Diploid hybrids between allotetraploid wild potato species <Emphasis Type="Italic">Solanum acaule</Emphasis> Bitt., <Emphasis Type="Italic">S. stoloniferum</Emphasis> Schltdl. and dihaploids of <Emphasis Type="Italic">S. tuberosum</Emphasis> L.

The possibility to obtain diploid hybrids by pollination of allotetraploid wild potato species Solanum acaule and S. stoloniferum plants with fertile pollen of S. tuberosum dihaploids was demonstrated for the first time. Dihaploid hybrids have arisen with comparatively high frequency (from 12.5 to 33.3%). They were characterized by high regularity of meiosis and high fertility. They easily crossed with S. tuberosum dihaploids, forming viable progeny. This seems prospective for effective introgression of valuable genetic gene pool of wild allotetraploid potato species in breeding material of S. tuberosum on the diploid level.     

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.     

Production of potato breeding material using somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. dihaploids and the wild diploid species <Emphasis Type="Italic">Solanum bulbocastanum</Emphasis> Dunal. from Mexico

An original approach to overcome interspecific incompatibility when backcrossing the tetraploid Solanum tuberosum + S. bulbocastanum somatic hybrids with cultivated potato was realized. This method is based on the decrease in their ploidy using anther culture and involvement of the haploid producer S. phureja IvP35. The feasibility of obtaining a diploid progeny from the somatic hybrids carrying genetic material of the wild species S. bulbocastanum and crossable with S. tuberosum dihaploids was demonstrated.     

Using disomic 4x(2EBN) potato species' germplasm via bridge species Solanum commersonii.

The cultivated potato Solanum tuberosum Dunal has many wild related species with desirable traits. Some of these wild tetraploids have disomic chromosome pairing, ready selfing with little inbreeding depression, but have strong crossing barriers with cultivars. They hybridize most easily with 2EBN forms (which include most diploid species). Chromosome doubling to the 8x level, use of 2n gametes, use of 2n gametes of 4x-2x triploid hybrids, and embryo rescue have been proposed to overcome the crossability barrier of these species with S. tuberosum. In this study, 2x S. commersonii (cmm) was used as a bridge species with S. acaule and series Longipedicellata species. Synthetic tetraploid 4x-cmm crossed readily to disomic 4x species, resulting in fertile F1 and F2 hybrids. Some of these had 2n gametes, which enabled direct crossing to tuberosum, resulting in 6x hybrids. The benefits of this scheme are (i) hybrids are relatively fertile, so many progeny may be produced for selection at each step, (ii) hybridization with cmm results in 2n gametes needed for crossing to tuberosum, and breaks up restricted recombination within disomic genomes, and (iii) simple techniques and tools are employed.     

Glycoalkaloid aglycone accumulations associated with infection by Clavibacter michiganensis ssp. sepedonicus in potato species Solanum acaule and Solanum tuberosum and their interspecific somatic hybrids

Solanum acaule Bitt., a wild potato species, is closely related to cultivated potato (Solanum. tuberosum L.). Incorporation of desirable traits from allotetraploid [2n=4x=48, 2 endosperm balance number (EBN)] S. acaule (acl) into autotetraploid (2n=4x=48, 4EBN) S. tuberosum (tbr) is difficult due to incongruity boundaries. In this study, three hybrid combinations, each with a specific genome constitution, were produced through protoplast fusion: (1) hexaploid 2x acl (+) 4x tbr, (2) tetraploid 2x acl (+) 2x tbr, and (3) hexaploid 4x acl (+) 2x tbr hybrids. In terms of glycoalkaloid aglycones, the hybrids produced demissidine, tomatidine and solanidine, similarly to the S. acaule parental species, but S. tuberosum synthesised only solanidine. Inoculations with Clavibacter michiganensis ssp. sepedonicus (Cms), which is the causal agent of bacterial ring rot in potato, yielded significantly lower total glycoalkaloid aglycone accumulation both in S. acaule plants and in interspecific hybrids in comparison with the corresponding mock-inoculated plants. However, in S. tuberosum the aglycone levels were either higher or unchanged as a result of infection by Cms. To incorporate the desirable traits of the interspecific somatic hybrids into 4EBN S. tuberosum, sexual backcrosses were carried out. The hexaploid 4x acl (+) 2x tbr hybrids with the hypothetical 4EBN showed the greatest capacity to undergo backcrosses with S. tuberosum.     

