Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids.     

Homeologous recombination in Solanum lycopersicoides introgression lines of cultivated tomato

A library of "introgression lines" containing Solanum lycopersicoides chromosome segments in the genetic background of cultivated tomato (Lycopersicon esculentum) was used to study factors affecting homeologous recombination. Recombination rates were estimated in progeny of 43 heterozygous introgressions and whole-chromosome substitution lines, together representing 11 of the 12 tomato chromosomes. Recombination within homeologous segments was reduced to as little as 0-10% of expected frequencies. Relative recombination rates were positively correlated with the length of introgressed segments on the tomato map. The highest recombination (up to 40-50% of normal) was observed in long introgressions or substitution lines. Double-introgression lines containing two homeologous segments on opposite chromosome arms were synthesized to increase their combined length. Recombination was higher in the double than in the single segment lines, despite a preference for crossovers in the region of homology between segments. A greater increase in homeologous recombination was obtained by crossing the S. lycopersicoides introgression lines to L. pennellii--a phylogenetically intermediate species--or to L. esculentum lines containing single L. pennellii segments on the same chromosome. Recombination rates were highest in regions of overlap between S. lycopersicoides and L. pennellii segments. The potential application of these results to breeding with introgression lines is discussed.     

A library of Solanum lycopersicoides introgression lines in cultivated tomato.

A set of introgression lines (ILs), containing individual chromosome segments from the wild nightshade Solanum lycopersicoides bred into the genetic background of cultivated tomato (Lycopersicon esculentum), has been developed. A primary group of 56 lines was selected for maximum representation of the S. lycopersicoides genome (approximately 96% of the total map units), homozygosity, and a minimum number of introgressed segments per line. A secondary set of 34 lines provides increased map resolution in certain regions. Approximately 34% of the lines were sterile in the homozygous condition, but could be maintained by heterozygotes. To facilitate identification of segregating ILs, restriction fragment length polymorphism probes were converted to higher throughput cleaved amplified polymorphic sequence markers, which supplement allozyme and morphological loci. Strong segregation distortion was observed in F2 progeny of heterozygous ILs, with an excess of L. esculentum alleles in most regions. For introgressions on distal chromosome 1L, a preferential transmission of S. lycopersicoides alleles was observed in the male germ line. Homozygous ILs generally yielded less seed from self pollination than corresponding heterozygotes, indicating that sterility effects were recessive. This IL library provides a novel resource for genetic studies of traits found in S. lycopersicoides.     

Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight

 Solanum bulbocastanum, a wild, diploid (2n=2x=24) Mexican species, is highly resistant to Phytophthora infestans, the fungus that causes late blight of potato. However this 1 EBN species is virtually impossible to cross directly with potato. PEG-mediated fusion of leaf cells of S. bulbocastanum PI 245310 and the tetraploid potato line S. tuberosum PI 203900 (2n=4x=48) yielded hexaploid (2n= 6x=72) somatic hybrids that retained the high resistance of the S. bulbocastanum parent. RFLP and RAPD analyses confirmed the hybridity of the materials. Four of the somatic hybrids were crossed with potato cultivars Katahdin or Atlantic. The BC₁ progeny segregated for resistance to the US8 genotype (A-2 mating type) of P. Infestans. Resistant BC₁ lines crossed with susceptible cultivars again yielded populations that segregated for resistance to the fungus. In a 1996 field-plot in Wisconsin, to which no fungicide was applied, two of the BC₁ lines, from two different somatic hybrids, yielded 1.36 and 1.32 kg/plant under a severe late-blight epidemic. In contrast, under these same conditions the cultivar Russet Burbank yielded only 0.86 kg/plant. These results indicate that effective resistance to the late-blight fungus in a sexually incompatible Solanum species can be transferred into potato breeding lines by somatic hybridization and that this resistance can then be further transmitted into potato breeding lines by sexual crossing. Received: 27 October 1997 / Accepted: 11 November 1997     

