Diego Fajardo

Species limits of Solanum berthaultii Hawkes and S. tarijense Hawkes and the implications for species boundaries in Solanum sect. Petota

Solanum berthaultii and S. tarijense are two wild potato (sect. Petota) species distributed from Bolivia to northern Argentina. All authors have accepted them as good species since their publication in 1944, but they have been hypothesized to hybridize extensively with each other and with other species, despite their classification into different series and superseries by some authors. This study is a molecular counterpart (AFLPs, plastid DNA restriction site data, survey of a plastid DNA deletion) to a prior morphological study of these two species. AFLP data show weak support for separate species status for some accessions, but with many exceptions. In agreement with the morphological results we place S. tarijense into synonymy of S. berthaultii, and use herbarium specimen data for a taxonomic treatment to include a description, synonymy, and mapping of all accessions. We show similar taxonomic problems in sect. Petota and suggest that there will be a continuing trend of reduction of names in wild potatoes.

The American cranberry: first insights into the whole genome of a species adapted to bog habitat

BackgroundThe American cranberry (Vaccinium macrocarpon Ait.) is one of only three widely-cultivated fruit crops native to North America- the other two are blueberry (Vaccinium spp.) and native grape (Vitis spp.). In terms of taxonomy, cranberries are in the core Ericales, an order for which genome sequence data are currently lacking. In addition, cranberries produce a host of important polyphenolic secondary compounds, some of which are beneficial to human health. Whereas next-generation sequencing technology is allowing the advancement of whole-genome sequencing, one major obstacle to the successful assembly from short-read sequence data of complex diploid (and higher ploidy) organisms is heterozygosity. Cranberry has the advantage of being diploid (2n = 2x = 24) and self-fertile. To minimize the issue of heterozygosity, we sequenced the genome of a fifth-generation inbred genotype (F ≥ 0.97) derived from five generations of selfing originating from the cultivar Ben Lear.ResultsThe genome size of V. macrocarpon has been estimated to be about 470 Mb. Genomic sequences were assembled into 229,745 scaffolds representing 420 Mbp (N50 = 4,237 bp) with 20X average coverage. The number of predicted genes was 36,364 and represents 17.7% of the assembled genome. Of the predicted genes, 30,090 were assigned to candidate genes based on homology. Genes supported by transcriptome data totaled 13,170 (36%).ConclusionsShotgun sequencing of the cranberry genome, with an average sequencing coverage of 20X, allowed efficient assembly and gene calling. The candidate genes identified represent a useful collection to further study important biochemical pathways and cellular processes and to use for marker development for breeding and the study of horticultural characteristics, such as disease resistance.

The American cranberry mitochondrial genome reveals the presence of selenocysteine (tRNA-Sec and SECIS) insertion machinery in land plants.

This is the first de novo assembly and annotation of a complete mitochondrial genome in the Ericales order from the American cranberry (Vaccinium macrocarpon Ait.). Moreover, only four complete Asterid mitochondrial genomes have been made publicly available. The cranberry mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with relatively little repetitive sequences and DNA of plastid origin. The complete mitochondrial genome of cranberry was annotated obtaining a total of 34 genes classified based on their putative function, plus three ribosomal RNAs, and 17 transfer RNAs. Maternal organellar cranberry inheritance was inferred by analyzing gene variation in the cranberry mitochondria and plastid genomes. The annotation of cranberry mitochondrial genome revealed the presence of two copies of tRNA-Sec and a selenocysteine insertion sequence (SECIS) element which were lost in plants during evolution. This is the first report of a land plant possessing selenocysteine insertion machinery at the sequence level.

Discrimination of American Cranberry Cultivars and Assessment of Clonal Heterogeneity Using Microsatellite Markers

Cranberries (Vaccinium macrocarpon Ait.) are an economically important fruit crop derived from a North American native species. We report the application of 12 simple sequence repeats (SSR) or microsatellite markers to assess the genetic diversity of cranberry cultivars. We studied 164 samples of 21 different cranberry cultivars, 11 experimental hybrids, and 6 representative accessions of wild species. Genetic cluster analysis, based on 117 SSR alleles, differentiated the major cranberry cultivars. However, some cranberry cultivar subclone variants and mislabeled samples were observed. Consensus genetic profiles identified the most likely clonal representatives of several important cranberry cultivars (e.g., “Ben Lear,” “Howes,” and “Stevens”). The markers were further used to confirm putative parents of several hybrid progenies. The long-term goal of our studies is to identify, preserve, and utilize unique genetic materials to breed improved cranberries. Attaining this goal will help growers maintain sustainability under changing economic and environmental conditions.

