Multi-environment association study highlights candidate genes for robust agronomic quantitative trait loci in a novel worldwide Capsicum core collection.

Investigating crop diversity through genome-wide association studies (GWAS) on core collections helps in deciphering the genetic determinants of complex quantitative traits. Using the G2P-SOL project world collection of 10 038 wild and cultivated Capsicum accessions from 10 major genebanks, we assembled a core collection of 423 accessions representing the known genetic diversity. Since complex traits are often highly dependent upon environmental variables and genotype-by-environment (G × E) interactions, multi-environment GWAS with a 10 195-marker genotypic matrix were conducted on a highly diverse subset of 350 Capsicum annuum accessions, extensively phenotyped in up to six independent trials from five climatically differing countries. Environment-specific and multi-environment quantitative trait loci (QTLs) were detected for 23 diverse agronomic traits. We identified 97 candidate genes potentially implicated in 53 of the most robust and high-confidence QTLs for fruit flavor, color, size, and shape traits, and for plant productivity, vigor, and earliness traits. Investigating the genetic architecture of agronomic traits in this way will assist the development of genetic markers and pave the way for marker-assisted selection. The G2P-SOL pepper core collection will be available upon request as a unique and universal resource for further exploitation in future gene discovery and marker-assisted breeding efforts by the pepper community.

CaMYB12-like underlies a major QTL for flavonoid content in pepper (Capsicum annuum) fruit.

The regulation of flavonoid biosynthesis is only partially explored in pepper (Capsicum annuum L.). The genetic basis underlying flavonoid variation in pepper fruit was studied. Variation of flavonoids in fruit of a segregating F2 population was studied using LC-MS followed by quantitative trait locus (QTL) analysis. Near-isogenic lines (NILs), BC1 S1 populations, virus-induced gene silenced (VIGS) and transgenic overexpression were used to confirm the QTL and the underlying candidate gene. A major QTL for flavonoid content was found in chromosome 5, and a CaMYB12-like transcription factor gene was identified as candidate gene. Near-isogenic lines (NILs) contrasting for CaMYB12-like confirmed its association with the flavonoid content variation. Virus-induced gene silencing (VIGS) of CaMYB12-like led to a significant decrease in the expression of several flavonoid pathway genes and a drastic decrease in flavonoid levels in silenced fruits. Expression of CaMYB12-like in the tomato slmyb12 mutant led to enhanced levels of several flavonoids in the fruit skin. Introgression of the CaMYB12-like allele into two cultivated varieties also increased flavonoid content in their fruits. A combination of metabolomic, genetic and gene functional analyses led to discovery of CaMYB12-like as a major regulator of flavonoid variation in pepper fruit and demonstrated its potential to breed for high-flavonoid content in cultivated pepper.

High-quality chromosome-scale genomes facilitate effective identification of large structural variations in hot and sweet peppers.

Pepper (Capsicum annuum) is an important vegetable crop that has been subjected to intensive breeding, resulting in limited genetic diversity, especially for sweet peppers. Previous studies have reported pepper draft genome assemblies using short read sequencing, but their capture of the extent of large structural variants (SVs), such as presence-absence variants (PAVs), inversions, and copy-number variants (CNVs) in the complex pepper genome falls short. In this study, we sequenced the genomes of representative sweet and hot pepper accessions by long-read and/or linked-read methods and advanced scaffolding technologies. First, we developed a high-quality reference genome for the sweet pepper cultivar 'Dempsey' and then used the reference genome to identify SVs in 11 other pepper accessions and constructed a graph-based pan-genome for pepper. We annotated an average of 42 972 gene families in each pepper accession, defining a set of 19 662 core and 23 115 non-core gene families. The new pepper pan-genome includes informative variants, 222 159 PAVs, 12 322 CNVs, and 16 032 inversions. Pan-genome analysis revealed PAVs associated with important agricultural traits, including potyvirus resistance, fruit color, pungency, and pepper fruit orientation. Comparatively, a large number of genes are affected by PAVs, which is positively correlated with the high frequency of transposable elements (TEs), indicating TEs play a key role in shaping the genomic landscape of peppers. The datasets presented herein provide a powerful new genomic resource for genetic analysis and genome-assisted breeding for pepper improvement.

