Widespread introgression of Solatium phureja DNA in potato (S. tuberosum) dihaploids

Dihaploids were assumed to be of parthenogenic origin but cv. ‘Pentland Crown’ dihaploids produced at the Scottish Crop Research Institute (SCRI), Dundee, Scotland, are known to contain DNA from the S. phureja dihaploid inducer. This study investigates whether inducer DNA occurs in dihaploids of cultivars ‘Aminca’, ‘Brio’, ‘Lizen’ and ‘Sirtema’, produced at INRA, Ploudaniel, France. Two microsatellite markers and five simple sequence repeat 5′-anchored polymerase chain reaction (PCR) primers were used to generate markers. Markers originating from the dihaploid inducer were detected in 13 of the 19 INRA dihaploids and in some dihaploids from each cultivar. Greater introgression occurred in ‘Lizen’ dihaploids than in ‘Brio’ dihaploids, suggesting that the female (S. tuberosum) parent influences introgression. The percentage of INRA dihaploids containing inducer DNA was similar to that for ‘Pentland Crown’ dihaploids (c. 65%). The micro-satellite markers provided the first evidence of the transfer of specific, potentially useful, genes from the inducer to the dihaploid offspring. Interspecific introgression during dihaploid induction is more widespread than previously thought and researchers should be aware that it may influence the results of research using dihaploids.     

The use of dihaploids in increasing the homozygosity of tetraploid potatoes

Summary Electrophoresis banding patterns of the tuber proteins of 12 dihaploids of the cultivar Pentland Crown showed that four had types of patatin unlike that of the parent. The patatin types of somatically chromosome-doubled clones derived from three of the dihaploids were identical to those of the dihaploid progenitors. Fourteen dihaploids produced from the chromosome-doubled derivative (PDH40X2) of one dihaploid, which had a variant patatin, had the patatin type of the parent dihaploid.The experiment showed that dihaploids and somatic chromosome doubling could be used to fix variation found in heterozygous tetraploid potatoes. Used together, haploidisation and chromosome doubling can generate highly homozygous tetraploids for use by plant breeders.     

AFLP analysis of Solanum phureja DNA introgressed into potato dihaploids

Introgression of Solanum phureja DNA into S. tuberosum dihaploids was analysed by the use of amplified fragment length polymorphism (AFLP) markers. Five dihaploids, derived from crosses between S. tuberosum cv. Pentland Crown and two different S. phureja pollinators (IVP48 and EC90) were investigated by use of 17 AFLP primer pairs. Also 30 dihaploids, derived from pollination of five different S. tuberosum seed parents with S. phureja IVP101, were investigated for the presence of S. phureja‐specific markers. In total approximately 680 and 850 AFLP products were detected in the diploid S. phureja clones and in the tetraploid S. tuberosum genotypes, respectively. A total of 68 S. phureja IVP48‐specific markers were detected, while the total number of S. phureja IVP101‐specific markers was in the range of 72‐96, depending on the S. tuberosum seed parent. Introgression of DNA in the S. tuberosum cv. Pentland Crown dihaploids, after pollination with S. phureja IVP48 and S. phureja EC90, was demonstrated by the detection of 14 of 68 IPV48‐specific markers in the dihaploids. However, no DNA introgression was found in any of the 30 S. tuberosum dihaploids derived from S. phureja IVP101. Hence, S. phureja IVP101 is regarded as an excellent pollinator in the production of S. tuberosum dihaploids in potato breeding programmes because of the high yield of dihaploids per 100 berries, and because no introgression of DNA into the S. tuberosum dihaploids was evidenced.     

Parthenogenetic monohaploids (2n=x=12) from Solanum tuberosum L. and S. verrucosum Schlechtd. and the production of homozygous potato diploids

Summary Dihaploid and dihaploid derived clones of Solanum tuberosum and diploid genotypes of S. verrucosum produced 85 viable monohaploids by female parthenogenesis. All were induced using diploid S. phureja clones, homozygous for embryo spot, as pollinator. Frequency of S. tuberosum monohaploids per 100 berries was rather constant in three successive years (14, 17 and 17 respectively). No male and female fertility was found in flowering monohaploids.Colchicine-induced chromosome doubling yielded homozygous s. tuberosum diploids with low pollen quality but good seed fertility.Two diploid self-incompatible species (S. multidissectum and S. berthaultii) produced no monohaploids. The presence of genes for female parthenogenesis in some dihaploids is discussed.     

