Rychc Confers Extreme Resistance to Potato virus Y in Potato.

The Potato virus Y (PVY) is responsible for huge economic losses for the potato industry worldwide and is the fifth most consequential plant virus globally. The main strategies for virus control are to limit aphid vectors, produce virus-free seed potatoes, and breed virus-resistant varieties. The breeding of PVY-resistant varieties is the safest and most effective method in terms of cost and environmental protection. Rychc, a gene that confers extreme resistance to PVY, is from S. chacoense, which is a wild diploid potato species that is widely used in many PVY-resistant breeding projects. In this study, Rychc was fine mapped and successfully cloned from S. chacoense accession 40-3. We demonstrated that Rychc encodes a TIR-NLR protein by stably transforming a diploid susceptible cultivar named AC142 and a tetraploid potato variety named E3. The Rychc conferred extreme resistance to PVYO, PVYN:O and PVYNTN in both of the genotypes. To investigate the genetic events occurring during the evolution of the Rychc locus, we sequenced 160 Rychc homologs from 13 S. chacoense genotypes. Based on the pattern of sequence identities, 160 Rychc homologs were divided into 11 families. In Family 11 including Rychc, we found evidence for Type I evolutionary patterns with frequent sequence exchanges, obscured orthologous relationships and high non-synonymous to synonymous substitutions (Ka/Ks), which is consistent with rapid diversification and positive selection in response to rapid changes in the PVY genomes. Furthermore, a functional marker named MG64-17 was developed in this study that indicates the phenotype with 100% accuracy and, therefore, can be used for marker-assisted selection in breeding programs that use S. chacoense as a breeding resource.

First-generation genome editing in potato using hairy root transformation.

Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.

Residual Heterozygosity and Epistatic Interactions Underlie the Complex Genetic Architecture of Yield in Diploid Potato.

Deconvolution of the genetic architecture underlying yield is critical for understanding bases of genetic gain in species of agronomic importance. To dissect the genetic components of yield in potato, we adopted a reference-based recombination map composed of four segregating alleles from an interspecific pseudotestcross F1 potato population (n = 90). Approximately 1.5 million short nucleotide variants were utilized during map construction, resulting in unprecedented resolution for an F1 population, estimated by a median bin length of 146 kb and 11 genes per bin. Regression models uncovered 14 quantitative trait loci (QTL) underpinning yield, average tuber weight, and tubers produced per plant in a population exhibiting a striking 332% average midparent-value heterosis. Nearly 80% of yield-associated QTL were epistatic, and contained between 0 and 44 annotated genes. We found that approximately one-half of epistatic QTL overlap regions of residual heterozygosity identified in the inbred parental parent (M6). Genomic regions recalcitrant to inbreeding were associated with an increased density of genes, many of which demonstrated signatures of selection and floral tissue specificity. Dissection of the genome-wide additive and dominance values for yield and yield components indicated a widespread prevalence of dominance contributions in this population, enriched at QTL and regions of residual heterozygosity. Finally, the effects of short nucleotide variants and patterns of gene expression were determined for all genes underlying yield-associated QTL, exposing several promising candidate genes for future investigation.

A common genetic mechanism underlies morphological diversity in fruits and other plant organs.

Shapes of edible plant organs vary dramatically among and within crop plants. To explain and ultimately employ this variation towards crop improvement, we determined the genetic, molecular and cellular bases of fruit shape diversity in tomato. Through positional cloning, protein interaction studies, and genome editing, we report that OVATE Family Proteins and TONNEAU1 Recruiting Motif proteins regulate cell division patterns in ovary development to alter final fruit shape. The physical interactions between the members of these two families are necessary for dynamic relocalization of the protein complexes to different cellular compartments when expressed in tobacco leaf cells. Together with data from other domesticated crops and model plant species, the protein interaction studies provide possible mechanistic insights into the regulation of morphological variation in plants and a framework that may apply to organ growth in all plant species.

Topological Data Analysis as a Morphometric Method: Using Persistent Homology to Demarcate a Leaf Morphospace.

Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied as a filtration across simplicial complexes (or more simply, a method to measure topological features of spaces across different spatial resolutions), to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. The approach predicts plant family above chance. The application of a persistent homology method, using topological features, to measure leaf shape allows for a unified morphometric framework to measure plant form, including shapes, textures, patterns, and branching architectures.

Plant Resistance to Colorado Potato Beetle (Coleoptera: Chrysomelidae) in Diploid F2 Families Derived From Crosses Between Cultivated and Wild Potato.

Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), is a serious global pest of potato, Solanum tuberosum L. Management of L. decemlineata has relied heavily on insecticides, but repeated evolution of insecticide resistance has motivated the exploration and development of alternative strategies, such as plant resistance. The recent development of two diploid potato families derived from crosses between cultivated and wild potato species (S. chacoense and S. berthaultii) has provided a unique opportunity to reexamine plant traits for resistance breeding. In this 2-yr study, we surveyed select F2 clones for the induction of L. decemlineata mortality and a reduction in defoliation in no-choice feeding assays when challenged with adults and larvae from three sites in Wisconsin. We tested for an association with glandular trichome density and foliar levels of the glycoalkaloids chaconine and solanine. Several potato clones demonstrated resistance in specific feeding assays, but none excelled consistently across experiments. Mortality and defoliation generally differed significantly among L. decemlineata populations, which could be indicative of heritable variation in beetle responses to plant defenses or variation in the physiological status of the beetle populations tested. Contrary to expectations, higher trichome density increased mortality or decreased defoliation in only a few cases, and levels of mortality and defoliation were unrelated to foliar glycoalkaloid content, warranting further investigation of the defense mechanisms of resistant clones. In addition to identifying several potential L. decemlineata resistance sources, this study underscores the need to include multiple insect populations in surveys of plant resistance to this diverse pest.

Genome sequence of M6, a diploid inbred clone of the high-glycoalkaloid-producing tuber-bearing potato species Solanum chacoense, reveals residual heterozygosity.

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.

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.

Quantitative Trait Loci for Resistance to Common Scab and Cold-Induced Sweetening in Diploid Potato.

The development of germplasm with resistance to common scab and cold-induced sweetening is a high priority for the potato ( L.) industry. A mapping population was developed from mating two individuals of a diploid family generated by crossing the susceptible cultivated potato clone US-W4 to the highly resistant wild relative ( Bitter) clone '524-8'. Progeny were evaluated in replicated field trials. Tubers were scored for percentage of surface area with scab lesions, scab lesion type, cold-induced sweetening, average tuber weight, and dry matter. Plants were evaluated for vine maturity. A genetic map was constructed, quantitative trait loci (QTLs) were identified, and the gene action of significant QTLs was characterized using 1606 single nucleotide polymorphisms (SNPs). Significant QTLs for common scab percentage of surface area covered with lesions and lesion type were identified in overlapping regions on chromosome 11 ( = 21.0 and 18.2%, respectively). Quantitative trait loci were identified on chromosomes 4 ( = 17.1%) and 6 ( = 19.4%) for cold-induced sweetening, chromosome 5 for maturity ( = 29.8%), and chromosome 1 ( = 26.3 and 22.0%) for average tuber weight. Identification of QTLs is the first step toward developing molecular markers for breeders to efficiently integrate these desirable traits into cultivars.

Amylose content decreases during tuber development in potato.

