Transcriptomic and lipidomic analysis of the differential pathway contribution to the incorporation of erucic acid to triacylglycerol during Pennycress seed maturation.

Thlaspi arvense (Pennycress) is an emerging feedstock for biofuel production because of its high seed oil content enriched in erucic acid. A transcriptomic and a lipidomic study were performed to analyze the dynamics of gene expression, glycerolipid content and acyl-group distribution during seed maturation. Genes involved in fatty acid biosynthesis were expressed at the early stages of seed maturation. Genes encoding enzymes of the Kennedy pathway like diacylglycerol acyltransferase1 (TaDGAT1), lysophosphatidic acid acyltransferase (TaLPAT) or glycerol 3-phosphate acyltransferase (TaGPAT) increased their expression with maturation, coinciding with the increase in triacylglycerol species containing 22:1. Positional analysis showed that the most abundant triacylglycerol species contained 18:2 at sn-2 position in all maturation stages, suggesting no specificity of the lysophosphatidic acid acyltransferase for very long chain fatty acids. Diacylglycerol acyltransferase2 (TaDGAT2) mRNA was more abundant at the initial maturation stages, coincident with the rapid incorporation of 22:1 to triacylglycerol, suggesting a coordination between Diacylglycerol acyltransferase enzymes for triacylglycerol biosynthesis. Genes encoding the phospholipid-diacylglycerol acyltransferase (TaPDAT1), lysophosphatidylcholine acyltransferase (TaLPCAT) or phosphatidylcholine diacylglycerolcholine phosphotransferase (TaPDCT), involved in acyl-editing or phosphatidyl-choline (PC)-derived diacylglycerol (DAG) biosynthesis showed also higher expression at the early maturation stages, coinciding with a higher proportion of triacylglycerol containing C18 fatty acids. These results suggested a higher contribution of these two pathways at the early stages of seed maturation. Lipidomic analysis of the content and acyl-group distribution of diacylglycerol and phosphatidyl-choline pools was compatible with the acyl content in triacylglycerol at the different maturation stages. Our data point to a model in which a strong temporal coordination between pathways and isoforms in each pathway, both at the expression and acyl-group incorporation, contribute to high erucic triacylglycerol accumulation in Pennycress.

Armeria maritima (Mill.) Willd. Flower Hydromethanolic Extract for Cucurbitaceae Fungal Diseases Control.

The cliff rose (Armeria maritima), like other halophytes, has a phenolics-based antioxidant system that allows it to grow in saline habitats. Provided that antioxidant properties are usually accompanied by antimicrobial activity, in this study we investigated the phytochemicals present in a hydromethanolic extract of A. maritima flowers and explored its antifungal potential. The main phytocompounds, identified by gas chromatography-mass spectrometry, were: hexadecanoic acid, octadecanoic acid, 9-octadecenoic acid, 3-(3,4-dihydroxy-phenyl)-acrylic acid ethyl ester, and benzeneacetaldehyde. The antifungal activity of the extract and its main constituents-alone and in combination with chitosan oligomers-was tested against six pathogenic taxa associated with soil-borne diseases of plant hosts in the family Cucurbitaceae: Fusarium equiseti, F. oxysporum f. sp. niveum, Macrophomina phaseolina, Neocosmospora falciformis, N. keratoplastica, and Sclerotinia sclerotiorum. In in vitro tests, EC90 effective concentrations in the 166-865 µg·mL-1 range were obtained for the chitosan oligomers-A. maritima extract conjugate complexes, lower than those obtained for fosetyl-Al and azoxystrobin synthetic fungicides tested for comparison purposes, and even outperforming mancozeb against F. equiseti. In ex situ tests against S. sclerotiorum conducted on artificially inoculated cucumber slices, full protection was achieved at a dose of 250 µg·mL-1. Thus, the reported results support the valorization of A. maritima as a source of biorationals for Cucurbitaceae pathogens protection, suitable for both organic and conventional agriculture.