Enhancement of diploid Solanum chacoense Bitt. using adapted clones of Solanum phureja Juz. & Buk.

Summary F₁ hybrids between Solanum phureja and S. chacoense were studied in a new approach to enhance wild diploid potato species using adapted S. phureja clones as the female parent. S. chacoense parents had few tubers, whereas S. phureja parents had been previously selected for tuberization under long days. Twenty hybrid families were evaluated for haulm vigor, vine maturity, stolon development, frequency of tuberization, tuber set, tuber yield and average tuber weight under naturally occurring increasing or decreasing photoperiods in spring and fall growing seasons in Morocco. The hybrid plants were more vigorous, later maturing with thicker stolons in the spring season. Frequency of tuberization and tuber set did not vary with season. Tuber yield and average tuber weight were higher in fall; the maximum yield was 2.4 and 4.6 kg per hill under spring and fall conditions, respectively. Remarkable variation was found both between and within hybrid families for all traits in this first cycle of enhancement. The selection of vigorous, fertile clones incorporating the S. chacoense genome is possible by using S. phureja as an adapted germplasm source.     

An assessment of the fertiliser response and other characters of three dihaploid potatoes and the characteristics of their tetraploid progenies

Summary Three dihaploids, two tetraploid cultivars and an unnamed tetraploid potato were subjected to three fertiliser treatments in a glasshouse. One dihaploid (PDH135), as well as being high yielding, responded to fertiliser dosage in a similar way to the cultivars by increasing tuber yield with each increase in fertiliser. The other two dihaploids increased shoot growth rather than tuber yield.An evaluation of tetraploid offspring obtained from dihaploid × tetraploid crosses, showed that PDH135 had the highest-yielding progenies in field trials. Other characters of the dihaploids were efficiently transferred into their tetraploid offspring. The use of dihaploids and unreduced gametes in breeding cultivars with useful quantitative characters is discussed.     

Monohaploids (n=x=12) from autotetraploid Solanum tuberosum (2n=4x=48) through two successive cycles of female parthenogenesis

Summary Viable monohaploids with 12 chromosomes in their somatic cells were obtained from an autotetraploid cultivar of Solanum tuberosum (2n=4x=48) by inducing female parthenogenesis successively in the tetraploid cultivar and in dihaploids from that cultivar. Both dihaploids and monohaploids were induced using the diploid S. phureja clones IvP 35 and IvP 48 as pollinators. The average frequencies of dihaploids and monohaploids in 1973 were 2.0 and 0.14 per berry respectively.Non-homologous chromosome associations (bivalents, trivalents and even quadrivalents) were observed at metaphase I of meiosis in pollen mother cells of the two monohaploids studied.The occurrence of non-homologous associations of chromosomes during meiosis is discussed. In addition the potential significance of monohaploids for basic research andfor breeding of potatoes is considered.     

Competition between 2x and x pollen in styles of Solanum tuberosum determined by a quick in vivo method

Summary Pollen competition in vivo was studied by placing two pollen samples on one stigma and comparing the length of the tubes under a U.V. microscope. Comparisons involving pollen of Solanum tuberosum and S. phureja showed that 2x pollen grew faster than x pollen, both in 2x and x styles. Consequences for induction of dihaploids in S. tuberosum are discussed.     

Breeding behavior for tuber protein in Solanum tuberosum and Tuberosum-phureja hybrids

Summary True protein content among tubers within a plant of Solanum tuberosum cv. Oneida was found to be negatively correlated with tuber size. A similar study of S. phureja genotype 148-17 revealed no correlation. Tuber protein was determined for 250 genotypes in each of four hybrid potato populations in a factorial mating design with four potato cultivars as stylar parents mated to four groups of pollen parents (4x cultivars, 4x high protein selections, 2x S. phureja, 2x high protein S. phureja). The hybrid population derived from the 4x high protein selections was significantly higher in protein content than the other populations. High estimates of general combining ability for tuber protein content were found for both stylar and pollen parents. Poor photoperiodic adaptation to growing conditions in northern latitudes was a possible explanation for the variable protein phenotypes typical of S. phureja and the lack of transmission of the high protein character in phureja to 4x-2x hybrids. Although protein content was negatively correlated with total yield, high protein segregates with good yield potential were identified in all four populations.Scientific Journal Series Article 11, 616 of the Minnesota Agricultural Experiment Station.     

