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Plants defend themselves chemically against herbivory through secondary metabolites and phytohormones. Few studies have investigated how constitutive variation in secondary metabolites contributes to systemic herbivory response. We hypothesized that plants with lower constitutive defenses would induce a stronger phytohormone response to spatially separated herbivory than plants with high constitutive defense. We used growth chamber bioassays to investigate how aboveground herbivory by Colorado potato beetle (Leptinotarsa decemlineata, CPB) and belowground herbivory by northern root-knot nematode (Meloidogyne hapla, RKN) altered phytohormones and glycoalkaloids in roots and shoots of two lines of wild potato (Solanum chacoense). These lines had different constitutive levels of chemical defense, particularly leptine glycoalkaloids, which are only present in aboveground tissues. We also determined how these differences influenced the preference and performance of CPB. The susceptible wild potato line responded to aboveground damage by CPB through induction of jasmonic acid (JA) and OPDA. However, when challenged by both RKN and CPB, the susceptible line retained high levels of JA, but not OPDA. Beetles gained more mass after feeding on the susceptible line compared to the resistant line, but were not affected by nematode presence. Belowground, JA, JA-Isoleucine, and OPDA were higher in the resistant line compared to the susceptible line, and some compounds demonstrated response to local herbivory. In contrast, the susceptible line did not induce phytohormone defenses belowground. These findings allow us to predict that constitutive level of defense may influence the threshold of herbivory that may lead to plant-mediated effects on spatially separated herbivores., (© 2024. The Author(s).)

A total of 3,860 accessions from the global in trust clonal potato germplasm collection w3ere genotyped with the Illumina Infinium SolCAP V2 12K potato SNP array to evaluate genetic diversity and population structure within the potato germplasm collection. Diploid, triploid, tetraploid, and pentaploid accessions were included representing the cultivated potato taxa. Heterozygosity ranged from 9.7% to 66.6% increasing with ploidy level with an average heterozygosity of 33.5%. Identity, relatedness, and ancestry were evaluated using hierarchal clustering and model-based Bayesian admixture analyses. Errors in genetic identity were revealed in a side-by-side comparison of in vitro clonal material with the original mother plants revealing mistakes putatively occurring during decades of processing and handling. A phylogeny was constructed to evaluate inter- and intraspecific relationships which together with a STRUCTURE analysis supported both commonly used treatments of potato taxonomy. Accessions generally clustered based on taxonomic and ploidy classifications with some exceptions but did not consistently cluster by geographic origin. STRUCTURE analysis identified putative hybrids and suggested six genetic clusters in the cultivated potato collection with extensive gene flow occurring among the potato populations, implying most populations readily shared alleles and that introgression is common in potato. Solanum tuberosum subsp. andigena (ADG) and S. curtilobum (CUR) displayed significant admixture. ADG likely has extensive admixture due to its broad geographic distribution. Solanum phureja (PHU), Solanum chaucha (CHA)/ Solanum stenotomum subsp. stenotomum (STN), and Solanum tuberosum subsp. tuberosum (TBR) populations had less admixture from an accession/population perspective relative to the species evaluated. A core and mini core subset from the genebank material was also constructed. SNP genotyping was also carried out on 745 accessions from the Seed Savers potato collection which confirmed no genetic duplication between the two potato collections, suggesting that the collections hold very different genetic resources of potato. The Infinium SNP Potato Array is a powerful tool that can provide diversity assessments, fingerprint genebank accessions for quality management programs, use in research and breeding, and provide insights into the complex genetic structure and hybrid origin of the diversity present in potato genetic resource collections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CR declared a past co-authorship with the author NA to the handling editor., (Copyright © 2024 Anglin, Chavez, Soto - Torres, Gomez, Panta, Vollmer, Durand, Meza, Azevedo, Manrique - Carpintero, Kauth, Coombs, Douches and Ellis.)

Inbred-hybrid breeding of diploid potatoes necessitates breeding lines that are self-compatible. One way of incorporating self-compatibility into incompatible cultivated potato ( Solanum tuberosum ) germplasm is to introduce the S-locus inhibitor gene ( Sli ), which functions as a dominant inhibitor of gametophytic self-incompatibility. To learn more about Sli diversity and function in wild species relatives of cultivated potato, we obtained Sli gene sequences that extended from the 5'UTR to the 3'UTR from 133 individuals from 22 wild species relatives of potato and eight diverse cultivated potato clones. DNA sequence alignment and phylogenetic trees based on genomic and protein sequences show that there are two highly conserved groups of Sli sequences. DNA sequences in one group contain the 533 bp insertion upstream of the start codon identified previously in self-compatible potato. The second group lacks the insertion. Three diploid and four polyploid individuals of wild species collected from geographically disjointed localities contained Sli with the 533 bp insertion. For most of the wild species clones examined, however, Sli did not have the insertion. Phylogenetic analysis indicated that Sli sequences with the insertion, in wild species and in cultivated clones, trace back to a single origin. Some diploid wild potatoes that have Sli with the insertion were self-incompatible and some wild potatoes that lack the insertion were self-compatible. Although there is evidence of positive selection for some codon positions in Sli , there is no evidence of diversifying selection at the gene level. In silico analysis of Sli protein structure did not support the hypothesis that amino acid changes from wild-type (no insertion) to insertion-type account for changes in protein function. Our study demonstrated that genetic factors besides the Sli gene must be important for conditioning a switch in the mating system from self-incompatible to self-compatible in wild potatoes., Competing Interests: The authors declare they have no conflict of interest., (© 2024 The Authors. Plant Direct published by John Wiley & Sons Ltd and American Society of Plant Biologists and the Society for Experimental Biology. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to minimize sprouting and losses due to disease. However, cold temperatures strongly induce the expression of the potato vacuolar invertase gene (VInv) and cause reducing sugar accumulation. This process, referred to as "cold-induced sweetening," is a major postharvest problem for the potato industry. We discovered that the cold-induced expression of VInv is controlled by a 200 bp enhancer, VInvIn2En, located in its second intron. We identified several DNA motifs in VInvIn2En that bind transcription factors involved in the plant cold stress response. Mutation of these DNA motifs abolished VInvIn2En function as a transcriptional enhancer. We developed VInvIn2En deletion lines in both diploid and tetraploid potato using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. VInv transcription in cold-stored tubers was significantly reduced in the deletion lines. Interestingly, the VInvIn2En sequence is highly conserved among distantly related Solanum species, including tomato (Solanum lycopersicum) and other non-tuber-bearing species. We conclude that the VInv gene and the VInvIn2En enhancer have adopted distinct roles in the cold stress response in tubers of tuber-bearing Solanum species., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Key Message: We find evidence of selection for local adaptation and extensive genotype-by-environment interaction in the potato National Chip Processing Trial (NCPT). We present a novel method for dissecting the interplay between selection, local adaptation and environmental response in plant breeding schemes. Balancing local adaptation and the desire for widely adapted cultivars is challenging for plant breeders and makes genotype-by-environment interactions (GxE) an important target of selection. Selecting for GxE requires plant breeders to evaluate plants across multiple environments. One way breeders have accomplished this is to test advanced materials across many locations. Public potato breeders test advanced breeding material in the National Chip Processing Trial (NCPT), a public-private partnership where breeders from ten institutions submit advanced chip lines to be evaluated in up to ten locations across the country. These clones are genotyped and phenotyped for important agronomic traits. We used these data to interrogate the NCPT for GxE. Further, because breeders submitting clones to the NCPT select in a relatively small geographic range for the first 3 years of selection, we examined these data for evidence of incidental selection for local adaptation, and the alleles underlying it, using an environmental genome-wide association study (envGWAS). We found genomic regions associated with continuous environmental variables and discrete breeding programs, as well as regions of the genome potentially underlying GxE for yield., (© 2024. The Author(s).)

