Database Construction For Chromosome-level Genome Assembly And Genome-wide Identification Of The ARF Gene Family And ARF3 Target Genes In Hazelnut(Corylus Heterophylla)

Hazelnut(Corylus spp.)is an economically important oil tree belonging to the genus Corylus in the family Betulaceae,but a satisfactory hazelnut genome database is absent.Hazelnut has a unique biological characteristic of ovary differentiation and development initiating from the ovary primordium after pollination;however,its detailed regulatory mechanism for fruit development remains unclear.In this study,the single-molecule real-time sequencing and the Hi-C sequencing were performed,followed by genome assisted assembly and database construction;Further,the hazelnut ARF(Ch ARF)gene family was identified,the expression characteristics of Ch ARFs were analyzed in the key period of ovule development,and the immunohistochemical localization analysis of Ch ARF3 was carried out.Finally,the target genes of Ch ARF3 were analyzed by chromatin immunoprecipitation followed by nextgeneration sequencing(Ch IP-Seq).The main results are as follows:(1)Using Pacbio sequencing and Hi-C assisted genome assembly strategy,a 343 Mb chromosome-scale genome sequence of C.heterophylla was obtained,with Scaffold N50 of32.88 Mb.In total,22,319 protein-coding genes were predicted,and 21,056(94.34%)were annotated.A comprehensive hazelnut genome database,Hazel Omics(http://122.9.151.76/),containing the assembled genome,CDS sequences,protein sequences,and various annotation information,was constructed to fill the vacuum of a satisfactory reference genome database for hazelnut.(2)Based on the published genome data of 15 closely related species and 14 key oil crops,comparative genomics analyses were carried out to identify the evolutionary status and expansion and contraction of C.heterophylla gene families.In total,352 and 439 contraction genes and 1,197 and 3,810 expansion genes were identified,respectively.The expansion genes FAD,SAD,and KAT were significantly enriched in ko01040(the unsaturated fatty acid synthesis)and highly up-regulated in the mature ovule,suggesting these genes may promote the synthesis of unsaturated fatty acids and improve the adaptability to the cold climate.(3)On the basis of the C.heterophylla genome,the genome-wide identification of the Ch ARF gene family and the key Ch ARF to regulate the ovule development were performed and screened by bioinformatics and expression analyses,respectively.In total,14 Ch ARF members were identified,and the molecular weight of the predicted proteins was 70.93–139.22 k D.Phylogenetic analysis clustered into four CLASSes,and Ch ARF3 from CLASS I showed the most significant differences over four ovary developmental stages,indicating that Ch ARF3 may play an essential role in the formation of hazelnut yield.(4)To analyze the Ch ARF3 immunohistochemical localization of female flowers and ovules,a specific polyclonal antibody against Ch ARF3 of high-efficiency and Ch IP-grade was prepared.Immunohistochemical analysis revealed that auxin should be essential for ovary initiation,and Ch ARF3 was located in the ovary primordium and funiculus,integument,endosperm,radicle,and cotyledon,suggesting Ch ARF3 potential regulatory roles in ovary differentiation and ovule development.(5)Through RNA-Seq and Ch IP-Seq correlation analysis,Ch ARF3 target genes and its regulatory network related to ovule development were systematically identified and revealed.In total,3,167 Ch ARF3 target genes were identified during ovule development and significantly enriched in the biosynthesis of secondary metabolites(ko01110),including phenylpropanoid biosynthesis(ko00940),and phytohormone signal transduction(ko04075),indicating that Ch ARF3 should be involved in coordinating auxin-induced genes and MADS,AP2/ERF,TCP,FT,and LFY transcription factors to mediate ovary initiation and ovule development.To summarize,the third-generation genome sequencing and chromosome-level assembly of C.heterophylla were performed,and the Hazel Omics database was constructed.This study identified the Ch ARF gene family and the target genes of the critical transcription factor Ch ARF3 for fruit development.It could provide a new data platform of great significance to preserve and obtain the genome sequences,and new insights into yield formation mechanism and molecular breeding for hazelnut.