Genetics of destemming in pepper: A step towards mechanical harvesting.

Introduction: The majority of peppers in the US for fresh market and processing are handpicked, and harvesting can account for 20-50% of production costs. Innovation in mechanical harvesting would increase availability; lower the costs of local, healthy vegetable products; and perhaps improve food safety and expand markets. Most processed peppers require removal of pedicels (stem and calyx) from the fruit, but lack of an efficient mechanical process for this operation has hindered adoption of mechanical harvest. In this paper, we present characterization and advancements in breeding green chile peppers for mechanical harvesting. Specifically, we describe inheritance and expression of an easy-destemming trait derived from the landrace UCD-14 that facilitates machine harvest of green chiles. Methods: A torque gauge was used for measuring bending forces similar to those of a harvester and applied to two biparental populations segregating for destemming force and rate. Genotyping by sequencing was used to generate genetic maps for quantitative trait locus (QTL) analyses. Results: A major destemming QTL was found on chromosome 10 across populations and environments. Eight additional population and/or environment-specific QTL were also identified. Chromosome 10 QTL markers were used to help introgress the destemming trait into jalapeño-type peppers. Low destemming force lines combined with improvements in transplant production enabled mechanical harvest of destemmed fruit at a rate of 41% versus 2% with a commercial jalapeno hybrid. Staining for the presence of lignin at the pedicel/fruit boundary indicated the presence of an abscission zone and homologs of genes known to affect organ abscission were found under several QTL, suggesting that the easy-destemming trait may be due to the presence and activation of a pedicel/fruit abscission zone. Conclusion: Presented here are tools to measure the easy-destemming trait, its physiological basis, possible molecular pathways, and expression of the trait in various genetic backgrounds. Mechanical harvest of destemmed mature green chile fruits was achieved by combining easy-destemming with transplant management.

Transcriptome Analysis of Tumor-Infiltrating Lymphocytes Identifies NK Cell Gene Signatures Associated With Lymphocyte Infiltration and Survival in Soft Tissue Sarcomas.

Purpose: Clinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS. Experimental Design: Using fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression. Results: Sorted intra-tumoral CD3+ T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45- tumor cells. TCGA analysis revealed that tumors with high TLR4 expression (P = 0.03) and low expression of STMN1 involved in microtubule polymerization (P < 0.001) were associated with significantly improved survival. Conclusions: Unlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.

Aging augments obesity-induced thymic involution and peripheral T cell exhaustion altering the "obesity paradox".

Introduction: The incidence of obesity, a condition characterized by systemic chronic inflammation, has reached pandemic proportions and is a poor prognostic factor in many pathologic states. However, its role on immune parameters has been diverse and at times contradictory. We have previously demonstrated that obesity can result in what has been called the "obesity paradox" which results in increased T cell exhaustion, but also greater efficacy of immune checkpoint blockade in cancer treatment. Methods: The role of obesity, particularly in the context of aging, has not been robustly explored using preclinical models. We therefore evaluated how age impacts the immune environment on T cell development and function using diet-induced obese (DIO) mice. Results: We observed that DIO mice initially displayed greater thymopoiesis but then developed greater thymic involution over time compared to their lean counterparts. Both aging and obesity resulted in increased T cell memory conversion combined with increased expression of T cell exhaustion markers and Treg expansion. This increased T cell immunosuppression with age then resulted in a loss of anti-tumor efficacy by immune checkpoint inhibitors (ICIs) in older DIO mice compared to the younger DIO counterparts. Discussion: These results suggest that both aging and obesity contribute to T cell dysfunction resulting in increased thymic involution. This combined with increased T cell exhaustion and immunosuppressive parameters affects immunotherapy efficacy reducing the advantage of obesity in cancer immunotherapy responses.

Comparative Immunogenomics of Canine Natural Killer Cells as Immunotherapy Target.

Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells have impeded translation. Outbred dogs offer an important link for studies of NK biology and immunotherapy. We analyzed gene expression of putative NK populations from healthy dogs and dogs with naturally-occurring cancers examining differential gene expression across multiple conditions, including steady-state, in vitro activation with cytokines and co-culture, and in vivo activation with inhaled IL-15 in dogs receiving IL-15 immunotherapy. We also compared dog, mouse and human CD3-NKp46+ NK cells using a novel orthologous transcriptome. Distinct transcriptional profiles between NK populations exist between conditions and in vitro versus in vivo treatments. In cross-species analysis, canine NK cells were globally more similar to human NK cells than mice. These data define canine NK cell gene expression under multiple conditions and across species, filling an important gap in translational NK studies.

Activation status dictates the function of unlicensed natural killer cells in mice and humans.

Natural killer (NK) cells are involved in innate defense against viral infection and cancer. NK cells can be divided into subsets based on the ability of different receptors to bind to major histocompatibility (MHC) class 1 molecules, resulting in differential responses upon activation in a process called "licensing" or "arming." NK cells expressing receptors that bind self-MHC are considered licensed due to an augmented effector lytic function capability compared with unlicensed subsets. However, we demonstrated that unlicensed NK subsets instead positively regulate the adaptive T-cell response during viral infections that are related to localization and cytokine production. In this study, the differential effects of the two types of NK subsets were contingent on the environment in viral infection and hematopoietic stem cell transplantation (HSCT) models. Infection of mice with high-dose (HD) murine cytomegalovirus (MCMC) led to a loss of licensing-associated differences, as compared with mice with low-dose (LD) infection: the unlicensed NK subset no longer localized in lymph nodes (LNs), but instead remained at the site of infection. Similarly, the patterns observed during HD infection paralleled the phenotypes of both human and mouse NK cells in an HSCT setting where NK cells exhibit an activated phenotype. However, in contrast to the effects of subset depletion in T-cell replete models, the licensed NK cell subsets still dominated antiviral responses after HSCT. Overall, our results highlight the intricate tuning of NK cells and how it affects overall immune responses with regard to licensing patterns and their dependency on the level of stimulation and activation status.

Surviving Under Pressure: The Role of Solvent, Crystal Size, and Morphology During Pelletization of Metal-Organic Frameworks.

As metal-organic frameworks (MOFs) gain traction for applications, such as hydrogen storage, it is essential to form the as-synthesized powder materials into shaped bodies with high packing densities to maximize their volumetric performance. Mechanical compaction, which involves compressing the materials at high pressure, has been reported to yield high monolith density but often results in a significant loss in accessible porosity. Herein, we sought to systematically control (1) crystal size, (2) solvation, and (3) compacting pressure in the pelletization process to achieve high packing density without compromising the porosity that makes MOFs functional. It was determined that solvation is the most critical factor among the three factors examined. Solvation that exceeds the pore volume prevents the framework from collapsing, allowing for porosity to be maintained through pelletization. Higher pelletization pressure results in higher packing density, with extensive loss of porosity being observed at a higher pressure if the solvation is below the pore volume. Lastly, we observed that the morphology and size of the MOF particles result in variation in the highest achievable packing efficiency, but these numbers (75%) are still greater than many existing techniques used to form MOFs. We concluded that the application of pressure through pelletization is a suitable and widely applicable technique for forming high-density MOF-monoliths.

Increased efficacy of dual proinflammatory cytokine blockade on acute GVHD while maintaining GVT effects.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a potential curative option for treating a variety of hematologic diseases, but acute and chronic graft-versus-host disease (GVHD) remain major barriers limiting efficacy. Acute gut GVHD occurs with marked increases in proinflammatory cytokines (including TNF and IL-6), which we recently demonstrated was exacerbated in obesity resulting in severe gastrointestinal pathology. Given the pleiotropic and overlapping effects of these 2 cytokines, we assessed the impact of dual TNF and IL-6R blockade on GVHD as well as graft-versus tumor (GVT) effects in different mouse GVHD models. Early administration of combined blockade resulted in greater protection and survival from acute gut GVHD compared with single blockade regimens and even development of later chronic skin GVHD. Importantly, double cytokine blockade preserved GVT effects reinforcing that GVT and GVHD can be delineated and may result in greater efficacy in allo-HSCT.

An anchored chromosome-scale genome assembly of spinach improves annotation and reveals extensive gene rearrangements in euasterids.