BACKGROUND: Potato starch is composed primarily of amylopectin and amylose in an approximately 3:1 ratio. Amylose is considered to be nutritionally desirable in North American and European markets, so there is interest in finding strategies to increase the amylose content of potato starch. There is also interest in marketing 'baby' potatoes, which are harvested when they are physiologically immature. This study was carried out to determine weekly changes in amylose content in potato tubers of 11 North American cultivars during the growing season. The trial was repeated across 3 years. RESULTS: We determined that amylose content is highest early and it decreases in a linear fashion as the growing season progresses. Mean amylose content across cultivars and years declined from 30.0% in late June to 26.8% in late August. The rate of decrease varied across years, with slopes of linear regression plots ranging from -0.17 in 2012 to -0.74 in 2011. Amylose content in tuber starch varied among cultivars, with the highest levels observed in Ranger Russet (30.7%) and White Pearl (31.6%); it was lowest in Kennebec (25.7%) and Langlade (25.6%). CONCLUSIONS: This study adds to a growing body of literature on the nutritional value of immature potato tubers. In addition to having higher levels of some phytonutrients, as reported in other studies, immature tubers have a higher proportion of amylose in the starch. This is nutritionally desirable in affluent regions where high fiber content is more important than calories from carbohydrates. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Solanum clarum and S. morelliforme as Novel Model Species for Studies of Epiphytism.

The natural history of epiphytic plant species has been extensively studied. However, little is known about the physiology and genetics of epiphytism. This is due to difficulties associated with growing epiphytic plants and the lack of tools for genomics studies and genetic manipulations. In this study, tubers were generated from 223 accessions of 42 wild potato Solanum species, including the epiphytic species S. morelliforme and its sister species S. clarum. Lyophilized samples were analyzed for 12 minerals using inductively coupled plasma optical emission spectrometry. Mineral levels in tubers of S. morelliforme and S. clarum were among the highest for 10 out of the 12 elements evaluated. These two wild potato relatives are native to southern Mexico and Central America and live as epiphytes or in epiphytic-like conditions. We propose the use of S. morelliforme and S. clarum as model organisms for the study of mineral uptake efficiency. They have a short life cycle, can be propagated vegetatively via tubers or cuttings, and can be easily grown in controlled environments. In addition, genome sequence data are available for potato. Transgenic manipulations and somatic fusions will allow the movement of genes from these epiphytes to cultivated potato.

Genetic mapping with an inbred line-derived F2 population in potato.

KEY MESSAGE: This is the first report of the production and use of a diploid inbred line-based F2 population for genetic mapping in potato. Potato (Solanum tuberosum L.) is an important global food crop, for which tetrasomic inheritance and self-incompatibility have limited both genetic discovery and breeding gains. We report here on the creation of the first diploid inbred line-derived F2 population in potato, and demonstrate its utility for genetic mapping. To create the population, the doubled monoploid potato DM1-3 was crossed as a female to M6, an S7 inbred line derived from the wild relative S. chacoense, and a single F1 plant was then self-pollinated. A genetic linkage map with 2264 single nucleotide polymorphisms was constructed and used to improve the physical anchoring of superscaffolds in the potato reference genome, which is based on DM1-3. Segregation was observed for skin and flesh color, skin and flesh pigment intensity, tuber shape, anther development, jelly end, and the presence of eye tubers instead of normal sprouts. Using the R/qtl software, we detected 10 genes, 7 of which have been previously mapped and 3 for which this is the first publication. The latter category includes tightly linked genes for the jelly end and eye tuber traits on chromosome 5. The development of recombinant inbred lines from this F2 population by single-seed descent is underway and should facilitate even better resolution of these and other loci.

Tuber starch amylose content is associated with cold-induced sweetening in potato.

Cold-induced sweetening (CIS) is the accumulation of reducing sugars in potato tubers at low storage temperatures. It is undesirable because it results in dark fry products. Our study evaluated the relationship between genetic resistance to CIS and two starch parameters, amylose content and starch granule size. We found that the amylose content in four CIS-resistant varieties was higher than that in five susceptible varieties. Amylose content was influenced not only by variety but also storage, production year, and field location. However, interactions between amylose content and environmental variables were not detected. In contrast, starch granule size was not associated with CIS resistance. No effect of storage on starch granule size was detected, and interactions among variety, production year, and field location were observed. Tuber starch amylose content should be considered a source of variability for CIS.

Rapid high throughput amylose determination in freeze dried potato tuber samples.

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones. 

Insertional mutagenesis using Tnt1 retrotransposon in potato.