Genetic and biotechnological tools to identify breeding targets for improving postharvest quality and extending shelf life of peppers.

Improved postharvest storage is a major target for pepper-crop production. The three main components of postharvest improvement of pepper fruit are reducing water-loss rate, reducing chilling susceptibility, and increasing resistance to pathogens. To date, a small number of Quantitative Trait Locus (QTL) studies have been reported for reduced water loss and enhanced tolerance to chilling and anthracnose. More effort is needed to screen germplasm collections for accessions with improved postharvest traits. Molecular studies have enabled the identification of candidate genes conferring reduced susceptibility to chilling injury and pathogen infection in pepper fruit, and in related crops such as tomato - which may be implemented in pepper. Manipulation of the activity of these genes by genome editing can improve postharvest pepper quality.

Pepper variome reveals the history and key loci associated with fruit domestication and diversification.

Pepper (Capsicum spp.) is an important vegetable crop that provides a unique pungent sensation when eaten. Through construction of a pepper variome map, we examined the main groups that emerged during domestication and breeding of C. annuum, their relationships and temporal succession, and the molecular events underlying the main transitions. The results showed that the initial differentiation in fruit shape and pungency, increase in fruit weight, and transition from erect to pendent fruits, as well as the recent appearance of large, blocky, sweet fruits (bell peppers), were accompanied by strong selection/fixation of key alleles and introgressions in two large genomic regions. Furthermore, we identified Up, which encodes a BIG GRAIN protein involved in auxin transport, as a key domestication gene that controls erect vs pendent fruit orientation. The up mutation gained increased expression especially in the fruit pedicel through a 579-bp sequence deletion in its 5' upstream region, resulting in the phenotype of pendent fruit. The function of Up was confirmed by virus-induced gene silencing. Taken together, these findings constitute a cornerstone for understanding the domestication and differentiation of a key horticultural crop.

Global range expansion history of pepper (Capsicum spp.) revealed by over 10,000 genebank accessions.

Genebanks collect and preserve vast collections of plants and detailed passport information, with the aim of preserving genetic diversity for conservation and breeding. Genetic characterization of such collections has the potential to elucidate the genetic histories of important crops, use marker-trait associations to identify loci controlling traits of interest, search for loci undergoing selection, and contribute to genebank management by identifying taxonomic misassignments and duplicates. We conducted a genomic scan with genotyping by sequencing (GBS) derived single nucleotide polymorphisms (SNPs) of 10,038 pepper (Capsicum spp.) accessions from worldwide genebanks and investigated the recent history of this iconic staple. Genomic data detected up to 1,618 duplicate accessions within and between genebanks and showed that taxonomic ambiguity and misclassification often involve interspecific hybrids that are difficult to classify morphologically. We deeply interrogated the genetic diversity of the commonly consumed Capsicum annuum to investigate its history, finding that the kinds of peppers collected in broad regions across the globe overlap considerably. The method ReMIXTURE-using genetic data to quantify the similarity between the complement of peppers from a focal region and those from other regions-was developed to supplement traditional population genetic analyses. The results reflect a vision of pepper as a highly desirable and tradable cultural commodity, spreading rapidly throughout the globe along major maritime and terrestrial trade routes. Marker associations and possible selective sweeps affecting traits such as pungency were observed, and these traits were shown to be distributed nonuniformly across the globe, suggesting that human preferences exerted a primary influence over domesticated pepper genetic structure.

Pepper Fruit Elongation Is Controlled by Capsicum annuum Ovate Family Protein 20.