Spinach (Spinacia oleracea L.) is a member of the Caryophyllales family, a basal eudicot asterid that consists of sugar beet (Beta vulgaris L. subsp. vulgaris), quinoa (Chenopodium quinoa Willd.), and amaranth (Amaranthus hypochondriacus L.). With the introduction of baby leaf types, spinach has become a staple food in many homes. Production issues focus on yield, nitrogen-use efficiency and resistance to downy mildew (Peronospora effusa). Although genomes are available for the above species, a chromosome-level assembly exists only for quinoa, allowing for proper annotation and structural analyses to enhance crop improvement. We independently assembled and annotated genomes of the cultivar Viroflay using short-read strategy (Illumina) and long-read strategies (Pacific Biosciences) to develop a chromosome-level, genetically anchored assembly for spinach. Scaffold N50 for the Illumina assembly was 389 kb, whereas that for Pacific BioSciences was 4.43 Mb, representing 911 Mb (93% of the genome) in 221 scaffolds, 80% of which are anchored and oriented on a sequence-based genetic map, also described within this work. The two assemblies were 99.5% collinear. Independent annotation of the two assemblies with the same comprehensive transcriptome dataset show that the quality of the assembly directly affects the annotation with significantly more genes predicted (26,862 vs. 34,877) in the long-read assembly. Analysis of resistance genes confirms a bias in resistant gene motifs more typical of monocots. Evolutionary analysis indicates that Spinacia is a paleohexaploid with a whole-genome triplication followed by extensive gene rearrangements identified in this work. Diversity analysis of 75 lines indicate that variation in genes is ample for hypothesis-driven, genomic-assisted breeding enabled by this work.

PD-1 Blockade Reverses Obesity-Mediated T Cell Priming Impairment.

Despite obesity reaching pandemic proportions, its impact on antigen-specific T cell responses is still unclear. We have recently demonstrated that obesity results in increased expression of PD-1 on T cells, and checkpoint blockade targeting PD-1/PD-L1 surprisingly resulted in greater clinical efficacy in cancer therapy. Adverse events associated with this therapy center around autoimmune reactions. In this study, we examined the impact of obesity on T cell priming and on autoimmune pathogenesis using the mouse model experimental autoimmune encephalomyelitis (EAE), which is mediated by autoreactive myelin-specific T cells generated after immunization. We observed that diet-induced obese (DIO) mice had a markedly delayed EAE onset and developed milder clinical symptoms compared to mice on control diet (CD). This delay was associated with impaired generation of myelin-specific T cell numbers and concurrently correlated with increased PD-L1 upregulation on antigen-presenting cells in secondary lymphoid organs. PD-1 blockade during the priming stage of EAE restored disease onset and severity and increased numbers of pathogenic CD4+ T cells in the central nervous system (CNS) of DIO mice to similar levels to those of CD mice. Administration of anti-PD-1 after onset of clinical symptoms did not increase EAE pathogenesis demonstrating that initial priming is the critical juncture affected by obesity. These findings demonstrate that obesity impairs antigen-specific T cell priming, but this can be reversed with PD-1 blockade. Our results further suggest that PD-1 blockade may increase the risk of autoimmune toxicities, particularly in obesity.

Obesity induces gut microbiota alterations and augments acute graft-versus-host disease after allogeneic stem cell transplantation.

The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by acute and chronic graft-versus-host disease (GVHD). The impact of obesity on allo-HSCT outcomes is poorly understood. Here, we report that obesity had a negative and selective impact on acute gut GVHD after allo-HSCT in mice with diet-induced obesity (DIO). These animals exhibited increased gut permeability, endotoxin translocation across the gut, and radiation-induced gastrointestinal damage after allo-HSCT. After allo-HSCT, both male and female DIO mouse recipients showed increased proinflammatory cytokine production and expression of the GVHD marker ST2 (IL-33R) and MHC class II molecules; they also exhibited decreased survival associated with acute severe gut GVHD. This rapid-onset, obesity-associated gut GVHD depended on donor CD4+ T cells and occurred even with a minor MHC mismatch between donor and recipient animals. Retrospective analysis of clinical cohorts receiving allo-HSCT transplants from unrelated donors revealed that recipients with a high body mass index (BMI, >30) had reduced survival and higher serum ST2 concentrations compared with nonobese transplant recipients. Assessment of both DIO mice and allo-HSCT recipients with a high BMI revealed reduced gut microbiota diversity and decreased Clostridiaceae abundance. Prophylactic antibiotic treatment protected DIO mouse recipients from endotoxin translocation across the gut and increased inflammatory cytokine production, as well as gut pathology and mortality, but did not protect against later development of chronic skin GVHD. These results suggest that obesity-induced alterations of the gut microbiota may affect GVHD after allo-HSCT in DIO mice, which could be ameliorated by prophylactic antibiotic treatment.

