Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

KEY MESSAGE: PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

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.

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.

Expression of a Large Single-Chain 13F6 Antibody with Binding Activity against Ebola Virus-Like Particles in a Plant System.

Pathogenic animal and human viruses present a growing and persistent threat to humans worldwide. Ebola virus (EBOV) causes zoonosis in humans. Here, two structurally different anti-Ebola 13F6 antibodies, recognizing the heavily glycosylated mucin-like domain (MLD) of the glycoprotein (GP), were expressed in transgenic Nicotiana tabacum plants and designed as inexpensive and effective diagnostic antibodies against Ebola virus disease (EVD). The first was anti-EBOV 13F6 full size antibody with heavy chain (HC) and light chain (LC) (monoclonal antibody, mAb 13F6-FULL), while the second was a large single-chain (LSC) antibody (mAb 13F6-LSC). mAb 13F6-LSC was constructed by linking the 13F6 LC variable region (VL) with the HC of mAb 13F6-FULL using a peptide linker and extended to the C-terminus using the endoplasmic reticulum (ER) retention motif KDEL. Agrobacterium-mediated plant transformation was employed to express the antibodies in N. tabacum. PCR, RT-PCR, and immunoblot analyses confirmed the gene insertion, transcription, and protein expression of these antibodies, respectively. The antibodies tagged with the KDEL motif displayed high-mannose type N-glycan structures and efficient binding to EBOV-like particles (VLPs). Thus, various forms of anti-EBOV plant-derived mAbs 13F6-FULL and LSC with efficient binding affinity to EBOV VLP can be produced in the plant system.

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.

Efficacy of topical 5-aminosalicylate monotherapy in patients with ulcerative proctitis with skip inflammation.

BACKGROUND AND AIM: In some patients with ulcerative proctitis (UP), skip inflammation is noted in the right side of the colon, but little is known about its clinical course. The aim of this study was to evaluate the clinical course of UP with skip inflammation and the efficacy of topical 5-aminosalicylate (5-ASA) monotherapy. METHODS: This study reviewed the data of 388 patients with an initial diagnosis of UP from January 2005 to October 2015. This study matched each UP patient with skip inflammation 1:2 with controls who had UP without skip inflammation; to reduce bias, this study matched the controls with the cases by age, gender, and initial disease activity. RESULTS: During the follow-up period (median: 69.5 months), the overall progression rates for the control group (n = 192) and the skip inflammation group (n = 96) were 24.0% and 32.9% at 10 years, respectively (log-rank P = 0.71). In the skip inflammation group, the progression rates were not significantly different between the 5-ASA combination group and the topical group, 33.4% and 26.6% at 10 years, respectively (log-rank P = 0.96). The overall acute exacerbation rates for the control and skip inflammation groups were 17.2% and 26.8% at 10 years, respectively (log-rank P = 0.68). In the skip inflammation group, the exacerbation rates were also not significantly different between the combination and topical treatment groups, 26.6% and 23.6% at 10 years, respectively (log-rank P = 0.88). CONCLUSION: The clinical course of UP with skip inflammation was not different from that of typical UP, and topical 5-ASA monotherapy for maintaining remission was as effective as 5-ASA combination therapy irrespective of the presence of skip lesions.

Reappraisal of hMLH1 promoter methylation and protein expression status in the serrated neoplasia pathway.

AIMS: The aim of this study was to determine whether human mutL homologue 1 (hMLH1) inactivation precedes the progression of sessile serrated lesion (SSL) into SSL with cytological dysplasia (SSL/D) and to define the histological stage at which promoter methylation and inactivation of hMLH1 occur. METHODS AND RESULTS: Using the MassARRAY EpiTYPER assay and immunohistochemistry, we examined methylation levels and the protein expression status of hMLH1 in 33 SSL/Ds with conventional epithelial dysplasia and compared the results with those of control hyperplastic polyps (HPs) and SSLs. The methylation level of hMLH1 was higher in the dysplastic component than in the non-dysplastic component of SSL/Ds (P = 0.005), and differed significantly with regard to the degree of dysplasia (P = 0.002). The methylation levels of hMLH1 in the dysplastic component of SSL/Ds tended to be higher than those of control SSLs and HPs (P = 0.063 and P = 0.017, respectively). The loss of hMLH1 protein expression was identified in only 13 of 33 (39.39%) dysplastic components of SSL/Ds. CONCLUSION: Promoter methylation and loss of protein expression of hMLH1 are not parallel processes that occur concurrently. hMLH1 methylation is an early molecular event which occurs even in HP. However, the loss of hMLH1 expression is a much later step, found in approximately 40% of SSL/Ds at various histological stages. Notably, the loss of hMLH1 protein expression does not necessarily precede the development of cytological dysplasia in SSL.

