Characterization of the F Locus Responsible for Floral Anthocyanin Production in Potato.

Anthocyanins are pigmented secondary metabolites produced via the flavonoid biosynthetic pathway and play important roles in plant stress responses, pollinator attraction, and consumer preference. Using RNA-sequencing analysis of a cross between diploid potato (Solanum tuberosum L.) lines segregating for flower color, we identified a homolog of the ANTHOCYANIN 2 (AN2) gene family that encodes a MYB transcription factor, herein termed StFlAN2, as the regulator of anthocyanin production in potato corollas. Transgenic introduction of StFlAN2 in white-flowered homozygous doubled-monoploid plants resulted in a recovery of purple flowers. RNA-sequencing revealed the specific anthocyanin biosynthetic genes activated by StFlAN2 as well as expression differences in genes within pathways involved in fruit ripening, senescence, and primary metabolism. Closer examination of the locus using genomic sequence analysis revealed a duplication in the StFlAN2 locus closely associated with gene expression that is likely attributable to nearby genetic elements. Taken together, this research provides insight into the regulation of anthocyanin biosynthesis in potato while also highlighting how the dynamic nature of the StFlAN2 locus may affect expression.

Genome-wide Inference of Somatic Translocation Events During Potato Dihaploid Production.

Potato ( L.) breeders often use dihaploids, which are 2× progeny derived from 4× autotetraploid parents. Dihaploids can be used in diploid crosses to introduce new genetic material into breeding germplasm that can be integrated into tetraploid breeding through the use of unreduced gametes in 4× by 2× crosses. Dihaploid potatoes are usually produced via pollination by haploid inducer lines known as in vitro pollinators (IVP). In vitro pollinator chromosomes are selectively degraded from initially full hybrid embryos, resulting in 2× seed. During this process, somatic translocation of IVP DNA may occur. In this study, a genome-wide approach was used to identify such events and other chromosome-scale abnormalities in a population of 95 dihaploids derived from a cross between potato cultivar Superior and the haploid inducing line IVP101. Most Superior dihaploids showed translocation rates of <1% at 16,947,718 assayable sites, yet two dihaploids showed translocation rates of 1.86 and 1.60%. Allelic ratios at translocation sites suggested that most translocations occurred in individual cell lineages and were thus not present in all cells of the adult plants. Translocations were enriched in sites associated with high gene expression and H3K4 dimethylation and H4K5 acetylation, suggesting that they tend to occur in regions of open chromatin. The translocations likely result as a consequence of double-stranded break repair in the dihaploid genomes via homologous recombination during which IVP chromosomes are used as templates. Additionally, primary trisomy was observed in eight individuals. As the trisomic chromosomes were derived from Superior, meiotic nondisjunction may be common in potato.

Genome diversity of tuber-bearing Solanum uncovers complex evolutionary history and targets of domestication in the cultivated potato.

Cultivated potatoes (Solanum tuberosum L.), domesticated from wild Solanum species native to the Andes of southern Peru, possess a diverse gene pool representing more than 100 tuber-bearing relatives (Solanum section Petota). A diversity panel of wild species, landraces, and cultivars was sequenced to assess genetic variation within tuber-bearing Solanum and the impact of domestication on genome diversity and identify key loci selected for cultivation in North and South America. Sequence diversity of diploid and tetraploid Stuberosum exceeded any crop resequencing study to date, in part due to expanded wild introgressions following polyploidy that captured alleles outside of their geographic origin. We identified 2,622 genes as under selection, with only 14-16% shared by North American and Andean cultivars, showing that a limited gene set drove early improvement of cultivated potato, while adaptation of upland (Stuberosum group Andigena) and lowland (S. tuberosum groups Chilotanum and Tuberosum) populations targeted distinct loci. Signatures of selection were uncovered in genes controlling carbohydrate metabolism, glycoalkaloid biosynthesis, the shikimate pathway, the cell cycle, and circadian rhythm. Reduced sexual fertility that accompanied the shift to asexual reproduction in cultivars was reflected by signatures of selection in genes regulating pollen development/gametogenesis. Exploration of haplotype diversity at potato's maturity locus (StCDF1) revealed introgression of truncated alleles from wild species, particularly Smicrodontum in long-day-adapted cultivars. This study uncovers a historic role of wild Solanum species in the diversification of long-day-adapted tetraploid potatoes, showing that extant natural populations represent an essential source of untapped adaptive potential.

