Biology subjects

Song, J.

Publications and source records attributed to Song, J..

At least 271 records · Page 15Linked to original sources

bioRxiv2020

Establishment of a stable zg6pdM118-144 transgenic zebrafish model of glucose-6-phosphate dehydrogenase deficiency

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common genetic defect and enzymopathy with a wide distribution and increased public health concern, predisposes subjects succumb to oxidative stress. G6PD deficiency has been associated with hemolysis. Clinically, G6PD deficiency is asymptomatic and the clinical manifestations occur with the exposure to certain agents. Due to the lack of suitable animal models that can predict the clinical hemolytic potential of drugs, it needs an appropriate research model to fully recapitulate the manifestations of G6PD deficiency in clinic, to optimize the malaria therapy and promote anti-malarias development. The present study has displayed a stable transgenic Tg(zgata1-g6pdM118-144-egfp) zebrafish model with G6PD deficiency which mimics the clinical features of G6PD deficiency phenotypically and functionally. The findings showed that there was an inadequate level of reduced GSH in the transgenic Tg(zgata1-g6pdM118-144-egfp) zebrafish line in the presence or absence of α-naphthol, compared to the wildtype zebrafish, indicating an attenuation of g6pd activity in the transgenic zebrafish line. In addition, the observations show that there is a less abundance of g6pd in the transgenic Tg(zgata1-g6pdM118-144-egfp) zebrafish line. On the other hand, there is no morphological abnormality in the transgenic Tg(zgata1-g6pdM118-144-egfp) zebrafish line. Taken together, our work has delivered a novel stable transgenic zebrafish model of G6PD deficiency that will facilitate the mechanistic and functional elucidation for the role of G6PD in erythrocytic pathophysiology. This model will promote the translational research for the drug development, in particular, for anti-malarias development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

zoology↗

bioRxiv2020

Comprehensive structure-function characterization of DNMT3B and DNMT3A reveals distinctive de novo DNA methylation mechanisms

Mammalian DNA methylation patterns are established by two de novo DNA methyltransferases DNMT3A and DNMT3B, which exhibit both redundant and distinctive methylation activities. However, the related molecular basis remains undetermined. Through comprehensive structural, enzymology and cellular characterization of DNMT3A and DNMT3B, we here report a multi-layered substrate-recognition mechanism underpinning their divergent genomic methylation activities. A hydrogen bond in the catalytic loop of DNMT3B causes a lower CpG specificity than DNMT3A, while the interplay of target recognition domain and homodimeric interface fine-tunes the distinct target selection between the two enzymes, with Lysine 777 of DNMT3B acting as a unique sensor of the +1 flanking base. The divergent substrate preference between DNMT3A and DNMT3B provides an explanation for site-specific epigenomic alterations seen in ICF syndrome with DNMT3B mutations. Together, this study reveals crucial and distinctive substrate-readout mechanisms of the two DNMT3 enzymes, implicative of their differential roles during development and pathogenesis.

genetics↗

bioRxiv2020

Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

genetics↗

bioRxiv2020

Endothelial basement membrane laminins as an environmental cue in monocyte differentiation to macrophages.

Monocyte differentiation to macrophages is triggered by migration across the endothelial barrier, which is constituted by both endothelial cells and their underlying basement membrane. We address here the role of the endothelial basement membrane laminins (laminins 411 and 511) in this monocyte to macrophage switch. Chimeric mice carrying CX3CR1-GFP bone marrow were employed to track CCL2-induced monocyte extravasation in a cremaster muscle model using intravital microscopy, revealing faster extravasation in mice lacking endothelial laminin 511 (Tek-cre::Lama5-/-) and slower extravasation in mice lacking laminin 411 (Lama4-/-). CX3CR1-GFPlow extravasating monocytes were found to have a higher motility at laminin 511 low sites and to preferentially exist vessels at these sites. However, in vitro experiments reveal that this is not due to effects of laminin 511 on monocyte migration mode nor on the tightness of the endothelial barrier. Rather, using an intestinal macrophage replenishment model and in vitro differentiation studies we demonstrate that laminin 511 together with the attached endothelium collectively promote monocyte differentiation to macrophages. Macrophage differentiation is associated with a change in integrin profile, permitting differentiating macrophages to distinguish between laminin 511 high and low areas and to migrate preferentially across laminin 511 low sites. These studies highlight the endothelial basement membrane as a critical site for monocyte differentiation to macrophages, which may be relevant to the differentiation of other cells at vascular niches.

