Metabolite biodiversity in pepper (Capsicum) fruits of thirty-two diverse accessions: variation in health-related compounds and implications for breeding

A comprehensive study on morphology and biochemical compounds of 32 Capsicum spp. accessions has been performed. Accessions represented four pepper species, Capsicum annuum, Capsicum frutescens, Capsicum chinense and Capsicum baccatum which were selected by their variation in morphological characters such as fruit color, pungency and origin. Major metabolites in fruits of pepper, carotenoids, capsaicinoids (pungency), flavonoid glycosides, and vitamins C and E were analyzed and quantified by high performance liquid chromatography. The results showed that composition and level of metabolites in fruits varied greatly between accessions and was independent of species and geographical location. Fruit color was determined by the accumulation of specific carotenoids leading to salmon, yellow, orange, red and brown colored fruits. Levels of both O- and C-glycosides of quercetin, luteolin and apigenin varied strongly between accessions. All non-pungent accessions were devoid of capsaicins, whereas capsaicinoid levels ranged from 0.07 up to 80 mg/100g fr. wt. in fruit pericarp. In general, pungent accessions accumulated the highest capsaicinoid levels in placenta plus seed tissue compared to pericarp. The non-pungent capsaicinoid analogs, capsiates, could be detected at low levels in some pungent accessions. All accessions accumulated high levels of vitamin C, up to 200 mg/100g fr. wt. The highest vitamin E concentration found was 16 mg/100g fr. wt. Based on these metabolic data, five accessions were selected for further metabolic and molecular analysis, in order to isolate key genes involved in the production of these compounds and to assist future breeding programs aimed at optimizing the levels of health-related compounds in pepper fruit.     

Analysis of microsatellite loci of the chloroplast genome in the genus Capsicum (Pepper)

Six plastome microsatellites were examined in 43 accessions of the genus Capsicum. In total, 33 allelic variants were detected. A specific haplotype of chloroplast DNA was identified for each Capsicum species. Species-specific allelic variants were found for most wild Capsicum species. The highest intraspecific variation was observed for the C. baccatum plastome. Low cpDNA polymorphism was characteristic of C. annuum: the cpSSRs were either monomorphic or dimorphic. The vast majority of C. annuum accessions each had alleles of one type. Another allele type was rare and occurred only in wild accessions. The results testified again to genetic conservation of C. annuum and especially its cultivated forms. The phylogenetic relationships established for the Capsicum species on the basis of plastome analysis were similar to those inferred from the morphological traits, isozyme patterns, and molecular analysis of the nuclear genome.     

Molecular mapping of the C locus for presence of pungency in Capsicum.

Pungency owing to the presence of capsaicinoids is a unique character of pepper (Capsicum spp.). Capsaicinoids are produced in the placenta and it has long been known that a single dominant gene, C, is required for pungent genotypes to produce capsaicinoids. We mapped C to pepper chromosome 2 in a cross between a pungent Capsicum frutescens wild accession and a non-pungent Capsicum annuum bell pepper. This position confirmed results from earlier studies. The RFLP marker TG 205 cosegregated with C and two additional RFLP markers were also located within 1 cM. The recessive allele at the C locus is used in breeding programs around the world focused on very diverse germplasm, hence any of these tightly linked markers may be of value as potential sources of useful markers for marker-assisted selection. To demonstrate this point, we developed a PCR-based CAPS (cleaved amplified polymorphic sequence) marker linked to C using the sequence of the Capsicum fibrillin gene located 0.4 cM from C. The use of molecular markers for high-throughput screening for the c allele in pepper breeding programs is discussed.     

Archaeobotanic evidence of the preincaic Chiribaya culture. Determination of capsaicinoids in archaeological samples of Capsicum frutescens and votive foods

The lack of Capsicum species or paprika (a basic ingredient of Peruvian foodstuff) in preincaic archaeological samples and votive foods, as evidenced by archaeobotanic studies, has stimulated the chemical analyses of these samples by HPLC methods. The results confirmed the absence of capsaicinoids in these samples whereas they were detected in more ancient fossil fruit.     

Tocopherol and fatty acid composition of twenty-five species of Onagraceae Juss.

