Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Genetic characterization of a locus responsible for low pungency using EMS-induced mutants in Capsicum annuum L.

KEY MESSAGE: The pepper mutants ('221-2-1a' and '1559-1-2h') with very low pungency were genetically characterized. The Pun4 locus, responsible for the reduced pungency of the mutant fruits, was localized to a 208 Mb region on chromosome 6. DEMF06G16460, encoding 3-ketoacyl-CoA synthase, was proposed as a strong candidate gene based on the genetic analyses of bulked segregants, DEG, and expression analyses. Capsaicinoids are unique alkaloids present in pepper (Capsicum spp.), synthesized through the condensation of by-products from the phenylpropanoid and branched-chain fatty acid pathways, and accumulating in the placenta. In this study, we characterized two allelic ethyl methanesulfonate-induced mutant lines with extremely low pungency ('221-2-1a' and '1559-1-2h'). These mutants, derived from the pungent Korean landrace 'Yuwolcho,' exhibited lower capsaicinoid content than Yuwolcho but still contained a small amount of capsaicinoid with functional capsaicinoid biosynthetic genes. Genetic crosses between the mutants and Yuwolcho or pungent lines indicated that a single recessive mutation was responsible for the low-pungency phenotype of mutant 221-2-1a; we named the causal locus Pungency 4 (Pun4). To identify Pun4, we combined genome-wide polymorphism analysis and transcriptome analysis with bulked-segregant analysis. We narrowed down the location of Pun4 to a 208-Mb region on chromosome 6 containing five candidate genes, of which DEMF06G16460, encoding a 3-ketoacyl-CoA synthase associated with branched-chain fatty acid biosynthesis, is the most likely candidate for Pun4. The expression of capsaicinoid biosynthetic genes in placental tissues in Yuwolcho and the mutant was consistent with the branched-chain fatty acid pathway playing a pivotal role in the lower pungency observed in the mutant. We also obtained a list of differentially expressed genes in placental tissues between the mutant and Yuwolcho, from which we selected candidate genes using gene co-expression analysis. In summary, we characterized the capsaicinoid biosynthesis-related locus Pun4 through integrated of genetic, genomic, and transcriptome analyses. These findings will contribute to our understanding of capsaicinoid biosynthesis in pepper.

Genome-wide association study of resistance to anthracnose in pepper (Capsicum chinense) germplasm.

BACKGROUND: Anthracnose is a fungal disease caused by Colletotrichum spp. that has a significant impact on worldwide pepper production. Colletotrichum scovillei is the most common pathogenic anthracnose-causing species in the Republic of Korea. RESULTS: The resistances of 197 pepper (Capsicum chinense) accessions deposited in Korea's National Agrobiodiversity Center were evaluated for their response against the virulent pathogens Colletotrichum acutatum isolate 'KSCa-1' and C. scovillei isolate 'Hana') in the field and in vitro methods for three consecutive years (2018 to 2020). The severity of the disease was recorded and compared between inoculation methods. Six phenotypically resistant pepper accessions were selected based on three years of disease data. All of the selected resistant pepper accessions outperformed the control resistant pepper in terms of resistance (PI 594,137). A genome-wide association study (GWAS) was carried out to identify single nucleotide polymorphisms (SNPs) associated with anthracnose resistance. An association analysis was performed using 53,518 SNPs and the disease score of the 2020 field and in vitro experiment results. Both field and in vitro experiments revealed 25 and 32 significantly associated SNPs, respectively. These SNPs were found on all chromosomes except Ch06 and Ch07 in the field experiment, whereas in the in vitro experiment they were found on all chromosomes except Ch04 and Ch11. CONCLUSION: In this study, six resistant C. chinense accessions were selected. Additionally, in this study, significantly associated SNPs were found in a gene that codes for a protein kinase receptor, such as serine/threonine-protein kinase, and other genes that are known to be involved in disease resistance. This may strengthen the role of these genes in the development of anthracnose resistance in Capsicum spp. As a result, the SNPs discovered to be strongly linked in this study can be used to identify a potential marker for selecting pepper material resistant to anthracnose, which will assist in the development of resistant varieties.

Exploring horticultural traits and disease resistance in Capsicum baccatum through segmental introgression lines.