Fruit shape is one of the most important quality traits of pepper (Capsicum spp.) and is used as a major attribute for the classification of fruit types. Wide natural variation in fruit shape exists among the major cultivated species Capsicum annuum, allowing the identification of several QTLs controlling the trait. However, to date, no genes underlying fruit shape QTLs have been conclusively identified, nor has their function been verified in pepper. We constructed a mapping population from a cross of round- and elongated-fruited C. annuum parents and identified a single major QTL on chromosome 10, termed fs10, explaining 68 and 70% of the phenotypic variation for fruit shape index and for distal fruit end angle, respectively. The QTL was mapped in several generations and was localized to a 5 Mbp region containing the ortholog of SlOFP20 that suppresses fruit elongation in tomato. Virus-induced gene silencing of the pepper ortholog CaOFP20 resulted in increased fruit elongation on two independent backgrounds. Furthermore, CaOFP20 exhibited differential expression in fs10 near-isogenic lines, as well as in an association panel of elongated- and round-fruited accessions. A 42-bp deletion in the upstream region of CaOFP20 was most strongly associated with fruit shape variation within the locus. Histological observations in ovaries and fruit pericarps indicated that fs10 exerts its effect on fruit elongation by controlling cell expansion and replication. Our results indicate that CaOFP20 functions as a suppressor of fruit elongation in C. annuum and is the most likely candidate gene underlying fs10.

CaFT-LIKE is a flowering promoter in pepper and functions as florigen in tomato.

We identified a pepper late-flowering mutant that is disrupted in the sequence of CaFT-LIKE, the ortholog of tomato SINGLE FLOWER TRUSS (SFT). Heterologous expression in tomato indicated that CaFT-LIKE has a conserved function as a flowering promoter and can rescue the wild-type phenotype of the tomato sft mutant. CaFT-LIKE confers a graft-transmissible signal for flowering initiation in tomato, implicating its function as a florigen. To test the relationship between CaFT-LIKE and FASCICULATE (FA), the ortholog of tomato SELF PRUNING (SP), we constructed the double mutant Caft-like fa. The phenotype of Caft-like fa resembled that of Caft-like, indicating epistasis of Caft-like over fa in controlling flowering time and sympodial shoot structure. To examine the association between the expression pattern of flowering genes and natural variation in flowering time, the expression levels of CaFT-LIKE and the flowering repressor CaAP2 were determined in a panel of early-flowering cultivars and late-flowering landraces and wild accessions. Strong positive and negative correlations between flowering time and expression levels of CaAP2 and CaFT-LIKE, respectively, were observed, indicating that high-expression alleles of CaFT-LIKE and low-expression alleles of CaAP2 were selected for early flowering during pepper domestication and breeding.

The zinc-finger transcription factor CcLOL1 controls chloroplast development and immature pepper fruit color in Capsicum chinense and its function is conserved in tomato.

Chloroplast development and chlorophyll content in the immature fruit has a major impact on the morphology and quality in pepper (Capsicum spp.) fruit. Two major quantitative trait loci (QTLs), pc1 and pc10 that affect chlorophyll content in the pepper fruit by modulation of chloroplast compartment size were previously identified in chromosomes 1 and 10, respectively. The pepper homolog of GOLDEN2-LIKE transcription factor (CaGLK2) has been found as underlying pc10, similar to its effect on tomato chloroplast development. In the present study, we identified the pepper homolog of the zinc-finger transcription factor LOL1 (LSD ONE LIKE1; CcLOL1) as the gene underlying pc1. LOL1 has been identified in Arabidopsis as a positive regulator of programmed cell death and we report here on its role in controlling fruit development in the Solanaceae in a fruit-specific manner. The light-green C. chinense parent used for QTL mapping was found to carry a null mutation in CcLOL1. Verification of the function of the gene was done by generating CRISPR/Cas9 knockout mutants of the orthologous tomato gene resulting in light-green tomato fruits, indicating functional conservation of the orthologous genes in controlling chlorophyll content in the Solanaceae. Transcriptome profiling of light and dark-green bulks differing for pc1, showed that the QTL affects multiple photosynthesis and oxidation-reduction associated genes in the immature green fruit. Allelic diversity of three known genes CcLOL1, CaGLK2, and CcAPRR2 that influence pepper immature fruit color, was found to be associated with variation in chlorophyll content primarily in C. chinense.

