Graph-Based Pangenome of Actinidia chinensis Reveals Structural Variations Mediating Fruit Degreening.

Fruit ripening is associated with the degreening process (loss of chlorophyll) that occurs in most fruit species. Kiwifruit is one of the special species whose fruits may maintain green flesh by accumulating a large amount of chlorophyll even after ripening. However, little is known about the genetic variations related to the fruit degreening process. Here, a graph-based kiwifruit pangenome by analyzing 14 chromosome-scale haplotype-resolved genome assemblies from seven representative cultivars or lines in Actinidia chinensis is built. A total of 49,770 non-redundant gene families are identified, with core genes constituting 46.6%, and dispensable genes constituting 53.4%. A total of 84,591 non-redundant structural variations (SVs) are identified. The pangenome graph integrating both reference genome sequences and variant information facilitates the identification of SVs related to fruit color. The SV in the promoter of the AcBCM gene determines its high expression in the late developmental stage of fruits, which causes chlorophyll accumulation in the green-flesh fruits by post-translationally regulating AcSGR2, a key enzyme of chlorophyll catabolism. Taken together, a high-quality pangenome is constructed, unraveled numerous genetic variations, and identified a novel SV mediating fruit coloration and fruit quality, providing valuable information for further investigating genome evolution and domestication, QTL genes function, and genomics-assisted breeding.

Haplotype-resolved genome assembly provides insights into evolutionary history of the Actinidia arguta tetraploid.

Actinidia arguta, known as hardy kiwifruit, is a widely cultivated species with distinct botanical characteristics such as small and smooth-fruited, rich in beneficial nutrients, rapid softening and tolerant to extremely low temperatures. It contains the most diverse ploidy types, including diploid, tetraploid, hexaploid, octoploid, and decaploid. Here we report a haplotype-resolved tetraploid genome (A. arguta cv. 'Longcheng No.2') containing four haplotypes, each with 40,859, 41,377, 39,833 and 39,222 protein-coding genes. We described the phased genome structure, synteny, and evolutionary analyses to identify and date possible WGD events. Ks calculations for both allelic and paralogous genes pairs throughout the assembled haplotypic individuals showed its tetraploidization is estimated to have formed ~ 1.03 Mya following Ad-α event occurred ~ 18.7 Mya. Detailed annotations of NBS-LRRs or CBFs highlight the importance of genetic variations coming about after polyploidization in underpinning ability of immune responses or environmental adaptability. WGCNA analysis of postharvest quality indicators in combination with transcriptome revealed several transcription factors were involved in regulating ripening kiwi berry texture. Taking together, the assembly of an A. arguta tetraploid genome provides valuable resources in deciphering complex genome structure and facilitating functional genomics studies and genetic improvement for kiwifruit and other crops.

Telomere-to-telomere and haplotype-resolved genome of the kiwifruit Actinidia eriantha.

Actinidia eriantha is a characteristic fruit tree featuring with great potential for its abundant vitamin C and strong disease resistance. It has been used in a wide range of breeding programs and functional genomics studies. Previously published genome assemblies of A. eriantha are quite fragmented and not highly contiguous. Using multiple sequencing strategies, we get the haplotype-resolved and gap-free genomes of an elite breeding line "Midao 31" (MD), termed MDHAPA and MDHAPB. The new assemblies anchored to 29 pseudochromosome pairs with a length of 619.3 Mb and 611.7 Mb, as well as resolved 27 and 28 gap-close chromosomes in a telomere-to-telomere (T2T) manner. Based on the haplotype-resolved genome, we found that most alleles experienced purifying selection and coordinately expressed. Owing to the high continuity of assemblies, we defined the centromeric regions of A. eriantha, and identified the major repeating monomer, which is designated as Ae-CEN153. This resource lays a solid foundation for further functional genomics study and horticultural traits improvement in kiwifruit.

quarTeT: a telomere-to-telomere toolkit for gap-free genome assembly and centromeric repeat identification.

