Genome-Wide Identification of HSF Gene Family in Kiwifruit and the Function of AeHSFA2b in Salt Tolerance.

Heat shock transcription factors (HSFs) play a crucial role in regulating plant growth and response to various abiotic stresses. In this study, we conducted a comprehensive analysis of the AeHSF gene family at genome-wide level in kiwifruit (Actinidia eriantha), focusing on their functions in the response to abiotic stresses. A total of 41 AeHSF genes were identified and categorized into three primary groups, namely, HSFA, HSFB, and HSFC. Further transcriptome analysis revealed that the expression of AeHSFA2b/2c and AeHSFB1c/1d/2c/3b was strongly induced by salt, which was confirmed by qRT-PCR assays. The overexpression of AeHSFA2b in Arabidopsis significantly improved the tolerance to salt stress by increasing AtRS5, AtGolS1 and AtGolS2 expression. Furthermore, yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays demonstrated that AeHSFA2b could bind to the AeRFS4 promoter directly. Therefore, we speculated that AeHSFA2b may activate AeRFS4 expression by directly binding its promoter to enhance the kiwifruit's tolerance to salt stress. These results will provide a new insight into the evolutionary and functional mechanisms of AeHSF genes in kiwifruit.

Comparative Transcriptomic Analysis Revealed the Suppression and Alternative Splicing of Kiwifruit (Actinidia latifolia) NAP1 Gene Mediating Trichome Development.

Kiwifruit (Actinidia chinensis) is commonly covered by fruit hairs (trichomes) that affect kiwifruit popularity in the commercial market. However, it remains largely unknown which gene mediates trichome development in kiwifruit. In this study, we analyzed two kiwifruit species, A. eriantha (Ae) with long, straight, and bushy trichomes and A. latifolia (Al) with short, distorted, and spare trichomes, by second- and third-generation RNA sequencing. Transcriptomic analysis indicated that the expression of the NAP1 gene, a positive regulator of trichome development, was suppressed in Al compared with that in Ae. Additionally, the alternative splicing of AlNAP1 produced two short transcripts (AlNAP1-AS1 and AlNAP1-AS2) lacking multiple exons, in addition to a full-length transcript of AlNAP1-FL. The defects of trichome development (short and distorted trichome) in Arabidopsis nap1 mutant were rescued by AlNAP1-FL but not by AlNAP1-AS1. AlNAP1-FL gene does not affect trichome density in nap1 mutant. The qRT-PCR analysis indicated that the alternative splicing further reduces the level of functional transcripts. These results indicated that the short and distorted trichomes in Al might be caused by the suppression and alternative splicing of AlNAP1. Together, we revealed that AlNAP1 mediates trichome development and is a good candidate target for genetic modification of trichome length in kiwifruit.

Application of developmental regulators to improve in planta or in vitro transformation in plants.

Plant genetic transformation is a crucial step for applying biotechnology such as genome editing to basic and applied plant science research. Its success primarily relies on the efficiency of gene delivery into plant cells and the ability to regenerate transgenic plants. In this study, we have examined the effect of several developmental regulators (DRs), including PLETHORA (PLT5), WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), ENHANCED SHOOT REGENERATION (ESR1), WUSHEL (WUS) and a fusion of WUS and BABY-BOOM (WUS-P2A-BBM), on in planta transformation through injection of Agrobacterium tumefaciens in snapdragons (Antirrhinum majus). The results showed that PLT5, WIND1 and WUS promoted in planta transformation of snapdragons. An additional test of these three DRs on tomato (Solanum lycopersicum) further demonstrated that the highest in planta transformation efficiency was observed from PLT5. PLT5 promoted calli formation and regeneration of transformed shoots at the wound positions of aerial stems, and the transgene was stably inherited to the next generation in snapdragons. Additionally, PLT5 significantly improved the shoot regeneration and transformation in two Brassica cabbage varieties (Brassica rapa) and promoted the formation of transgenic calli and somatic embryos in sweet pepper (Capsicum annum) through in vitro tissue culture. Despite some morphological alternations, viable seeds were produced from the transgenic Bok choy and snapdragons. Our results have demonstrated that manipulation of PLT5 could be an effective approach for improving in planta and in vitro transformation efficiency, and such a transformation system could be used to facilitate the application of genome editing or other plant biotechnology application in modern agriculture.

