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.

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.

CRISPR/Cas9-Mediated HY5 Gene Editing Reduces Growth Inhibition in Chinese Cabbage (Brassica rapa) under ER Stress.

Various stresses can affect the quality and yield of crops, including vegetables. In this study, CRISPR/Cas9 technology was employed to examine the role of the ELONGATED HYPOCOTYL 5 (HY5) gene in influencing the growth of Chinese cabbage (Brassica rapa). Single guide RNAs (sgRNAs) were designed to target the HY5 gene, and deep-sequencing analysis confirmed the induction of mutations in the bZIP domain of the gene. To investigate the response of Chinese cabbage to endoplasmic reticulum (ER) stress, plants were treated with tunicamycin (TM). Both wild-type and hy5 mutant plants showed increased growth inhibition with increasing TM concentration. However, the hy5 mutant plants displayed less severe growth inhibition compared to the wild type. Using nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) staining methods, we determined the amount of reactive oxygen species (ROS) produced under ER stress conditions, and found that the hy5 mutant plants generated lower levels of ROS compared to the wild type. Under ER stress conditions, the hy5 mutant plants exhibited lower expression levels of UPR- and cell death-related genes than the wild type. These results indicate that CRISPR/Cas9-mediated editing of the HY5 gene can mitigate growth inhibition in Chinese cabbage under stresses, improving the quality and yield of crops.

CRISPR/Cas9-mediated gene editing to confer turnip mosaic virus (TuMV) resistance in Chinese cabbage (Brassica rapa).

Genome editing approaches, particularly the CRISPR/Cas9 technology, are becoming state-of-the-art for trait development in numerous breeding programs. Significant advances in improving plant traits are enabled by this influential tool, especially for disease resistance, compared to traditional breeding. One of the potyviruses, the turnip mosaic virus (TuMV), is the most widespread and damaging virus that infects Brassica spp. worldwide. We generated the targeted mutation at the eIF(iso)4E gene in the TuMV-susceptible cultivar "Seoul" using CRISPR/Cas9 to develop TuMV-resistant Chinese cabbage. We detected several heritable indel mutations in the edited T0 plants and developed T1 through generational progression. It was indicated in the sequence analysis of the eIF(iso)4E-edited T1 plants that the mutations were transferred to succeeding generations. These edited T1 plants conferred resistance to TuMV. It was shown with ELISA analysis the lack of accumulation of viral particles. Furthermore, we found a strong negative correlation (r = -0.938) between TuMV resistance and the genome editing frequency of eIF(iso)4E. Consequently, it was revealed in this study that CRISPR/Cas9 technique can expedite the breeding process to improve traits in Chinese cabbage plants.

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 mapping revealed that the Pun2 gene in Capsicum chacoense encodes a putative aminotransferase.

Several genes regulating capsaicinoid biosynthesis including Pun1 (also known as CS), Pun3, pAMT, and CaKR1 have been studied. However, the gene encoded by Pun2 in the non-pungent Capsicum chacoense is unknown. This study aimed to identify the Pun2 gene by genetic mapping using interspecific (C. chacoense × Capsicum annuum) and intraspecific (C. chacoense × C. chacoense) populations. QTL mapping using the interspecific F2 population revealed two major QTLs on chromosomes 3 and 9. Two bin markers within the QTL regions on two chromosomes were highly correlated with the capsaicinoid content in the interspecific population. The major QTL, Pun2_PJ_Gibbs_3.11 on chromosome 3, contained the pAMT gene, indicating that the non-pungency of C. chacoense may be attributed to a mutation in the pAMT gene. Sequence analysis revealed a 7 bp nucleotide insertion in the 8th exon of pAMT of the non-pungent C. chacoense. This mutation resulted in the generation of an early stop codon, resulting in a truncated mutant lacking the PLP binding site, which is critical for pAMT enzymatic activity. This insertion co-segregated with the pungency phenotype in the intraspecific F2 population. We named this novel pAMT allele pamt11 . Taken together, these data indicate that the non-pungency of C. chacoense is due to the non-functional pAMT allele, and Pun2 encodes the pAMT gene.

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.

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 Derived Genetic Linkage Map and Quantitative Trait Loci for Sugar Content in Onion (Allium cepa L.).

