David S. Douches

Integration of Two Diploid Potato Linkage Maps with the Potato Genome Sequence

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

Construction of Reference Chromosome-Scale Pseudomolecules for Potato: Integrating the Potato Genome with Genetic and Physical Maps

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

Analysis of the Brassica oleracea genome by the generation of B. campestris-oleracea chromosome addition lines: characterization by isozymes and rDNA genes

SummaryThis study aimed at generating chromosome addition lines and disclosing genome specific markers in Brassica. These stocks will be used to study genome evolution in Brassica oleracea L., B. campestris L. and the derived amphidiploid species B. napus L. B. campestris-oleracea monosomic and disomic chromosome addition plants were generated by crossing and backcrossing the natural amphidiploid B. napus to the diploid parental species B. campestris. The pollen viability of the derived sesquidiploid and hyperploid ranged from 63% to 88%, while the monosomic and disomic addition plants had an average pollen fertility of 94% and 91%, respectively. The addition lines were genetically characterized by genome specific markers. The isozymes for 6PGD, LAP, PGI and PGM, and rDNA Eco RI restriction fragments were found to possess the desired genome specificity. Duplicated loci for several of these markers were observed in B. campestris and B. oleracea, supporting the hypothesis that these diploid species are actually secondary polyploids. A total of eight monosomic and eight disomic addition plants were identified and characterized on the basis of these markers. Another 51 plants remained uncharacterized due to the lack of additional markers. rDNA genes were found to be distributed in more than one chromosome, differing in its restriction sites. Intergenomic recombination for some of the markers was detected at frequencies between 6% and 20%, revealing the feasibility of intergenomic gene transfer.

Comparison of four molecular markers in measuring relationships among the wild potato relatives Solanum section Etuberosum (subgenus Potatoe)

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.

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.

Biochemical and folk assessment of variability of Andean cultivated potatoes

Isozyme markers were used to survey the genetic variability of non-bitter potatoes in 10 subsistence fields of Andean farmers at 3600-3850 m above sea level. Sixty-seven percent of the varieties were tetraploids corresponding to the speciesSolanum tuberosum ssp.andigena, 14% were triploids, probably corresponding to the speciesS. x chaucha, and 13% were diploids corresponding to the speciesS. stenotomum, S. phureja, andS. goniocalyx. The isozyme information served to determine the consistency of the folk naming system. We found a high degree of correspondence between farmer identification and electrophoretic phenotypes. The consistency of the folk system in electrophoretic terms depended on the farmer who was interviewed. The most common incongruity consisted of calling different electrophoretic phenotypes by the same variety name, leading to a slight underestimation of genetic variability present in the fields. The amount of variability observed in the sample of the Andean potato population was superior to that present in North American and European varieties. This was measured in terms of number ofalleles, number of electrophoretic phenotypes and percent of heterozygosity. This finding supports the impression that a substantial amount of yet unexploited variability remains in Andean potato populations.ResumenEn este trabajo se da a conocer los resultados de un estudio genético sobre variedades de papa dulce en los Andes, realizada por medio de marcadores isoenzimáticos en parcelas de subsistencia localizados entre 3600 y 3850 metros sobre el nivel del mar. Se encontró que 67% de las variedades muestreadas eran tetraploides de la especie S. tuberosum ssp. andigena, 14% triploides probablemente de la especie S. x chaucha, y 13% diploides de las especiesS. stenotomum, S. phureja andS. goniocalyx. La información isoenzimática fue útil en la evaluatión de la precisión del sistema folklorico para identificar variedades. Se encontró un alto grado de asociación entre el sistema de clasificación usado por el campesino para denominar sus variedades, y los fenotipos electroforéticos. La precisión del sistema de identidad folklorico en tℰminos electroforéticos dependió del campesino entrevistado. La discrepancia mas frecuente entre los dos sistemas de nomenclatura consistió en llamar diferentes fenotipos electroforéticos con el mismo nombre varietal, lo que resultó en una subestimación de la variabilidad genética presente en los campos. El nivel de variabilidad observado en la muestra de papas de la población andina fue superior al observado en variedades nortea-mericanas y europeas. La variabilidad se midió en base al número de alelos, número de fenotipos electroforéticos y porcentaje de heterocigosidad. Estos resultados están de acuerdo con la impresión general de que todavía existe mucha variabilidad en variedades de papa andinas que no ha sido aún explotada.