Chemotaxonomic relationships in Onagraceae have been investigated at the level of leaf and flower compounds but not for seed traits. The objective of this study was to characterize a set of 26 accessions (25 species, 7 genera) of this family for seed oil content, fatty acid composition, tocopherol content and composition, and to evaluate the chemotaxonomic implications of the results. The accessions showed a large variation for all the traits. Gamma-linolenic acid was exclusively found in Oenothera spp. where two groups, showing different concentrations of gamma-linolenic acid and alpha-tocopherol, were observed. The species of Clarkia Pursh were markedly different to the rest of species, showing low alpha-linolenic acid and low gamma-tocopherol concentration. Circaea lutetiana L. had a characteristic tocopherol profile, with high levels (13%) of beta-tocopherol. Variation for fatty acid and tocopherol composition in Epilobium spp. suggested some phylogenetic relationships. The annual species E. pankulatum Nutt. showed a characteristic composition very similar to the closely related Boisduvalia densiflora (Lindl.) S. Watson. The accessions of the phylogenetically related E. fleischeri Hochst. and E. dodonaei Vill., also showed similar fatty acid and tocopherol composition. The results of this study suggest a potential chemotaxonomic value of seed fatty acids and tocopherols in Onagraceae.     

Cuticular wax variation in the tomato (Solanum lycopersicum L.), related wild species and their interspecific hybrids

The tomato (Solanum lycopersicum L.) is one of the world's most important vegetable crop species. Among the many tomato accessions available, only a few are tolerant to abiotic stresses, which are responsible for the majority of the crop losses worldwide. Wild tomato species are then secondary gene pool in the breeding of more resistant tomato cultivars. In the current study, the composition of leaf cuticular waxes from fourteen tomato accessions, including S. lycopersicum, Solanum pennellii, Solanum pimpinellifolium, and their interspecific hybrids was studied in order to select the most adequate chemotaxonomic markers. Total cuticular wax load of S. pennellii plants was much higher than in the other plant species. Hydrocarbons were usually the most abundant wax components, followed by minor quantities of triterpenes and other compounds. Interspecific hybrids showed intermediate wax characteristics. The amount and composition of surface waxes were not correlated with the abiotic stress tolerance in S. lycopersicum. The composition of the hydrocarbon fraction was the least variable both within a single accession and between all the plants studied. Based on the results, cuticular hydrocarbons are proposed as potential chemotaxonomic markers in the classification of tomato and related species.     

辣椒种质遗传多样性的RAPD和ISSR及其表型数据分析

用RAPDI、SSR分子标记及28个表型性状数据对辣椒属5个栽培种的13份材料进行了分析,结果表明:23条RAPD引物共扩增出209条带,平均每个引物扩增出9.09条,多态性位点比率为83.73%;16条ISSR引物共扩增出94条带,平均每个引物扩增出5.88条,多态性位点比率为79.79%.与RAPD相比,ISSR标记检测到的有效等位基因数(Ne)及Shannon多样性指数(I)、遗传离散度(Ht)和遗传分化系数(Gst)等遗传多样性参数都较大,多态性位点比例在亲缘关系较近的一年生辣椒(Capsicum annuum)种内较高,说明ISSR有更高的多态性检测效率,并且适合亲缘关系较近的种群间遗传多样性分析.基于RAPDI、SSR的聚类与基于表型数据的聚类之间存在极显著正相关,且都能将C.annuum与其它栽培种区分开来.     

Biosynthesis of capsinoid is controlled by the Pun1 locus in pepper

Pungency in pepper (Capsicum annuum L.) has unique characteristics due to the alkaloid compound group, capsaicinoids, which includes capsaicin. Although capsaicinoids have been proved to have pharmacological and physiological effects on human health, the application of capsaicinoids has been limited because of their pungency. Capsinoids found in non-pungent peppers share closely related structures with capsaicinoids and show similar biological effects. Previous studies demonstrated that mutations in the p-AMT gene were related to the production of capsinoids; however, the pathway of capsinoid synthesis has not yet been fully elucidated. In this study, we performed genetic analysis to determine the mechanism of capsinoid synthesis using a F6 recombinant inbred line population. In this population, the presence/absence of capsinoids co-segregated with the genotype of the Pun1 locus, without exception. In addition, we screened the patterns of capsinoid synthesis and the correlation between the Pun1 locus and capsinoid synthesis in p-AMT mutant accessions. In Capsicum germplasms, we selected amino-acid-substituted mutants in the PLP binding domain of the p-AMT gene. Capsinoids were not synthesized with the recessive pun1 gene, regardless of the p-AMT genotype, and no relationship was found between p-AMT mutant type and capsinoid content. We concluded that the Pun1 gene, which is responsible for capsaicinoid synthesis, also controls capsinoid synthesis.     