KEY MESSAGE: Segmental introgression and advanced backcross lines were developed and validated as important tools for improving agronomically important traits in pepper, offering improved sensitivity in detecting quantitative trait loci for breeding. Segmental introgression lines (SILs) and advanced backcross lines (ABs) can accelerate genetics and genomics research and breeding in crop plants. This study presents the development of a complete collection of SILs and ABs in pepper using Capsicum annuum cv. 'CM334' as the recipient parent and Capsicum baccatum 'PBC81', which displays various agronomically important traits including powdery mildew and anthracnose resistance, as donor parent. Using embryo rescue to overcome abortion in interspecific crosses, and marker-assisted selection with genotyping-in-thousands by sequencing (GT-seq) to develop SILs and ABs containing different segments of the C. baccatum genome, we obtained 63 SILs and 44 ABs, covering 94.8% of the C. baccatum genome. We characterized them for traits including powdery mildew resistance, anthracnose resistance, anthocyanin accumulation, trichome density, plant architecture, and fruit morphology. We validated previously known loci for these traits and discovered new sources of variation and quantitative trait loci (QTLs). A total of 15 QTLs were identified, including four for anthracnose resistance with three novel loci, seven for plant architecture, and four for fruit morphology. This is the first complete collection of pepper SILs and ABs validated for agronomic traits and will enhance QTL detection and serve as valuable breeding resources. Further, these SILs and ABs will be useful for comparative genomics and to better understand the genetic mechanisms underlying important agronomic traits in pepper, ultimately leading to improved crop productivity and sustainability.

A mutation in Zeaxanthin epoxidase contributes to orange coloration and alters carotenoid contents in pepper fruit (Capsicum annuum).

Phytoene synthase (PSY1), capsanthin-capsorubin synthase (CCS), and pseudo-response regulator 2 (PRR2) are three major genes controlling fruit color in pepper (Capsicum spp.). However, the diversity of fruit color in pepper cannot be completely explained by these three genes. Here, we used an F2 population derived from Capsicum annuum 'SNU-mini Orange' (SO) and C. annuum 'SNU-mini Yellow' (SY), both harboring functional PSY1 and mutated CCS, and observed that yellow color was dominant over orange color. We performed genotyping-by-sequencing and mapped the genetic locus to a 6.8-Mb region on chromosome 2, which we named CaOr. We discovered a splicing mutation in the zeaxanthin epoxidase (ZEP) gene within this region leading to a premature stop codon. HPLC analysis showed that SO contained higher amounts of zeaxanthin and total carotenoids in mature fruits than SY. A color complementation assay using Escherichia coli harboring carotenoid biosynthetic genes showed that the mutant ZEP protein had reduced enzymatic activity. Transmission electron microscopy of plastids revealed that the ZEP mutation affected plastid development with more rod-shaped inner membrane structures in chromoplasts of mature SO fruits. To validate the role of ZEP in fruit color formation, we performed virus-induced gene silencing of ZEP in the yellow-fruit cultivar C. annuum 'Micropep Yellow' (MY). The silencing of ZEP caused significant changes in the ratios of zeaxanthin to its downstream products and increased total carotenoid contents. Thus, we conclude that the ZEP genotype can determine orange or yellow mature fruit color in pepper.

Phytoene synthase 2 can compensate for the absence of PSY1 in the control of color in Capsicum fruit.

Phytoene synthase 1 (PSY1) and capsanthin-capsorubin synthase (CCS) are two major genes responsible for fruit color variation in pepper (Capsicum spp.). However, the role of PSY2 remains unknown. We used a systemic approach to examine the genetic factors responsible for the yellow fruit color of C. annuum 'MicroPep Yellow' (MY) and to determine the role of PSY2 in fruit color. We detected complete deletion of PSY1 and a retrotransposon insertion in CCS. Despite the loss of PSY1 and CCS function, both MY and mutant F2 plants from a cross between MY and the 'MicroPep Red' (MR) accumulated basal levels of carotenoids, indicating that other PSY genes may complement the loss of PSY1. qRT-PCR analysis indicated that PSY2 was constitutively expressed in both MR and MY fruits, and a color complementation assay using Escherichia coli revealed that PSY2 was capable of biosynthesizing a carotenoid. Virus-induced gene silencing of PSY2 in MY resulted in white fruits. These findings indicate that PSY2 can compensate for the absence of PSY1 in pepper fruit, resulting in the yellow color of MY fruits.

