John R. Stommel

Effects of Anthocyanin and Carotenoid Combinations on Foliage and Immature Fruit Color of Capsicum annuum L.

Shades ranging from violet to black pigmentation in pepper (Capsicum annuum L.) are attributed to anthocyanin accumulation. High-performance liquid chromatography and mass spectrometry analysis of violet and black fruit tissue identified a single anthocyanin that was determined to be delphinidin-3-p-coumaroyl-rutinoside-5-glucoside. Leaf tissue of a black-pigmented foliage genotype contained the same anthocyanin found in fruit but at a considerably higher concentration in comparison to violet and black fruit tissue. Fruit chlorophyll concentration was approximately 14-fold higher in black fruit in comparison to violet fruit that contained relatively little chlorophyll. Beta-carotene, lutein, violaxanthin, and neoxanthin carotenoid concentrations in black fruit were also significantly greater in comparison to violet fruit. High concentrations of delphinidin in combination with chlorophyll and accessory carotenoid pigments produced the characteristic black pigmentation observed in fruits and leaves of selected genotypes. Anthocyanins were accumulated in the outer mesocarp of violet and black fruit and in the palisade and mesophyll cells of black leaves. Consistent with chlorophyll content of respective genotypes, chloroplast density was greater in cells of black fruits. Utilizing Capsicum pigment variants, we determine the biochemical factors responsible for violet versus black-pigmented pepper tissue in the context of described pepper color genes.

Genetic diversity and population structure of Capsicum baccatum genetic resources

Capsicum baccatum is one of five domesticated pepper species which, despite its morphological and ecological variability, has been underexploited for germplasm improvement. Utilizing a broad spectrum of domesticated and wild C. baccatum germplasm, we utilize AFLP markers to describe the species’ molecular diversity and population structure in the South American gene pool. Analysis of molecular variance (AMOVA) revealed greater genetic diversity in the wild form of C. baccatum (C. baccatum var. baccatum) than in the domesticated form of the species (C. baccatum var. pendulum). Both Bayesian and distance based clustering analysis, as well as principal coordinates analysis (PCA), concordantly demonstrated admixture/shared ancestry between wild and cultivated C. baccatum botanical varieties. Two principal genetic groups were identified in the domesticated C. baccatum accessions largely based on their geographic distribution in South America. One group was predominated by accessions from the western territories of the species’ distribution (Peru, Colombia, Ecuador, Bolivia, Chile and northwestern Argentina) and the second by accessions from the eastern regions, Paraguay and eastern Argentina). The two genetic groups overlapped in the geographic location of present-day Bolivia. The grouping pattern suggested that C. baccatum was domesticated in multiple sites and that its evolution took two lineages followed by lineage differentiation. The wild accessions most closely related to the cultigens were found in the highlands of Peru and Bolivia, which support the early hypothesis that this region is one of the domestication sites of this species. A Bayesian assignment analysis demonstrated that Brazilian wild forms of C. baccatum were genetically distant to all other accessions and made little to no contribution to the domesticated genepool. Moreover, results of clustering analysis suggested that C. baccatum likely originated from present day Paraguay. Analysis of inter-specific relationships across selected Capsicum species supported independent lineages for the two crossability groups within Capsicum, the baccatum species-complex (including C. baccatum) and the annuum species-complex (including C. annuum, C. chinense and C. frutescens). However, the results did not support taxonomic distinction of C. baccatum var. umbilicatum from C. baccatum var. pendulum. The present study provides new insights into the domestication of C. baccatum. The results will be useful for identifying accessions for crop improvement and guiding the development of in situ and ex situ conservation programs.

Multiple forms of invertase from Daucus carota cell cultures.

Abstract A 400-fold purification of two isoenzymes of acid invertase (pH optima 4.5) and a two- to three-fold purification of alkaline (pH optima 7.5) invertase from suspension cultured carrot cells was obtained using ion-exchange (DEAE cellulose) and affinity (concanavalin-A) column chromatography. SDS polyacrylamide gel electrophoresis resolved major polypeptide bands at 61 000 and 49000 corresponding to the two isoenzymes of acid invertase, designated acid- 1 and acid-2, respectively. The acid invertase isoenzymes, in contrast with alkaline invertase, are likely glycoproteins based on their ability to bind the lectin concanavalin-A.