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.     

Introgression of resistance to root-knot nematodes from wild Central American Solanum species into S. tuberosum ssp. tuberosum

 Crossing experiments were conducted to introduce resistance to the root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from various polyploid Central American Solanum spp. into the cultivated potato, S. tuberosum ssp. tuberosum. The most effort was put into producing tetraploid hybrids through inter-EBN (Endosperm Balance Number) crosses. From the crosses of tetraploid S. tuberosum (4 EBN) with tetraploid S. stoloniferum and S. fendleri (both 2 EBN), few seeds were derived that led to viable plants. In vitro culture of immature seeds also yielded several hybrid plants. From crosses of diploid S. tuberosum (2 EBN) with hexaploid S. hougasii (4 EBN) four hybrids were obtained through in vitro culture. Backcrosses were made with selected hybrids and a variable number of seeds was produced depending on the hybrid genotype. The successful introgression of resistance into backcross populations is shown. A scheme is presented for the introgression of traits at a tetraploid level from allotetraploid Solanum species into autotetraploid S. tuberosum through sexual crosses. The relevance of EBN for potato breeding is discussed. Received: 25 November 1996 / Accepted: 14 February 1997     

Development and characterization of potato-Solanum brevidens chromosomal addition/substitution lines

Solanum brevidens is a wild diploid potato species possessing high levels of resistances to several major potato diseases. We previously developed fertile somatic hybrids between S. brevidens and the cultivated potato (Solanum tuberosum) in order to introgress disease resistances from this wild species into potato. A series of backcross progenies was developed from a hexaploid somatic hybrid A206. Using a combination of S. brevidens-specific randomly amplified polymorphic DNA (RAPD) markers and a sequential genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) technique, we identified all 12 S. brevidens chromosomes in the backcross progenies. Seven potato-S. brevidens monosomic chromosome addition lines (chromosomes 1, 3, 4, 5, 8, 9 and 10) and one monosomic substitution line (chromosome 6) were identified, and the remaining four S. brevidens chromosomes (2, 7, 11, and 12) were included in two other lines. These chromosomal addition/substitution stocks provide valuable tools for potato cytogenetic research, and can be used to introgress disease resistances from S. brevidens into potato.     

Overcoming crossing barriers between nontuber-bearing and tuber-bearing Solanum species: towards potato germplasm enhancement with a broad spectrum of solanaceous genetic resources.

The first direct sexual hybrids between diploid nontuber-bearing species and diploid potato breeding lines are reported here. Three nontuberous species of Solanum, S. brevidens, S. etuberosum, and S. fernandezianum, were used for sexual crosses, achieved by a combination of rescue pollinations and embryo rescue. Initial hybrid selection was made using an embryo spot marker, followed by the evaluation of morphological and reproductive traits. Putative hybrids were first tested for resistance to potato leaf roll virus derived from the wild species, and then were tested with molecular markers using species-specific DNA probes. Finally, the tuberization of several 2x hybrids was tested for actual potato germplasm enhancement. These hybrids are unique in terms of their potential to enhance recombination between chromosomes of wild species and those of cultivated potatoes in germplasm utilization, and to exploit the genetic nature of tuber formation. The finding that nontuber-bearing Solanum spp. can be directly crossed with tuber-bearing species also has important implications for the regulatory aspects of the use of genetically modified organisms.     

Origin and evolution of Andigena potatoes revealed by chloroplast and nuclear DNA markers.

Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are important, native-farmer-selected cultivars in the Andes, which form a primary gene pool for improving a worldwide grown potato (S. tuberosum subsp. tuberosum). To elucidate the origin of Andigena, 196 Andigena accessions were compared with 301 accessions of 33 closely related cultivated and wild species using several types of chloroplast DNA (ctDNA) markers and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Fourteen ctDNA types (haplotypes) and 115 RFLP bands were detected in Andigena, of which the main haplotypes and frequent RFLP bands were mostly shared with a cultivated diploid species, S. stenotomum Juz. et Buk. Principal component analysis of nDNA polymorphisms revealed a progressive and continuous variation from Peruvian wild species with C-type ctDNA to a group of wild species having S-type ctDNA in its variation range (S. bukasovii, S. canasense, S. candolleanum, and S. multidissectum), to cultivated diploid potatoes (S. phureja and S. stenotomum), and to cultivated tetraploid potatoes (Andigena and Chilean S. tuberosum subsp. tuberosum). These results suggest that the initial Andigena population arose with multiple origins exclusively from S. stenotomum. The overall evolutionary process toward the present-day Andigena was discussed.     