Gene Functional Networks from Time Expression Profiles: A Constructive Approach Demonstrated in Chili Pepper (Capsicum annuum L.).

Gene co-expression networks are powerful tools to understand functional interactions between genes. However, large co-expression networks are difficult to interpret and do not guarantee that the relations found will be true for different genotypes. Statistically verified time expression profiles give information about significant changes in expressions through time, and genes with highly correlated time expression profiles, which are annotated in the same biological process, are likely to be functionally connected. A method to obtain robust networks of functionally related genes will be useful to understand the complexity of the transcriptome, leading to biologically relevant insights. We present an algorithm to construct gene functional networks for genes annotated in a given biological process or other aspects of interest. We assume that there are genome-wide time expression profiles for a set of representative genotypes of the species of interest. The method is based on the correlation of time expression profiles, bound by a set of thresholds that assure both, a given false discovery rate, and the discard of correlation outliers. The novelty of the method consists in that a gene expression relation must be repeatedly found in a given set of independent genotypes to be considered valid. This automatically discards relations particular to specific genotypes, assuring a network robustness, which can be set a priori. Additionally, we present an algorithm to find transcription factors candidates for regulating hub genes within a network. The algorithms are demonstrated with data from a large experiment studying gene expression during the development of the fruit in a diverse set of chili pepper genotypes. The algorithm is implemented and demonstrated in a new version of the publicly available R package "Salsa" (version 1.0).

Different Cis-Regulatory Elements Control the Tissue-Specific Contribution of Plastid ω-3 Desaturases to Wounding and Hormone Responses.

Trienoic fatty acids are essential constituents of biomembranes and precursors of jasmonates involved in plant defense responses. Two ω-3 desaturases, AtFAD7 and AtFAD8, synthetize trienoic fatty acids in the plastid. Promoter:GUS and mutagenesis analysis was used to identify cis-elements controlling AtFAD7 and AtFAD8 basal expression and their response to hormones or wounding. AtFAD7 promoter GUS activity was much higher than that of AtFAD8 in leaves, with specific AtFAD7 expression in the flower stamen and pistil and root meristem and vasculature. This specific tissue and organ expression of AtFAD7 was controlled by different cis-elements. Thus, promoter deletion and mutagenesis analysis indicated that WRKY proteins might be essential for basal expression of AtFAD7 in leaves. Two MYB target sequences present in the AtFAD7 promoter might be responsible for its expression in the flower stamen and stigma of the pistil and in the root meristem, and for the AtFAD7 wound-specific response. Two MYB target sequences detected in the distal region of the AtFAD8 gene promoter seemed to negatively control AtFAD8 expression, particularly in true leaves and flowers, suggesting that MYB transcription factors act as repressors of AtFAD8 gene basal expression, modulating the different relative abundance of both plastid ω-3 desaturases at the transcriptional level. Our data showed that the two ABA repression sequences detected in the AtFAD7 promoter were functional, suggesting an ABA-dependent mechanism involved in the different regulation of both ω-3 plastid desaturases. These results reveal the implication of different signaling pathways for the concerted regulation of trienoic fatty acid content in Arabidopsis.

Transcriptome Analyses Throughout Chili Pepper Fruit Development Reveal Novel Insights into the Domestication Process.

Chili pepper (Capsicum spp.) is an important crop, as well as a model for fruit development studies and domestication. Here, we performed a time-course experiment to estimate standardized gene expression profiles with respect to fruit development for six domesticated and four wild chili pepper ancestors. We sampled the transcriptomes every 10 days from flowering to fruit maturity, and found that the mean standardized expression profiles for domesticated and wild accessions significantly differed. The mean standardized expression was higher and peaked earlier for domesticated vs. wild genotypes, particularly for genes involved in the cell cycle that ultimately control fruit size. We postulate that these gene expression changes are driven by selection pressures during domestication and show a robust network of cell cycle genes with a time shift in expression, which explains some of the differences between domesticated and wild phenotypes.