immunology↗

bioRxiv2020

Acoustic regularities in infant-directed vocalizations across cultures

The forms of many species vocal signals are shaped by their functions1-15. In humans, a salient context of vocal signaling is infant care, as human infants are altricial16, 17. Humans often alter their vocalizations to produce "parentese", speech and song produced for infants that differ acoustically from ordinary speech and song18-35 in fashions that have been proposed to support parent-infant communication and infant language learning36-39; modulate infant affect33, 40-45; and/or coordinate communicative interactions with infants46-48. These theories predict a form-function link in infant-directed vocalizations, with consistent acoustic differences between infant-directed and adult-directed vocalizations across cultures. Some evidence supports this prediction23, 27, 28, 32, 49-52, but the limited generalizability of individual ethnographic reports and laboratory experiments53 and small stimulus sets54, along with intriguing reports of counterexamples55-62, leave the question open. Here, we show that people alter the acoustic forms of their vocalizations in a consistent fashion across cultures when speaking or singing to infants. We collected 1,615 recordings of infant- and adult-directed singing and speech produced by 410 people living in 21 urban, rural, and small-scale societies, and analyzed their acoustic forms. We found cross-culturally robust regularities in the acoustics of infant-directed vocalizations, such that infant-directed speech and song were reliably classified from acoustic features found across the 21 societies studied. The acoustic profiles of infant-directedness differed across language and music, but in a consistent fashion worldwide. In a secondary analysis, we studied whether listeners are sensitive to these acoustic features, playing the recordings to 51,065 people recruited online, from many countries, who guessed whether each vocalization was infant-directed. Their intuitions were largely accurate, predictable in part by acoustic features of the recordings, and robust to the effects of linguistic relatedness between vocalizer and listener. By uniting rich cross-cultural data with computational methods, we show links between the production of vocalizations and cross-species principles of bioacoustics, informing hypotheses of the psychological functions and evolution of human communication.

animal behavior and cognition↗

bioRxiv2020

Analysis of insertion mutants in distal α9 portion of C-terminal heptad repeat (CHR) of HIV-1 gp41 subunit

We have made insertion mutants in 9 of HXB2 gp41 and observed similar phenotypes like recent JRFL mutants: insertion of alanine (653+A), but not glutamine (653+Q), severely attenuated membrane fusion. To understand the underlying mechanism, we performed the fusion inhibition assay by corresponding mutant C34 peptides. Both mutant C34 peptides added at the beginning of the coculture of the effector and target cells showed less efficient inhibition of membrane fusion, which was similar to wildtype C34 added after 30 min of coculture, indicating slow association of mutant C34 peptides with the N-terminal heptad region of gp41. Due to uninterpretable CD profiles of C34 and N36, we tested the longer peptide pairs (N46 and C42) and observed CD profiles indicative of weak -helix formation. The melting temperatures for N46-C42 pairs of 653+A, 653+Q, and wild type were 56.8 {degrees}C, 59.8{degrees}C, and 96{degrees}C, respectively. Taken together, our data suggested that the phenotypic difference in membrane fusion between 653+A and 653+Q (or wild type) was not based on the stability of the six-helix bundle (6HB), but due to differences in the kinetics of 6HB formation. Further, we examined additional insertions (E, R, I, and L) at position 653, for which only I and L showed fusion recovery similar to Q, suggesting that the polar nature of glutamine was not a phenotypic determinant.

microbiology↗

bioRxiv2020

Evolutionary patterns of 64 vertebrate genomes (species) revealed by phylogenomics analysis of protein-coding gene families

BackgroundRecent studies have demonstrated that phylogenomics is an important basis for answering many fundamental evolutionary questions. With more high-quality whole genome sequences published, more efficient phylogenomics analysis workflows are required urgently. ResultsTo this end and in order to capture putative differences among evolutionary histories of gene families and species, we developed a phylogenomics workflow for gene family classification, gene family tree inference, species tree inference and duplication/loss events dating. Our analysis framework is on the basis of two guiding ideas: 1) gene trees tend to be different from species trees but they influence each other in evolution; 2) different gene families have undergone different evolutionary mechanisms. It has been applied to the genomic data from 64 vertebrates and 5 out-group species. And the results showed high accuracy on species tree inference and few false-positives in duplication events dating. ConclusionsBased on the inferred gene duplication and loss event, only 9[~]16% gene families have duplication retention after a whole genome duplication (WGD) event. A large part of these families have ohnologs from two or three WGDs. Consistent with the previous study results, the gene function of these families are mainly involved in nervous system and signal transduction related biological processes. Specifically, we found that the gene families with ohnologs from the teleost-specific (TS) WGD are enriched in fat metabolism, this result implyng that the retention of such ohnologs might be associated with the environmental status of high concentration of oxygen during that period.