Onion (2n = 2x = 16) has been a nutritional, medicinal and economically valuable vegetable crop all over the world since ancient times. To accelerate the molecular breeding in onion, genetic linkage maps are prerequisite. However, construction of genetic linkage maps of onion remains relatively rudimentary due to a large genome (about 16.3 Gbp) as well as biennial life cycle, cross-pollinated nature, and high inbreeding depression. In this study, we constructed single nucleotide polymorphism (SNP)-based genetic linkage map of onion in an F2 segregating population derived from a cross between the doubled haploid line '16P118' and inbred line 'Sweet Green' through genotyping by sequencing (GBS). A total of 207.3 Gbp of raw sequences were generated using an Illumina HiSeq X system, and 24,341 SNPs were identified with the criteria based on three minimum depths, lower than 30% missing rate, and more than 5% minor allele frequency. As a result, an onion genetic linkage map consisting of 216 GBS-based SNPs were constructed comprising eight linkage groups spanning a genetic length of 827.0 cM. Furthermore, we identified the quantitative trait loci (QTLs) for the sucrose, glucose, fructose, and total sugar content across the onion genome. We identified a total of four QTLs associated with sucrose (qSC4.1), glucose (qGC5.1), fructose (qFC5.1), and total sugar content (qTSC5.1) explaining the phenotypic variation (R2%) ranging from 6.07-11.47%. This map and QTL information will contribute to develop the molecular markers to breed the cultivars with high sugar content in onion.

Genome-Wide Analysis of MYB10 Transcription Factor in Fragaria and Identification of QTLs Associated with Fruit Color in Octoploid Strawberry.

The genus Fragaria encompass fruits with diverse colors influenced by the distribution and accumulation of anthocyanin. Particularly, the fruit colors of strawberries with different ploidy levels are determined by expression and natural variations in the vital structural and regulatory genes involved in the anthocyanin pathway. Among the regulatory genes, MYB10 transcription factor is crucial for the expression of structural genes in the anthocyanin pathway. In the present study, we performed a genome wide investigation of MYB10 in the diploid and octoploid Fragaria species. Further, we identified seven quantitative trait loci (QTLs) associated with fruit color in octoploid strawberry. In addition, we predicted 20 candidate genes primarily influencing the fruit color based on the QTL results and transcriptome analysis of fruit skin and flesh tissues of light pink, red, and dark red strawberries. Moreover, the computational and transcriptome analysis of MYB10 in octoploid strawberry suggests that the difference in fruit colors could be predominantly influenced by the expression of MYB10 from the F. iinumae subgenome. The outcomes of the present endeavor will provide a platform for the understanding and tailoring of anthocyanin pathway in strawberry for the production of fruits with aesthetic colors.

Chromosome Level Assembly of Homozygous Inbred Line 'Wongyo 3115' Facilitates the Construction of a High-Density Linkage Map and Identification of QTLs Associated With Fruit Firmness in Octoploid Strawberry (Fragaria × ananassa).

Strawberry is an allo-octoploid crop with high genome heterozygosity and complexity, which hinders the sequencing and the assembly of the genome. However, in the present study, we have generated a chromosome level assembly of octoploid strawberry sourced from a highly homozygous inbred line 'Wongyo 3115', using long- and short-read sequencing technologies. The assembly of 'Wongyo 3115' produced 805.6 Mb of the genome with 323 contigs scaffolded into 208 scaffolds with an N50 of 27.3 Mb after further gap filling. The whole genome annotation resulted in 151,892 genes with a gene density of 188.52 (genes/Mb) and validation of a genome, using BUSCO analysis resulted in 94.10% complete BUSCOs. Firmness is one of the vital traits in strawberry, which facilitate the postharvest shelf-life qualities. The molecular and genetic mechanisms that contribute the firmness in strawberry remain unclear. We have constructed a high-density genetic map based on the 'Wongyo 3115' reference genome to identify loci associated with firmness in the present study. For the quantitative trait locus (QTL) identification, the 'BS F2' populations developed from two inbred lines were genotyped, using an Axiom 35K strawberry chip, and marker positions were analyzed based on the 'Wongyo 3115' genome. Genetic maps were constructed with 1,049 bin markers, spanning the 3,861 cM. Using firmness data of 'BS F2' obtained from 2 consecutive years, five QTLs were identified on chromosomes 3-3, 5-1, 6-1, and 6-4. Furthermore, we predicted the candidate genes associated with firmness in strawberries by utilizing transcriptome data and QTL information. Overall, we present the chromosome-level assembly and annotation of a homozygous octoploid strawberry inbred line and a linkage map constructed to identify QTLs associated with fruit firmness.

Fine Mapping and Candidate Gene Identification for the CapUp Locus Controlling Fruit Orientation in Pepper (Capsicum spp.).

