Single-base cytosine methylation analysis in fruits of three Capsicum species

Single-base cytosine methylation analysis across fruits of Capsicum annuum, C. chinense and C. frutescens showed global average methylation ranging from 82.8–89.1%, 77.6–83.9%, and 22.4–25% at CG, CHG and CHH contexts, respectively. High gene-body methylation at CG and CHG was observed across Capsicum species. The C. annuum showed the highest proportion (>80%) of mCs at different genomic regions compared to C. chinense and C. frutescens. Cytosine methylation for transposable-elements were lower in C. frutescens compared to C. annuum and C. chinense. A total of 510,165 CG, 583112 CHG and 277,897 CHH DMRs were identified across three Capsicum species. The differentially methylated regions (DMRs) distribution analysis revealed C. frutescens as more hypo-methylated compared to C. annuum and C. chinense, and also the presence of more intergenic DMRs in Capsicum genome. At CG and CHG context, gene expression and promoter methylation showed inverse correlations. Furthermore, the observed correlation between methylation and expression of genes suggested the potential role of methylation in Capsicum fruit development/ripening.     

Genetic diversity in Capsicum germplasm based on microsatellite and random amplified microsatellite polymorphism markers

A sound knowledge of the genetic diversity among germplasm is vital for strategic germplasm collection, maintenance, conservation and utilisation. Genomic simple sequence repeats (SSRs) and random amplified microsatellite polymorphism (RAMPO) markers were used to analyse diversity and relationships among 48 pepper (Capsicum spp.) genotypes originating from nine countries. These genotypes covered 4 species including 13 germplasm accessions, 30 improved lines of 4 domesticated species and 5 landraces derived from natural interspecific crosses. Out of 106 SSR markers, 25 polymorphic SSR markers (24 %) detected a total of 76 alleles (average, 3.04; range, 2–5). The average polymorphic information content (PIC) was 0.69 (range, 0.29–0.92). Seventeen RAMPO markers produced 87 polymorphic fragments with average PIC of 0.63 (range, 0.44–0.81). Dendrograms based on SSRs and RAMPOs generated two clusters. All 38 Capsicum annuum genotypes and an interspecific landrace clustered together, whereas nine non-annuum (three Capsicum frutescens, one Capsicum chinense, one Capsicum baccatum and four interspecific landraces) genotypes clustered separately. Genetic variation within non-annuum genotypes was greater than the C. annuum genotypes. Distinctness of interspecific derivative landraces grown in northeast India was validated; natural crossing between sympatric Capsicum species has been proposed as the mechanism of their origin.     

Metabolomics and molecular marker analysis to explore pepper (Capsicum sp.) biodiversity

An overview of the metabolic diversity in ripe fruits of a collection of 32 diverse pepper (Capsicum sp.) accessions was obtained by measuring the composition of both semi-polar and volatile metabolites in fruit pericarp, using untargeted LC–MS and headspace GC–MS platforms, respectively. Accessions represented C. annuum, C. chinense, C. frutescens and C. baccatum species, which were selected based on variation in morphological characters, pungency and geographic origin. Genotypic analysis using AFLP markers confirmed the phylogenetic clustering of accessions according to Capsicum species and separated C. baccatum from the C. annuum–C. chinense–C. frutescens complex. Species-specific clustering was also observed when accessions were grouped based on their semi-polar metabolite profiles. In total 88 semi-polar metabolites could be putatively identified. A large proportion of these metabolites represented conjugates of the main pepper flavonoids (quercetin, apigenin and luteolin) decorated with different sugar groups at different positions along the aglycone. In addition, a large group of acyclic diterpenoid glycosides, called capsianosides, was found to be highly abundant in all C. annuum genotypes. In contrast to the variation in semi-polar metabolites, the variation in volatiles corresponded well to the differences in pungency between the accessions. This was particularly true for branched fatty acid esters present in pungent accessions, which may reflect the activity through the acyl branch of the metabolic pathway leading to capsaicinoids. In addition, large genetic variation was observed for many well-established pepper aroma compounds. These profiling data can be used in breeding programs aimed at improving metabolite-based quality traits such as flavour and health-related metabolites in pepper fruits.     

A versatile PCR marker for pungency in Capsicum spp.

