Diploid hybrids between allotetraploid wild potato species Solanum acaule Bitt., S. stoloniferum Schltdl. and dihaploids of S. tuberosum 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.     

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.     

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.     

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.     

Rescuing abortive inter-EBN potato hybrids through double pollination and embryo culture

Summary Regeneration of inter-EBN hybrids among potato species was achieved using embryo rescue techniques. Tetraploid hybrids between 4x(2EBN) Solanum stoloniferum x 4x(4EBN) S. tuberosum Gp. Andigena as well as diploid hybrids between 2x(1EBN) S. chancayense x 2x(2EBN) S. chacoense were obtained by culturing immature hybrid embryos in nutrient medium. Identification of appropriate embryo developmental stages was critical in developing a suitable protocol for rescuing viable hybrid embryos. The use of IVP clones as the second pollinator in 4x(2EBN) x 4x(4EBN) crosses reduced premature fruit drop and helped to identify triploid hybrids. Morphological and cytological examination confirmed true hybridity for a few of the regenerated plants. Male sterility and meiotic abnormalities were characteristic of the hybrids. Several S. stoloniferum-Andigena hybrids were successfully backcrossed to Gp. Andigena.Cooperative investigation of Vegetable Crops Research Unit, USDA, Agricultural Research Service, and the Wisconsin Agricultural Experiment Station Note: Reference to a specific brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of similar nature not mentioned     

Transfer of resistance to PLRV titer buildup from Solanum etuberosum to a tuber-bearing Solanum gene pool

Summary Hybrids between Solanum etuberosum and S. pinnatisectum harboring resistance to titer buildup of potato leafroll virus (PLRV) were reciprocally crossed with tuber-bearing wild species S. acaule and S. verrucosum. A total of 47 hybrids with acaule were obtained with the aid of embryo rescue and sterile culturing of embryos from imbibing seeds. All but two hybrids with acaule had low pollen stainabilities or were pollen sterile. Hybrid seeds from crosses with verrucosum were easily obtained, and the triploid progenies were sterile. Hybrid progeny were screened for resistance to PLRV infection by viruliferous green peach aphid and for resistance to titer buildup. Although hybrids did not exhibit resistance to infection, PLRV was not detectable using ELISA. Virus was detected, however, by graft transmission to Datura stramonium. Crosses of fertile acaule-etuberosum-pinnatisectum hybrids with S. phureja, a cultivated diploid, using the latter as pollen parent, produced berries but seed did not complete development and was aborted. Rescue of immature embryos 25 days after pollination by excision from berries and sterile culture produced vigorous, pot-cultured plants. Segregation of susceptible (virus detected) and resistant (virus undetected) progenies suggests simple inheritance.     

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.     

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.     

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.     

Resistance to Phytophthora infestans in somatic hybrids of Solanum nigrum L. and diploid potato

In breeding for resistance to late blight, ( Phytophthora infestans Mont. de Bary), an economically important disease affecting potatoes, the search for new sources of durable resistance includes the non-host wild Solanum species. The aim of this work was to evaluate the resistance to P. infestans in the somatic hybrids between S. nigrum L. and diploid potato clone ZEL-1136. Sixteen somatic hybrids, their fusion parents, and three standard potato cultivars were screened for resistance to P. infestans in two types of tests-on whole plants and detached leaves-with two highly aggressive and virulent isolates of P. infestans, US8 and MP322. In the whole plant assay, the foliage of the somatic hybrids showed no symptoms of infection, while the foliage of the potato fusion parent and the standard cultivars was infected with P. infestans. In the detached leaflet assay, the breaking-down of resistance of the S. nigrum L. parent and the variable response of individual hybrid clones were noted. Nine S. nigrum L. (+) ZEL-1136 hybrids showed a resistance that was significantly higher than that of S. nigrum, while six clones expressed a resistance to P. infestans similar to that of S. nigrum. The results confirm the effective transfer of late blight resistance of S. nigrum into its somatic hybrids with potato.     