A high-quality genome is the basis for studies on functional, evolutionary, and comparative genomics. The majority of attention has been paid to the solution of complex chromosome structures and highly repetitive sequences, along with the emergence of a new 'telomere-to-telomere (T2T) assembly' era. However, the bioinformatic tools for the automatic construction and/or characterization of T2T genome are limited. Here, we developed a user-friendly web toolkit, quarTeT, which currently includes four modules: AssemblyMapper, GapFiller, TeloExplorer, and CentroMiner. First, AssemblyMapper is designed to assemble phased contigs into the chromosome-level genome by referring to a closely related genome. Then, GapFiller would endeavor to fill all unclosed gaps in a given genome with the aid of additional ultra-long sequences. Finally, TeloExplorer and CentroMiner are applied to identify candidate telomere and centromere as well as their localizations on each chromosome. These four modules can be used alone or in combination with each other for T2T genome assembly and characterization. As a case study, by adopting the entire modular functions of quarTeT, we have achieved the Actinidia chinensis genome assembly that is of a quality comparable to the reported genome Hongyang v4.0, which was assembled with the addition of manual handling. Further evaluation of CentroMiner by searching centromeres in Arabidopsis thaliana and Oryza sativa genomes showed that quarTeT is capable of identifying all the centromeric regions that have been previously detected by experimental methods. Collectively, quarTeT is an efficient toolkit for studies of large-scale T2T genomes and can be accessed at http://www.atcgn.com:8080/quarTeT/home.html without registration.

Telomere-to-telomere and gap-free reference genome assembly of the kiwifruit Actinidia chinensis.

Kiwifruit is an economically and nutritionally important fruit crop with extremely high contents of vitamin C. However, the previously released versions of kiwifruit genomes all have a mass of unanchored or missing regions. Here, we report a highly continuous and completely gap-free reference genome of Actinidia chinensis cv. 'Hongyang', named Hongyang v4.0, which is the first to achieve two de novo haploid-resolved haplotypes, HY4P and HY4A. HY4P and HY4A have a total length of 606.1 and 599.6 Mb, respectively, with almost the entire telomeres and centromeres assembled in each haplotype. In comparison with Hongyang v3.0, the integrity and contiguity of Hongyang v4.0 is markedly improved by filling all unclosed gaps and correcting some misoriented regions, resulting in ~38.6-39.5 Mb extra sequences, which might affect 4263 and 4244 protein-coding genes in HY4P and HY4A, respectively. Furthermore, our gap-free genome assembly provides the first clue for inspecting the structure and function of centromeres. Globally, centromeric regions are characterized by higher-order repeats that mainly consist of a 153-bp conserved centromere-specific monomer (Ach-CEN153) with different copy numbers among chromosomes. Functional enrichment analysis of the genes located within centromeric regions demonstrates that chromosome centromeres may not only play physical roles for linking a pair of sister chromatids, but also have genetic features for participation in the regulation of cell division. The availability of the telomere-to-telomere and gap-free Hongyang v4.0 reference genome lays a solid foundation not only for illustrating genome structure and functional genomics studies but also for facilitating kiwifruit breeding and improvement.

A Comparative Analysis of the Microbiome of Kiwifruit at Harvest Under Open-Field and Rain-Shelter Cultivation Systems.

The composition of microbial communities can directly affect fruit quality, health status, and storability. The present study characterized the epiphytes and endophytes of "Hongyang" and "Cuiyu" kiwifruit at harvest under grown under open-field (OF) and rain-shelter (RS) cultivation systems. Disease incidence in kiwifruit was significantly lower (p < 0.05) under the RS system than it was under the OF system. High-throughput sequencing [16S V3-V4 ribosomal region and the fungal internal transcribed spacer (ITS2)] was conducted to compare the composition of the epiphytic and endophytic microbial community of kiwifruit under the two cultivation systems. Results indicated that the abundance of Actinobacteria, Bacteroidetes, Enterobacteriales, Acetobacterales, Sphingomonas, Pseudomonas, and Sphingobacterium was higher under the RS system, relative to the OF system, while the abundance of Capnodiales, Hypocreales, Vishniacozyma, and Plectosphaerella was also higher under the RS system. Some of these bacterial and fungal taxa have been reported to as act as biocontrol agents and reduce disease incidence. Notably, the α-diversity of the epiphytic bacterial and fungal communities on kiwifruit was higher under RS cultivation. In summary, RS cultivation reduced natural disease incidence in kiwifruit, which may be partially attributed to differences in the structure and composition of the microbial community present in and on kiwifruit.