Autologous adipose tissue-derived stem cells treatment demonstrated favorable and sustainable therapeutic effect for Crohn's fistula.

Fistula is a representative devastating complication in Crohn's patients due to refractory to conventional therapy and high recurrence. In our phase I clinical trial, adipose tissue-derived stem cells (ASCs) demonstrated their safety and therapeutic potential for healing fistulae associated with Crohn's disease. This study was carried out to evaluate the efficacy and safety of ASCs in patients with Crohn's fistulae. In this phase II study, forty-three patients were treated with ASCs. The amount of ASCs was proportioned to fistula size and fistula tract was filled with ASCs in combination with fibrin glue after intralesional injection of ASCs. Patients without complete closure of fistula at 8 weeks received a second injection of ASCs containing 1.5 times more cells than the first injection. Fistula healing at week 8 after final dose injection and its sustainability for 1-year were evaluated. Healing was defined as a complete closure of external opening without any sign of drainage and inflammation. A modified per-protocol analysis showed that complete fistula healing was observed in 27/33 patients (82%) by 8 weeks after ASC injection. Of 27 patients with fistula healing, 26 patients completed additional observation study for 1-year and 23 patients (88%) sustained complete closure. There were no adverse events related to ASC administration. ASC treatment for patients with Crohn's fistulae was well tolerated, with a favorable therapeutic outcome. Furthermore, complete closure was well sustained. These results strongly suggest that autologous ASC could be a novel treatment option for the Crohn's fistula with high-risk of recurrence.

Can endoscopic submucosal dissection technique be an alternative treatment option for a difficult giant (≥ 30 mm) pedunculated colorectal polyp?

BACKGROUND: Snare polypectomy of a giant pedunculated colorectal polyp is sometimes technically demanding, and, therefore, piecemeal resection is inevitable, despite the relative risk of invasive cancer and postpolypectomy bleeding. OBJECTIVE: The aim of this study was to evaluate the efficacy and safety of endoscopic submucosal dissection in comparison with conventional snare polypectomy for giant pedunculated polyps DESIGN AND SETTINGS: We retrospectively reviewed the clinical outcomes and complications of endoscopic polypectomy for giant pedunculated polyps from October 2006 to November 2011. PATIENTS: All the patients who underwent endoscopic submucosal dissection (n = 23) or snare polypectomy (n = 20) for pedunculated polyps ≥ 3 cm were enrolled consecutively. In the case of a giant pedunculated polyp with 1) poor visualization of the stalk, 2) technical difficulties in snare positioning for en bloc resection, or 3) need for trimming of the head, we did not attempt piecemeal snare polypectomy, and we performed endoscopic submucosal dissection instead. (These were arbitrarily defined as "difficult" giant pedunculated polyps.) MAIN OUTCOME MEASURES: Data on the patient's demography, endoscopic and histopathologic findings, clinical outcomes, and complications were analyzed. RESULTS: Among the 43 giant pedunculated polyps, 23 polyps were defined as "difficult" polyps and were removed with endoscopic submucosal dissection. Subpedunculated (stalk <1 cm) type was more common in the "difficult" polyp group (p = 0.01). The overall incidence of cancer was 18.6% (8/43). En bloc resection rates were 100% (23/23) in the endoscopic submucosal dissection group and 90% (18/20) in the snare polypectomy group. The procedure times of snare polypectomy and endoscopic submucosal dissection group did not differ significantly (41.7 ± 13.7 minutes vs 44.9 ± 35.6 minutes, p = 0.70). Postpolypectomy bleeding was noted in 1 case (4.3%) in the endoscopic submucosal dissection group and in 3 cases (15%) in the snare polypectomy group. CONCLUSIONS: Endoscopic submucosal dissection, as well as the snare polypectomy for giant pedunculated polyps, appeared to be effective without major complications and can be an alternative option to achieve en bloc resection, particularly for difficult cases, such as giant subpedunculated polyps.

Endoscopic submucosal dissection for colorectal tumors--1,000 colorectal ESD cases: one specialized institute's experiences.

