RNA sequencing and mechanistic data suggested that reactivation may occur through activation of the activator protein 1 signaling pathway. Stored samples from a prior clinical trial aimed at analyzing the effect of alendronate on bone mineral density, provided further evidence of alendronate-mediated latency reversal and activation of immune effector cells. Decay of the reservoir measured by IPDA was however not detected. Our results demonstrate the novel use of N-BPs to reverse HIV-1 latency while inducing immune effector functions. This preliminary evidence merits further investigation in a controlled clinical setting possibly in combination with therapeutic vaccination.Antiretroviral therapy (ART) is not curative due to the existence of cellular reservoirs of latent HIV-1 that persist during therapy. Current research efforts to cure HIV-1 infection include “shock and kill” strategies to disrupt latency using small molecules or latency-reversing agents (LRAs) to induce expression of HIV-1 enabling cytotoxic immune cells to eliminate infected cells. The modest success of current LRAs urges the field to identify novel drugs with increased clinical efficacy. Aminobisphosphonates (N-BPs) that include pamidronate, zoledronate, or alendronate, are the first-line treatment of bone-related diseases including osteoporosis and bone malignancies. Here, we show the use of N-BPs as a novel class of LRA: we found in  Despite viremia , 4. Bothex vivo expanded Vδ2 T cells to target and eliminate latently HIV-1-infected resting CD4 T cells upon ex vivo latency reversal (Aminobisphosphonates (N-BPs), like pamidronate (PAM), zoledronate (Zol), or alendronate (ALN), function by inhibiting the enzyme farnesyl pyrophosphate synthase in the mevalonate pathway, which results in 1) inhibition of the production of the key intermediate farnesyl pyrophosphate (FPP) and 2) accumulation of upstream isopentenyl pyrophosphate , 6. FPP reversal , 19. In reversal –24. We hex vivo exposure to N-BPs reverses HIV-1 latency most likely through the activator protein 1 (AP-1) signaling pathway. In vivo treatment of ART-suppressed people living with HIV (PLWH) with ALN induced viral reactivation although a significant reduction in the reservoir size was not detected in our small cohort. This study provides evidence of the novel use of N-BPs as LRAs and warrants further investigations with the specific objective to assess latency reversal and infected cell clearance in a larger cohort.Here, we show that ex vivo studies, frozen samples from PLWH on ART with undetectable plasma viral load (<50 copies/mL) for at least one consecutive year before inclusion in this study were obtained at the University of North Carolina, USA. Participants’ characteristics have been previously reported  doses  doses , 27. PAMex vivo analysis of HIV-1 caRNA levels, magnetically isolated rCD4T cells from PLWH on suppressive ART were exposed to N-BPs, PHA and IL-2, or left untreated. In some experiments, cells were exposed to VOR. After six hours of exposure, rCD4 T cells were washed and plated in 6-15 replicates of 1x106 cells per condition, depending on cell availability. After further washes, cells were pelleted and stored at -80°C, until RNA isolation. For the in vivo part of the study, thawed PBMCs were rested overnight, plated in 3-5 replicates of 1x106 cells (depending on cell availability), and stored at -80°C. Automated RNA extraction was performed , cDNA was synthesized using standardized protocols, and qPCR performed in triplicate using specific and validated primers, probes, and standard curves to amplify unspliced HIV-1 gag, as previously described  T cells (Stemcell Technologies) were rested in the presence of antiretroviral drugs . Cells were washed, counted and plated in limiting dilution from 1x106 to 0.1x106 cells. Cells were treated with 5U/mL IL-2 as the negative control or exposed 24 hours to PHA and IL-2, or to N-BPs PAM, Zol and ALN. After washing, isolated PHA-activated CD4 cells from uninfected individuals were added twice during the 19-day culture as targets of new infections to outgrowth the virus. After 15 days of culture, supernatants were harvested and HIV-1p24 ELISA performed and confirmed at day 19 of culture. The frequency of infection was calculated and expressed as infectious units per million (IUPM) cultured cells, as explained in Statistical Methods.HIV-1 p24 protein production (replication-competent virus as measure of infectious particles production) was analyzed in quantitative viral outgrowth assays (QVOA) , 29 afteIn order to explore the mechanism of action of N-BPs related to HIV latency reactivation, RNA was isolated from rCD4 T cells exposed to PAM or VOR for 6 hours, or left untreated.RNA was isolated using the automated KingFisher . All RNA samples were assayed for integrity, concentration, and fragment size on a TapeStation system .Library was constructed using Truseq RNA Exome library construction following the steps: Fragmenting RNA, Synthesizing 1st Strand cDNA, Synthesizing 2nd Strand cDNA, Adenylating 3’ Ends, Ligating Adaptors, Enriching DNA fragments, Hybridizing Probes to library, capturing Hybridized Probes, performing a 2nd Hybridization, performing a 2nd capture, Clean-up of captured library was performed via AMPure XP Beads followed by amplification of the enriched library, and a final clean-up of amplified enriched library using AMPureXP beads. Libraries were prepared on an Agilent Bravo Automated Liquid Handling System. Indexed libraries were prepared and run on HiSeq4000 to generated paired end 75 base pair reads which generated approximately 90 million reads per sample library with a target of greater than 90% mapped reads.RNA-seq reads were aligned to the human genome GRCh38.d1.vd1 from Genomic Data Commons using STAR v2.4.2a  . TranscrPhosphorylation status of p65 and c-JUN were analyzed by flow cytometry in isolated CD4 T cells (StemCell Technologies) as previously described . Briefly6 PBMCs , and concentrated using an ethanol precipitation protocol with GlycoBlue following manufacturer’s instructions . DNA concentration and integrity were measured by Nanodrop spectrophotometer (Thermofisher). Intact, hypermutated and/or 3’ defective, and 5’ defective HIV-1 copies/million cells were determined by droplet digital PCR (ddPCR) using the IPDA (env double-positive droplets) were corrected for DNA shearing based on the frequency of RPP30 and RPP30-Shear double-positive droplets.Total DNA was extracted from frozen cell pellets of 0.3-1x10the IPDA , where Hthe IPDA , and, inMass cytometry was performed in eight individuals treated with ALN and seven who took placebo using an optimized panel of 32 markers.Pairing of metals with antibodies was conducted using Fluidigm software . When commercial antibodies were not available, custom conjugations were performed using MaxPar X8 Antibody Labeling Kit (Fluidigm). Cell populations were phenotypically defined as previously reported , 37 and 6 PBMCs (pre-stained with CD45/89Y) were combined with 5x105 spiked control PBMCs from a single HIV-1-infected control donor that was consistently used in all the experiments (pre-stained with CD45/115In), as described (6 cells/mL with addition of 10% (v/v) EQ Four Element Calibration Beads (Fluidigm).In each experiment, 3x10escribed . Prior tData were acquired on a Helios model mass cytometer (Fluidigm) equipped with a WB injector.Data files were bead normalized and debarcoded using software provided by Fluidigm. Analysis was performed in collaboration with Astrolabe Diagnostics . Single-in vivo with ALN or PLACEBO were thawed and rested overnight then added to ELISpot plates  in quadruplicate (1 or 1.25 × 105 PBMC/well) with peptides pools of optimal CD8 T cell epitopes from either HIV-1-1 Gag/Nef  or Influenza, HCMV, and Epstein-Barr Viruses . PHA positive controls and media-only controls were also run. Samples were counted independently three times, and cell counts were averaged resulting in <10% variation. No minimum cell viability or cell recovery criteria was applied. Timepoints for each participant were run together. Antigens in thawed peptide plates were mixed with 1:1 with PBMCs in the ELISpot plate to a final concentration of 2 μg/ml and incubated for 18-20 hours at 37°C, 5% CO2. Coating, development (MabTech), and reading of ELISpot plates (AID Reader) has been described previously. Positive T cell responses were defined as ≥30 SFU per million, > 4 times the average of replicate background wells. Zero values were not accepted in any replicate of antigen-stimulated wells.ELISPOT was performed as previously reported . Brieflyex vivo analysis of HIV-1 gag caRNA below the limit of quantification (LOQ) were assigned a value of half the limit of quantification (LOQ=5 copies/mL). SLDAssay , and statistical analyses were performed in either GraphPad Prism v.9.4 or R version 3.5.3 (RStudio version 1.2).All participants provided written informed consent prior to inclusion in the study, and studies were approved by the George Washington and University of North Carolina Institutional Review Board.ex vivo studies and compared to Zol and ALN when sufficient cells were available. We utilized N-BP concentrations  that mimic plasma concentrations achieved in the blood with FDA-approved doses  T cells from 23 PLWH on suppressive ART , while PAM induced production of replication competent HIV-1 in all individuals with a group mean of 0.24 IUPM (range: 0.78 to 0.46)  , 49, 51.ex vivo untreated or 2.5μg/mL PAM-treated paired samples from six ART-suppressed PLWH. To exclude changes in gene expression, due to toxicity, a dose-curve analysis of PAM toxicity was performed showing that the selected dose did not impact cell viability  from the clinical trial ACTG A5163 that previously assessed the efficacy of ALN vs. placebo to increase bone mineral density in PLWH on ART , total HIV-1 DNA , total defective HIV-1 DNA , hypermutated/3’ defective , and 5’ defective . The mean proportion of intact proviruses out of total HIV-1 DNA at baseline was 11.5% for all participants, 9.9% for the ALN group and 13.5% for the placebo group  was performed on PBMCs, and no assay detection failures were observed with all 15 participants showing amplification of the Packaging Signal (Ψ) and HIV-1 DNA copies decayed from baseline to week 2 and remained stable in longitudinal measures until week 48 in both groups (We performed mass cytometry in eight participants from the ALN group and six from the placebo group. Over the course of ALN treatment frequencies of Vδ2 and Vδ1 T cells remained stable whereas effector memory (EM)-CD8 T cell frequencies decreased  can be found below: GSE224546 (GEO).The studies involving human participants were reviewed and approved by George Washington University and University of North Carolina. The patients/participants provided their written informed consent to participate in this study.Conception of the study: NSS. Data collection: MS, MLC, BTM, MAI, CPW, AC, CG, JK, SS, YX, DC. Data analysis and interpretation: MS, AMKW, ARW, MLC, SGR, SRS, MAI, CPW, ARC, YHT, YX, MGH, AB, BRJ, JSP, NG, NSS. Drafting the article: MS, AMKW, SGR, SRS, BTM, AW, MGH, NG, NSS. Critical revision of the article: MS, AMKW, ARW, JK, ARC, CG, AB, MGH, JSP, NG. Final approval of the version to be published: MS, AMKW, ARW, SGR, BTM, AW, AB, BRJ, JSP, MGH, NG, NSS. All authors contributed to the article and approved the submitted version.


