Substitutes for genome differentiation in tuber-bearing Solanum: interspecific pollen-pistil incompatibility, nuclear-cytoplasmic male sterility, and endosperm.

The cultivated potato, Solanum tuberosum L. (2n=4x=48), has a very large number of related wild and cultivated tuber-bearing species widely distributed in the Americas. These species, grouped in 16 taxonomic series, range from the diploid to the hexaploid level. Polyploid species are either disomic or polysomic, and sexual polyploidization via genetically controlled 2 n gametes has played a major role in their evolution. Species are separated in nature by geographical and ecological barriers. However, there are several examples of sympatric species that share the same niches but do not readily cross (i.e., the diploids S. commersonii and S. chacoense in certain areas of Argentina). External barriers alone are, therefore, not sufficient to explain species integrity. In addition, there is no strong evidence indicating that genome differentiation is important in the group. In this review we present evidence supporting the assertion that interspecific pollen-pistil incompatibility, nuclear-cytoplasmic male sterility, and the endosperm are major forces that strengthen the external hybridization barriers allowing, at the same time and under specific circumstances, a certain amount of gene exchange without jeopardizing the integrity of the species.

Chromosome regions between centromeres and proximal crossovers are the physical sites of major effect loci for yield in potato: genetic analysis employing meiotic mutants.

Meiotic mutant (2n) gametes formed by first-division restitution without crossover (FDR-NCO) are expected to be superior to FDR with crossover (FDR-CO) because they transmit to the progeny, without disruption by recombination, almost 100% of the parental genotype. FDR-CO transfers approximately 80% of the parental heterozygosity and a large fraction of the epistatic interactions. Another genetic expectation associated with both FDR gametes is their equivalence for the phenotypic expression of traits controlled by genes residing between centromeres and proximal crossover sites. This set of unique cytogenetic features of FDR mutants was employed here as a tool to infer physical location of quantitative trait loci controlling total tuber yield (TTY) in potato. Two assays were conducted to verify the superiority of FDR-NCO over FDR-CO gametes for TTY by using progenies from 4x-2x factorial crosses. Male clones were 2n-pollen producers by either FDR-CO or FDR-NCO mechanisms. Compared with the 4x parents, TTY of the progenies ranged from 41% to 175% (i.e., high-parent heterosis). However, no significant TTY differences were observed between FDR-CO and FDR-NCO families. In addition, the size of variance components of males was smaller than females and near zero. Our results reinforce the hypothesis that genes controlling yielding ability have a predominant physical location between centromeres and proximal chiasmata. Quantitative trait loci in chromosome regions with reduced levels of recombination may provide a partial explanation for the slow progress in increasing TTY through conventional 4x-4x crosses and for the often high degree of heterosis obtained by introgressing genetic diversity via 4x-2x crosses in potato.

Endosperm balance number manipulation for direct in vivo germplasm introgression to potato from a sexually isolated relative (Solanum commersonii Dun.).

Diploid (2n = 2x = 24) Solanum species with endosperm balance number (EBN) = 1 are sexually isolated from diploid 2EBN species and both tetraploid (2n = 4x = 48, 4EBN) and haploid (2n = 2x = 24, 2EBN) S. tuberosum Group Tuberosum. To sexually overcome these crossing barriers in the diploid species S. commersonii (1EBN), the manipulation of the EBN was accomplished by scaling up and down ploidy levels. Triploid F1 hybrids between an in vitro-doubled clone of S. commersonii (2n = 4x = 48, 2EBN) and diploid 2EBN clones were successfully used in 3x x 4x crosses with S. tuberosum Group Tuberosum, resulting in pentaploid/near pentaploid BC1 progenies. This provided evidence of 2n (3x) egg formation in the triploid female parents. Two selected BC1 pentaploid hybrids were successfully backcrossed both as male and as female parents with S. tuberosum Group Tuberosum. The somatic chromosome number varied greatly among the resulting BC2 progenies, which included hyperaneuploids, but also a number (4.8%) of 48-chromosome plants. The introgression of S. commersonii genomes was confirmed by the presence of S. commersonii-specific randomly amplified polymorphic DNA markers in the BC2 population analyzed. The results clearly demonstrate the feasibility of germplasm introgression from sexually isolated diploid 1EBN species into the 4x (4EBN) gene pool of the cultivated potato using sexual hybridization. Based on the amount and type of genetic variation generated, cumbersomeness, general applicability, costs, and other factors, it would be interesting to compare the approach reported here with other in vitro or in vivo, direct or indirect, approaches previously reported.