Before the commercialization of genetically modified crops, the events carrying the novel DNA must be thoroughly evaluated for agronomic, nutritional, and molecular characteristics. Over the years, polymerase chain reaction-based methods, Southern blot, and short-read sequencing techniques have been utilized for collecting molecular characterization data. Multiple genomic applications are necessary to determine the insert location, flanking sequence analysis, characterization of the inserted DNA, and determination of any interruption of native genes. These techniques are time-consuming and labor-intensive, making it difficult to characterize multiple events. Current advances in sequencing technologies are enabling whole-genomic sequencing of modified crops to obtain full molecular characterization. However, in polyploids, such as the tetraploid potato, it is a challenge to obtain whole-genomic sequencing coverage that meets the regulatory approval of the genetic modification. Here we describe an alternative to labor-intensive applications with a novel procedure using Samplix Xdrop ® enrichment technology and next-generation Nanopore sequencing technology to more efficiently characterize the T-DNA insertions of four genetically modified potato events developed by the Feed the Future Global Biotech Potato Partnership: DIA_MSU_UB015, DIA_MSU_UB255, GRA_MSU_UG234, and GRA_MSU_UG265 (derived from regionally important varieties Diamant and Granola). Using the Xdrop® /Nanopore technique, we obtained a very high sequence read coverage within the T-DNA and junction regions. In three of the four events, we were able to use the data to confirm single T-DNA insertions, identify insert locations, identify flanking sequences, and characterize the inserted T-DNA. We further used the characterization data to identify native gene interruption and confirm the stability of the T-DNA across clonal cycles. These results demonstrate the functionality of using the Xdrop® /Nanopore technique for T-DNA characterization. This research will contribute to meeting regulatory safety and regulatory approval requirements for commercialization with small shareholder farmers in target countries within our partnership., Competing Interests: Author LJ was employed by the company Samplix, Birkerød, Denmark. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zarka, Jagd and Douches.)

Camelina sativa (L.) Crantz, a member of the Brassicaceae, has potential as a biofuel feedstock which is attributable to the production of fatty acids in its seeds, its fast growth cycle, and low input requirements. While a genome assembly is available for camelina, it was generated from short sequence reads and is thus highly fragmented in nature. Using long read sequences, we generated a chromosome-scale, highly contiguous genome assembly (644,491,969 bp) for the spring biotype cultivar 'Suneson' with an N50 contig length of 12,031,512 bp and a scaffold N50 length of 32,184,682 bp. Annotation of protein-coding genes revealed 91,877 genes that encode 133,355 gene models. We identified a total of 4,467 genes that were significantly up-regulated under cold stress which were enriched in gene ontology terms associated with "response to cold" and "response to abiotic stress". Coexpression analyses revealed multiple coexpression modules that were enriched in genes differentially expressed following cold stress that had putative functions involved in stress adaptation, specifically within the plastid. With access to a highly contiguous genome assembly, comparative analyses with Arabidopsis thaliana revealed 23,625 A. thaliana genes syntenic with 45,453 Suneson genes. Of these, 24,960 Suneson genes were syntenic to 8,320 A. thaliana genes reflecting a 3 camelina homeolog to 1 Arabidopsis gene relationship and retention of all three homeologs. Some of the retained triplicated homeologs showed conserved gene expression patterns under control and cold-stressed conditions whereas other triplicated homeologs displayed diverged expression patterns revealing sub- and neo-functionalization of the homeologs at the transcription level. Access to the chromosome-scale assembly of Suneson will enable both basic and applied research efforts in the improvement of camelina as a sustainable biofuel feedstock., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fang, Hamilton, Vaillancourt, Wang, Wood, Deans, Scroggs, Carlton, Mailloux, Douches, Nadakuduti, Jiang and Buell.)

The Gametophytic Self-Incompatibility (GSI) system in diploid potato ( Solanum tuberosum L.) poses a substantial barrier in diploid potato breeding by hindering the generation of inbred lines. One solution is gene editing to generate self-compatible diploid potatoes which will allow for the generation of elite inbred lines with fixed favorable alleles and heterotic potential. The S-RNase and HT genes have been shown previously to contribute to GSI in the Solanaceae family and self-compatible S. tuberosum lines have been generated by knocking out S-RNase gene with CRISPR-Cas9 gene editing. This study employed CRISPR-Cas9 to knockout HT-B either individually or in concert with S-RNase in the diploid self-incompatible S. tuberosum clone DRH-195. Using mature seed formation from self-pollinated fruit as the defining characteristic of self-compatibility, HT-B- only knockouts produced little or no seed. In contrast, double knockout lines of HT-B and S-RNase displayed levels of seed production that were up to three times higher than observed in the S-RNase -only knockout, indicating a synergistic effect between HT-B and S-RNase in self-compatibility in diploid potato. This contrasts with compatible cross-pollinations, where S-RNase and HT-B did not have a significant effect on seed set. Contradictory to the traditional GSI model, self-incompatible lines displayed pollen tube growth reaching the ovary, yet ovules failed to develop into seeds indicating a potential late-acting self-incompatibility in DRH-195. Germplasm generated from this study will serve as a valuable resource for diploid potato breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Lee, Enciso-Rodriguez, Behling, Jayakody, Panicucci, Zarka, Nadakuduti, Buell, Manrique-Carpintero and Douches.)

The relationships of interspecific compatibility and incompatibility in Solanum section Petota are complex. Inquiry into these relationships in tomato and its wild relatives has elucidated the pleiotropic and redundant function of S-RNase and HT which tandemly and independently mediate both interspecific and intraspecific pollen rejection. Our findings presented here are consistent with previous work conducted in Solanum section Lycopersicon showing that S-RNase plays a central role in interspecific pollen rejection. Statistical analyses also demonstrated that HT-B alone is not a significant factor in these pollinations; demonstrating the overlap in gene function between HT-A and HT-B , as HT-A , was present and functional in all genotypes used. We were not able to replicate the general absence of prezygotic stylar barriers observable in S. verrucosum , which has been attributed to the lack of S-RNase, indicating that other non- S-RNase factors play a significant role. We also demonstrated that Sli played no significant role in these interspecific pollinations, directly conflicting with previous research. It is possible that S. chacoense as a pollen donor is better able to bypass stylar barriers in 1EBN species such as S. pinnatisectum . Consequently, S. chacoense may be a valuable resource in accessing these 1EBN species regardless of Sli status.

