The use of RAPD markers for the detection of gene introgression in potato

Randomly Amplified Polymorphic DNAs were employed to demonstrate that potato dihaploids generated after interspecific pollination of a tetraploid Solanum tuberosum cultivar (Pentland Crown) by Solanum phureja dihaploid inducer clones could not be of parthenogenetic origin. Of six different 10-mer oligonucleotides, four generated products from total potato dihaploid genomic DNAs which were not derived from the S. tuberosum parent. Gel electrophoresis and Southern analysis indicated that these amplified bands originated from S. phureja. The results are discussed in the context of recent cytological and molecular evidence which demonstrates that potato dihaploids are aneusomatic (Clulow et al. 1991) and emphasises this approach as a general methodology for the detection of alien gene introgression in both natural and cultivated plant populations.     

Cytological and molecular observations on Solanum phureja-induced dihaploid potatoes

Summary Seventeen potato dihaploids, produced by pollinating the tetraploid (2n = 48) cv Pentland Crown with pollen from Solanum phureja (2n = 24) dihaploid inducer clones, were studied. Since dihaploids are thought to develop parthenogenetically from unfertilized ovules they were expected to be euploid (2n = 24), but somatic chromosome counts showed that 15 of the 17 dihaploids were aneusomatic. Ten of the clones were predominantly diploid (2n = 24) with a proportion of hyperploid cells that contained 25 or 26 chromosomes. Five of the dihaploids contained variable numbers of triploid cells (2n = 36). RFLP analysis was used to determine whether the additional chromosomes were from S. phureja or S. tuberosum. Unique hybridizing fragments present in S. phureja but not in Pentland Crown were identified. These S. phureja-specific restriction fragments were present in some of the dihaploid offspring of Pentland Crown. Of the 5 clones that contained triploid cells 4 had S. phureja type banding. Four of the 10 aneusomatic clones that contained hyperploid cells had the unique S. phureja hybridizing fragments. We propose that ovules of Pentland Crown were fertilized by pollen from S. phureja and that the aneusomatic clones were derived from triploid zygotes from which some of the S. phureja chromosomes were eliminated. We consider that this is an additional mechanism of dihaploid formation in potato.     

Periderm cell size as a ploidy indicator in potato (Solatium tuberosum L. subspecies tuberosum)

Using the tubers of glasshouse-grown potato plants, periderm cell dimensions were found to indicate ploidy in comparisons of Solanum tuberosum subspecies tuberosum dihaploids and their somatically-chromosome-doubled derivatives. The mean ratio of dihaploid to tetraploid cell sizes, determined as the product of cell length x breadth, was 0.60:1. There were differences between 34 tetraploid cultivars in their mean periderm cell sizes and between clones within six dihaploid families derived parthenogen-etically from tetraploids. There was variation in mean cell size but dihaploid cells were always smaller than those of their parents. The mean cell size of the parent was usually in the next highest size class of its largest-celled dihaploids. As there was overlap between dihaploid and tetraploid ranges it was concluded that in order to identify dihaploids the mean cell size in tubers of the parent grown under the same conditions should be used for comparison. Clones with mean periderm cell sizes no greater than 60% of the parent's cell size could be provisionally classed as dihaploid.     

Interspecific somatic hybrids between wild potato Solanum acaule Bitt. and anther-derived dihaploid potato (Solanum tuberosum L.)

Solanum acaule Bitt. is a disomic tetraploid (4x) wild potato species which is resistant to several potato diseases. Introgression of disease resistance and abiotic stress tolerance to the tetrasomic tetraploid (4x) cultivated potato (S. tuberosum L.) gene pool via crossing has been limited due to the difference in the endosperm balance number. In the present study, protoplast fusion was applied to produce hexaploid (6x) somatic hybrids between the parental lines, tetraploid (4x) S. acaule and two anther-derived dihaploid (2x) lines of S. tuberosum cv. White Lady. One callus (0.4%) of a total of 229 calli obtained regenerated into shoots in the fusion combination S. acaule (+) White Lady 15.dh.8.2.2. All the regenerated shoots were confirmed to be interspecific somatic hybrids using species-specific RAPD markers. In another fusion combination, S. acaule (+) White Lady 7.dh.23.1.1, fifteen calli (5%) regenerated into a total of sixteen shoots from 289 calli. All the analysed somatic hybrids between S. acaule and S. tuberosum were hexaploid. The mean DNA content (2C value) of the combination S. acaule (+) White Lady 15.dh.8.2.2 somatic hybrids (4.55 pg), was approximately the sum (4.69 pg) of the DNA contents of the parental lines, S. acaule (2.95 pg) and S. tuberosum (1.74 pg). In the greenhouse, the two somatic hybrids analysed were normal in their morphological characteristics and more vigorous than their parental lines. Most of the morphological characteristics were closer to the tetraploid S. acaule than to the dihaploid S. tuberosum. The interspecific somatic hybrids are currently being tested for frost tolerance and glycoalkaloid composition. Received: 19 January 1998 / Revision received: 27 March 1998 / Accepted: 20 April 1998     

