Pan-Genomic Analyses Of Fraxinus Tree Species And Salt-Tolerance Gene Mining

The genus Fraxinus,belonging to the family Oleaceae,comprises about 40 deciduous trees and shrubby species widely distributed in the warm temperate and subtropical regions of the Northern Hemisphere.Fraxinus tree species exhibit rich genetic diversity and extensive ecological adaptability.Several Fraxinus tree species,such as Fraxinus velutina and F.americana,were introduced into China and have been widely planted as salt-tolerant tree plants in saline-alkali land afforestation and landscape greening.With the development of genomics,it is essential to analyze the evolutionary adaptability of Fraxinus tree species and explore the salt-tolerance genes by pan-genomic,which is of great significance for the genetic improvement of salt tolerance in Fraxinus tree species.Herein,we sequenced and assembled whole genome assemblies for 11 Fraxinus tree species,including F.sogdiana,F.mandschurica,F.hupehensis,F.chinensis,F.baroniana,F.velutina,F.americana,F.excelsior,F.angustifolia,F.ornus,and F.pennsylvanica.Phylogenetic analysis of Fraxinus tree species was performed.A gene family-based and structural variation(SV)-based pan-genome architectures were separately constructed.Though whole-genome resequencing of an inter-species cross F1-population of F.velutina‘Lula 3’(salt-tolerant)×F.pennsylvanica‘Lula 5’(salt-sensitive)and two parents,we performed a salt tolerance presence-absence variation(PAV)-based quantitative trait loci(QTL)mapping based on PAV and salt-tolerance index and pinpointed two PAV-QTLs associated with Fraxinus salt tolerance.Then,the salt-tolerant genes were identified,and the salt-tolerance mechanisms were further investigated.The main results are as follows:1.Whole genome sequencing,assembly,and annotation:Whole genome sequences of 11Fraxinus tree species were sequenced and assembled by integrating ONT long-read sequencing and Illumina short-read sequencing data.The results showed that the genome size of 11Fraxinus species was 678–868 Mb,including 486–1,027 contigs,and the length of contig N50 was 1.14–3.6 Mb.Using the published genome sequence of F.pennsylvanica(v1.4)as a reference,the contig sequence of the genome was assembled at the chromosome level.More than 99.5%of the contig sequence was attached to 23 chromosomes,and the final genome size was 678–868 Mb,containing 23–46 scaffolds,and the length of the scaffold N50 was 30.32–39.75 Mb.The integrity of genome assembly was 97.9%–98.9%;the genome sequence contiguity index(LAI)was 10.84–16.12,which reached the level of the reference genome.By integrating de novo prediction,homology prediction,and transcription assembly strategies,48,389–53,800 protein-coding genes were annotated and identified from the genome assemblies of 11 Fraxinus tree species,and the integrity of gene annotation reached 91.0%to97.2%.2.Phylogenetic analysis of Fraxinus tree species:Phylogenetic orthology inference was made on 11 Fraxinus tree species and 10 other related plant species.A phylogenetic tree based on 290 one-to-one orthologs was identified across the 21 plant species.The results showed that the Fraxinus species was closely related to Olea europaea.Analysis of species divergent time showed that Fraxinus species diverged from the most recent common ancestor of O.europaea about 40.57 million years ago(Ma,late Eocene)and the Sect.Melioides,represented by North American species,diverged at the earliest.Subsequently,during the Oligocene and Miocene periods,there were Sect.Ornus,Sect.Sciadanthus,and Sect.Fraxinus.3.Whole-genome duplication events:An intra-genomic analysis was performed on gene synteny and collinearity of each Fraxinus assembly.It revealed that each assembly could be divided into two sub-genomes exhibiting unequal chromosome numbers,with sub-genome A boasting 10 chromosomes and sub-genome B containing 13.An inter-genomic comparison of Fraxinus species to Coffea canephora with no whole-genome duplication(WGD)event,Sesamum indicum with a single WGD event,and Solanum lycopersicum with a single whole-genome triplication(WGT)event was performed to explore the polyploidy events occurred in Fraxinus species.It revealed a 6:1,6:2,and 6:3 syntenic depths pattern between each Fraxinus assembly and C.canephora,S.indicum,as well as S.lycopersicum,respectively,suggesting that Fraxinus tree species experienced at least two WGT events.Analyzing K_s distributions within syntenic gene pairs of WGD or segmental duplications also confirmed 2 WGT events.The estimated divergent time of 2 WGTs was 13.02 Ma(middle Miocene)and 37.93 Ma(late Eocene).4.A gene family-based pan-genome architecture:A pan-genome architecture for Fraxinus was constructed by clustering 1,608,427 protein-coding genes from 39 Fraxinus genome assemblies(11 assembled in this study and 28 previously released).The pan-genome of Fraxinus contains 12,156 core genes,40,664 dispensable genes,and 1215 specific genes.The sequence length of core genes was significantly higher than that of dispensable genes and specific genes.The K_a/K_s ratio of the homologous gene pairs of dispensable genes was significantly higher than that of core genes.There were