Development and Validation of 697 Novel Polymorphic Genomic and EST-SSR Markers in the American Cranberry (Vaccinium macrocarpon Ait.)

The American cranberry, Vaccinium macrocarpon Ait., is an economically important North American fruit crop that is consumed because of its unique flavor and potential health benefits. However, a lack of abundant, genome-wide molecular markers has limited the adoption of modern molecular assisted selection approaches in cranberry breeding programs. To increase the number of available markers in the species, this study identified, tested, and validated microsatellite markers from existing nuclear and transcriptome sequencing data. In total, new primers were designed, synthesized, and tested for 979 SSR loci; 697 of the markers amplified allele patterns consistent with single locus segregation in a diploid organism and were considered polymorphic. Of the 697 polymorphic loci, 507 were selected for additional genetic diversity and segregation analyses in 29 cranberry genotypes. More than 95% of the 507 loci did not display segregation distortion at the p < 0.05 level, and contained moderate to high levels of polymorphism with a polymorphic information content >0.25. This comprehensive collection of developed and validated microsatellite loci represents a substantial addition to the molecular tools available for geneticists, genomicists, and breeders in cranberry and Vaccinium.

Generation and Characterization of a Sugarbeet Transcriptome and Transcript-Based SSR Markers

Sugarbeet is a major source of refined sucrose and increasingly grown for biofuel production. Demand for higher productivity for this crop requires greater knowledge of sugarbeet physiology, pathology, and genetics, which can be advanced by the development of new genomic resources. Towards this end, a sugarbeet transcriptome of expressed genes from leaf and root tissues at varying stages of development and production, and after elicitation with jasmonic acid (JA) or salicylic acid (SA), was constructed and used to generate simple sequence repeat (SSR) markers. The transcriptome was generated via paired‐end RNA sequencing and contains 82,404 unigenes. A total of 37,207 unigenes were annotated, of which 9480 were functionally classified using clusters of orthologous groups (COG) annotations, 17,191 were classified into biological process, molecular function, or cellular component using gene ontology (GO) terms, and 17,409 were assigned to 126 metabolic pathways using Kyoto Encyclopedia of Genes and Genomes (KEGG) identifiers. A SSR search of the transcriptome identified 7680 SSRs, including 6577 perfect SSRs, of which 3834 were located in unigenes with ungapped sequence. Primer‐pairs were designed for 288 SSR loci, and 72 of these primer‐pairs were tested for their ability to detect polymorphisms. Forty‐three primer‐pairs detected single polymorphic loci and effectively distinguished diversity among eight B. vulgaris genotypes. The transcriptome and SSR markers provide additional, public domain genomic resources for an important crop plant and can be used to increase understanding of the functional elements of the sugarbeet genome, aid in discovery of novel genes, facilitate RNA‐sequencing based expression research, and provide new tools for sugarbeet genetic research and selective breeding.

Phylogenetic Relationships of Solanum Series Conicibaccata and Related Species in Solanum Section Petota Inferred from Five Conserved Ortholog Sequences

Abstract Solanum series Conicibaccata is the second largest series in section Petota, containing 40 species widely distributed from southern Mexico to central Bolivia. It contains diploids (2n = 2x = 24), tetraploids (2 = 4x = 48) and hexaploids (2n = 6x = 72), and the limited number of species examined have been shown to be allopolyploids. Previous morphological and molecular studies using plastid DNA failed to discriminate clear species boundaries. Conserved orthologous nuclear DNA sequences (COSII) were used to compare the relationships among 72 accessions from 22 species from series Conicibaccata and 42 additional accessions from related series. The results supported previous studies showing the diploid members of series Conicibaccata to be related to other South American “clade 4” species, and showed all of the polyploids to be allopolyploids among members of clade 4 and other South American species of “clade 3” (series Piurana and related species). Low bootstrap support values and morphological similarity suggest recent origins and the need for a reduction in number of recognized species in series Conicibaccata.