The chromosome-scale reference genome of Rubus chingii Hu provides insight into the biosynthetic pathway of hydrolyzable tannins.

Rubus chingii Hu (Fu-Pen-Zi), a perennial woody plant in the Rosaceae family, is a characteristic traditional Chinese medicinal plant because of its unique pharmacological effects. There are abundant hydrolyzable tannin (HT) components in R. chingii that provide health benefits. Here, an R. chingii chromosome-scale genome and related functional analysis provide insights into the biosynthetic pathway of HTs. In total, sequence data of 231.21 Mb (155 scaffolds with an N50 of 8.2 Mb) were assembled into seven chromosomes with an average length of 31.4 Mb, and 33 130 protein-coding genes were predicted, 89.28% of which were functionally annotated. Evolutionary analysis showed that R. chingii was most closely related to Rubus occidentalis, from which it was predicted to have diverged 22.46 million years ago (Table S8). Comparative genomic analysis showed that there was a tandem gene cluster of UGT, carboxylesterase (CXE) and SCPL genes on chromosome 02 of R. chingii, including 11 CXE, eight UGT, and six SCPL genes, which may be critical for the synthesis of HTs. In vitro enzyme assays indicated that the proteins encoded by the CXE (LG02.4273) and UGT (LG02.4102) genes have tannin hydrolase and gallic acid glycosyltransferase functions, respectively. The genomic sequence of R. chingii will be a valuable resource for comparative genomic analysis within the Rosaceae family and will be useful for understanding the biosynthesis of HTs.

Kiwifruit Genome Database (KGD): a comprehensive resource for kiwifruit genomics.

Kiwifruit (Actinidia spp.) plants produce economically important fruits containing abundant, balanced phytonutrients with extraordinarily high vitamin C contents. Since the release of the first kiwifruit reference genome sequence in 2013, large volumes of genome and transcriptome data have been rapidly accumulated for a handful of kiwifruit species. To efficiently store, analyze, integrate, and disseminate these large-scale datasets to the research community, we constructed the Kiwifruit Genome Database (KGD; http://kiwifruitgenome.org/). The database currently contains all publicly available genome and gene sequences, gene annotations, biochemical pathways, transcriptome profiles derived from public RNA-Seq datasets, and comparative genomic analysis results such as syntenic blocks and homologous gene pairs between different kiwifruit genome assemblies. A set of user-friendly query interfaces, analysis tools and visualization modules have been implemented in KGD to facilitate translational and applied research in kiwifruit, which include JBrowse, a popular genome browser, and the NCBI BLAST sequence search tool. Other notable tools developed within KGD include a genome synteny viewer and tools for differential gene expression analysis as well as gene ontology (GO) term and pathway enrichment analysis.

Low pH-shifting treatment would improve functional properties of black turtle bean (Phaseolus vulgaris L.) protein isolate with immunoreactivity reduction.

Low pH-shifting was firstly applied in the black turtle bean (Phaseolus vulgaris L.) protein isolate treatment by acidic (pH 1.0-3.0) buffer incubation for 8 h, then was adjusted to pH 7.2 and kept 3 h for protein stabilizing. Mild loss of secondary structure was confirmed in the protein isolate after low pH-shifting treatment by CD and FT-IR analyses. Intrinsic fluorescence, UV spectra, surface hydrophobicity, SH content and SDS-PAGE analyses indicated the protein conformation was unfolded with the exposure of much more buried hydrophobic residues, which would result in the enhancement of emulsifying properties, foaming properties and fat holding capacity, and lead to the reduction of solubility and water holding capacity. Furthermore, lower immunoreactivity was observed by the ELISA, and improved digestibility was found in in vitro digestion assay. Our results suggested the low pH-shifting treatments would broaden the application of bean protein isolate with better hydrophobic processing functions and safety.