A MYB transcription factor is a candidate to control pungency in Capsicum annuum.

KEY MESSAGE: Identification of a novel pungency-controlling gene Pun3, which acts as a master regulator of capsaicinoid biosynthetic genes in Capsicum annuum. Capsaicinoid is a unique compound that gives hot peppers (Capsicum spp.) their spicy taste. The Pun1 and Pun2 loci are known to control pungency in Capsicum species. Whereas Pun1 encodes an acyltransferase, the identity of Pun2 is currently unknown. Here, we used recombinant inbred lines and F2 plants derived from a cross between the non-pungent C. annuum accession 'YCM334' and the pungent C. annuum cultivar 'Tean' to identify a novel non-pungency locus. Inheritance studies showed that non-pungency in C. annuum 'YCM334' is controlled by a single recessive gene, which we named Pun3. Using a high-density SNP map derived from genotyping-by-sequencing, Pun3 was mapped to chromosome 7. By comparing physical information about the Pun3 region in the C. annuum 'Zunla-1' and C. chinense 'PI159236' reference genomes, we identified candidate genes in this target region. One cDNA sequence from 'PI159236' was homologous to an unannotated gene in 'Zunla-1.' This sequence was also homologous to CaMYB31, which is expressed only in 'Tean' and harbors one stop codon in the non-pungent accession 'YCM334.' RNA-Seq analysis showed that major structural genes in the capsaicinoid biosynthetic pathway were significantly downregulated in 'YCM334' compared to pungent pepper. Therefore, CaMYB31 is a candidate gene for Pun3, which may act as a master regulator of capsaicinoid biosynthetic genes in pepper.

QTL mapping and GWAS reveal candidate genes controlling capsaicinoid content in Capsicum.

Capsaicinoids are unique compounds produced only in peppers (Capsicum spp.). Several studies using classical quantitative trait loci (QTLs) mapping and genomewide association studies (GWAS) have identified QTLs controlling capsaicinoid content in peppers; however, neither the QTLs common to each population nor the candidate genes underlying them have been identified due to the limitations of each approach used. Here, we performed QTL mapping and GWAS for capsaicinoid content in peppers using two recombinant inbred line (RIL) populations and one GWAS population. Whole-genome resequencing and genotyping by sequencing (GBS) were used to construct high-density single nucleotide polymorphism (SNP) maps. Five QTL regions on chromosomes 1, 2, 3, 4 and 10 were commonly identified in both RIL populations over multiple locations and years. Furthermore, a total of 109 610 SNPs derived from two GBS libraries were used to analyse the GWAS population consisting of 208 C. annuum-clade accessions. A total of 69 QTL regions were identified from the GWAS, 10 of which were co-located with the QTLs identified from the two biparental populations. Within these regions, we were able to identify five candidate genes known to be involved in capsaicinoid biosynthesis. Our results demonstrate that QTL mapping and GBS-GWAS represent a powerful combined approach for the identification of loci controlling complex traits.

Genetic diversity and population structure analysis to construct a core collection from a large Capsicum germplasm.