Pungency in Capsicum spp. is an important quality trait for pepper breeding. The perception of pungency in pepper is due to the presence of a group of compounds named capsaicinoids, only found within the Capsicum genus. How pungency is controlled at genetic and molecular levels has not been completely elucidated. The use of molecular markers to assess pungency trait is required for molecular breeding, despite the difficulty of development of universal markers for this trait. In this work, a DNA sequence possibly related to pungency with a high similarity to Pun1 locus was studied, and sequence analysis of this homolog revealed a 15?bp deletion in non-pungent pepper accessions. An allele-specific pair of primers was designed and specific fragments of 479?bp from non-pungent and 494?bp from pungent accessions were obtained. Polymorphism of this marker, named MAP1, was tested in a wide range of accessions, belonging to several Capsicum species, including pungent and non-pungent accessions of C. annuum L., and pungent accessions of C. chinense, C. baccatum, C. frutescens, C. pubescens, C. galapagoense, C. eximium, C. tovarii, C. cardenasii, and C. chacoense. All these Capsicum accessions were correctly discriminated. The marker suitability to assess pungency in domesticated and wild Capsicum species was demonstrated, and therefore it will be very useful in marker assisted selection (MAS). Moreover, MAP1 was located in a saturated pepper linkage map and its possible relationship with the Pun1 locus has been discussed. Among the available markers for this complex quality trait, the marker developed in this study is the most universal so far.     

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.     

Successful Wide Hybridization and Introgression Breeding in a Diverse Set of Common Peppers (Capsicum annuum) Using Different Cultivated Ají (C. baccatum) Accessions as Donor Parents

Capsicum baccatum, commonly known as ají, has been reported as a source of variation for many different traits to improve common pepper (C. annuum), one of the most important vegetables in the world. However, strong interspecific hybridization barriers exist between them. A comparative study of two wide hybridization approaches for introgressing C. baccatum genes into C. annuum was performed: i) genetic bridge (GB) using C. chinense and C. frutescens as bridge species; and, ii) direct cross between C. annuum and C. baccatum combined with in vitro embryo rescue (ER). A diverse and representative collection of 18 accessions from four cultivated species of Capsicum was used, including C. annuum (12), C. baccatum (3), C. chinense (2), and C. frutescens (1). More than 5000 crosses were made and over 1000 embryos were rescued in the present study. C. chinense performed as a good bridge species between C. annuum and C. baccatum, with the best results being obtained with the cross combination [C. baccatum (♀) × C. chinense (♂)] (♀) × C. annuum (♂), while C. frutescens gave poor results as bridge species due to strong prezygotic and postzygotic barriers. Virus-like-syndrome or dwarfism was observed in F₁ hybrids when both C. chinense and C. frutescens were used as female parents. Regarding the ER strategy, the best response was found in C. annuum (♀) × C. baccatum (♂) crosses. First backcrosses to C. annuum (BC₁s) were obtained according to the crossing scheme [C. annuum (♀) × C. baccatum (♂)] (♀) × C. annuum (♂) using ER. Advantages and disadvantages of each strategy are discussed in relation to their application to breeding programmes. These results provide breeders with useful practical information for the regular utilization of the C. baccatum gene pool in C. annuum breeding.     

Patterns of genetic variation of the genusCapsicum (Solanaceae) in Mexico

The evolutionary relationships of 186 accessions ofCapsicum from Mexico were studied through enzyme electrophoresis. A total of 76 alleles representing 20 genetic loci coding for nine enzyme systems were observed and the allelic variations of enzymes were studied for geographical distribution. Allele frequencies were used to estimate the apportionment of gene diversity within and between populations and to construct a dendrogram based on a similarity matrix containingNei genetic distances. — The gene diversity estimates suggest that the structure ofCapsicum populations in Mexico consists of predominantly homozygous genotypes presumably due to a self-pollinated breeding system and population bottlenecks. Significant genetic differentiation was found mainly between populations of differing geographical regions.—Based on the results of this study, three species of domesticatedCapsicum can be identified in Mexico,C. annuum var.annuum, C. chinense, andC. pubescens. Semidomesticated and wild forms include two species,C. frutescens andC. annuum var.glabriusculum. A sharp geographical division results between the latter species;C. frutescens was collected exclusively in the southeastern states of Oaxaca, Chiapas, and Tabasco; whereas wild and semidomesticated forms from the rest of the country areC. annuum. Based upon the similarity of enzyme genotypes of semidomesticated and wild forms, the primary center of domestication of cultivatedC. annuum was estimated to be the region comprising the states of Tamaulipas, Nuevo Leon, San Luis Potosi, Veracruz, and Hidalgo in eastern Mexico. A possible second center of domestication is suggested to be localized in the state of Nayarit, western Mexico.     