Availability of readily transformable germplasm, as well as efficient pipelines for gene discovery are notable bottlenecks in the application of genome editing in potato. To study and introduce traits such as resistance against biotic and abiotic factors, tuber quality traits and self-fertility, model germplasm that is amenable to gene editing and regeneration is needed. Cultivated potato is a heterozygous autotetraploid and its genetic redundancy and complexity makes studying gene function challenging. Genome editing is simpler at the diploid level, with fewer allelic variants to consider. A readily transformable diploid potato would be further complemented by genomic resources that could aid in high throughput functional analysis. The heterozygous Solanum tuberosum Group Phureja clone 1S1 has a high regeneration rate, self-fertility, desirable tuber traits and is amenable to Agrobacterium-mediated transformation. We leveraged its amenability to Agrobacterium-mediated transformation to create a Cas9 constitutively expressing line for use in viral vector-based gene editing. To create a contiguous genome assembly, a homozygous doubled monoploid of 1S1 (DM1S1) was sequenced using 44 Gbp of long reads generated from Oxford Nanopore Technologies (ONT), yielding a 736 Mb assembly that encoded 31,145 protein-coding genes. The final assembly for DM1S1 represents a nearly complete genic space, shown by the presence of 99.6% of the genes in the Benchmarking Universal Single Copy Orthologs (BUSCO) set. Variant analysis with Illumina reads from 1S1 was used to deduce its alternate haplotype. These genetic and genomic resources provide a toolkit for applications of genome editing in both basic and applied research of potato., Competing Interests: Conflicts of interest The authors have no conflicts of interest to declare that are relevant to the content of this article., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America.)

Knowledge regarding genetic diversity and population structure of breeding materials is essential for crop improvement. The Texas A&M University Potato Breeding Program has a collection of advanced clones selected and maintained in-vitro over a 40-year period. Little is known about its genetic makeup and usefulness for the current breeding program. In this study, 214 potato clones were genotyped with the Infinium Illumina 22 K V3 Potato Array. After filtering, a total of 10,106 single nucleotide polymorphic (SNP) markers were used for analysis. Heterozygosity varied by SNP, with an overall average of 0.59. Three groups of tetraploid clones primarily based on potato market classes, were detected using STRUCTURE software and confirmed by discriminant analysis of principal components. The highest coefficient of differentiation observed between the groups was 0.14. Signatures of selection were uncovered in genes controlling potato flesh and skin color, length of plant cycle and tuberization, and carbohydrate metabolism. A core set of 43 clones was obtained using Core Hunter 3 to develop a sub-collection that retains similar genetic diversity as the whole population, minimize redundancies, and facilitates long-term conservation of genetic resources. The comprehensive molecular characterization of our breeding clone bank collection contributes to understanding the genetic diversity of existing potato resources. This analysis could be applied to other breeding programs and assist in the selection of parents, fingerprinting, protection, and management of the breeding collections.

Standard food safety assessments of genetically modified crops require a thorough molecular characterization of the novel DNA as inserted into the plant that is intended for commercialization, as well as a comparison of agronomic and nutritional characteristics of the genetically modified to the non-modified counterpart. These characterization data are used to identify any unintended changes in the inserted DNA or in the modified plant that would require assessment for safety in addition to the assessment of the intended modification. An unusual case of an unintended effect discovered from the molecular characterization of a genetically modified late blight resistant potato developed for growing in Bangladesh and Indonesia is presented here. Not only was a significant portion of the plasmid vector backbone DNA inserted into the plant along with the intended insertion of an R-gene for late blight resistance, but the inserted DNA was split into two separate fragments and inserted into two separate chromosomes. One fragment carries the R-gene and the other fragment carries the NPTII selectable marker gene and the plasmid backbone DNA. The implications of this for the food safety assessment of this late blight resistant potato are considered.

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., (© 2019 The Author(s).)

Genome-editing is being implemented in increasing number of plant species using engineered sequence specific nucleases (SSNs) such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9), Transcription activator like effector nucleases (TALENs), and more recently CRISPR/Cas12a. As the tissue culture and regeneration procedures to generate gene-edited events are time consuming, large-scale screening methodologies that rapidly facilitate validation of genome-editing reagents are critical. Plant protoplast cells provide a rapid platform to validate genome-editing reagents. Protoplast transfection with plasmids expressing genome-editing reagents represents an efficient and cost-effective method to screen for in vivo activity of genome-editing constructs and resulting targeted mutagenesis. In this study, we compared three existing methods for detection of editing activity, the T7 endonuclease I assay (T7EI), PCR/restriction enzyme (PCR/RE) digestion, and amplicon-sequencing, with an alternative method which involves tagging a double-stranded oligodeoxynucleotide (dsODN) into the SSN-induced double stranded break and detection of on-target activity of gene-editing reagents by PCR and agarose gel electrophoresis. To validate these methods, multiple reagents including TALENs, CRISPR/Cas9 and Cas9 variants, eCas9(1.1) (enhanced specificity) and Cas9-HF1 (high-fidelity1) were engineered for targeted mutagenesis of Acetolactate synthase1 ( ALS1 ), 5-Enolpyruvylshikimate- 3-phosphate synthase1 ( EPSPS1 ) and their paralogs in potato. While all methods detected editing activity, the PCR detection of dsODN integration provided the most straightforward and easiest method to assess on-target activity of the SSN as well as a method for initial qualitative evaluation of the functionality of genome-editing constructs. Quantitative data on mutagenesis frequencies obtained by amplicon-sequencing of ALS1 revealed that the mutagenesis frequency of CRISPR/Cas9 reagents is better than TALENs. Context-based choice of method for evaluation of gene-editing reagents in protoplast systems, along with advantages and limitations associated with each method, are discussed.

Cultivated potato, Solanum tuberosum Group Tuberosum L. (2n = 4x = 48) is a heterozygous tetraploid crop that is clonally propagated, thereby resulting in identical genotypes. Due to the lack of sexual reproduction and its concomitant segregation of alleles, genetic engineering is an efficient way of introducing crop improvement traits in potato. In recent years, genome-editing via the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system for targeted genome modifications has emerged as the most powerful method due to the ease in designing and construction of gene-specific single guide RNA (sgRNA) vectors. These sgRNA vectors are easily reprogrammable to direct Streptococcus pyogenes Cas9 (SpCas9) to generate double stranded breaks (DSBs) in the target genomes that are then repaired by the cell via the error-prone non-homologous end-joining (NHEJ) pathway or by precise homologous recombination (HR) pathway. CRISPR/Cas9 technology has been successfully implemented in potato for targeted mutagenesis to generate knockout mutations (by means of NHEJ) as well as gene targeting to edit an endogenous gene (by HR). In this chapter, we describe procedures for designing sgRNAs, protocols to clone sgRNAs for CRISPR/Cas9 constructs to generate knockouts, design of donor repair templates and use geminivirus replicons (GVRs) to facilitate gene-editing by HR in potato. We also describe tissue culture procedures in potato for Agrobacterium-mediated transformation to generate gene-edited events along with their molecular characterization.