Somatic hybrids <Emphasis Type="Italic">Solanum nigrum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis>: morphological assessment and verification of hybridity

Somatic hybrids between the cultivated potato diploid hybrid clone, ZEL-1136, and hexaploid non-tuber-bearing wild species Solanum nigrum L. exhibiting resistance to Phytophthora infestans were regenerated after PEG-mediated fusion of mesophyll protoplasts. The objective was to transfer the late-blight resistance genes from the wild species into plants of the cultivated potato clone. From a total of 59 regenerants, 40 clones survived and have been maintained in vitro on hormone-free MS/2 medium. Thirty-two somatic hybrids were identified by their intermediate morphology (leaves of nigrum type and flowers of tuberosum type) and verified by flow cytometry and random amplified polymorphic DNA (RAPD) patterns. The RAPD analysis of nuclear DNA confirmed the hybrid nature of 29 clones. Flow cytometry revealed a wide range of ploidy in the generated hybrids, from nearly the tetra- to decaploid level. Most of the hybrid clones were stable in vitro, grew vigorously in soil, and set flowers and parthenocarpic berries. However, all of the flowering hybrids were male-sterile. Nine hybrid clones produced tuber-like structures in soil. The most vigorous flowering somatic hybrids were selected for assessment of the late-blight resistance.     

Identification of mitotic chromosomes of tuberous and non-tuberous solanum species (Solanum tuberosum and Solanum brevidens) by GISH in their interspecific hybrids.

GISH (genomic in situ hybridization) was applied for the analysis of mitotic chromosome constitutions of somatic hybrids and their derivatives between dihaploid clones of cultivated potato (Solanum tuberosum L.) (2n = 2x = 24, AA genome) and the diploid, non-tuberous, wild species Solanum brevidens Phil. (2n = 2x = 24, EE genome). Of the primary somatic hybrids, both tetraploid (2n = 4x) and hexaploid (2n = 6x) plants were found with the genomic constitutions of AAEE and AAEEEE, respectively. Androgenic haploids (somatohaploids) derived from the tetraploid somatic hybrids had the genomic constitutions of AE (2n = 2x = 24) and haploids originating from the hexaploid hybrids were triploid AEE (2n = 3x = 33 and 2n = 3x = 36). As a result of subsequent somatic hybridization from a fusion between dihaploid S. tuberosum (2n = 2x = 24, genome AA) and a triploid somatohaploid (2n = 3x = 33, genome AEE), second-generation somatic hybrids were obtained. These somatic hybrids were pentaploids (2n = 5x, genome AAAEE), but had variable chromosome numbers. GISH analysis revealed that both primary and second-generation somatic hybrids had lost more chromosomes of S. brevidens than of S. tuberosum.     

Allopolyploid speciation of the Mexican tetraploid potato species Solanum stoloniferum and S. hjertingii revealed by genomic in situ hybridization

Thirty-six percent of the wild potato (Solanum L. section Petota Dumort.) species are polyploid, and about half of the polyploids are tetraploid species (2n = 4x = 48). Determination of the type of polyploidy and development of the genome concept for members of section Petota traditionally has been based on the analysis of chromosome pairing in species and their hybrids and, most recently, DNA sequence phylogenetics. Based on these data, the genome designation AABB was proposed for Mexican tetraploid species of series Longipedicellata Buk. We investigated this hypothesis with genomic in situ hybridization (GISH) for both representatives of the series, S. stoloniferum Schltdl. and S. hjertingii Hawkes. GISH analysis supports an AABB genome constitution for these species, with S. verrucosum Schltdl. (or its progenitor) supported as the A genome donor and another North or Central American diploid species (S. cardiophyllum Lindl., S. ehrenbergii (Bitter) Rydb., or S. jamesii Torrey) as the B genome donor. GISH analysis of chromosome pairing of S. stoloniferum also confirms the strict allopolyploid nature of this species. In addition, fluorescence in situ hybridization data suggest that 45S rDNA regions of the two genomes of S. stoloniferum were changed during coevolution of A and B genomes of this allotetraploid species.     