High-Density SNP Genotyping of Tomato (Solanum lycopersicum L.) Reveals Patterns of Genetic Variation Due to Breeding

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.

Geographical approaches to crop conservation: The partitioning of genetic diversity in andean potatoes

The geographical concepts of spatial scale and the human-geographic region offer significant contributions to the conservation of crop genetic resources. They are used in the present study to examine the partitioning of genetic diversity along two axes: geographical location and landrace population.Locations in the study consist of three micro-regions within the highland Paucartambo region of southern Peru. Six widely distributed landraces of the potato species Solatium stenotomum Juz. et Buk. and S. tuberosum subsp. andigena (Juz. et Buk.) Hawkes are evaluated. Electrophoretic analysis of isozyme loci demonstrates that the majority of allelic variation is contained within the geographical and landrace populations. Geographically, greater than 99% of total variation is found within single micro-regions. Taxonomically, approximately 75% of variation occurs within individual landraces. The weak geographical partitioning of allelic variation is due in part to formerly high rates of seed-tuber exchange. The weak-moderate taxonomic partitioning of variation is attributed to common parentage and shared introgression. Unique genotypes are microgeographically concentrated.Findings recommend that conservation strategies focus on intensive sampling or preservation in micro-regional areas due to the concentration of unique genotypes. Evaluation of the spatial patterning of diversity and recognition of the taxonomic specificity of results (not necessarily applicable even to related potato landraces) rely on biogeographical and human-geographic concepts.

INTERSPECIFIC SOMATIC HYBRIDIZATION BETWEEN SOLANUM TUBEROSUM L. AND S. BULBOCASTANUM DUN. AS A MEANS OF TRANSFERRING NEMATODE RESISTANCE

Interspecific somatic hybrids were produced between tetraploidSolanum tuberosum and a nematode-resistant accession of the diploid speciesSolanum bulbocastanum by protoplast fusion. Hybrid cells were selected using dual fluorescent labeling of protoplast preparations prior to fusion. Hybridity of regenerated plants was confirmed with a combination of morphological assessment, chromosome counting and isozyme analysis. Somatic hybrids had the same level of resistance to infection by race 1 of the nematodeMeloidogyne chitwoodi as theS. bulbocastanum parent used in the fusion. Some of the somatic hybrids were fertile as females when crossed with tetraploidS. tuberosum breeding lines. Thus, these hybrids can be used in a potato improvement program to incorporate a valuable pest resistanceCompendioHíbridos somáticos interespecíficos fueron producidos, por fusión de protoplastos, entre el tetraploideSolanum tuberosum y una entrada de la especie diploideSolanum bulbocastanum resistente a los nematodos. Se seleccionaron células híbridas utilizando la marcación con sondas fluorescentes duales en las preparaciones de protoplastos antes de la fusión. Se confirmó la hibridación de las plantas regeneradas con una combinación de evaluación morfológica, contaje de cromosomas y análisis de isozimas.Los híbridos somáticos tuvieron el mismo nivel de resistencia a la infectión por la raza 1 del nematodoMeloidogyne chitwoodi como el de los padresS. bulbocastanum utilizados en la fusión. Algunos de los híbridos somáticos fueron fértiles cuando se les usó como madres en cruzamientos con líneas de mejoramiento del tetraploideS. tuberosum. Por lo tanto, estos híbridos pueden ser usados en un programa relacionado al mejoramiento de la papa, para incorporar resistencia valiosa a la plaga.

Generation and inheritance of targeted mutations in potato (Solanum tuberosum L.) Using the CRISPR/Cas System

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.