Genome-editing has revolutionized biology. When coupled with a recently streamlined regulatory process by the U.S. Department of Agriculture and the potential to generate transgene-free varieties, genome-editing provides a new avenue for crop improvement. For heterozygous, polyploid and vegetatively propagated crops such as cultivated potato, Solanum tuberosum Group Tuberosum L., genome-editing presents tremendous opportunities for trait improvement. In potato, traits such as improved resistance to cold-induced sweetening, processing efficiency, herbicide tolerance, modified starch quality and self-incompatibility have been targeted utilizing CRISPR/Cas9 and TALEN reagents in diploid and tetraploid clones. However, limited progress has been made in other such crops including sweetpotato, strawberry, grapes, citrus, banana etc., In this review we summarize the developments in genome-editing platforms, delivery mechanisms applicable to plants and then discuss the recent developments in regulation of genome-edited crops in the United States and The European Union. Next, we provide insight into the challenges of genome-editing in clonally propagated polyploid crops, their current status for trait improvement with future prospects focused on potato, a global food security crop.

Crop genetic engineering involves transformation in which transgenic plants are regenerated through tissue culture manipulations that can elicit somaclonal variation due to mutations, translocations, and/or epigenetic alterations. Here, we report on alterations in the transcriptome in a panel of transgenic potato plants engineered to be herbicide resistant. Using an inbred diploid potato clone (DMRH S5 28-5), ten single-insert transgenic lines derived from independent Agrobacterium-mediated transformation events were selected for herbicide resistance using an allelic variant of acetolactate synthase (mALS1). Expression abundances of the single-copy mALS1 transgene varied in individual transgenic lines was correlated with the level of phenotypic herbicide resistance, suggesting the importance of transgene expression in transgenic performance. Using RNA-sequencing, differentially expressed genes were identified with the proportion of genes up-regulated significantly higher than down-regulated genes in the panel, suggesting a differential impact of the plant transformation on gene expression activation compared to repression. Not only were transcription factors among the differentially expressed genes but specific transcription factor binding sites were also enriched in promoter regions of differentially expressed genes in transgenic lines, linking transcriptomic variation with specific transcription factor activity. Collectively, these results provide an improved understanding of transcriptomic variability caused by plant transformation., Competing Interests: The authors have declared that no competing interests exist.

Background: Genome-wide single nucleotide polymorphism (SNP) markers coupled with allele dosage information has emerged as a powerful tool for studying complex traits in cultivated autotetraploid potato (Solanum tuberosum L., 2n = 4× = 48). To date, this approach has been effectively applied to the identification of quantitative trait loci (QTLs) underlying highly heritable traits such as disease resistance, but largely unexplored for traits with complex patterns of inheritance., Results: In this study, an F 1 tetraploid russet mapping population (162 individuals) was evaluated for multiple quantitative traits over two years and two locations to identify QTLs associated with tuber sugar concentration, processing quality, vine maturity, and other high-value agronomic traits. We report the linkage maps for the 12 potato chromosomes and the QTL location with corresponding genetic models and candidate SNPs explaining the highest phenotypic variation for tuber quality and maturity related traits. Significant QTLs for tuber glucose concentration and tuber fry color were detected on chromosomes 4, 5, 6, 10, and 11. Collectively, these QTLs explained between 24 and 46% of the total phenotypic variation for tuber glucose and fry color, respectively. The QTL on chromosome 10 was associated with apoplastic invertases, with 'Premier Russet' contributing the favorable allele for fry processing quality. On chromosome 5, minor-effect QTLs for tuber glucose concentration and fry color co-localized with various major-effect QTLs, including vine maturity, growth habit, tuber shape, early blight (Altenaria tenuis), and Verticillium wilt (Verticillium spp.)., Conclusions: Linkage analysis and QTL mapping in a russet mapping population (A05141) using SNP dosage information successfully identified favorable alleles and candidate SNPs for resistance to the accumulation of tuber reducing sugars. These novel markers have a high potential for the improvement of tuber processing quality. Moreover, the discovery of different genetic models for traits with overlapping QTLs at the maturity locus clearly suggests an independent genetic control.

The cultivated potato ( Solanum tuberosum ) has a complex genetic structure due to its autotetraploidy and vegetative propagation which leads to accumulation of mutations and a highly heterozygous genome. A high degree of heterozygosity has been considered to be the main driver of fitness and agronomic trait performance in potato improvement efforts, which is negatively impacted by genetic load. To understand the genetic landscape of cultivated potato, we constructed a gynogenic dihaploid (2 n = 2 x = 24) population from cv. Superior, prior to development of a high-density genetic map containing 12,753 single nucleotide polymorphisms (SNPs). Common quantitative trait loci (QTL) were identified for tuber traits, vigor and height on chromosomes 2, 4, 7, and 10, while specific QTL for number of inflorescences per plant, and tuber shape were present on chromosomes 4, 6, 10, and 11. Simplex rather than duplex loci were mainly associated with traits. In general, the Q allele (main effect) detected in one or two homologous chromosomes was associated with lower mean trait values suggesting the importance of dosage allelic effects, and the presence of up to two undesired alleles in the QTL region. Loss of heterozygosity has been associated with a lower rate of fitness, yet no correlation between the percent heterozygosity and increased fitness or agronomic performance was observed. Based upon linkage phase, we reconstructed the four homologous chromosome haplotypes of cv. Superior. revealing heterogeneity throughout the genome yet nearly duplicate haplotypes occurring among the homologs of particular chromosomes. These results suggest that the potentially deleterious mutations associated with genetic load in tetraploid potato could be mitigated by multiple loci which is consistent with the theory that epistasis complicates the identification of associations between markers and phenotypic performance.

Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid that presents challenges in genome analyses and breeding. Wild potato species serve as a resource for the introgression of important agronomic traits into cultivated potato. One key species is Solanum chacoense and the diploid, inbred clone M6, which is self-compatible and has desirable tuber market quality and disease resistance traits. Sequencing and assembly of the genome of the M6 clone of S. chacoense generated an assembly of 825 767 562 bp in 8260 scaffolds with an N50 scaffold size of 713 602 bp. Pseudomolecule construction anchored 508 Mb of the genome assembly into 12 chromosomes. Genome annotation yielded 49 124 high-confidence gene models representing 37 740 genes. Comparative analyses of the M6 genome with six other Solanaceae species revealed a core set of 158 367 Solanaceae genes and 1897 genes unique to three potato species. Analysis of single nucleotide polymorphisms across the M6 genome revealed enhanced residual heterozygosity on chromosomes 4, 8 and 9 relative to the other chromosomes. Access to the M6 genome provides a resource for identification of key genes for important agronomic traits and aids in genome-enabled development of inbred diploid potatoes with the potential to accelerate potato breeding., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