Cucumis melo L.) is an important crop that is cultivated worldwide for its fleshy fruit. Understanding the genetic basis of a plant’s qualitative and quantitative traits is essential for developing consumer-favored varieties. This review presents genetic and molecular advances related to qualitative and quantitative phenotypic traits and biochemical compounds in melons. This information guides trait incorporation and the production of novel varieties with desirable horticultural and economic characteristics and yield performance. This review summarizes the quantitative trait loci, candidate genes, and development of molecular markers related to plant architecture, branching patterns, floral attributes , fruit attributes , and seed attributes (seed coat color and size). The findings discussed in this review will enhance demand-driven breeding to produce cultivars that benefit consumers and melon breeders.Melon ( Cucumis melo L.; 2n = 2x = 24) is an important diploid, cucurbit vegetable crop grown on approximately 1.5 million hectares worldwide. The total production of melons exceeds 30 million metric tons on a commercial scale in over 100 countries [Citrullus lanatus [Thunb.] Matsun. & Nakai), cucumber (Cucumis sativus L.), and squash (Cucurbita spp.), belongs to the Cucurbitaceae family, which consists of approximately 90 genera and 750 species [Cucumis genus is divided into two subgenera, Cucumis and Melo. Melon has been categorized into two subspecies, C. melo ssp. melo and C. melo ssp. agrestis, based on differences in ovary pubescence [Melon ; sugars ; organic acids (myristic and pantothenic acid); amino acids; phytochemical compounds, such as carotenoids (α- and β-carotene); vitamins ; fiber; and antioxidant enzymes. Two significant factors that affect fruit quality are the aroma profile and amount of carotenoids in the flesh. Carotenoids function as photoprotectors, visual attractants, and the precursors of fragrance compounds and phytohormones; carotene is a highly abundant carotenoid and is a primary source of vitamin A in food ,9.Melons exhibit the highest genetic diversity of phenotypic and biochemical traits, depending on the climatic zone and local preferences of the Cucurbitaceae family, allowing plant breeders to develop superior cultivars . The vitFruit traits are crucial attributes that affect consumer preferences and the concomitant selection and breeding of market-preferred varieties. Melon fruit yield is an important trait that differs substantially among cultivars, influenced by genetics, environmental factors, and cultivation practices. Melon fruit yields have been documented to vary between 6101 and 25,173 kg per hectare. In a study of 85 diverse melon varieties, the average yield per plant was found to range from 2.47 to 6.76 kg . Melon sThe advent of numerous molecular markers has enabled genetic analyses and molecular marker-assisted breeding (MAB) for qualitative and quantitative traits. Numerous commercially important traits of melons, including the yield and fruit quality, exhibit polygenic inheritance. Identifying quantitative trait loci (QTLs) using traditional breeding methods is challenging because of their complexity and interactions with the environment. In marker-assisted selection (MAS), a molecular marker associated with a particular characteristic is indirectly selected to supplement conventional breeding. Thus, genes that influence desirable characteristics can be selected using MAS. When used in conjunction with conventional selection methods, MAS has been confirmed to be an effective tool for selecting plants with desirable traits. Several genomic regions and candidate genes associated with essential horticultural traits, such as fruit quality attributes, ripening behavior, and carotene content, have been identified using QTL mapping analysis in molecular breeding programs ,14,15,16C. melo and C. agrestis. Numerous research projects have successfully located and created markers for traits such as plant architecture, floral biology, fruit and seed quality traits, and biochemical compounds [Genetic map construction is a reliable and crucial step in QTL detection and gene mapping. A limited number of markers, such as cleaved amplified polymorphic sequences (CAPS), simple sequence repeats (SSRs), restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), and random amplified polymorphic DNA (RAPD), have been exploited for generating genetic linkage maps for QTLs for various phenotypes in several populations ,25,26,27ompounds .Markers created for MAS applications must be closely related to genes or QTLs. It has been widely acknowledged that MAS must consider QTL validation, QTL effects, or fine-mapping with high resolution . MoleculThis review outlines the genetic regulation of qualitative and quantitative traits in melons to assist trait selection and integration, as well as the development of novel varieties with enhanced yield and other desirable horticultural characteristics. This review discusses the QTLs, major and minor functional genes, and the development of molecular markers related to horticultural traits, including plant architecture, branching patterns, floral biology, fruit and seed traits, and biochemical compounds.C. melo ssp. Cantalupensis), with the inbred, wild-type melon line, PNU-WT1 (C. melo ssp. Agrestis). In their study, the primary stem length of the F2 progenies indicated that a single recessive gene, mdw1, controlled dwarfism in this population. A genetic linkage map was constructed using SSR markers to determine the chromosomal location of mdw 1 and 76 SSR markers positioned on 15 linkage groups (LGs) spanning 462.84 cM localized the mdw1 to Chr. 7. They identified the candidate genes, ERECTA (serine/threonine kinase) and UBI (ubiquitin), in the genomic regions flanking mdw1 at distances of 0.6 and 1.2 cM, respectively.Dwarf and bush melon varieties are commercially important because of their concentrated fruit sets, lodging resistance, early maturity, tillering ability, and potential for planting at higher densities than regular vining types. Several dwarf crop traits, including short internode (SI) length, short mainstem length, and bush-type growth habits, have been reported in cucurbits. Plant dwarfism is primarily caused by mutations in the genes associated with hormonal biosynthetic pathways, including those that produce gibberellins (Gas) , cytokin2 and F2:3 populations derived from a cross between a “Bush Sugar Baby”  and a PCL-J1  cultivar and identified the QTL for semi-dwarfism (sdw) on Chr. 9 in watermelons using GBS. Furthermore, they performed QTL sequencing and identified a genomic region that matched the sdw-1 flanking region in the linkage map. After narrowing down the genomic region, they identified a candidate gene, ClCG09G018320, encoding the ATP-binding cassette (ABC) transporter B family member 19, which controls semi-dwarfism in BSB.In watermelons, Cho et al.  construccp locus at Chr. 4. After narrowing the cp locus to a genomic DNA region spanning 220 kb, an annotated gene with a predicted function was identified. This gene is a homolog of the cytokinin oxidase (CKX) gene and can serve as a candidate compact gene [2 plants derived from a cross between the pumpkin inbred lines, “Rimu” and SQ026. The F2 plants were genotyped using a GBS approach. A high-density genetic map containing 458 binary markers was constructed. Three QTLs in LGs 1, 3, and 4 were identified. One QTL, qCmB2, which was located at an interval of 0.42 Mb on LG 3, explained 21.4% of the phenotypic variations and a candidate gene, Cma_004516, encoding gibberellin (GA) 20-oxidase in the GA biosynthesis pathway was identified, suggesting that this gene is a possible candidate gene controlling the dwarf vine in pumpkin [In cucumber, genome-wide molecular mapping with SSR markers was conducted using two inbred cucumber lines, PI 308915 (compact vining) and PI 249561 (regular vining), and a linkage map consisting of 187 SSR markers covering 537.5 cM was identified. Linkage analysis placed the act gene  generate pumpkin .Melons are monoecious or andromonoecious, with male flowers located on the main stem and female or hermaphroditic flowers on lateral branches; the plant grows on a long, trailing vine. Therefore, lateral branching is a key element in melon plant architecture. Three main growth patterns are evident in muskmelons: vining, SIs, and bird nests (multiple branches). Fruit is often set on vining types away from the center of the plant. As the fruits do not mature consistently, they are harvested separately. Cultivating and controlling weeds is challenging as the vines extend between rows. SI muskmelons have fewer and shorter internodes and more restricted leaf surfaces per plant than vining muskmelons . Accordi2 plants resulting from a hybrid between “Chukanbohon Nou 4 Go” (Nou-4) and “Earl’s Favorite” (Harukei-3) to create a linkage map to uncover molecular markers for MAS of the short lateral-branching (Slb) gene. Nou-4 is a weedy melon with a short, lateral-branching trait. This characteristic is governed by a single recessive or partially dominant major gene called slb. QTL analysis of the F2 population revealed two loci for short, lateral branching. With an LOD score of 12.5, a major QTL in LG XI, in which the slb allele from Nou-4 has been associated with short, lateral branching, explained 50.9% of the phenotypic variance. With an LOD score of 4.2, another minor QTL in LG III, where the slb allele from Harukei-3 explained 9.9% of the phenotypic variance, was related to short, lateral branching. This study also showed that a QTL in LG XI (slb) coupled to an SSR marker could be utilized to select short, lateral branching.Fukino et al.  used 94 SHORT VEGETATIVE PHASE (SVP)-like genes, including CmSVPa, CmSVPb, CmSVPc, and CmSVPd, in melons; the gene structure of CmSVPc differed from those of the other CmSVP-like genes. Overexpression of CmSVPc in Arabidopsis leads to increased lateral branches. However, CmSVPc-RNAi plants in melons demonstrated the repressed formation of lateral branches, shorter internodes, reduced petal size, and more compact fruits compared to the control plants, suggesting that CmSVPc might be a candidate gene for maintaining the growth of lateral branches in melons. A lateral branchless trait was also identified in watermelons from the inbred WCZ, and genetic analysis revealed that it was controlled by a single recessive gene, Clbl (Citrullus lanatus branchless) [Clbl in the watermelon Chr. 4. This demonstrated that the candidate gene of branchless watermelons, Cla018392, belongs to the TERMINAL FLOWER 1 (TFL1) gene family, implying a genetic control for lateral branching that is different from that in melons.Fang et al.  identifinchless) . Linkageandromonoecious (a) and gynoecious (g), or CmACS-7 and CmWIP1, respectively. The allele “a” regulates the presence or absence of the pistil in female flowers, whereas the allele “g” regulates the presence or absence of two different types of flowers on the same plant [aagg), gynoecious (Aagg), monoecious (A_ G_), and andromonoecious (aaG_) [a encodes the enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), which prevents the development of stamens in female flowers [ACS, which is encoded by the CmACS-7 on Chr. 2 and is homologous to the ACS-7 gene in Arabidopsis [The floral biology and sexual expression of melon flowers are crucial characteristics in melon improvement programs. Commercial melon cultivars are typically andromonoecious and produce male and bisexual (perfect) blooms in the developmental stages. To avoid artificial emasculation and ensure hybrid purity, gynoecious sexual expression, which produces female or pistillate flowers, is extremely valuable in melon heterosis breeding. In melons, two key genes have been implicated in regulating sex expression: me plant . Four dis (aaG_) . Locus a flowers . The pribidopsis .CmACS-7, which is required for ethylene synthesis. Complete sequences of CmACS-7 were cloned from monoecious (MO23) and andromonoecious (AM24) lines, and the alignment of these CmACS-7 sequences revealed an SNP (C170T) in exon 1 and an 18 bp indel in the 3′-untranslated region (UTR) between MO23 and AM24. Subsequently, the SNP and indel were used to