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.

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.

Baicalin attenuates dexamethasone-induced apoptosis of bone marrow mesenchymal stem cells by activating the hedgehog signaling pathway

Background:Perturbations in bone marrow mesenchymal stem cell(BMSC)differentiation play an important role in steroid-induced osteonecrosis of the femoral head(SONFH).At present,studies on SONFH concentrate upon the balance within BMSC osteogenic and adipogenic differentiation.However,BMSC apoptosis as well as proliferation are important prerequisites in their differentiation.The hedgehog(HH)signaling pathway regulates bone cell apoptosis.Baicalin(BA),a well-known compound in traditional Chinese medicine,can affect the proliferation and apoptosis of numerous cell types via HH signaling.However,the potential role and mechanisms of BA on BMSCs are unclear.Thus,we aimed to explore the role of BA in dexamethasone(Dex)-induced BMSC apoptosis in this study.Methods:Primary BMSCs were treated with 10-6 mol/L Dex alone or with 5.0μmol/L,10.0μmol/L,or 50.0μmol/L BA for 24 hours followed by co-treatment with 5.0μmol/L,10.0μmol/L,or 50.0μmol/L BA and 10-6 mol/L Dex.Cell viability was assayed through the Cell Counting Kit-8(CCK-8).Cell apoptosis was evaluated using Annexin V-fluorescein isothiocyanate/propidium iodide(PI)staining followed by flow cytometry.The imaging and counting,respectively,of Hochest 33342/PI-stained cells were used to assess the morphological characteristics and proportion of apoptotic cells.To quantify the apoptosis-related proteins(e.g.,apoptosis regulator BAX[Bax],B-cell lymphoma 2[Bcl-2],caspase-3,and cleaved caspase-3)and HH signaling pathway proteins,western blotting was used.A HH-signaling pathway inhibitor was used to demonstrate that BA exerts its anti-apoptotic effects via the HH signaling pathway.Results:The results of CCK-8,Hoechst 33342/PI-staining,and flow cytometry showed that BA did not significantly promote cell proliferation(CCK-8:0μmol/L,100%;2.5μmol/L,98.58%;5.0μmol/L,95.18%;10.0μmol/L,98.11%;50.0μmol/L,99.38%,F=2.33,P>0.05),but it did attenuate the effect of Dex on apoptosis(Hoechst 33342/PI-staining:Dex+50.0μmol/L BA,12.27%vs.Dex,39.27%,t=20.62;flow cytometry:Dex+50.0μmol/L BA,12.68%vs.Dex,37.43%,t=11.56;Both P<0.05).The results of western blotting analysis showed that BA reversed Dex-induced apoptosis by activating the HH signaling pathway,which down-regulated the expression of Bax,cleaved-caspase 3,and suppressor of fused(SUFU)while up-regulating Bcl-2,sonic hedgehog(SHH),and zinc finger protein GLI-1(GLI-1)expression(Bax/Bcl-2:Dex+50.0μmol/L BA,1.09 vs.Dex,2.76,t=35.12;cleaved caspase-3/caspase-3:Dex+50.0μmol/L BA,0.38 vs.Dex,0.73,t=10.62;SHH:Dex+50.0μmol/L BA,0.50 vs.Dex,0.12,t=34.01;SUFU:Dex+50.0μmol/L BA,0.75 vs.Dex,1.19,t=10.78;GLI-1:Dex+50.0μmol/L BA,0.40 vs.Dex,0.11,t=30.68.All P<0.05).Conclusions:BA antagonizes Dex-induced apoptosis of human BMSCs by activating the HH signaling pathway.It is a potential candidate for preventing SONFH.

Genome wide association analysis identifies candidate genes for fruit quality and yield in Actinidia eriantha

Quality and yield are the primary concerns in kiwifruit breeding,but research on the genetic mechanisms of fruit size,shape,and ascorbic acid(ASA)content is currently very limited,which restricts the development of kiwifruit molecular breeding.In this study,we obtained a total of 8.88 million highly reliable single nucleotide polymorphism(SNP)markers from 140 individuals from the natural hybrid offspring of Actinidia eriantha cv.‘White’using whole genome resequencing technology.A genome-wide association study was conducted on eight key agronomic traits,including single fruit weight,fruit shape,ASA content,and the number of inflorescences per branch.A total of 59 genetic loci containing potential functional genes were located,and candidate genes related to single fruit weight,fruit length,ASA content,number of inflorescences per branch and other traits were identified within the candidate interval,such as AeWUSCHEL,AeCDK1(cell cycle dependent kinase),AeAO1(ascorbic oxidase)and AeCO1(CONSTANS-like 4).After constructing an RNAi vector for AeAO1 and injecting it into the fruit of cv.‘Midao 31’to interfere with the expression of the AeAO1 gene,the results showed that the activity of ascorbic oxidase in the fruit of‘Midao 31’significantly decreased,while the content of ASA significantly increased.This study provides valuable insights into the genetic basis of variation in A.eriantha fruit traits,which may benefit molecular marker-assisted breeding efforts.