Cytological basis of 2n pollen formation in a wide range of 2x, 4x, and 6x taxa from tuber-bearing Solanum species.

Cytological mechanisms of 2n pollen formation were investigated in microsporogenesis and sporad development in 2x, 4x, and 6x taxa of tuber-bearing Solanum species that were originally collected from Mexico, and Central and South America. Parallel spindles and tripoles were seen at anaphase II in the majority of genotypes observed. The occurrence of parallel spindles and tripoles were well correlated with the occurrence of dyads and triads at the sporad stage. Further, the frequency of 2n pollen predicted by frequencies of parallel spindles and tripoles and by frequencies of dyads and triads were also well correlated with observed 2n pollen frequencies. Premature cytokinesis at telophase I to prophase II was seen only in 2x and 4x species from series Conicibaccata. The frequency of premature cytokinesis corresponded well with the frequency of dyads leading to 2n pollen formation. It was concluded that the major mechanisms of 2n pollen formation in tuber-bearing Solanum species is parallel spindles.

Efficiency of potato breeding using FDR 2n gametes for multitrait selection and progeny testing.

The objective of this research was to compare the efficiency of the 4x×2x breeding scheme with the traditional 4x×4x method with respect to potato improvement. The basis for such a comparison was the parental value of four 2x and four 4x male parents from the International Potato Center (CIP) as measured by multitrait selection and progeny testing. The 2x parents produced 2n pollen by parallel spindles at anaphase II, which is genetically equivalent to a first division restitution (FDR) mechanism. Both 2x and 4x parents were crossed with four common 4x female parents. Thus, 32 families were evaluated over 2 years at four Peruvian locations. A selection index which considered tuber yield, tuber number, average tuber weight and specific gravity was used for multitrait selection. Three FDR 2x parents had better selection index scores than the 4x parents over the four locations. Estimates of broad-sense heritability for total yield using different number of replications and locations were calculated by using the variance components. The 4x × 2x breeding scheme was found to be better than the traditional 4x × 4x method since fewer replications and locations are required to evaluate tuber yield in 4x × 2x progenies than in 4x × 4x progenies. The FDR 2x parents were also better material than the 4x parents for testing combining ability for tuber yield of the 4x progenitors. This could be the result of the mode of FDR 2n pollen formation. The pollen of FDR 2x parents is more heterozygous, but more homogenous than n pollen from 4x parents.

The occurrence and frequency of 2n pollen in 2x, 4x, and 6x wild, tuber-bearing Solanum species from Mexico, and Central and South America.

The occurrence of 2n pollen-producing plants was investigated in 187 plant introductions (PIs) of 38 wild species of tuber-bearing Solanum. These 2x, 4x, and 6x species are from Mexico, and Central and South America. The determination of 2n pollen-producing plants was conducted using acetocarmine glycerol. Plants with more than 1% large-size pollen were regarded as 2n pollen-producing plants. 2n pollen-producing plants were identified in the following species: 10 out of 12 Mexican 2x species, seven of nine South American 2x species, seven of seven Mexican and Central American 4x species, five of five South American 4x species, and five of five Mexican 6x species. The frequency of 2n pollen-producing plants varied among species at the same ploidy level, but the range of frequency, generally between 2 and 10% among species, was similar over different ploidy levels. The general occurrence of 2n pollen in both 2x and polyploid species, which are evolutionarily related, is evidence that the mode of polyploidization in tuber-bearing Solanums is sexual polyploidization. Furthermore, the frequencies of 2n pollen-producing plants in autogamous disomic polyploid species were not markably different from those of their related diploid species. It is thought that the frequent occurrence of 2n gametes with autogamy tends to disturb the fertility and consequently reduce fitness of polyploids. Thus, we propose that the breeding behavior of polyploids and the occurrence of 2n gametes may be genetically balanced in order to conserve high fitness in polyploid species in tuberbearing Solanum.