Meiotic crossovers are associated with open chromatin and enriched with Stowaway transposons in potato.

BACKGROUND: Meiotic recombination is the foundation for genetic variation in natural and artificial populations of eukaryotes. Although genetic maps have been developed for numerous plant species since the late 1980s, few of these maps have provided the necessary resolution needed to investigate the genomic and epigenomic features underlying meiotic crossovers. RESULTS: Using a whole genome sequencing-based approach, we developed two high-density reference-based haplotype maps using diploid potato clones as parents. The vast majority (81%) of meiotic crossovers were mapped to less than 5 kb. The fine-scale accuracy of crossover detection was validated by Sanger sequencing for a subset of ten crossover events. We demonstrate that crossovers reside in genomic regions of "open chromatin", which were identified based on hypersensitivity to DNase I digestion and association with H3K4me3-modified nucleosomes. The genomic regions spanning crossovers were significantly enriched with the Stowaway family of miniature inverted-repeat transposable elements (MITEs). The occupancy of Stowaway elements in gene promoters is concomitant with an increase in recombination rate. A generalized linear model identified the presence of Stowaway elements as the third most important genomic or chromatin feature behind genes and open chromatin for predicting crossover formation over 10-kb windows. CONCLUSIONS: Collectively, our results suggest that meiotic crossovers in potato are largely determined by the local chromatin status, marked by accessible chromatin, H3K4me3-modified nucleosomes, and the presence of Stowaway transposons.

Lanosterol synthase-like is involved with differential accumulation of steroidal glycoalkaloids in potato.

MAIN CONCLUSION: Phytosterol homeostasis may be maintained in leaves through diversion of intermediates into glycoalkaloid biosynthesis, whereas in tuber flesh, excess intermediates are catalyzed by tuber-specific StLAS - like , resulting in low tuber glycoalkaloids. Lanosterol synthase (LAS) and cycloartenol synthase (CAS) are phylogenetically related enzymes. Cycloartenol is the accepted precursor leading to cholesterol and phytosterols, and in potato, to steroidal glycoalkaloid (SGA) biosynthesis. LAS was also shown to synthesize some plant sterols, albeit at trace amounts, questioning its role in sterol homeostasis. Presently, a potato LAS-related gene (StLAS-like) was identified and its activity verified in a yeast complementation assay. A transgenic approach with targeted gene expression and metabolic profiling of sterols and SGAs was used. Analyses of StLAS-like transcript levels and StLAS-like-promoter::GUS reporter assays indicated specific expression in tuber flesh tissue. Overexpression of Arabidopsis AtLAS in leaves where the endogenic StLAS-like is not expressed, resulted with increased SGA level and reduced phytosterol level, while in the tuber flesh SGA level was reduced. StLAS-like expression only in tuber flesh may explain the differential accumulation of SGAs in commercial cultivars-low in tubers, high in leaves. In leaves, to maintain phytosterol homeostasis, an excess of intermediates may be diverted into SGA biosynthesis, whereas in tuber flesh these intermediates are catalyzed by tuber-specific StLAS-like instead, resulting in low levels of SGA.

Self-Fertility in a Cultivated Diploid Potato Population Examined with the Infinium 8303 Potato Single-Nucleotide Polymorphism Array.

Within a population of F hybrids between two genotypes ( L. Group Phureja DM 1-3 516 R44 [DM] and L. Group Tuberosum RH89-039-16 [RH]) used in the potato genome sequencing project, we observed fruit set after self-pollination on many plants. Examination of pollen tube growth in self-fertile and self-unfruitful F plants after controlled self-pollinations revealed no difference in the ability of pollen tubes to reach the ovary. To identify genomic regions linked with self-fertility, we genotyped the F population using a genome-wide single-nucleotide polymorphism (SNP) array. Polymorphic and robust SNPs were analyzed to identify allelic states segregating with the self-fertile phenotype. All 88 highly significant SNPs occurred on chromosome 12. Seeds obtained after self-pollination of self-fertile individuals were used to advance the population for four generations. Genotyping 46 self-fruitful and 46 self-unfruitful S plants on the Infinium 8303 Potato SNP array revealed eight SNPs segregating with self-fertility on chromosomes 4, 9, 11, and 12. Three times more heterozygosity than expected was found in the S generation. Estimates of heterozygosity were influenced by copy number variation (CNV) in the potato genome leading to spurious heterozygous genotyping calls. Some spurious heterozygosity could be removed by application of a CNV filter developed from alignment of additional monoploid potato genomic sequence to the DM reference genome. The genes responsible for fruit set in self-fertile plants in the F generation were restricted to chromosome 12, whereas new genomic regions contributed to the ability of S plants to set fruit after self-pollination.