Analysis of ribosomal RNA genes in somatic hybrids between wild and cultivated Solanum species

Ribosomal RNA genes were exploited as markers to identify somatic hybrids between Solanum tuberosum cv. Brodick and wild diploid Solanum species, S. megistacrolobum, S. sanctae-rosae and S. sparsipilum and DNA methylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probe combinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions of the ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas others were characteristic of S. tuberosum cv. Brodick. This evidence is suggestive of somatic exchange/re-arrangement between the NOR region of S. sanctae-rosae and S. tuberosum cv. Brodick. Ribosomal RNA gene copy number analysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetric hybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation of ribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, others show an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.     

Reduced recombination and distorted segregation in a Solanum tuberosum (2x) x S. spegazzinii (2x) hybrid.

In this paper we describe the reduced recombination and distorted segregation in an interspecific hybrid between Solanum tuberosum and Solanum spegazzinii. To study these phenomena, a cross was made between a (di)haploid S. tuberosum, used as a female parent, and a diploid wild potato species, S. spegazzinii, used as a male parent. Next, a backcross (BC) population was made with F1 genotype 38 that was backcrossed to S. tuberosum. In the backcross, S. tuberosum was used as the male parent. RFLP linkage maps were made using the F1 and the BC populations, yielding linkage maps of the interspecific hybrid, S. spegazzinii, and S. tuberosum from which male and female linkage maps could be constructed. The computer program JOINMAP was used to construct and combine the separate linkage maps. Subsequently, the separate linkage maps were compared with each other, and reduced recombination was observed in the linkage maps of the male S. tuberosum and the interspecific hybrid. The reason for this reduced recombination is discussed. Another common feature in linkage maps is the observation of distorted segregation. The distorted segregation of alleles from the interspecific hybrid was studied in more detail in the BC population. Most of the distortion was probably caused by gamete selection, but for 3 loci, on chromosomes 2, 3, and 4, we found evidence for the presence of a strong selection force acting at the zygote level against homozygous genotypes.     

RAPD analysis of the interspecific somatic hybrids: Solanum bulbocastanum (+) S. tuberosum

The diploid Mexican species S. bulbocastanum (blb) was used as a source of late blight resistance in somatic hybridization with the potato (S. tuberosum, tbr) dihaploid H-8105. The produced 2x blb (+) 2x tbr H-8105 somatic hybrids did not retain the blb parent's characteristic high resistance to P. infestans. The revealed aneuploidy of blb (+) tbr H-8105 hybrids indicated a possible loss of individual blb chromosome(s) carrying the resistance genes. Four hybrid clones differing in terms of their ploidy, morphology and growth potential were analysed for the presence of all twelve blb chromosomes (linkage groups). The RAPD markers assigned to particular chromosomes were selected on the basis of the linkage map of S. bulbocastanum constructed by Naess et al., Mol. Gen. Genom. 265 (2001) 694-704. Of the 86 markers analysed, twelve (14%) were common for blb and H-8105, while 34 (40%) and 40 (46%) markers were specific for the blb and H-8105 genome, respectively; this confirms the differences between the nuclear genomes of the two species. Seventeen markers (20%) present in one or the other parent were absent in the hybrids, and only one new marker was found in the hybrids. The poorly growing, aneuploid and chimeric hybrids had the same band profiles as the well growing, morphologically normal hybrids, except for two bands that were present only in normal hybrids. The presence of eleven blb linkage groups in the blb (+) tbr H-8105 hybrids was confirmed. The markers specific for the second linkage group (chromosome 2) of blb were not present in the RAPD patterns of the somatic hybrids analysed, suggesting a loss or rearrangement of this chromosome in the combined nuclear genome of the hybrids.     