bioinformatics↗

bioRxiv2020

DNA Encoded Glycan Libraries as a next-generation tool for the study of glycan-protein interactions

Interactions between glycans and glycan-binding proteins (GBPs) mediate diverse cellular functions, and therefore are of diagnostic and therapeutic significance. Current leading strategies for studying glycan-GBP interactions require specialized knowledge and instrumentation. In this study, we report a strategy for studying glycan-GBP interactions that uses PCR, qPCR and next-generation sequencing (NGS) technologies that are more routinely accessible. Our headpiece conjugation-code ligation (HCCL) strategy couples glycans with unique DNA codes that specify glycan sugar moieties and glycosidic linkages when sequenced. We demonstrate the technology by synthesizing a DNA encoded glycan library of 50 biologically relevant glycans (DEGL-50) and probing interactions against 25 target proteins including lectins and antibodies. Data show glycan-GPB interactions in solution that are consistent with lower content, lower throughput ELISA assays. Data further demonstrate how monovalent and multivalent headpieces can be used to increase glycan-GPB interactions and enrich signals while using smaller sample sizes. The flexibility of our modular HCCL strategy has potential for producing large glycan libraries, facilitating high content-high throughput glycan binding studies, and increasing access to lower cost glyco-analyses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/017012v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@fb1c1dorg.highwire.dtl.DTLVardef@1f2c3ceorg.highwire.dtl.DTLVardef@11546e7org.highwire.dtl.DTLVardef@1e21a83_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Substrate deformation regulates DRM2-mediated DNA methylation in plants

DNA methylation is an important epigenetic mechanism that critically regulates gene expression and genomic stability. In plants, Domains Rearranged Methyltransferase 2 (DRM2) preferentially mediates CHH methylation (H=C, T, A), a substrate specificity distinct from that of mammalian DNA methyltransferases. However, the underlying mechanism is unknown. Here, we report structure-function characterizations of DRM2-mediated methylation. An arginine finger from the catalytic loop intercalates into DNA minor groove, inducing large DNA deformation that impacts the substrate specificity of DRM2. To accommodate the substrate deformation, the target recognition domain of DRM2 embraces the enlarged DNA major groove via shape complementarity, disruption of which via C397R mutation shifts the substrate specificity of DRM2 toward CHG DNA. This study uncovers DNA deformation as a mechanism in regulating the substrate specificity of DRM2, implicative of transposon-specific repression in plants.

biochemistry↗

bioRxiv2020

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis

Scutellaria baicalensis and Scutellaria barbata, common medicinal plants of the Lamiaceae family, produce specific flavonoid compounds with antioxidant and antitumor activities, including baicalein, scutellarein, norwogonin, wogonin, and their glycosides. Here, we reported two chromosome-level genome assemblies of S. baicalensis and S. barbata with significant quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. The comparative genome analysis of congeneric species elucidated the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of the phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes, and the S. barbata-specific duplication of 4-CoA ligase (4CL) genes. In addition, the paralogous duplication, collinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of flavone hydroxylase genes between S. baicalensis and S. barbata. These Scutellaria genomes highlight the common and species-specific evolution of flavone biosynthetic genes, promoting the development of molecular breeding and the study of the biosynthesis and regulation of bioactive compounds.

genomics↗

bioRxiv2020

Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots

Plant cells undergo two types of cell cycles - the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Here, we compare sequence-based RT profiles and found that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small, with a median size of 135 kb, and shift to a later RT in the endocycle. However, we found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere in each case, which are [~]1-2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells, but primarily in late S phase of the endocycle. Strikingly, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in nuclei of different ploidies suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and reduced CENH3 enrichment after endocycle replication is consistent with the hypothesis that centromeres are being inactivated as their function is no longer needed. AUTHOR SUMMARYIn traditional cell division, or mitosis, a cells genetic material is duplicated and then split between two daughter cells. In contrast, in some specialized cell types, the DNA is duplicated a second time without an intervening division step, resulting in cells that carry twice as much DNA - a phenomenon called an endocycle, which is common during plant development. At each step, DNA replication follows an ordered program, in which highly compacted DNA is unraveled and replicated in sections at different times during the synthesis (S) phase. In plants, it is unclear whether traditional and endocycle programs are the same. Using root tips of maize, we found a small portion of the genome whose replication in the endocycle is shifted in time, usually to later in S phase. Some of these regions are scattered around the genome, and mostly coincide with active genes. However, the most prominent shifts occur in centromeres. This location is noteworthy because centromeres orchestrate the process of separating duplicated chromosomes into daughter cells, a function that is not needed in the endocycle. Our observation that centromeres replicate later in the endocycle suggests there is an important link between the time of replication and the function of centromeres.