Natural variation in the HAN1 gene confers chilling tolerance in rice and allowed adaptation to a temperate climate.

Rice (Oryza sativa L.) is a chilling-sensitive staple crop that originated in subtropical regions of Asia. Introduction of the chilling tolerance trait enables the expansion of rice cultivation to temperate regions. Here we report the cloning and characterization of HAN1, a quantitative trait locus (QTL) that confers chilling tolerance on temperate japonica rice. HAN1 encodes an oxidase that catalyzes the conversion of biologically active jasmonoyl-L-isoleucine (JA-Ile) to the inactive form 12-hydroxy-JA-Ile (12OH-JA-Ile) and fine-tunes the JA-mediated chilling response. Natural variants in HAN1 diverged between indica and japonica rice during domestication. A specific allele from temperate japonica rice, which gained a putative MYB cis-element in the promoter of HAN1 during the divergence of the two japonica ecotypes, enhances the chilling tolerance of temperate japonica rice and allows it to adapt to a temperate climate. The results of this study extend our understanding of the northward expansion of rice cultivation and provide a target gene for the improvement of chilling tolerance in rice.

Genome-Wide Expression Profiling Analysis of Kiwifruit GolS and RFS Genes and Identification of AcRFS4 Function in Raffinose Accumulation.

The raffinose synthetase (RFS) and galactinol synthase (GolS) are two critical enzymes for raffinose biosynthesis, which play an important role in modulating plant growth and in response to a variety of biotic or abiotic stresses. Here, we comprehensively analyzed the RFS and GolS gene families and their involvement in abiotic and biotic stresses responses at the genome-wide scale in kiwifruit. A total of 22 GolS and 24 RFS genes were identified in Actinidia chinensis and Actinidia eriantha genomes. Phylogenetic analysis showed that the GolS and RFS genes were clustered into four and six groups, respectively. Transcriptomic analysis revealed that abiotic stresses strongly induced some crucial genes members including AcGolS1/2/4/8 and AcRFS2/4/8/11 and their expression levels were further confirmed by qRT-PCR. The GUS staining of AcRFS4Pro::GUS transgenic plants revealed that the transcriptionlevel of AcRFS4 was significantly increased by salt stress. Overexpression of AcRFS4 in Arabidopsis demonstrated that this gene enhanced the raffinose accumulation and the tolerance to salt stress. The co-expression networks analysis of hub transcription factors targeting key AcRFS4 genes indicated that there was a strong correlation between AcNAC30 and AcRFS4 expression under salt stress. Furthermore, the yeast one-hybrid assays showed that AcNAC30 could bind the AcRFS4 promoter directly. These results may provide insights into the evolutionary and functional mechanisms of GolS and RFS genes in kiwifruit.

Cell Wall Matrix Polysaccharides Contribute to Salt-Alkali Tolerance in Rice.

Salt-alkali stress threatens the resilience to variable environments and thus the grain yield of rice. However, how rice responds to salt-alkali stress at the molecular level is poorly understood. Here, we report isolation of a novel salt-alkali-tolerant rice (SATR) by screening more than 700 germplasm accessions. Using 93-11, a widely grown cultivar, as a control, we characterized SATR in response to strong salt-alkali stress (SSAS). SATR exhibited SSAS tolerance higher than 93-11, as indicated by a higher survival rate, associated with higher peroxidase activity and total soluble sugar content but lower malonaldehyde accumulation. A transcriptome study showed that cell wall biogenesis-related pathways were most significantly enriched in SATR relative to 93-11 upon SSAS. Furthermore, higher induction of gene expression in the cell wall matrix polysaccharide biosynthesis pathway, coupled with higher accumulations of hemicellulose and pectin as well as measurable physio-biochemical adaptive responses, may explain the strong SSAS tolerance in SATR. We mapped SSAS tolerance to five genomic regions in which 35 genes were candidates potentially governing SSAS tolerance. The 1,4-ß-D-xylan synthase gene OsCSLD4 in hemicellulose biosynthesis pathway was investigated in details. The OsCSLD4 function-disrupted mutant displayed reduced SSAS tolerance, biomass and grain yield, whereas the OsCSLD4 overexpression lines exhibited increased SSAS tolerance. Collectively, this study not only reveals the potential role of cell wall matrix polysaccharides in mediating SSAS tolerance, but also highlights applicable value of OsCSLD4 and the large-scale screening system in developing SSAS-tolerant rice.