Variation among and within Capsicum species revealed by RAPD markers

 Germplasm characterization is an important link between the conservation and utilization of plant genetic resources. A total of 134 accessions from six Capsicumspecies maintained at the Asian Vegetable Research and Development Center were characterized using 110 randomly amplified polymorphic DNA (RAPD) markers. Ten pairs of potentially duplicated accessions were identified. Multidimensional scaling analysis of the genetic distances among accessions resulted in clustering corresponding to a previous species assignment except for six accessions. Diagnostic RAPDs were identified which discriminate among the Capsicumspecies. The diagnostic markers were employed for improved taxonomic identification of accessions since many morphological traits used in the identification of Capsicumare difficult to score. Three Capsicumaccessions, misclassified based on morphological traits, were reassigned species status based on diagnostic RAPDs. Three accessions, not previously classified, were assigned to a species based on diagnostic RAPDs. Definitive conclusions about the species assignment of three other accessions were not possible. The level of diversity between Capsicum annuumaccessions from the genebank and the breeding program were compared and no differences were observed either for RAPD variation or diversity. The utilization of genetic resources as a source of variance for useful traits in the breeding program may be the reason for the similarity of these two groups. Received: 1 September 1998 / Accepted: 28 December 1998     

Screening Capsicum germplasm for resistance to tomato spotted wilt virus (TSWV)

Resistance conferred by the Tsw locus from Capsicum chinense against Tomato spotted wilt virus (TSWV) has been widely used in breeding programmes. Nevertheless, this resistance depends on inoculation conditions, and isolates able to overcome it have already been detected. In this work 29 accessions of several Capsicum species have been mechanically inoculated with TSWV to identify new sources of resistance. Five accessions showed variable percentages of resistant plants, two of which did not show local lesions on inoculated leaves, suggesting that the response was not mediated through hypersensitivity. Two of these accessions also had a remarkable reduced viral accumulation compared to susceptible control. ECU‐973., a C. chinense accession, showed the best performance against TSWV, with 100% resistant plants. This response was confirmed after mechanical inoculation with three different TSWV isolates. The resistance was maintained when the accession was inoculated with TSWV using a high pressure of viruliferous thrips. These results open new possibilities in the development of a durable resistance to TSWV in pepper.     

Identification of Capsicum species using SNP markers based on high resolution melting analysis

Single nucleotide polymorphisms (SNPs) derived from both nuclear and cytoplasmic DNA sequences were developed to identify distinct species of Capsicum. Species identification was achieved by detecting allelic variations of these type of markers via high resolution melting analysis (HRM). We used the HRM polymorphisms of COSII markers and the Waxy gene from the nuclear sequence, in addition to the intergenic spacer between trnL and trnF from cytoplasmic DNA as our SNP markers. A total of 31 accessions of Capsicum, representing six species, were analyzed using this method. As single markers were insufficient for identifying Capsicum species, combinations of all markers unambiguously identified all six. A phylogeny based on the SNP markers was consistent with the current taxonomy of Capsicum species. These observations demonstrate that the markers developed in this study are useful for rapid identification of new germplasm for management of Capsicum species.     

Induction of capsaicinoid synthesis in <Emphasis Type="Italic">Capsicum chinense</Emphasis> cell cultures by salicylic acid or methyl jasmonate

Suspension cultures of Habanero pepper (Capsicum chinense Jacq.) were exposed to salicylic acid or methyl jasmonate to change secondary metabolism. Both treatments led to the accumulation of capsaicinoids and their late biosynthetic intermediate, vanillin. Both elicitors had a positive effect on the activities of phenylalanine ammonia lyase and coumarate O-methyltransferase, but none of them represented the main limiting step for capsaicinoid accumulation since vanillin contents were two orders of magnitude higher than those of capsaicinoids.     