BACKGROUND: Conservation of genetic diversity is an essential prerequisite for developing new cultivars with desirable agronomic traits. Although a large number of germplasm collections have been established worldwide, many of them face major difficulties due to large size and a lack of adequate information about population structure and genetic diversity. Core collection with a minimum number of accessions and maximum genetic diversity of pepper species and its wild relatives will facilitate easy access to genetic material as well as the use of hidden genetic diversity in Capsicum. RESULTS: To explore genetic diversity and population structure, we investigated patterns of molecular diversity using a transcriptome-based 48 single nucleotide polymorphisms (SNPs) in a large germplasm collection comprising 3,821 accessions. Among the 11 species examined, Capsicum annuum showed the highest genetic diversity (HE = 0.44, I = 0.69), whereas the wild species C. galapagoense showed the lowest genetic diversity (HE = 0.06, I = 0.07). The Capsicum germplasm collection was divided into 10 clusters (cluster 1 to 10) based on population structure analysis, and five groups (group A to E) based on phylogenetic analysis. Capsicum accessions from the five distinct groups in an unrooted phylogenetic tree showed taxonomic distinctness and reflected their geographic origins. Most of the accessions from European countries are distributed in the A and B groups, whereas the accessions from Asian countries are mainly distributed in C and D groups. Five different sampling strategies with diverse genetic clustering methods were used to select the optimal method for constructing the core collection. Using a number of allelic variations based on 48 SNP markers and 32 different phenotypic/morphological traits, a core collection 'CC240' with a total of 240 accessions (5.2 %) was selected from within the entire Capsicum germplasm. Compared to the other core collections, CC240 displayed higher genetic diversity (I = 0.95) and genetic evenness (J' = 0.80), and represented a wider range of phenotypic variation (MD = 9.45 %, CR = 98.40 %). CONCLUSIONS: A total of 240 accessions were selected from 3,821 Capsicum accessions based on transcriptome-based 48 SNP markers with genome-wide distribution and 32 traits using a systematic approach. This core collection will be a primary resource for pepper breeders and researchers for further genetic association and functional analyses.

Tomato Male sterile 1035 is essential for pollen development and meiosis in anthers.

Male fertility in flowering plants depends on proper cellular differentiation in anthers. Meiosis and tapetum development are particularly important processes in pollen production. In this study, we showed that the tomato male sterile (ms10(35)) mutant of cultivated tomato (Solanum lycopersicum) exhibited dysfunctional meiosis and an abnormal tapetum during anther development, resulting in no pollen production. We demonstrated that Ms10(35) encodes a basic helix-loop-helix transcription factor that is specifically expressed in meiocyte and tapetal tissue from pre-meiotic to tetrad stages. Transgenic expression of the Ms10(35) gene from its native promoter complemented the male sterility of the ms10(35) mutant. In addition, RNA-sequencing-based transcriptome analysis revealed that Ms10(35) regulates 246 genes involved in anther development processes such as meiosis, tapetum development, cell-wall degradation, pollen wall formation, transport, and lipid metabolism. Our results indicate that Ms10(35) plays key roles in regulating both meiosis and programmed cell death of the tapetum during microsporogenesis.

Development of virus-induced genome editing methods in Solanaceous crops.

Genome editing (GE) using CRISPR/Cas systems has revolutionized plant mutagenesis. However, conventional transgene-mediated GE methods have limitations due to the time-consuming generation of stable transgenic lines expressing the Cas9/single guide RNA (sgRNA) module through tissue cultures. Virus-induced genome editing (VIGE) systems have been successfully employed in model plants, such as Arabidopsis thaliana and Nicotiana spp. In this study, we developed two VIGE methods for Solanaceous plants. First, we used the tobacco rattle virus (TRV) vector to deliver sgRNAs into a transgenic tomato (Solanum lycopersicum) line of cultivar Micro-Tom expressing Cas9. Second, we devised a transgene-free GE method based on a potato virus X (PVX) vector to deliver Cas9 and sgRNAs. We designed and cloned sgRNAs targeting Phytoene desaturase in the VIGE vectors and determined optimal conditions for VIGE. We evaluated VIGE efficiency through deep sequencing of the target gene after viral vector inoculation, detecting 40.3% and 36.5% mutation rates for TRV- and PVX-mediated GE, respectively. To improve editing efficiency, we applied a 37°C heat treatment, which increased the editing efficiency by 33% to 46% and 56% to 76% for TRV- and PVX-mediated VIGE, respectively. To obtain edited plants, we subjected inoculated cotyledons to tissue culture, yielding successful editing events. We also demonstrated that PVX-mediated GE can be applied to other Solanaceous crops, such as potato (Solanum tuberosum) and eggplant (Solanum melongena). These simple and highly efficient VIGE methods have great potential for generating genome-edited plants in Solanaceous crops.

Evolution of the large genome in Capsicum annuum occurred through accumulation of single-type long terminal repeat retrotransposons and their derivatives.