Combined effects of pH and thermal treatments on IgE-binding capacity and conformational structures of lectin from black kidney bean (Phaseolus vulgaris L.).

Combined effects of pH and thermal treatments on black kidney bean lectin (BKBL) were investigated by response surface methodology (RSM). Low-pH (1.0, 2.0, 3.0) incubation decreased hemagglutination activity (HA) and IgE-binding capacity, but the activities would be restored when the lectin was treated by pH shifting to 7.2. Conformational structure analyses indicated that low-pH induced protein unfolding and pH-shifting treatment resulted in a limited structural rearrangement. Mild heating, such as 60 °C for 3 min, slightly increased the HA and IgE-binding activities of pH shifted BKBL, but no obvious effects in the pH 1.0 incubated BKBL. High-temperature and long-time treatment might induce the protein aggregation, further decreased HA and IgE-binding capacities. RSM results showed both IgE-binding capacity and HA were the lowest under the combination of pH 1.0 incubation with 80 °C heating for 15 min or pH shifting from 1.0 to 7.2 with 100 °C heating for 10 min.

Full-length transcriptome sequencing provides insights into the evolution of apocarotenoid biosynthesis in Crocus sativus.

Crocus sativus, containing remarkably amounts of crocin, picrocrocin and safranal, is the source of saffron with tremendous medicinal, economic and cultural importance. Here, we present a high-quality full-length transcriptome of the sterile triploid C. sativus, using the PacBio SMRT sequencing technology. This yields 31,755 high-confidence predictions of protein-coding genes, with 50.1% forming paralogous gene pairs. Analysis on distribution of Ks values suggests that the current genome of C. sativus is probably a product resulting from at least two rounds of whole-genome duplication (WGD) events occurred at ~28 and ~114 million years ago (Mya), respectively. We provide evidence demonstrating that the recent ß WGD event confers a major impact on family expansion of secondary metabolite genes, possibly leading to an enhanced accumulation of three distinct compounds: crocin, picrocrocin and safranal. Phylogenetic analysis unravels that the founding member (CCD2) of CCD enzymes necessary for the biosynthesis of apocarotenoids in C. sativus might be evolved from the CCD1 family via the ß WGD event. Based on the gene expression profiling, CCD2 is found to be expressed at an extremely high level in the stigma. These findings may shed lights on further genomic refinement of the characteristic biosynthesis pathways and promote germplasm utilization for the improvement of saffron quality.

Genome Sequence, Assembly, and Characterization of the Antagonistic Yeast Candida oleophila Used as a Biocontrol Agent Against Post-harvest Diseases.

Candida oleophila is an effective biocontrol agent used to control post-harvest diseases of fruits and vegetables. C. oleophila I-182 was the active agent used in the first-generation yeast-based commercial product, Aspire®, for post-harvest disease management. Several action modes, like competition for nutrients and space, induction of pathogenesis-related genes in host tissues, and production of extracellular lytic enzymes, have been demonstrated for the biological control activity exhibited by C. oleophila through which it inhibits post-harvest pathogens. In the present study, the whole genome of C. oleophila I-182 was sequenced using PacBio and Illumina shotgun sequencing technologies, yielding an estimated genome size of 14.73 Mb. The genome size is similar in length to that of the model yeast strain Saccharomyces cerevisiae S288c. Based on the assembled genome, protein-coding sequences were identified and annotated. The predicted genes were further assigned with gene ontology terms and clustered in special functional groups. A comparative analysis of C. oleophila proteome with the proteomes of 11 representative yeasts revealed 2 unique and 124 expanded families of proteins in C. oleophila. Availability of the genome sequence will facilitate a better understanding the properties of biocontrol yeasts at the molecular level.

Sophorolipid biosynthesis and production from diverse hydrophilic and hydrophobic carbon substrates.