Starch Characteristics of Modern and Heirloom Potato Cultivars

In a number of ways, modern potato breeding efforts differ from those that created heirloom cultivars. As a result of the rapid expansion of the potato processing industry in the mid twentieth century, potato breeders shifted their focus from fresh market varieties to those intended for the fry and chip markets. Selection for higher dry matter content and lower reducing sugar levels in tubers was successful. This study sought to determine whether modern processing cultivars differ from heirloom cultivars for two major characteristics of tuber starch, amylose content and starch granule morphology. Since breeding efforts for processing potatoes have focused on the maintenance of tuber quality during storage, these parameters were measured in both fresh and stored tubers. Twenty cultivars selected to span the range of cultivar release dates in the U.S. were grown at Hancock, WI in 2009 and 2010 and evaluated for tuber amylose content and measurements of starch granule surface area, length, width, length to width ratio, and smoothness. These traits were found to be stable across years and during storage. Significant differences in the amylose content and starch granule morphology exist among cultivars, but there is no clear distinction between modern and heirloom cultivars. Genetic variation for amylose content and starch granule morphology was found within the set of clones sampled for this study. However, germplasm with higher amylose content will need to be identified and utilized in breeding if the nutritional benefits associated with high fiber and low glycemic index are to be realized.ResumenDe diferentes maneras los esfuerzos modernos en el mejoramiento de la papa difieren de aquellos que crearon las variedades clásicas. Como resultado de la rápida expansión de la industria del procesado de la papa a mediados del siglo veinte, los fitomejoradores de la papa se reenfocaron de variedades de mercado fresco a aquellas para mercado de las frituras. La selección para alto contenido de materia seca y bajos niveles de azúcares reductores en tubérculos fue exitosa. Este estudio buscó determinar si las variedades modernas para procesamiento difieren de las clásicas en dos características principales de almidón en el tubérculo, contenido de amilosa y morfología del gránulo de almidón. Considerando que los esfuerzos de mejoramiento para papa de proceso se ha enfocado en el mantenimiento de la calidad el tubérculo durante el almacenamiento, se midieron estos parámetros tanto en tubérculos frescos como en almacenados. Se sembraron veinte variedades para aclarar la amplitud de fechas de liberación de variedades en los EUA en Hancock, WI, en 2009 y 2010 y se evaluaron para el contenido de amilosa en el tubérculo y se hicieron mediciones del área de la superficie del gránulo de almidón, longitud, ancho, la relación largo-ancho, y la suavidad. Se encontró que estos caracteres eran estables a lo largo de los años y durante el almacenamiento. Existen diferencias significativas en el contenido de amilosa y en la morfología del gránulo de almidón entre variedades, pero no hay una distinción clara entre las variedades modernas y las clásicas. Se encontró variación genética en el contenido de amilosa y para la morfología del gránulo de almidón entre el juego de clones muestreado para este estudio. No obstante, el germoplasma con el mas alto contenido de amilosa requerirá ser identificado y utilizado en mejoramiento si se consideran los beneficios nutrimentales asociados con alta fibra y bajo índice glicémico.

A Morphometric Study of Species Boundaries of the Wild Potato Solanum Series Conicibaccata: a Replicated Field Trial in Andean Peru

Abstract Solanum series Conicibaccata contains about 40 wild potato (section Petota) species distributed from southern Mexico to central Bolivia. It contains diploids (2n = 2x = 24), tetraploids (2n = 4x = 48) and hexaploids (2n = 6x = 72) and some polyploids are likely allopolyploids. Our morphological phenetic study in an Andean site in central Peru (12°S, 3200 m altitude) is a replicated study from one done in the north central United States (45°N, 180 m elevation) but uses more species (28 vs. 25), accessions (173 vs. 100), and morphological characters (72 vs. 45) and also includes members of related series Piurana. Both US and Peruvian studies provide phenetic support with Canonical Discriminant Analyses (but poorly if at all with Principal Components Analyses) to distinguish the following species or species groups in series Conicibaccata: 1) S. agrimonifolium and S. oxycarpum as a possible single species, and 2) S. longiconicum (tetraploids from Mexico and Central America), 3) the South American Conicibaccata diploids as a possible single species, except for 4) S. trinitense that is distinctive, 5) the South American tetraploids as a group except for 6) S. flahaultii that is distinctive. However, character states among these species or species groups are often present only by using a range of widely overlapping character states (polythetic support). We suspect that our continuing molecular studies will support the synonymy of many of these species.

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.