Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation.

Auxin inactivation is critical for plant growth and development. To develop plant growth regulators functioning in auxin inactivation pathway, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that partially mimicked the effects of auxin. Genetic, pharmacological, and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture.

The chromatin remodeller MdRAD5B enhances drought tolerance by coupling MdLHP1-mediated H3K27me3 in apple.

RAD5B belongs to the Rad5/16-like group of the SNF2 family, which often functions in chromatin remodelling. However, whether RAD5B is involved in chromatin remodelling, histone modification, and drought stress tolerance is largely unclear. We identified a drought-inducible chromatin remodeler, MdRAD5B, which positively regulates apple drought tolerance. Transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analysis showed that MdRAD5B affects the expression of 466 drought-responsive genes through its chromatin remodelling function in response to drought stress. In addition, MdRAD5B interacts with and degrades MdLHP1, a crucial regulator of histone H3 trimethylation at K27 (H3K27me3), through the ubiquitin-independent 20S proteasome. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that MdRAD5B modulates the H3K27me3 deposition of 615 genes in response to drought stress. Genetic interaction analysis showed that MdRAD5B mediates the H3K27me3 deposition of drought-responsive genes through MdLHP1, which causes their expression changes under drought stress. Our results unravelled a dual function of MdRAD5B in gene expression modulation in apple in response to drought, that is, via the regulation of chromatin remodelling and H3K27me3.

The RNA-binding protein MdHYL1 modulates cold tolerance and disease resistance in apple.

Apple (Malus domestica) trees often experience various abiotic and biotic stresses. However, due to the long juvenile period of apple and its high degree of genetic heterozygosity, only limited progress has been made in developing cold-hardy and disease-resistant cultivars through traditional approaches. Numerous studies reveal that biotechnology is a feasible approach to improve stress tolerance in woody perennial plants. HYPONASTIC LEAVES1 (HYL1), a double-stranded RNA-binding protein, is a key regulator involved in apple drought stress response. However, whether HYL1 participates in apple cold response and pathogen resistance remains unknown. In this study, we revealed that MdHYL1 plays a positive role in cold tolerance and pathogen resistance in apple. MdHYL1 acted upstream to positively regulate freezing tolerance and Alternaria alternata resistance by positively modulating transcripts of MdMYB88 and MdMYB124 in response to cold stress or A. alternata infection. In addition, MdHYL1 regulated the biogenesis of several miRNAs responsive to cold and A. alternata infection in apple. Furthermore, we identified Mdm-miRNA156 (Mdm-miR156) as a negative regulator of cold tolerance and Mdm-miRNA172 (Mdm-miR172) as a positive regulator of cold tolerance, and that Mdm-miRNA160 (Mdm-miR160) decreased plant resistance to infection by A. alternata. In summary, we highlight the molecular role of MdHYL1 regarding cold tolerance and A. alternata infection resistance, thereby providing candidate genes for breeding apple with freezing tolerance and A. alternata resistance using biotechnology.

A multifaceted module of BRI1 ETHYLMETHANE SULFONATE SUPRESSOR1 (BES1)-MYB88 in growth and stress tolerance of apple.

The R2R3 transcription factor MdMYB88 has previously been reported to function in biotic and abiotic stress responses. Here, we identify BRI1 ETHYLMETHANE SULFONATE SUPRESSOR1 (MdBES1), a vital component of brassinosteroid (BR) signaling in apple (Malus × domestica) that directly binds to the MdMYB88 promoter, regulating the expression of MdMYB88 in a dynamic and multifaceted mode. MdBES1 positively regulated expression of MdMYB88 under cold stress and pathogen attack, but negatively regulated its expression under control and drought conditions. Consistently, MdBES1 was a positive regulator for cold tolerance and disease resistance in apple, but a negative regulator for drought tolerance. In addition, MdMYB88 participated in BR biosynthesis by directly regulating the BR biosynthetic genes DE ETIOLATED 2 (MdDET2), DWARF 4 (MdDWF4), and BRASSINOSTEROID 6 OXIDASE 2 (MdBR6OX2). Applying exogenous BR partially rescued the erect leaf and dwarf phenotypes, as well as defects in stress tolerance in MdMYB88/124 RNAi plants. Moreover, knockdown of MdMYB88 in MdBES1 overexpression (OE) plants decreased resistance to a pathogen and C-REPEAT BINDING FACTOR1 expression, whereas overexpressing MdMYB88 in MdBES1 OE plants increased expression of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 （MdSPL3） and BR biosynthetic genes, suggesting that MdMYB88 contributes to MdBES1 function during BR biosynthesis and the stress response. Taken together, our results reveal multifaceted regulation of MdBES1 on MdMYB88 in BR biosynthesis and stress tolerance.