Occurrence of 2n pollen and ps gene frequencies in cultivated groups and their related wild species in tuber-bearing Solanums.

The gene frequency for parallel spindles (ps) was estimated from the frequency of plants producing 2n pollen in three cultivated groups: 2x Phureja (phu), 2x Stenotomum (stn), and 4x Andigena (adg), as well as in four related wild taxa: 2x Solanum brevicaule (brc), 2x S. sparsipilum (spl), 4x S. gourlayi (grl) and 4x S. gourlayi-S. infundibuliforme hybrids (grl-ifd). Plants with more than 1% large pollen were considered as 2n pollen producers. Observations of meiosis in a sample of 2n pollen-producing plants indicated that parallel spindles is the mechanism of 2n pollen formation. The number of plants with 2n pollen among the total examined was 228 plants (15.5%) of 1,473 in 2x spl, 31 (26.7%) of 116 in 2x brc, 92 (17.4%) of 528 in 2x stn, 665 (22.1%) of 3,008 in 2x phu, 731 (51.4%) of 1,421 in 4x adg, 591 (41.2%) of 1,436 in 4x grl, and 36 (64.3%) out of 56 in 4x grl-ifd. The ps gene frequencies assuming Hardy-Weinberg equilibrium were: 0.393 for 2x spl, 0.462 for 2x brc, 0.417 for 2x stn, 0.470 for 2x phu, 0.847 for 4x adg, 0.801 for 4x grl, and 0.895 for 4x grl-ifd. Twenty-five adg clones were randomly selected from a large population and were crossed with 2x clone W5295.7, which produces 2n pollen by parallel spindles (ps). The 4x progenies from 4x×2x crosses were used to determine the genotypes at the ps locus by screening 10-20 plants in each family for 2n pollen. Based on chromosome segregation at the ps locus, 9, 14, 1, and 1 clones were nulliplex, simplex, simplex or duplex, and duplex, respectively. The frequency of the ps gene in the adg population was estimated to be 0.825 and 0.815 for chromosome and chromatid segregation, respectively. The high frequencies of 2n pollen and the ps gene in cultivated 2x and 4x groups, and in wild taxa closely related to them, provide evidence for sexual polyploidization in the tuber-bearing Solanums.

Mayer's hemalum-methyl salicylate: a stain-clearing technique for observations within whole ovules.

Nondissected ovaries of tuber-bearing Solanum sp. were stained with Mayer's hemalum, a positive stain for chromatin and nucleoli, and then optically cleared with methyl salicylate, a clearing agent. Clarity, resolution and contrast within the ovules dissected from ovaries were comparable to those of sectioned, paraffin embedded ovaries. Contrast within ovules greatly exceeded that of unstained and nonspecifically stained clearings, and eliminated the need of special optics, i.e., Nomarski interference-contrast optics, for optimal viewing and photography. Much less time and labor were required than for embedded specimens. Usefulness of the technique for cytogenetic and cytological research was verified by analyzing meiosis and other features of megasporogenesis and megagametogenesis in normal, and in two meiotic mutants, of Solanum. The results illustrate the usefulness of combined Mayer's hemalum staining and methyl salicylate clearing, and suggest additional stain-clearing agent combinations have potential for cytological and cytogenetic research. Preliminary results with other species suggest the technique may also be useful for classroom instruction.

Origin and evolution of cultivated tetraploid potatoes via 2n gametes.

A high gene frequency for ps (parallel spindles) is expected in cultivated tetraploid potatoes, S. tuberosum Group Tuberosum, if 2n pollen produced by ancestral diploid plants which were psps was involved in the origin and evolution of the potato. Fifty-six North American cultivars (varieties and advanced selections) were pollinated by diploid clones, either W 5295.7 or W 5337.3 which are homozygous recessive for ps. The segregation ratios in regard to 2n pollen production in derived tetraploid progenies, from 4x×2x crosses, reveal the genotype of ps in the cultivars. Microsporogenesis of 2n pollen producing 4x progeny was observed to avoid an overestimation of the frequency of 2n pollen producing plants due to mechanisms other than parallel spindles. More than 50% of the 56 cultivars are simplex (Pspspsps), since in each of these cultivars about 50% of their progeny produced 2n pollen. The ps gene frequency in the 56 cultivars was estimated as high as 0.69. The high frequency of ps in the tetraploid cultivars clearly supports the hypothesis that 2n pollen produced by plants homozygous recessive for ps have been involved in the origin of cultivated tetraploid potatoes, since a higher frequency of ps in the tetraploid than in the ancestral diploid population can be expected from sexual polyploidization but not from somatic doubling. The importance of meiotic mutants such as ps for the successful evolution of polysomic polyploids is emphasized.