Transfer of resistance to potato leaf roll virus from Solanum brevidens into Solanum tuberosum by somatic fusion

Tetraploid somatic hybrids were produced by protoplast fusion between Solanum brevidens, a diploid non-tuber-bearing wild species and a diploid tuber-bearing potato line derived from an S. tuberosum Gp. Phureja-Stenotumum population. S. brevidens has resistance to potato leaf roll virus (PLRV) and frost but is difficult to cross sexually with cultivated potato. Hybridity was verified by morphological characteristics and cytological observations. Nine of ten hybrids tested showed resistance to PLRV. Hybrids produced fertile pollen and eggs which may allow beneficial traits of S. brevidens to be incorporated into a conventional potato breeding programme.     

RAPD and pedigree-based genetic diversity estimates in cultivated diploid potato hybrids

In this study, RAPD and pedigree data were used to investigate the genetic relationships in a group of 45 diploid hybrid potato clones used in the breeding and genetics program of the Agriculture and Agri-Food Canada Potato Research Centre in Fredericton, New Brunswick, and used for the potato after-cooking darkness program at the Nova Scotia Agricultural College. These hybrids were derived from crossing primitive cultivated South American diploid species such as Solanum phureja or Solanum stenotomum and wild diploid species such as Solanum chacoense and other wild Argentine species with haploids of Solanum tuberosum. These hybrids have subsequently undergone up to 30 years of breeding and selection, for adaptation to local growing and storage conditions, processing traits and pest resistances. The objectives of this study were to estimate the level of genetic similarity (GS) among these sets of clones and to investigate the correlation between RAPD-based GS and f, based on pedigree information. Genetic similarity coefficients varied from 0.29 to 0.90 with a mean of 0.65 when based on the RAPD data, whereas the coefficient of parentage varied from zero to 0.75 with a mean of 0.11. The degree of relationship between the similarity matrices based on RAPD and pedigree was measured by comparing the similarity matrices with the normalized Mantel test. A low positive correlation (R = 0.104, p = 0.999) between the two matrices was observed. Cluster analysis using GS divided the clones into many subgroups that did not correspond well with the grouping based on pedigree. The level of genetic variation present in this set of potato clones is very high. Rigorous selection pressure aimed at different breeding purposes may result in the genetic differentiation of the clones from the same origin.     

Hybridization and polyploidization effects on LTR-retrotransposon activation in potato genome

Hybridization and polyploidization are major forces in plant evolution and potatoes are not an exception. It is proposed that the proliferation of Long Terminal Repeat-retrotransposons (LTR-RT) is related to genome reorganization caused by hybridization and/or polyploidization. The main purpose of the present work was to evaluate the effect of interspecific hybridization and polyploidization on the activation of LTR-RT. We evaluated the proliferation of putative active LTR-RT in a diploid hybrid between the cultivated potato Solanum tuberosum and the wild diploid potato species S. kurtzianum, allotetraploid lines derived from this interspecific hybrid and S. kurtzianum autotetraploid lines (ktz-autotetraploid) using the S-SAP (sequence-specific amplified polymorphism) technique and normalized copy number determination by qPCR. Twenty-nine LTR-RT copies were activated in the hybrid and present in the allotetraploid lines. Major LTR-RT activity was detected in Copia-27, Copia-12, Copia-14 and, Gypsy-22. According to our results, LTR-RT copies were activated principally in the hybrid, there was no activation in allotetraploid lines and only one copy was activated in the autotetraploid.

     

Interspecific somatic hybrids <Emphasis Type="Italic">Solanum bulbocastanum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis> H-8105

Interspecific somatic hybrids between a dihaploid potato clone H-8105 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuberizing species Solanum bulbocastanum were generated and analysed. Only ten regenerants displaying the intermediate morphology with dominating characteristics of the wild parent (simple leaves, anthocyanin pigmentation) were produced in 15 weeks after a single PEG-mediated fusion event. The RAPD patterns confirmed the hybridity of all of them. The hybrids rooted poorly and grew slowly in vitro. The cytological analysis revealed a high degree of aneuploidy in the hybrids with morphological and growth anomalies in vitro, while the morphologically normal hybrids were tetraploids. All the S. bulbocastanum (+) H-8105 hybrids were unstable in culture and three of them were consequently lost during three years of propagation in vitro. The possible reasons for instability of somatic hybrids between the distantly related species are discussed.