develop a CAPS (EX1-C170T) and a sequence-characterized amplified region (SCAR) marker (T1ex). Sex expression was determined in 442 F2 plants obtained from a cross between the MO23 and AM24 plants. The sex types of 429 plants (13 not classified) co-segregated with the SCAR marker, indicating that sex expression mediated by CmACS-7 is controlled by a single dominant gene that confers monoecy in the line, MO23. Daryono and Prasetya [CmACS-7 in different sexual forms of melon flowers using RT-PCR and found a higher expression in female and hermaphrodite flowers than that in male flowers. Moreover, the expression of CmACS-7 was higher in male flowers with an andromonoecious sexual type than in male flowers with a monoecious sexual type. Recently, a GWAS revealed the SNPs on Chr. 1 and 8 associated with sex expression in the oriental melon and identified four promising candidate genes: MELO3C015898 (transport inhibitor response 1), MELO3C015904 (SWR1-complex protein 4/DNA methyltransferase 1-associated protein 1), MELO3C024563 (putative UDP-N-acetylglucosamine—peptide N-acetylglucosaminyltransferase SPINDLY), and MELO3C024565 (MRNA-decapping enzyme-like protein) [Kim et al.  developePrasetya  investigprotein) .CmACS-7. Additionally, environmental factors and growth regulators can influence sexual expression, highlighting the plasticity of this trait in melons.Sex expression in developing flower buds is plastic; therefore, the sex of any particular flower at a given node depends on various genetic, developmental, environmental, and hormonal factors. External variables, such as mineral nutrition, temperature, water regulation, illuminance, photoperiod, mechanical trauma, and the use of growth regulators, can alter sex expression in melons . Growth Dysfunctional Tapetum 1 (DYT1) [ABORTED MICROSPORES (AMS) [basic Helix–Loop–Helix (bHLH) [Male sterility (MS) is a key feature observed in various higher plants and is essential for producing low-cost hybrid seeds because it eliminates the requirement for cross-pollination and emasculation. Disorders in tapetum formation and abnormal pollen growth are closely related to the occurrence of MS. The abortion of pollen results from mutations in the genes at various pollen developmental stages such as 1 (DYT1) , ABORTEDES (AMS) , and basx (bHLH)  transcrims-1 to ms-5. ”Punjab anmol” [1 hybrids, were successfully developed using the ms-1 GMS inbred line. To introduce MS into fertile melon cultivars and breeding lines to construct parents for F1 hybrid seed production, Park and Crosby [SOAM08.644) that was tightly linked to the ms-3 gene at 2.1 cM on the LGs. A linkage map was constructed based on SSR markers for the ms-1 locus using an F2 population from a cross between MS-1 and KP4HM-15 [2 plants mapped three SSR markers, DM0187, DM0038, and TJ14, to ms-1 on Chr 6. Of these, marker DM0187 was closest to the gene at a genetic distance of 4.8 cM and was successfully used to transfer the ms-1  gene into elite muskmelon genotypes through MABC [ms-5 was mapped to a 30 kb genomic region on Chr. 9 using BSA combined with specific length amplified fragment sequencing (SLAF-seq) [2 individuals.Genetic male sterility (GMS) in melons involves five recessive genes, b anmol” , and “MHb anmol” , which ad Crosby  developeKP4HM-15 . Linkageugh MABC . SubsequLAF-seq) . The CAPLOC103498166 AMS gene was expressed differentially during the pollen tetrad formation period in MS lines (ms-5) and male-fertile (HM1-1) lines, suggesting that AMS is a possible candidate gene for MS in melon [Furthermore, six genes were annotated in the 30 kb genomic region, of which the in melon .2 segregation population from a cross between green and yellow rind accessions [7 generation, resulting from a cross between a light rind, honeydew parent (“Tam Dew”), and a dark rind, reticulatus parent (“Dulce”). The second population comprised 114 families at the F3:4 generations, obtained by crossing “Dulce” with another light rind accession of yellow casaba, inodorous melon. The GWAS identified a genomic region (640–930 kb) on Chr. 4 that is associated with the rind color. Furthermore, they mapped the target genomic region and found 33 putative genes, including MELO3C003375 annotated as an ARABIDOPSIS PSEUDO-RESPONSE REGULATOR2-LIKE gene (APRR2), that were responsible for the light rind phenotype in melons [w) mutation [The rind color in melons varies from yellow-green, light to dark green, and orange. Melons can also have mottled or striped rinds. The dark- and light-green rinds of melon fruit exhibit qualitative variations in the chlorophyll content. Prior research has found that in a biparentally segregated population, a light immature rind color exhibits recessive single-gene inheritance . Green rcessions . Oren etcessions  performen melons . The afomutation .CmKFB (MELO3C011980). Yellow and white rind accessions were genotyped using a PCR marker at CmKFB to validate SNP3541676 and SNP3541866; co-segregation was demonstrated using the QTL marker for yellow and white rind color.Gur et al.  characteMELO3C003375 on Chr. 4 encoding the two-component response regulator-like protein, APRR2, and the previously identified gene, CmKFB (MELO3C011980), on Chr. 10, which negatively regulated naringenin chalcone accumulation. Naringenin chalcone is a yellow flavonoid pigment that is considered one of the primary pigments affecting melon rind color. MELO3C003375 was expressed at much higher levels during the gene expression test than in the white and yellow rind-colored lines. However, CmKFB was hardly expressed in yellow rind color lines, which was consistent with its function of negatively regulating flavonoid accumulation, suggesting that MELO3C003375 and CmKFB are associated with green and yellow rind colors, respectively. An additional gene, MELO3C003097, was detected by Zhao et al. [Arabidopsis SG1, which encodes the protein SLOW GREEN 1 and is required for chloroplast development, suggesting that MELO3C003097 might be a minor gene involved in the formation of green, white, and yellow rind colors.Later, Zhao et al.  detectedo et al.  on Chr. CmMt1 and CmMt2, regulate the formation of mottled rinds by mapping the F2 population derived from SC (mottled rind) and MG (non-mottled rind). The observed phenotypic segregation ratio suggested that immature rind color exerted an epistatic influence on the mottled rind phenotype. This effect was regulated by a specific gene, CmAPRR2. To further validate this, a Kompetitive allele-specific PCR (KASP) DNA marker, CmAPRR2SNP(G/T), was developed, which demonstrated co-segregation with the rind color trait, suggesting that CmAPRR2 corresponded to CmMt1. Using BSA-seq and KASP markers, CmMt2 was successfully fine-mapped to a specific region of 40.6 kb, containing six predicted genes. MELO3C026282, a homolog of AtCPSFL1, emerged as the top candidate gene for CmMt2 based on functional annotation, expression analysis, and sequence variation analysis. Additionally, investigations involving pigment content measurements and transmission electron microscopy have revealed that CmMt2 is likely to play a role in chloroplast development, subsequently leading to reduced chlorophyll accumulation [Shen et al.  reportedmulation . Shen etmulation  attribut2 population from the cross “Piel de Sapo” × PI124112, Diaz et al. [C. agrestis and melo). Pereira et al. [FW5.1, FD5.1, and FP5.1, within a 496 kb genomic region containing 48 predicted genes. Among these genes, MELO3C014402, which encodes the protein FANTASTIC FOUR 2, was suggested to be a potential QTL for FW [CmSUN family genes and found that the expression of most CmSUNs was specifically enriched in the reproductive organs of melons, including young flowers and ovaries. Furthermore, the overexpression of CmSUN23-24 and CmSUN25-26-27c resulted in increased FSI, indicating that these genes are important regulators of melon fruit shape variation. Using a BSA-seq from B8 (long-horn fruit) and HP22 (flat-round fruit) melon inbreds, Ma et al. [CmFSI8/CmOFP13, which encodes the OVATE family protein (OFP)  and the ratio of fruit length (FL) to fruit diameter (FD). Various QTLs for FSI have been identified and are related to QTLs for FL and FD ,27,68,69z et al.  found SNa et al.  identifiL for FW . Later, L for FW  used a ga et al.  identifiin (OFP) . In addiThe color of melon fruit flesh is determined by chlorophyll and carotenoid pigments, resulting in distinctive white, green, and orange colors . β-carotgf) and white flesh (wf). The presence of Gf determines the orange flesh, which is dominant over the green flesh (gf). Melons with gfgf have either white flesh (wfwf) or green flesh (Wf-) [CmOr, formerly known as the gf (green flesh) locus in melons, was found to co-segregate with fruit flesh color in segregating F2 and backcrossing populations derived from a cross between light-green (“Tam Dew”) and orange (“Dulce”) flesh melon genotypes and presented two haplotypes  of the CmOr-encoding protein ORANGE-ORANGE, chloroplastic, one of which was associated with orange flesh and the other with either white or green flesh [CmOr and was pivotal in determining the orange and non-orange phenotypes observed in melon fruit [CmOr in the Arabidopsis callus system stimulated the accumulation of β-carotene. Through site-directed mutagenesis of the CmOr green/white allele, the substitution of arginine with histidine resulted in a marked increase in β-carotene accumulation.Fruit flesh color in melon is a complex trait that is quantitatively controlled by two major epistatic genes: green flesh (sh (Wf-) ,80. The en flesh . A specion fruit . OverexpC. agrestis and ssp. melo) and found that color phenotypes were uniformly distributed within the GWAS panel, with green, white, and orange color categories representing substantial proportions. Using GWAS, they identified a single SNP on Chr. 9  that had a substantial effect; this SNP was located within the causative CmOr gene (MELO3C005449), indicating that ~70% of the flesh color variation was across the panel and effectively distinguished between orange and non-orange. Additionally, all lines were genotyped for polymorphisms in CmOr to validate SNP20550439, exhibiting a complete co-segregation of CmOr with orange and green flesh colors  on Chr. 8 as a candidate for white flesh. CmPPR1, which encodes a member of the pentatricopeptide protein family, is involved in RNA processing in plastids, where carotenoids and chlorophyll pigments accumulate. The CmPPR1 gene is positioned within the 2–5 Mb region previously associated with the wf locus in white flesh [Gf gene (CmOr) controlling orange flesh [Wf locus, which regulates white and green flesh, as reported by Perin et al. [Galpaz et al.  conductete flesh . Zhao ette flesh  conductege flesh . Notablyn et al. .Wf locus on Chr. 8. When the QTL mapping and GWAS results were combined by Zhao et al. [MELO3C003069 (CmPPR1), by Galpaz et al. [Wf, was 202 kb away from the mapping interval found in the study. One of the 11 genes, MELO3C003097, which is the ortholog of SG1 in Arabidopsis and has been reported to be essential for chloroplast development and chlorophyll biosynthesis [MELO3C003097 is a promising candidate gene for the Wf locus in green flesh. CAPS markers have been utilized for the MAS of fruit flesh color, rind color, and shape attributes [To further identify candidate genes, researchers developed an RIL population from a cross between an orange flesh line, “Védrantais,” and a white flesh parental line, “Piel de Sapo,” for QTL mapping. They identified a significant QTL corresponding to the o et al. , a 96 kbz et al.  for Wf, ynthesis , showed tributes .The physical appearance of fruits of different melon varieties varies depending on the skin surface; fruits of some varieties have a smooth skin appearance, whereas others exhibit different types of reticulation decorations, referred to as netting. Reticulation, or rind netting, is a term for ligno-suberized patterns that vary in degree and beautify the melon cuticle . NettingqND6 on LG6 was the sole QTL identified for net density (ND). Notably, this QTL co-localized with qNW6, one of the four QTLs mapped for net width (NW) [qND2, qND4, and qND7) in LG2, LG4, and LG7, respectively, were found to co-localize with QTLs for NW . These