The cloning and characterization of hypersensitive to salt stress mutant, affected in quinolinate synthase, highlights the involvement of NAD in stress-induced accumulation of ABA and proline.

Nicotinamide adenine dinucleotide (NAD), a ubiquitous coenzyme, is required for many physiological reactions and processes. However, it remains largely unknown how NAD affects plant response to salt stress. We isolated a salt-sensitive mutant named hypersensitive to salt stress (hss) from an ethyl methanesulfonate-induced mutation population. A point mutation was identified by MutMap in the encoding region of Quinolinate Synthase (QS) gene required for the de novo synthesis of NAD. This point mutation caused a substitution of amino acid in the highly-conserved NadA domain of QS, resulting in an impairment of NAD biosynthesis in the mutant. Molecular and chemical complementation have restored the response of the hss mutant to salt stress, indicating that the decreased NAD contents in the mutant were responsible for its hypersensitivity to salt stress. Furthermore, the endogenous levels of abscisic acid (ABA) and proline were also reduced in stress-treated hss mutant. The application of ABA or proline could alleviate stress-induced oxidative damage of the mutant and partially rescue its hypersensitivity to salt stress, but not affect NAD concentration. Taken together, our results demonstrated that the NadA domain of QS is important for NAD biosynthesis, and NAD participates in plant response to salt stress by affecting stress-induced accumulation of ABA and proline.

Unraveling microbial community diversity and succession of Chinese Sichuan sausages during spontaneous fermentation by high-throughput sequencing.

The microbial community diversity and succession of Chinese Sichuan sausages during the spontaneous fermentation were demonstrated using high-throughput sequencing technology. The bacterial diversity was abundant and the succession of bacterial community along the direction of Lactobacillus spp. increased and Weissella spp. decreased. While fungal diversity was single and trace fungal population was detected. The core functional microbiota were lactic acid bacteria, including Lactobacillus spp., Weissella spp. and Pediococcus spp. In initial fermentation, Weissella spp. was the dominant bacteria and its relative abundance was 49.84%, but then its relative abundance decreased to 11.96% during fermentation before recovering to 26.74% at the end of fermentation. Meanwhile, Lactobacillus spp. rose from 24.70 to 55.74% and became the dominant genus. Moreover, Pediococcus spp. increased from 0.06 to 18.05% on day 20 but then decreased to 1.89% on day 30. These results revealed that the primary microorganisms contributing to spontaneous fermentation of Chinese Sichuan sausages were bacteria, while eukaryotic microorganisms such as yeast scarcely contributed to fermentation.

Correction to: Heat stress alters genome-wide profiles of circular RNAs in Arabidopsis.

Due to an unfortunate turn of events, the first name of the fifth author appeared incorrectly in the original publication and should have read Guangbing. The correct representation of the authors' names and their affiliation is listed here and should be treated as definitive.

Heat stress alters genome-wide profiles of circular RNAs in Arabidopsis.

KEY MESSAGE: 1599 novel circRNAs and 1583 heat stress-specific circRNAs were identified in Arabidopsis. Heat stress enhanced accumulation of circRNAs remarkably. Heat stress altered the sizes of circRNAs, numbers of circularized exons and alterative circularization events. A putative circRNA-mediated ceRNA networks under heat stress was established. Heat stress retards plant growth and destabilizes crop yield. The noncoding RNAs were demonstrated to be involved in plant response to heat stress. As a newly-characterized class of noncoding RNAs, circular RNAs (circRNAs) play important roles in transcriptional and post-transcriptional regulation. A few recent investigations indicated that plant circRNAs were differentially expressed under abiotic stress. However, little is known about how heat stress mediates biogenesis of circRNAs in plants. Here, we uncovered 1599 previously-unknown circRNAs and 1583 heat-specific circRNAs, by RNA-sequencing and bioinformatic analysis. Our results indicated that much more circRNAs were expressed under heat stress than in control condition. Besides, heat stress also increased the length of circRNAs, the quantity of circularized exons, and alternative circularization events. Moreover, we observed a positive correlation between expression patterns of some circRNAs and their parental genes. The prediction of ceRNA (competing endogenous RNA) networks indicated that differentially-expressed circRNAs could influence expression of many important genes, that participate in response to heat stress, hydrogen peroxide, and phytohormone signaling pathways, by interacting with the corresponding microRNAs. Together, our observations indicated that heat stress had great impacts on the biogenesis of circRNAs. Heat-induced circRNAs might participate in plant response to heat stress through the circRNA-mediated ceRNA networks.