Conservation of floral,fruit and chromosomal diversity: a review on diploid and polyploid Capsicum annuum complex in India

Capsicum as a spice crop, has wild and cultivated forms admired globally, including Indian subcontinent with vast climatic ranges. Systematic representation of the Indian Capsicum is required to address species relationships and sustainable agriculture, in face of unpredictable climatic conditions. We have updated the catalogue of Indian ‘C. annuum complex’ with 28 landraces and populations from different agro-climatic regions. The agro-climatic influence on the origin of stable chili landraces in India is remarkable, especially in the North East. The floral and fruit morphotype standards and chromosomal attributes have been considered for four distinct ‘C. annuum complex’ members under three species. The highlights of study are: (1) comparative profiling of Indian Capsicum species revealing less infraspecific variation within C. frutescens and C. chinense than C. annuum, at par with cultivation status, (2) karyotype analysis of some unique diploid landraces of C. annuum, (3) karyotypic confirmation of the polyploid Dalle Khursani landraces exclusive to India. To obtain more information, we attempted to correlate diversity of fruit and floral morphotype with chromosomal diversity. Existence of elite and rare germplasm found in the regional pockets offer great scope for enriching the agricultural tradition. The present dataset may serve as a template to be continuously upgraded by taxonomists, genomicists and breeders.

     

Molecular Profiling for Genetic Variability in <Emphasis Type="Italic">Capsicum</Emphasis> Species Based on ISSR and RAPD Markers

The taxonomic identity of Capsicum species is found to be difficult as it displays variations at morpho-chemical characters. Twenty-two accessions of six Capsicum species, namely, C. annuum, C. baccatum, C. chinense, C. eximium, C. frutescens, and C. luteum were investigated for phenotypic diversity based on flower color and for genetic differences by molecular makers. The genetic cluster analyses of 27 RAPD and eight ISSR primers, respectively, revealed genetic similarities in the ranges of 23–88% and 11–96%. Principal component analysis of the pooled RAPD and ISSR data further supports the genetic similarity and groupings. Different species showed variations in relation to corolla shade of flower. C. annuum accessions formed a single cluster in the molecular analysis as maintaining their flower characteristic. C. chinense accession shared flower features with the accessions of C. frutescens and were found to be closer at genotypic level. C. luteum was found to be rather closer to C. baccatum complex, both phenotypically and genetically. The only accession of C. eximium presenting purple flowers falls apart from the groupings. The floral characteristics and the molecular markers are found to be useful toward the delineation of the species specificity in Capsicum collection and identification of genetic stock.     

Polymorphic Microsatellite Markers Transferable Across <Emphasis Type="Italic">Capsicum</Emphasis> Species

Pepper (Capsicum annuum L.) is one of the most important crops in the family Solanaceae. However, the number of polymorphic molecular loci detected in this important crop is far behind that of other cultivated plant species. In the present study, a total of 45 microsatellite primer pairs were developed using Capsicum expressed sequence tags databases. Microsatellite primer pairs were tested using several species of Capsicum and several genera in the family Solanaceae including tomato, potato, eggplant, and tobacco. Results indicated that microsatellite primer pairs amplified genomic targets of C. annuum L., Capsicum baccatum L., Capsicum chacoense L., Capsicum chinense L., Capsicum frutescens L., and Capsicum pubescens Ruiz et Pavon, indicating species transferability within Capsicum. Further analyses revealed that amplicons of these primer pairs segregated 1:2:1 or 3:1 Mendelian fashions in 38 F₂ individuals of pepper. It was also noted that markers derived from sequences containing dinucleotide repeats were generally more polymorphic at the intraspecific level than sequences containing trinucleotide repeats. All the microsatellite primer pairs developed in this study will be useful for marker-assisted selection and mapping studies in pepper.     