Sophorolipids (SLs), mainly synthesized by yeasts, were a sort of biosurfactant with the highest fermentation level at present. In recent years, SLs have drawn extensive attention for their excellent physiochemical properties and physiological activities. Besides, issues such as economics, sustainability, and use of renewable resources also stimulate the shift from chemical surfactants towards green or microbial-derived biosurfactants. SLs' large-scale production and application were restricted by the relatively high production costs. Currently, waste streams from agriculture, food and oil refining industries, etc., have been exploited as low-cost renewable substrates for SL production. Advanced cultivation method, uncommonly used substrates, and new genetically modified SL-producing mutants were also designed and applied to improve the productivity or the special properties of SLs. In this review, a systematic and detailed description of primary and secondary metabolism pathways involved in SL biosynthesis was summarized firstly. Furthermore, based on the pathways of SL biosynthesis from different carbon substrates, we reviewed the current knowledge and advances in the exploration of cost-effective and infrequently used hydrophilic and hydrophobic substrates for large or specialized SL production.

A novel oriented antibody immobilization based voltammetric immunosensor for allergenic activity detection of lectin in kidney bean by using AuNPs-PEI-MWCNTs modified electrode.

As a well-known allergenic indicator in kidney beans, lectins have always been the serious threats for human health. Herein, we introduced a new label-free voltammetric immunosensor for the direct determination of kidney bean lectin (KBL) with potential allergenic activity. Gold nanoparticles-polyethyleneimine-multiwalled carbon nanotubes nanocomposite was one-pot synthesized and modified onto the glass carbon electrode to enhance catalytic currents of oxygen reduction reaction. The KBL polyclonal antibody, acquired from rabbit immunization, was orientedly immobilized on the electrode modified with recombinant staphylococcal protein A via fragment crystallizable (Fc) region of antibody. Under the optimized condition, the immunosensor displayed a good linear response (R2 = 0.978) to KBL with a range from 0.05 to 100 µg/mL and a detection limit of 0.023 µg/mL. Simultaneously, the immunosensor exhibited well selectivity, interference-resistant ability, stability (4 °C) and reproducibility. Compared with the conventional enzyme-linked immunosorbent assay (ELISA) method, the immunosensor was successfully applied to quantify allergenic activity of lectin in raw and cooked (boiled for 30 min) kidney bean milk samples. This new approach provides new perspectives both for rapid quantification of lectin in kidney beans-derived foodstuffs and as a real-time monitoring tool for the allergenic potential during the whole production and consumption process.

Chromosome-scale genome assembly of kiwifruit Actinidia eriantha with single-molecule sequencing and chromatin interaction mapping.

BACKGROUND: Kiwifruit (Actinidia spp.) is a dioecious plant with fruits containing abundant vitamin C and minerals. A handful of kiwifruit species have been domesticated, among which Actinidiaeriantha is increasingly favored in breeding owing to its superior commercial traits. Recently, elite cultivars from A. eriantha have been successfully selected and further studies on their biology and breeding potential require genomic information, which is currently unavailable. FINDINGS: We assembled a chromosome-scale genome sequence of A. eriantha cultivar White using single-molecular sequencing and chromatin interaction map-based scaffolding. The assembly has a total size of 690.6 megabases and an N50 of 21.7 megabases. Approximately 99% of the assembly were in 29 pseudomolecules corresponding to the 29 kiwifruit chromosomes. Forty-three percent of the A. eriantha genome are repetitive sequences, and the non-repetitive part encodes 42,988 protein-coding genes, of which 39,075 have homologues from other plant species or protein domains. The divergence time between A. eriantha and its close relative Actinidia chinensis is estimated to be 3.3 million years, and after diversification, 1,727 and 1,506 gene families are expanded and contracted in A. eriantha, respectively. CONCLUSIONS: We provide a high-quality reference genome for kiwifruit A. eriantha. This chromosome-scale genome assembly is substantially better than 2 published kiwifruit assemblies from A. chinensis in terms of genome contiguity and completeness. The availability of the A. eriantha genome provides a valuable resource for facilitating kiwifruit breeding and studies of kiwifruit biology.

A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang.