Grafting Snake Melon [Cucumis melo L. subsp. melo Var. flexuosus (L.) Naudin] in Organic Farming: Effects on Agronomic Performance; Resistance to Pathogens; Sugar, Acid, and VOC Profiles; and Consumer Acceptance.

The performance of snake melon [Cucumis melo var. flexuosus (L.)] in organic farming was studied under high biotic and salt stress conditions. Soilborne diseases (mainly caused by Macrophomina phaseolina and Neocosmospora falciformis), combined with virus incidence [Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), and Tomato leaf curl New Delhi virus (ToLCNDV)] and Podosphaera xanthii attacks, reduced yield by more than 50%. Snake melon susceptibility to M. phaseolina and Monosporascus cannonballus was proved in pathogenicity tests, while it showed some degree of resistance to Neocosmospora keratoplastica and N. falciformis. On the contrary, salt stress had a minor impact, although a synergic effect was detected: yield losses caused by biotic stress increased dramatically when combined with salt stress. Under biotic stress, grafting onto the melon F1Pat81 and wild Cucumis rootstocks consistently reduced plant mortality in different agroecological conditions, with a better performance compared to classic Cucurbita commercial hybrids. Yield was even improved under saline conditions in grafted plants. A negative effect was detected, though, on consumer acceptability, especially with the use of Cucurbita rootstocks. Cucumis F1Pat81 rootstock minimized this side effect, which was probably related to changes in the profile of sugars, acids, and volatiles. Grafting affected sugars and organic acid contents, with this effect being more accentuated with the use of Cucurbita rootstocks than with Cucumis. In fact, the latter had a higher impact on the volatile organic compound profile than on sugar and acid profile, which may have resulted in a lower effect on consumer perception. The use of Cucumis rootstocks seems to be a strategy to enable organic farming production of snake melon targeted to high-quality markets in order to promote the cultivation of this neglected crop.

Mapping a Partial Andromonoecy Locus in Citrullus lanatus Using BSA-Seq and GWAS Approaches.

The sexual expression of watermelon plants is the result of the distribution and occurrence of male, female, bisexual and hermaphrodite flowers on the main and secondary stems. Plants can be monoecious (producing male and female flowers), andromonoecious (producing male and hermaphrodite flowers), or partially andromonoecious (producing male, female, bisexual, and hermaphrodite flowers) within the same plant. Sex determination of individual floral buds and the distribution of the different flower types on the plant, are both controlled by ethylene. A single missense mutation in the ethylene biosynthesis gene CitACS4, is able to promote the conversion of female into hermaphrodite flowers, and therefore of monoecy (genotype MM) into partial andromonoecy (genotype Mm) or andromonoecy (genotype mm). We phenotyped and genotyped, for the M/m locus, a panel of 207 C. lanatus accessions, including five inbreds and hybrids, and found several accessions that were repeatedly phenotyped as PA (partially andromonoecious) in several locations and different years, despite being MM. A cosegregation analysis between a SNV in CitACS4 and the PA phenotype, demonstrated that the occurrence of bisexual and hermaphrodite flowers in a PA line is not dependent on CitACS4, but conferred by an unlinked recessive gene which we called pa. Two different approaches were performed to map the pa gene in the genome of C. lanatus: bulk segregant analysis sequencing (BSA-seq) and genome wide association analysis studies (GWAS). The BSA-seq study was performed using two contrasting bulks, the monoecious M-bulk and the partially andromonoecious PA-bulk, each one generated by pooling DNA from 20 F2 plants. For GWAS, 122 accessions from USDA gene bank, already re-sequenced by genotyping by sequencing (GBS), were used. The combination of the two approaches indicates that pa maps onto a genomic region expanding across 32.24-36.44 Mb in chromosome 1 of watermelon. Fine mapping narrowed down the pa locus to a 867 Kb genomic region containing 101 genes. A number of candidate genes were selected, not only for their function in ethylene biosynthesis and signalling as well as their role in flower development and sex determination, but also by the impact of the SNPs and indels differentially detected in the two sequenced bulks.