CaVIL1, a plant homeodomain gene that promotes flowering in pepper.

KEY MESSAGE: CaVIL1 is a homolog of VIL1, a regulator of vernalization response in Arabidopsis and acts as a flowering promoter in pepper which does not respond to vernalization and photoperiod. As part of our goal to study the genetic and molecular basis of transition to flowering in pepper, we isolated the late-flowering mutant E-2698. Aside from late flowering, multiple pleiotropic alterations of the shoot structure, such as enlarged and distorted leaves, weak apical dominance, and reduced angle of the lateral branches were observed, indicating a broad role for the mutated gene in pepper development. Genetic mapping and sequence analyses revealed that the disrupted gene in E-2698 is the pepper homolog of VERNALIZATION INSENSITIVE 3-LIKE 1 (VIL1) that acts as a regulator of vernalization in Arabidopsis through chromatin modification. The pepper gene, CaVIL1, contains a plant homeodomain motif associated with chromatin modification and a VERNALIZATION INSENSITIVE 3-interacting domain that is truncated in E-2698 and in two other allelic mutants. Because pepper flowering does not respond to vernalization, we postulate that CaVIL1 regulates flowering time via chromatin modification of unknown targets. Expression analysis indicated that CaVIL1 activates the flowering promoter CaFLOWERING LOCUS T and represses the flowering repressor CaAPETALA2. Furthermore, CaVIL1 represses several genes from the FLOWERING LOCUS C (FLC)-LIKE clade that are clustered together in the pepper genome. This indicates their possible involvement in flowering regulation in this species. Our results show that CaVIL1 is a major regulator of flowering and interacts with other flowering promoters and repressors, as well as with FLC-LIKE genes whose function in flowering regulation is not yet known in pepper.

Pathogenic Variability of Meloidogyne incognita Populations Occurring in Pepper-Production Greenhouses in Israel Toward Me1, Me3 and N Pepper Resistance Genes.

Natural variation in the root-knot nematode Meloidogyne incognita is problematic for breeding programs: populations possessing similar morphological characteristics can produce different reactions on the same host. We collected 30 widely dispersed M. incognita populations from protected pepper production systems in major pepper-growing regions of Israel and accurately identified their virulence characteristics by modified differential host test in a growth chamber on tomato, tobacco, cotton, melon, pepper, and peanut. Galling indices and reproduction were determined on the different hosts. All populations fit the published scheme for M. incognita race 2, except for reproduction on cotton plants by five out of 25 tested M. incognita populations, indicating host-range variations. Reaction of three genes that confer resistance to M. incognita-Me1, Me3 and N-to the collected populations was evaluated. Several M. incognita populations induced galling and reproduced successfully on pepper genotypes carrying Me3 and N, whereas plant resistance conferred by Me1 was more robust for all examined populations. Moreover, the effect of genetic background on Me1 resistance demonstrated a relative advantage of several genotypes in nematode infestations. Efficiency of Me3 under local nematode infestation was further studied with a homozygous line carrying two Me3 alleles. Reproduction of virulent populations on the homozygotes (Me3/Me3) and heterozygotes (Me3/Me3+) was similar, suggesting a limited quantitative effect of Me3. These results present the first characterization of host range, reproduction, and molecular aspects of M. incognita from Israel and highlight the importance of taking a multidimensional approach in pepper-breeding programs for resistance to M. incognita.

Genetic and biochemical analysis reveals linked QTLs determining natural variation for fruit post-harvest water loss in pepper (Capsicum).