Application of somatic hybrids between dihaploids of potato Solanum tuberosum L. and wild diploid species from Mexico in breeding: Generation and backcrossing of dihaploids of somatic hybrids

The efficiency of an original approach to involvement of the valuable genetic pool of wild diploid potato species from Mexico is estimated. The essence of this method is in generation of dihaploids (2n = 2x = 24) of tetraploid somatic hybrids (2n = 4x = 48) followed by backcrossing with dihaploids of Solanum tuberosum. A haploid producer, S. phureja IvP35, was used to generate ten dihaploids of S. tuberosum + S. pinnatisectum, all of which crossed with fertile S. tuberosum dihaploids and developed plump viable seeds. This gives the possibility of an efficient introgression of the genes valuable for breeding from wild species to the bred plants at a diploid level, which has several advantages compared with the corresponding procedure at a tetraploid level. A part of the dihaploids produced was compatible (the pollen tubes reached the ovary) with diploid and tetraploid forms of S. pinnatisectum; however, no viable seeds were developed. The attempt to generate the dihaploids of S. tuberosum + S. bulbocastanum somatic hydrides using the haploid producer S. phureja IvP35 was unsuccessful.     

Strategy differences of two potato species in response to nitrogen starvation. Do plants have a genetic switch for nitrogen signalling?

Survival responses to nitrogen starvation are well known in micro‐organisms but little studied in plants. To construct a framework for study of the plant responses, we investigated the strategy differences of tubers from two closely related potato species. Solanum tuberosum conserves tuber nitrogen by inhibiting shoot growth, but S. phureja mobilizes tuber nitrogen to grow shoots, flowers and seeds. Genetic analysis of progeny from S. phureja–haploid S. tuberosum crosses uncovered segregation of a single dominant gene for the S. tuberosum inhibition strategy. Within S. tuberosum, haploid progeny closely resembled their tetraploid parents, suggesting strong genetic control of the inhibition. Growth of the inhibited shoots was proportional to sub‐optimal levels of added nitrate, and was triggered by exogenous gibberellic acid (GA₃). These observations support the notion that potato plants can closely tie shoot growth to ambient nitrogen levels – probably by a root–shoot nitrogen signal transduction pathway, and that this can be overridden by emergency mobilization of nitrogen reserves, perhaps by GA signalling from the tuber. Furthermore, genes for such developmental switches can be identified by classical genetic analysis of closely related species, such as S. tuberosum and S. phureja, that exhibit opposite survival strategies.     

RAPD markers for confirmation of somatic hybrids in the dihaploid breeding of potato (Solanum tuberosum L.)

Summary The applicability and reliability of RAPD markers were evaluated for an examination of the possible use of RAPD markers to confirm hybridity of somatic hybrids between dihaploids of potato (Solanum tuberosum L.). Most of the primers examined detected polymorphism among either tetraploids or dihaploids, and polymorphism was easily detected even among closely related clones. Most of the examples of polymorphism were confirmed as being the result of amplification from the nuclear genome by a comparison of patterns generated by PCR of clones that carried the same cytoplasm. All the bands of dihaploids were transmitted stably to the respective hybrids. In the absence of primers that generated complementary polymorphic bands for both parents, a mixture of two appropriate primers, each of which generated a band specific to one parent, permitted the simple confirmation of hybridity. Hybridity of all the fusion-derived regenerants of 32 fusion combinations was unequivocally confirmed, a result that suggests that RAPD analysis could be universally applicable to the confirmation of hybridity in the dihaploid breeding of potato.     