Characterization of interspecific hybrids and first backcross generations from crosses between two cultivated eggplants (Solanum melongena and S. aethiopicum Kumba group) and implications for eggplant breeding

Common (Solanum melongena L.) and scarlet (S. aethiopicum L.) eggplants are cultivated for their fruits and form part of the same genepool. We have studied plant and fruit characteristics, pollen viability and seed set, phenolics content, and fruit flesh browning in accessions of S. melongena and S. aethiopicum Kumba group, as well as interspecific hybrids between these species and first backcross generations to each parental species. Respective genotypes were also characterized with seven polymorphic SSR markers. The results demonstrate that many differences exist for plant and fruit morphology among S. melongena, S. aethiopicum and the interspecific hybrids. The latter are very vigorous and generally intermediate between the two parents, except for fruit size which is smaller (and parthenocarpic due to a high pollen sterility) than those of any of the parents. Backcross progenies also exhibited morphological variation with moderate heritability values for the attributes evaluated. Variation for fruit size was present in the backcross generations but fruits were small resulting in little variation for fruit shape. Backcross plants with moderate fertility produced seeded fruits. Primary hybrids had fruit phenolics content similar to that of S. aethiopicum, the parent with lowest phenolics concentration, and were heterotic for fruit flesh browning. Backcross progenies were quite variable for both traits. SSR markers did not reveal segregation distortion in the backcross generations for these interspecific hybrids. The results demonstrate that generations derived from sexual interspecific hybridization can be a powerful tool for S. melongena and S. aethiopicum Kumba group breeding.

Viral Satellite RNA Expression in Transgenic Tomato Confers Field Tolerance to Cucumber Mosaic Virus

Field trials of transgenic tomato plants expressing an ameliorative satellite RNA of cucumber mosaic virus (CMV) were conducted to test the efficacy of satellite-transgenic technology to protect against CMV infection. Three transgenic tomato lines derived from two susceptible genotypes were evaluated over two growing seasons for viral symptoms and titers, satellite RNA expression, and fruit yield. Satellite-transgenic lines exhibited mild or no CMV symptoms and low viral titers relative to nontransformed plants. A significant negative correlation between satellite RNA levels and disease severity was evident in transgenic lines. Total marketable yield of CMV-infected satellite-transgenic lines was 40 to 84% greater than that of CMV-infected parent lines. Importantly, yield of CMV-infected satellite-transgenic lines did not differ significantly from mock-inoculated parent lines. Risk assessment results demonstrated low levels of satellite RNA transmission within the test site and no evidence of satellite RNA-induced damage on surrounding plants.

Genetic diversity in Capsicum baccatum is significantly influenced by its ecogeographical distribution

BackgroundThe exotic pepper species Capsicum baccatum, also known as the aji or Peruvian hot pepper, is comprised of wild and domesticated botanical forms. The species is a valuable source of new genes useful for improving fruit quality and disease resistance in C. annuum sweet bell and hot chile pepper. However, relatively little research has been conducted to characterize the species, thus limiting its utilization. The structure of genetic diversity in a plant germplasm collection is significantly influenced by its ecogeographical distribution. Together with DNA fingerprints derived from AFLP markers, we evaluated variation in fruit and plant morphology of plants collected across the species native range in South America and evaluated these characters in combination with the unique geography, climate and ecology at different sites where plants originated.ResultsThe present study mapped the ecogeographic distribution, analyzed the spatial genetic structure, and assessed the relationship between the spatial genetic pattern and the variation of morphological traits in a diverse C. baccatum germplasm collection spanning the species distribution. A combined diversity analysis was carried out on the USDA-ARS C. baccatum germplasm collection using data from GIS, morphological traits and AFLP markers. The results demonstrate that the C. baccatum collection covers wide geographic areas and is adapted to divergent ecological conditions in South America ranging from cool Andean highland to Amazonia rainforest. A high level of morphological diversity was evident in the collection, with fruit weight the leading variable. The fruit weight distribution pattern was compatible to AFLP-based clustering analysis for the collection. A significant spatial structure was observed in the C. baccatum gene pool. Division of the domesticated germplasm into two major regional groups (Western and Eastern) was further supported by the pattern of spatial population structure.ConclusionsThe results reported improve our understanding of the combined effects of geography, ecology and human intervention on organization of the C. baccatum genepool. The results will facilitate utilization of C. baccatum for crop improvement and species conservation by providing a framework for efficient germplasm collection management and guidance for future plant acquisitions.