Although plant genome sizes are extremely diverse, the mechanism underlying the expansion of huge genomes that did not experience whole-genome duplication has not been elucidated. The pepper, Capsicum annuum, is an excellent model for studies of genome expansion due to its large genome size (2700 Mb) and the absence of whole genome duplication. As most of the pepper genome structure has been identified as constitutive heterochromatin, we investigated the evolution of this region in detail. Our findings show that the constitutive heterochromatin in pepper was actively expanded 20.0-7.5 million years ago through a massive accumulation of single-type Ty3/Gypsy-like elements that belong to the Del subgroup. Interestingly, derivatives of the Del elements, such as non-autonomous long terminal repeat retrotransposons and long-unit tandem repeats, played important roles in the expansion of constitutive heterochromatic regions. This expansion occurred not only in the existing heterochromatic regions but also into the euchromatic regions. Furthermore, our results revealed a repeat of unit length 18-24 kb. This repeat was found not only in the pepper genome but also in the other solanaceous species, such as potato and tomato. These results represent a characteristic mechanism for large genome evolution in plants.

Development of a speed breeding protocol with flowering gene investigation in pepper (Capsicum annuum).

Pepper (Capsicum spp.) is a vegetable and spice crop in the Solanaceae family with many nutritional benefits for human health. During several decades, horticultural traits, including disease resistance, yield, and fruit quality, have been improved through conventional breeding methods. Nevertheless, cultivar development is a time-consuming process because of the long generation time of pepper. Recently, speed breeding has been introduced as a solution for shorting the breeding cycle in long-day or day-neutral field crops, but there have been only a few studies on speed breeding in vegetable crops. In this study, a speed breeding protocol for pepper was developed by controlling the photoperiod and light quality. Under the condition of a low red (R) to far-red (FR) ratio of 0.3 with an extended photoperiod (Epp) of 20 h (95 ± 0 DAT), the time to first harvest was shortened by 75 days after transplant (DAT) compared to that of the control treatment (170 ± 2 DAT), suggesting that Epp with FR light is an essential factor for flowering in pepper. In addition, we established the speed breeding system in a greenhouse with a 20 h photoperiod and a 3.8 R:FR ratio and promoted the breeding cycle of C. annuum for 110 days from seed to seed. To explain the accelerated flowering response to the Epp and supplemented FR light, genome-wide association study (GWAS) and gene expression analysis were performed. As a result of the GWAS, we identified a new flowering gene locus for pepper and suggested four candidate genes for flowering (APETALA2 (AP2), WUSCHEL-RELATED HOMEOBOX4 (WOX4), FLOWERING LOCUS T (FT), and GIGANTEA (GI)). Through expression analysis with the candidate genes, it appeared that Epp and FR induced flowering by up-regulating the flowering-promoting gene GI and down-regulating FT. The results demonstrate the effect of a combination of Epp and FR light by genetic analysis of flowering gene expression. This is the first study that verifies gene expression patterns associated with the flowering responses of pepper in a speed breeding system. Overall, this study demonstrates that speed breeding can shorten the breeding cycle and accelerate genetic research in pepper through reduced generation time.

Identification of Genetic Factors Controlling the Formation of Multiple Flowers Per Node in Pepper (Capsicum spp.).

Flower production provides the foundation for crop yield and increased profits. Capsicum annuum is a pepper species with a sympodial shoot structure with solitary flowers. By contrast, C. chinense produces multiple flowers per node. C. annuum accounts for 80% of pepper production worldwide. The identification of C. chinense genes that control multiple flowers and their transfer into C. annuum may open the way to increasing fruit yield. In this study, we dissected the genetic factors were dissected controlling the multiple-flower-per-node trait in Capsicum. 85 recombinant inbred lines (RILs) between the contrasting C. annuum 'TF68' and C. chinense 'Habanero' accessions were phenotyped and genotyped. Quantitative Trait Loci (QTL) analysis identified four novel QTLs on chromosomes 1, 2, 7, and 11 that accounted for 65% of the total phenotypic variation. Genome-wide association study was also performed on a panel of 276 genotyped and phenotyped C. annuum accessions, which revealed 28 regions significantly associated with the multiple-flower trait, of which three overlapped the identified QTLs. Five candidate genes involved in the development of the shoot and flower meristems were identified and these genes could cause multiple flowers per node in pepper. These results contribute to our understanding of multiple flower formation in Capsicum and will be useful to develop high-yielding cultivars.

Genotyping-by-sequencing-based QTL mapping reveals novel loci for Pepper yellow leaf curl virus (PepYLCV) resistance in Capsicum annuum.

