Study On Whole Genome And Molecular Basis Of Drought Resistance And Dimorphic Flower Differentiation In Lespedeza Potaninii

Breedinding new varieties of stress-resistant forage plants is important to ensure food security,sustainable agricultural development,and ecological security around the world.In the long-term evolution,native forage species has formed a strong ability to resist climatic adversity,and is an ideal plant for near-natural restoration of degraded grassland and desertification areas.Native forages have been proven to be an important gene pool for stress resistance gene mining,which could be used to improve the stress resistance of legume crops through genetic engineering.Lespedeza potaninii is the representative xerophyte in the genus Lespedeza.However,there is poorly understanding about the molecular basis of drought tolerance in L.potaninii.To comprehensively analyze the molecular basis of drought tolerance and dimorphic flower differentiation in L.potaninii,were studied the full genome sequencing,regulatory network of drought tolerance,functional gene identification,positively selected gene,and dimorphic flower differentiation were studied.The main results are as follows:1.High-quality chromosome-scale reference genome of the allotetraploid L.potaninii was assembed.The genome size of the assembled L.potaninii genome was1.34 Gb.A total of 99.4 % of the total contig bases were anchored and oriented to the21 chromosomes by Hi-C technique,with contig N50 length of 15.7 Mb.Phylogenetic analysis indicated that the allotetraploid L.potaninii genome originated about 4 million years ago and occurred chromosomal rearrangements.The subgenome analysis showed that the subgenomes of L.potaninii were functional differentiation during the evolution.The expansion gene families of L.potaninii were significantly enriched in metabolic pathways related to abiotic stress.which indicated that the expanded gene family contributed to the stress adaptability of L.potaninii.2.The core regulatory network was constructed in L.potaninii under drought stress and the functions of three unknown functional genes were identified.The gene expression profiling of leaves and roots were obtained under three drought treatments and rehydration treatments.Based on the weighted gene co-expression analysis,the core regulatory networks and hub genes of leaves and roots under drought stress were constructed and identified by correlating the expression profiles and physiological traits,respectively.A high-efficiency hairy root transformation system was established in L.potaninii,which was used to identify the functions of three unknown functional genes in the core regulatory network of roots under drought stress.Yeast heterologous expression and overexpression of three unknown function genes indicated that three genes of unknown function were positively regulated genes involved in the drought stress response of L.potaninii.3.The positively selected ASPARTIC PROTEASE IN GUARD CELL（ASPG）gene were identified to be involved in ABA-induced stomatal closure by regulating by ABF transcription factors under drought stress.L.potaninii was sensitive to ABAinduced stomatal closure than the other three Lespedeza species under exogenous ABA treatment,and had stronger drought tolerance.The positive selection analysis was showed that two ASPG genes were positively selected during the evolution of L.potaninii.The expression levels of positively selected ASPG1 genes were significantly induced by drought stress and ABA treatment in L.potaninii.Based on dual-luciferase reporter system and yeast one-hybrid system,it was demonstrated that the positively selected ASPG1 gene is regulated by the ABF transcription factor in the ABAdependent pathway through the ABRE cis-acting element,thereby participating in ABA-induced stomatal closure under drought stress.4.The regulatory network of dimorphic flower differentiation was analyzed in L.potaninii.Chasmogamy and cleistogamy flowers were developed in a single plant of L.potaninii.The phenotypic analysis of the dimorphic flower showed that the petals of the cleistogamy flowers were not exposed and were tightly wrapped by sepals.Anatomical analysis showed that the petals and other floral organs of the cleistogamy flowers were smaller compared to the chasmogamy flowers.A total of 37 ABCE model genes that involoved in flower development were identified in L.potaninii.Expression analysis showed that the expression levels of class B genes and class E genes were significantly downregulated in cleistogamy flowers.A total of 49 genes involved in anthocyanin synthesis were also significantly downregulated in cleistogamy flowers.Co-expression network analysis identified a module that was significantly negatively correlated with cleistogamy flowers.Among them,the class B genes that involved in petal development play the role of core genes.Furthermore,the result indicated that the class B genes,class E genes and genes involved in anthocyanin synthesis were coexpressed in the regulatory network.