Methods and strategies for detecting Solanum tuberosum dihaploids in interspecific crosses with S. phureja

The frequency of dihaploid production following the interspecific pollination of Solanum tuberosum clones with Solanum phureja has been investigated. Seventeen hybrid combinations were tested and the crosses differed significantly in the number of seed produced, the proportions that were viable and the proportions that were dihaploid. The overall frequency of dihaploid production was 62%. The dominant embryo spot marker of the pollinator (IVP 48) in conjunction with seed diameter was used to classify the seed samples. Number of days taken to germinate was also recorded. It was therefore possible to examine retrospectively the effect of selecting sub-sets of the seed based on the presence or absence of the embryo spot, seed size and germination time. A seed selection system based on the absence of the embryo spot in conjunction with seed greater than 1.25 mm in diameter resulted in 93.6% of the potential dihaploids being identified.     

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability

In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers (HIs) yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of HI DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three HIs: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n = 2x − 1 = 23 to 2n = 2x + 3 = 27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the HI parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual HI DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.

A large potato progeny population produced by crossing tetraploid cultivated clones to diploid Phureja lines displays rare instances of haploid inducer chromosomes, which are frequently damaged.     

Production of androgenic dihaploid lines of the disomic tetraploid potato species Solanum acaule ssp. acaule

Over 250 dihaploid lines derived from a disomic tetraploid genotype of Solanum acaule ssp. acaule Bitt. (acc. PI 472655) were produced via androgenesis. The anther donor plant had previously shown immunity to bacterial ring rot caused by Clavibacter michiganensis ssp. sepedonicus (Spieck. and Kotth.) Davis et al., and has now been shown to have high embryogenic capacity in anther culture. In total, 370 shoots were regenerated from 4,011 anthers cultured. The ploidy level of the 287 regenerants was determined from greenhouse-grown plants using flow cytometry. Of these plants, 274 (95%) were dihaploids with an average DNA content of 1.68 pg, approximately half that of the tetraploid anther donor (2.95 pg). The remainder of the anther-derived regenerants (5%) were tetraploid, hexaploid or mixoploid. Chromosome counts confirmed the results obtained by flow cytometry. In the greenhouse, none of the 33 dihaploid lines analysed produced berries but showed low (2%) male fertility. This contrasted with five greenhouse-grown tetraploid anther-derived plants which produced berries and seeds. Comparison of the general leaf morphology and floral characteristics of the tetraploid anther donor, S. acaule, and the dihaploids indicated that little variation exists in this species. Received: 28 August 1997 / Revision received: 22 December 1997 / Accepted: 27 July 1998     

The ploidy composition of offspring from Solanum tuberosum (4×) ×S. phureja (2×) crosses with special reference to triploid frequencies

Data on the production of offspring of 4×× 2× crosses in potato are presented. The ploidy composition of seedlings varied from year to year and parent to parent. Products of dihaploid induction crosses were chiefly 2× or 3× whereas crosses between S. tuberosum cultivars and unreduced gamete-producing S. phureja clones were mainly 3× or 4×. Substantial percentages of 3× seedlings were produced by some crosses in some years. It was concluded that the so-called ‘triploid block’ in potatoes is a variable phenomenon and that factors which increase seed production may suppress the proportion of triploids produced.     

The novel, major locus Rpi-phu1 for late blight resistance maps to potato chromosome IX and is not correlated with long vegetation period

Despite the long history of breeding potatoes resistant to Phytophthora infestans, this oomycete is still economically the most important pathogen of potato worldwide. The correlation of high levels of resistance to late blight with a long vegetation period is one of the bottlenecks for progress in breeding resistant cultivars of various maturity types. Solanum phureja was identified as a source of effective late blight resistance, which was transferred to the cultivated gene pool by interspecific crosses with dihaploids of Solanum tuberosum. A novel major resistance locus, Rpi-phu1, derived most likely from S. phureja and conferring broad-spectrum resistance to late blight, was mapped to potato chromosome IX, 6.4 cM proximal to the marker GP94. Rpi-phu1 was highly effective in detached leaflet, tuber slice and whole tuber tests during 5 years of quantitative phenotypic assessment. The resistance did not show significant correlation with vegetation period length. Our findings provide a well-characterized new source of resistance for breeding early and resistant-to-P. infestans potatoes.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Expression of reducing sugar accumulation in interspecific somatic hybrids of potato