Comparative Analysis of Regions with Distorted Segregation in Three Diploid Populations of Potato.

Genes associated with gametic and zygotic selection could underlie segregation distortion, observed as alterations of expected Mendelian genotypic frequencies in mapping populations. We studied highly dense genetic maps based on single nucleotide polymorphisms to elucidate the genetic nature of distorted segregation in potato. Three intra- and interspecific diploid segregating populations were used. DRH and D84 are crosses between the sequenced doubled monoploid DM 1-3 516 R44 Solanum tuberosum Group Phureja and either RH89-039-16 S. tuberosum or 84SD22, a S. tuberosum × S. chacoense hybrid. MSX902 is an interspecific cross between 84SD22 and Ber83 S. berthaultii × 2 × species mosaic. At the 0.05 significance level, 21%, 57%, and 51% of the total markers mapped in DRH, D84, and MSX902 exhibited distorted segregation, respectively. Segregation distortion regions for DRH were located on chromosomes 9 and 12; for D84 on chromosomes 2, 3, 4, 6, 7, and 8; and on chromosomes 1, 2, 7, 9, and 12 for MSX902. In general, each population had unique segregation distortion regions and directions of distortion. Interspecific crosses showed greater levels of distorted segregation and lower recombination rates as determined from the male parents. The different genomic regions where the segregation distortion regions occurred in the three populations likely reflect unique genetic combinations producing distorted segregation.

Genome Reduction Uncovers a Large Dispensable Genome and Adaptive Role for Copy Number Variation in Asexually Propagated Solanum tuberosum.

Clonally reproducing plants have the potential to bear a significantly greater mutational load than sexually reproducing species. To investigate this possibility, we examined the breadth of genome-wide structural variation in a panel of monoploid/doubled monoploid clones generated from native populations of diploid potato (Solanum tuberosum), a highly heterozygous asexually propagated plant. As rare instances of purely homozygous clones, they provided an ideal set for determining the degree of structural variation tolerated by this species and deriving its minimal gene complement. Extensive copy number variation (CNV) was uncovered, impacting 219.8 Mb (30.2%) of the potato genome with nearly 30% of genes subject to at least partial duplication or deletion, revealing the highly heterogeneous nature of the potato genome. Dispensable genes (>7000) were associated with limited transcription and/or a recent evolutionary history, with lower deletion frequency observed in genes conserved across angiosperms. Association of CNV with plant adaptation was highlighted by enrichment in gene clusters encoding functions for environmental stress response, with gene duplication playing a part in species-specific expansions of stress-related gene families. This study revealed unique impacts of CNV in a species with asexual reproductive habits and how CNV may drive adaption through evolution of key stress pathways.

Characterization of a potato activation-tagged mutant, nikku, and its partial revertant.

MAIN CONCLUSION: A potato mutant with a strong stress-response phenotype, and a partial mutant revertant, were characterized. Gene expression patterns and DNA cytosine methylation varied between these and wild-type, indicating a role for DNA cytosine methylation changes in the gene expression and visible phenotypes. Morphological and molecular studies were conducted to compare potato cv. Bintje, a Bintje activation-tagged mutant (nikku), and nikku revertant phenotype plants. Morphological studies revealed that nikku plants exhibited an extremely dwarf phenotype, had small hyponastic leaves, were rootless, and infrequently produced small tubers compared to wild-type Bintje. The overall phenotype was suggestive of a constitutive stress response, which was further supported by the greater expression level of several stress-responsive genes in nikku. Unlike the nikku mutant, the revertant exhibited near normal shoot elongation, larger leaves and consistent rooting. The reversion appeared partial, and was not the result of a loss of 35S enhancer copies from the original nikku mutant. Southern blot analyses indicated the presence of a single T-DNA insertion on chromosome 12 in the mutant. Gene expression studies comparing Bintje, nikku and revertant phenotype plants indicated transcriptional activation/repression of several genes flanking both sides of the insertion in the mutant, suggesting that activation tagging had pleiotropic effects in nikku. In contrast, gene expression levels for many, but not all, of the same genes in the revertant were similar to Bintje, indicating some reversion at the gene expression level as well. DNA methylation studies indicated differences in cytosine methylation status of the 35S enhancers between the nikku mutant and its revertant. In addition, global DNA cytosine methylation varied between Bintje, the nikku mutant and the revertant, suggesting involvement in gene expression changes, as well as mutant phenotype.