Cytokinin regulates apical hook development via the coordinated actions of EIN3/EIL1 and PIF transcription factors in Arabidopsis.

The apical hook is indispensable for protecting the delicate shoot apical meristem while dicot seedlings emerge from soil after germination in darkness. The development of the apical hook is co-ordinately regulated by multiple phytohormones and environmental factors. Yet, a holistic understanding of the spatial-temporal interactions between different phytohormones and environmental factors remains to be achieved. Using a chemical genetic approach, we identified kinetin riboside, as a proxy of kinetin, which promotes apical hook development of Arabidopsis thaliana in a partially ethylene-signaling-independent pathway. Further genetic and biochemical analysis revealed that cytokinin is able to regulate apical hook development via post-transcriptional regulation of the PHYTOCHROME INTERACTING FACTORs (PIFs), together with its canonical roles in inducing ethylene biosynthesis. Dynamic observations of apical hook development processes showed that ETHYLENE INSENSITVE3 (EIN3) and EIN3-LIKE1 (EIL1) are necessary for the exaggeration of hook curvature in response to cytokinin, while PIFs are crucial for the cytokinin-induced maintenance of hook curvature in darkness. Furthermore, these two families of transcription factors display divergent roles in light-triggered hook opening. Our findings reveal that cytokinin integrates ethylene signaling and light signaling via EIN3/EIL1 and PIFs, respectively, to dynamically regulate apical hook development during early seedling development.

Insights into the effect of human civilization on Malus evolution and domestication.

The evolutionary history of the Malus genus has not been well studied. In the current study, we presented genetic evidence on the origin of the Malus genus based on genome sequencing of 297 Malus accessions, revealing the genetic relationship between wild species and cultivated apples. Our results demonstrated that North American and East Asian wild species are closer to the outgroup (pear) than Central Asian species, and hybrid species including natural (separated before the Pleistocene, about 2.5 Mya) and artificial hybrids (including ornamental trees and rootstocks) are between East and Central Asian wild species. Introgressions from M. sylvestris in cultivated apples appeared to be more extensive than those from M. sieversii, whose genetic background flowed westward across Eurasia and eastward to wild species including M. prunifolia, M. × asiatica, M. × micromalus, and M. × robust. Our results suggested that the loss of ancestral gene flow from M. sieversii in cultivated apples accompanied the movement of European traders around the world since the Age of Discovery. Natural SNP variations showed that cultivated apples had higher nucleotide diversity than wild species and more unique SNPs than other apple groups. An apple ERECTA-like gene that underwent selection during domestication on 15th chromosome was identified as a likely major determinant of fruit length and diameter, and an NB-ARC domain-containing gene was found to strongly affect anthocyanin accumulation using a genome-wide association approach. Our results provide new insights into the origin and domestication of apples and will be useful in new breeding programmes and efforts to increase fruit crop productivity.

Abscisic acid homeostasis is mediated by feedback regulation of MdMYB88 and MdMYB124.

The phytohormone abscisic acid (ABA) is involved in various plant processes. In response to drought stress, plants quickly accumulate ABA, but the regulatory mechanism of ABA accumulation is largely unknown, especially in woody plants. In this study, we report that MdMYB88 and MdMYB124 are myeloblastosis (MYB) transcription factors critical for ABA accumulation in apple trees (Malus x domestica) following drought, and this regulation is negatively controlled by ABA. MdMYB88 and MdMYB124 positively regulate leaf water transpiration, photosynthetic capacity, and stress endurance in apple trees under drought conditions. MdMYB88 and MdMYB124 regulate the expression of biosynthetic and catabolic genes of ABA, as well as drought- and ABA- responsive genes. MdMYB88 associates with promoter regions of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase 3 (NCED3). Finally, expression of MdMYB88 and MdMYB124 is repressed by ABA. Our results identify a feedback regulation of MdMYB88 and MdMYB124 in modulating ABA homeostasis in apple trees.