Delaying chloroplast turnover increases water-deficit stress tolerance through the enhancement of nitrogen assimilation in rice.

Abiotic stress-induced senescence in crops is a process particularly affecting the photosynthetic apparatus, decreasing photosynthetic activity and inducing chloroplast degradation. A pathway for stress-induced chloroplast degradation that involves the CHLOROPLAST VESICULATION (CV) gene was characterized in rice (Oryza sativa) plants. OsCV expression was up-regulated with the age of the plants and when plants were exposed to water-deficit conditions. The down-regulation of OsCV expression contributed to the maintenance of the chloroplast integrity under stress. OsCV-silenced plants displayed enhanced source fitness (i.e. carbon and nitrogen assimilation) and photorespiration, leading to water-deficit stress tolerance. Co-immunoprecipitation, intracellular co-localization, and bimolecular fluorescence demonstrated the in vivo interaction between OsCV and chloroplastic glutamine synthetase (OsGS2), affecting source-sink relationships of the plants under stress. Our results would indicate that the OsCV-mediated chloroplast degradation pathway is involved in the regulation of nitrogen assimilation during stress-induced plant senescence.

Stress-induced chloroplast degradation in Arabidopsis is regulated via a process independent of autophagy and senescence-associated vacuoles.

Two well-known pathways for the degradation of chloroplast proteins are via autophagy and senescence-associated vacuoles. Here, we describe a third pathway that was activated by senescence- and abiotic stress-induced expression of Arabidopsis thaliana CV (for chloroplast vesiculation). After targeting to the chloroplast, CV destabilized the chloroplast, inducing the formation of vesicles. CV-containing vesicles carrying stromal proteins, envelope membrane proteins, and thylakoid membrane proteins were released from the chloroplasts and mobilized to the vacuole for proteolysis. Overexpression of CV caused chloroplast degradation and premature leaf senescence, whereas silencing CV delayed chloroplast turnover and senescence induced by abiotic stress. Transgenic CV-silenced plants displayed enhanced tolerance to drought, salinity, and oxidative stress. Immunoprecipitation and bimolecular fluorescence complementation assays demonstrated that CV interacted with photosystem II subunit PsbO1 in vivo through a C-terminal domain that is highly conserved in the plant kingdom. Collectively, our work indicated that CV plays a crucial role in stress-induced chloroplast disruption and mediates a third pathway for chloroplast degradation. From a biotechnological perspective, silencing of CV offers a suitable strategy for the generation of transgenic crops with increased tolerance to abiotic stress.

Tomato MBD5, a methyl CpG binding domain protein, physically interacting with UV-damaged DNA binding protein-1, functions in multiple processes.

In tomato (Solanum lycopersicum), high pigment mutations (hp-1 and hp-2) were mapped to genes encoding UV-damaged DNA binding protein 1 (DDB1) and de-etiolated-1 (DET1), respectively. Here we characterized a tomato methyl-CpG-binding domain protein SlMBD5 identified by yeast two-hybrid screening using SlDDB1 as a bait. Yeast two-hybrid assay demonstrated that the physical interaction of SlMBD5 with SlDDB1 is mediated by the C-termini of SlMBD5 and the ß-propeller-C (BPC) of SlDDB1. Co-immunoprecipitation analyses revealed that SlMBD5 associates with SlDDB1-interacting partners including SlDET1, SlCUL4, SlRBX1a and SlRBX1b in vivo. SlMBD5 was shown to target to nucleus and dimerizes via its MBD motif. Electrophoresis mobility shift analysis suggested that the MBD of SlMBD5 specifically binds to methylated CpG dinucleotides but not to methylated CpHpG or CpHpH dinucleotides. SlMBD5 expressed in protoplast is capable of activating transcription of CG islands, whereas CUL4/DDB1 antagonizes this effect. Overexpressing SlMBD5 resulted in diverse developmental alterations including darker green fruits with increased plastid level and elevated pigmentation, as well as enhanced expression of SlGLK2, a key regulator of plastid biogenesis. Taken together, we hypothesize that the physical interaction of SlMBD5 with the CUL4-DDB1-DET1 complex component may affect its binding activity to methylated DNA and subsequently attenuate its transcription activation of downstream genes.