Evolution of Capsaicinoids in Peter Pepper (Capsicum annuum var. annuum) During Fruit Ripening

The evolution of individual and total contents of capsaicinoids present in Peter peppers (Capsicum annuum var. annuum) at different ripening stages has been studied. Plants were grown in a glasshouse and the new peppers were marked in a temporal space of ten days. The extraction of capsaicinoids was performed by ultrasound‐assisted extraction with MeOH. The capsaicinoids nordihydrocapsaicin (n‐DHC), capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin were analyzed by ultraperformance liquid chromatography (UHPLC)‐fluorescence and identified by UHPLC‐Q‐ToF‐MS. The results indicate that the total capsaicinoids increase in a linear manner from the first point of harvest at ten days (0.283 mg/g FW) up to 90 days, at which point they reach a concentration of 1.301 mg/g FW. The evolution as a percentage of the individual capsaicinoids showed the initial predominance of capsaicin, dihydrocapsaicin, and n‐DHC. Dihydrocapsaicin was the major capsaicinoid up to day 50 of maturation. After 50 days, capsaicin became the major capsaicinoid as the concentration of dihydrocapsaicin fell slightly. The time of harvest of Peter pepper based on the total capsaicinoids content should be performed as late as possible. In any case, harvesting should be performed before overripening of the fruit is observed.     

Molecular characterization of 'Bhut Jolokia' the hottest chilli

The northeast region of India, considered as 'hot spot' of biodiversity, having unique ecological environment with hot and high-humidity conditions, has given rise to the world's hottest chilli, 'Bhut Jolokia', which is at least two times hotter than Red Savina Habanero in terms of Scoville heat units (SHU). This study was undertaken to determine the distinctiveness of 'Bhut Jolokia' from Capsicum frutescens or Capsicum chinense through sequencing of the ribosomal RNA (rRNA) gene-internal transcribed ((ITS) region along with its phylogenetic analysis. Although a compensatory base change (CBC) in the ITS2 region was not observed between the closely related species of C. frutescens and C. chinense when compared with Bhut Jolokia; phylogenetic analysis using ITS1, 5.8S and ITS2 sequences indicated a distinct clade for all the accessions of 'Bhut Joloikia', while C. frutescens and C. chinense occupied discrete lineages. Further, a unique 13-base deletion was observed in all the representative accessions of 'Bhut Jolokia', making it distinct from all other members within the genus and beyond. The degree of genetic variations along with its extreme pungency might be related to ambient environmental factors of northeastern India.     

Identification of Gene-Specific Polymorphisms and Association with Capsaicin Pathway Metabolites in Capsicum annuum L. Collections

Pepper (Capsicum annuum L.) is an economically important crop with added nutritional value. Production of capsaicin is an important quantitative trait with high environmental variance, so the development of markers regulating capsaicinoid accumulation is important for pepper breeding programs. In this study, we performed association mapping at the gene level to identify single nucleotide polymorphisms (SNPs) associated with capsaicin pathway metabolites in a diverse Capsicum annuum collection during two seasons. The genes Pun1, CCR, KAS and HCT were sequenced and matched with the whole-genome sequence draft of pepper to identify SNP locations and for further characterization. The identified SNPs for each gene underwent candidate gene association mapping. Association mapping results revealed Pun1 as a key regulator of major metabolites in the capsaicin pathway mainly affecting capsaicinoids and precursors for acyl moieties of capsaicinoids. Six different SNPs in the promoter sequence of Pun1 were found associated with capsaicin in plants from both seasons. Our results support that CCR is an important control point for the flux of p-coumaric acid to specific biosynthesis pathways. KAS was found to regulate the major precursors for acyl moieties of capsaicinoids and may play a key role in capsaicinoid production. Candidate gene association mapping of Pun1 suggested that the accumulation of capsaicinoids depends on the expression of Pun1, as revealed by the most important associated SNPs found in the promoter region of Pun1.     