As one of the world's most important food crops, the potato ( Solanum tuberosum L.) has spurred innovation in autotetraploid genetics, including in the use of SNP arrays to determine allele dosage at thousands of markers. By combining genotype and pedigree information with phenotype data for economically important traits, the objectives of this study were to (1) partition the genetic variance into additive vs. nonadditive components, and (2) determine the accuracy of genome-wide prediction. Between 2012 and 2017, a training population of 571 clones was evaluated for total yield, specific gravity, and chip fry color. Genomic covariance matrices for additive ( G ), digenic dominant ( D ), and additive × additive epistatic ( G # G ) effects were calculated using 3895 markers, and the numerator relationship matrix ( A ) was calculated from a 13-generation pedigree. Based on model fit and prediction accuracy, mixed model analysis with G was superior to A for yield and fry color but not specific gravity. The amount of additive genetic variance captured by markers was 20% of the total genetic variance for specific gravity, compared to 45% for yield and fry color. Within the training population, including nonadditive effects improved accuracy and/or bias for all three traits when predicting total genotypic value. When six F 1 populations were used for validation, prediction accuracy ranged from 0.06 to 0.63 and was consistently lower (0.13 on average) without allele dosage information. We conclude that genome-wide prediction is feasible in potato and that it will improve selection for breeding value given the substantial amount of nonadditive genetic variance in elite germplasm., (Copyright © 2018 by the Genetics Society of America.)

Understanding the impacts of climate change on agriculture is essential to ensure adequate future food production. Controlled growth experiments provide an effective tool for assessing the complex effects of climate change. However, a review of the use of climate projections in 57 previously published controlled growth studies found that none considered within-season variations in projected future temperature change, and few considered regional differences in future warming. A fixed, often arbitrary, temperature perturbation typically was applied for the entire growing season. This study investigates the utility of employing more complex climate change scenarios in growth chamber experiments. A case study in potato was performed using three dynamically downscaled climate change projections for the mid-twenty-first century that differ in terms of the timing during the growing season of the largest projected temperature changes. The climate projections were used in growth chamber experiments for four elite potato cultivars commonly planted in Michigan's major potato growing region. The choice of climate projection had a significant influence on the sign and magnitude of the projected changes in aboveground biomass and total tuber count, whereas all projections suggested an increase in total tuber weight and a decrease in specific gravity, a key market quality trait for potato, by mid-century. These results demonstrate that the use of more complex climate projections that extend beyond a simple incremental change can provide additional insights into the future impacts of climate change on crop production and the accompanying uncertainty.

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 S tuberosum 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 ( S tuberosum 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 S microdontum 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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Relative to homozygous diploids, the presence of multiple homologs or homeologs in polyploids affords greater tolerance to mutations that can impact genome evolution. In this study, we describe sequence and structural variation in the genomes of six accessions of cultivated potato (Solanum tuberosum L.), a vegetatively propagated autotetraploid and their impact on the transcriptome. Sequence diversity was high with a mean single nucleotide polymorphisms (SNP) rate of approximately 1 per 50 bases suggestive of high levels of allelic diversity. Additive gene expression was observed in leaves (3605 genes) and tubers (6156 genes) that contrasted the preferential allele expression of between 2180 and 3502 and 3367 and 5270 genes in the leaf and tuber transcriptome, respectively. Preferential allele expression was significantly associated with evolutionarily conserved genes suggesting selection of specific alleles of genes responsible for biological processes common to angiosperms during the breeding selection process. Copy number variation was rampant with between 16 098 and 18 921 genes in each cultivar exhibiting duplication or deletion. Copy number variable genes tended to be evolutionarily recent, lowly expressed, and enriched in genes that show increased expression in response to biotic and abiotic stress treatments suggestive of a role in adaptation. Gene copy number impacts on gene expression were detected with 528 genes having correlations between copy number and gene expression. Collectively, these data suggest that in addition to allelic variation of coding sequence, the heterogenous nature of the tetraploid potato genome contributes to a highly dynamic transcriptome impacted by allele preferential and copy number-dependent expression effects., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Key Message: New software to make tetraploid genotype calls from SNP array data was developed, which uses hierarchical clustering and multiple F1 populations to calibrate the relationship between signal intensity and allele dosage. SNP arrays are transforming breeding and genetics research for autotetraploids. To fully utilize these arrays, the relationship between signal intensity and allele dosage must be calibrated for each marker. We developed an improved computational method to automate this process, which is provided as the R package ClusterCall. In the training phase of the algorithm, hierarchical clustering within an F1 population is used to group samples with similar intensity values, and allele dosages are assigned to clusters based on expected segregation ratios. In the prediction phase, multiple F1 populations and the prediction set are clustered together, and the genotype for each cluster is the mode of the training set samples. A concordance metric, defined as the proportion of training set samples equal to the mode, can be used to eliminate unreliable markers and compare different algorithms. Across three potato families genotyped with an 8K SNP array, ClusterCall scored 5729 markers with at least 0.95 concordance (94.6% of its total), compared to 5325 with the software fitTetra (82.5% of its total). The three families were used to predict genotypes for 5218 SNPs in the SolCAP diversity panel, compared with 3521 SNPs in a previous study in which genotypes were called manually. One of the additional markers produced a significant association for vine maturity near a well-known causal locus on chromosome 5. In conclusion, when multiple F1 populations are available, ClusterCall is an efficient method for accurate, autotetraploid genotype calling that enables the use of SNP data for research and plant breeding.

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., (Copyright © 2016 Crop Science Society of America.)

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., (Copyright © 2016 Manrique-Carpintero et al.)

Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species.

Genome-wide association studies (GWAS) are widely used in diploid species to study complex traits in diversity and breeding populations, but GWAS software tailored to autopolyploids is lacking. The objectives of this research were to (i) develop an R package for autopolyploids based on the + mixed model, (ii) validate the software with simulated data, and (iii) analyze a diversity panel of tetraploid potatoes. A unique feature of the R package, called GWASpoly, is its ability to model different types of polyploid gene action, including additive, simplex dominant, and duplex dominant. Using a simulated tetraploid population, we confirmed our hypothesis that statistical power is higher when the assumed gene action in the GWAS model matches the gene action at unobserved quantitative trait loci (QTL). Thirteen traits were analyzed in the Solanaceae Coordinated Agricultural Project (SolCAP) potato diversity panel and, consistent with previous studies, significant QTL for tuber shape and eye depth co-localized on chromosome 10. For the other traits, only marginally significant QTL were detected, most likely due to insufficient statistical power: for simulated traits with a heritability () of 0.3, the median genome-wide power was only 0.01. Our results indicate that both marker density and population size were limiting factors for GWAS with the SolCAP panel., (Copyright © 2016 Crop Science Society of America.)

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., (© 2016 American Society of Plant Biologists. All rights reserved.)

Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3-60% per transformation and from 0-29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87-100%. This demonstration of CRISPR/Cas in potato extends the range of plant species modified using CRISPR/Cas and provides a framework for future studies.