findings suggest a strong correlation between ND and NW, with NW influencing ND. However, only two QTLs associated with NW (qNW2 and qNW7) were consistently detected in both trials [qND4, qND6, and qND7) and netting width  [NDen2.1 and NDen2.2, were identified on Chr. 2 across 9 and 2 Mb, respectively, with a genetic interval of 12 cM [NDen9.1, was reported on Chr. 9, with a genetic interval of 12 cM across 734 kb [NDen2.1, NDen2.2, and NDen9.1) are responsible for the variation in rind ND [Few studies have been conducted to identify the QTL associated with rind netting in melons. Based on the QTL analysis in Trial 1, dth (NW) . In Triah trials . Furthernd qNW7) . Two QTLof 12 cM . A thirds 734 kb . All thr rind ND .makuwa, of which Rth.6 for rind thickness was co-localized on Chr. 6 with net.6, which accounted for the less netted fruits. Additionally, two other QTLs, net.5 and net.7, were associated with the reduced netting variety in makuwa [CmSN) locus in melons using GWAS by crossbreeding the H906 line with smooth fruit and the H58 line with netted fruit. They observed that CmSN was controlled by a single dominant gene located on Chr. 2 within an area of ~351 kb. Furthermore, they delimited the genomic region to a 71 kb region containing five genes. Among these, MELO3C010288, encoding a protein in the EamA-like transporter family, was the most likely gene responsible for the netted phenotype [Various QTLs for rind thickness and netting have been identified in the introgression lines of melon var. n makuwa . Wang etn makuwa  identifin makuwa  studied henotype .Melon fruit yield is an important trait that differs substantially among cultivars. According to FAOSTAT , the glo2 population derived from the thick-skinned line, “Elizabeth,” (M4-5), and a thin-skinned line, M1-15. Derived-CAPS (dCAPS) markers have been developed for the MAB of fruit-related traits in melons. Most QTLs associated with fruit-related traits  were clustered on Chr. 6 and 9 [FWt2.1, FWt4.1, and FWt9.1) and one major QTL for fruit length (FL12.1) were mapped, based on SNP-derived CAPS markers in melon [Zhang et al.  construc 6 and 9 . The pos 6 and 9 . Amanull 6 and 9  identifiin melon .2 populations, WAP and MAP, using WGRS. These populations originated from different crosses involving cultivated and wild agrestis melons. From this study, 871,671 and 1,976,589 high-quality SNPs were identified in WAP and MAP, respectively. They identified two key loci influencing melon fruit size: one on Chr. 11 in the WAP population and another on Chr. 5 in the MAP population. In the WAP population, two QTLs linked to FD and weight were located on Chr. 11, sharing a common 130.8 Kb region. Notably, within this overlap, the MELO3C025758 gene, encoding an auxin response factor (ARF), was pinpointed as a possible candidate gene in melon fruit size. For the MAP population, a region on Chr. 5 was identified that impacted FW and length. Within this region, the gene, MELO3C004493, which encodes a YABBY transcription factor—a type previously linked with tomato fruit size—was found. As a result, both MELO3C025758 (ARF) and MELO3C004493 (YABBY transcription factor) are postulated to be candidate genes for melon fruit size [Lian et al.  developeuit size Melon seeds are rich in vital bioactive compounds, including tocopherols, phospholipids, and sterols, and offer substantial health benefits . Melon s2:3 families were produced using cantaloupe inbred ms5 and muskmelon inbred HM1-1 as the parental lines. QTL mapping of the F2:3 families showed that a single dominant locus (SCC) controlled the seed coat color, and the white color was dominant over yellow [2 segregation population and SLAF markers and constructed 12 LGs with a map distance of 1356.49 cM (an average genetic distance of 0.37 cM). The locus, SC12.1, responsible for the seed coat color (SC) in muskmelon, was located on Chr. 12 within the genomic region, spanning 12,785,226 to 15,029,452 bp. These findings lay the groundwork for future gene cloning and functional analyses to elucidate SC traits in muskmelon [To explore the genetic inheritance of seed-related variables using major gene and poly-gene genetic models, Fr yellow . Li et ar yellow  used an uskmelon .2 population derived from IC2508 with yellow and IC2518 with brown seed coats and identified a genomic region of 11,860,000–15,890,000 bp (4.03 Mb) on Chr. 9. Subsequently, a 233.98 kb region harboring 12 annotated genes was finely mapped and a single dominant gene CmBS-1 (MELO3C019554), encoding homeobox protein (PHD transcription factor), was identified as a candidate gene for yellow SC. MELO3C019554 was expressed at lower levels in yellow seed coats than in brown seed coats. It has been reported that MELO3C019554 is related to 12 flavonoid metabolites.Hu et al.  carried 2:3 population resulting from crossing large-seeded inbred ms-5 and small-seeded inbred HM-1 to carry out QTL analysis for seed size. Four chromosomes  had seven QTLs for seed length (SL), seed width (SW), and 100 seed weight (100SWT). Two major-effect QTLs, Sl11.1 and SW11.1, controlling SL and SW, were identified in the same genomic region on Chr. 11. They exhibited a marked phenotypic variation explained (PVE) of 17.5% and 19.5%, respectively. Ye et al. [1 population from a cross between MR-1 (long seed) and M1-15 (short/narrow seed). Two QTLs for seed length and seed diameter, SL5.1 and SD5.1, were located on Chr. 5, explaining a PVE of 9.9–11.2%, and two QTLs, SL1.1 and TGW1.2, were positioned on Chr. 1. Later, Pereira et al. [SWQU8.1) positioned on Chr. 8 with 19.9% PVE.Seed size plays a crucial role in determining the evolutionary fitness and agronomic characteristics of plants during domestication and breeding. Seed size substantially affects the seed yield, eating quality, and tolerance to environmental stresses ,101. Thee et al.  identifia et al.  identifi2:3 population by crossing ms-5 and HM1-1. The major QTLs, sl11.1 and sl11. 2, for SL and 100swt11.1 for 100SWT were identified on Chr. 11, while the QTL, sd2.1, for SW was detected on Chr. 2. Furthermore, Amanullah et al. [2 plants derived from M4−75  and X207  to map QTLs for SL, SW, seed weight, and seed thickness. They identified three QTLs for SL, SW, and thickness on Chrs 3 and 9. A major QTL, SW3.1, was detected on Chr. 3 with an 2R of 13.32%. Jiao [Cm11.1 is likely to play a substantial role in regulating the seed size and weight variation in melons. Candidate genes for the seed size in melons have been cloned from the 43.47 kb–1.89 Mb genomic region of the seed trait QTLs (qSL3 and qSWD3) on Chr. 3, using the QTL mapping approach of Zhang et al. [qSL3 and qSWD3 on Chr. 3, they designed and utilized 18 KASP markers within the QTL region. Employing regional QTL mapping analysis, they successfully confirmed the existence of qSL3 and qSWD3 within the genomic region delimited by the KASP markers, A288 and A291, spanning approximately 522 kb in length  [EVM0009818-encoded cytokinin-activated signaling was selected as a candidate gene. Based on RNA-seq data, this gene was differentially expressed in the young fruits of the parent plants. These findings lay the foundation for future research to elucidate the genetics of seed size and ultimately provide more efficient and targeted breeding strategies for melons [Zhang et al.  mapped sh et al.  utilized2%. Jiao  and Zhan2%. Jiao  detectedg et al. . To vali,031 bp) . Furtherr melons .Cucumis melo subsp. melo var. flexuosus) with “Noy Yizre’el” (subsp. melo var. reticulatus), a cultivar with a high sucrose and total sugar content, F1 plants had a slightly higher sucrose content than the low-sucrose parent. High sucrose buildup in melon fruit flesh was proposed to be mediated by a single recessive gene based on the segregation of F2 and backcross progenies. Diaz et al. [Sucrose accumulation during the last phases of fruit development is primarily responsible for the increase in the total sugar content in mature melon fruit. Given that sucrose dominates sugar composition, it is crucial for potential genetic advancements to elucidate the molecular mechanisms underlying sucrose accumulation. Burger et al.  discoverz et al.  construc2 extreme phenotypes, and discovered 6 sweet trait-related regions containing 13 differential SLAF markers and 62 genes and 23 source-related trait regions with 48 differential SLAF markers and 185 genes. Furthermore, fine-mapping mapped sweet traits to genomic regions on Chr. 6, 10, 11, and 12, and sour traits in the genomic region of Chr. 2, 3, 5, 9, and 12. Subsequently, they located 9 of the 61 differential markers on the parental genome in the sweet and sour trait candidate regions. Of these nine markers, one marker corresponded to sweetness, and eight markers were associated with source-related genes.Later, Zhang et al.  performeN-acetylglucosaminyl transferase III (MELO3C011944T1), was correlated with sweetness. Harel-Beja et al. [SUCQSC5.1., on Chr. 5. The authors determined that it was responsible for reducing the soluble solid content (SSC) and sucrose content, and MELO3C014519 was considered a candidate gene [The marker, SLAF18745, located in a gene encoding a et al.  construcate gene .CmTST1, CmTST2, and CmTST3, from melon plants and found that CmTST 2 played a crucial role in sugar accumulation in melon fruits.Plants synthesize sugar in the mesophyll cells of leaves, which is then transported to other plant parts. Shin et al.  identifiMelons are known for their refreshing aromas, which are attributed to the presence of a mixture of volatile compounds. Esters, aldehydes, alcohols, ketones, and terpenes predominantly contribute to a distinct melon aroma. The profile and concentration of these volatile compounds can vary substantially among different melon varieties, contributing to a wide range of aromatic characteristics from fruity to floral, green, and even herbaceous notes. For instance, in honeydew melon, the principal volatile compound is -2,6-nonadienal, which gives it a fresh, cucumber-like aroma . CantaloLOX genes in the melon genome. Their findings, derived from RT-PCR and qPCR expression analyses, indicated varying expression levels across the 18 CmLOX genes. These differences were noted in both vegetative and reproductive tissue samples throughout the multiple developmental phases of melon fruit. This suggests that specific CmLOX genes play a critical role in producing volatile compounds in oriental melons. Furthermore, Galpaz et al. [CmThAT1 (MELO3C024190), encoding S-methyl-thioacetate (thiol acyltransferase), was identified in 10 genes proximal to the QTL genomic region; S-methyl-thioacetate, a main constituent of melon fruit aroma, indicated that fruit aroma was controlled by the CmThAT1 gene.Zhang et al.  have idez et al.  identifiMayobre et al.  construcMELO3C024886) encoding 4-coumarate:CoA ligase, which is potentially a part of the eugenol pathway. The study also highlighted other genes, including CmLOX18, CmAAT, CmBAMT, and CmCRTISO1, which have been reported in previous studies as key players in the production of melon VOCs from sources such as fatty acids, amino acids, and carotenoids. Candidate gene and gene-based molecular markers linked with horticultural traits in melons identified in previous studies are reported in Later, Perpina et al.  performeAlthough molecular markers facilitate MAB in melons, they are characterized by several limitations and challenges. Marker density in melons is relatively low, which hampers the fine-mapping of crucial agronomic genes. In the realm of crop improvement through molecular techniques, several critical limitations arise. Firstly, there is the intricate challenge of dissecting traits using markers, especially when dealing with gene–environment (G × E) interactions. These interactions often complicate the clarity of results and make the identification of influential genes more arduous. Additionally, a constrained gene pool presents issues, as limited genetic diversity can impede the potential for groundbreaking advancements. From an operational perspective, the financial costs associated with genome analysis and the development of markers and genotyping procedures, particularly in MAS, can be substantial. Even when finances are available, the science itself is not