PURPOSE: Endoscopic submucosal dissection (ESD) is a very useful endoscopic technique, making it possible to perform en bloc resection regardless of lesion size. Since the introduction of ESD at our hospital, we have performed 1,000 colorectal ESDs during 56 months. The purpose of this study was to evaluate the clinical outcomes of our colorectal ESD experience and to access the efficacy and safety of colorectal ESD. METHODS: Between October 2006 and August 2011, we performed ESD on 1,000 consecutive colorectal tumors in 966 patients. We evaluated the clinical outcomes of all said cases. RESULTS: The mean resected tumor size was 24.1 ± 13.3 (3-145) mm. Our overall endoscopic en bloc resection rate was 97.5% (975/1,000), and our R0 resection rate was 91.2% (912/1,000) respectively. Our perforation rate was 5.3% (53/1,000). Of these 53 perforations, 50 cases were treated through conservative management with/without endoscopic clipping, whereas the remaining 3 patients received laparoscopic operation. Pathological examination showed adenocarcinoma in 37.2% of cases (372/1,000) and neuroendocrine tumors in 11.2% (112/1,000). We recommended additional radical surgery to 82 patients who had a risk of lymph node metastasis. Follow-up colonoscopies were performed on 722 patients. During the median follow-up period of 13 (1-62) months, there were three recurrences (0.4%). CONCLUSIONS: ESD is technically difficult, with a substantial risk of perforation. However, ESD enabled en bloc resection and pathologically complete resection of large colorectal epithelial tumors and submucosal tumors. As experience with the technique increases, ESD may gradually replace piecemeal endoscopic mucosal resection and radical colon resection in the treatment of colorectal tumors.

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.

Clinical risk factors for perforation during endoscopic submucosal dissection (ESD) for large-sized, nonpedunculated colorectal tumors.

BACKGROUND: The possible risk of colonic perforation during endoscopic submucosal dissection (ESD) for colorectal tumors is a barrier to wide application. This retrospective study was performed to evaluate the risk and the predictive factors for perforation during ESD procedure. METHODS: Between October 2006 and November 2010, a total of 499 consecutive patients (mean age 60.0 ± 11.3 years) who underwent ESD for large-sized (≥ 20 mm), nonpedunculated colorectal tumor were analyzed. First, incidence rate and clinical course of perforation were evaluated. Second, patient-related variables (age, sex, history of aspirin or antiplatelet agents, and comorbidity), endoscopic variables (tumor size, location, and type), procedure-related variables (experience of procedures, procedure time, and materials of submucosal injection), and pathologic diagnosis were analyzed. RESULTS: The mean size of the lesions was 28.9 mm. The overall en bloc resection rate was 95.0%. Perforation occurred in 37 out of 499 patients (7.4%). Thirty-four patients could be successfully treated conservatively. The type (laterally spreading tumor) and the location (right-sided colon) of the tumors, less experience of the procedure (<100 cases) in each endoscopist, and submucosal injection without hyaluronic acid were associated with higher frequency of perforation (all P < 0.05). On multivariate analysis, laterally spreading type of tumor [odds ratio (OR) 4.10, 95% confidence interval (CI) 1.17-14.34] and submucosal injection with hyaluronic acid (OR 0.31, 95% CI 0.13-0.72) were independent predictive factors. CONCLUSIONS: Perforation rate was 7.4%, and most cases could be successfully managed nonsurgically. In case of laterally spreading type of tumor, more caution is needed during submucosal dissection and long-lasting submucosal cushion is important for preventing perforation.

Immune Responses to Plant-Derived Recombinant Colorectal Cancer Glycoprotein EpCAM-FcK Fusion Protein in Mice.

Epidermal cell adhesion molecule (EpCAM) is a tumor-associated antigen (TAA), which has been considered as a cancer vaccine candidate. The EpCAM protein fused to the fragment crystallizable region of immunoglobulin G (IgG) tagged with KDEL endoplasmic reticulum (ER) retention signal (EpCAM-FcK) has been successfully expressed in transgenic tobacco (Nicotiana tabacum cv. Xanthi) and purified from the plant leaf. In this study, we investigated the ability of the plant-derived EpCAM-FcK (EpCAM-FcKP) to elicit an immune response in vivo. The animal group injected with the EpCAM-FcKP showed a higher differentiated germinal center (GC) B cell population (~9%) compared with the animal group injected with the recombinant rhEpCAM-Fc chimera (EpCAM-FcM). The animal group injected with EpCAM-FcKP (~42%) had more differentiated T follicular helper cells (Tfh) than the animal group injected with EpCAM-FcM (~7%). This study demonstrated that the plant-derived EpCAM-FcK fusion antigenic protein induced a humoral immune response in mice.

Clinical outcomes of stapled transanal rectal resection (STARR) for obstructed defecation syndrome (ODS): a single institution experience in South Korea.