An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease for potato cultivation. Here, we describe the positional cloning of the Rpi-blb1 gene from the wild potato species Solanum bulbocastanum known for its high levels of resistance to late blight. The Rpi-blb1 locus, which confers full resistance to complex isolates of P. infestans and for which race specificity has not yet been demonstrated, was mapped in an intraspecific S. bulbocastanum population on chromosome 8, 0.3 cM from marker CT88. Molecular analysis of a bacterial artificial chromosome (BAC) clone spanning the Rpi-blb1 locus identified a cluster of four candidate resistance gene analogues of the coiled coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) class of plant resistance (R) genes. One of these candidate genes, designated the Rpi-blb1 gene, was able to complement the susceptible phenotype in a S. tuberosum and tomato background, demonstrating the potential of interspecific transfer of broad-spectrum late blight resistance to cultivated Solanaceae from sexually incompatible host species. Paired comparisons of synonymous and non-synonymous nucleotide substitutions between different regions of Rpi-blb1 paralogues revealed high levels of synonymous divergence, also in the LRR region. Although amino acid diversity between Rpi-blb1 homologues is centred on the putative solvent exposed residues of the LRRs, the majority of nucleotide differences in this region have not resulted in an amino acid change, suggesting conservation of function. These data suggest that Rpi-blb1 is relatively old and may be subject to balancing selection.     

GISH analysis of meiotic chromosome pairing in Solanum lycopersicoides introgression lines of cultivated tomato.

Meiotic chromosome pairing was studied in introgression lines of cultivated tomato, Lycopersicon esculentum (= Solanum lycopersicum), containing 1 or 2 chromosome segments from the wild species Solanum lycopersicoides. Genomic in situ hybridization (GISH) was used to compare the relative lengths at diakinesis of the different introgressed segments and to measure the chiasmate arm frequency for the chromosome pair involved in the introgression(s). Longer segments generally produced stronger GISH signals than shorter segments. GISH signal intensity also depended on whether or not an introgressed segment encompassed the centromeric region. For example, a 29 cM segment that included the centromeric region produced a stronger GISH signal than a 42 cM segment that did not. In each line the chromosome arm containing the homeologous segment showed a reduction in chiasmate arm frequency that was most pronounced in lines with long segments. This reduction was accompanied by an increased chiasmate arm frequency on the other arm. Double introgression lines, heterozygous in repulsion phase for 2 introgressions on opposite chromosome arms, showed a lower frequency of chiasmata than single introgression lines. Pairing failure, indicated by the presence of univalents, was highest in the double introgression and whole chromosome substitution lines. These results are discussed with respect to observations of suppressed recombination in these stocks and potential practical implications for reducing linkage drag in breeding programs.     

Insights of section-wide pan-genome into hybrid potato breeding

Science China Life Sciences -     

The Rpi-blb2 gene from Solanum bulbocastanum is an Mi-1 gene homolog conferring broad-spectrum late blight resistance in potato

The necessity to develop potato and tomato crops that possess durable resistance against the oomycete pathogen Phytophthora infestans is increasing as more virulent, crop-specialized and pesticide resistant strains of the pathogen are rapidly emerging. Here, we describe the positional cloning of the Solanum bulbocastanum-derived Rpi-blb2 gene, which even when present in a potato background confers broad-spectrum late blight resistance. The Rpi-blb2 locus was initially mapped in several tetraploid backcross populations, derived from highly resistant complex interspecific hybrids designated ABPT (an acronym of the four Solanum species involved:S. acaule, S. bulbocastanum, S. phureja and S. tuberosum), to the same region on chromosome 6 as the Mi-1 gene from tomato, which confers resistance to nematodes, aphids and white flies. Due to suppression of recombination in the tetraploid material, fine mapping was carried out in a diploid intraspecific S. bulbocastanum F1 population. Bacterial artificial chromosome (BAC) libraries, generated from a diploid ABPT-derived clone and from the resistant S. bulbocastanum parent clone, were screened with markers linked to resistance in order to generate a physical map of the Rpi-blb2 locus. Molecular analyses of both ABPT- and S. bulbocastanum-derived BAC clones spanning the Rpi-blb2 locus showed it to harbor at least 15 Mi-1 gene homologs (MiGHs). Of these, five were genetically determined to be candidates for Rpi-blb2. Complementation analyses showed that one ABPT- and one S. bulbocastanum-derived MiGH were able to complement the susceptible phenotype in both S. tuberosum and tomato. Sequence analyses of both genes showed them to be identical. The Rpi-blb2 protein shares 82% sequence identity to the Mi-1 protein. Significant expansion of the Rpi-blb2 locus compared to the Mi-1 locus indicates that intrachromosomal recombination or unequal crossing over has played an important role in the evolution of the Rpi-blb2 locus. The contrasting evolutionary dynamics of the Rpi-blb2/Mi-1 loci in the two related genomes may reflect the opposite evolutionary potentials of the interacting pathogens.     