The objective of this study was to construct a single nucleotide polymorphism (SNP)-based genetic map at the cultivated tetraploid level to locate quantitative trait loci (QTL) contributing to economically important traits in potato (Solanum tuberosum L.). The 156 F1 progeny and parents of a cross (MSL603) between "Jacqueline Lee" and "MSG227-2" were genotyped using the Infinium 8303 Potato Array. Furthermore, the progeny and parents were evaluated for foliar late blight reaction to isolates of the US-8 genotype of Phytophthora infestans (Mont.) de Bary and vine maturity. Linkage analyses and QTL mapping were performed using a novel approach that incorporates allele dosage information. The resulting genetic maps contained 1972 SNP markers with an average density of 1.36 marker per cM. QTL mapping identified the major source of late blight resistance in "Jacqueline Lee." The best SNP marker mapped ~0.54 Mb from a resistance hotspot on the long arm of chromosome 9. For vine maturity, the major-effect QTL was located on chromosome 5 with allelic effects from both parents. A candidate SNP marker for this trait mapped ~0.25 Mb from the StCDF1 gene, which is a candidate gene for the maturity trait. The identification of markers for P. infestans resistance will enable the introgression of multiple sources of resistance through marker-assisted selection. Moreover, the discovery of a QTL for late blight resistance not linked to the QTL for vine maturity provides the opportunity to use marker-assisted selection for resistance independent of the selection for vine maturity classifications., (Copyright © 2015 Massa et al.)

Because of their adaptation to a diverse set of habitats and stresses, wild species of cultivated crops offer new sources of genetic diversity for germplasm improvement. Using an Infinium array representing a genome-wide set of 8303 single nucleotide polymorphisms (SNPs), phylogenetic relationships and allelic diversity were evaluated within a diversity panel of germplasm from Solanum sect. Petota. This panel consists of 74 plant introductions (PIs) representing 25 species and provides a diverse representation of tuber-bearing Solanum germplasm. Unlike other molecular markers, genome-wide SNPs have not been widely implemented in potato. To determine relatedness between current species classifications and SNP-based genetic distances, a phylogeny was generated based on random individuals from each core collection PI. With few exceptions, SNP-based estimates of species relationships revealed general agreement with the existing taxonomic grouping of species in Solanum sect. Petota. Genotype comparisons between the Solanum sect. Petota diversity panel and a panel of 213 tetraploid cultivars and breeding lines indicated a greater extent of diversity between populations of native Andean landraces than among modern cultivated varieties. Comparison of SNP allele frequencies between the Solanum sect. Petota panel and tetraploid cultivars identified loci with extreme divergence between cultivated potato and its tuber-bearing relatives. Several of these loci are associated with genes related to carbohydrate metabolism and tuber development, suggesting potential roles in potato domestication. The Infinium SNP data offer a new taxonomic view of potato germplasm, while further identifying candidate alleles likely to differentiate wild germplasm and cultivated potato, possibly underlying key agronomic traits., (© 2015 The Authors.)

Perennial plants undergo repression of meristematic activity in a process called dormancy. Dormancy is a complex metabolic process with implications for plant breeding and crop yield. Endodormancy, a specific subclass of dormancy, is characteristic of internal physiological mechanisms resulting in growth suppression. In this study, we examine transcriptional changes associated with the natural cessation of endodormancy in potato tuber meristems and in endodormant tubers treated with the cytokinin analog 1-(α-ethylbenzyl)-3-niroguanidine (NG), which terminates dormancy. RNA-sequencing was used to examine transcriptome changes between endodormant and non-dormant meristems from four different harvest years. A total of 35,091 transcripts were detected with 2132 differentially expressed between endodormant and non-dormant tuber meristems. Endodormant potato tubers were treated with the synthetic cytokinin NG and transcriptome changes analyzed using RNA-seq after 1, 4, and 7 days following NG exposure. A comparison of natural cessation of dormancy and NG-treated tubers demonstrated that by 4 days after NG exposure, potato meristems exhibited transcriptional profiles similar to the non-dormant state with elevated expression of multiple histones, a variety of cyclins, and other genes associated with proliferation and cellular replication. Three homologues encoding for CYCD3 exhibited elevated expression in both non-dormant and NG-treated potato tissues. These results suggest that NG terminates dormancy and induces expression cell cycle-associated transcripts within 4 days of treatment.

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".

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.

The potato (Solanum tuberosum L.) cultivar 'SpuntaG2' contains a single copy of the Bacillus thuringiensis (Bt) cry1Ia1 gene and controls potato tuberworm (Phthorimaea operculella Zeller, Lepidoptera: Gelechiidae). Two potato cultivars and two breeding lines were crossed with SpuntaG2 creating four populations used to study cry1Ia1 segregation and efficacy. The cry1Ia1 gene segregated in each of the four populations with a 1:1 ratio. All progeny that were polymerase chain reaction positive for the cry1Ia1 gene had no surviving larvae and no leaf mining in detached leaf assays after 72 h. These results support previous evidence that SpuntaG2 carries a single copy of the cry1Ia1 gene and that transmission of the transgene from parent to progeny is not restricted and follows expected Mendelian segregation ratios. Based on detached leaf assays, the efficacy of the cry1Ia1 gene is retained through sexual transmission. If the SpuntaG2 cry1Ia1 insertion event is deregulated for commercial use, SpuntaG2 could be used for conventional breeding and the progeny carrying the SpuntaG2 event would also be available for commercial use.

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.

The effects of selection on genome variation were investigated and visualized in tomato using a high-density single nucleotide polymorphism (SNP) array. 7,720 SNPs were genotyped on a collection of 426 tomato accessions (410 inbreds and 16 hybrids) and over 97% of the markers were polymorphic in the entire collection. Principal component analysis (PCA) and pairwise estimates of F(st) supported that the inbred accessions represented seven sub-populations including processing, large-fruited fresh market, large-fruited vintage, cultivated cherry, landrace, wild cherry, and S. pimpinellifolium. Further divisions were found within both the contemporary processing and fresh market sub-populations. These sub-populations showed higher levels of genetic diversity relative to the vintage sub-population. The array provided a large number of polymorphic SNP markers across each sub-population, ranging from 3,159 in the vintage accessions to 6,234 in the cultivated cherry accessions. Visualization of minor allele frequency revealed regions of the genome that distinguished three representative sub-populations of cultivated tomato (processing, fresh market, and vintage), particularly on chromosomes 2, 4, 5, 6, and 11. The PCA loadings and F(st) outlier analysis between these three sub-populations identified a large number of candidate loci under positive selection on chromosomes 4, 5, and 11. The extent of linkage disequilibrium (LD) was examined within each chromosome for these sub-populations. LD decay varied between chromosomes and sub-populations, with large differences reflective of breeding history. For example, on chromosome 11, decay occurred over 0.8 cM for processing accessions and over 19.7 cM for fresh market accessions. The observed SNP variation and LD decay suggest that different patterns of genetic variation in cultivated tomato are due to introgression from wild species and selection for market specialization.