always straightforward; there are inherent limitations in accurately identifying genes and in the subsequent development of gene-based markers. Finally, with the advancements in the field, new traits emerge that await thorough analysis, adding to the list of challenges to be addressed.Quantitative traits are inherently complex owing to the involvement of multiple genes, making their improvement through molecular markers challenging. Accurate phenotyping is a major obstacle, as environmental factors can cause variations in phenotypic and genotypic traits. Furthermore, short-range linkage disequilibrium complicates marker-trait associations, and functional genomic resources are limited, impeding the precise identification of gene function. Finally, the cost and time-intensive nature of high-throughput genotyping restrict its widespread use, particularly in developing countries. Overcoming these challenges will necessitate concentrated efforts to increase marker density, improve our comprehension of the genetic basis of complex characteristics, enhance phenotyping capabilities, and develop cost-effective genotyping technologies.In the rapidly evolving field of melon research, genomic tools and markers have provided valuable insights into the intricate genetic underpinnings of various traits. Plant architecture, a fundamental aspect influencing cultivation practices, and sex expression, crucial for reproductive efficiency, have been dissected at the genomic level, enabling the identification of markers that can predict these traits with enhanced precision. Fruit characteristics, including shape, rind color, netting, and flesh color, which substantially influence consumer preferences, have been mapped to specific genomic regions. Notably, markers associated with fruit yield—a critical trait for commercial viability—offer breeders a refined tool to enhance productivity. Seed attributes, encompassing seed size and coat color, have similarly benefited from genomic investigations, with markers elucidating the genetic basis for these variances. Perhaps the most exciting advancements lie in the realm of biochemical traits. Genomic markers linked to sugar content and volatile aromatic components—direct determinants of flavor profiles—are revolutionizing the way we perceive melon quality. These markers provide a roadmap for breeders, enabling them to tailor melon cultivars for optimum sweetness and aromatic appeal.The past century has witnessed considerable advancements in melon breeding, and we anticipate that this trend will continue. Crop improvement depends largely on conventional breeding approaches. The advent and application of MAS have ushered in a transformative era in melon breeding. By enabling the precise identification and incorporation of desirable genes into breeding lines, MAS has heightened the potential for genetic gains. Traditional breeding often demands extensive time and resources, relying heavily on phenotypic evaluations that may not always translate to the desired genotypic modifications. MAS circumvents this by pinpointing specific genetic markers linked to beneficial traits, allowing breeders to make informed decisions early in the breeding cycle. This precision not only reduces the guesswork inherent in conventional breeding but also substantially expedites the overall breeding process. By accelerating the development of robust melon varieties and enhancing the predictability of desired outcomes, MAS plays a pivotal role in addressing food security challenges and fulfilling the increasing global demand for resilient crops. In essence, through the power of genetic markers and informed breeding strategies, MAS stands as a cornerstone for the future of efficient, effective, and advanced melon cultivation.Although MAS has increased the effectiveness of crossing and selection, it is limited for crops with complex genetics. The use of DNA microarrays and restriction site-associated DNA (RAD) sequencing will accelerate the genome mapping and tagging of additional QTLs as the cost of genome sequencing decreases. These QTLs may be utilized to introduce resistance into genotypes of high-yielding melons, and several QTLs may be combined with genes with established resistance to other races. A large amount of resequencing data have facilitated the discovery of an increasing number of polymorphic SNPs in melons. The current large-scale sequencing of melon genomes will enable the collection of accurate data on genes with crucial biochemical properties.Current breeding programs need to incorporate the latest approaches, including whole-genome selection, mutation detection, high-throughput genotyping, genetic transformation, omics, and genome editing, to create critical opportunities to improve fruit quality and accelerate future melon breeding . Genome


MYBH and RSM in the MYB family play crucial roles during critical transitions in the culm neck development, such as changes in the angle between the rhizome and the culm neck. Our new findings provide essential insights into the cellular roadmaps, transcriptional networks, and key genes involved in the culm neck development.The Moso bamboo culm neck, connected with the rhizome and the shoot bud, is an important hub for connecting and transporting the aboveground and belowground systems of bamboo for the shoot bud development and rapid growth. Our previous study revealed that the culm neck generally undergoes six different developmental stages (CNS1–CNS6), according to the primary thickening growth of the underground shoot bud. However, the molecular mechanism of the culm neck development remains unknown. The present study focused on the developmental process of the CNS3–CNS5 stages, representing the early, middle, and late elongation stages, respectively. These stages are densely packed with vascular tissues and consist of epidermis, hypodermis, cortex, and ground tissue. Unlike the hollow structure of the culms, the culm necks are solid structures. As the culm neck continues to grow, the lignin deposition increases noticeably, contributing to its progressive strengthening. For the transcriptome analysis, a total of 161,160 transcripts with an average length of 2373 were obtained from these stages using both PacBio and Illumina sequencing. A total of 92.2% of the reads mapped to the Moso bamboo reference genome. Further analysis identified a total of 5524 novel genes and revealed a dynamic transcriptome. Secondary-metabolism- and transport-related genes were upregulated particularly with the growth of the culm neck. Further analysis revealed the molecular processes of lignin accumulation in the culm neck, which include differentially expressed genes (DEGs) related to cell wall loosening and remodeling and secondary metabolism. Moreover, the upregulations of transcription factors such as  Phyllostachys edulis) is one of the fastest growing plants [Moso bamboo  [Several studies have reported that the shoot growth is controlled at multiple levels, such as cell division, rapid division, rapid elongation, and secondary cell wall thickening. Hormones, such as auxin and gibberellin, and their possible downstream target genes, are closely associated with the shoot growth; signal transduction, DNA synthesis, and RNA transcription are the key regulators of the rapid growth ,12,13,14T, etc.) . This suIn addition to the shoot developmental stages, our previous study  on the cOur previous work reported that the culm necks at the CNS3, CNS4, and CNS5 stages primarily undergo elongation, with the CNS3 stage being the most active growth stage [The culm necks in the CNS3, CNS4, and CNS5 stages, each with five pooled samples, were sequenced using the PacBio Sequel platform to obtain the full-length transcript sequences. A total of 15,229,362 subreads (16.68 Gb) were obtained, with an average read length of 1096 bp and N50 of 2137 bp . In addiThe study compared all the corrected transcripts against the Moso bamboo reference genome  using GMFurther analysis identified a total of 32,394 lncRNAs divided into four groups: 10,867 (22%) long intergenic non-coding RNAs (lincRNAs), 1676 (20%) intronic sense RNAs, 947 (11%) intronic antisense RNAs, and 3980 sense-overlapping lncRNAs (47%). These classifications were determined using the CNCI, Pfam, PLEK, and CPC methods e. MoreovIn-depth gene expression analysis revealed that totals of 30,338, 30,841, and 29,267 genes were expressed in the CNS3, CNS4, and CNS5 stages, respectively a. Venn dPAL, C4H, C3H, CCoAOMT, F5H, and CAD. In addition, there were cellulose-related novel genes, encompassing precursors, cellulose synthesis, hemicellulose synthesis, cell wall proteins, and genes involved in degradation and modification   and leavTo identify differentially expressed genes (DEGs) within the culm neck, the study analyzed the differential expressions across these developmental stages via comparisons. The results showed that 63, 470, and 574 genes were differentially expressed in the CNS4/CNS3, CNS5/CNS3, and CNS5/CNS4 stages, respectively a,b. AmonINV (Invertase), PMEPCRA (Pectin Methylesterase), PE (PE protein), XTH9 (Xyloglucan Endotransglucosylase/Hydrolase 9), PNP (Polynucleotide Phosphorylase), EXPAs (Alpha Expansin), TCH4 (Touch 4), XTRs (Xyloglucan Endo-Transglycosylase-Related), PGIP2 , GH5 (Glycoside Hydrolase), GDSL (Gly-Asp-Ser-Leu), LRX1 (Leucine-Rich Repeat/Extensin 1), LRR (Leucine-Rich Repeat), and FLA2 (Flowering Locus A2) (COBL1 (COBRA Like 1) was upregulated in both the CNS4 and CNS5 stages. Notably, genes such GH3 (Glycoside hydrolase 3), GH9B8 (Glycosyl Hydrolase 9B8), and CSLD2 (Cellulose Synthase Like D2) showed initial upregulation in the CNS4 stage, followed by subsequent downregulation in the CNS5 stage  a. In conS5 stage a.UGT73B5 (UDP-Glucosyl Transferase 73B5), F5H (Ferulate 5-Hydroxylase), HCT (Hydroxycinnamoyl Transferase), PRR1 (Pinoresinol Reductase 1), OMT-1 (O-methyltransferases 1), HAT (Histone Acetyltransferase), and PSY (Phytoene Synthase). In contrast, the expressions of SDR (Short-chain dehydrogenase/reductase) and OMT-2 (O-methyltransferases 2) followed an opposite pattern (AAP (Amino Acid Permease), AAP3, AAP6, and PROT1 (Proline Transporter 1) exhibited continuous upregulation with the culm neck growth. In contrast, PTR6 (Peptide Transporter 6), LAX2 (Like Auxin resistant 2), PILS5 (PIN-Likes 5), OCT2 (Organic Cation Transporter 2), ALF5 , PLT5 (Polyol Transporter 5), and STP1 (Sugar Transporter 1) were downregulated in the CNS4 stage. Interestingly, some of the genes downregulated in the CNS4 stage, such as PTR6, ALF5, and PLT5, were inversely upregulated in the CNS5 stage , and RSM1 exhibited upregulation in the CNS4 and CNS5 stages  had fully differentiated, with epidermis, hypodermis, cortex, and ground tissues , indicatPMEPCRA [EXPA1 [EXPA4 [F5H [HCT [PRR1 [PALs, C4Hs, C3Hs, 4CLs, HCTs, CCRs, CCoAOMTs, CAD, F5Hs, and COMT, in the lignin biosynthesis process in Moso bamboo [AAP3, AAP6 [PROT1 [AAP6, expressed in the xylem parenchyma or phloem in Arabidopsis [To gain a deeper insight into the molecular mechanisms regulating the developmental process of the CNS3–CNS5 stages, the current study performed a transcriptome analysis. Interestingly, the study found 5524 novel genes, accounting for 10% of the Moso bamboo genome, that are involved in specialized cellular processes that fine-tune biological pathways involved in the culm neck development , which hPMEPCRA , EXPA1 [A [EXPA1 , EXPA4 [1 [EXPA4 , etc., sPA4 [F5H , HCT [32CT [PRR1 , were siCT [PRR1 . Notablyo bamboo . FurtherP3, AAP6 ,37, and 6 [PROT1 . In partbidopsis ,39, has bidopsis . These rWRKY18, a member of the WRKY TF family, was upregulated in the CNS5 stage and plays a crucial role in regulating the rapid responses to sugar metabolism [Arabidopsis, MYBH, a member of the MYB TF family, has been reported to be involved in the positive regulation of darkness-induced hypocotyl elongation [MYBH in the CNS4 and CNS5 stages suggests that it is a molecular component responsible for promoting the elongation of the culm neck in the underground darkness. In addition, as members of the MYB TF family, both MYB59 and MYR2 are involved in distinct roles. While MYB59 is involved in the regulation of nutrient transport [MYR2 has been reported to regulate