Comparison of diploid and tetraploid potato families derived from Solanum phureja x dihaploid S. tuberosum hybrids and their vegetatively doubled counterparts

Summary To determine whether in potatoes the tetraploid level is preferable to the diploid level, especially regarding tuber yield, four diploid (2n=2×=24) Solanum phureja x dihaploid S. tuberosum hybrid parents and their vegetatively doubled, tetraploid (2n=4×=48) counterparts were intermated, which resulted in two F₁ hybrid families at both levels of ploidy. The parents and clones of the F₁ families and their offspring were used in crosses in such a way that in addition Sib₁, Sib₂, F₁×Sib₁, BC₁ and Sib₁×Sib₁ families were produced. Of the first clonal generation of the 12 2 x families and their 12 counterpart 4 x families two tubers per clone were planted in three replications in a field experiment at Sturgeon Bay in 1969; of the parents six tubers were planted in each replication. Data were recorded on 16 characters, including plant height at four different times.The ANOVA's showed significant clone effects within each family for all characters. Computed from all family means as well as from the family means per ploidy level, differences due to family were also significant for all characters except one.As at the 2 x level and at the 4 x level of ploidy the mean phenotypic correlations between characters were of similar magnitude, it is concluded that they are independent of ploidy level.With the exception of eye depth, the mean coefficient of variation was greater at the 2 x than at the 4 x level of ploidy, indicating the greatest response to selection for those characters at the 2 x level. From the differences in family means between the 4 x and the 2 x level of ploidy it was apparent that the 4 x families generally had significantly taller plants, later maturity, fewer tubers, higher mean tuber weight, more tuber yield and more dry matter yield than their counterpart 2 x families. In contrast, the 4 x parents had on the average shorter plants, lower mean tuber weight, much lower tuber yield and lower dry matter yield than their 2 x counterparts.The phenotypic correlation and Spearman's rank correlation between the family means of the 2 x and the 4 x level of ploidy were positive for almost all characters and significant for nearly half of them.From the results it is concluded that 1. in potatoes the 4 x level of ploidy is preferable to the 2 x level, and 2. the performance of 4 x families is predictable from the performance of their counterpart 2 x families.Based on results mentioned in the literature and on the present results, a continued use of S. tuberosum dihaploids in potato breeding needs to be dissuaded.     

Breeding behavior of yield components and hollow heart in tetraploid-diploid vs. conventionally derived potato hybrids

Summary A factorial mating design with six 4x cultivars as stylar parents mated to three groups of pollen parents (4x cultivars, 4x high protein selections, and 2x S. phureja) was used to study breeding behavior of yield, tuber set, marketable yield and hollow heart in potatoes. Mean yield for the 4x-2x hybrids was equivalent to that of their cultivar parents although yield components, tuber set and average tuber size, were greater and less, respectively, than the cultivar parents. High estimates of general combining ability were found in both pollen and stylar parents for yield, tuber set, marketable yield, number of marketable tubers and hollow heart in all three hybrid populations. Specific combining ability estimates were significant only for yield and hollow heart. Frequency of white-fleshed 4x-2x progeny of five heterozygous yellow-fleshed 2x phureja genotypes was used to confirm cytological identification of a first division restitution mechanism operative in the 2x parents. Although three of these phureja genotypes generated high-yielding progeny, two others produced 4x-2x progeny with mean yield similar to that of hybrids among 4x cultivars.Scientific journal series article 11,601 of the Minnesota Agricultural Experiment Station.     

Assessment of genetic and phenotypic variation among intraspecific somatic hybrids of potato, Solanum tuberosum L.

Intraspecific somatic hybrids have been produced by protoplast fusion in eight combinations involving 10 dihaploids (2n= 2x= 24) in an attempt to provide new material for potato breeding. Cytological analysis revealed extensive variation in chromosome number, such as aneuploid, aneusomatic and mixoploid hybrids. Most of the hybrids represented the expected chromosome number of 48; however, the frequency of aneuploids reached 50% in some combinations. Some hybrids carried structurally rearranged chromosomes and exhibited a high frequency of aberrant anaphases. Isozyme and random amplified polymorphic DNA (RAPD) patterns of the hybrids from the same fusion combination were uniform. In the field, somatic hybrids showed wide phenotypic variation in 20 morphological characters. There was a significant correlation between certain leaf characters and the ploidy level, which may be used to distinguish the tetraploid hybrids from hexaploids and octoploids. Tuber yield and flowering intensity were highest in tetraploid hybrids (2n= 4x= 48). Eighteen of the 73 hybrids reached higher yields than the standard variety ‘Adretta’. Floral development and fertility were restored in hybrids derived from fusions between non-flowering or sterile dihaploids.     