PURPOSE: The purpose of this study was to assess both the short- and long-term functional outcomes of stapled transanal rectal resection (STARR) in ODS patients. PATIENTS AND METHODS: We performed a retrospective review of data that were collected from January 2005 to October 2008. Between January 2005 to June 2006, 58 patients who underwent STARR were enrolled in this study. Follow-up was scheduled for 3 months and 1 year after surgery with the Cleveland Clinic Florida (CCF) constipation score and satisfaction grade. To evaluate the long-term functional outcome, we interviewed the patients by telephone using questionnaires for the CCF score and satisfaction grade on October 2008. RESULTS: The median follow-up period was 34 months (range, 27-46 months). The mean age and sex ratio were 54 years (range, 19-85 years) and 8:50 (M/F). The mean CCF constipation scores were 17.6 before the surgery, 9.5 at 3 months, 9.6 at 12 months, and 10.3 at the time of the latest interview. The satisfaction grade, which was rated as excellent and good by 63.4% of the patients at the time of the latest interview, was worse than that at 3 months (37.8%). Among the cases of the excellent group (19 cases) at postoperative 3 months, 13 cases (68.4%) were classified as excellent or good at the time of the latest interview. CONCLUSION: The STARR is a safe and effective surgical procedure for restoring the anatomy and function in ODS patients. Strict selection of patients is needed in enhancing and maintaining the patients' satisfaction after the procedure.

Laparoscopic Ventral Mesh Rectopexy (LVMR) for Internal and External Rectal Prolapse: An Analysis of 122 Consecutive Patients.

BACKGROUND: Even though several reports have been published on the results of laparoscopic ventral mesh rectopexy (LVMR) in Asia, there are few mid-term or long-term results of LVMR. The authors aimed to evaluate the results of LVMR in patients with internal rectal prolapse (IRP) external rectal prolapse (ERP). MATERIALS AND METHODS: From September 2013 to January 2019, 122 patients with IRP (n=48) or ERP (n=74) underwent LVMR. Constipation and fecal incontinence (FI) scores were evaluated using the Cleveland Clinic Florida score preoperatively and postoperatively. The questionnaire for the change of obstructed defecation or FI symptoms after surgery was also administered to grade the results as cured, improved, unchanged, or worsened for each survey. RESULTS: The mean age of the patients was 61.9 years. The mean operation time was 116.5 minutes, and the mean hospital stay was 5.1 days. The mean follow-up was 42.1 months. There were no mesh-related complications. Eight patients (10.7%) among the ERP group required additional surgery for recurrent full-thickness prolapse. Eleven patients (14.7%) who had mucosal prolapse within 2 cm underwent stapled hemorrhoidopexy after LVMR. In the postoperative 6-month period, the overall constipation score (7.12) significantly improved compared with the preoperative score (13.03) (P<0.001), whereas the FI score significantly improved after surgery (12.16 to 8.92; P<0.001). CONCLUSION: LVMR is a feasible and safe technique and favorable recurrence for ERP. Functional outcomes of obstructed defecation and FI were improved and the satisfaction of LVMR was high after the surgery. LVMR can be considered a recommended surgical option to treat ERP and IRP.

Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant.

Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HT-GA733-FcK was infiltrated into the leaves of Nicotiana benthamiana plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1-10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated N. benthamiana leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration.

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.

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.

Genotyping by Sequencing-Based Discovery of SNP Markers and Construction of Linkage Map from F5 Population of Pepper with Contrasting Powdery Mildew Resistance Trait.

Powdery mildew (PM) is a common fungal disease infecting pepper plants worldwide. Molecular breeding of pepper cultivars with powdery mildew resistance is desirable for the economic improvement of pepper cultivation. In the present study, 188 F5 population derived from AR1 (PM resistant) and TF68 (PM sensitive) parents were subjected to high-throughput genotyping by sequencing (GBS) for the identification of single nucleotide polymorphism (SNP) markers. Further, the identified SNP markers were utilized for the construction of genetic linkage map and QTL analysis. Overall read mapping percentage of 87.29% was achieved in this study with the total length of mapped region ranging from 2,956,730 to 25,537,525 bp. A total of 41,111 polymorphic SNPs were identified, and a final of 1,841 SNPs were filtered for the construction of a linkage map. A total of 12 linkage groups were constructed corresponding to each chromosome with 1,308 SNP markers with the map length of 2506.8 cM. Further, two QTLs such as Pm-2.1 and Pm-5.1 were identified in chromosomes 2 and 5, respectively, for the PM resistance. Overall, the outcomes of the present endeavor can be utilized for the marker-assisted selection of pepper with powdery mildew-resistant trait.

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.