Discovery Of Candidate Resistance Genes And Cloning Of TaCOI1 Related To Fusurium Head Blight Resistance Of Wangshuibai

Fusarium head blight(FHB)or scab is an important disease of wheat in warm and humid regions of China which causes a serious threat to grain yield,quality and food safety.F.graminearum(sexual stage:Gibberellazeae)is the dominant pathogen responsible for wheat scab epidemics in China.Growing of resistant cultivars is the most economical and effective approach for FHB control.The major and most stable QTL for FHB resistance have been located in the deletion bin 3BS 0.78-0.87,which can explain the phenotypic variation of 25-40%.Cloning of Fhb1 candidate gene will be of great significance not only for marker development but also to provide gene resources for the wheat improvement by genetic engineering.Until now no major genes/QTLs associated with FHB resistance was cloned.In the present research,by using Wangshuibai and its FHB susceptible mutant NAUH117,which is deficit in the major FHB resistance QTL Fhbl on chromosome 3BS,Fhb1 candidate genes were screened by high-throughput sequencing.TaCOI1,a key gene involved in JA signaling pathway,was cloned and functional analyzed.The main results obtained are as following:1.Discovery of Fhbl candidate genes using Wangshuibai Fhb1-deficit mutantWangshuibai and its Fhb1-deficit mutant NAUH117 was profiled with RNA-seq of the spikes inoculated with F.graminearum.The assembled sequence of Wangshuibai 3B,which was obtained by flow sorting and followed by next generation sequencing by Illumina Hi-seq3000,was compared with the Chinese spring 3B reference sequence.A total of 360 genes were annotated.These annotated genes were compared for their expression in Wangshuibai and its mutant NAUH117.The RNA-seq reads which expressed in Wangshuibai but not in NAUH117 were selected and predicted to be located in Fhb1.A total of 14 candidate genes were identified,including 3 receptor-like protein kinases,1 glucosyl transferase,1 tRNA synthetase,1 lipid transfer protein,1 ubiquitin protein ligase and 7 unnamed proteins.RT-PCR were used to validate the expression of the 14 genes in Wangshuibai and a FHB susceptible wheat variety Alondra’s.It was found that 2 of the 14 genes was not expressed in Alondra’s but expressed normaly in Wangshuibai,and 3 of the 14 expressed more in Alondra’s than in Wangshuibai.2.JA pathway was involved in the regulation of wheat resistance to FHBIn a previous study,the transcription profiles of Wangshuibai and NAUH117 upon F.graminearum inoculation were analyzed using the Affymetrix Wheat Genome GeneChip and compared.Genes associated with JA pathway were found showing different expression patterns,indicating the JA pathway may involved in the FHB resistance response in wheat.Exogenous application of MeJA showed that in Alondra’s,the scabby spikelet rate was decreased to 14.89%compared to 29.01%in water treatment;in NAUH117 the scabby spikelet rate was decreased to 12.36%compared to 24.57%in water treatment;in Wangshuibai the scabby spikelet rate was decreased to 4.28%compared to 7.30%in water treatment;These indicated the application of JA could improve FHB resistance,both in resistant and susceptible wheat materials.Using ELISA assay,it was found that in the F.graminearum-challenged spikes,endogenous JA content was increased only in Wangshuibai while not in NAUH117.This result indicated that,in the susceptible wheat,the JA pathway was not activated when induced by the pathogen.These physiological experiments confirmed that the JA pathway may participate in the wheat resistance to FHB.3.Cloning of wheat TaCOI1 associated with FHB resistanceA key gene of JA pathway,TaCOI1,was further cloned from Wangshuibai.The full-length cDNA was 2300bp.When TaCOI1 was silenced using VIGS in a FHB resistant wheat variety Sumai 3,the scabby spikelet rate was 5.43%after 15 dpi of E graminearum.While in the control,the the scabby spikelet rate was 1.83%,indicating the silencing of TaCOI1 compromised the FHB resistance of Sumai 3.In order to elucidate the function of TaCOI1,an over expression vector of TaCOI1 was constructed and transformed into the callus of a FHB susceptible variety Alondra’s by the Genegun bombardment method.Totally,180 regenerated plants were obtained,in which 7 were identified as the positive transgenic plants by PCR.The preliminary evaluation result of the plants at T0 generation showed that none of the 7 plants showed significantly enhanced FHB resistance.