars represent the different genotypic classes, based on the presence of the mmcd allele (solid black), or mtbr allele (netted) at the four RFLP loci TG536, TG481, TG434 and TG346 of chromosome 8. The number under each bar represents the number of plants per genotypic class.

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The 2n-eggs of the mcd×tbr hybrid comprised 58 nonrecombinant genotypes (columns 1 and 2 in Fig. 3), 72 genotypes with one crossover (columns 3–8 in Fig. 3), and eight genotypes with two crossovers (columns 9 and 10 in Fig. 3). The logical explanation for these genotypes is that chromosomes in haploid products of normal meiosis were doubled in order to give 2n-eggs in the mcd×tbr hybrid. This means that the genotypes of the 2n-eggs were equivalent to the genotypes of doubled haploids and that the genetic distances between the four RFLP loci of chromosome 8 in the mcd×tbr hybrid could be estimated using the doubled haploid option of the computer package JoinMap. The order of genes and the genetic distances were consistent with the tomato map of Tanksley et al. (1992) (Fig. 4).

Fig. 4
figure 4

Genetic map distances of chromosome 8 in tomato and the mcd×tbr hybrid.

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Discussion

Detection and confirmation of different restitution mechanisms of 2n-eggs

With the use of multiple genetic markers for chromosome 8 of potato, it has been demonstrated that the diploid mcd×tbr hybrid produced completely homozygous 2n-eggs. The amf marker was very useful for the detection of this genotype, allowing quick assessment of the mode of 2n-egg formation, because of the full classification of the genotypes in the tetraploid progeny of Amf/amf×amf/amf/amf/amf crosses, and the prediction of the segregation ratio for different modes of origins of 2n-eggs based on the distal position of this marker locus and the occurrence of a high degree of chiasma interference (Bastiaanssen et al., 1996). This enabled detection of the homozygous 2n-eggs of the mcd×tbr hybrid, that clearly deviated from the segregation expected for SDR 2n-egg formation (Table 2). The homozygosity of the 2n-eggs of this hybrid was demonstrated for 71 of the 73 progeny plants of the cross Amf/amf×amf/amf/amf/amf, and further confirmed in RFLP analysis of progeny plants of the same hybrid using four appropriate markers covering most of chromosome 8. In view of the complete homozygosity of all progeny plants at all four RFLP loci tested, the two simplex plants for the amf marker probably resulted from errors in the classification of duplex genotypes. The usefulness of the amf marker in facilitating the selection of clones with distinct restitution mechanisms was also demonstrated in progenies of the tbr×spg hybrid, in which the suggested SDR 2n-egg formation (Table 2) was confirmed through RFLP analysis (Bastiaanssen, 1997).

Discovery of completely homozygous 2n-eggs

In potato, highly heterozygous to relatively homozygous 2n-eggs that originated through FDR and SDR, respectively, have been detected previously (Iwanaga & Peloquin, 1979; Stelly & Peloquin, 1986; Douches & Quiros, 1988; Jongedijk et al., 1991; Werner et al., 1992; Barone et al., 1995). However, diploid genotypes with completely homozygous 2n-eggs have not been described before. The discovery of the mcd×tbr hybrid is therefore a valuable addition which completes a spectrum of genotypes giving rise to highly heterozygous to completely homozygous 2n-eggs.

Evidence for postmeiotic restitution in 2n-egg formation

In order to provide genetic evidence for postmeiotic doubling of chromosomes, it is essential to show genotypes of 2n-eggs that are recombinant as well as completely homozygous. These genotypes can only be shown in multilocus analysis, as was demonstrated in the progeny of the mcd×tbr hybrid segregating for the mmcd and mtbr alleles at the four RFLP loci covering most of chromosome 8.

In sugarcane, Bremer (1959, 1961) presented cytological evidence for the occurrence of endomitotic division of chromosomes in a reduced nucleus during megasporogenesis, which is expected to be genetically equivalent to postmeiotic doubling of reduced gametes. In potato, genetic indications for the occurrence of postmeiotic doubling of reduced megaspores, which is expected to generate completely homozygous 2n-eggs, were reported by Stelly & Peloquin (1986) as well as by Douches & Quiros (1988). In these genetic analyses, however, it was not possible to rule out the possibility of SDR 2n-egg formation in which no crossover had occurred between the marker locus and its centromere because of the use of a single marker gene per chromosome. Douches & Quiros (1988) described the suggested postmeiotic doubling of the haploid products of normal meiosis as a type of SDR 2n-gamete formation. Because both the cytological events and the genetic consequences of postmeiotic doubling of the unreduced megaspores are very distinct from omission of the second division of the megaspore, it is preferable to characterize postmeiotic doubling as an additional mechanism called ‘postmeiotic restitution (PMR)’.

The presence of only PMR 2n-egg formation in the mcd×tbr hybrid

In addition to the discovery of a new restitution mechanism, the multilocus analysis of the 2n-eggs of the mcd×tbr hybrid elucidated the number of restitution mechanisms involved. Because of the complete homozygosity of the 2n-eggs for the four markers of chromosome 8, it was concluded that all 2n-eggs were derived from PMR. The absence of other restitution mechanisms could not have been demonstrated using a single marker locus per chromosome.

Implications of PMR 2n-gametes

For the breeding of potato at the diploid level, it is important to realize that 2n-eggs are not always of SDR origin. This is because the genetic consequences of PMR are quite distinct from those of SDR. Postmeiotic restitution 2n-eggs are highly relevant for potato genetics because of the possibility of generating homozygous diploid genotypes through, for example, ‘prickle’ pollinations (Hermsen & Verdenius, 1973) of PMR 2n-egg producers to induce parthenogenetic development of the homozygous 2n-eggs. Because the cultivated potato is a highly heterozygous crop with a high degree of inbreeding depression and sterility, it is difficult to achieve homozygosity through repeated selfings. Alternatively, homozygous genotypes can be generated through the production of doubled monoploids by in vitro culture of anthers (Meyer et al., 1993; Veilleux et al., 1995), or stem explants of monohaploids (Uijtewaal et al., 1987). Because these genotypes are fixed, they can be propagated sexually as well as vegetatively. They can be used as tester lines in inheritance studies for analysing the extent of meiotic recombination, and for the generation of hybrids with maximal level of heterozygosity. Furthermore, the generation of both anther-culture-derived and PMR 2n-egg-derived progenies would allow the use of gametic samples for genetic mapping to analyse sex differences in recombination (Rivard et al., 1996). Finally, the PMR 2n-eggs might be especially attractive when a F1 hybrid involving wild species, such as the mcd×tbr hybrid, is to be crossed with a tetraploid male and the undesirable genes are to be eliminated rapidly; for example, it is relatively easy to detect genotypes that are homozygous for a desired locus of mcd, in which a substantial amount of the chromosomes are derived from S. tuberosum (Fig. 3).