Genome-wide Diversity and Association Mapping for Capsaicinoids and Fruit Weight in Capsicum annuum L

Accumulated capsaicinoid content and increased fruit size are traits resulting from Capsicum annuum domestication. In this study, we used a diverse collection of C. annuum to generate 66,960 SNPs using genotyping by sequencing. The study identified 1189 haplotypes containing 3413 SNPs. Length of individual linkage disequilibrium (LD) blocks varied along chromosomes, with regions of high and low LD interspersed with an average LD of 139 kb. Principal component analysis (PCA), Bayesian model based population structure analysis and an Euclidean tree built based on identity by state (IBS) indices revealed that the clustering pattern of diverse accessions are in agreement with capsaicin content (CA) and fruit weight (FW) classifications indicating the importance of these traits in shaping modern pepper genome. PCA and IBS were used in a mixed linear model of capsaicin and dihydrocapsaicin content and fruit weight to reduce spurious associations because of confounding effects of subpopulations in genome-wide association study (GWAS). Our GWAS results showed SNPs in Ankyrin-like protein, IKI3 family protein, ABC transporter G family and pentatricopeptide repeat protein are the major markers for capsaicinoids and of 16 SNPs strongly associated with FW in both years of the study, 7 are located in known fruit weight controlling genes.

Influence of 2-deoxy-D-glucose upon growth and invertase activity of carrot (Daucus carota L. ) cell suspension cultures

Carrot (Daucus carota L.) cell suspension cultures grew well when provided with glucose, fructose, sucrose or raffinose. Galactose and melibiose supported less growth unless supplemented with glucose or fructose. In combination with ten different sugar mixtures, 2-deoxy-D-glucose (dGlc) inhibited culture growth. Inhibitory effects of dGlc were more marked with fructose, melibiose, raffinose or mixtures of these sugars in the culture medium. The presence of glucose or galactose reduced the inhibitory effects of dGlc on culture growth. Experiments with radioactive labelled sugars demonstrated that dGLc uptake was greater in the presence of fructose than glucose, and that growth inhibition of dGlc coincided with its uptake. Reduced protein content was also associated with the inhibitory effects of dGlc. Cultured cells contained lower levels of invertase (EC 3.2.1.26) activity during the active phase of culture growth (up to 25 days after subculture) than when growth had peaked and subsequently declined. Acid and alkaline invertase activities were not greatly reduced by exogenous hexoses. Invertase activity was greatest during periods of low protein content in all cultures and was inhibited by dGlc during the latter phases of the culture period. Free intracellular sugars throughout the culture period consisted mainly of glucose and fructose.

Genome-Wide Divergence and Linkage Disequilibrium Analyses for Capsicum baccatum Revealed by Genome-Anchored Single Nucleotide Polymorphisms

Principal component analysis (PCA) with 36,621 polymorphic genome-anchored single nucleotide polymorphisms (SNPs) identified collectively for Capsicum annuum and Capsicum baccatum was used to characterize population structure and species domestication of these two important incompatible cultivated pepper species. Estimated mean nucleotide diversity (π) and Tajimas D across various chromosomes revealed biased distribution toward negative values on all chromosomes (except for chromosome 4) in cultivated C. baccatum, indicating a population bottleneck during domestication of C. baccatum. In contrast, C. annuum chromosomes showed positive π and Tajimas D on all chromosomes except chromosome 8, which may be because of domestication at multiple sites contributing to wider genetic diversity. For C. baccatum, 13,129 SNPs were available, with minor allele frequency (MAF) ≥0.05; PCA of the SNPs revealed 283 C. baccatum accessions grouped into 3 distinct clusters, for strong population structure. The fixation index (FST) between domesticated C. annuum and C. baccatum was 0.78, which indicates genome-wide divergence. We conducted extensive linkage disequilibrium (LD) analysis of C. baccatum var. pendulum cultivars on all adjacent SNP pairs within a chromosome to identify regions of high and low LD interspersed with a genome-wide average LD block size of 99.1 kb. We characterized 1742 haplotypes containing 4420 SNPs (range 9–2 SNPs per haplotype). Genome-wide association study (GWAS) of peduncle length, a trait that differentiates wild and domesticated C. baccatum types, revealed 36 significantly associated genome-wide SNPs. Population structure, identity by state (IBS) and LD patterns across the genome will be of potential use for future GWAS of economically important traits in C. baccatum peppers.