A phenotype-directed chemical screen identifies ponalrestat as an inhibitor of the plant flavin monooxygenase YUCCA in auxin biosynthesis.

Plant development is regulated by both synergistic and antagonistic interactions of different phytohormones, including a complex crosstalk between ethylene and auxin. For instance, auxin and ethylene synergistically control primary root elongation and root hair formation. However, a lack of chemical agents that specifically modulate ethylene or auxin production has precluded precise delineation of the contribution of each hormone to root development. Here, we performed a chemical genetic screen based on the recovery of root growth in ethylene-related Arabidopsis mutants with constitutive "short root" phenotypes (eto1-2 and ctr1-1). We found that ponalrestat exposure recovers root elongation in these mutants in an ethylene signal-independent manner. Genetic and pharmacological investigations revealed that ponalrestat inhibits the enzymatic activity of the flavin-containing monooxygenase YUCCA, which catalyzes the rate-limiting step of the indole-3-pyruvic acid branch of the auxin biosynthesis pathway. In summary, our findings have identified a YUCCA inhibitor that may be useful as a chemical tool to dissect the distinct steps in auxin biosynthesis and in the regulation of root development.

MdMYB88 and MdMYB124 Enhance Drought Tolerance by Modulating Root Vessels and Cell Walls in Apple.

Water deficit is one of the main limiting factors in apple (Malus × domestica Borkh.) cultivation. Root architecture plays an important role in the drought tolerance of plants; however, research efforts to improve drought tolerance of apple trees have focused on aboveground targets. Due to the difficulties associated with visualization and data analysis, there is currently a poor understanding of the genetic players and molecular mechanisms involved in the root architecture of apple trees under drought conditions. We previously observed that MdMYB88 and its paralog MdMYB124 regulate apple tree root morphology. In this study, we found that MdMYB88 and MdMYB124 play important roles in maintaining root hydraulic conductivity under long-term drought conditions and therefore contribute toward adaptive drought tolerance. Further investigation revealed that MdMYB88 and MdMYB124 regulate root xylem development by directly binding MdVND6 and MdMYB46 promoters and thus influence expression of their target genes under drought conditions. In addition, MdMYB88 and MdMYB124 were shown to regulate the deposition of cellulose and lignin root cell walls in response to drought. Taken together, our results provide novel insights into the importance of MdMYB88 and MdMYB124 in root architecture, root xylem development, and secondary cell wall deposition in response to drought in apple trees.

An atypical R2R3 MYB transcription factor increases cold hardiness by CBF-dependent and CBF-independent pathways in apple.

Apple (Malus × domestica) trees are vulnerable to freezing temperatures. However, there has been only limited success in developing cold-hardy cultivars. This lack of progress is due at least partly to lack of understanding of the molecular mechanisms of freezing tolerance in apple. In this study, we evaluated the potential roles for two R2R3 MYB transcription factors (TFs), MYB88 and the paralogous FLP (MYB124), in cold stress in apple and Arabidopsis. We found that MYB88 and MYB124 positively regulate freezing tolerance and cold-responsive gene expression in both apple and Arabidopsis. Chromatin-Immunoprecipitation-qPCR and electrophoretic mobility shift assays showed that MdMYB88/MdMYB124 act as direct regulators of the COLD SHOCK DOMAIN PROTEIN 3 (MdCSP3) and CIRCADIAN CLOCK ASSOCIATED 1 (MdCCA1) genes. Dual luciferase reporter assay indicated that MdCCA1 but not MdCSP3 activated the expression of MdCBF3 under cold stress. Moreover, MdMYB88 and MdMYB124 promoted anthocyanin accumulation and H2 O2 detoxification in response to cold. Taken together, our results suggest that MdMYB88 and MdMYB124 positively regulate cold hardiness and cold-responsive gene expression under cold stress by C-REPEAT BINDING FACTOR (CBF)-dependent and CBF-independent pathways.

Melatonin regulates proteomic changes during leaf senescence in Malus hupehensis.