Identification of 2n breeding lines and 4n varieties of potato (Solanum tuberosum,ssp. tuberosum) with RFLP-fingerprints

Summary The possibility of genotype identification with RFLP fingerprints was examined with 20 tetraploid potato varieties and 38 diploid potato lines. By using a sensitive detection system for small restriction fragment length differences and highly variable potato sequences as probes, all genotypes (diploids and tetraploids) were distinguished by a minimum of two probe/enzyme combinations. The best single probe/enzyme combination distinguished 19 out of 20 4n varieties and 33 out of 38 2n lines. Intravarietal variability was very small compared to the intervarietal variability, and patterns obtained with different DNA sources of the same genotype were identical.     

Polymorphisms and Evolution of Solanum bulbocastanum Genes for Broad-Spectrum Resistance to Phytophthora infestans

Russian Journal of Plant Physiology - For two decades, Solanum bulbocastanum Dun. has been challenging potato geneticists with its durable resistance to Phytophthora infestans Mont. de Bary, the...     

Application of interspecific sesquiploidy to introgression of PLRV resistance from non-tuber-bearing Solanum etuberosum to cultivated potato germplasm

Summary Hybridization of synthetic allotetraploids of S. pinnatisectum with S. etuberosum (4x-EP) with S. acaule (2n = 4x = 48) resulted in two individuals that were highly fertile, in contrast to all other progenies. The unique individuals are hexaploids, 2n = 72, while the other progenies are tetraploids, 2n = 48. They are thought to be the products of a union between 2n eggs of S. acaule and normal 1n microspores of 4x-EP. The fertile hexaploids (designated 6x-AEP) produced abundant selfed seed and viable hybrids with cultivated diploid potato, S. phureja, when developing embryos were rescued from berries and cultured before transplanting to pot culture. The extreme variability in chromosome constitution of the hybrids with S. phureja and selfed progenies indicates that addition and substitution lines of etb chromosomes bearing genes of interest to breeders could easily be produced from this material. The production of sesquiploids, as the 6x-AEP hybrids are called, is discussed as a useful bridging step in the introduction of alien genes from genomes that share little homology with the cultivated genome.This work was part of the requirement for a Ph.D. degree of the senior author R. Chavez at The University of Birmingham, UK     

Solanum lycopersicoides gene introgression to tomato,Lycopersicon esculentum,through the systematic avoidance and suppression of breeding barriers