Screening Genetic Resources of Capsicum Peppers in Their Primary Center of Diversity in Bolivia and Peru

For most crops, like Capsicum, their diversity remains under-researched for traits of interest for food, nutrition and other purposes. A small investment in screening this diversity for a wide range of traits is likely to reveal many traditional varieties with distinguished values. One objective of this study was to demonstrate, with Capsicum as model crop, the application of indicators of phenotypic and geographic diversity as effective criteria for selecting promising genebank accessions for multiple uses from crop centers of diversity. A second objective was to evaluate the expression of biochemical and agromorphological properties of the selected Capsicum accessions in different conditions. Four steps were involved: 1) Develop the necessary diversity by expanding genebank collections in Bolivia and Peru; 2) Establish representative subsets of ~100 accessions for biochemical screening of Capsicum fruits; 3) Select promising accessions for different uses after screening; and 4) Examine how these promising accessions express biochemical and agromorphological properties when grown in different environmental conditions. The Peruvian Capsicum collection now contains 712 accessions encompassing all five domesticated species (C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens). The collection in Bolivia now contains 487 accessions, representing all five domesticates plus four wild taxa (C. baccatum var. baccatum, C. caballeroi, C. cardenasii, and C. eximium). Following the biochemical screening, 44 Bolivian and 39 Peruvian accessions were selected as promising, representing wide variation in levels of antioxidant capacity, capsaicinoids, fat, flavonoids, polyphenols, quercetins, tocopherols, and color. In Peru, 23 promising accessions performed well in different environments, while each of the promising Bolivian accessions only performed well in a certain environment. Differences in Capsicum diversity and local contexts led to distinct outcomes in each country. In Peru, mild landraces with high values in health-related attributes were of interest to entrepreneurs. In Bolivia, wild Capsicum have high commercial demand.     

A SNP-based genetic linkage map of <Emphasis Type="Italic">Capsicum baccatum</Emphasis> and its comparison to the <Emphasis Type="Italic">Capsicum annuum</Emphasis> reference physical map

Capsicum baccatum L., one of five domesticated species of Capsicum, is a valuable species in chili pepper breeding. In particular, it is a source of disease resistance against anthracnose and powdery mildew. Genetic maps and molecular markers are important to improve the efficiency of crop breeding programs. Recently, using genetic maps several researchers have identified quantitative trait loci (QTLs) for important horticultural traits and have cloned genes of interest. In this study, we constructed a genetic map of C. baccatum in an intraspecific population from a cross between ‘Golden-aji’ and ‘PI594137.’ A total of 395 high-resolution melting markers were developed based on single-nucleotide polymorphisms identified by comparing genome sequences generated through next-generation resequencing of the parents, ‘Golden-aji’ and ‘PI594137.’ The genetic linkage map contained 12 linkage groups, covered a total distance of 1056.2 cM, and had an average distance of 2.67 cM between markers. In addition, the final map was compared to the reference physical map of C. annuum ‘CM334.’ Interestingly, two major reciprocal translocations between chromosomes 3 and 5 and between chromosomes 3 and 9 were found, suggesting that these translocations might act as a genetic barrier between C. annuum and C. baccatum. Translocations between chromosomes 1 and 8 were also observed, as were previously reported in C. chinense, C. frutescens, and wild C. annuum. The synteny of other chromosomes was maintained, on the whole, except for several small inversions. The information on this genetic map will be helpful to analyze QTLs for important traits such as anthracnose resistance in C. baccatum and to study the causes of genetic barriers between C. annuum and C. baccatum.     

Comparative Analysis of SSR Markers Developed in Exon,Intron, and Intergenic Regions and Distributed in Regions Controlling Fruit Quality Traits in <Emphasis Type="Italic">Prunus</Emphasis> Species: Genetic Diversity and Association Studies

Simple sequence repeats (SSRs) are genome domains located in both coding and non-coding regions in eukaryotic genomes. Although SSRs are often characterized by low polymorphism, their DNA-flanking sequences could be a useful source of DNA markers, which could help in genetic studies and breeding because they are associated with genes that control traits of interest. In this study, 56 genotypes from different Prunus species were used, including peach, apricot, plum, and almond (already phenotyped for several agronomical traits, including self-compatibility, flowering and ripening time, fruit type, skin and flesh color, and shell hardness). These Prunus genotypes were molecularly characterized using 28 SSR markers developed in exons, introns, and intergenic regions. All these genes were located in specific regions where quantitative trait loci (QTLs) for certain fruit quality traits were also located, including flowering and ripening times and fruit flesh and skin color. A sum of 309 SSR alleles were identified in the whole panel of analyzed cultivars, with expected heterozygosity values of 0.61 (upstream SSRs), 0.17 (exonic SSRs), 0.65 (intronic SSRs), and 0.58 (downstream SSRs). These values prove the low level of polymorphism of the exonic (gene-coding regions) markers. Cluster and structural analysis based on SSR data clearly differentiated the genotypes according to either specie (for the four species) and pedigree (apricot) or geographic origin (Japanese plum). In addition, some SSR markers mainly developed in intergenic regions could be associated with genes that control traits of interest in breeding and could therefore help in marker-assisted breeding. These findings highlight the importance of using molecular markers able to discriminate between the functional roles of the gene allelic variants.     