Genetic-cytoplasmic male sterility in progeny of 4x-2x crosses in cultivated potatoes.

Forty-three percent of the progeny from 4x × 2x crosses [Group Tuberosum cultivar x diploid hybrid (Groups Phureja or Stenotomum x Group Tuberosum haploid)] were male sterile. In contrast only four percent of the progeny from the reciprocal crosses were male sterile. Male sterility among the former progeny is presumed to result from the interaction of Tuberosum cytoplasm with dominant genes from the cultivated diploids, Groups Phureja and Stenotomum, an interaction known to occur in crosses of Tuberosum haploid x cultivated diploid species (2n = 2x = 24). The frequency of fertile progeny from the 4x × 2x crosses (57%) was significantly higher than that from the 2x × 2x crosses (Tuberosum haploid x cultivated diploid), (28%). The frequency of male fertility among progeny from different cultivars in 4x × 2x crosses varied from 31-82 percent. The difference between cultivars strongly suggests that some cultivars may have dominant male fertility restorer genes.

Cross-incompatibility between two sympatric polyploid Solanum species.

Preliminary results from a large number of reciprocal crosses between the closely related sympatric species S. gourlayi Hawkes (2n=4x=48) and S. oplocense Hawkes (2n=6x=72) indicated that they are difficult to hybridize. Pollen-pistil incompatibility barriers were detected via fluorescent microscopy. The cross incompatibility reaction occurred at three sites in 6x×4x crosses; on the stigma, in the first one-third of the style, and in the first two-thirds of the style. In the reciprocal 4x×6x crosses the incompatibility reaction invariably occurred in the ovary. Backcrosses of interspecific pentaploid hybrids (that were occasionally formed) to both parental populations were fully compatible, partially compatible, and fully incompatible with three sites of cross-incompatibility reaction similar to those observed in 6x×4x crosses, respectively. Both polyploid species were found to be selfcompatible, whereas their F1 hybrids were found to be self-incompatible. An hypothesis based on interactions of dominant cross-incompatibility (CI) genes in pistils and dominant specific complementary genes in pollen grains is postulated to explain these observations. The cross-incompatibility system that appears to be operating in nature between 4x S. gourlayi and 6x S. oplocense provides a way for gene exchange between sympatric populations without threatening the identity of either species.

Inheritance of electrophoretic variants of tuber proteins in Solanum tuberosum haploids.

Soluble tuber proteins were separated by discontinuous polyacrylamide gel electrophoresis on vertical slabs. Banding patterns of proteins stained with Coomassie Blue in 7.5% acrylamide gels (pH 4.3) were few and distinctive for haploids (2n = 2x = 24) derived from several cultivars (2n = 4x = 48). Katahdin and Chippewa haploids have only three different banding patterns for the eight fastest moving bands. The haploids have either the parental pattern (all eight bands) or one of two complementary banding patterns (four bands). The frequency of these patterns among the haploids indicates that the eight bands are controlled by one locus which is duplex (A1 A1 A2 A2) in the parents. Haploids with the genotype A1 A2 have eight bands. A1 A1 haploids have four bands, and A2 A2 have the other four bands. Tawa haploids have in equal numbers either the eight (A1 A2) or four (A2 A2) band pattern. Thus the genotype of Tawa is A1 A2 A2 A2. The control of four bands by one allele could be explained by assuming that these alleles are involved in posttranslational modification or assembly of one or two protein species. Another explanation is that pseudoalleles or redundant genes produce the groups of protein bands. The eight proteins studied apparently are of similar molecular weight but differ in charge.

The significance of genic balance to endosperm development in interspecific crosses.