Background: Current breeding approaches in potato rely almost entirely on phenotypic evaluations; molecular markers, with the exception of a few linked to disease resistance traits, are not widely used. Large-scale sequence datasets generated primarily through Sanger Expressed Sequence Tag projects are available from a limited number of potato cultivars and access to next generation sequencing technologies permits rapid generation of sequence data for additional cultivars. When coupled with the advent of high throughput genotyping methods, an opportunity now exists for potato breeders to incorporate considerably more genotypic data into their decision-making., Results: To identify a large number of Single Nucleotide Polymorphisms (SNPs) in elite potato germplasm, we sequenced normalized cDNA prepared from three commercial potato cultivars: 'Atlantic', 'Premier Russet' and 'Snowden'. For each cultivar, we generated 2 Gb of sequence which was assembled into a representative transcriptome of ~28-29 Mb for each cultivar. Using the Maq SNP filter that filters read depth, density, and quality, 575,340 SNPs were identified within these three cultivars. In parallel, 2,358 SNPs were identified within existing Sanger sequences for three additional cultivars, 'Bintje', 'Kennebec', and 'Shepody'. Using a stringent set of filters in conjunction with the potato reference genome, we identified 69,011 high confidence SNPs from these six cultivars for use in genotyping with the Infinium platform. Ninety-six of these SNPs were used with a BeadXpress assay to assess allelic diversity in a germplasm panel of 248 lines; 82 of the SNPs proved sufficiently informative for subsequent analyses. Within diverse North American germplasm, the chip processing market class was most distinct, clearly separated from all other market classes. The round white and russet market classes both include fresh market and processing cultivars. Nevertheless, the russet and round white market classes are more distant from each other than processing are from fresh market types within these two groups., Conclusions: The genotype data generated in this study, albeit limited in number, has revealed distinct relationships among the market classes of potato. The SNPs identified in this study will enable high-throughput genotyping of germplasm and populations, which in turn will enable more efficient marker-assisted breeding efforts in potato.

The Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), is the most destructive insect pest of potato, Solanum tuberosum (L.), in North America. Avidin sequesters available biotin, thereby causing abnormal growth and development of insects. We expressed avidin in two potato lines: MSE149-5Y, a susceptible potato line, and ND5873-15, a line with S. chacoense-derived insect resistance. A preliminary study was conducted to determine the bioactivity of the transgene in each background. A single transgenic line was selected in each background for further studies. Detached leaf bioassays were performed on transgenic and nontransgenic clones of the susceptible and S. chacoense lines by using first-stage Colorado potato beetle larvae. Consumption, survival, and survivor growth were measured after 5 d. Larvae consumed significantly less on the two avidin-expressing lines compared with the nontransgenic lines. Survival was also significantly less for larvae feeding on transgenic avidin lines compared with the nontransgenic lines. The mass of survivors was significantly reduced on two transgenic avidin lines compared with the nontransgenic lines. Further studies examined the development from first-stage larvae to adulthood on greenhouse-grown whole plants in a no-choice setting for larvae fed on the four potato lines. Development from first stage to pupation was significantly prolonged for larvae fed on the avidin line compared with larvae fed on the susceptible line. Significantly fewer larvae fed on transgenic avidin plants, avidin or avidin + S. chacoense-derived line survived to adulthood compared with survival on nontransgenic plants, susceptible or S. chacoense-derived line. Avidin-based resistance may be useful in managing Colorado potato beetle populations in commercial planting by reducing the population size.

Vitamin E (tocopherol) is a powerful antioxidant essential for human health and synthesized only by photosynthetic organisms. The effects of over-expression of tocopherol biosynthetic enzymes have been studied in leaves and seeds, but not in a non-photosynthetic, below-ground plant organ. Genetic and molecular approaches were used to determine if increased levels of tocopherols can be accumulated in potato (Solanum tuberosum L.) tubers through metabolic engineering. Two transgenes were constitutively over-expressed in potato: Arabidopsis thaliana p-hydroxyphenylpyruvate dioxygenase (At-HPPD) and A. thaliana homogentisate phytyltransferase (At-HPT). alpha-Tocopherol levels in the transgenic plants were determined by high-performance liquid chromatography. In potato tubers, over-expression of At-HPPD resulted in a maximum 266% increase in alpha-tocopherol, and over-expression of At-HPT yielded a 106% increase. However, tubers from transgenic plants still accumulated approximately 10- and 100-fold less alpha-tocopherol than leaves or seeds, respectively. The results indicate that physiological and regulatory constraints may be the most limiting factors for tocopherol accumulation in potato tubers. Studying regulation and induction of tocopherol biosynthesis should reveal approaches to more effectively engineer crops with enhanced tocopherol content.

The Colorado potato beetle, Leptinotarsa decemlineata Say, is the major insect pest of potato, Solanum tuberosum L., in eastern North America and is renowned for resistance development, currently resistant to >40 insecticides worldwide. Host plant resistance may assist in delaying in resistance development to insecticides. We evaluated natural host plant resistance mechanisms (glandular trichomes and Solanum chacoense Bitter-derived resistance) and engineered resistance mechanisms (Bacillus thuringiensis [Bt] Berliner cry3A and cry1Ia1) in a no-choice cage study. Six different potato lines representing four host plant resistance mechanisms were evaluated over 2 yr. Egg masses were placed in each cage (one egg mass per plant). Almost no feeding was observed in the Bt-cry3A lines, and only minor feeding was observed in the Bt-cry1Ia1 lines in either year. On the S. chacoense-derived line, there was significantly less defoliation than on either the susceptible line or the glandular trichome line in 2003. In 2004, there was significantly higher defoliation on the S. chacoense-derived line than on the susceptible line or glandular trichome line. The defoliation of the Solanum chacoense-derived line was largely due to larvae clipping the petioles, rather than consumption of the leaves. Defoliation on the glandular trichome line did not differ significantly from the defoliation of the susceptible line, suggesting glandular trichomes may not be effective in controlling larvae and preventing defoliation. This study suggested that Bt can provide high levels of resistance, but the natural resistance mechanisms tested here are variable for control of Colorado potato beetle larvae in no-choice situations.

Colorado potato beetle, Leptinotarsa decemlineata (Say), is a destructive pest of potato, Solanum tuberosum (L.), in North America. It is renowned for adapting to insecticides. With the arsenal of effective insecticides decreasing, it is important to consider alternative forms of control. Biotin is an essential coenzyme for insect growth and development. Avidin is a protein found in chicken egg that sequesters biotin and has shown insecticidal properties against a range of insect. We assessed the effectiveness of avidin against the Colorado potato beetle neonates in a no-choice detached leaf bioassay at 0, 17, 34, 51, 102, and 204 microg avidin/ml over 12 d. The LC50 was 136 microg avidin/ml (108-188 95% CL). The combined effects of avidin (136 microg avidin/ml) with Bt-Cry3A or leptines were evaluated with neonates and third instars over 12 and 6 d, respectively. Three potato lines were used: susceptible line, a line engineered to express Cry3A from Bacillus thuringiensis, and a line expressing the natural resistance factor leptines. The addition of avidin at the LC50 concentration significantly reduced consumption by neonates, but it did not affect consumption by third instars feeding on the susceptible line and the leptine line. Survival of neonates feeding on the susceptible line with avidin was significantly reduced compared with the susceptible line. Survival of third instars on the Bt-Cry3A with avidin was significantly reduced after 3 d compared with survival on the Bt-Cry3A, suggesting the addition of avidin may increase susceptibility to Bt-Cry3A.