KEY MESSAGE: Molecular markers linked to QTLs controlling post-harvest fruit water loss in pepper may be utilized to accelerate breeding for improved shelf life and inhibit over-ripening before harvest. Bell pepper (Capsicum annuum L.) is an important vegetable crop world-wide. However, marketing is limited by the relatively short shelf life of the fruit due to water loss and decay that occur during prolonged storage. Towards breeding pepper with reduced fruit post-harvest water loss (PWL), we studied the genetic, physiological and biochemical basis for natural variation of PWL. We performed quantitative trait locus (QTL) mapping of fruit PWL in multiple generations of an interspecific cross of pepper, which resulted in the identification of two linked QTLs on chromosome 10 that control the trait. We further developed near-isogenic lines (NILs) for characterization of the QTL effects. Transcriptome analysis of the NILs allowed the identification of candidate genes associated with fruit PWL-associated traits such as cuticle biosynthesis, cell wall metabolism and fruit ripening. Significant differences in PWL between the NILs in the immature fruit stage, differentially expressed cuticle-associated genes and differences in the content of specific chemical constituents of the fruit cuticle, indicated a likely influence of cuticle composition on the trait. Reduced PWL in the NILs was associated with delayed over-ripening before harvest, low total soluble solids before storage, and reduced fruit softening after storage. Our study enabled a better understanding of the genetic and biological processes controlling natural variation in fruit PWL in pepper. Furthermore, the genetic materials and molecular markers developed in this study may be utilized to breed peppers with improved shelf life and inhibited over-ripening before harvest.

CaAP2 transcription factor is a candidate gene for a flowering repressor and a candidate for controlling natural variation of flowering time in Capsicum annuum.

KEY MESSAGE: The APETALA2 transcription factor homolog CaAP2 is a candidate gene for a flowering repressor in pepper, as revealed by induced-mutation phenotype, and a candidate underlying a major QTL controlling natural variation in flowering time. To decipher the genetic control of transition to flowering in pepper (Capsicum spp.) and determine the extent of gene function conservation compared to model species, we isolated and characterized several ethyl methanesulfonate (EMS)-induced mutants that vary in their flowering time compared to the wild type. In the present study, we report on the isolation of an early-flowering mutant that flowers after four leaves on the primary stem compared to nine leaves in the wild-type 'Maor'. By genetic mapping and sequencing of putative candidate genes linked to the mutant phenotype, we identified a member of the APETALA2 (AP2) transcription factor family, CaAP2, which was disrupted in the early-flowering mutant. CaAP2 is a likely ortholog of AP2 that functions as a repressor of flowering in Arabidopsis. To test whether CaAP2 has an effect on controlling natural variation in the transition to flowering in pepper, we performed QTL mapping for flowering time in a cross between early and late-flowering C. annuum accessions. We identified a major QTL in a region of chromosome 2 in which CaAP2 was the most significant marker, explaining 52 % of the phenotypic variation of the trait. Sequence comparison of the CaAP2 open reading frames in the two parents used for QTL mapping did not reveal significant variation. In contrast, significant differences in expression level of CaAP2 were detected between near-isogenic lines that differ for the flowering time QTL, supporting the putative function of CaAP2 as a major repressor of flowering in pepper.

CaGLK2 regulates natural variation of chlorophyll content and fruit color in pepper fruit.