Transposon based activation tagging in diploid strawberry and monoploid derivatives of potato.

KEY MESSAGE: Diploid strawberry and potato transformed with a transposon tagging construct exhibited either global (strawberry) or local transposition (potato). An activation tagged, compact-sized strawberry mutant overexpressed the gene adjacent to Ds. As major fruit and vegetable crops, respectively, strawberry and potato are among the first horticultural crops with draft genome sequences. To study gene function, we examined transposon-tagged mutant strategies in model populations for both species, Fragaria vesca and Solanum tuberosum Group Phureja, using the same Activation/Dissociation (Ac/Ds) construct. Early somatic transposition during tissue culture occurred at a frequency of 18.5% in strawberry but not in potato transformants. Green fluorescent protein under a monocot promoter was a more reliable selectable marker in strawberry compared to potato. BASTA (gluphosinate herbicide) resistance served as an effective selectable marker for both species (80 and 85% reliable in strawberry and potato, respectively), although the effective concentration differed (0.5% for strawberry and 0.03% for potato). Transposons preferentially reinserted within genes (exons and introns) in both species. Real-time quantitative PCR revealed enhanced gene expression (670 and 298-fold expression compared to wild type in petiole and leaf tissue, respectively) for an activation tagged strawberry mutant with Ds inserted about 0.6 kb upstream from a gene coding for an epidermis-specific secreted glycoprotein EP1. Our data also suggested that endopolyploid (diploid) cells occurring in leaf explants of monoploid potato were the favored targets of T-DNA integration during transformation. Mutants obtained in these studies provide a useful resource for future genetic studies.

Characterization and RNA-seq analysis of underperformer, an activation-tagged potato mutant.

The potato cv. Bintje and a Bintje activation-tagged mutant, underperformer (up) were compared. Mutant up plants grown in vitro were dwarf, with abundant axillary shoot growth, greater tuber yield, altered tuber traits and early senescence compared to wild type. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant remained, but the up plants exhibited a lower tuber yield and fewer axillary shoots compared to wild type. Southern blot analyses indicated a single T-DNA insertion in the mutant, located on chromosome 10. Initial PCR-based gene expression studies indicated transcriptional activation/repression of several genes in the mutant flanking the insertion. The gene immediately flanking the right border of the T-DNA insertion, which encoded an uncharacterized Broad complex, Tramtrac, Bric-a-brac; also known as Pox virus and Zinc finger (BTB/POZ) domain-containing protein (StBTB/POZ1) containing an Armadillo repeat region, was up-regulated in the mutant. Global gene expression comparisons between Bintje and up using RNA-seq on leaves from 60 day-old plants revealed a dataset of over 1,600 differentially expressed genes. Gene expression analyses suggested a variety of biological processes and pathways were modified in the mutant, including carbohydrate and lipid metabolism, cell division and cell cycle activity, biotic and abiotic stress responses, and proteolysis.

Allelic variation in genes contributing to glycoalkaloid biosynthesis in a diploid interspecific population of potato.

