Whole Genome Sequencing Of Oxytropis Ochrocephala And Its Symbiotic Relationship With Endophyte Alternaria Oxytropis

Oxytropis ochrocephala Bunge(Leguminosae,Oxytropis sp.),is a perennial poisonous forb.It forms symbiosis with the endophytic fungi Alternaria oxytropis,which produces an alkaloid mycotoxin swainsonine(SW),making the plants toxic to grazing anmials especially horses and sheep.When those animals consume the toxic O.ochrocephala plants unintentionally,they are often poisoned or even die,causing a great threat to the development of animal husbandry.At present,relevant research on the species evolution of Oxytropis plants and the symbiotic relationship between Oxytropis and A.oxytropis is still at the exploratory stage,and genomic resources have been lacking in the Oxytropis genus,which limits the progress of relevant research.In this study,we performed a de novo whole genome sequencing of O.ochrocephala by combining Illumina,Oxford Nanopore(ONT),and chromosome conformation capture technology(Hi-C).Then a high-quality and chromosome-level O.ochrocephala genome was assembled.After I analyzed the evolutionary background of O.ochrocephala by comparative genomics analysis.Finally,I explored the differential gene expression and functional gene enrichment between O.ochrocephala and A.oxytropis in their symbiotic and non-symbiotic states by transcriptomic analysis,in an aim to further reveal the symbiotic meachanism at the genetic level.The main results of this study were as follows:(1)Genome survey(K-mer analysis)and flow cytometry showed that the genome(2n =16)size of O.ochrocephala was about 780 Mb ～ 914 Mb.Heterozygosity was ～ 0.43 %,the proportion of repetitive sequences was ～ 66.47 %,and the GC content was ～ 38.34 %.It was speculated that O.ochrocephala has a highly repetitive and complex genome,and therefore a combination of the third-generation and the second-generation sequencing methods was recommended for in-depth sequencing and analysis.(2)Whole genome sequencing,assembly and annotation showed that the genome size of O.ochrocephala was 958.83 Mb,of which 930.94 Mb(97.09 %)were able to be anchored and oriented onto 8 chromosomes.Contig N50 was 1.40 Mb and the scaffold N50 was121.79 Mb.After genome annotation,a total of 31,700 protein-coding genes,75.66 % of the repeats,104 mi RNAs,1,477 r RNAs,852 t RNAs were predicted,and 31,218(98.48 %)protein-coding genes were annotated.(3)Comparative genomics analysis showed that O.ochrocephala differentiated about42.62 million years(Mya)ago.It had a close genetic relationship with Medicago truncatula and Glycine max,and had better gene collinearity with M.truncatula,in which had some between-chromosome fragment rearrangment.Through functional annotation,it was found that these rearranged genes were mainly related to metabolic function.Gene family expansion and contraction analysis showed that 6,938 gene families contracted and1,862 gene families expanded in the O.ochrocephala genome,of which 62 gene families expanded rapidly(p < 0.01).It was found that the rapidly expanded gene families were mainly involved in nucleoside excision repair,DNA replication,homologous recombination,metabolic process,cellular processes,etc.Ks analysis showed that O.ochrocephala,like M.truncatula,experienced a whole-genome duplication event(WGD)during evolution.(4)Transcriptomic analysis of the symbiotic and non-symbiotic samples between O.ochrocephala and A.oxytropis showed that the gene expression patterns of both sides in the symbiotic state were significantly different from those in the non-symbiotic state.For O.ochrocephala,after forming symbiosis with A.oxytropis,an up-regulated expression pattern of the differential expressed genes(DEGs)was observed.A large number of the DEGs involved in plant-pathogen interaction and defense were induced or up-regulated,such as CML(calmodulin or Ca M like),the WRKY and CYP450 gene families,genes related to the cutins,suberine and wax biosynthesis and etc.The up-regulated expression of these related genes may indicate that A.oxytropis provides benefits to plant growth,development and environmental adaptation when it coexists in plants.For A.oxytropis,in its symbiosis in O.ochrocephala,a large number of DEGs involved in fatty acid metabolism,nitrogen metabolism and degradation of cell wall were also induced or up-regulated.In this way,A.oxytropis could maintain the symbiotic relationship with the host through self-regulation and transfer of nutrients with plants,which also reflects the influence of O.ochrocephala on A.oxytropis.