Content of Capsaicinoids and Capsiate in "Filius" Pepper Varieties as Affected by Ripening.

Peppers are fruits with wide genetic variability and multiple ways of being consumed that hold a relevant position in the human diet. Nowadays, consumers are interested in new gastronomic experiences provided by pepper cultivars that present new shapes, colors, and flavors while preserving their bioactive compounds, such as their capsaicinoids and capsinoids. However, numerous changes take place during their development that may alter their biological properties. Therefore, this work evaluates the capsaicinoid and capsiate contents in two traditional varieties of ornamental peppers ("Filius Blue" and "Filius Green'") during fruit maturation. The aim is to determine the ideal harvesting moment depending on the farmer's objective (e.g., achieving a specific color, shape, or flavor; achieving the maximum concentrations of bioactive compounds). The capsaicinoid contents followed different patterns in the two varieties analyzed. The "Filius Blue" variety exhibited increasing concentrations of capsaicinoids up to the 41st day post-anthesis (dpa), from which point on this trend was reversed. The concentrations in the "Filius Green" variety increased and decreased several times, reaching maximum concentrations on the 69th dpa. Regarding capsiate contents, both varieties varied in the same way, reaching maximum concentrations on the 34th dpa and then decreasing.

Melon Genome Regions Associated with TGR-1551-Derived Resistance to Cucurbit yellow stunting disorder virus.

Cucurbit yellow stunting disorder virus (CYSDV) is one of the main limiting factors of melon cultivation worldwide. To date, no commercial melon cultivars resistant to CYSDV are available. The African accession TGR-1551 is resistant to CYSDV. Two major quantitative trait loci (QTLs) have been previously reported, both located near each other in chromosome 5. With the objective of further mapping the gene or genes responsible of the resistance, a recombinant inbred line (RIL) population derived from the cross between TGR-1551 and the susceptible cultivar 'Bola de Oro' was evaluated for resistance to CYSDV in five different assays and genotyped in a genotyping by sequencing (GBS) analysis. The major effect of one of the two QTLs located on chromosome 5 was confirmed in the multienvironment RIL assay and additionally verified through the analysis of three segregating BC1S1 populations derived from three resistant RILs. Furthermore, progeny test using the offspring of selected BC3 plants allowed the narrowing of the candidate interval to a 700 kb region. The SNP markers identified in this work will be useful in marker-assisted selection in the context of introgression of CYSDV resistance in elite cultivars.

Functional analysis of ß-ketoacyl-CoA synthase from biofuel feedstock Thlaspi arvense reveals differences in the triacylglycerol biosynthetic pathway among Brassicaceae.

KEY MESSAGE: Differences in FAE1 enzyme affinity for the acyl-CoA substrates, as well as the balance between the different pathways involved in their incorporation to triacylglycerol might be determinant of the different composition of the seed oil in Brassicaceae. Brassicaceae present a great heterogeneity of seed oil and fatty acid composition, accumulating Very Long Chain Fatty Acids with industrial applications. However, the molecular determinants of these differences remain elusive. We have studied the ß-ketoacyl-CoA synthase from the high erucic feedstock Thlaspi arvense (Pennycress). Functional characterization of the Pennycress FAE1 enzyme was performed in two Arabidopsis backgrounds; Col-0, with less than 2.5% of erucic acid in its seed oil and the fae1-1 mutant, deficient in FAE1 activity, that did not accumulate erucic acid. Seed-specific expression of the Pennycress FAE1 gene in Col-0 resulted in a 3 to fourfold increase of erucic acid content in the seed oil. This increase was concomitant with a decrease of eicosenoic acid levels without changes in oleic ones. Interestingly, only small changes in eicosenoic and erucic acid levels occurred when the Pennycress FAE1 gene was expressed in the fae1-1 mutant, with high levels of oleic acid available for elongation, suggesting that the Pennycress FAE1 enzyme showed higher affinity for eicosenoic acid substrates, than for oleic ones in Arabidopsis. Erucic acid was incorporated to triacylglycerol in the transgenic lines without significant changes in their levels in the diacylglycerol fraction, suggesting that erucic acid was preferentially incorporated to triacylglycerol via DGAT1. Expression analysis of FAE1, AtDGAT1, AtLPCAT1 and AtPDAT1 genes in the transgenic lines further supported this conclusion. Differences in FAE1 affinity for the oleic and eicosenoic substrates among Brassicaceae, as well as their incorporation to triacylglycerol might explain the differences in composition of their seed oil.