nitrogen reutilization [Arabidopsis, RSM1, another member of the MYB TF family, has been reported to negatively regulate the apical hook bending [RSM1 and RSM3 may serve as a key factor in the angle change between the rhizome and the culm neck  has a humid subtropical climate with an average annual precipitation of 1631.5 mm (64 inches). The soil at the collection point primarily consists of red soil and paddy soil. First, the underground shoot buds of Moso bamboo were collected at different developmental stages. Subsequently, the culm necks, used for the anatomical and transcriptome analyses, were carefully separated from the shoot buds. The developmental stages of the shoot buds of Moso bamboo were described according to our previous report [3 of the young fresh tissues of Moso bamboo culm necks at different developmental stages were collected and then fixed in formalin–acetic acid–70% alcohol  buffer. The paraffin sectioning and observation were performed and observed under a Leica DM2500 light microscope , as described in our previous report [Approximately 0.5 cms report .The fresh tissues of the culm necks at different developmental stages  ,52. BrieIn order to obtain full-length transcripts, full-length transcriptome sequencing was also performed in the present study. Already quality-passed RNA samples were further selected, with each stage having five biological replicates. Subsequently, the samples were carefully pooled together in equal quantities to create one sample for PacBio SMRT sequencing. The full-length cDNAs were synthesized using the SMRTer PCR cDNA Synthesis Kit . A fraction containing fragments > 4 kb was enriched and collected, using the BluePippin Size-Selection System . Finally, the enriched fraction was mixed equally (equimolar) with the unscreened fractions to create a mixed SMRT cell that was run on the PacBio Sequel platform. The experiment was performed at Novogene Biotechnology Company . The raw reads from Illumina sequencing were first checked for quality using FastQC , and theAfter transcriptome assembly, the transcripts were finally annotated by comparing their sequences against several databases, such as Gene Ontology (GO) , SWISS-Pk-mer scheme) [Transcription factors (TFs) were annotated using the iTAK program . The non scheme)  and CNCI scheme) . The res scheme)  and Pfam scheme)  to finalp-values < 0.05 and absolute fold changes no less than 2 were identified as DEGs. The expression patterns of the DEGs were visualized using MapMan (v.3.5.1R2) [For the differential gene expression analysis, the clean reads from Illumina sequencing were aligned to the non-redundant consensus transcript using Bowtie2 . After t3.5.1R2) . In addi3.5.1R2) .http://www.premierbiosoft.com/ (accessed on 24 September 2023)); their sequences are listed in TIP41) gene was used as an internal control [ΔCq values between the target gene and the reference gene, with three replicates for each gene [To verify the transcriptome results, 4 genes for the culm neck were randomly selected, and gene-specific primers were designed using Primer Premier 5 software  was represented by three biological replicates for anatomical analysis and five biological replicates for transcriptome sequencing. For qRT-PCR, three replicates were performed for each gene, providing additional validation of the transcriptome data. The data obtained from these replicates were subjected to appropriate statistical tests to determine the significance of the differences in the gene expression levels.In conclusion, our study focused on the anatomical and transcriptome analyses of the CNS3, CNS4, and CNS5 stages. The culm neck is a solid structure and differentiates in the CNS3 stage and further elongates in the CNS4 or CNS5 stages. The increased angle between the rhizome and the culm neck in the CNS5 stage facilitates the upward growth of the shoot bud . The ligMYBH and MYBLU, both members of the MYB TF family, in the CNS4 and CNS5 stages, contributes to the elongation of the culm neck, leading to a significant increase in the angle between the culm neck and the rhizome. The changing angle between the culm neck and the rhizome, particularly marked in the CNS5 stage, enables the emergence of the shoot bud in an upward direction. Further we identified a total of 5524 novel genes related to the cell wall, secondary metabolism, and nutrient transport. Among these, 51, 102, and 34 novel genes exhibited specific expressions in the CNS3, CNS4, and CNS5 stages, respectively. These novel genes are related to lignin synthesis, cellulose synthesis, hemicellulose synthesis, nutrient transporters, etc. In addition, the genes involved in these stages are also involved in the fast-growing internodes and leaves. The members of WRKY and MYB TFs exhibited elevated expression levels, particularly during the transition from the CNS4 to CNS5 stages. The upregulation of Overall, our study revealed that the WRKY and MYB families play distinct roles in the elongation of the culm neck and provide valuable insights into the complex processes controlling the shoot bud emergence and the culm neck elongation . The int


Prunus salicina Lindl.) fruit softening. Exogenous MT treatment increased the endogenous MT content in plum fruits before fruit ripening. However, in mature plum fruits, exogenous MT treatment decreased the fruit hardness, pulp hardness, fruit elasticity, contents of ion-bound pectin, covalently-bound pectin, hemicellulose, and cellulose, and activities of xyloglucan endotransglycosylase/hydrolase and endo-β-1,4-glucanase, and increased the water-soluble pectin content, and activities of pectin methyl esterase, pectin lyase, polygalacturonase, β-galactopyranosidase, and α-L-arabinofuranosidase. Transcriptome analysis revealed that the differentially expressed genes (DEGs) associated with cell wall metabolism in the exogenous MT-treated plum fruits were mainly enriched in the pentose and glucuronate interconversions, phenylpropanoid biosynthesis, cyanoamino acid metabolism, and galactose metabolism pathways. Analysis of these DEGs revealed that exogenous MT treatment affected the expression of genes regulating the cell wall metabolism. Overall, exogenous MT treatment promotes the fruit softening of Chinese plum.In this study, we investigated the effect of exogenous melatonin (MT) on cell wall metabolism leading to Chinese plum ( The change in cell wall metabolism is an important indicator of fruit ripening, which plays a critical role in fruit softening . The celMelatonin (MT) is a tryptophan-indole derivative widely found in plants and animals. It was first extracted from the pineal gland of cattle in 1958, hence the name pinealectin . In vertPpPME and PpPG transcripts, up-regulates some arabinogalactan-like protein family genes, and alleviates cell wall modification by positively regulating the activity of cell wall modifying enzymes and protein synthesis, which alleviates fruit pulverization [In fruits, high pectin methyl esterase (PME) and low polygalacturonase (PG) activities significantly increase the accumulation of demethylated pectin, resulting in fruit pulverization . Howeverrization . In pearrization . HoweverMaACO1 and MaACS1, which delays the post-harvest ripening of fruits. More importantly, the exogenous MT (100 μM) treatment affects the endogenous hormone level in sweet cherries. Interestingly, the MT trend is consistent with that of jasmonic acid and salicylic acid in plants. At the same time, MT inhibits sweet cherry fruit ripening by crosstalk with cytokinins [2O2 accumulation and up-regulates TDC, SNAT, T1000H, ASMT, and abscisic acid content to promote fruit ripening [Ethylene plays an important role in fruit ripening. However, Hu et al. 2017)  pointed tokinins . In straripening . Similarripening ,17.  pointedPrunus salicina Lindl.) is a common temperate fruit with a high economic value and is widely loved by consumers, but the content of lignin in the fruit affects its commercial value [Chinese plum  were analyzed on the Illumina platform. The sequencing results revealed that the GC content was greater than 45%, and the percentage of Q30 was greater than 94%  implyingThe GO enrichment analyses revealed that 350 DEGs were enriched at 21 days and 306 DEGs at 28 days . The DEGSubsequently, KEGG pathway enrichment analysis detected 302 and 260 DEGs enriched at 21 and 28 days, respectively . At 21 dDEGs analysis identified 11 genes related to cell wall metabolism in the exogenous MT-treated group at 21 days. These cell wall metabolism-related DEGs were mainly enriched in the pentose and glucuronate interconversions and phenylpropanoid biosynthesis pathways . PG, galAt 28 days, 16 genes related to cell wall metabolism in the exogenous MT-treated plum fruits were mainly enriched in the pentose and glucuronate interconversions, phenylpropanoid biosynthesis, cyanoamino acid metabolism, and galactose metabolism pathways. Pectinesterase and pectate lyase were down-regulated in the pentose and glucuronate interconversions pathway, and galacturan 1,4-alpha-galacturonidase was up-regulated C. In thePPE8B, XTH23, At4g24780, and GSVIVT0002692001 were up-regulated, and RCA and PME were down-regulated at 21 days post-MT treatment. At 28 days, PGIP, Xyl2, BGLU41, and BGLU11 were up-regulated, and PPE8B, RCA, BGAL8, XTH23, and PECS-1.1 were down-regulated in the MT-treated group.The expression patterns of the six and nine DEGs at 21 and 28 days, respectively, screened by qRT-PCR analysis, were consistent with the RNA-seq results . NotablyIn eggplants, exogenous MT treatment up-regulates the MT synthesis-related genes and the endogenous MT content . In applThe changes in the cell wall components are directly related to the changes in the fruit texture . Pectin The cell wall-modifying hydrolases play an important role in fruit texture changes. Notably, the synergy of different hydrolases catalyzes cell wall degradation, a complex physiological process . For exaXTH genes are associated with tomato fruit ripening in tomato fruits. Moreover, XET and XEH activities are higher during fruit development and may mainly play a role in maintaining the structural integrity of the cell wall. Thus, the down-regulation of XTH during fruit ripening may facilitate fruit softening [On the other hand, XTH, a bifunctional enzyme, mainly played an integrative role in plum fruits, integrating newly secreted xyloglucan chains into the cell wall and participating in the formation of a complex xyloglucan–cellulose structure, ultimately increasing fruit hardness 21 days after exogenous MT treatment. Only 2 of the 10 oftening . In thisoftening . This maXTH and Exps, significantly changes with decreasing pear fruit hardness after ripening. Moreover, the high expression levels of genes encoding pectin-degrading enzymes induce an increase in the pectic oligomers, which favors the increase in ethylene yield [PG, pectin methyl esterase 1 (PE1), and β-galactosidase (TBG4) in tomato fruits, which accelerate pectin hydrolysis and promote fruit ripening and softening [PPE8B, XTH23, At4g24780, and GSVIVT0002692001 in exogenous MT-treated plum fruits were up-regulated at 21 days, while RCA and PME were down-regulated. At the same time, PGIP, Xyl2, BGLU41, and BGLU11 were up-regulated at 28 days in the exogenous MT-treated plum fruits, while PME activity inhibitor, PPE8B, RCA, BGAL8, XTH23, and PECS-1.1 were down-regulated. This implies that the exogenous MT treatment mediated the conversion of ion-bound and covalently-bound pectin to water-soluble pectin and the degradation of cellulose and hemicellulose through the pentose and glucuronate interconversions, phenylpropanoid biosynthesis, cyanoamino acid metabolism, and galactose metabolism pathways at the mature stage of plum fruits, thus promoting the fruit softening.In the present study, the DEGs in MT-treated plum fruits regulated the pentose and glucuronate interconversions, phenylpropanoid biosynthesis, cyanoamino acid metabolism, and galactose metabolism pathways. These four pathways are mainly regulated by genes related to cell wall metabolism. A previous study revealed that the cell wall metabolism-related genes in jujube fruits treated with MT are enriched in the interconversion of pentose and glucuronide and galactose