Reduced Seed Shattering Of SH4 And QSH1 Genes Edited Weedy Rice And Its Regulatory Mechanisms

Weedy rice(Oryza sativa f.spontanea)is a worldwide noxius weed in rice fields and poses a great challenge for world rice production.Weed rice is characterised by strong seed shattering,which can promote seed dispersal and the establishment of weedy rice populations,making the control of this weed very difficult.Also,weedy rice may obtain herbicide-resistant and stress-tolerant traits or other genes through pollen-mediated gene flow(PMGF)from cultivated rice,causing serious damage to rice production.However,the transgene-mitigating strategy can prevent the spread of the weedy rice seeds/plants containing these genes through PMGF by reduced seed shattering(mitigation).This strategy has great application potential for weed control.Some studies have reported that modifction on the shattering-controlling genes reduced seed shattering of weedy rice,but the related studies are still lmited.To answer the above questions,we produced 13 gene editing weedy rice lines,of which the seed-shattering controlling genes,SH4 and q SH1,single or double edited usig the CRISPR-Cas9 system.We studied the impacts on seed shattering and production of SH4 and q SH1 gene-editing and their mechanisms by seed-shattering examination,histological observation,seed weight and size examination,transcriptome sequencing,gene expression content examination,and plant hormone examination.The main results obtained from the study are as follows:1.Editing a single gene or both of the SH4 and q SH1 genes significantly decreased seed shattering of the weedy rice.Compared with the wild-type parental weedy rice line with seed-shattering-index of 100%,seed-shattering-index of the SH4-edited,q SH1-edited,and SH4&q SH1-edited lines decreased to 11%～28%,even lower than that of the cultivated rice control line shouxiang-1 with seed-shattering-index of 54%.The edited weedy rice lines were also lack of the entire abscission layers.Therefore,the edition on SH4 and q SH1 genes has considerable effects on seed shattering control of the weedy rice.2.Editing a single gene SH4 or q SH1,has no significantly effect on the seed-production related traits of the weedy rice.Editing a single gene SH4 or q SH1,did not significantly effected seed weight,but decreased seed length by 2%～17%.However,editing both of the SH4 and q SH1 genes significantly decreased the seed weight and length of the gene-editing weedy rice lines.Therefore,editing a single gene,SH4 or q SH1,has no negative impact on seed production related traits of the weedy rice,thus it may not reduce the seed production of cultivated rice,if the same technique is applied to cultivated rice.The SH4 and q SH1 genes is the reasonable potential interference target on transgene-mitigating strategy that can be conducted on cultivated rice.3.Editing a single gene or both of the SH4 and q SH1 genes significantly inhibited the expression of the seed shattering controlling genes on weedy rice lines.The result of the transcriptome sequencing,gene expression content examination showed that compared with the wild-type parental weedy rice line,the shattering-controlling QTLs(CPL1,SH4,SHAT1,Os SH1,Os SH15,q SH1,SH5,SH8),genes encoding the key enzymes of ABA(abscisic acid)(ZEP,NCED1.1,NCED1.2,SDR1,SDR),ABA-signaling-pathway-related genes(PYL3,PYL4,PP2C39,SAPK5,BZIP23),genes encoding the cell-wall hydrolases(PME34,PG,XTH28,MAN4)of the edited lines significantly reduced expression.Therefore,the interferences on SH4 and q SH1 genes decreased the expression of cell-wall hydrolases through the down-regulation of ABA biosynthesis and signaling-pathway,negatively affected the development of abscission layers of the spikelet pedicles,and thus decreased seed shattering of the weedy rice.4.Editing a single gene or both of the SH4 and q SH1 genes significantly decreased the biosynthesis of ABA.The ABA contents at the late stage of seed development in the edited weedy rice lines were significantly lower than their wild-type parental weedy rice line.In addition,no consistent and significant variation were detected on the contents of the seed size related IAAs(auxin a,auxin b)and cytokinins(t Z,c Z,t ZR,c ZR).Therefore,decrease on the content of ABA is the main factor of seed shattering decrease of the weedy rice containing the edited SH4 and q SH1 genes.In conclusion,editing the SH4 and q SH1 genes in the weedy rice will inhibit ABA biosynthesis and signaling-pathway,stunted development of abscission layers,and thus decreased seed shattering.Editing a single gene,SH4 or q SH1,may have no negative effect on the production of cultivated rice.The SH4 and q SH1 genes thus has valuable application prospect on risk control of herbicide-resistant transgene flow based on transgene-mitigating strategy.Our results from the study on edited weedy rice lines not only provide an important basis for revealing the controlling-mechanism on rice seed shattering,but also provide strategy for controlling weedy rice as a weed.