Insertional mutagenesis using transfer DNA or transposable elements, which is an important tool in functional genomics and is well established in several crops, has not been developed in potato (Solanum tuberosum). Here, we report the application of the tobacco (Nicotiana tabacum) Tnt1 retrotransposon as an insertional mutagen in potato. The Tnt1 retrotransposon was introduced into a highly homozygous and self-compatible clone, 523-3, of the diploid wild potato species Solanum chacoense. Transposition of the Tnt1 elements introduced into 523-3 can be efficiently induced by tissue culture. Tnt1 preferentially inserted into genic regions in the potato genome and the insertions were stable during sexual reproduction, making Tnt1 an ideal mutagen in potato. Several distinct phenotypes associated with plant stature and leaf morphology were discovered in mutation screening from a total of 38 families derived from Tnt1-containing lines. We demonstrate that the insertional mutagenesis system based on Tnt1 and the 523-3 clone can be expanded to the genome-wide level to potentially tag every gene in the potato genome.

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.

A test of taxonomic and biogeographic predictivity: resistance to soft rot in wild relatives of cultivated potato.

The concept that traits should be associated with related organisms and that nearby populations of the same species are likely to be more similar to each other than to populations spread far apart has long been accepted. Consequently, taxonomic relationships and biogeographical data are commonly believed to have the power to predict the distribution of disease resistance genes among plant species. In this study, we test claims of such predictivity in a group of widely distributed wild potato species. There was no clear association between resistance to soft rot and taxonomic relationships. However, we have found some associations between resistance to soft rot and environmental data such as annual precipitation and annual mean temperature. In addition, we have noted that high levels of resistance are mostly found in species with high levels of phenotypic plasticity. The three most resistant species were Solanum paucijugum, S. brevicaule, and S. commersonii.

Suppression of the vacuolar invertase gene prevents cold-induced sweetening in potato.

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to prevent sprouting, minimize disease losses, and supply consumers and the processing industry with high-quality tubers throughout the year. Unfortunately, cold storage triggers an accumulation of reducing sugars in tubers. High-temperature processing of these tubers results in dark-colored, bitter-tasting products. Such products also have elevated amounts of acrylamide, a neurotoxin and potential carcinogen. We demonstrate that silencing the potato vacuolar acid invertase gene VInv prevents reducing sugar accumulation in cold-stored tubers. Potato chips processed from VInv silencing lines showed a 15-fold acrylamide reduction and were light in color even when tubers were stored at 4°C. Comparable, low levels of VInv gene expression were observed in cold-stored tubers from wild potato germplasm stocks that are resistant to cold-induced sweetening. Thus, both processing quality and acrylamide problems in potato can be controlled effectively by suppression of the VInv gene through biotechnology or targeted breeding.

Hybrid origins of cultivated potatoes.

Solanum section Petota is taxonomically difficult, partly because of interspecific hybridization at both the diploid and polyploid levels. The taxonomy of cultivated potatoes is particularly controversial. Using DNA sequence data of the waxy gene, we here infer relationships among the four species of cultivated potatoes accepted in the latest taxonomic treatment (S. ajanhuiri, S. curtilobum, S. juzepczukii and S. tuberosum, the latter divided into the Andigenum and Chilotanum Cultivar Groups). The data support prior ideas of hybrid origins of S. ajanhuiri from the S. tuberosum Andigenum Group (2x = S. stenotomum) × S. megistacrolobum; S. juzepczukii from the S. tuberosum Andigenum Group (2x = S. stenotomum) × S. acaule; and S. curtilobum from the S. tuberosum Andigenum Group (4x = S. tuberosum subsp. andigenum) × S. juzepczukii. For the tetraploid cultivar-groups of S. tuberosum, hybrid origins are suggested entirely within much more closely related species, except for two of three examined accessions of the S. tuberosum Chilotanum Group that appear to have hybridized with the wild species S. maglia. Hybrid origins of the crop/weed species S. sucrense are more difficult to support and S. vernei is not supported as a wild species progenitor of the S. tuberosum Andigenum Group.