The orientation of fruits is a distinguishing morphological feature of pepper (Capsicum spp.) varieties. The pendent (downward curved) growth of the fruit stalks, known as pedicels, is highly correlated with fruit weight and pedicel length. A previous genetic analysis revealed that the pendent fruit orientation is governed by a dominant gene, and incomplete inheritance is also observed in some Capsicum accessions. To identify and localize this gene, a single quantitative trait locus (QTL) analysis was performed on one F2 and two recombinant inbred line (RIL) populations, and a genome-wide association study (GWAS) was performed using a core collection. Common QTL regions associated with fruit orientation were detected on chromosome 12. A total of 187,966 SNPs were identified in a genotyping-by-sequencing (GBS) for GWAS analysis of 196 Capsicum annuum, 25 Capsicum baccatum, 21 Capsicum chinense, and 14 Capsicum frutescens accessions, representing the germplasm collection of South Korea. The results of these analyses enabled us to narrow down the CapUp region of interest to 200-250 Mbp on chromosome 12. Seven candidate genes were found to be located between two markers that were completely cosegregated with the fruit orientation phenotype. The findings and markers developed in this study will be helpful for additional understanding of pepper fruit development and breeding for fruit orientation.

QTL Mapping for Gummy Stem Blight Resistance in Watermelon (Citrullus spp.).

Watermelon (Citrulluslanatus) is an economically important fruit crop worldwide. Gummy stem blight (GSB) is one of the most damaging diseases encountered during watermelon cultivation. In the present study, we identified quantitative trait loci (QTLs) associated with GSB resistance in an F2 population derived from a cross between maternal-susceptible line '920533' (C. lanatus) and the paternal-resistant line 'PI 189225' (C. amarus). The resistance of 178 F2 plants was assessed by two different evaluation methods, including leaf lesion (LL) and stem blight (SB). To analyze the QTLs associated with GSB resistance, a linkage map was constructed covering a total genetic distance of 1070.2 cM. QTL analysis detected three QTLs associated with GSB resistance on chromosome 8 and 6. Among them, two QTLs, qLL8.1 and qSB8.1 on chromosome 8 identified as major QTLs, explaining 10.5 and 10.0% of the phenotypic variations localizing at same area and sharing the same top markers for both LL and SB traits, respectively. A minor QTL, qSB6.1, explains 9.7% of phenotypic variations detected on chromosome 6 only for the SB trait. High-throughput markers were developed and validated for the selection of resistant QTLs using watermelon accessions, and commercial cultivars. Four potential candidate genes were predicted associated with GSB resistance based on the physical location of flanking markers on chromosome 8. These findings will be helpful for the development of watermelon cultivars resistant to GSB.

Versatile Nutraceutical Potentials of Watermelon-A Modest Fruit Loaded with Pharmaceutically Valuable Phytochemicals.

Watermelon (Citrulus lantus) is an important horticultural crop which belongs to the Curcubitaceae family. The nutraceutical potential of watermelon has been illustrated by several researchers, which makes it a better choice of functional food. Watermelon has been used to treat various ailments, such as cardio-vascular diseases, aging related ailments, obesity, diabetes, ulcers, and various types of cancers. The medicinal properties of watermelon are attributed by the presence of important phytochemicals with pharmaceutical values such as lycopene, citrulline, and other polyphenolic compounds. Watermelon acts as vital source of l-citrulline, a neutral-alpha amino acid which is the precursor of l-arginine, an essential amino acid necessary for protein synthesis. Supplementation of l-citrulline and lycopene displayed numerous health benefits in in vitro and in vivo studies. Similarly, the dietary intake of watermelon has proven benefits as functional food in humans for weight management. Apart from the fruits, the extracts prepared from the seeds, sprouts, and leaves also evidenced medicinal properties. The present review provides a comprehensive overview of benefits of watermelon for the treatment of various ailments.

Development and Characterization of an Ethyl Methane Sulfonate (EMS) Induced Mutant Population in Capsicum annuum L.

Plant breeding explores genetic diversity in useful traits to develop new, high-yielding, and improved cultivars. Ethyl methane sulfonate (EMS) is a chemical widely used to induce mutations at loci that regulate economically essential traits. Additionally, it can knock out genes, facilitating efforts to elucidate gene functions through the analysis of mutant phenotypes. Here, we developed a mutant population using the small and pungent ornamental Capsicum annuum pepper "Micro-Pep". This accession is particularly suitable for mutation studies and molecular research due to its compact growth habit and small size. We treated 9500 seeds with 1.3% EMS and harvested 3996 M2 lines. We then selected 1300 (32.5%) independent M2 families and evaluated their phenotypes over four years. The mutants displayed phenotypic variations in plant growth, habit, leaf color and shape, and flower and fruit morphology. An experiment to optimize Targeting Induced Local Lesions IN Genomes (TILLING) in pepper detected nine EMS-induced mutations in the eIF4E gene. The M2 families developed here exhibited broad phenotypic variation and should be valuable genetic resources for functional gene analysis in pepper molecular breeding programs using reverse genetics tools, including TILLING.