Disease caused by Pepper yellow leaf curl virus (PepYLCV) is one of the greatest threats to pepper (Capsicum spp.) cultivation in the tropics and subtropics. Resistance to PepYLCV was previously identified in a few Capsicum accessions, but no resistance QTLs have been mapped. This study aimed to elucidate the genetics of PepYLCV resistance in C. annuum L. Augmented inoculation by the viruliferous whitefly Bemisia tabaci was used to evaluate parental lines and an F2 segregating population derived from a cross between resistant C. annuum line LP97 and susceptible C. annuum line ECW30R. Final evaluation was performed six weeks after inoculation using a standardized 5-point scale (0 = no symptoms to 4 = very severe symptoms). A high-density linkage map was constructed using genotyping-by-sequencing (GBS) to identify single-nucleotide polymorphism (SNP) markers associated with PepYLCV resistance in the F2 population. QTL analysis revealed three QTLs, peplcv-1, peplcv-7, and peplcv-12, on chromosomes P1, P7, and P12, respectively. Candidate genes associated with PepYLCV resistance in the QTL regions were inferred. In addition, single markers Chr7-LCV-7 and Chr12-LCV-12 derived from the QTLs were developed and validated in another F2 population and in commercial varieties. This work thus provides not only information for mapping PepYLCV resistance loci in pepper but also forms the basis for future molecular analysis of genes involved in PepYLCV resistance.

Genomic selection with fixed-effect markers improves the prediction accuracy for Capsaicinoid contents in Capsicum annuum.

Capsaicinoids provide chili peppers (Capsicum spp.) with their characteristic pungency. Several structural and transcription factor genes are known to control capsaicinoid contents in pepper. However, many other genes also regulating capsaicinoid contents remain unknown, making it difficult to develop pepper cultivars with different levels of capsaicinoids. Genomic selection (GS) uses genome-wide random markers (including many in undiscovered genes) for a trait to improve selection efficiency. In this study, we predicted the capsaicinoid contents of pepper breeding lines using several GS models trained with genotypic and phenotypic data from a training population. We used a core collection of 351 Capsicum accessions and 96 breeding lines as training and testing populations, respectively. To obtain the optimal number of single nucleotide polymorphism (SNP) markers for GS, we tested various numbers of genome-wide SNP markers based on linkage disequilibrium. We obtained the highest mean prediction accuracy (0.550) for different models using 3294 SNP markers. Using this marker set, we conducted GWAS and selected 25 markers that were associated with capsaicinoid biosynthesis genes and quantitative trait loci for capsaicinoid contents. Finally, to develop more accurate prediction models, we obtained SNP markers from GWAS as fixed-effect markers for GS, where 3294 genome-wide SNPs were employed. When four to five fixed-effect markers from GWAS were used as fixed effects, the RKHS and RR-BLUP models showed accuracies of 0.696 and 0.689, respectively. Our results lay the foundation for developing pepper cultivars with various capsaicinoid levels using GS for capsaicinoid contents.

Investigation of genetic factors regulating chlorophyll and carotenoid biosynthesis in red pepper fruit.