The Role of Polyphenol Oxidase and Peroxidase in Potato Tuber Resistance to Soft Rot Caused by Erwinia carotovora

Tubers from somatic hybrids produced by protoplast fusion between Solanum brevidens, a diploid, non-tuber-bearing wild species, and a tetraploid S. tuberosum showed resistance to decay caused by soft rot Erwinia. Tubers of the S. tuberosum fusion parent and potato cultivar Russet Burbank are susceptible to bacterial soft rot. Tubers of somatic hybrids indicated higher levels of activities of polyphenol oxidase and peroxidase than tubers of the parental line of S. tuberosum and cultivar Russet Burbank. This is true for intact tubers and also for injured or inoculated tubers. Polish commercial potato cultivars indicated a higher susceptibility to soft rot than somatic hybrids. However, there were some differences in susceptibility to soft rot between Polish commercial potato cultivars, only slight differences were observed in the activities of the polyphenol oxidase and peroxidase between Polish cultivars. A relation between soft rot resistance and the activity of each enzyme was not found for intact, injured or inoculated tissue of commercial cultivars. On the contrary, the activities of both enzymes were significantly higher in the periderm than in the medullary tissue of somatic hybrids, the parental line and the commercial cultivars.     

Analysis of the introgression of Solanum bulbocastanum DNA into potato breeding lines

Somatic hybrids have been obtained between potato and Solanum bulbocastanum PI 245310, a Mexican diploid (2n=2x=24) species. Through restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) analyses it was found that the somatic hybrids contain each chromosome of the diploid parent and that the synteny of RFLP markers noted with tomato, potato and S. brevidens is largely maintained in S. bulbocastanum. RFLP analyses of BC1 progeny of two different hybrids indicated that a substantial number of markers were either lost or were heterozygous, in marked contrast with results previously noted with S. brevidens. A RAPD map for all 12 chromosomes of S. bulbocastanum was prepared and marker transmission was followed in three BC2 populations. Results with chromosomes 3, 8 and 10 from these populations are compared.     

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.     

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.     

Cryopreservation of the Norway spruce tissue culture line able to produce extracellular lignin

Somatic hybridization has been used in potato to overcome the sexual barriers between the cultivated (Solanum tuberosum L.) and wild species. To date hundreds of inter/intra-specific somatic hybrids have been produced via protoplast fusions using 23 Solanum species and characterized for multiple traits such as agronomic, disease/pest resistance, salinity, frost and others. With increasing success in recovery of fusion products, somatic hybrids have been exploited in potato genetics, breeding and genomics studies. Here, we report on progress in somatic hybridization research in potato during the past 40 years.     

Mapping QTLs for resistance to the cyst nematode Globodera pallida derived from the wild potato species Solanum vernei

Resistance to the potato cyst nematode (PCN) species Globodera pallida, derived from the wild diploid potato species Solanum vernei, has been investigated. This source of resistance, which is effective against all of the major pathotypes of G. pallida and Globodera rostochiensis, has been assumed to be due to several genetic factors, but it has proved difficult to deploy effectively in breeding strategies for potato cultivars. Diploid and tetraploid potato populations segregating for 'vernei' resistance were analysed. At the tetraploid level, a bulk segregant analysis (BSA) approach was employed and detected AFLP markers linked to a resistance QTL on potato linkage group V. Conventional linkage analysis of a diploid population identified QTL on linkage groups V and IX. A marker linked to a QTL on linkage group V has been converted to a single-locus PCR-based marker, which can be used to detect the presence of the QTL in diploid and tetraploid potato germplasm. Moreover, there is evidence that one of the AFLPs detected by BSA appears to be specific to an introgressed segment of DNA from S. vernei. These results are compared with those obtained from other studies on resistance to the PCN species G. pallida.     

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.     

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.     

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.