differences in gene function between core genes and dispensable genes.The core genes were mainly involved in biological regulatory processes such as transcriptional regulation activity and metabolic process regulation,while the variable genes were mainly involved in catalytic activity,hydrolase activity,transport,and cell localization.5.An SV-based pan-genome architecture:A syntenic orthology map of 11 Fraxinus genome assemblies was analyzed and showed a high synteny and collinearity at the physical position across 11 Fraxinus genome assemblies.Using the F.pennsylvanica genome as a reference,424,102 non-redundant PAVs were identified across 11 Fraxinus genome assemblies.An SV-based pan-genome based on those non-redundant PAVs was constructed,resulting in a graph with 11.87 Gb in length boasting 371,617,125 nodes and 372,200,666 edges.The analysis of the SV-based pan-genomic feature showed that 47%–55%of PAVs were distributed in the genic and genic flanking regions.These PAV-affected genes across 11 Fraxinus tree species exhibited distinctive differences in gene function,especially for salt-tolerant tree species F.velutina and F.americana,specifically enriched PAV-affected genes mainly involved in transmembrane transport,transmembrane transporter activity,and oxidoreductase activity.6.A salt tolerance PAV-based QTL mapping and salt-tolerant gene identification:The whole-genome re-sequencing of 125 individuals from an inter-species cross F1-population of F.velutina‘Lula 3’(salt-tolerant)×F.pennsylvanica‘Lula 5’(salt-sensitive)together with their parents was performed and produced more than 1.105 Tb high-quality sequencing data.A population PAV analysis was conducted using the SV-based pan-genome,and 38,456 PAVs were identified.The genetic linkage map based on effective PAV was constructed.The total length of the map was 2,526 c M,consisting of 23 linkage groups and 2,550 PAV markers.Based on the genetic map constructed,integrating the genotypes and Fv/Fm ratios of those F1progenies and two parents,a salt tolerance PAV-based QTL mapping was performed.Two PAV-QTLs,Q6-45.2 and Q8-61.8,significantly related to salt tolerance,were identified,explaining14.70 and 16.81%of salt-tolerance phenotypic variance,respectively.Eight candidate genes with known functional annotation were identified within 500 kb of the prominent peak of the Q6-45.2 locus,and the Fvb HLH85 gene was significantly up-regulated under salt stress.The prominent peak of the Q8-61.8 locus was in the promoter region of the Fv SWEET5 gene(-663to transcription start site),and the expression level of the Fv SWEET5 gene was significantly increased under salt stress.7.Analysis of salt tolerance of Fvb HLH85 and Fv SWEET5 genes:The salt tolerance of Fvb HLH85 and Fv SWEET5 genes was analyzed by VIGS,gene transient overexpression,yeast one-hybrid,EMSA,and dual luciferase assays.The results showed that the Fvb HLH85 gene targeted the binding G-box element in the Fv AKT1 promoter region,positively regulated its expression,and mediated Na~+/K~+balance.Fvb HLH85 also targeted G-box elements that bind to the Fv PNC1 promoter region and mediated reactive oxygen species(ROS)homeostasis by positively regulating its expression.Fv SWEET5 enhanced salt tolerance by promoting the accumulation of soluble sugars.Collectively,the eleven high-quality chromosome-level genome sequences for Fraxinus tree species were assembled and annotated.Fraxinus species diverged from the most recent common ancestor of O.europaea about 40.57 Ma(late Eocene).Each Fraxinus genome assembly exhibited two sub-genomes exhibiting unequal chromosome numbers,with sub-genome A boasting 10 chromosomes and sub-genome B containing 13.Fraxinus tree species experienced at least two WGT events during its evolution.The gene family-based pan-genome analysis showed that dispensable genes played crucial roles in the evolutionary adaptation of Fraxinus species.The SV-based pan-genome constructed based on non-redundant PAV sequences analysis showed that 47%–55%of PAVs were distributed in the genic and genic flanking regions.These PAV-affected genes across 11 Fraxinus tree species exhibited distinctive differences in gene function,especially for salt-tolerant tree species F.velutina and F.americana,specifically enriched PAV-affected genes mainly involved in transmembrane transport,transmembrane transporter activity,and oxidoreductase activity.The PAV-based genetic linkage map of an inter-species cross F1-population of‘Lula 3’בLula 5’was constructed and pinpointed 2 PAV-QTLs,Q6-45.2 and Q8-61.8,significantly associated with salt tolerance.Two salt-tolerance genes,Fvb HLH85 and Fv SWEET5,were identified and functionally analyzed,of which the Fvb HLH85 gene mediated Na~+/K~+balance and ROS homeostasis,and the Fv SWEET5 gene promoted the accumulation of soluble sugar,thereby enhancing the salt tolerance of Fraxinus tree species.The results of this paper enrich the genomic data of Fraxinus species,deepen the understanding of the evolutionary adaptability of Fraxinus tree species,mine and analyze the function of salt-tolerance genes,and lay a foundation for genomics research and genetic improvement of salt-tolerance of Fraxinus tree species.