Summary A somatic hybridisation programme was undertaken to evaluate the expression of reducing sugar accumulation in potato. Interspecific hybrids created between the Solanum tuberosum cultivar Record and the diploid species Solanum phureja were evaluated at the morphological and molecular levels. These analyses indicated that the protoplast regenerants were partial (asymmetric) hybrids which had undergone elimination of S. phureja chromosomes. Tubers of the parents exhibited significant differences for reducing sugar accumulation during cold storage with S. phureja having lower levels of glucose and fructose than Record. The somatic hybrids resembled the S. phureja parent in terms of reducing sugar accumulation demonstrating that low reducing sugar accumulation is dominant to high reducing sugar accumulation in these particular genotypes. These results are discussed in relation to the exploitation of asymmetric hybridisation for the production of potato genotypes for the potato processing industry.Abbreviations BSA (Bovine serum albumin) - GA₃ (Gibberellic acid) - MS (Murashige and Skoog) - NAA (-Naphthaleneacetic acid) - RAPD (Randomly Amplified Polymorphic DNA)     

R6 and R7 alleles of potato conferring race-specific resistance to Phytophthora infestans (Mont.) de Bary identified genetic loci clustering with the R3 locus on chromosome XI

In potato, 11 resistance alleles (R1–R11) are known which confer race-specific resistance to the fungus Phytophthora infestans. R1 has been mapped previously to potato chromosome V and R3 to chromosome XI. Here we report on the localization of the R6 and R7 alleles on the genetic map of potato. Differential resistant strains of tetraploid Solanum tuberosum, clones MaR6 and MaR7, were used as parental plants for the parthenogenetic induction and selection of diploid genotypes containing the R6 or the R7 resistance allele to P. infestans. One resistant dihaploid from MaR7 could be used directly as a parent to produce diploid F₁ progeny suitable for phenotypic and RFLP analysis. MaR6 did not produce useful dihaploids directly. After crossing MaR6 with a tetraploid susceptible genotype, resistant F₁ clones were selected. The resistant genotypes were then used as parents for the induction of dihaploids. Six dihaploids bearing R6 were identified that could be crossed with a diploid susceptible genotype. Two diploid F₁ populations, segregating for R6 and R7, respectively, were analysed with RFLP markers known to be linked with previously identified R genes. Markers linked with R3 were found also to be linked with R6 and R7. The resistance alleles R6 and R7 mapped to a similar distal position on chromosome XI as the R3 allele.     

Elimination of Solanum phureja nucleolar chromosomes in S. tuberosum + S. phureja somatic hybrids

Summary The karyotype of the dihaploid SVP1 line of S. tuberosum (2n=2x=24) showed two nucleolar chromosomes with differently sized satellites. The diploid SVP5 line (2n=2x=24) and tetraploid regenerants of S. phureja had larger but similar satellites. Somatic hybrids between the diploid lines of these potato species with genome combinations 4 tub + 2 ph (plants 1–3), 2 tub + 4 ph (plants 4–7) and 4 tub + 4 ph (plant 8) had lost 2 phureja nucleolar chromosomes if 4 phureja genomes were present. One phureja nucleolar chromosome of plants 1–3 and both of plants 5 and 7 had rearranged satellites. Elimination of the two nucleolar chromosomes occurred preferentially, was under genetic control, and probably took place during early callus development. NOR activity resulting in rear-rangements between NORs may have caused the elimination.     

Evidence for gametoclonal variation in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

Gametoclonal variation that occurs in gametic cells in culture and is recovered in their regenerated derivatives has not been reported in potato (Solanum tuberosum L.). Based on a set of 24 differentiating phenotypic traits, canonical variates analysis genetically distinguished the androgenic (di)haploid (2n = 2x = 24) D4 from its tetraploid (2n = 4x = 48) anther-derived sibs and anther donor JTH/C-107. Nuclear microsatellite analysis over six polymorphic loci indicated that meiotic rearrangements and mutant alleles were primarily associated with the release of gametoclonal variation. The incidence of null alleles in D4 at the loci STACCAS3 and STM0031 was also indicative of mutations occurring within the priming sequence. Microsatellite results were supported by random amplified polymorphic DNA (RAPD) assays that characterized a total of 567 loci (bins) representing 4,258 amplified fragments. Sixty-five new RAPDs that were absent in the anther donor and in either of its parents, viz., S. phureja Juz. & Buk. IVP-35 and S. tuberosum cv. Kufri Jyoti were present in D4, indicating the occurrence of extensive recombinational events. The results have been discussed in the context of microsatellite null alleles providing the most conclusive evidence for gametoclonal variation.     