Despite the relationship between melatonin and aging, the overall changes and regulation of proteome profiling by long-term melatonin exposure during leaf senescence is not well understood. In this study, leaf senescence in Malus hupehensis plants was delayed when exogenous melatonin was regularly applied to the roots for 2 months compared with natural leaf senescence. Proteins of samples 0 and 50 day for both treatments were extracted and labeled with TMT regents before being examined via NanoLC-MS/MS. The proteomics data showed that 622 and 309 proteins were altered by senescence and melatonin, respectively. Our GO analysis by Blast2GO revealed that most of the altered proteins that are involved in major metabolic processes exhibited hydrolase activity and were mainly located in the plastids. These proteins were classified into several senescence-related functional categories, including degradation of macromolecules, redox and stress responses, transport, photosynthesis, development, and other regulatory proteins. We found that melatonin treatment led to the downregulation of proteins that are normally upregulated during senescence. The melatonin-related delay in senescence might have occurred due to the altering of proteins involved in processes associated with senescence. And as well, there are many unknown regulatory proteins possibly being involved in the melatonin's function. This study is the first to demonstrate changes at the proteome level in response to exogenous melatonin in plants. Our findings provide a set of informative and fundamental data about the role of melatonin in apple leaf senescence.

Identification of DNA methylation and genetic alteration simultaneously from a single blood biopsy.

BACKGROUND: High-throughput sequencing of blood cell-free DNA (cfDNA) techniques offer an opportunity to characterize and monitor cancer rapidly in a non-invasive and real-time manner. Nonetheless, there lacks a tool within therapeutic arsenal to identify multi-omics alterations simultaneously from a single biopsy. In current times, bisulfite-based sequencing detects 5mC and 5hmC at single-base resolution is the golden standard of DNA methylation, while the degradation of DNA and biased sequencing data are the problems of this method. OBJECTIVE: To identify the consistency analysis of methylation and genetic variation with single library, we presented a platform detecting multi-omics data simultaneously from a single blood biopsy using bisulfite-free method of genomic methylation sequencing (GM-seq) mediated by TET enzyme. METHODS: We detected methylomic and genetic changes simultaneously from a single blood biopsy in NA12878 and randomly chose ten blood biopsies from colorectal cancer or lung cancer patients to validate the ability of GM-seq. RESULTS: Similar cytosine methylation level between whole genome bisulfite sequencing (WGBS) and GM-seq were identified in NA12878. Moreover, longer insert size, CpGs coverage and GC distribution were outperformed than WGBS. In addition, the comparison of the single nucleotide polymorphism (SNP), insertion-deletion (Indel) and copy number variation (CNV) in NA12878 or ctDNA from liver cancer between GM-seq and whole genome sequencing (WGS) show a good consistency, indicating that this method is feasible for detecting genetic variation in blood. CONCLUSION: In conclusion, our work demonstrated a method for identification of the methylated modification and genetic variations simultaneously from a single blood biopsy.

New Wine in an Old Bottle: Utilizing Chemical Genetics to Dissect Apical Hook Development.

The apical hook is formed by dicot seedlings to protect the tender shoot apical meristem during soil emergence. Regulated by many phytohormones, the apical hook has been taken as a model to study the crosstalk between individual signaling pathways. Over recent decades, the roles of different phytohormones and environmental signals in apical hook development have been illustrated. However, key regulators downstream of canonical hormone signaling have rarely been identified via classical genetics screening, possibly due to genetic redundancy and/or lethal mutation. Chemical genetics that utilize small molecules to perturb and elucidate biological processes could provide a complementary strategy to overcome the limitations in classical genetics. In this review, we summarize current progress in hormonal regulation of the apical hook, and previously reported chemical tools that could assist the understanding of this complex developmental process. We also provide insight into novel strategies for chemical screening and target identification, which could possibly lead to discoveries of new regulatory components in apical hook development, or unidentified signaling crosstalk that is overlooked by classical genetics screening.

Maximum Somatic Allele Frequency-Adjusted Blood-Based Tumor Mutational Burden Predicts the Efficacy of Immune Checkpoint Inhibitors in Advanced Non-Small Cell Lung Cancer.