Assessment of Capsaicinoid and Capsinoid Accumulation Patterns during Fruit Development in Three Chili Pepper Genotypes (Capsicum spp.) Carrying Pun1 and pAMT Alleles Related to Pungency.

Quantification, using an accurate analytical approach, of capsinoids and capsaicinoids was performed on three chili pepper (Capsicum spp.) genotypes: "Chiltepín", "Tampiqueño 74", and "Bhut Jolokia" at various stages of fruit development. The accumulation of capsinoids, in all these peppers started between 10 to 20 days post-anthesis (dpa), increased and reached the highest capsinoid amount at 40 dpa, and then decreased until 60 dpa. Conversely, capsaicinoids could already be determined at 10 dpa in "Bhut Jolokia" and their accumulation pattern was different from that of the capsinoids in this genotype. The capsiate/dihydrocapsiate ratio presented a higher variation between genotypes and developmental stages than the capsaicin/dihydrocapsaicin ratio. Capsinoid ratios (4-24%) and Pun1/pAMT genotyping were determined. These results provide information on the progress of the accumulation of capsinoids in the aforementioned pungent and superhot cultivars and could support future breeding studies toward the understanding of the factors affecting their accumulation.

Quantitation of capsiate and dihydrocapsiate and tentative identification of minor capsinoids in pepper fruits (Capsicum spp.) by HPLC-ESI-MS/MS(QTOF).

A highly sensitive, selective method has been developed and validated for determination of capsiate and dihydrocapsiate for the first time using HPLC-ESI/MS(QTOF). For both capsinoids, LODs and LOQs were 0.02 and 0.05 µmol/l, respectively. The intra- and interday repeatability values (RSD %) were 0.26-0.41% for retention time, and 2.25-2.11% for peak area. Recoveries were up to 98% and 97% for capsiate and dihydrocapsiate, respectively. This method was successfully applied to quantify capsiate and dihydrocapsiate in eight pepper fruit accessions. Capsinoids were found in the range of 1.21-544.59 µg/g DW for capsiate and of 0.61-81.95 µg/g DW for dihydrocapsiate. In the 'Tabasco' accession, capsiate and dihydrocapsiate were quantified for the first time, ranging from 3.09 to 58.76 and 1.80 to 6.94 µg/g DW, respectively. Additionally, the ESI-MS/MS(QTOF) analysis has allowed the tentative identification of two other minor capsinoids by exact mass and fragmentation pattern, in the 'Bhut Jolokia' accession.

Quantitative trait loci analysis of melon (Cucumis melo L.) domestication-related traits.