Many Actinidia cultivars are characterized by anthocyanin accumulation, specifically in the inner pericarp, but the underlying regulatory mechanism remains elusive. Here we report two interacting transcription factors, AcMYB123 and AcbHLH42, that regulate tissue-specific anthocyanin biosynthesis in the inner pericarp of Actinidia chinensis cv. Hongyang. Through transcriptome profiling analysis we identified five MYB and three bHLH transcription factors that were upregulated in the inner pericarp. We show that the combinatorial action of two of them, AcMYB123 and AcbHLH42, is required for activating promoters of AcANS and AcF3GT1 that encode the dedicated enzymes for anthocyanin biosynthesis. The presence of anthocyanin in the inner pericarp appears to be tightly associated with elevated expression of AcMYB123 and AcbHLH42. RNA interference repression of AcMYB123, AcbHLH42, AcF3GT1 and AcANS in 'Hongyang' fruits resulted in significantly reduced anthocyanin biosynthesis. Using both transient assays in Nicotiana tabacum leaves or Actinidia arguta fruits and stable transformation in Arabidopsis, we demonstrate that co-expression of AcMYB123 and AcbHLH42 is a prerequisite for anthocyanin production by activating transcription of AcF3GT1 and AcANS or the homologous genes. Phylogenetic analysis suggests that AcMYB123 or AcbHLH42 are closely related to TT2 or TT8, respectively, which determines proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than regulators in dicots. All these experimental results suggest that AcMYB123 and AcbHLH42 are the components involved in spatiotemporal regulation of anthocyanin biosynthesis specifically in the inner pericarp of kiwifruit.

Transcriptome analysis of differentially expressed unigenes involved in flavonoid biosynthesis during flower development of Chrysanthemum morifolium 'Chuju'.

Chrysanthemum morifolium is an ornamentally and medicinally important plant species. Up to date, molecular and genetic investigations have largely focused on determination of flowering time in the ornamental species. However, little is known about gene regulatory networks for the biosynthesis of flavonoids in the medicinal species. In the current study, we employed the high-throughput sequencing technology to profile the genome-wide transcriptome of C. morifolium 'Chuju', a famous medicinal species in traditional Chinese medicine. A total of 63,854 unigenes with an average length of 741 bp were obtained. Bioinformatic analysis has identified a great number of structural and regulatory unigenes potentially participating in the flavonoid biosynthetic pathway. According to the comparison of digital gene expression, 8,370 (3,026 up-regulated and 5,344 down-regulated), 1,348 (717 up-regulated and 631 down-regulated) and 944 (206 up-regulated and 738 down-regulated) differentially expressed unigenes (DEUs) were detected in the early, middle and mature growth phases, respectively. Among them, many DEUs were implicated in controlling the biosynthesis and composition of flavonoids from the budding to full blooming stages during flower development. Furthermore, the expression patterns of 12 unigenes involved in flavonoid biosynthesis were generally validated by using quantitative real time PCR. These findings could shed light on the molecular basis of flavonoid biosynthesis in C. morifolium 'Chuju' and provide a genetic resource for breeding varieties with improved nutritional quality.

DFR1-Mediated Inhibition of Proline Degradation Pathway Regulates Drought and Freezing Tolerance in Arabidopsis.

Proline accumulation is one of the most important adaptation mechanisms for plants to cope with environmental stresses, such as drought and freezing. However, the molecular mechanism of proline homeostasis under these stresses is largely unknown. Here, we identified a mitochondrial protein, DFR1, involved in the inhibition of proline degradation in Arabidopsis. DFR1 was strongly induced by drought and cold stresses. The dfr1 knockdown mutants showed hypersensitivity to drought and freezing stresses, whereas the DFR1 overexpression plants exhibited enhanced tolerance, which was positively correlated with proline levels. DFR1 interacts with proline degradation enzymes PDH1/2 and P5CDH and compromises their activities. Genetic analysis showed that DFR1 acts upstream of PDH1/2 and P5CDH to positively regulate proline accumulation. Our results demonstrate a regulatory mechanism by which, under drought and freezing stresses, DFR1 interacts with PDH1/2 and P5CDH to abrogate their activities to maintain proline homeostasis, thereby conferring drought and freezing tolerance.