INTRODUCTION: Recent studies exhibited the unstable prediction ability of blood-based tumor mutational burden (bTMB) when predicting the response of immune checkpoint inhibitors (ICIs) therapy in patients with non-small cell lung cancer (NSCLC). Circulating tumor DNA (ctDNA) abundance, usually represented by maximum somatic allele frequency (MSAF), was one possible confounding factor influencing bTMB ability in ICIs response prediction. METHODS: MSAF-adjusted bTMB (Ma-bTMB) was established and validated in patients with advanced NSCLC among Geneplus Cancer Genome Database (GCGD, n = 1679), Zhuo (n = 35), Wang (n = 45), POPLAR (NCT01903993, n = 211) and OAK (NCT02008227, n = 642) cohorts. RESULTS: MSAF demonstrated a modest positive correlation with bTMB and a negative one with survival benefit. Improved survival outcomes of ICIs therapy have been observed among patients with high-Ma-bTMB compared to those with low-Ma-bTMB in Zhuo and Wang cohorts. In addition, compared to low-Ma-bTMB, high-Ma-bTMB was associated with more positive clinical benefits from ICIs therapy than chemotherapy both in POPLAR and OAK cohorts. Further exploration suggested that Ma-bTMB could precisely identify more potential ICIs beneficiaries compared to bTMB and LAF-bTMB, complementary to PD-L1 expression. CONCLUSIONS: We developed Ma-bTMB, a convenient, readily available, non-invasive predictive biomarker effectively differentiates beneficiaries of ICIs therapy in advanced NSCLC, warranting future clinical trials.

Apple TIME FOR COFFEE contributes to freezing tolerance by promoting unsaturation of fatty acids.

Freezing stress is a major environmental factor that threatens the growth and development of fruit trees. MdMYB88 and its paralogue MdMYB124 have been identified as pivotal regulators in apple (Malus × domestica) freezing stress tolerance. Here, we demonstrated that a target of MdMYB88 and MdMYB124, TIME FOR COFFEE (TIC), contributes to freezing tolerance in apple. MdMYB88 and MdMYB124 directly bound the MdTIC promoter and positively regulated its expression under cold conditions. MdTIC RNAi plants displayed reduced freezing tolerance when MdTIC expression was repressed. Moreover, MdTIC RNAi plants lowered antioxidant enzyme activity. Transcriptome profiling revealed altered expression of cold-responsive genes in MdTIC RNAi plants under cold conditions, including MdPLC2, MdMKK2, and MdICE1. We also discovered that disordered MdTIC expression changed the saturation of fatty acids. Taken together, our data suggest that MdTIC is required for apple to tolerate freezing by mediating the expression of cold-responsive genes and fatty acid composition.

Apple SERRATE negatively mediates drought resistance by regulating MdMYB88 and MdMYB124 and microRNA biogenesis.

The function of serrate (SE) in miRNA biogenesis in Arabidopsis is well elucidated, whereas its role in plant drought resistance is largely unknown. In this study, we report that MdSE acts as a negative regulator of apple (Malus × domestica) drought resistance by regulating the expression levels of MdMYB88 and MdMYB124 and miRNAs, including mdm-miR156, mdm-miR166, mdm-miR172, mdm-miR319, and mdm-miR399. MdSE interacts with MdMYB88 and MdMYB124, two positive regulators of apple drought resistance. MdSE decreases the transcript and protein levels of MdMYB88 and MdMYB124, which directly regulate the expression of MdNCED3, a key enzyme in abscisic acid (ABA) biosynthesis. Furthermore, MdSE is enriched in the same region of the MdNECD3 promoter where MdMYB88/MdMYB124 binds. Consistently, MdSE RNAi transgenic plants are more sensitive to ABA-induced stomatal closure, whereas MdSE OE plants are less sensitive. In addition, under drought stress, MdSE is responsible for the biogenesis of mdm-miR399, a negative regulator of drought resistance, and negatively regulates miRNAs, including mdm-miR156, mdm-miR166, mdm-miR172, and mdm-miR319, which are positive regulators of drought resistance. Taken together, by revealing the negative role of MdSE, our results broaden our understanding of the apple drought response and provide a candidate gene for apple drought improvement through molecular breeding.

Regulation of phenylpropanoid biosynthesis by MdMYB88 and MdMYB124 contributes to pathogen and drought resistance in apple.