Analysis of tumor-infiltrating NK and T cells highlights IL-15 stimulation and TIGIT blockade as a combination immunotherapy strategy for soft tissue sarcomas.

PURPOSE: Given the unmet need for novel immunotherapy in soft tissue sarcoma (STS), we sought to characterize the phenotype and function of intratumoral natural killer (NK) and T cells to identify novel strategies to augment tumor-infiltrating lymphocyte (TIL) function. EXPERIMENTAL DESIGN: Using prospectively collected specimens from dogs and humans with sarcomas, archived specimens, and The Cancer Genome Atlas (TCGA) data, we evaluated blood and tumor NK and T cell phenotype and function and correlated those with outcome. We then assessed the effects of interleukin 15 (IL-15) stimulation on both NK and T cell activation and TIGIT upregulation. Finally, we evaluated cytotoxic effects of IL-15 combined with TIGIT blockade using a novel anti-TIGIT antibody. RESULTS: TILs were strongly associated with survival outcome in both archived tissue and TCGA, but higher TIL content was also associated with higher TIGIT expression. Compared with blood, intratumoral NK and T cells showed significantly higher expression of both activation and exhaustion markers, in particular TIGIT. Ex vivo stimulation of blood and tumor NK and T cells from patients with STS with IL-15 further increased both activation and exhaustion markers, including TIGIT. Dogs with metastatic osteosarcoma receiving inhaled IL-15 also exhibited upregulation of activation markers and TIGIT. Ex vivo, combined IL-15 and TIGIT blockade using STS blood and tumor specimens significantly increased cytotoxicity against STS targets. CONCLUSION: Intratumoral NK and T cells are prognostic in STS, but their activation is marked by significant upregulation of TIGIT. Our data suggest that combined IL-15 and TIGIT blockade may be a promising clinical strategy in STS.

Transcriptional analyses of differential cultivars during resistant and susceptible interactions with Peronospora effusa, the causal agent of spinach downy mildew.

Downy mildew of spinach is caused by the obligate oomycete pathogen, Peronospora effusa. The disease causes significant economic losses, especially in the organic sector of the industry where the use of synthetic fungicides is not permitted for disease control. New pathotypes of this pathogen are increasingly reported which are capable of breaking resistance. In this study, we took advantage of new spinach genome resources to conduct RNA-seq analyses of transcriptomic changes in leaf tissue of resistant and susceptible spinach cultivars Solomon and Viroflay, respectively, at an early stage of pathogen establishment (48 hours post inoculation, hpi) to a late stage of symptom expression and pathogen sporulation (168 hpi). Fold change differences in gene expression were recorded between the two cultivars to identify candidate genes for resistance. In Solomon, the hypersensitive inducible genes such as pathogenesis-related gene PR-1, glutathione-S-transferase, phospholipid hydroperoxide glutathione peroxidase and peroxidase were significantly up-regulated uniquely at 48 hpi and genes involved in zinc finger CCCH protein, glycosyltransferase, 1-aminocyclopropane-1-carboxylate oxidase homologs, receptor-like protein kinases were expressed at 48 hpi through 168 hpi. The types of genes significantly up-regulated in Solomon in response to the pathogen suggests that salicylic acid and ethylene signaling pathways mediate resistance. Furthermore, many genes involved in the flavonoid and phenylpropanoid pathways were highly expressed in Viroflay compared to Solomon at 168 hpi. As anticipated, an abundance of significantly down-regulated genes was apparent at 168 hpi, reflecting symptom development and sporulation in cultivar Viroflay, but not at 48 hpi. In the pathogen, genes encoding RxLR-type effectors were expressed during early colonization of cultivar Viroflay while crinkler-type effector genes were expressed at the late stage of the colonization. Our results provide insights on gene expression in resistant and susceptible spinach-P. effusa interactions, which can guide future studies to assess candidate genes necessary for downy mildew resistance in spinach.