KEY MESSAGE: Variation for allelic state within genes of both primary and secondary metabolism influences the quantity and quality of steroidal glycoalkaloids produced in potato leaves. Genetic factors associated with the biosynthesis and accumulation of steroidal glycoalkaloids (SGAs) in potato were addressed by a candidate gene approach and whole genome single nucleotide polymorphism (SNP) genotyping. Allelic sequences spanning coding regions of four candidate genes [3-hydroxy-3-methylglutaryl coenzyme A reductase 2 (HMG2); 2,3-squalene epoxidase; solanidine galactosyltransferase; and solanidine glucosyltransferase (SGT2)] were obtained from two potato species differing in SGA composition: Solanum chacoense (chc 80-1) and Solanum tuberosum group Phureja (phu DH). An F2 population was genotyped and foliar SGAs quantified. The concentrations of α-solanine, α-chaconine, leptine I, leptine II and total SGAs varied broadly among F2 individuals. F2 plants with chc 80-1 alleles for HMG2 or SGT2 accumulated significantly greater leptines and total SGAs compared to plants with phu DH alleles. Plants with chc 80-1 alleles at both loci expressed the greatest levels of total SGAs, α-solanine and α-chaconine. A significant positive correlation was found between α-solanine and α-chaconine accumulation as well as between leptine I and leptine II. A whole genome SNP genotyping analysis of an F2 subsample verified the importance of chc 80-1 alleles at HMG2 and SGT2 for SGA synthesis and accumulation and suggested additional candidate genes including some previously associated with SGA production. Loci on five and seven potato pseudochromosomes were associated with synthesis and accumulation of SGAs, respectively. Two loci, on pseudochromosomes 1 and 6, explained phenotypic segregation of α-solanine and α-chaconine synthesis. Knowledge of the genetic factors influencing SGA production in potato may assist breeding for pest resistance.

Construction of reference chromosome-scale pseudomolecules for potato: integrating the potato genome with genetic and physical maps.

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker-based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal "pseudomolecules".

Sequence diversity in coding regions of candidate genes in the glycoalkaloid biosynthetic pathway of wild potato species.

Natural variation in five candidate genes of the steroidal glycoalkaloid (SGA) metabolic pathway and whole-genome single nucleotide polymorphism (SNP) genotyping were studied in six wild [Solanum chacoense (chc 80-1), S. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, S. stoloniferum] and cultivated S. tuberosum Group Phureja (phu DH) potato species with contrasting levels of SGAs. Amplicons were sequenced for five candidate genes: 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2) and 2.3-squalene epoxidase (SQE) of primary metabolism, and solanidine galactosyltransferase (SGT1), and glucosyltransferase (SGT2) of secondary metabolism. SNPs (n = 337) producing 354 variations were detected within 3.7 kb of sequenced DNA. More polymorphisms were found in introns than exons and in genes of secondary compared to primary metabolism. Although no significant deviation from neutrality was found, dN/dS ratios < 1 and negative values of Tajima's D test suggested purifying selection and genetic hitchhiking in the gene fragments. In addition, patterns of dN/dS ratios across the SGA pathway suggested constraint by natural selection. Comparison of nucleotide diversity estimates and dN/dS ratios showed stronger selective constraints for genes of primary rather than secondary metabolism. SNPs (n = 24) with an exclusive genotype for either phu DH (low SGA) or chc 80-1 (high SGA) were identified for HMG2, SQE, SGT1 and SGT2. The SolCAP 8303 Illumina Potato SNP chip genotyping revealed eight informative SNPs on six pseudochromosomes, with homozygous and heterozygous genotypes that discriminated high, intermediate and low levels of SGA accumulation. These results can be used to evaluate SGA accumulation in segregating or association mapping populations.

Retrospective view of North American potato (Solanum tuberosum L.) breeding in the 20th and 21st centuries.

Cultivated potato (Solanum tuberosum L.), a vegetatively propagated autotetraploid, has been bred for distinct market classes, including fresh market, pigmented, and processing varieties. Breeding efforts have relied on phenotypic selection of populations developed from intra- and intermarket class crosses and introgressions of wild and cultivated Solanum relatives. To retrospectively explore the effects of potato breeding at the genome level, we used 8303 single-nucleotide polymorphism markers to genotype a 250-line diversity panel composed of wild species, genetic stocks, and cultivated potato lines with release dates ranging from 1857 to 2011. Population structure analysis revealed four subpopulations within the panel, with cultivated potato lines grouping together and separate from wild species and genetic stocks. With pairwise kinship estimates clear separation between potato market classes was observed. Modern breeding efforts have scarcely changed the percentage of heterozygous loci or the frequency of homozygous, single-dose, and duplex loci on a genome level, despite concerted efforts by breeders. In contrast, clear selection in less than 50 years of breeding was observed for alleles in biosynthetic pathways important for market class-specific traits such as pigmentation and carbohydrate composition. Although improvement and diversification for distinct market classes was observed through whole-genome analysis of historic and current potato lines, an increased rate of gain from selection will be required to meet growing global food demands and challenges due to climate change. Understanding the genetic basis of diversification and trait improvement will allow for more rapid genome-guided improvement of potato in future breeding efforts.