MdMYB88 and MdMYB124 have been demonstrated to be responsible for lignin accumulation in apple under drought stress. In this study, using a metabolomic approach, we identified differentially accumulated phenylpropanoid and flavonoid metabolites in MdMYB88/124 transgenic RNAi plants under control and long-term drought stress conditions in apple roots. We confirmed the regulation of phenylalanine by MdMYB88 and MdMYB124 via UPLC-MS in apple roots under both control and drought conditions. Using Electrophoretic Mobility Shift Assay (EMSA) and ChIP-quantitative PCR (qPCR) analyses, we found that MdMYB88 positively regulates the MdCM2 gene, which is responsible for phenylalanine biosynthesis, through binding to its promoter region. Under long-term drought conditions, MdMYB88/124 RNAi plants consistently accumulated increased amounts of H2O2 and MDA, while MdMYB88 and MdMYB124 overexpression plants accumulated decreased amounts of H2O2 and MDA. We also examined the accumulation of metabolites in the phenylpropanoid biosynthesis pathway in the leaves of MdMYB88 and MdMYB124 transgenic apple plants after long-term drought stress. We found that metabolites responsible for plant defense, including phenylpropanoids and flavonoids, accumulated less in the RNAi plants but more in the overexpression plants under both control and drought conditions. We further demonstrated that MdMYB88/124 RNAi plants were more sensitive to Alternaria alternata f. sp. mali and Valsa mali, two pathogens that currently severely threaten apple production. In contrast, MdMYB88 and MdMYB124 overexpression plants were more tolerant to these pathogens. The cumulative results of this study provided evidence for secondary metabolite regulation by MdMYB88 and MdMYB124, further explained the molecular roles of MdMYB88 and MdMYB124 in drought resistance, and provided information concerning molecular aspects of their roles in disease resistance.

Genome-wide identification of drought-responsive microRNAs in two sets of Malus from interspecific hybrid progenies.

Drought stress can negatively impact apple fruit quality and yield. Apple microRNAs (miRNAs) participate in apple tree and fruit development, as well as in biotic stress tolerance; however, it is largely unknown whether these molecules are involved in the drought response. To identify drought-responsive miRNAs in Malus, we first examined the drought stress tolerance of ten F1 progenies of R3 (M. × domestica) × M. sieversii. We performed Illumina sequencing on pooled total RNA from both drought-tolerant and drought-sensitive plants. The sequencing results identified a total of 206 known miRNAs and 253 candidate novel miRNAs from drought-tolerant plants and drought-sensitive plants under control or drought conditions. We identified 67 miRNAs that were differentially expressed in drought-tolerant plants compared with drought-sensitive plants under drought conditions. Under drought stress, 61 and 35 miRNAs were differentially expressed in drought-tolerant and drought-sensitive plants, respectively. We determined the expression levels of seven out of eight miRNAs by stem-loop qPCR analysis. We also predicted the target genes of all differentially expressed miRNAs and identified the expression of some genes. Gene Ontology analyses indicated that the target genes were mainly involved in stimulus response and cellular and metabolic processes. Finally, we confirmed roles of two miRNAs in apple response to mannitol. Our results reveal candidate miRNAs and their associated mRNAs that could be targeted for improving drought tolerance in Malus species, thus providing a foundation for understanding the molecular networks involved in the response of apple trees to drought stress.

Improved hybrid de novo genome assembly of domesticated apple (Malus x domestica).

BACKGROUND: Domesticated apple (Malus × domestica Borkh) is a popular temperate fruit with high nutrient levels and diverse flavors. In 2012, global apple production accounted for at least one tenth of all harvested fruits. A high-quality apple genome assembly is crucial for the selection and breeding of new cultivars. Currently, a single reference genome is available for apple, assembled from 16.9 × genome coverage short reads via Sanger and 454 sequencing technologies. Although a useful resource, this assembly covers only ~89 % of the non-repetitive portion of the genome, and has a relatively short (16.7 kb) contig N50 length. These downsides make it difficult to apply this reference in transcriptive or whole-genome re-sequencing analyses. FINDINGS: Here we present an improved hybrid de novo genomic assembly of apple (Golden Delicious), which was obtained from 76 Gb (~102 × genome coverage) Illumina HiSeq data and 21.7 Gb (~29 × genome coverage) PacBio data. The final draft genome is approximately 632.4 Mb, representing ~ 90 % of the estimated genome. The contig N50 size is 111,619 bp, representing a 7 fold improvement. Further annotation analyses predicted 53,922 protein-coding genes and 2,765 non-coding RNA genes. CONCLUSIONS: The new apple genome assembly will serve as a valuable resource for investigating complex apple traits at the genomic level. It is not only suitable for genome editing and gene cloning, but also for RNA-seq and whole-genome re-sequencing studies.