genomics↗

bioRxiv2019

The effect of feeding behavior of Monochamus alternatus (Coleoptera: Cerambycidae) on the departure of pine wood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

In order to study the causes of pine wood nematode (PWN) departure from Monochamus alternatus, the effects of the feeding behavior of M. alternatus on the start date of the departure of PWN were studied. The start date of the departure of PWN carried by the directly fed M. alternatus was 5—13 d after beetle emergence, mainly concentrated within 6—10 d, with a mean (±SD) of 8.02 ± 1.96 d. The start date of the departure of PWN carried by the M. alternatus fed after starvation was 5—14 d after beetle emergence, mainly concentrated within 6—9 d, with a mean of 7.76 ± 2.28 d. The results show that there was no significant difference in the start departure date of PWN between the two treatments. This shows that the feeding behavior of M. alternatus is not the trigger for PWN departure. At the same time, it was found that the motility of the PWN carried by M. alternatus at 8 d after emergence was significantly greater than that of the PWN carried by the newly emerged M. alternatus. And the PWN carried by M. alternatus at 8 d after emergence was extracted more easily than the PWN carried by newly emerged beetles. These results show that greater motility was associated with easier departure of PWN from M. alternatus. In addition, transcriptome sequencing found that the level of oxidative phosphorylation metabolism of PWN carried by beetles at 8 d after emergence was significantly higher than that in the PWN carried by newly emerged beetle. High oxidative phosphorylation was associated with increased energy production and motility by the PWN and were the internal cause of the start of nematode departure.Competing Interest StatementThe authors have declared no competing interest.View Full Text

animal behavior and cognition↗

bioRxiv2019

A cryo-tomography-based volumetric model of the actin core of mouse vestibular hair cell stereocilia lacking plastin 1

Electron cryo-tomography allows for high-resolution imaging of stereocilia in their native state. Because their actin filaments have a higher degree of order, we imaged stereocilia from mice lacking the actin crosslinker plastin 1 (PLS1). We found that while stereocilia actin filaments run 13 nm apart in parallel for long distances, there were gaps of significant size that were stochastically distributed throughout the actin core. Actin crosslinkers were distributed through the stereocilium, but did not occupy all possible binding sites. At stereocilia tips, protein density extended beyond actin filaments, especially on the side of the tip where a tip link should anchor. Along the shaft, repeating density was observed that corresponds to actin-to-membrane connectors. In the taper region, most actin filaments terminated near the plasma membrane. The remaining filaments twisted together to make a tighter bundle than was present in the shaft region; the spacing between them decreased from 13 nm to 9 nm. Our models illustrate detailed features of distinct structural domains that are present within the stereocilium.

neuroscience↗

bioRxiv2019

Diversity and complexity of the large surface protein family in the compacted genomes of various Pneumocystis species

Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multi-copy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins, and provide detailed description of the classification, unique characteristics and phylogenetic relatedness of five Msg families. We further describe the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests a wide variety of functions for this superfamily, not only conferring antigenic variation to allow immune evasion but also mediating life-stage development, optimizing cell mobility and adhesion, and adapting to specific host niches or environmental conditions. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.

molecular biology↗

bioRxiv2019

DHHC20 palmitoyl-transferase reshapes the membrane to foster catalysis

Cysteine palmitoylation, a form of S-acylation, is a key post-translational modification in cellular signaling. This type of reversible lipidation is catalyzed by a family of integral membrane proteins known as DHHC acyltransferases. The first step in the S-acylation process is the recognition of free acyl-CoA from the lipid bilayer. The DHHC enzyme then becomes auto-acylated, at a site defined by a conserved Asp-His-His-Cys motif. This reaction entails ionization of the catalytic Cys. Intriguingly, in known DHHC structures this catalytic Cys appears to be exposed to the hydrophobic interior of the lipid membrane, which would be highly unfavorable for a negatively charged nucleophile, thus hindering auto-acylation. Here, we use biochemical and computational methods to reconcile these seemingly contradicting facts. First, we experimentally demonstrate that human DHHC20 is active when reconstituted in POPC nanodiscs. Microsecond-long all-atom molecular dynamics simulations are then calculated for hDHHC20 and for different acyl-CoA forms, also in POPC. Strikingly, we observe that hDHHC20 induces a drastic deformation in the membrane, particularly on the cytoplasmic side where auto-acylation occurs. As a result, the catalytic Cys becomes hydrated and optimally positioned to encounter the cleavage site in acyl-CoA. In summary, we hypothesize that DHHC enzymes locally reshape the membrane to foster a morphology that is specifically adapted for acyl-CoA recognition and auto-acylation.\n\nSignificance StatementPalmitoylation, the most common form of S-acylation and the only reversible type of protein lipidation, is ubiquitous in eukaryotic cells. In humans, for example, it has been estimated that as much as [~]10% of the proteome becomes palmitoylated, i.e. thousands of proteins. Accordingly, protein palmitoylation touches every important aspect of human physiology, both in health and disease. Despite its biological and biomedical importance, little is known about the molecular mechanism of the enzymes that catalyze this post-translational modification, known as DHHC acyltransferases. Here, we leverage the recently-determined atomic-resolution structure of human DHHC20 to gain novel insights into the mechanism of this class of enzymes, using both experimental and computational approaches.