The endosperm has played a significant role in the evolution of angiosperms because of its physiological and genetic relationships to the embryo. One manifestation of this evolutionary role is its abnormal development in interploidy crosses. It is now established that the endosperm develops abnormally in interploidy-intraspecific crosses when the maternal: paternal genome ratio deviates from 2∶1 in the endosperm itself. We propose an Endosperm Balance Number (EBN) hypothesis to explain endosperm development in both interploidy-intraspecific and interspecific crosses. Each species is assigned an EBN on the basis of its crossing behavior to a standard species. It is the EBN which determines the effective ploidy in the endosperm of each species, and it is the EBNs which must be in a 2∶1, maternal:paternal ratio. The EBN of a species may be determined by a few genes rather than the whole genome. This hypothesis brings most intraspecific-interploidy and interspecific crossing data under a single concept with respect to endosperm function. The implications of this hypothesis to isolating mechanisms, 2n gametes, the evolution of disomic polyploids, and reciprocal differences in seed development are discussed.

The occurrence and frequency of 2n pollen in three diploid solanums from Northwest Argentina.

A group of wild, tuber-bearing species from Northwest Argentina, belonging to the series Tuberosa, Solarium spegazzini Bitt. (spg, 2n=2x=24), S. gourlayi Hawkes (grl, 2n=2x=24 and 2n=4x=48) and S. oplocense Hawkes (opl, 2n=6x=72), and Cuneolata, S. infundibuliforme Phil (ifd, 2n=2x=24), is being used to investigate the mode of origin of polyploids in the genus Solanum. 2n gametes have been detected in the diploid species ifd and spg and in a diploid race of grl, using cytological and breeding approaches. Twenty-two introductions of spg, 8 of grl and 26 of ifd have been tested for 2n pollen; 59%, 63% and 54% of them, respectively, had at least one 2n pollen producing plant. These introductions comprised 238, 76 and 235 plant respectively, of which 20, 16, and 32 plant produced 5% or more 2n pollen. The mechanism of 2n pollen formation was determined in several plant of 2x spg, 2x grl and 2x ifd. All of them were found to form diplandroids via parallel spindles. This mechanism, which gives meiotic products genetically equivalent to first division restitution gametes, is under control of the Mendelian recessive ps. The results suggest that the allele ps is widely distributed in natural populations of the three diploids, and that its frequency is very high. These species are seen as valuable material for population genetic studies, and for the eventual incorporation into a breeding scheme involving sexual polyploidization via 2n gametes.

Gene-centromere mapping by 4x-2x matings in potatoes.

Two of the four strands of a bivalent are recovered together in tetraploid progeny arising from 4x-2x matings. This provides a method for gene and centromere mapping. The cross pppp x Pp produced 62 nulliplex tetraploid individuals in a total of 951, i.e., 6.5%. The diploid clone was found to produce diplandrous gametes through first division restitution (FDR). The map distance P-centromere was estimated as 13.0 units, the limiting values at the 95% binomial confidence interval being 10.1 and 16.3. The mapping method is explained in detail and a formula is deduced to obtain genotypic series of 2n gametes under particular assumptions. The direction 4x×2x is advantageous, since high seed set diploid clones which give 99% tetraploid progeny, and whose method of diplandroid formation is known are already available. Diploid clones heterozygous for many markers and tetraploids nulliplex for the same markers are needed to fully exploit this method.

The influence of paternal species on the origin of callus in anther culture of Solanum hybrids.

Stamen culture of several Solanum species and interspecific hybrids was performed and each stamen was scored for presence and origin of callus. Each species and hybrid has a characteristic callus growth pattern - either no growth (0), callus growth from the filament (F), or callus growth from the anther (A). Characteristic growth types of the interspecific hybrids indicate that callus growth takes precedence over no growth. Hybrids between F and A species exhibit mainly paternal passage of either characteristic callus type. Possible explanations for this pattern of inheritance are male plastome factors or imprinting of paternal genes. The latter explanation is presently preferred.

The significance of 2N gametes in potato breeding.