KEY MESSAGE: Loci on LGIV, VI, and VIII of melon genome are involved in the control of fruit domestication-related traits and they are candidate to have played a role in the domestication of the crop. The fruit of wild melons is very small (20-50 g) without edible pulp, contrasting with the large size and high pulp content of cultivated melon fruits. An analysis of quantitative trait loci (QTL) controlling fruit morphology domestication-related traits was carried out using an in vitro maintained F2 population from the cross between the Indian wild melon "Trigonus" and the western elite cultivar 'Piel de Sapo'. Twenty-seven QTL were identified in at least two out of the three field trials. Six of them were also being detected in BC1 and BC3 populations derived from the same cross. Ten of them were related to fruit morphological traits, 12 to fruit size characters, and 5 to pulp content. The Trigonus alleles decreased the value of the characters, except for the QTL at andromonoecious gene at linkage group (LG) II, and the QTL for pulp content at LGV. QTL genotypes accounted for a considerable degree of the total phenotypic variation, reaching up to 46%. Around 66% of the QTL showed additive gene action, 19% exhibited dominance, and 25% consisted of overdominance. The regions on LGIV, VI, and VIII included the QTL with more consistent and strong effects on domestication-related traits. QTLs on those regions were validated in BC2S1, BC2S2, and BC3 families, with "Trigonus" allele decreasing the fruit morphological traits in all cases. The validated QTL could represent loci involved in melon domestication, although further experiments as genomic variation studies across wild and cultivated genotypes would be necessary to confirm this hypothesis.

Ontogenetic Variation of Individual and Total Capsaicinoids in Malagueta Peppers (Capsicum frutescens) during Fruit Maturation.

The ontogenetic variation of total and individual capsaicinoids (nordihydrocapsaicin (n-DHC), capsaicin (C), dihydrocapsaicin (DHC), homocapsaicin (h-C) and homodihydrocapsaicin (h-DHC)) present in Malagueta pepper (Capsicum frutescens) during fruit ripening has been studied. Malagueta peppers were grown in a greenhouse under controlled temperature and humidity conditions. Capsaicinoids were extracted using ultrasound-assisted extraction (UAE) and the extracts were analyzed by ultra-performance liquid chromatography (UHPLC) with fluorescence detection. A significant increase in the total content of capsaicinoids was observed in the early days (between 12 and 33). Between day 33 and 40 there was a slight reduction in the total capsaicinoid content (3.3% decrease). C was the major capsaicinoid, followed by DHC, n-DHC, h-C and h-DHC. By considering the evolution of standardized values of the capsaicinoids it was verified that n-DHC, DHC and h-DHC (dihydrocapsaicin-like capsaicinoids) present a similar behavior pattern, while h-C and C (capsaicin-like capsaicinoids) show different evolution patterns.

Identification of target genes and processes involved in erucic acid accumulation during seed development in the biodiesel feedstock Pennycress (Thlaspi arvense L.).

We studied erucic acid accumulation in the biodiesel feedstock Pennycress (Thlaspi arvense L.) as a first step towards the development of a sustainable strategy for biofuel production in the EU territory. To that end, two inbred Pennycress lines of European origin, "NASC" and "French," were cultivated in a controlled chamber and in experimental field plots, and their growth, seed production and seed oil characteristics analyzed. Differences in some agronomical traits like vernalization (winter-French versus spring-NASC), flowering time (delayed in the French line) and seed production (higher in the French line) were detected. Both lines showed a high amount (35-39%) of erucic acid (22:1Δ13) in their seed oil. Biochemical characterization of the Pennycress seed oil indicated that TAG was the major reservoir of 22:1Δ13. Incorporation of 22:1Δ13 to TAG occurred very early during seed maturation, concomitant with a decrease of desaturase activity. This change in the acyl fluxes towards elongation was controlled by different genes at different levels. TaFAE1 gene, encoding the fatty acid elongase, seemed to be controlled at the transcriptional level with high expression at the early stages of seed development. On the contrary, the TaFAD2 gene that encodes the Δ12 fatty acid desaturase or TaDGAT1 that catalyzes TAG biosynthesis were controlled post-transcriptionally. TaWRI1, the master regulator of seed-oil biosynthesis, showed also high expression at the early stages of seed development. Our data identified genes and processes that might improve the biotechnological manipulation of Pennycress seeds for high-quality biodiesel production.