Minimal PD-1 expression in mouse and human NK cells under diverse conditions.

PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.

Sequencing-Based Bin Map Construction of a Tomato Mapping Population, Facilitating High-Resolution Quantitative Trait Loci Detection.

Genotyping-by-sequencing (GBS) was employed to construct a highly saturated genetic linkage map of a tomato ( L.) recombinant inbred line (RIL) population, derived from a cross between cultivar NC EBR-1 and the wild tomato L. accession LA2093. A pipeline was developed to convert single nucleotide polymorphism (SNP) data into genomic bins, which could be used for fine mapping of quantitative trait loci (QTL) and identification of candidate genes. The pipeline, implemented in a python script named SNPbinner, adopts a hidden Markov model approach for calculation of recombination breakpoints followed by genomic bins construction. The total length of the newly developed high-resolution genetic map was 1.2-fold larger than previously estimated based on restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-based markers. The map was used to verify and refine QTL previously identified for two fruit quality traits in the RIL population, fruit weight (FW) and fruit lycopene content (LYC). Two well-described FW QTL ( and ) were localized precisely at their known underlying causative genes, and the QTL intervals were decreased by two- to tenfold. A major QTL for LYC content () was verified at high resolution and its underlying causative gene was determined to be ζ (). The RIL population, the high resolution genetic map, and the easy-to-use genotyping pipeline, SNPbinner, are made publicly available.

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.

Paradoxical effects of obesity on T cell function during tumor progression and PD-1 checkpoint blockade.

The recent successes of immunotherapy have shifted the paradigm in cancer treatment, but because only a percentage of patients are responsive to immunotherapy, it is imperative to identify factors impacting outcome. Obesity is reaching pandemic proportions and is a major risk factor for certain malignancies, but the impact of obesity on immune responses, in general and in cancer immunotherapy, is poorly understood. Here, we demonstrate, across multiple species and tumor models, that obesity results in increased immune aging, tumor progression and PD-1-mediated T cell dysfunction which is driven, at least in part, by leptin. However, obesity is also associated with increased efficacy of PD-1/PD-L1 blockade in both tumor-bearing mice and clinical cancer patients. These findings advance our understanding of obesity-induced immune dysfunction and its consequences in cancer and highlight obesity as a biomarker for some cancer immunotherapies. These data indicate a paradoxical impact of obesity on cancer. There is heightened immune dysfunction and tumor progression but also greater anti-tumor efficacy and survival after checkpoint blockade which directly targets some of the pathways activated in obesity.

Comparative transcriptomics and genomic patterns of discordance in Capsiceae (Solanaceae).

The integration of genomics and phylogenetics allows new insight into the structure of gene tree discordance, the relationships among gene position, gene history, and rate of evolution, as well as the correspondence of gene function, positive selection, and gene ontology enrichment across lineages. We explore these issues using the tribe Capsiceae (Solanaceae), which is comprised of the genera Lycianthes and Capsicum (peppers). In combining the annotated genomes of Capsicum with newly sequenced transcriptomes of four species of Lycianthes and Capsicum, we develop phylogenies for 6747 genes, and construct a backbone species tree using both concordance and explicit phylogenetic network approaches. We quantify phylogenetic discordance among individual gene trees, measure their rates of synonymous and nonsynonymous substitution, and test whether they were positively selected along any branch of the phylogeny. We then map these genes onto the annotated Capsicum genome and test whether rates of evolution, gene history, and gene ontology vary significantly with gene position. We observed substantial discordance among gene trees. A bifurcating species tree placing Capsicum within a paraphyletic Lycianthes was supported over all phylogenetic networks. Rates of synonymous and nonsynonymous substitution varied 41-fold and 130-fold among genes, respectively, and were significantly lower in pericentromeric regions. We found that results of concordance tree analyses vary depending on the subset of genes used, and that genes within the pericentromeric regions only capture a portion of the observed discordance. We identified 787 genes that have been positively selected throughout the diversification history of Capsiceae, and discuss the importance of these genes as targets for investigation of economically important traits in the domesticated peppers.