The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress.

OsWRKY47 is a divergent rice transcription factor belonging to the group II of the WRKY family. A transcriptomic analysis of the drought response of transgenic rice plants expressing P SARK ::IPT, validated by qPCR, indicated that OsWRKY47 expression was induced under drought stress in P SARK ::IPT plants. A PCR-assisted site selection assay (SELEX) of recombinant OsWRKY47 protein showed that the preferred sequence bound in vitro is (G/T)TTGACT. Bioinformatics analyses identified a number of gene targets of OsWRKY47; among these two genes encode a Calmodulin binding protein and a Cys-rich secretory protein. Using Oswrk47 knockout mutants and transgenic rice overexpressing OsWRKY47 we show that the transcription of these putative targets were regulated by OsWRKY47. Phenotypic analysis carried out with transgenic rice plants showed that Oswrky47 mutants displayed higher sensitivity to drought and reduced yield, while plants overexpressing OsWRKY47 were more tolerant.

Salt stress-induced disassembly of Arabidopsis cortical microtubule arrays involves 26S proteasome-dependent degradation of SPIRAL1.

The dynamic instability of cortical microtubules (MTs) (i.e., their ability to rapidly alternate between phases of growth and shrinkage) plays an essential role in plant growth and development. In addition, recent studies have revealed a pivotal role for dynamic instability in the response to salt stress conditions. The salt stress response includes a rapid depolymerization of MTs followed by the formation of a new MT network that is believed to be better suited for surviving high salinity. Although this initial depolymerization response is essential for the adaptation to salt stress, the underlying molecular mechanism has remained largely unknown. Here, we show that the MT-associated protein SPIRAL1 (SPR1) plays a key role in salt stress-induced MT disassembly. SPR1, a microtubule stabilizing protein, is degraded by the 26S proteasome, and its degradation rate is accelerated in response to high salinity. We show that accelerated SPR1 degradation is required for a fast MT disassembly response to salt stress and for salt stress tolerance.

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.

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.

Agrobacterium rhizogenes-mediated marker-free transformation and gene editing system revealed that AeCBL3 mediates the formation of calcium oxalate crystal in kiwifruit.

The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis 'Hongyang' and A.eriantha 'White'). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.

An endogenous promoter LpSUT2 discovered in duckweed: a promising transgenic tool for plants.

Promoters are one of the most critical elements in regulating gene expression. They are considered essential biotechnological tools for heterologous protein production. The one most widely used in plants is the 35S promoter from cauliflower mosaic virus. However, our study for the first time discovered the 35S promoter reduced the expression of exogenous proteins under increased antibiotic stress. We discovered an endogenous strong promoter from duckweed named LpSUT2 that keeps higher initiation activity under antibiotic stress. Stable transformation in duckweed showed that the gene expression of eGFP in the LpSUT2:eGFP was 1.76 times that of the 35S:eGFP at 100 mg.L-1 G418 and 6.18 times at 500 mg.L-1 G418. Notably, with the increase of G418 concentration, the gene expression and the fluorescence signal of eGFP in the 35S:eGFP were weakened, while the LpSUT2:eGFP only changed slightly. This is because, under high antibiotic stress, the 35S promoter was methylated, leading to the gene silencing of the eGFP gene. Meanwhile, the LpSUT2 promoter was not methylated and maintained high activity. This is a previously unknown mechanism that provides us with new insights into screening more stable promoters that are less affected by environmental stress. These outcomes suggest that the LpSUT2 promoter has a high capacity to initiate the expression of exogenous proteins. In conclusion, our study provides a promoter tool with potential application for plant genetic engineering and also provides new insights into screening promoters.

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.