biophysics↗

bioRxiv2019

Hormone Levels Are Related to Altered Functional Connectivity in Prolactinomas

Background and ObjectiveProlactinomas may cause drastic hormone fluctuations throughout the body. It is not fully understood how endogenous hormone disorders such as prolactinomas reshape the patients brain. By employing the resting-state functional magnetic resonance imaging technique, we aimed to investigate the whole-brain functional connectivity (FC) and its relationship with hormone levels in patients with prolactinomas.\n\nMethodsUsing whole-brain and seed-based functional connectivity analyses, we compared FC metrics between 33 prolactinoma patients and 31 healthy controls matched with age, sex, and handedness. Then we performed partial correlation analysis to examine the relationship between FC metrics and hormone levels.\n\nResultsCompared to healthy controls, we found that prolactinoma patients showed significantly increased thalamocortical (visual cortex) and cerebellar-cerebral connectivity. In addition, endogenous hormone levels were positively correlated with the increased FC, and the hormone-FC relationships showed sex difference in prolactinoma patients.\n\nConclusionsOur findings are the first to reveal the altered FC patterns and sex-dependent hormone-FC relationships in prolactinoma patients, indicating the important role of hormone levels in the neural mechanism of brain reorganization and hyperactive intrinsic connections in prolactinomas.

neuroscience↗

bioRxiv2019

ATP emerged to induce protein folding, inhibit aggregation and increase stability

By NMR characterization of effects of ATP and related molecules on the folding and dynamics of the ALS-causing C71G-PFN1 and nascent hSOD1, we reveal for the first time that ATP has a general capacity in inducing protein folding with the highest efficiency known so far. This capacity was further identified to result from triphosphate, a key intermediate in prebiotic chemistry, which, however, can severely trigger protein aggregation. Remarkably, by joining adenosine and triphosphate together, ATP integrates three abilities to simultaneously induce protein folding, inhibit aggregation and increase thermodynamic stability. Our study implies that the emergence of ATP might represent an irreplaceable step essential for the Origin of Life, and decrypts a principle for engineering small molecules with three functions to treat aggregation-associated ageing and diseases. One sentence summaryBy joining adenosine and triphosphate, ATP integrates three abilities to control protein homeostasis for the Origin of Life.

biochemistry↗

bioRxiv2019

Allelic diversity of NAC18.1 is a major determinant of fruit firmness and harvest date in apple (Malus domestica)

Softening is a hallmark of ripening in fleshy fruits, and has both desirable and undesirable implications for texture and postharvest stability. Accordingly, the timing and extent of ripening and associated textural changes are key targets for improving fruit quality through breeding. Previously, we identified a large effect locus associated with harvest date and firmness in apple (Malus domestica) using genome-wide association studies (GWAS). Here, we present additional evidence that polymorphisms in or around a transcription factor gene, NAC18.1, may cause variation in these traits. First, we confirmed our previous findings with new phenotype and genotype data from [~]800 apple accessions. In this population, we compared a genetic marker within NAC18.1 to markers targeting three other firmness-related genes currently used by breeders (ACS1, ACO1, and PG1), and found that the NAC18.1 marker was the strongest predictor of both firmness at harvest and firmness after three months of cold storage. By sequencing NAC18.1 across 18 accessions, we revealed two predominant haplotypes containing the single nucleotide polymorphism (SNP) previously identified using GWAS, as well as dozens of additional SNPs and indels in both the coding and promoter sequences. NAC18.1 encodes a protein with high similarity to the NON-RIPENING (NOR) transcription factor, a regulator of ripening in tomato (Solanum lycopersicum). To test whether these genes are functionally orthologous, we introduced both NAC18.1 transgene haplotypes into the tomato nor mutant and showed that both haplotypes complement the nor ripening deficiency. Taken together, these results indicate that polymorphisms in NAC18.1 may underlie substantial variation in apple firmness through modulation of a conserved ripening program.

plant biology↗