Doubled haploid production from Spanish onion (Allium cepa L.) germplasm: embryogenesis induction, plant regeneration and chromosome doubling.

The use of doubled haploids in onion breeding is limited due to the low gynogenesis efficiency of this species. Gynogenesis capacity from Spanish germplasm, including the sweet cultivar Fuentes de Ebro, the highly pungent landrace BGHZ1354 and the two Valenciana type commercial varieties Recas and Rita, was evaluated and optimized in this study. The OH-1 population, characterized by a high gynogenesis induction, was used as control. Growing conditions of the donor plants were tested with a one-step protocol and field plants produced a slightly higher percentage of embryogenesis induction than growth chamber plants. A one-step protocol was compared with a two-step protocol for embryogenesis induction. Spanish germplasm produced a 2-3 times higher percentage of embryogenesis with the two-step protocol, Recas showing the highest percentage (2.09%) and Fuentes de Ebro the lowest (0.53%). These percentages were significantly lower than those from the OH-1 population, with an average of 15% independently of the protocol used. The effect of different containers on plant regeneration was tested using both protocols. The highest percentage of acclimated plants was obtained with the two-step protocol in combination with Eco2box (70%), whereas the lowest percentage was observed with glass tubes in the two protocols (20-23%). Different amiprofos-methyl (APM) treatments were applied to embryos for chromosome doubling. A similar number of doubled haploid plants were recovered with 25 or 50 µM APM in liquid medium. However, the application of 25 µM in solid medium for 24 h produced the highest number of doubled haploid plants. Somatic regeneration from flower buds of haploid and mixoploid plants proved to be a successful approach for chromosome doubling, since diploid plants were obtained from the four regenerated lines. In this study, doubled haploid plants were produced from the four Spanish cultivars, however further improvements are needed to increase their gynogenesis efficiency.

New insights into Capsicum spp relatedness and the diversification process of Capsicum annuum in Spain.

The successful exploitation of germplasm banks, harbouring plant genetic resources indispensable for plant breeding, will depend on our ability to characterize their genetic diversity. The Vegetable Germplasm Bank of Zaragoza (BGHZ) (Spain) holds an important Capsicum annuum collection, where most of the Spanish pepper variability is represented, as well as several accessions of other domesticated and non-domesticated Capsicum spp from all over the five continents. In the present work, a total of 51 C. annuum landraces (mainly from Spain) and 51 accessions from nine Capsicum species maintained at the BGHZ were evaluated using 39 microsatellite (SSR) markers spanning the whole genome. The 39 polymorphic markers allowed the detection of 381 alleles, with an average of 9.8 alleles per locus. A sizeable proportion of alleles (41.2%) were recorded as specific alleles and the majority of these were present at very low frequencies (rare alleles). Multivariate and model-based analyses partitioned the collection in seven clusters comprising the ten different Capsicum spp analysed: C. annuum, C. chinense, C. frutescens, C. pubescens, C. bacatum, C. chacoense and C. eximium. The data clearly showed the close relationships between C. chinense and C. frutescens. C. cardenasii and C. eximium were indistinguishable as a single, morphologically variable species. Moreover, C. chacoense was placed between C. baccatum and C. pubescens complexes. The C. annuum group was structured into three main clusters, mostly according to the pepper fruit shape, size and potential pungency. Results suggest that the diversification of C. annuum in Spain may occur from a rather limited gene pool, still represented by few landraces with ancestral traits. This ancient population would suffer from local selection at the distinct geographical regions of Spain, giving way to pungent and elongated fruited peppers in the South and Center, while sweet blocky and triangular types in Northern Spain.

Identification, validation and survey of a single nucleotide polymorphism (SNP) associated with pungency in Capsicum spp.