Integration of two diploid potato linkage maps with the potato genome sequence.

To facilitate genome-guided breeding in potato, we developed an 8303 Single Nucleotide Polymorphism (SNP) marker array using potato genome and transcriptome resources. To validate the Infinium 8303 Potato Array, we developed linkage maps from two diploid populations (DRH and D84) and compared these maps with the assembled potato genome sequence. Both populations used the doubled monoploid reference genotype DM1-3 516 R44 as the female parent but had different heterozygous diploid male parents (RH89-039-16 and 84SD22). Over 4,400 markers were mapped (1,960 in DRH and 2,454 in D84, 787 in common) resulting in map sizes of 965 (DRH) and 792 (D84) cM, covering 87% (DRH) and 88% (D84) of genome sequence length. Of the mapped markers, 33.5% were in candidate genes selected for the array, 4.5% were markers from existing genetic maps, and 61% were selected based on distribution across the genome. Markers with distorted segregation ratios occurred in blocks in both linkage maps, accounting for 4% (DRH) and 9% (D84) of mapped markers. Markers with distorted segregation ratios were unique to each population with blocks on chromosomes 9 and 12 in DRH and 3, 4, 6 and 8 in D84. Chromosome assignment of markers based on linkage mapping differed from sequence alignment with the Potato Genome Sequencing Consortium (PGSC) pseudomolecules for 1% of the mapped markers with some disconcordant markers attributable to paralogs. In total, 126 (DRH) and 226 (D84) mapped markers were not anchored to the pseudomolecules and provide new scaffold anchoring data to improve the potato genome assembly. The high degree of concordance between the linkage maps and the pseudomolecules demonstrates both the quality of the potato genome sequence and the functionality of the Infinium 8303 Potato Array. The broad genome coverage of the Infinium 8303 Potato Array compared to other marker sets will enable numerous downstream applications.

Steroidal glycoalkaloids in Solanum chacoense.

Potato (Solanum tuberosum L.), a domesticated species that is the fourth most important world agricultural commodity, requires significant management to minimize the effects of herbivore and pathogen damage on crop yield. A wild relative, Solanum chacoense Bitt., has been of interest to plant breeders because it produces an abundance of novel steroidal glycoalkaloid compounds, leptines and leptinines, which are particularly effective deterrents of herbivory by the Colorado potato beetle (Leptinotarsa decemlineata Say). Biochemical approaches were used in this study to investigate the formation and accumulation of SGAs in S. chacoense. SGA contents were determined in various organs at different stages of organ maturity during a time course of plant development. Leptines and leptinines were the main contributors to the increased levels in SGA concentration measured in the aerial versus the subterranean organs of S. chacoense accession 8380-1. Leptines were not detected in aboveground stolons until the stage where shoots had formed mature chlorophyllous leaves. To gain insights into SGA biosynthesis, the abundance of SGAs and steady-state transcripts of genes coding for enzymes of the central terpene and SGA-specific pathways in various plant organs at anthesis were compared. For two genes of primary terpene metabolism, transcript and SGA abundances were correlated, although with some discrepancies. For genes associated with SGA biosynthesis, transcripts were not detected in some tissues containing SGAs; however these transcripts were detected in the progenitor tissues, indicating the possibility that under our standard growth conditions, SGA biosynthesis is largely limited to highly proliferative tissues such as shoot, root and floral meristems.

Induction of potato steroidal glycoalkaloid biosynthetic pathway by overexpression of cDNA encoding primary metabolism HMG-CoA reductase and squalene synthase.