Summary While Lycopersicon esculentum and Solanum lycopersicoides have been successfully hybridized, attempts at further direct gene introgression have been unsuccessful due to the presence of incompatibility barriers. A systematic study of the initial hybridization and subsequent backcrosses has identified multiple barriers to introgression. These barriers are expressed as pollen tube inhibition in the upper style and lower pistil, and failures in syngamy, zygote development, and sporogenesis. Upper style cross-incompatibility barriers were successfully avoided by bud pollinations using a stigma complementation procedure to allow pollen germination on otherwise unreceptive stigmas. The inhibition of pollen tube growth was observed in the lower pistil. A combination of environmental, plant, and genetic manipulations facilitated consistent pollen tube growth to the ovule micropyles in all crosses attempted. Failures at syngamy and early zygote formation proved to be the most difficult barriers to overcome — these were particularly severe in crosses to F₁ hybrid plants. Progeny were obtained in all crossing combinations attempted except in the initial hybridization with S. lycopersicoides as the pistillate parent. Although the strong pre-zygotic barriers were overcome in this cross, further progress was restricted by post-zygotic failures. The capability to overcome pre-zygotic barriers and to excise and culture very young embryos has allowed plantlet recovery from male sterile F₁ plants. Partially pollen-fertile F₁ plants were recovered when relatively large F₁ populations were generated from different _{S. lycopersicoides accessions}. In general, barriers to introgression diminished with increased backcrossing, though exceptions were noted. Progeny from the second backcross to L. esculentum possessed adequate fertility to set self-seed under field conditions. Although all backcross progeny were developed from only a few F₁ individuals, considerable genetic variability was recovered for fruit and vegetative characteristics. Potentially useful levels of disease resistance, particularly to Botrytis cinerea, were also recovered.     

Chloroplast DNA evolution in potato (Solanum tuberosum L.)

Summary A deletion specific to chloroplast (ct) DNA of potato (Solanum tuberosum ssp. tuberosum) was determined by comparative sequence analysis. The deletion was 241 bp in size, and was not flanked by direct repeats. Five small, open reading frames were found in the corresponding regions of ctDNAs from wild potato (S. tuberosum ssp. andigena) and tomato (Lycopersicon esculentum). Comparison of the sequences of 1.35-kbp HaeIII ctDNA fragments from potato, tomato, and tobacco (Nicotiana tabacum) revealed the following: the locations of the 5 ends of both rubisco large subunit (rbcL) and ATPase beta subunit (atp) mRNAs were probably the same as those of spinach (Spinacia oleracea); the promoter regions of the two genes were highly conserved among the four species; and the 5 untranslated regions diverged at high rates. A phylogenetic tree for the three potato cultivars, one tomato cultivar, and one tobacco cultivar has been constructed by the maximum parsimony method from DNA sequence data, demonstrating that the rate of nucleotide substitution in potato ctDNA is much slower than that in tomato ctDNA. This fact might be due to the differences in the method of propagation between the two crops.     

Complete chloroplast genome sequences of Solanum bulbocastanum, Solanum lycopersicum and comparative analyses with other Solanaceae genomes

Despite the agricultural importance of both potato and tomato, very little is known about their chloroplast genomes. Analysis of the complete sequences of tomato, potato, tobacco, and Atropa chloroplast genomes reveals significant insertions and deletions within certain coding regions or regulatory sequences (e.g., deletion of repeated sequences within 16S rRNA, ycf2 or ribosomal binding sites in ycf2). RNA, photosynthesis, and atp synthase genes are the least divergent and the most divergent genes are clpP, cemA, ccsA, and matK. Repeat analyses identified 33–45 direct and inverted repeats ≥30 bp with a sequence identity of at least 90%; all but five of the repeats shared by all four Solanaceae genomes are located in the same genes or intergenic regions, suggesting a functional role. A comprehensive genome-wide analysis of all coding sequences and intergenic spacer regions was done for the first time in chloroplast genomes. Only four spacer regions are fully conserved (100% sequence identity) among all genomes; deletions or insertions within some intergenic spacer regions result in less than 25% sequence identity, underscoring the importance of choosing appropriate intergenic spacers for plastid transformation and providing valuable new information for phylogenetic utility of the chloroplast intergenic spacer regions. Comparison of coding sequences with expressed sequence tags showed considerable amount of variation, resulting in amino acid changes; none of the C-to-U conversions observed in potato and tomato were conserved in tobacco and Atropa. It is possible that there has been a loss of conserved editing sites in potato and tomato.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

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.     