KEY MESSAGE: We provide multiple evidences that CaGLK2 underlies a quantitative trait locus controlling natural variation in chlorophyll content and immature fruit color of pepper via modulating chloroplast compartment size. Pepper fruit quality is attributed to a variety of traits, affecting visual appearance, flavor, chemical composition and nutritional value. Among the quality traits, fruit color is of primary importance because the pigments that confer color are associated with nutrition, health and flavor. Although gene models have been proposed for qualitative aspects of fruit color, large natural variation in quantitative pigment content and fruit color exists in pepper. However, its genetic basis is largely unknown which hampers its utilization for plant improvement. We studied the role of GLK2, a GOLDEN2-like transcription factor that regulates chloroplast development in controlling natural variation for chlorophyll content and immature fruit color of pepper. The role of GLK2 in regulating fruit development has been studied previously in tomato using ectopic expression and the uniform ripening mutant analyses. However, pepper provides a unique opportunity to further study the function of this gene because of the wide natural variation of fruit colors in this species. Segregation, sequencing and expression analyses indicated that pepper GLK2 (CaGLK2) corresponds to the recently reported pc10 QTL that controls chloroplast development and chlorophyll content in pepper. CaGLK2 exerts its effect on chloroplast compartment size predominantly during immature fruit development. We show that the genetic background, sequence variation and expression pattern confer a complex and multi-level regulation of CaGLK2 and fruit color in Capsicum. The positive effect on fruit quality predominantly at the green stage conferred by CaGLK2 can be utilized to breed green pepper varieties with improved nutritional values and taste.

Capsicum annuum S (CaS) promotes reproductive transition and is required for flower formation in pepper (Capsicum annuum).

The genetic control of the transition to flowering has mainly been studied in model species, while few data are available in crop species such as pepper (Capsicum spp.). To elucidate the genetic control of the transition to flowering in pepper, mutants that lack flowers were isolated and characterized. Genetic mapping and sequencing allowed the identification of the gene disrupted in the mutants. Double mutants and expression analyses were used to characterize the relationships between the mutated gene and other genes controlling the transition to flowering and flower differentiation. The mutants were characterized by a delay in the initiation of sympodial growth, a delay in the termination of sympodial meristems and complete inhibition of flower formation. Capsicum annuum S (CaS), the pepper (Capsicum annuum) ortholog of tomato (Solanum lycopersicum) COMPOUND INFLORESCENCE and petunia (Petunia hybrida) EVERGREEN, was found to govern the mutant phenotype. CaS is required for the activity of the flower meristem identity gene Ca-ANANTHA and does not affect the expression of CaLEAFY. CaS is epistatic over other genes controlling the transition to flowering with respect to flower formation. Comparative homologous mutants in the Solanaceae indicate that CaS has uniquely evolved to have a critical role in flower formation, while its role in meristem maturation is conserved in pepper, tomato and petunia.

Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species.

Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species.

Fruit cuticle lipid composition and water loss in a diverse collection of pepper (Capsicum).

Pepper (Capsicum spp.) fruits are covered by a relatively thick coating of cuticle that limits fruit water loss, a trait previously associated with maintenance of postharvest fruit quality during commercial marketing. To shed light on the chemical-compositional diversity of cuticles in pepper, the fruit cuticles from 50 diverse pepper genotypes from a world collection were screened for both wax and cutin monomer amount and composition. These same genotypes were also screened for fruit water loss rate and this was tested for associations with cuticle composition. Our results revealed an unexpectedly large amount of variation for the fruit cuticle lipids, with a more than 14-fold range for total wax amounts and a more than 16-fold range for cutin monomer amounts between the most extreme accessions. Within the major wax constituents fatty acids varied from 1 to 46%, primary alcohols from 2 to 19%, n-alkanes from 13 to 74% and triterpenoids and sterols from 10 to 77%. Within the cutin monomers, total hexadecanoic acids ranged from 54 to 87%, total octadecanoic acids ranged from 10 to 38% and coumaric acids ranged from 0.2 to 8% of the total. We also observed considerable differences in water loss among the accessions, and unique correlations between water loss and cuticle constituents. The resources described here will be valuable for future studies of the physiological function of fruit cuticle, for the identification of genes and QTLs associated with fruit cuticle synthesis in pepper fruit, and as a starting point for breeding improved fruit quality in pepper.

CaHAM is autoregulated and regulates CaSTM expression and is required for shoot apical meristem organization in pepper.