Potato tuberworm, Phthorimaea operculella (Zeller), is the most serious insect pest of potatoes worldwide. The introduction of the Bacillus thuringiensis (Bt) toxin gene through genetic engineering offers host plant resistance for the management of potato tuberworm. We report on the field and storage studies to evaluate Bt-cry5 potato lines for resistance to potato tuberworm in Egypt under natural infestations and their agronomic performance in both Egypt and Michigan. From 1997 to 2001, field experiments were conducted at the International Potato Center (CIP) Research Station, Kafr El-Zyat, Egypt, and/or Agricultural Genetic Engineering Institute (AGERI), Giza, Egypt, to evaluate resistance to tuberworm. A total of 27 Bt-transgenic potato lines from six different Bt constructs were evaluated over a 5-yr period. After harvest and evaluation of the agronomic trials, storage evaluation of potato tuberworm damage was done at the CIP Research Station. The 1997 field trial was the first field test of genetically engineered crops in Egypt. Field tests to assess potato tuberworm resistance in Egypt were able to differentiate between the Bt-transgenic lines and the nontransgenic lines/cultivars in 1999, 2000, and 2001. The Bt-cry5-Spunta lines (Spunta-G2, Spunta-G3, and Spunta-6a3) were the most resistant lines in field with 99-100% of tubers free of damage. In the 2001 storage study, these lines were also over 90% free of tuberworm damage after 3 mo. NYL235-4.13, which combines glandular trichomes with the Bt-cry5/gus fusion construct, also had a high percentage of clean tubers in the field studies. In agronomic field trials in Michigan from 1997 to 2001, the Bt-transgenic lines in most instances performed similar to the nontransgenic line in the agronomic trials; however, in Egypt (1998-1999), the yields were less than one-half of those in Michigan. Expression of the Bt-cry5 gene in the potato tuber and foliage will provide the seed producer and grower a tool in which to reduce potato tuberworm damage to the tuber crop in the field and storage.

Field experiments were conducted during 1998 to 2000 to determine the response of commercial potato cultivars and advanced breeding lines (ABL) differing in susceptibility to foliar late blight (caused by Phytophthora infestans) to reduced rates and frequencies of residual, contact fungicide applications. When environmental conditions were most favorable for the development of late blight, the lowest application rate of the fungicides chlorothalonil or fluazinam (33% of the manufacturers' recommended application rate [MRAR]) gave unsatisfactory control of potato late blight. Under conditions moderately conducive for late blight development, effective control was achieved with 33 to 66% MRAR with either fungicide. The Michigan State University advanced selection, MSG274-3, was the least susceptible ABL tested and, during 1998 to 2000, late blight was effectively managed using reduced rates of fungicides. Application rates of chlorothalonil (33 to 100% MRAR) significantly reduced late blight in the cultivar Snowden (5-day application interval) compared with the nontreated control; whereas, late blight was not effectively controlled in Snowden even at 100% MRAR of chlorothalonil at either 10- or 15-day application intervals in 1999 or 2000. The ABL MSG274-3 was the least susceptible of all cultivars and ABL used in this study, and required minimal chemical protection against late blight. The study demonstrates that ABL with reduced susceptibility to late blight can be managed with reduced fungicide rates and longer application intervals, thus offering more economical control of this disease.

The potato tuber moth, Phthorimaea operculella (Zeller), in tropical and subtropical countries, is the most destructive pest of potato, Solanum tuberosum L. The larvae attack foliage and tubers in the field and in storage. The purpose of this study was to evaluate the efficacy of a Bt-cry5 transgene to control the potato tuber moth in tuber tissues. Tuber bioassays using stored (11-12 mo old) and newly harvested tubers of Bt-cry5-Lemhi Russet and Bt-cry5-Atlantic potato lines showed up to 100% mortality of 1st instars. Mortality was lowest in the newly harvested tubers of Bt-cry5-Atlantic lines (47.1-67.6%). Potato tuber moth mortality was 100% in the Bt-cry5-Spunta lines that were transformed with Bt-cry5 gene controlled by the CaMV 35S promoter (pBIML5 vector) and in 2 of 3 lines transformed with Bt-cry5 gene controlled by the Gelvin super promoter (pBIML1 vector). The transgenic Spunta lines expressing Bt-cry5 controlled by the patatin promoter (pBMIL2 vector) showed the lowest tuber moth mortality (25.6 and 31.1%). The Bt-cry5 transgenic lines with high tuber expression of B. thuringiensis have value in an integrated pest management system to control potato tuber moth.

We evaluated chloroplast DNA (cpDNA), isozymes, single to low-copy nuclear DNA (RFLPs), and random amplified polymorphic DNAs (RAPDs) in terms of concordance for genetic distance of 15 accessions each of Solanum etuberosum and S. palustre, and 4 accessions of S. fernandezianum. These self-compatible, diploid (2n=24), and morphologically very similar taxa constitute all species in Solanum sect. Etuberosum, a group of non-tuber-bearing species closely related to Solanum sect. Petota (the potato and its wild relatives). Genetic distance and multidimentional scaling results show general concordance of isozymes, RFLPs and RAPDs between all three taxa; cpDNA shows S. etuberosum and S. palustre to be more similar to each other than to S. fernandezianum. Interspecific sampling variance shows a gradation of resolution from allozyme (low) to RAPD to RFLP (high); while intraspecific comparisons graded from RFLPs (low) to RAPDs (high; lack of sufficient allozyme variability within species precluded comparisons for allozymes). Experimental error was low in RFLPs and RAPDs.

Quantitative trait locus (QTL) analysis for tuber dormancy was performed in a diploid potato population (TRP133) consisting of 110 individuals. The female parent was a hybrid between haploid S. tuberosum (2x) and S. chacoense, while the male parent was a S. phureja clone. The population was characterized for ten isozyme loci, 44 restriction fragment length polymorphisms (RFLPs) and 63 random amplified polymorphic DNAs (RAPDs). Eighty-seven of these loci segregating from the female parent were utilized to develop a linkage map that comprised 10 of the 12 chromosomes in the genome. Dormancy, as measured by days-to-sprouting after harvest, ranged from 10 to 90 days, with a mean of 19 days. QTLs were mapped by conducting one-way analyses of variance for each marker locus by dormancy combination. Twenty-two markers had a significant association with dormancy, identifying six putative QTLs localized on each of chromosomes 2, 3, 4, 5, 7 and 8. The QTL with the strongest effect on dormancy was detected on chromosome 7. A multilocus model was developed using the locus with highest R(2) value in each QTL. This model explained 57.5% of the phenotypic variation for dormancy. Seven percent of possible epistatic interactions among significant markers were significant when tested through two-way analyses of variance. When these were included in the main-effects model, it explained 72.1% of the phenotypic variation for dormancy. QTL analysis in potato, the methodology to transfer traits and interactions into the 4x level, and QTLs of value for marker-assisted selection, are discussed.