Phureja-haploid Tuberosum hybrids, which produce 2n gametes in addition to n gametes, were used to obtain diploid progenies in 2x - 2x matings, and tetraploid progeny in 4x - 2x matings. Seven of these diploid clones were intermated in a modified diallel design, and also crossed to seven cultivars, to obtain 21, 16-clone diploid; and 49 (35, 2x X 4x and 14, 4x X 2x) 24-clone tetraploid families, respectively. These progenies were included together with the 14 parental clones, in a tuber yield trial conducted in two locations in Wisconsin. Heterotic responses were striking in 4x X 2x progenies. Three of these entries had mean yields of 5.0, 5.0 and 5.2 lbs/hill, for 24 unselected clones, thus outyielding not only the mid-parent but also the best tetraploid cultivars in the experiment, which are highly selected; Wis 643 (4.8 lbs/hill), and Kennebec (4.8 lbs/hill). As a group, the 14, 4x X 2x families averaged 4.4 lbs/hill, while the mean of all seven cultivars was 4.0 lbs/hill, and the mid-parent was at 3.4 lbs/hill. This unusually high mean performance of 336 unselected clones represented in the 14, 4x X 2x progenies, is interpreted as a manifestation of the capacity of 2n pollen, formed by first meiotic division restitution (FDR), to pass onto the progeny the already heterotic diploid genotype in a largely intact array. Beneficial intra and inter-locus interactions are presumably compounded upon syngamy with an unrelated n egg from the tetraploid parent. -The performance of 2x X 4x progenies was at or below that of the midparent. The failure of these families to perform as well as 4x X 2x families may be a reflection of the incapacity of 2n megasporogenesis to avoid meiotic reassortment as efficiently as FDR does, which would result in inbred 2n gametes. However, the method of 2n megasporogenesis is not known.Hereditary variances were large, both within and among families, and indicated considerable nonadditivity in the action of genes affecting tuber yield at both levels of ploidy. Inbreeding was strongly depressing at both ploidy levels.

Sexual polyploidization and depolyploidization: some terminology and definitions.

Sexual polyploidization (deployploidization) is the process through which a euploid zygote is formed whose chromosome number is increased above (decreased below) the level that would be expected if each parent contributed a gamete carrying one-half the parental premeiotic chromosome number. One or both gametes may contribute (lack) the extra genome (s) so that these processes may be unilateral or bilateral. Sexual polyploidization has the distinctive characteristic of being a genetically innovative mechanism. This is believed to be its most significant difference with respect to somatic doubling. Sexual depolyploidization, which may be conceived to arise in some rare situations, also has the capacity for innovation. Several cytological mechanisms may be envisioned which give rise to polyploidizing gametes. The genetic consequences of these mechanisms can be markedly different in regards to gene reassortment. Short of a mitotic-like method, FDR (first division restitution) appears to be the most appropriate mechanism to attain such a goal. Other modified meiotic processes generally result in more disturbance of the premeiotic genotypes. Under these circumstances, sexual polyploidization is probably associated with mitigated heterotic responses and a more extensive release of genetic variability.As a cytological device, FDR has the unique property of producing balanced 2n gametes even after a numerical nondisjunction has occurred. This simple mechanism may restore fertility to, e.g., monoploids and triploids, so that odd-ploids may possess more evolutionary significance by acquiring a means of producing balanced gametes systematically.Some new terminology is proposed to designate gametes and gametophytes which conserve the parental number of chromosomes, and for polyploids arising in conjunction with their functioning in fertilization.

Analysis of stylar self-incompatibility competence by use of heat induced inactivation.

Immersion of Lilium longiflorum pistils in 49 °C water for increasing durations of 1,2,3, or 4 minutes immediately prior to incompatible pollination resulted in a correspondingly progressive decrease in the stylar self-incompatibility competence, as determined from the lengths attained by pollen tubes during 48 hours growth in the styles at 24 °C. Neither pistils remaining on the plant nor those detached from the plant which were immersed after anthesis in 49 °C water for 5 minutes regained self-incompatibility competence during a 48 hour incubation at 24 °C prior to incompatible pollination. Heat treatment of detached pistils as early as 39 hours prior to bud anthesis also resulted in an inactivation of stylar self-incompatibility competence when incompatible pollination was made at 24 hours after anthesis. Experiments utilizing heat treatment of partial lengths of detached whole styles revealed that pollen tubes which have grown through as much at 45 millimeters of either a physiologically incompatible or compatible portion of the style are still capable of shifting to either a higher growth rate or lower growth rate upon entry into respectively either a physiologically compatible or incompatible portion of the style.