Molecular marker-assisted introgression of the wild Solanum commersonii genome into the cultivated S. tuberosum gene pool

A breeding scheme involving ploidy and EBN manipulations was set up to overcome the interspecific barriers existing between the cultivated Solanum tuberosum and the wild species S. commersonii. Three backcross generations were obtained that were analyzed by means of molecular markers in order to verify the occurrence of recombination between homeologous chromosomes, the extent of the wild genome carried in each backcross, and the efficiency of introgressing useful genes. Twenty commersonii-specific RFLPs provided evidence for recombination on 5 out of 12 chromoso-mes; for the others no more than 1 commersonii-specific marker was found. Thirty-four commersonii-specific RAPDs and 61 commersonii-specific AFLPs were used to estimate the wild genome content in the BC₁, BC₂, and BC₃ generations. The mean value of commersonii-specific markers was 93% and 91% in the BC₁, 74% and 76% in the BC₂, and 31% and 26% in the BC₃ using RAPDs or AFLPs, respectively. Efficiency of the breeding scheme was evaluated by monitoring across these progenies the introgression of S. commersonii resistance to tuber soft rot caused by Erwinia carotovora. Eleven and five resistant genotypes were found among BC₂ and BC₃ hybrids, respectively. The same progenies were also evaluated for chromosome number and tuber traits. For all analyzed traits except stolon length, all BC₂ and BC₃ hybrids resembled the tuberosum type. In order to choose the best genotypes to obtain the following backcrosses, we performed, in each generation, a negative molecular-assisted selection against the wild genome combined with selection for resistance to Erwinia spp. and other traits. Received: 3 April 2000 / Accepted: 31 August 2000     

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

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

Cisgenesis strongly improves introgression breeding and induced translocation breeding of plants

There are two ways for genetic improvement in classical plant breeding: crossing and mutation. Plant varieties can also be improved through genetic modification; however, the present GMO regulations are based on risk assessments with the transgenes coming from non-crossable species. Nowadays, DNA sequence information of crop plants facilitates the isolation of cisgenes, which are genes from crop plants themselves or from crossable species. The increasing number of these isolated genes, and the development of transformation protocols that do not leave marker genes behind, provide an opportunity to improve plant breeding while remaining within the gene pool of the classical breeder. Compared with induced translocation and introgression breeding, cisgenesis is an improvement for gene transfer from crossable plants: it is a one-step gene transfer without linkage drag of other genes, whereas induced translocation and introgression breeding are multiple step gene transfer methods with linkage drag. The similarity of the genes used in cisgenesis compared with classical breeding is a compelling argument to treat cisgenic plants as classically bred plants. In the case of the classical breeding method induced translocation breeding, the insertion site of the genes is a priori unknown, as it is in cisgenesis. This provides another argument to treat cisgenic plants as classically bred plants, by exempting cisgenesis of plants from the GMO legislations.     

The late blight resistance locus Rpi-bib3 from Solanum bulbocastanum belongs to a major late blight R gene cluster on chromosome 4 of potato

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases in cultivated potato. Breeding of new potato cultivars with high levels of resistance to P. infestans is considered the most durable strategy for future potato cultivation. In this study, we report the identification of a new late-blight resistance (R) locus from the wild potato species Solanum bulbocastanum. Using several different approaches, a high-resolution genetic map of the new locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome 4. One amplification fragment length polymorphism marker was identified that cosegregated in 1,396 progeny plants of an intraspecific mapping population with Rpi-blb3. For comparative genomics purposes, markers linked to Rpi-blb3 were tested in mapping populations used to map the three other late-blight R loci Rpi-abpt, R2, and R2-like also to chromosome 4. Marker order and allelic conservation suggest that Rpi-blb3, Rpi-abpt, R2, and R2-like reside in the same R gene cluster on chromosome 4 and likely belong to the same gene family. Our findings provide novel insights in the evolution of R gene clusters conferring late-blight resistance in Solanum spp.