Analysis of Microsatellite Loci of the Chloroplast Genome in the Genus Capsicum (Pepper)

Six plastome microsatellites were examined in 43 accessions of the genus Capsicum. In total, 33 allelic variants were detected. A specific haplotype of chloroplast DNA was identified for eachCapsicum species. Species-specific allelic variants were found for most wild Capsicum species. The highest intraspecific variation was observed for the C. baccatum plastome. Low cpDNA polymorphism was characteristic of C. annuum:the cpSSRs were either monomorphic or dimorphic. The vast majority of C. annuum accessions each had alleles of one type. Another allele type was rare and occurred only in wild accessions. The results testified again to genetic conservation of C. anuum and especially its cultivated forms. The phylogenetic relationships established for the Capsicum species on the basis of plastome analysis were similar to those inferred from the morphological traits, isozyme patterns, and molecular analysis of the nuclear genome.     

Genetic variation in three species of Hordeum,and the selection of accessions for the Barley Core Collection

Starch gel electrophoresis of isozymes was used to investigate the level and distribution of genetic variation in accessions of three wild, diploid Hordeum species. Allelic variation was assessed for seven isozyme loci and used in the statistical analysis of 18 accessions of Hordeum brachyantherum subsp. californicum, 29 accessions of H. murinum subsp. glaucum and 11 accessions of H. pusillum. A major purpose with this study was to evaluate the selection of accessions for the international Barley Core Collection, complementing existing passport data. Allelic variation was found in all three species. The variation was mainly between accessions, as most accessions were fixed for certain alleles. Since all three species are self-pollinated, this was anticipated. The fact that the accessions have undergone one or several regeneration cycles from a rather limited number of individuals, after being collected, likely also have reduced the variation. The accessions were clustered by subjecting the genetic distances between them to UPGMA statistical treatment. The clusters in the dendrograms displayed for H. brachyantherum subsp. californicum and H. pusillum distinct patterns related to the geographical origin of the accessions. The accessions of H. murinum subsp. glaucum were less variable than the other two, and did not reveal such a clear pattern. By comparing the already made, geographically based, selection of accessions with the results from this study, it was evident that the selection had worked fairly well for H. brachyantherum subsp. californicum, but less so for the other two species.     

Genetic diversity and similarity revealed via molecular analysis among and within an in situ population and ex situ accessions of chiltepín (Capsicum annuum var. glabriusculum)

Molecular analysis of genetic diversity amongand within phenotypically similar wild Capsicum annuum var. glabriusculum(chile) populations revealed geneticdifferences among accessions spread over abroad geographic range. These chiles areregionally known as chiltepíns and are a 50metric ton per year wild harvest for the spiceindustry, as well as a genetic resource forcrop improvement. Understanding geneticvariability in this species providesinformation related to conservation efforts. The objective of this research was to surveygenetic diversity among and within an insitu population and ex situ accessionsof chiltepíns. Random AmplifiedPolymorphic DNA (RAPD) molecular markers wereused to study the genetic structure of an in situ population found at the nothernmostrange of this species and ex situaccessions collected from Mexico and Guatemala. Novel genetic variation was found in both thein situ northern disjunct population, aswell as some ex situ accessions, thussupporting conservation of this species viaboth in situ and ex situ strategies The evidence presented here supports effortsto conserve outlier populations via insitu management practices.     

Seed isozyme variation in Petrocoptis A. Braun (Caryophyllaceae)

The electrophoretic patterns of seven isozyme systems (ADH, AMY, AAT, GDH, LAP, MDH, and SOD) obtained from dormant seeds from 44 accessions belonging to 12 Petrocoptis taxa were compared in order to clarify taxonomic relationships within the genus. Overall, electrophoretic zymograms showed the presence of up 28 electromorphs, of which 26 were polymorphic among accessions. Mantel tests revealed a moderate level of correlation between the geographic distance matrix and several dissimilarity matrices based on the isozyme data (r=0.3052-0.3376). The electrophoretic profiles of seed isozymes did not match closely the analytical taxonomic framework drawn from morphology. Many electromorphs are widely distributed among Petrocoptis species, and since isozyme polymorphism is present within taxa, few species-specific markers have been found. However, a relationship between the geographic origin of the accessions and several electromorphs has been noticed. Isozyme data gave moderate support to the splitting of the genus into two groups previously defined on the basis of morphology and geographic distribution (western and eastern taxa). However, some samples belonging to P. hispanica and P. pseudoviscosa were somewhat intermediate between both groups as revealed by multivariate ordination techniques. Seed isozymes did not reveal any clear taxonomic grouping among western Petrocoptis species. In fact, no single segregate of this group is supported by the electrophoretic data.