| Manchurian walnut(Juglans mandshurica Maxim.)is an important timber tree,medicinal and woody oil plant species,and has crucial economic value.In this study,the whole genome sequencing and assembly of J.mandshurica were carried out based on three generation sequencing technology,and we also systematically studied its genomic characteristics,evolutionary status,and history.Combing the growth,physiological indexes,and multi-group technology,we studied the dynamic development law of J.mandshurica fruit.Also,the genes related to juglone and lipid synthesis and regulation were analyzed,and a preliminary network model was constructed for fruit development of J.mandshurica.The main results are as follows:(1)The high-quality genome of J.mandshurica was obtained.In this study,combing the high-quality Hi Fi reads obtained by the CCS sequencing mode of Pac Bio Sequel II platform and Hi-C chromosome interaction capture technology,the de novo genome of J.mandshurica was obtained,and the chromosomes were anchored to 16 chromosomes.The raw data were14.62 Gb,and the sequencing depth was 26×.The final assembly genome was 548.7 Mb,contig N50 was 21.39 Mb,scaffold N50 was 35.38 Mb.BUSCO analysis showed that the genome of J.mandshurica covered 1588(98.3%)eukaryotic single copy gene sets,and 40,453protein-coding genes were obtained.(2)The evolutionary status and evolutionary history of J.mandshurica was identified according to the of comparative genomics analysis J.mandshurica and other 14 representative species.Based on the comparative genomics analysis of 15 representative species,558 singlecopy gene families were identified,and the phylogenetic tree was constructed,and the divergence time was estimated.The proportion of collinearity genes of J.mandshurica and J.cathayensis are 60%,and they diverged in about 13.8(10.6～17.3)mya,thus they are two independent species.Based on the Ks and 4d TV values,we identified a ’Juglandoid’ WGD event originated prior to the radiation of Juglandaceae in the Paleocene.(3)The dynamic changes of fruit development and metabolites of J.mandshurica were revealed.The fruit length,fruit width,fruit weight and fruit shape index at different developmental stages showed significant differences(P < 0.01).The fruit development of J.mandshurica showed an ’S’ growth pattern.Plant abscisic acid,cytokinin,auxin content and brassinolide had significant changes in the whole development stage of fruit(P < 0.05).Ethylene participates in the walnut exocarp and seed kernel development.During the development of walnut exocarp and seed kernel,the number of metabolites decreased first,then increased and then decreased.(4)The key genes and transcription factors related to walnut exocarp development and juglone biosynthesis were identified by multi-omics technology.Phenolic acids and flavonoids are the main metabolic components in walnut exocarp.The ERF,NAC,GRAS and C2H2 have important regulatory functions during the development of walnut exocarp.A total of 310 differentially expressed genes were involved in plant hormone signal transduction pathway,and salicylic acid content was significantly up-regulated.Furthermore,183 DEGs were positively correlated with juglone,and 248 DEGs were involved in the biosynthesis of juglone.2-ODD and P450 genes are key structural genes involved in the juglone biosynthesis.(5)Functional genes and transcription factors related to seed kernel development and lipid biosynthesis were identified based on multi-omics technology.Linoleic acid,α-linolenic acid and γ-linolenic acid are key metabolites in the lipid synthesis of seed kernel.In which,the content of linoleic acid was the highest content in lipid compounds,and it significantly upregulated in the unsaturated fatty acid synthesis pathway,which was closely related to the lipid accumulation of seed kernel in J.mandshurica.A total of 152 DEGs related to lipid synthesis were obtained from transcriptome data,including 60 DEGs involved in fatty acid biosynthesis,72 DEGs involved in triacylglycerol assembly,and 20 DEGs involved in oil formation.WRI1,ABI3 and VAL1 transcription factors are key regulators of lipid biosynthesis in J.mandshurica.Gene-jman006g0242100,gene-JMAN014G0153600 and geneJMAN014G0141000 are key regulators of linoleic acid,α-linolenic acid and γ-linolenic acid accumulation.(6)NAC transcription factors play key regulatory roles during fruit development and ripening of J.mandshurica.Based on the high-quality assembled reference genome of J.mandshurica,114 Jm NACs genes were identified.According to the phylogenetic tree,Jm NACs were divided into eight subgroups.In which,Jm NAC011,Jm NAC097,Jm NAC064,Jm NAC050 and Jm NAC052 are key genes involved in fruit development and morphogenesis of J.mandshurica.The expression analysis of Jm NACs gene showed that 35 and 20 Jm NACs played an important role during the development of walnut exocarp and seed kernel,respectively,and12 of which were specifically involved in the development and ripening of J.mandshurica fruit. |