Intraspecific somatic and sexual hybridisation between dihaploid lines of Solanum tuberosum L.: evaluation of morphological traits and resistance to late blight Phytophthora infestans (Mont.) de Bary in the foliage

Intraspecific tetraploid somatic and sexual hybrid plants have been resynthesised following protoplast fusion and by sexual crosses between two dihaploid potato (Solanum tuberosum) lines each possessing complementary agronomic traits. The dihaploid PDH 40 possesses good tuber shape and yield but has foliage susceptibility to late blight (Phytophthora infestans). On the other hand, the dihaploid PDH 727 possesses resistance to blight in the foliage but has a low yield of small and irregular shaped tubers. Since it was only possible to use a partial selection strategy based on culture media to facilitate recovery of somatic hybrid plants-further morphological and esterase isozyme based characterisations were performed to identify somatic hybrid plants from amongst the non-hybrid plant material. When the blight resistance of both the intraspecific somatic and sexual hybrid plants was assessed there was no significant difference in the mean resistance value and it was intermediate between those of their parents. However, the range of resistance was much wider among the sexual hybrids than among the plants derived from somatic fusion. An assessment of tuber yield between tetraploid sexual and somatic hybrids showed no significant difference and it was higher than that of either parent value. The implication of these results in the context of potato genetics and breeding is discussed.     

Breeding for quantitative resistance to potato cyst nematode (Globodera pallida) in tetraploid potatoes using dihaploids and unreduced gametes

Summary Dihaploids were produced from tetraploids resistant to potato cyst nematode (Globodera pallida (Stone)). High levels of resistance were found in the dihaploids and three were used to produce tetraploid progenies by crossing them with susceptible tetraploid cultivars. One dihaploid, PDH505, produced more highly resistant offspring than the other two, PDHs 417 and 418. The latter gave progenies whose levels of resistance were similar to those obtained from susceptible dihaploids crossed with resistant tetraploids.The differences between the progenies of the resistant dihaploids were probably due to different modes of unreduced gamete formation (PDH505 producing gametes by first division restitution (FDR) and PDHs 417 and 418 by second division restitution (SDR)) although cytological studies would be necessary to confirm this. The methods by which dihaploids could be utilised in a tetraploid potato breeding programme are discussed in relation to the mode of unreduced gamete formation.     

Monohaploid plants from anthers of a dihaploid genotype of Solanum tuberosum L.

Summary Monohaploid Solanum tuberosum plants were produced from the anthers of a dihaploid genotype. From another dihaploid genotype plants containing 36 chromosomes were obtained. For plantlet production anthers containing pollen at the uninucleate microspore stage were inoculated on a Linsmaier and Skoog-based medium supplemented with 1 mg/1 indole-3-acetic acid and 1 mg/1 benzyl aminopurine.Anthers from donor plants grown either in the summer or in the winter responded similarly on a range of media. Anther response in the form of callus induction and root formation was at least partly dependent on the genotype of the donor dihaploid plant.     

Breaking the crossability barriers between disomic tetraploid Solanum acaule and tetrasomic tetraploid S. tuberosum

Summary A combination of compatible second pollinations and embryo rescue was applied for systematic production of true tetraploid hybrids from crosses between disomic tetraploid Solanum acaule and tetrasomic tetraploid potato, S. tuberosum. Several genotypes of tetraploid potatoes were pollinated with S. acaule, and the compatible second pollinations were made on the following day, with a genotype of S. phureja, IvP 35 to promote fruit development. Embryo rescue was carried out in 21 families, 14 to 27 days after the first pollination. A total of eight plants were obtained from the embryo rescue and their chromosome numbers were counted in the root tips. Three of the eight plants were identified as tetraploid, and five others as diploid. Morphology, isozyme banding patterns, and pollen stainability, as well as potato spindle tuber viroid (PSTVd) resistance, indicated the hybrid nature of the three plants. This is the first report of successful tetraploid hybrid production between disomic tetraploid S. acaule (4x) and tetrasomic tetraploid potatoes. Seed set from the crosses between one of hybrids and diploid potatoes indicated workable levels of both male and female fertility for introgression of valuable genes from S. acaule into the cultivated potato gene pool. The methodology used may be applied to other disomic tetraploid tuber-bearing Solanum species and with some modifications also to distantly related solanaceous species and genera.     

