Multi-omics Identification And Characterization Of Genes Related To Self-incompatibility In ’Wuzishatangju’ Mandarin

Self-incompatibility（SI）is a genetic mechanism evolved in angiosperm to prevent inbreeding depression.Citrus belongs to Citrinae of Rutaceae,and the SI reaction is mediated by S-RNase.‘Wuzishatangju’（Citrus reticulata Blanco）is derived from a bud sport of a self-compatible cultivar‘Shatangju’,and its seedlessness is caused by SI reaction.In this paper,S-locus F-box proteins,non-S-locus SKP1-like and CUL1 were identified from‘Shatangju’and‘Wuzishatangju’mandarins.The SCF complex involved in S-RNase-based pathway was analyzed by yeast two hybridization,GST-pull down and luciferase complementary imaging methods.The S-RNase candidates were characterized by in silico method based on pistil transcriptome,and the function of S-RNase candidates was analyzed by proteomics,transgenic tobacco and S-genotype dependent analyses.To investigate the main factors contributed to the SI reaction in‘Wuzishatangju’,proteomics was used to compare the proteome in pollen between‘Shatangju’and‘Wuzishatangju’mandarins.The main results were as follows:（1）Genome-wide identification of F-box protein was conducted based on the genome of C.clementina,and a total of 298 F-box proteins were identified.Among which 46 F-box proteins contained the FBA domain in the C-terminal region,called FBA subfamily in this paper.In FBA subfamily,16 pollen-specific F-box genes（CrFBX1-CrFBX16）were cloned from‘Shatangju’and‘Wuzishatangju’.Sequence alignment showed that there are no differences between‘Shatangju’and‘Wuzishatangju’mandarins.（2）The genome sizes of‘Shatangju’and‘Wuzishatangju’were 355.47±4.25 Mbp and378.15±7.52 Mbp according to flow cytometry analyses,respectively.No significant differences were detected between the two cultivars.Phylogenetic analysis indicated that thirteen CrFBXs are clustered individually and are homologous with S-locus F-box brothers（SFBB）genes and S-locus F-box like（SLFL）genes from Rosaceae.Furthermore,ten CrFBXs（CrFBX1-CrFBX10）were physically anchored into the S-locus.However,Southern blotting analyses showed that ten CrFBXs which were located into S-locus were no difference between‘Shatangju’and‘Wuzishatangju’mandarins.（3）SKP1-like and CUL1 genes possibly involved in SI reaction were identified by in silico method based on the pistil transcriptome datasets of‘Shatangju’and‘Wuzishatangju’and the reference genome of C.clementina.A pollen-specific SKP1-like gene orthologous with the SSK1（SLF-interacting-SKP1-like）involved in SI reaction,CrSKP1-e,was cloned and encoded 176 amino acid residues containing the conserved domain“WAFE”and a typical domain“GVDPDDDDV”in the C-terminal region.Two CUL1 family genes:CrCUL1A and CrCUL1B,were characterized.Higher expression level of CrCUL1A was detected in pollen compared to lower expression of CrCUL1B in pollen.Y2H and in vitro binding assays indicated that CrSKP1-e interacted with CrFBX2,CrFBX7 and CrFBX9proteins located into S-locus,and with CrFBX13,CrFBX15 located into non-S-locus.Luciferase complementary imaging and in vitro binding assays showed that CrSKP1-e interacted with the N-termini of both CrCUL1A and CrCUL1B proteins.Those results collectively indicated that CrSKP1-e serves as a functional member of the SCF-type E3complex in‘Wuzishatangju’.（4）A total of five T2 family RNase genes（CrRNS1-CrRNS5）were identified by in silico method based on the pistil transcriptome datasets of‘Shatangju’and‘Wuzishatangju’.CrRNS5 was constitutive expression and encoded a ClassⅡsubclass acidic protein,CrRNS1and CrRNS4 were lower expression level in pistil and encoded ClassⅠsubclass acidic protein.Pistil-expressed CrRNS1 and CrRNS3 were homologous with S-RNase encoding ClassⅢsubclass basic proteins,respectively.However,sequence alignment indicated that two proteins encoded by CrRNS1 and CrRNS3 had identical sequence in‘Shatangju’and‘Wuzishatangju’.In addition,the average fold difference values of two proteins were 0.95（p=0.03）and 0.87（p=0.06）,respectively,suggesting that there are no significant accumulation differences between‘Shatangju’and‘Wuzishatangju’mandarins.CrRNS1 and CrRNS3 without negative function for pollen tube growth in pistil of transgenic tobacco were located at the non-S-locus and existed in the genome of citrus cultivar in an S-haplotype independent manner.Those results suggested that CrRNS1 to CrRNS5 functioned as S-like RNase genes in mandarin.（5）Self-and cross-pollination showed that self-pollen tube of‘Wuzishatangju’was inhibited elongation at the upper site of the style.Pollen tube from‘Shatangju’reached the base of style,suggesting that mutations played a role in pollen of‘Wuzishatangju’.Therefore,protein differences between the pollen of‘Shatangju’and‘Wuzishatangju’were compared by the relative quantitative proteomic technique.A total of 67 differential abundance proteins were identified,of which 24 were up-regulated and 43 down-regulated.A polyamine oxidase2（CrPAO2）was up-accumulated in pollen of‘Wuzishatangju’and catalyzed the spermine and spermidine oxidation leading to produce H₂O₂.