Quantitative Trait Loci Controlling Fruit Size and Other Horticultural Traits in Bell Pepper (Capsicum annuum).

Bell pepper ( L.) is a group of fruit vegetables that has large variation in fruit shape, fruit size, and horticultural traits. Using unadapted sources of germplasm to bring in novel alleles while maintaining favorable quality and horticultural traits is challenging for breeding in pepper. A genetic map with 318 loci from genotype-by-sequencing (GBS) and single nucleotide polymorphism assays was generated from a recombinant inbred line population derived from a cultivated bell-type 'Maor' and a landrace highly resistant to , 'Criollo de Morelos-334'. Forty-nine quantitative trait loci (QTLs) were detected for fruit, leaf, and horticultural traits with the scantwo permutation and stepwiseqtl methods from R/qtl. With the availability of a pepper reference genome and GBS data, candidate genes for pepper organ size and other horticultural traits were predicted. , , and genes were candidate genes for controlling organ sizes on chromosome 1, 2, and 3, respectively. Two candidate genes controlling trichome formation in pepper are located at chromosome 10: and . The locus on chromosome 10, which encodes a member of the R2R3 MYB-domain family of proteins, has a function in anthocyanin accumulation. These QTL results and the candidate genes for each trait emphasize the genetic basis of the important traits for breeding with unadapted parents in bell pepper.

Reference quality assembly of the 3.5-Gb genome of Capsicum annuum from a single linked-read library.

Linked-Read sequencing technology has recently been employed successfully for de novo assembly of human genomes, however, the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5-gigabase (Gb) diploid pepper (Capsicum annuum) genome with a single Linked-Read library. Plant genomes, including pepper, are characterized by long, highly similar repetitive sequences. Accordingly, significant effort is used to ensure that the sequenced plant is highly homozygous and the resulting assembly is a haploid consensus. With a phased assembly approach, we targeted a heterozygous F1 derived from a wide cross to assess the ability to derive both haplotypes and characterize a pungency gene with a large insertion/deletion. The Supernova software generated a highly ordered, more contiguous sequence assembly than all currently available C. annuum reference genomes. Over 83% of the final assembly was anchored and oriented using four publicly available de novo linkage maps. A comparison of the annotation of conserved eukaryotic genes indicated the completeness of assembly. The validity of the phased assembly is further demonstrated with the complete recovery of both 2.5-Kb insertion/deletion haplotypes of the PUN1 locus in the F1 sample that represents pungent and nonpungent peppers, as well as nearly full recovery of the BUSCO2 gene set within each of the two haplotypes. The most contiguous pepper genome assembly to date has been generated which demonstrates that Linked-Read library technology provides a tool to de novo assemble complex highly repetitive heterozygous plant genomes. This technology can provide an opportunity to cost-effectively develop high-quality genome assemblies for other complex plants and compare structural and gene differences through accurate haplotype reconstruction.

Regions Underlying Population Structure and the Genomics of Organ Size Determination in Capsicum annuum.

Fruits, as an important part of the human diet, have been under strong selection during domestication. In general, continued directed selection has led to varieties having larger fruit with greater shape variation and tremendous increases in fruit mass. Common cultivated peppers ( L.) are found in a wide range of sizes and shapes. Analysis of genetic relatedness and population structure has shown that the large-fruited, nonpungent types have reduced diversity and comprise a highly structured group. To explore this population structure, a statistical method for detecting fixation within subpopulations was applied to a set of 21 pungent and 19 nonpungent lines that represent the pepper breeding germplasm. We have identified 17 blocks within the pepper genome that are conserved among nonpungent large-fruited varieties. To determine if these regions were fixed by selection on fruit size or pungency, quantitative trait loci (QTLs) from seven studies along with capsaicin biosynthesis genes and homologs of organ size regulatory genes were mapped onto the current pepper genome assembly. Of the 17 fixed regions, 14 overlapped with fruit size or shape QTLs. There were seven putative organ size regulators and seven capsaicin biosynthetic genes within these regions. This work defines genomic regions that underly structure within the nonpungent pepper germplasm and QTLs or genes that may have been selected for during the development of large-fruited nonpungent pepper varieties.