Gene Identification And Evolution Of Self-Incompatibility Of Citrus

Self-incompatibility（SI）is an important mechanism preventing self-fertilization and avoiding inbreeding in flowering plants.The most widespread SI system is found in the Solanaceae,Rosaceae,and Plantaginaceae and utilizes S-ribonuclease（S-RNase）and S-locus F-box（SLF）as female and male S-determinants respectively.Citrus belongs to the Citrinae tribe of the Rutaceae family and is grown worldwide due to its commercial importance.In citrus,pummelo is self-incompatible,while mandarin and its hybrids are predominantly self-compatible（SC）.However,the genes encode the S-determinants has not been demonstrated to date.Here,we identified the pistil S-determinants using the RNA-seq from different pummelo and demonstrated that they functioned as the female S-determinants.The corresponding pollen S-determinants were identified through the bacterial artificial chromosome（BAC）technology.Moreover,we obtained a nonfunctional S-RNase conferring the loss of SI and then illustrated the evolutionary relationship between the SC and apomixis in citrus.The main results are as follows:1.The physiological responses of SI in pummeloTo test whether the SI system was adopted by the pummelo,manual pollinations comprising self-and cross-pollinations,were performed on nine pummelo varieties.Results demonstrated that four pummelo accessions（HB pummelo,‘Wanbai’pummelo,‘Shuijingmiyou’pummelo and‘Guanximiyou’pummelo）produced seedless fruit in the absence of pollination,which were identified as parthenocarpic.All cross pollinations showed pollen tubes that extended all the way down the style and resulted in fruits,while self-pollinations showed the inhibition of pollen tube growth near the top of the style and resulted in no seed set.Moreover,half-compatible pollinations were observed and showed pollen tube inhibition in the upper style as well as arrays of long pollen tubes.These data provide good evidence that pummelo is self-incompatible and the SI system is gametophytically controlled.2.Genome-wide analysis of RNase T2 in sweet orange and clementine mandarinA total of 16 RNase T2 genes were identified in the genomes of sweet orange and clementine mandarin.Among them,CgRNS₃ possessed several common characteristics of the pistil S-determinant of self-incompatibility and is specifically expressed in style.In an in vitro pollen assays,the CgRNS₃ recombinant protein significantly inhibited the growth of self-pollen tubes,but after a heat treatment,this protein did not significantly inhibit the elongation of self-or non-self-pollen tubes.However,CgRNS₃ did not segregate with S-haplotype and had not the polymorphic alleles,suggesting CgRNS₃ did not function as the pistil S-determinant.3.The identification and function demonstration of the pistil S-determinantUsing 64 transcriptomic profiles between style and anther from pummelos,we identified nine S-RNases that their sequence and structure features were similar to previously reported S-RNases and were specifically expressed in pistil.Notably,we identified a mutated S-RNase（named S_m-RNase）,which revealed a single nucleotide deletion,resulting in a frameshift mutation and premature stop codon.The level of expression of the S_m-RNase in the pistil was much lower than that of the nomal S-RNase.The frequency analysis of these nine S-RNases within natural pummelo populations in China,comprising 391 accessions showed that these S-haplotypes were abundant and found in 77%of the accessions and their frequency ranged from 2.3%to 30.4%.The F1 populations of pummelos demonstrate that the nine S-RNases segregated with the S-haplotypes of the pummelos.The recombinant S₁-RNase and S₂-RNase protein specifically inhibited the growth of pollen carrying the same S-haplotype in a self-incompatibility bioassay and induced the actin cytoskeleton rearrangement within pollen tubes.Interestingly,S_m-RNase lacks pollen inhibitory activity,while the“recovered”S_m^R-RNase exhibits inhibitory activity.These results provide definitive evidences that S-RNase serves as the pistil S-determinant of SI in citrus.4.The identification of the S-locus and the pollen S-determinant in citrusNine S-loci spanning 198 to 370 kb were obtained based on the BAC library and the available citrus genomes.The genome locations of S-loci indicate that the S-locus of citrus is anchored toward the telomere of chromosome 1.A total of 113 SLF/SLFL were identified on these S-loci.There are approximately eight polymorphic SLFs and three conserved SLFLs at each S-locus.Each SLF was specifically expressed in pollen and tightly linked to the S-RNase.All SLF/SLFL were clustered into 12 types based on the phylogenetic analysis.The S-RNases and the SLFs/SLFLs at each S-haplotype are highly polymorphic and share the comparable synonymous（Ks）and non-synonymous（Ka）substitution rates between them,suggesting that they are equivalent in their age and degree of coevolution.However,the inter-allelic SLF/SLFL of each type are conserve and have extremely lower Ks and Ka values,indicating that the divergence of these genes occurred more recently.We therefore speculate that the S-locus of citrus harbors multi-SLF genes and the SI system adopts the non-self recognition mechanism.5.Evolution analysis of the SC in citrusThe genome sequences of 153 citrus accessions showed that 90 accessions contained the S_m-RNase and all of those with the S_m-RNase were SC.We proposed that the SI-SC transition arose initially in mandarin and then spread to its hybrids through mating or introgression from mandarin,while pummelo and citron retained SI.Moreover,apomixis/polyembryony（which naturally occurs in citrus）helped to fix the transition of SI-SC in citrus SC populations.In summary,we show here that SI in citrus utilizes an S-RNase-based SI system.Our identification of a new genus utilizing this SI system is a milestone for evolutionary comparative studies（Ramanauskas and Igi?,2017）.As citrus is>117m.y.separated from the nearest S-RNase family,this provides important new information that will help clarify the distribution of S-RNase-based SI systems and their evolution.We provide evidence that SI is ancestral and show that a truncated S_m-RNase is responsible for the loss of SI.This has allowed us to decipher the evolutionary history of the SI to SC transition in 153 citrus accessions.Use of apomixis in conjunction with selection of SC makes this an interesting example of evolution of plant reproductive strategies.