metabolism pathways . Transcrne yield . Exogenooftening . Many proftening . In this2 orchard at Sichuan Huijia Seven Colorful Fields Agricultural Technology Co., Chengdu, China  in February 2018. The seedling spacing was 4 m (plant space) × 3 m (row space). MT was obtained from Beijing Solarbio Science & Technology Co., Ltd. .The Chinese plum variety ‘Qiangcuidali’ grafted on a wild peach rootstock was used in this study. The grafted plum seedlings were planted in a 2000 mIn May 2022, when the plum fruits were at the second rapid expansion period (90 days after flowering), 18 plum trees were selected for the present study. Among them, nine plum trees were treated with 100 μmol/L of MT ,33, and The plum fruit flesh was cut with a 10 mm diameter punch, and a 9 mm-thick section of pulp below the peel was retained. Next, the texture profile analysis (TPA) was performed using a TA/2R disc probe attached to a TA.XTC-18 texture analyzer . The test type was downward pressure, the test target was displacement, the test depth was 6 mm, and the test speed was 0.8 mm/s to determine the elasticity of the fruits . SubsequThe samples were dried and passed through a 0.425 mm sieve. Next, 1 g per sample was weighed in a centrifuge tube, and 30 mL of an 80% ethanol solution was added before boiling for 25 min. Subsequently, the precipitate was washed 1–2 times with 30 mL of 80% ethanol after cooling. The samples were then filtered under vacuum, and the residue was rinsed with 30 mL of the 80% ethanol solution. The obtained filter residue was soaked with 30 mL of 90% dimethyl sulfoxide overnight. Subsequently, the residue was soaked in acetone for 10–20 min to remove the dimethyl sulfoxide, vacuum filtered fully, rinsed in three changes of 30 mL of acetone each time, and dried to a constant weight. The final weight obtained was the cell wall substance .The supernatant containing water-soluble pectin was obtained by weighing 0.05 g of the cell wall substance in a 10 mL capped centrifuge tube, adding 5 mL of deionized water, and shaking for 6 h. Next, 5 mL of sodium acetate buffer was added to the residue to obtain the supernatant containing ion-bound pectin after 6 h of shaking. Subsequently, 5 mL of a sodium carbonate solution was added to the residue to obtain the supernatant containing covalently-bound pectin after 6 h of shaking . FinallyFor hemicellulose quantification, 5 mL of a sodium hydroxide solution was added to the extracted pectin residue and shaken for 6 h to obtain the supernatant containing hemicellulose. Next, 2 mL of the hemicellulose supernatant was mixed with 3 mL of sulfuric acid and hydrolyzed in a boiling water bath at 100 °C for 2 h to obtain the hydrolyzed hemicellulose and the extracted hemicellulose residue (cellulose). Subsequently, 1.5 mL of sulfuric acid was added to the residue and left for 2 h. An appropriate amount of activated carbon and 3 mL of ultrapure water was then added to the residue and hydrolyzed in a boiling water bath at 100 °C for 2 h. The residue was then filtered to obtain the cellulose-containing solution. Finally, the cellulose and hemicellulose contents were determined by the anthrone and ethyl acetate colorimetric method .The lignin content was determined by the acetylation method using the lignin content determination kit  following the manufacturer’s instructions.The MT content was determined by the enzyme-linked immunosorbent assay using the plant MT ELISA kit  following the manufacturer’s instructions.PME, pectin lyase (PL), PG, β-galactosidase (β-GAL), xyloglucan endo glycosyltransferase/hydrolase (XTH), α-L-arabinofuranosidase (α-AF), and endo-β-1,4-glucanase (EG) activities were determined using the PME, PL, PG, β-GAL, XTH, α-AF, and EGase kits , respectively, following the manufacturer’s instructions.RNA was extracted from the fruit pulp using the RNAprep Pure Plant Kit  according to the instructions provided by the manufacturer. Next, the extracted RNA integrity was assessed using the RNA Nano 6000 Assay Kit on the Agilent Bioanalyzer 2100 system  according to the manufacturer’s protocol. RNA sequencing libraries were then generated using the Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China) following the manufacturer’s instructions.p < 0.01 and fold change ≥ 2 were denoted as differentially expressed. Finally, the gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs were performed.After sequencing, the obtained raw data (raw reads) were processed to obtain high-quality clean data. Next, sequence comparison to the reference genome of plum was performed using the Hisat2 tools. Subsequently, the differential expression analysis between treatments was performed using DESeq2. Genes with an adjusted −ΔΔCT method [The first strand cDNA synthesis of the selected DEGs was performed using the M5 Super plus qPCR RT kit with gDNA remover from Mei5 Biotechnology Co., Ltd., Beijing, China, referring to the manufacturer’s instructions. The primers were designed using Primer Premier 5.0 software according to the reference gene sequences in plums . RT-qPCRT method .t-test (0.01 ≤ p < 0.05 or p < 0.01) using the SPSS 20.0.0 software .The data were analyzed in triplicate by Student’s Exogenous MT treatment increases the endogenous MT in plum fruits at the early stage of fruit development. At maturity, exogenous MT treatment accelerates the plum fruit softening by decreasing the fruit hardness, promoting the conversion of ion-bound and covalently-bound pectin to water-soluble pectin, and degrading the cellulose and hemicellulose. Furthermore, exogenous MT treatment increases the PME, PG, PL, β-GAL, and α-AF activities and decreases XTH and EG activities during plum fruit ripening. Exogenous MT treatment also mediates the pentose and glucuronate interconversions, phenylpropanoid biosynthesis, cyanoamino acid metabolism, and galactose metabolism pathways, regulating the expression of genes related to cell wall metabolism.


Venezuelan equine encephalitis virus (VEEV) causes a febrile illness that can progress to neurological disease with the possibility of death in human cases. The evaluation and optimization of therapeutics that target brain infections demands knowledge of the host’s response to VEEV, the dynamics of infection, and the potential for within-host evolution of the virus. We hypothesized that selective pressures during infection of the brain may differ temporally and spatially and so we investigated the dynamics of the host response, viral transcript levels, and genetic variation of VEEV TC-83 in eight areas of the brain in mice over 7 days post-infection (dpi). Viral replication increased throughout the brain until 5–6 dpi and decreased thereafter with neurons as the main site of viral replication. Low levels of genetic diversity were noted on 1 dpi and were followed by an expansion in the genetic diversity of VEEV and nonsynonymous (Ns) mutations that peaked by 5 dpi. The pro-inflammatory response and the influx of immune cells mirrored the levels of virus and correlated with substantial damage to neurons by 5 dpi and increased activation of microglial cells and astrocytes. The prevalence and dynamics of Ns mutations suggest that the VEEV is under selection within the brain and that progressive neuroinflammation may play a role in acting as a selective pressure.Treatment of encephalitis in humans caused by Venezuelan equine encephalitis virus (VEEV) from natural or aerosol exposure is not available, and hence, there is a great interest to address this gap. In contrast to natural infections, therapeutic treatment of infections from aerosol exposure will require fast-acting drugs that rapidly penetrate the blood-brain barrier, engage sites of infection in the brain and mitigate the emergence of drug resistance. Therefore, it is important to understand not only VEEV pathogenesis, but the trafficking of the viral population within the brain, the potential for within-host evolution of the virus, and how VEEV might evolve resistance. Alphavirus that has caused a number of epidemic/epizootic outbreaks of disease in humans and equines in the past century in the Americas  is a mosquito-borne, New World Americas , 2. EpizAmericas -5. In Americas , 6. PathAmericas -9. TheAmericas , 11. TheAmericas . Treatme+ T cells are protective  following its intranasal exposure of C3H/HeN mice. We focused on the main olfactory bulb, piriform cortex, striatum, motor cortex, sensory cortex, hippocampus, thalamus, and cerebellum. VEEV TC-83 infection is lethal in the mouse strain, C3H/HeN, when administered intranasally (i.n.), intracranially, or by aerosol exposure, and mice become moribund from 7 to 9 dpi -33. We2 in complete medium containing Dulbecco’s modified Eagle medium with Hi-glucose and L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. VEEV TC-83 was obtained from Connie Schmaljohn . Virus seed stocks were amplified in Bovine hamster kidney (BHK)cells in minimum essential medium with Earle’s salts Eagle's minimum essential medium (EMEM) with 2% FBS and 1% penicillin-streptomycin. All cell culture reagents were purchased from Thermo Fisher Scientific unless otherwise specified. VEEV TC-83 was sequenced as a reference genome as previously reported (Vero 76 cells (ATCC CRL-1587) were maintained at 37°C and 5% COreported .5 Vero 76 cells and a series of 10-fold dilutions of virus samples were made to measure titer as described in reference (Twelve-well plates were seeded with 2.5 × 10eference .7 plaque-forming units (PFU) of VEEV TC-83 or mock inoculated with Dulbecco’s phosphate-buffered saline (PBS) using 15 µL per naris. After inoculation, all mice were weighed daily and checked twice daily for clinical signs and morbidity. All mice were humanely sacrificed using isoflurane, followed by cervical dislocation. All animal studies and methods were conducted under the protocol 18-044 or 21-0258, authorized by the Institutional Animal Care and Use Committee at the University of Tennessee Health Science Center.C3H/HeN mice were purchased from Charles River Laboratories for all studies. In all studies, mice were inoculated i.n. with 1 × 10Twenty-eight, 5- to 6-week-old, female, C3H/HeN mice were randomly assigned to groups. Each group of VEEV TC-83 infected mice (four mice per group) were humanely sacrificed at 1, 3, 5, 6, or 7 dpi. Mock-inoculated mice were sacrificed on 1 and 5 dpi. Following euthanasia, the whole brain, including the olfactory bulb, was removed from each mouse. Each brain was separated into eight portions: main olfactory bulb, piriform cortex, caudate-putamen, motor cortex, sensory cortex, thalamus, hippocampus, and cerebellum. Each section was homogenized in 1 mL of lysis buffer from the MagMAX mirVana Total RNA Isolation Kit (Thermo Fisher Scientific). Total RNA was isolated from each brain section using the MagMAX mirVana Total RNA Isolation Kit and the KingFisher Flex System (Thermo Fisher Scientific).Mus musculus  and sequenced lab strain of VEEV TC-83. Gene count results were used to assess differential gene expression using DESeq2 and mock-inoculated mice defined baseline gene expression. Determined differentially expressed genes  were used to assess canonical pathway activation as well as perform comparison analysis using Ingenuity Pathway Analysis . RNA-Seq data were uploaded to the NIH Gene Expression Omnibus (GSE213725).Sequencing of total RNA was performed by Azenta Life Sciences. Total RNA was quantified and qualified using a Qubit (Thermo Fisher Scientific) and TapeStation (Agilent), whereafter sequencing libraries were constructed by mRNA enrichment using NEBNext Ultra II RNA Kits (New England Biolabs). Constructed libraries were sequenced on Illumina’s HiSeq platform . Sequence read results were processed with CLC Genomics Workbench . Sequencing reads from each sample underwent trimming and filtering using default parameters. Read mapping was conducted using default parameters and performed to a concatenated genome genome consisting of Sequencing reads (described above) that mapped to the genome sequence of our lab seed stock VEEV TC-83 was used to assess virus mutations using CLC Genomics Workbench . Viral SNPs were identified using low-frequency variant detection was used with the following parameters, minimum coverage = 100, minimum count = 