The angiosperm shoot apical meristem (SAM) is characterized by tightly organized cell layers and zones. The SAM's organization allows it to maintain its indeterminate nature while producing determinate lateral organs. Alterations in SAM gene expression partly account for the immense diversity in plant architecture. The GRAS protein family gene HAIRY MERISTEM (HAM) is an important regulator of SAM organization in Petunia and Arabidopsis. Here we describe CaHAM loss-of-function pepper mutants characterized by an arrested SAM following the formation of several leaves on the primary stem, complete inhibition of axillary meristem development, an expanded tunica domain and trichome formation on the SAM epidermis. CaHAM is expressed in the periphery of the SAM and in the vasculature of young leaves throughout plant development, reaching its highest level in the reproductive growth stage. Analysis of the effect of CaHAM loss-of-function on its own expression showed that CaHAM is negatively autoregulated. Furthermore, CaHAM negatively regulates the expression level and pattern of pepper SHOOT MERISTEMLESS (CaSTM), which is required to maintain the SAM in an undifferentiated state. We conclude that CaHAM is regulated to achieve adjusted functional levels and has a conserved role in controlling SAM maintenance, organization and axillary meristem formation.

Induced mutation in ß-CAROTENE HYDROXYLASE results in accumulation of ß-carotene and conversion of red to orange color in pepper fruit.

Pepper fruit is typically red, but green, orange and yellow cultivars are gaining consumer acceptance. This color variation is mainly due to variations in carotenoid composition. Orange color in pepper can result from a number of carotenoid profiles, but its genetic basis is only partly known. We identified an EMS-induced orange-fruited mutant using the wild-type blocky red-fruited cultivar 'Maor' as progenitor. This mutant accumulates mainly ß-carotene in its fruit, instead of the complex pattern of red and yellow carotenoids in 'Maor'. We identified an A(709) to G transition in the cDNA of ß-CAROTENE HYDROXYLASE2 in the orange pepper and complete co-segregation of this single-nucleotide polymorphism with the mutated phenotype. We therefore hypothesized that ß-CAROTENE HYDROXYLASE2 controls the orange mutation in pepper. Interestingly, the expression of ß-CAROTENE HYDROXYLASE2 and additional carotenogenesis genes was elevated in the orange fruit compared with the red fruit, indicating possible feedback regulation of genes in the pathway. Because carotenoids serve as precursors for volatile compounds, we compared the volatile profiles of the two parents. The orange pepper contained more volatile compounds than 'Maor', with predominant elevation of norisoprenoids derived from ß-carotene degradation, while sesquiterpenes predominated in the red fruit. Because of the importance of ß-carotene as a provitamin A precursor in the human diet, the orange-fruited mutant might serve as a natural source for pepper fruit biofortification. Moreover, the change in volatile profile may result in a fruit flavor that differs from other pepper cultivars.

CaJOINTLESS is a MADS-box gene involved in suppression of vegetative growth in all shoot meristems in pepper.

In aiming to decipher the genetic control of shoot architecture in pepper (Capsicum spp.), the allelic late-flowering mutants E-252 and E-2537 were identified. These mutants exhibit multiple pleiotropic effects on the organization of the sympodial shoot. Genetic mapping and sequence analysis indicated that the mutants are disrupted at CaJOINTLESS, the orthologue of the MADS-box genes JOINTLESS and SVP in tomato and Arabidopsis, respectively. Late flowering of the primary and sympodial shoots of Cajointless indicates that the gene functions as a suppressor of vegetative growth in all shoot meristems. While CaJOINTLESS and JOINTLESS have partially conserved functions, the effect on flowering time and on sympodial development in pepper, as well as the epistasis over FASCICULATE, the homologue of the major determinant of sympodial development SELF-PRUNING, is stronger than in tomato. Furthermore, the solitary terminal flower of pepper is converted into a structure composed of flowers and leaves in the mutant lines. This conversion supports the hypothesis that the solitary flowers of pepper have a cryptic inflorescence identity that is suppressed by CaJOINTLESS. Formation of solitary flowers in wild-type pepper is suggested to result from precocious maturation of the inflorescence meristem.