Identifying candidate genes for Phytophthora capsici resistance in pepper (Capsicum annuum) via genotyping-by-sequencing-based QTL mapping and genome-wide association study.

Phytophthora capsici (Leon.) is a globally prevalent, devastating oomycete pathogen that causes root rot in pepper (Capsicum annuum). Several studies have identified quantitative trait loci (QTL) underlying resistance to P. capsici root rot (PcRR). However, breeding for pepper cultivars resistant to PcRR remains challenging due to the complexity of PcRR resistance. Here, we combined traditional QTL mapping with GWAS to broaden our understanding of PcRR resistance in pepper. Three major-effect loci (5.1, 5.2, and 5.3) conferring broad-spectrum resistance to three isolates of P. capsici were mapped to pepper chromosome P5. In addition, QTLs with epistatic interactions and minor effects specific to isolate and environment were detected on other chromosomes. GWAS detected 117 significant SNPs across the genome associated with PcRR resistance, including SNPs on chromosomes P5, P7, and P11 that colocalized with the QTLs identified here and in previous studies. Clusters of candidate nucleotide-binding site-leucine-rich repeat (NBS-LRR) and receptor-like kinase (RLK) genes were predicted within the QTL and GWAS regions; such genes often function in disease resistance. These candidate genes lay the foundation for the molecular dissection of PcRR resistance. SNP markers associated with QTLs for PcRR resistance will be useful for marker-assisted breeding and genomic selection in pepper breeding.

Genetic diversity and population structure of Ethiopian Capsicum germplasms.

We established a collection of 142 Capsicum genotypes from different geographical areas of Ethiopia with the aim of capturing genetic diversity. Morphological traits and high-resolution melting analysis distinguished one Capsicum baccatum, nine Capsicum frutescens and 132 Capsicum annuum accessions in the collection. Measurement of plant growth parameters revealed variation between germplasms in parameters including plant height, stem thickness, internode length, number of side branches, fruit width, and fruit length. Broad-sense heritability was maximum for fruit weight, followed by length and width of leaves. We used genotyping by sequencing (GBS) to identify single-nucleotide polymorphisms (SNPs) in the panel of 142 Capsicum germplasms and found 2,831,791 genome-wide SNP markers. Among these, we selected 53,284 high-quality SNPs and used them to estimate the level of genetic diversity, population structure, and phylogenetic relationships. From model-based ancestry analysis, the phylogenetic tree and principal-coordinate analysis (PCoA), we identified two distinct genetic populations: one comprising 132 C. annuum accessions and the other comprising the nine C. frutescens accessions. GWAS analysis detected 509 SNP markers that were significantly associated with fruit-, stem- and leaf-related traits. This is the first comprehensive report of the analysis of genetic variation in Ethiopian Capsicum species involving a large number of accessions. The results will help breeders utilize the germplasm collection to improve existing commercial cultivars.

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.

A major QTL and candidate genes for capsaicinoid biosynthesis in the pericarp of Capsicum chinense revealed using QTL-seq and RNA-seq.

KEY MESSAGE: A major QTL and candidate genes controlling capsaicinoid content in the pericarp were identified by QTL-seq and RNA-seq in Capsicum chinense. Capsaicinoid biosynthesis was previously thought to be restricted to the placental tissue; however, the recent discovery of their biosynthesis in the pericarp provides new opportunities to increase the capsaicinoid content in pepper fruits. Currently, the genetic mechanisms regulating capsaicinoid biosynthesis in the pericarp remain unknown. Here, we performed quantitative trait loci (QTL) mapping and RNA sequencing (RNA-seq) to reveal the genes controlling capsaicinoid biosynthesis in the pericarp. A whole-genome sequencing-based QTL-seq strategy was employed, identifying a major QTL on chromosome 6. To validate the QTL on chromosome 6, we performed traditional QTL mapping using the same population in QTL-seq with an additional biparental population. A total of 15 QTLs for capsaicinoid content distributed on chromosomes 3, 6, and 11 were newly identified. Among these QTLs, the genetic loci on the lower arm of chromosome 6 were commonly detected in the two mapping populations, corresponding to the location of the major QTL detected using whole-genome sequencing-based QTL-seq. Our RNA-seq analysis identified candidate genes within the common QTL that were differentially expressed in the pungent and non-pungent pericarp tissues. Our results are expected to contribute to the elucidation of the regulation of capsaicinoid biosynthesis. We also demonstrated that a combination of QTL mapping and RNA-seq is helpful for refining the candidate genes of a complicated trait of interest.

Single-molecule real-time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.).

The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene ß-cyclase, ß-carotene hydroxylase, zeaxanthin epoxidase and capsanthin-capsorubin synthase (CCS) genes, in 94 pepper accessions by single-molecule real-time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra-performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.