50, and minimum frequency = 1.0%. Ns mutations were determined by using the Amino Acid Change tool to filter away SNPs that did not result in nonsynonymous changes.Twenty-eight, 5- to 6-week-old, female, C3H/HeN mice were randomly assigned to groups based on day of sacrifice, i.e., 1, 2, 3, 4, 5, 6, and 7 dpi, and VEEV TC-83 or mock inoculated. Following euthanasia, the whole brain with the olfactory bulb was removed from each mouse and fixed in 20 mL of 10% formalin for 24 h at 4°C. One sagittal section of each brain was made and then the tissue was cryoprotected with 30% sucrose in 0.1 M phosphate buffer pH 7.4. Sagittal sections of brain were made with a Leica CM3050 at 40 µM thickness. To collect nasal turbinate, each mouse skull was fixed with 10% buffered formalin, dissected, decalcified with 10% ethylenediaminetetraacetic acid (EDTA), and embedded in paraffin for samples taken on 1, 3, and 5 dpi. About 4 μm sections were cut using a Leica microtome.For immunohistochemical (IHC) staining and imaging of cryosections, brain sections were washed with PBS and endogenous peroxidases were quenched with 1% hydrogen peroxide in PBS for 5 min, rinsed with PBS three times and blocked with 1% bovine serum albumin in PBS and 0.3% Triton X-100. Floating sections were incubated with goat anti-VEE GP antibody  at a 1:12,000 dilution for 2 days at 4°C on a shaker, for rabbit AIF-1Iba-1 antibody , with dilution of 1:1,000, rabbit anti-GFAP antibody  with dilution of 1:2,000, rinsed with PBS three times and incubated with biotinylated rabbit anti-goat IgG at 1:200  or biotinylated horse anti-rabbit IgG  for overnight. After rinsing three times with PBS, sections were incubated with avidin-biotin complex (ABC) using the Vectastain ABC Elite Kit PK-6100  at a 1:100 dilution and developed with 3,3ʹ-diaminobenzidine (DAB) . Sections were mounted onto gelatin-coated glass slides and air dried, counterstained with methyl green solution or without counterstain, dehydrated through serial alcohol rinses and xylene and then mounted using Permount Mounting Media (Thermo Fisher Scientific).For IHC with paraffin-embedded sections, we used heat-induced antigen retrieval for OMP with Tris/EDTA buffer pH 9.0 (H3301) and for LDLRAD3 we used citric buffer pH 6 (H3300). Both products are from Vector Laboratories. For OMP , we used a 1:2,000 dilution for LDLRAD3 , we used a 1:50 dilution, and for the VEEV GP, we used a 1:1,000 dilution. Incubations with primary antibodies were done overnight followed by ABC method as described above.About 5 µm sections of formalin-fixed nasal turbinates were cut, mounted on Fisherbrand Superfrost Plus Microscope Slides (Fisher Scientific), and stained with hematoxylin and eosin (H&E). For paraffin-embedded nasal mucosa/epithelium sections, heat-induced antigen retrieval was performed. Sections were incubated with goat anti-VEEV GP antibody at a 1:1,000 dilution and treated using ABC and DAB as described above.Cryostat sections of mock and VEEV infected mice brains were incubated with a cocktail of goat anti-VEEV capsid antibody  at 1:8,000 dilution with rabbit anti-Iba-1 antibody at 1:1,000  or rabbit anti-GFAP at 1:2,000 NB300-141, Novus Biologicals) for 2 days, after which slide sections were rinsed with PBS, and then incubated with a cocktail of Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 (Invitrogen) or Donkey anti-Goat IgG (H + L) Secondary Antibody, Alexa Fluor 594 (Invitrogen) at 1:500 overnight. Slides were rinsed with PBS counterstained with DAPI, mounted and coverslip with Fluoromount-G . Images captured with Zeiss 710 confocal microscope.All slides were scanned with an Olympus VS200 by scanning slides with a 40× objective. Images were captured and processed with the OlyVia v. 3.2 software.n = 4/day) were humanely sacrificed at 1, 4, or 7 dpi. Whole brains, including olfactory bulbs, were harvested and brain cells were isolated using 0.1 mg/mL Collagenase D and 5 µg/mL DNase for 30 min at 37°C. The cells were filtered through a 70-µm cell strainer and then resuspended in 37% Percoll, followed by centrifugation at 860 × g.Twenty-four, 5- to 6-week-old, female, C3H/HeN mice (The cell pellet obtained from Percoll as stated above was resuspended in fluorescence-activated cell sorting (FACS) buffer and cell surface flow cytometry performed using fluorochrome-conjugated antibodies to CD11b (clone M1/700), CD206 (clone C068C2), CD45 (clone 30-F11), CD4 (clone RM4-5), TcRβ(clone H57-597), CD80 (clone 16-10A1), or CD69 (clone H1.2F3). All antibodies were from BioLegend, San Diego, CA, USA or BD Biosciences, Franklin Lake, NJ, USA. A minimum of 10,000 events/sample were collected on a Bio-Rad ZE5 Cell Analyzer . The expression of cell surface markers was analyzed using FlowJo v10.7.2 software.7 PFU of VEEV TC-83 on day 0, and then mice were sacrificed on 1, 3, 5, 6, and 7 dpi. Each day, eight areas of the brain were dissected for RNA-Seq: the main olfactory bulb, piriform cortex, striatum, motor cortex, sensory cortex, thalamus, hippocampus, and cerebellum. The number of viral genome transcripts was normalized during library preparation and then by assessment of transcripts per million (TPM). The TPM values increased by 3 dpi for all areas of the brain, and by 5 dpi, the main olfactory bulb and piriform cortex showed a slight decrease that continued to 7 dpi . A total number of 329 Ns mutations were discovered and plotted for each area on each day post-infection . Of the P value = 0.7031; 5 dpi adjusted P value = 0.7031; 6 dpi adjusted P value = 0.7031; and 7 dpi adjusted P value = 0.7031).The distribution of Ns mutations over time was similar in the nonstructural and structural genes . Ns mutaOf the 102 unique Ns mutations (Table S1), only 6  were observed across multiple days, brain portions, and mice. Two Ns mutations (NSP2:F286L and NSP3:T333I) were observed across multiple brain areas and multiple mice, and 49 Ns mutations were only observed in multiple brain areas of the same individual mouse but not on multiple days. Finally, we measured 47 Ns mutations in only one area of the brain.7 PFU of VEEV TC-83 or PBS (mock) on day 0, and then sacrificed to evaluate infection of the nasal epithelium  which includes lymphocytes, cells that were CD45hi CD11bhi (R2 gate) which contains myeloid cells, such as monocytes, macrophages, DCs, and neutrophils, and finally, the cells that were CD45int CD11b+ gate (R3 gate) which includes microglia  that infiltrated the brain revealed three subpopulations that changed in frequency from 4 to 7 dpi . CD80 is+ T cells. Although a small percentage of the CD4+ T cells were activated as measured by CD69 expression (14 ± 3.3%), the majority of the CD69+ T cells were CD4− (37 ± 15%).In addition to myeloid cells increasing in the brains of TC-83 mice, there was also an increase in lymphoid cells by 7 dpi . ClassicEvaluation of microglial cells by IHC in the piriform and motor cortex showed a morphology with thicker and shorter processes following infection . MicroglTo study the spatial and dynamic response of the host response of the brain to VEEV TC-83 infection, we used the RNA-Seq data from 1, 3, 5, 6, and 7 dpi for each of the eight areas of the brain from virus and mock-inoculated mice. Analysis of the differential gene expression was assessed by comparison of brain regions from infected mice from 1, 3, 5, 6, or 7 dpi to mock-inoculated mice . At 1 dpThe canonical activated pathways were defined based on the DESeq2 results using Ingenuity Pathway Analysis for 1, 3, 5, 6, and 7 dpi, which compared infected to mock-inoculated mice. The most significantly activated pathways associated with infection were pathogen-induced cytokine storm signaling pathway, phagosome formation, CREB signaling in neurons, role of hypercytokinemia/hyperchemokinemia in the pathogenesis of influenza, and macrophage classical activation signaling pathway . SeveralIFNB1, IFNG, TNF, IL10, and IL6  and future studies would benefit from additional mice to define the probability of occurrence of individual Ns mutations and noted consensus sequences.Herein, we examined the distribution of viral antigen, viral RNA levels, and genetic variation of viral genomes over time in the mouse brain following infection with VEEV TC-83 along with the host’s response to infection. The RNA-Seq and the IHC analysis of VEEV TC-83-infected brains from C3H/HeN mice show that the virus reaches the olfactory bulbs and most other parts of the brain  within 24 h after intranasal infection. Notably, there was a low level of genetic diversity at 1 dpi within the olfactory bulbs which was followed by an expansion of genetic diversity which peaked at 5 dpi. By 7 dpi, diversity fell. Viral RNA levels peaked in the main olfactory bulbs and piriform cortex by 3 dpi and decreased slightly thereafter through 7 dpi, but this did not correlate with genetic diversity. By 3 dpi, the piriform cortex shows signs of cellular damage, which continued to increase as noted by perivascular cuffing, and diffused meningitis. By 5 dpi, numerous areas show lesions, and RNA-Seq suggested a significant increase in the neuroinflammatory response. The rise in pathology and inflammation correlated with a dramatic rise in Ns mutations. Interestingly, by 7 dpi, the number of Ns mutations decreased in the olfactory bulbs, piriform cortex, thalamus, and hippocampus, but remained elevated in the motor cortex, striatum, and sensory cortex. This may reflect continued replication in motor cortex, striatum, and sensory cortex in contrast to other areas with fewer viable neurons and increased cell death. The distribution of the viral antigen matches well with published data on the distribution of viral antigen in this mouse model, in which the time points for the appearance of virus in the piriform cortex, caudate putamen, thalamus, and hippocampus were the same , 36. The+ T cells are protective in this model  non-CD4+ T cells which are presumably cytotoxic CD8+ T cells. Increased cytotoxic activity against virally infected neurons may contribute to the cell death and pathology observed at the later time points; however, the role of CD8+ T cells in the disease process has not been firmly elucidated for any of the VEEV strains. It is likely that NK cells, B cells, or T cells make up the remainder of the cells in the lymphocyte gate. The increase in myeloid cells is also evident at 4 dpi and approximately half of the cells express the co-stimulatory marker CD80 which is typically expressed on DCs and macrophages and indicative of a pro-inflammatory state. This is consistent with the RNA-Seq data which demonstrated an increase in numerous pro-inflammatory cytokines, chemokines, and other inflammatory host response genes by 5 dpi. These data also confirm results from other with studies demonstrating an increase in pro-inflammatory cytokines such as TNF, IL-6, IL-1β, and IFN- in VEEV-infected mice  and monocyte/macrophage (R2) gates which correlates well with the kinetics of viral invasion of the brain and the RNA-Seq data. It has been suggested that CD4is model , 24 and ted mice -43. InThis model suggests three stages of infection and disease to consider during therapeutic treatment of brain infection, the early stage of infection (1–3 dpi), midway (4–5 dpi), and late (6–8 dpi). The earliest stages of infection are characterized by low levels of infectious virus and minimal inflammation. During this period, antivirals that attack the virus may well show the greatest efficacy and protection from pathogenesis. High levels of virus and inflammation characterize the timeframe midway during infection and will therefore require greater exposure of the drug in the brain. It is unknown if elimination of the virus during this stage will permit adequate healing and regeneration of damaged brain tissue. Treatment of the disease in the last stage which is characterized by widespread cell death, continued inflammation, and infectious virus represents the greatest challenge. The levels of drug exposure required may change again due to alterations of the BBB and deterioration of the endothelial cell network resulting in difficulty in reaching all areas of the brain. Future efforts will seek to assess efficacy and drug exposure in each of these stages with VEEV antivirals , 35.