Potato steroidal glycoalkaloids (SGAs) are toxic secondary metabolites whose total content in tubers must be regulated. SGAs are biosynthesized by the sterol branch of the mevalonic acid/isoprenoid pathway. In a previous study, we showed a correlation between SGA levels and the abundance of transcript coding for HMG-CoA reductase 1 (HMG1) and squalene synthase 1 (SQS1) in potato tissues and potato genotypes varying in SGA content. Here, Solanum tuberosum cv. Desirée (low SGA producer) was transformed with a gene construct containing the coding region of either HMG1 or SQS1 of Solanum chacoense Bitt. clone 8380-1, a high SGA producer. SGA levels in transgenic HMG-plants were either greater than (in eight of 14 plants) or no different from untransformed controls, whereas only four of 12 SQS-transgenics had greater SGA levels than control, as determined by HPLC. Quantitative real-time PCR was used to estimate relative steady-state transcript levels of isoprenoid-, steroid-, and SGA-related genes in leaves of the transgenic plants compared to nontransgenic controls. HMG-transgenic plants exhibited increased transcript accumulation of SQS1, sterol C24-methyltransferase type1 (SMT1), and solanidine glycosyltransferase 2 (SGT2), whereas SQS-transgenic plants, had consistently lower transcript levels of HMG1 and variable SMT1 and SGT2 transcript abundance among different transgenics. HMG-transgenic plants exhibited changes in transcript accumulation for some sterol biosynthetic genes as well. Taken together, the data suggest coordinated regulation of isoprenoid metabolism and SGA secondary metabolism.

Retrotransposon-based markers from potato monoploids used in somatic hybridization.

In an attempt to remove lethal and deleterious genes and enhance the heterozygosity of the potato genome, we developed several diverse somatic hybrids through the electrofusion of selected monoploids. Somatic hybrids and somaclones resulting from fused and unfused protoplasts, respectively, were verified with microsatellites. Molecular markers anchored in the Tst1 retrotransposon were used to examine polymorphisms in the regenerated plants and to reveal any somaclonal variation. Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon display (sequence-specific amplified polymorphism (S-SAP), anchored in a retransposon) were examined on an ALFexpress DNA sequencer. Because of inconsistencies in the number and quality of bands revealed by the combination of either class of marker in combination with the ALFexpress, we cloned and sequenced 11 S-SAP bands to use as restriction fragment length polymorphism (RFLP) probes in Southern blot analyses of genetic relationships in our potato populations and among related Solanaceae. Readily scorable bands (n = 27) that separated somatic hybrids from parental monoploids and somaclones and grouped monoploids according to known genetic relationships were produced. Some of the probes could be used to differentiate tomato and Datura from potato. Sequence analysis of 5 cloned IRAP and 11 cloned S-SAP markers confirmed that they were anchored in the Tst1 retrotransposon. BLAST searches within GenBank produced 10 highly significant hits (5 nucleotide, 4 expressed sequence tag (EST), and 1 protein) within closely related Solanaceae, suggesting that Tst1 represents an old retroelement that was inserted before the diversion of genera within Solanaceae; however, most sequences were undescribed.

Phenotypic and transcriptomic changes associated with potato autopolyploidization.

Polyploidy is remarkably common in the plant kingdom and polyploidization is a major driving force for plant genome evolution. Polyploids may contain genomes from different parental species (allopolyploidy) or include multiple sets of the same genome (autopolyploidy). Genetic and epigenetic changes associated with allopolyploidization have been a major research subject in recent years. However, we know little about the genetic impact imposed by autopolyploidization. We developed a synthetic autopolyploid series in potato (Solanum phureja) that includes one monoploid (1x) clone, two diploid (2x) clones, and one tetraploid (4x) clone. Cell size and organ thickness were positively correlated with the ploidy level. However, the 2x plants were generally the most vigorous and the 1x plants exhibited less vigor compared to the 2x and 4x individuals. We analyzed the transcriptomic variation associated with this autopolyploid series using a potato cDNA microarray containing approximately 9000 genes. Statistically significant expression changes were observed among the ploidies for approximately 10% of the genes in both leaflet and root tip tissues. However, most changes were associated with the monoploid and were within the twofold level. Thus, alteration of ploidy caused subtle expression changes of a substantial percentage of genes in the potato genome. We demonstrated that there are few genes, if any, whose expression is linearly correlated with the ploidy and can be dramatically changed because of ploidy alteration.