Genome Sequencing Of Quercus Mongolica And Functional Studies Of Brassinosteroid Receptor Genes During Its Secondary Xylem Development

Mongolian oak(Quercus mongolica Fisch.ex Ledeb.)is a deciduous tree in the genus Quercus(Fagaceae).It is a well-known timber and ornamental tree species with economic,ecological,and ornamental values.It has an important phylogenetic position among the global white oaks(Quercus section Quercus).High-quality genome sequences are an important information basis for genome-wide selection breeding,genetic,and evolutionary studies.The whole genome sequencing study of Q.mongolica has important theoretical and practical significance to deepen the excavation of its excellent trait genes and accelerate its molecular breeding process.Previous studies by our team found that altering exogenous and endogenous brassinosteroid(BR)levels could affect secondary xylem development in Q.mongolica.Studies on the role of BR in secondary xylem development in herbaceous plants have also demonstrated that functional BR receptors play an important role.However,research on the role of BR in the secondary xylem development of woody plants is relatively scarce.Therefore,in this study,the whole genome of Q.mongolica was sequenced using second-generation Illumina,third-generation Pac Bio sequencing platform,and Hi-C sequencing technology,and the evolutionary features of Q.mongolica were resolved.On this basis,the effects of BR on the secondary xylem development of Q.mongolica were investigated by exogenous applying 2,4-epibrassinolide(EBL)and propiconazole(PCZ)through the lanolin method;the key BR receptor in the BR-induced secondary xylem development of Q.mongolica was screened by identifying and analyzing the spatiotemporal expression patterns of functional BR receptors;its functions were verified.It is expected to clarify the phylogenetic position and evolutionary process of representative section Quercus species in East Asia,and to resolve the protein structure and evolution of functional BR receptors and their roles in Q.mongolica secondary xylem development,which is important for further excavation of important trait genes and breeding of excellent species with high wood biomass yield and ornamental value.The representative results are as follows:1.The chromosome-level genome of Q.mongolica was obtained.Based on Illumina and Pac Bio sequencing platforms,the Q.mongolica genome size was 809.84 Mb,with a contig N50 of 2.64 Mb.Using Hi-C technology,95.65% of the genome sequence was anchored to 12 chromosomes.After annotation,transposon elements(TEs)accounted for 51.78% of the genome;a total of 36,553 protein-coding genes were predicted,of which 34,684(94.89%)protein-coding genes were functionally annotated through public databases;129 mi RNAs,768 t RNAs,4,896 r RNAs,and 371 sn RNAs were also annotated.2.The phylogenetic position and evolutionary process of Q.mongolica in Quercus section Quercus were clarified.Comparative genomic analyses showed that within the Quercus section Quercus,the Roburoid lineage in which Q.mongolica is located diverged later than the Dumosae lineage;Q.mongolica is closely related to Q.robur from Europe,and they shared a common ancestor ～11.8 million years ago,during which 151 gene families of Q.mongolica underwent significant expansion and 227 gene families underwent significant contraction;Q.mongolica experienced only one whole genome duplication(WGD)event,the ancestral triplication shared among the eudicots(γ);the chromosomes of Q.mongolica and Q.lobata from North America were relatively conserved throughout evolution.3.Two functional BR receptor genes were identified in Q.mongolica,namely Qm BRI1 and Qm BRL1,among which Qm BRI1 played a key role in BR-induced secondary xylem development in Q.mongolica.Bioinformatics analysis revealed that the methionine(Met)residue was replaced by an isoleucine(Ile)residue on the Asn-Gly-Ser-Met(NGSM)motif in the conserved BR-binding region of angiosperm BRI1 s in Qm BRI1.This natural mutation was shared among BRI1 s of Quercus species.Among functional BR receptors in angiosperms,the diversity of BRI1-type proteins was relatively high.Local stem and girdling stem treatments with 1 μM and 10 μM concentration EBL lanolin mixtures significantly promoted vascular development and xylem differentiation at the treated site in Q.mongolica seedlings,while girdling stem treatment with 500 μM and 1 m M concentration PCZ lanolin mixtures had the opposite effect.Compared with Qm BRL1,Qm BRI1 showed higher expression levels in Q.mongolica young leaves,shoots,the phloem and xylem of young stems,and BR-induced stem vascular development and xylem differentiation.4.Qm BRI1 was functionally validated in secondary xylem development.Qm BRI1 showed similar physiological,molecular,and genetic functions to At BRI1,a functional BR receptor in Arabidopsis,by mediating BR signal regulation of elongation growth in Arabidopsis petioles,siliques,and inflorescence stems,as well as the vascular patterning,xylem differentiation,and cell wall thickening in inflorescence stems.In this study,the Q.mongolica genome and its evolutionary characteristics were resolved,and the role of Qm BRI1 in mediating BR signaling to regulate xylem differentiation in Q.mongolica was clarified.These results would provide important information resources for Q.mongolica genome-wide selection breeding and a theoretical basis for the phylogenetic reconstruction of section Quercus species and molecular mechanism analysis of BR-promoted secondary xylem development in Q.mongolica.