Evolutionary Analysis Of Aux/IAA Gene Family In Rice And Functional Identification Of OsIAA23

The Auxin/Indole-3-Acetic Acid(Aux/IAA)gene family plays a critical role in auxin signal transduction.It is closely related to cell elongation,cell division and differentiation,tropic responses to light and gravity,general root and shoot architecture,organ patterning,vascular development,growth in tissue culture,and apical dominance.The signaling pathway of this phytohormone have been intensively studied in Arabidopsis,however,relatively limited information is available in rice.In this study,the evolutionary process of Aux/IAA gene family in rice was systematically studied through the evolutionary and population genetics analysis of Aux/IAA gene family.Furthermore,its function was verified to provide theoretical support for high-yield rice breeding.The Aux/IAA genes pertain to an auxin response gene family that encode short-lived nuclear proteins comprising four highly conserved domains,designated as domains I,?,?,and IV.Our research indicates that there were 31 members of Aux/IAA gene family in rice.The genetic polymorphisms of Aux/IAA homologous gene sequences in rice samples with different ecotypes were systematically compared by population genetics method.The results showed that the CDS regions of several Aux/IAA genes showed relatively high polymorphism(?2‰)such as OsIAA2,OsIAA23,and OsIAA27.For some genes,including OsIAA2,OsIAA22,OsIAA23,and OsIAA29,strong selection was detected in their Domain ?,which plays a major role in the stability of Aux/IAA proteins.In this study,OsIAA23(Os06t0597000)was taken as the representative to discuss the relationship between its functions and its evolution.In rice,the gene OsIAA23(Os06t0597000)is known to affect development of roots and shoots,but previous knockouts in OsIAA23 have been sterile and difficult for research.Here,we isolate new Osiaa23 mutants using the CRISPR/Cas9 system in japonica(Wuyunjing24)and indica(Kasalath)rice,with extensive genome re-sequencing to confirm the absence of off-target effects.Two mutation types were identified in the Kasalath mutants,namely,a 3-bp deletion that we designated as KaOsiaa23-3,and a 39-bp deletion named KaOsiaa23-39.Similarly,for the Wuyunjing24 mutants,two mutations were also identified,namely,a G-insertion that we designated as WuOsiaa23+1(g)and a T-insertion we called WuOsiaa23+1(t).In Kasalath,mutants with a 13-amino acid deletion showed profoundly greater dwarfing,lateral root developmental disorder,and fertility deficiency,relative to mutants with a single amino acid deletion,demonstrating that those 13 amino acids in Kasalath are essential to gene function.In Wuyunjing24,we predicted that mutants with a single base-pair frameshift insertion would experience premature termination and strong phenotypic defects,but instead these lines exhibited negligible phenotypic difference and normal fertility.Through RNA-seq,we show here that new mosaic transcripts of OsIAA23 were produced de novo,which circumvented the premature termination and thereby preserved the wild-type phenotype.This finding is a notable demonstration in plants that mutants can mask loss of function CRISPR/Cas9 editing of the target gene through de novo changes in alternative splicing.