Chlorophylls and carotenoids are synthesized in the chloroplast and chromoplast, respectively. Even though the two pigments are generated from the same precursor, the genetic correlation between chlorophyll and carotenoid biosynthesis has not yet been fully understood. We investigated the genetic correlation of chlorophyll and carotenoid biosynthesis during fruit ripening. Two recombinant inbred lines populations, "Long Sweet" × "AC2212" ("LA") RILs derived from a cross between Capsicum annuum "Long Sweet" with light-green and light-red fruit and C. annuum "AC2212" with dark-green and brown-fruit and "3501 (F)" × "3509 (C)" ("FC") RILs from C. annuum "3501" with dark-green and dark-red fruit and C. annuum "3509" with intermediate green and light-red fruit, were used. As the fruit ripened, three accessions produced high levels of xanthophyll. The dark-green immature fruit accumulated more total carotenoids than the light-green fruit. This trend corresponded to the expression pattern of 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and CaGLK2 genes during fruit development. The expression levels of DXS and CaGLK2 in the dark-green accession "3501" were significantly higher than those of "3509" and "Long Sweet" during the early stages of fruit development. Furthermore, the genotype analysis of the transcription factor controlling chloroplast development (CaGLK2) in LA RILs revealed that CaGLK2 expression affected both carotenoid and chlorophyll contents. The single nucleotide polymorphism (SNP) linkage maps were constructed using genotyping-by-sequencing (GBS) for the two populations, and QTL analysis was performed for green fruit color intensity and carotenoid content. The QTL (LA_BG-CST10) for capsanthin content in LA RILs located at 24.4 to 100.4 Mbp on chromosome 10 was overlapped with the QTL (FC15-Cap10) for capsanthin content in FC RILs. Three QTLs for capsanthin content, American spice trade association (ASTA) value, and immature green fruit color intensity were also overlapped from 178.2 to 204 Mbp on chromosome 10. At the location, 151.6 to 165 Mbp on chromosome 8, QTLs (FC15-tcar8, FC17-ASTA8.1, and FC17-ASTA8.2) for total carotenoid content and ASTA value were discovered, and this region contained 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (MCT), which is involved in the MEP pathway. This result is the first report to show the correlation between carotenoid and chlorophyll biosynthesis in pepper. This research will expand our understanding of the mechanism of the chloroplast-to-chromoplast transition and the development of high pigment pepper varieties.

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.

Comparative analysis of pepper and tomato reveals euchromatin expansion of pepper genome caused by differential accumulation of Ty3/Gypsy-like elements.

BACKGROUND: Among the Solanaceae plants, the pepper genome is three times larger than that of tomato. Although the gene repertoire and gene order of both species are well conserved, the cause of the genome-size difference is not known. To determine the causes for the expansion of pepper euchromatic regions, we compared the pepper genome to that of tomato. RESULTS: For sequence-level analysis, we generated 35.6 Mb of pepper genomic sequences from euchromatin enriched 1,245 pepper BAC clones. The comparative analysis of orthologous gene-rich regions between both species revealed insertion of transposons exclusively in the pepper sequences, maintaining the gene order and content. The most common type of the transposon found was the LTR retrotransposon. Phylogenetic comparison of the LTR retrotransposons revealed that two groups of Ty3/Gypsy-like elements (Tat and Athila) were overly accumulated in the pepper genome. The FISH analysis of the pepper Tat elements showed a random distribution in heterochromatic and euchromatic regions, whereas the tomato Tat elements showed heterochromatin-preferential accumulation. CONCLUSIONS: Compared to tomato pepper euchromatin doubled its size by differential accumulation of a specific group of Ty3/Gypsy-like elements. Our results could provide an insight on the mechanism of genome evolution in the Solanaceae family.

Characterization and genetic analysis of a low-temperature-sensitive mutant, sy-2, in Capsicum chinense.

A temperature-sensitive mutant of Capsicum chinense, sy-2, shows a normal developmental phenotype when grown above 24°C. However, when grown at 20°C, sy-2 exhibits developmental defects, such as chlorophyll deficiency and shrunken leaves. To understand the underlying mechanism of this temperature-dependent response, phenotypic characterization and genetic analysis were performed. The results revealed abnormal chloroplast structures and cell collapse in leaves of the sy-2 plants grown at 20°C. Moreover, an excessive accumulation of reactive oxygen species (ROS) resulting in cell death was detected in the chlorophyll-deficient sectors of the leaves. However, the expression profile of the ROS scavenging genes did not alter in sy-2 plants grown at 20°C. A further analysis of fatty acid content in the leaves showed the impaired pathway of linoleic acid (18:2) to linolenic acid (18:3). Additionally, the Cafad7 gene was downregulated in sy-2 plants. This change may lead to dramatic physiological disorder and alteration of leaf morphology in sy-2 plants by losing low-temperature tolerance. Genetic analysis of an F(2) population from a cross between C. chinense 'sy-2' and wild-type C. chinense 'No. 3341' showed that the sy-2 phenotype is controlled by a single recessive gene. Molecular mapping revealed that the sy-2 gene is located at a genomic region of the pepper linkage group 1, corresponding to the 300 kb region of the Ch1_scaffold 00106 in tomato chromosome 1. Candidate genes in this region will reveal the identity of sy-2 and the underlying mechanism of the temperature-dependent plant response.