A single nucleotide polymorphism (SNP) associated with pungency was detected within an expressed sequence tag (EST) of 307 bp. This fragment was identified after expression analysis of the EST clone SB2-66 in placenta tissue of Capsicum fruits. Sequence alignments corresponding to this new fragment allowed us to identify an SNP between pungent and non-pungent accessions. Two methods were chosen for the development of the SNP marker linked to pungency: tetra-primer amplification refractory mutation system-PCR (tetra-primer ARMS-PCR) and cleaved amplified polymorphic sequence. Results showed that both methods were successful in distinguishing genotypes. Nevertheless, tetra-primer ARMS-PCR was chosen for SNP genotyping because it was more rapid, reliable and less cost-effective. The utility of this SNP marker for pungency was demonstrated by the ability to distinguish between 29 pungent and non-pungent cultivars of Capsicum annuum. In addition, the SNP was also associated with phenotypic pungent character in the tested genotypes of C. chinense, C. baccatum, C. frutescens, C. galapagoense, C. eximium, C. tovarii and C. cardenasi. This SNP marker is a faster, cheaper and more reproducible method for identifying pungent peppers than other techniques such as panel tasting, and allows rapid screening of the trait in early growth stages.

Inheritance of capsaicin and dihydrocapsaicin, determined by HPLC-ESI/MS, in an intraspecific cross of Capsicum annuum L.

The quantitative inheritance of capsaicin and dihydrocapsaicin contents in fruits has been studied in an intraspecific cross of Capsicum annuum L. across two different environments, namely, fruits developed in spring and summer. A liquid chromatography-electrospray ionization/time-of-flight mass spectrometry [HPLC-ESI/MS(TOF)] method was used to identify and quantify capsaicin and dihydrocapsaicin in extracts of pepper fruits. The analytical method used was able to determine the pungency of genotypes that, using other methods, would have been classified as non-pungent. Capsaicin and dihydrocapsaicin contents varied largely among families, and families did not respond similarly in producing these capsaicinoids when their fruits were grown in spring and summer, with some families showing no increase, whereas in others, the increase was more than 2-fold. Heterosis for the pungency trait, assessed by the capsaicin and dihydrocapsaicin contents in fruits, was found, indicating the existence of epistasis, over-dominance, or dominance complementation. Non-pungent parent alleles contributed to the capsaicin and dihydrocapsaicin contents since transgressive segregation did occur. Furthermore, the type of gene action varied between capsaicin and dihydrocapsaicin, and a seasonal effect during fruit development could affect gene action.

Determination of capsaicin and dihydrocapsaicin in capsicum fruits by liquid chromatography-electrospray/time-of-flight mass spectrometry.

A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/time-of-flight mass spectrometry method has been developed for the direct and simultaneous determination of capsaicin and dihydrocapsaicin in Capsicum fruit extracts. Capsaicin and dihydrocapsaicin are the two major members of the so-called capsaicinoid family, which includes other minor analogues, and usually account for at least 90% of the pungency trait in Capsicum fruits. Chromatographic separation of capsaicin and dihydrocapsaicin was achieved with a reversed-phase chromatography column, using a gradient of methanol and water. Quantification was done using as an internal standard (4,5-dimethoxybenzyl)-4-methyloctamide, a synthetic capsaicin analogue not found in nature. Analytes were base-peak resolved in less than 16 min, and limits of detection were 20 pmol for capsaicin and 4 pmol for dihydrocapsaicin. The intraday repeatability values were lower than 0.5 and 12% for retention time and peak area, respectively, whereas the interday repeatability values were lower than 0.6 and 14% for retention time and peak area, respectively. Analyte recoveries found were 86 and 93% for capsaicin and dihydrocapsaicin, respectively. The method developed has been applied to the identification and quantification of capsaicin and dihydrocapsaicin in fruit extracts from different Capsicum genotypes, and concentrations found ranged from 2 to 6639 mg kg(-1).