Use of <Emphasis Type="Italic">Capsicum frutescens</Emphasis> L. by the Indigenous Peoples of Taiwan and the Batanes Islands

Use of Capsicum frutescens L. by the Indigenous Peoples of Taiwan and the Batanes Islands. The local nomenclature, use, and distribution of C. frutescens among indigenous peoples in Taiwan and the Batanes Islands were studied. Among Taiwanese indigenous peoples, the distribution, frequency of use, and importance of C. frutescens were found to increase with decreasing latitude, which appears to have affected the local names of Capsicum and C. frutescens. The local name for Capsicum in the Batanes Islands—“sili”—is used by several indigenous peoples in Taiwan, suggesting that Capsicum was brought from the south to the north. Indigenous peoples in Taiwan and the Batanes Islands used C. frutescens fruits as condiments, medicines, ornaments, or for ritual uses; also, they used its leaves for soup. A complex of both green and yellowish-green types possessing ShDH-B was introduced from Indonesia into the Batanes Islands and Taiwan, and later only the type with yellowish-green immature fruit was introduced to the Ryukyu Islands under the bottleneck effect.     

Fluorescent chromosome banding in the cultivated species ofCapsicum (Solanaceae)

Fluorochrome chromosome banding is applied for the first time to 15 samples of five cultivatedCapsicum species, all with 2n = 24, and allows a detailed analysis of the karyotypes (Tables 2–3, Fig. 8). Banding patterns differ between cytotypes, species and groups, reflecting the dynamics of chromosomal differentiation and evolutionary divergence. Taxa have from 1 to 4 NOR-bearing satellited chromosome pairs and exhibit increasing numbers of terminal (rarely intercalary and indistinct centromeric) heterochromatic fluorescent bands. Amounts of heterochromatin (expressed in % of karyotype length) increase from the group withC. annuum (1.80–2.88),C. chinense (3.91–5.52), andC. frutescens (5.55) toC. baccatum (7.30–7.56), and finally toC. pubescens (18.95). In all taxa CMA+DAPI—(GC-rich) constitutive heterochromatin dominates, onlyC. pubescens has an additional CMAo DAPI+ (AT-rich) band. The fluorochrome bands generally (but not completely) correspond to the Giemsa C-bands. Structural heterozygosity can be demonstrated but is not prominent. The independent origin of at least three evolutionary lines leading to the cultivated taxa ofCapsicum is supported.Chromosome studies inCapsicum (Solanaceae), V. For the fourth part seeMoscone & al. 1995.     

Development of sequence characterized amplified region (SCAR) primers for the detection of Phyto.5.2, a major QTL for resistance to Phytophthora capsici Leon. in pepper

Phytophthora capsici causes devastating disease on many crop species, including Capsicum. Resistance in Capsicum annuum is genetically and physiologically complex. A panel of Capsicum germplasm that included genotypes from both C. annuum and C. chinense showing highly resistant, highly susceptible and intermediate or tolerant responses to the pathogen, respectively, was screened with a series of randomly amplified polymorphic sequence primers to determine which genomic regions contribute to the highest level of resistance. One primer, OpD04, amplified a single band only in those C. annuum and C. chinense genotypes showing the highest level of resistance. The amplified product was cloned, sequenced and used to design longer primers in order to generate a sequence characterized amplified region marker which was then mapped in a reference mapping population and a screened population segregating for resistance to P. capsici. These primers were observed to define a locus on pepper chromosome 5 tightly linked to Phyto.5.2, one of six quantitative trait loci (QTL) previously reported to contribute to P. capsici resistance. These results indicate that the Phyto.5.2 QTL may be widely distributed in highly resistant germplasm and provide improved resolution for this QTL. This work also defines the first breeding tools for this system, allowing for the rapid selection of genotypes likely to be highly resistant to P. capsici.     

Exploitation of pepper EST–SSRs and an SSR-based linkage map

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F₂ individuals from an interspecific cross of TF68 × Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.Gibum Yi and Je Min Lee equally contributed to this work.