Analysis of glutamate oxaloacetate transaminase (GOT) isozyme variants in diploid tuberous Solanum; inheritance and linkage relationships to ds1 (desynapsis), y (tuber flesh colour), cr (crumpled) and yc (yellow cotyledon)

Summary Employing polyacrylamide gradient-gel electrophoresis, the genetic control of leaf glutamate oxaloacetate transaminase (GOT) isozyme variants and their linkage to ds1 (desynapsis), y (tuber flesh colour), cr (crumpled) and yc (yellow cotyledon) were studied in diploid tuberous Solanum species hybrids.Leaf GOT isozymes proved to be dimeric and under the control of two independently segregating loci (Got-1 and Got-2) with two (1 ^s , 1 ^f ) and three (2 ^s , 2 ^f , 2 ⁿ )alleles respectively. The 2 ⁿ allele was concluded to encode a 2 ^fallozyme subunit capable of forming heterodimeric but no homodimeric active molecules. As expected with Got-1 and Got-2 isozymes being localized in different cellular compartments, no intergenic heterodimer formation was observed.In contrast to earlier studies, a gradual rather than discrete transition between the tuber flesh colour classes white and yellow was observed in segregating progenies. Adopting a standardized classification method, however, monogenic recessive inheritance of white tuber flesh was confirmed. Got-1, ds1 and cr were found to represent a single linkage group. Among the presently studied marker loci no further linkages were detected. As to linkage analysis, the difficulties imposed by gametophytic incompatibility in nearly all diploid potato species and by the frequent occurrence of recessive (sub)lethal genes are discussed.     

Somatic Fusion for Breeding of Tetraploid Potatoes

From two tetraploid, one Transformed tetraploid, one triploid and 11 dihaploid clones of Solanum tuberosum somatic hybrids were produced by polyethylene glycol mediated somatic fusion. The inter-dihaploid clones comprised clones of agronomic value, homozygous doubled monohaploids, and in vitro selected clones resistant t0 Fusarium or Phytophthora toxins. Presumptive hybrids were enriched at the callus Stage in vitro by using differentiating media and by growth characteristics; further identification was performed by chromosome counting in vitro shoots and by isozyme analysis of in vitro plants. Final analysis was made from morphological characteristic of plant and tuber phenotypes. From 15 different combinations, 6009 plantlets have been regenerated. From five combinations, 310 reentrants were checked for hybrid nature by morphology and cytology and 88 by peroxidase and esterase isozyme analyses. Amongst these, from two combinations, a total of 17 different hybrids were confirmed by all methods. The procedures described are general enough to allow genome combination of interdihaploids resulting in tetraploids of practical breeding value.     

Structure and chromosomal location of malate dehydrogenase (zone 2) isozymes in common and durum wheats

Summary This paper describes the phenotype for MDH-2 isozymes of leaf tissues of Triticum turgidum, T. aestivum and the nulli-tetrasomic lines of homoeologous group 1 of T. aestivum. The results obtained support the hypothesis that the MDH-2 isozymes are monomers coded by genes located in the 1A, 1B and 1D chromosomes.     

Potential and limitations of isozymes for chromosomal localization of resistance genes against barley mild mosaic virus (BaMMV)

Summary To assess the possibilities offered by isozymes to locate resistance genes against barley mild mosaic virus (BaMMV), the isozyme patterns of 19 barley (Hordeum vulgare L.) genotypes carrying genes different from ym4 were determined. Of the 15 isozyme systems tested, only three were polymorphic, namely aconitate hydratase, esterase, phosphogluconate dehydrogenase, providing markers on four of the seven barley chromosomes. Studies of F₂ progenies derived from three crosses between resistant genotypes and susceptible varieties failed to reveal linkage between resistance genes and isozymes. Another goal of the experiment was to study the linkage relationships between ym4 and the esterase locus (Est1-Est2-Est4). Our estimates of the recombination rate between these two loci (3.41 and 8.32%) were in the range of those reported between these esterases and one of the resistance genes of the Chinese variety Mokusekko 3.