Somatic hybrids between potato <Emphasis Type="Italic">Solanum tuberosum</Emphasis> and wild species <Emphasis Type="Italic">Solanum verneï</Emphasis> exhibit a recombination in the plastome

In the present survey, interspecific somatic protoplast fusion between a dihaploid cultivated potato Solanum L. tuberosum (BF15) and a wild species Solanum verneï (V3) has been performed using the PEG method. Five putative hybrids were first selected. Among them, only two were retained. DNA analysis, using flow cytometry, showed that the first hybrid (VB2) was dihaploid (2n=2x=24 chromosomes), whereas the second (VB1) was hexaploid (2n=6x=72 chromosomes). In the greenhouse, these putative hybrids showed that they were able of raising and producing large tubers but with a modified shape compared to parental ones. Furthermore, they harbored an intermediate leaf morphology compared to their parents. The hybrid nature of these plants was first confirmed according to their esterase and peroxidase isoenzyme patterns. The RAPD analysis of genomic DNA and microsatellite based amplification (I-SSR) showed that both clones VB1 and VB2 are asymmetric somatic hybrids. They seem to have eliminated the major part of V3 parental nucleus but not the totality. The analysis of chloroplast DNA suggests that both hybrid plastomes were the result of a recombination between parental ones.     

Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs

Summary The techniques of microspore and protoplast regeneration starting from dihaploid Solanum tuberosum plants has been improved to such an extent that the production of more than 2000 microspore derived A₁ plant lines and of several hundred protoplast derived plantlets has become possible. Further, from the dihaploid Solanum species S. phureja the regeneration of microspores to plants, and from the species S. infundibuliforme, S. sparsipilum and S. tarijense the regeneration of protoplasts to calluses, has been achieved. The plants descending from the two single cell culture systems are compared with reference to phenotypic markers and economic qualities. Some principles characteristic for either microspore or protoplast derived plants are examined and their significance is discussed. The results are compiled into an extended analytical synthetic breeding scheme based on a stepwise reduction of the autotetraploid to the monohaploid level and a subsequent controlled combination to a new synthetic completely heterozygous tetraploid potato.     

The inheritance and expression of sterility in hybrids of dihaploid and cultivated diploid potatoes

When dihaploids of EuropeanSolanum tuberosum are used as female parents in crosses with South American cultivated diploid potatoes (Group Phureja/Stenotomum), various kinds and degrees of male sterility are found in the offspring. The effect of using different dihaploid and cultivated diploid parents on shrivelled microspore sterility of F₁ hybrid progenies was studied. Variation in the character was continuous and statistical analyses showed high general combining ability for dihaploid parents but not for cultivated diploids. A significant but non-linear relationship was found between percent of stainable pollen and seed set in crosses with female tester parents, provided that some degree of functional male fertility was present. F₁ clones with pollen of normal appearance but with no functional fertility probably represent a hitherto unclassified cytoplasmic male sterility. The results are discussed from the point of view of methods to be adopted in improving potatoes at the diploid level.     

Chromosome doubling via tuber disc culture in dihaploid potato as determined by confocal microscopy

Summary The potential of tuber disc culture for chromosome doubling was investigated in somaclonal populations of four dihaploid genotypes and one tetraploid cultivar of potato (Solanum tuberosum). Laser scanning confocal microscopy (LSCM) was used for rapid determination of the ploidy level based on the number of chloroplasts in stomatal guard cells of leaves. Factorial analysis of chloroplast number in 58 clones and two leaf types showed that somaclones were clearly divided in two groups. Clones with 5–7 chloroplasts per cell as observed in tuber derived diploid controls were classified as 2X (not doubled), while those with 9–14 chloroplasts resembled the tuber derived tetraploid controls and were considered 4X (doubled). A high frequency of spontaneous chromosome doubling, 42% – 50%, was detected in 3 dihaploid genotypes, whereas no doubling was observed in one of the dihaploids as well as the tetraploid cultivar Yukon Gold. Effects of leaf type on chloroplast number was also significant. The middle leaf showed significantly higher chloroplast number than the younger leaves.