Cloning And Functional Analysis Of The Capsule Spine Gene RcMYB106 In Castor(Ricinus Communis L.)

Castor bean（Ricinus communis L.）is a member of Euphorbia family,genus castor.Due to the high oil content in seed,the castor is known as one of the top ten oil crops.Further,the castor could generally resist to abiotic stresses,such as drought and saline-alkali.Natural capsule is spiny in natural,while some were glabrous.The spine structure on the castor capsule can warn phytophagous animals,self-protection and suitable for wide distributions of seeds.During the agricultural harvesting,however,the castor spines will cause potential stabbing risk to the harvesters.The number of capsules will be greatly reduced due to the space occupied by spines,thus reducing the harvest efficiency.In addition,the spines will increase the amount of dust when the castor capsules are crushed and shelled,which have adverse effects on the environment.To further investigate the molecular mechanisms underlying capsule spine phenotype in castor,we focused on examination of the genetic information,which will provide certain reference significance on revealing capsule spine phenotype variation in castor.Several studies showed R2R3 MYB transcription factor involved in spine-like development in various species,but no report about spine phenotype in castor so far.Herein,we detected the target gene Rc MYB106 transcription factor in two independent populations LYY5×DL01 F₂ and LYY9×DL01 F₂ by map-based cloning and we found the Rc MYB106gene has two mutation alleles in different glabrous accessions.From a cross between LYY5 as donor and LYY9 as recipient,F₁ plants showed a spineless phenotype.Besides,the comparison of 268 castor accessions resequencing revealed that only these two mutation alleles in spineless castor varieties.Rc MYB106 gene function has been identified in castor and Arabidopsis.Then we discovered the putative downstream target gene Rc WIN1 by RNA sequencing and further verified it.The results showed Rc MYB106 gene could positively regulate Rc WIN1 gene by binding the promoter.The findings are as follows:（1）Segregation analysis of the cross between DL01 and LYY5 showed the 434 spiny,830less spine,441 spineless plants in F₂ population and follow the 1:2:1（p>0.05,Chi-square test）.Map-based cloning by 441 spineless plants from F₂ population on capsule spiny phenotype in castor narrowed the gene on chromosome 10 between RC9-7.295 and RC9-7.411 indel markers.Five genes were located within this region,and only three non-synonymous genes,Rc10G022840,Rc10G022841,and Rc10G022843,were identified in this region.Compared to the spiny DL01,Rc10G022841 DNA sequence contain one single nucleotide polymorphism（SNP）in the third exon in the spineless LYY5.We found that the SNP locus transition from C-to-T then due to a premature codon,which resided in the third exon（+2228 bp）.Rc10G022841is orthologous to Arabidopsis MYB106,which encodes a MIXTA-like R2R3-MYB family TFs.In Arabidopsis,the MIXTA-like gene MYB106,also known as NOECK or NOK,encodes a TF that is involved in leaf trichome development as a negative regulator in trichome branch.（2）Parallelly,we constructed another genetic population between DL01 and another spineless germplasm LYY9 independently.Genetic linkage analysis within 133 F₂ recessive spineless individuals derived from the cross between DL01 and LYY9 showed that controlling capsule spine gene located between indels RC9-6.861 and RC9-7.373 on chromosome 10,containing the Rc10G022841,which was repeatedly identified in two independent mapping populations.Based on comparison of the genomic sequences of Rc10G022841 in DL01 and LYY9,we detected a 4353 bp deletion in the promoter from-4607 to-254 bp region,furthermore,LYY9 exhibited no change in the non-synonymous bases within the coding region.GUS activity analysis demonstrated that a 2000 bp promoter sequence from Rc10G022841^DL01resulted in higher GUS activity than did the 253 bp promoter of Rc10G022841^LYY9,suggesting that the 253 bp promoter of Rc10G022841^LYY9 carries fewer cis-elements related to transcriptional activation.（3）To further analyze whether Rc10G022841 was involved in the formation of capsule spine phenotypes in castor,we examined the types of mutation present in the Rc10G022841locus based on resequencing data from 225 spiny and 43 spineless castor varieties.Interestingly,39 spineless accessions clustered together with LYY9 that from the northern of regions Shandong or Northeast China.LYY3 and LYY4 were more related to LYY5 from southern regions such as Yunnan Province.However,224 accessions with spiny capsules consistently clustered along with DL01.Together,these findings suggested that spine identity required Rc10G022841 function in castor.（4）To further examine whether mutations in the Rc10G022841 gene cause the capsule phenotypes characteristic of LYY5 and LYY9,we tested their alleles for complementation of function.From a cross between LYY5 as donor and LYY9 as recipient,F₁ plants showed a spineless phenotype.This indicated that the mutations that cause these capsule phenotypes could complement each other as alleles,although we did not an excellent genetic transformation system.Hence that we overexpressed Rc MYB106 in myb106 mutant（SALK-025449）,and the transgenic T₃ homozygous plant showed over branched trichome phenotype consistent with myb106,suggesting the diverse functions of Rc MYB106 in different organisms.（5）To probe the regulatory networks that could underlie spine formation on castor capsules,we compared differentially expressed genes（DEGs）in young aril tissues of DL01,LYY5,LYY9 plants by RNA sequencing（RNA-Seq）.Among these DEGs,we detected and focused on the ethylene-responsive TF WIN1（Rc07G016896）,which is an ortholog of the Arabidopsis WIN1/SHN1 gene that encodes an ERF（ethylene response factor）in the B-6subfamily of ERF/AP2 TFs.There was a higher transcript abundance for Rc WIN1 in DL01compared to LYY5 and LYY9,and it was consistent with the q RT-PCR results.A dual-luciferase assay indicated that Rc MYB106 could activate gene expression from the promoter of Rc WIN1 in castor cotyledons.In additional,the results were also confirmed by electrophoretic mobility shift assay（EMSA）and yeast one-hybrid（Y1H）assay.We also found that Rc MYB106^DL01 and Rc MYB106^LYY5 could bind to the promotor of Rc WIN1.In conclusion,we used a map-and sequencing-based cloning strategy to identify Rc MYB106 as a causative gene likely responsible for differences in the spine phenotypes of castor capsules and identified its function.Then we detected the putatively downstream target gene Rc WIN1.Results of biochemical experiment illustrated that Rc MYB106 could positively activate Rc WIN1 expression.This further explains the regulatory networks that could underlie spine formation on castor capsules.Rc MYB106 therefore likely represents the first gene to be successfully cloned in castor using this kind of mapping strategy,which could also provide a way to further study important traits and their causative genes in other non-model plants.At the same time,it also revealed the regulatory pathway for castor spine phenotype,and further provided a novel idea for castor breeding.