Fine Mapping Of Wheat Powdery Mildew Resistance Gene Pm6 And Cloning Of The Candidate Gene TiNLR-G In Triticum Timopheevii

Wheat powdery mildew is a main disease which causes significant reduction of grain yield as well as kernel quality in wheat production.Breeding for resistant varieties is widely accepted as the most economical and environmentally safe approach for disease control.Pm6,one of wheat powdery mildew resistance gene from wheat relative species Triticum timopheevii,has been used in wheat breeding program.The resistance characteristics of Pm6 displayed a developmental stage-dependent manner,shown as being moderately effective at the juvenile seedling stage and highly resistant from the fourth leaf stage and thereafter.Pm6 is still effective in breeding,especially when used in combination with Pm2.Fine mapping and cloning of Pm6 would facilitate the understanding of its resistance mechanism and its use in wheat breeding for powdery mildew resistance.In previous study,the introgressed fragment sizes of 2GL in seven wheat-T.timopheevii introgression lines harboring Pm6 were determined by linkage markers.Lines IGV1-465 and IGV1-466 contain the smallest and largest introgression 2G fragments,respectively.To fine map Pm6,two segregation populations from the crosses IGV1-465/Prins and IGV1-466/Prins were used to construct the genetic map of Pm6.Combined with comparative genomics,the Pm6 was mapped to a genetic region flanked by markers CINAU127 and CINAU123.Two members of LRR-RLK residing in this region were cloned and proved to play positive role in wheat powdery mildew resistance.However,the two TaLRR-RLKs were not considered as Pm6 candidate because they were located outside the introgressed 2G fragment in IGV1-465.We suspect the presence of recombination inhibition between introgressed 2G fragment and its wheat 2B counterpart impeded our effort in approaching to the Pm6.In the present study,to overcome 2B/2G recombination suppression,a ph1b-based strategy was employed to produce introgressions with reduced 2G fragments.Recombinants carrying the size of introgression fragment smaller than that in IGV 1-465 were obtained,this allowed us to narrow Pm6 into a much smaller physical region.Based on the fine mapping of Pm6,we cloned the candidate gene of Pm6 and validate its function in Pm resistance.The main results obtained were as follows:1.Fine mapping and candidate gene prediction of wheat powdery mildew resistance gene Pm6（1）The microcolinearity analysis of Pm6 region among Triticeae speciesReferring to 2B of Chinese Spring genome sequence,our previous research defined the introgression 2G fragment in IGV1-465 into a physical interval of 57 Mb,flanked by markers CINAU134 and CINAU140.Microcolinearity analysis among Triticeae species was performed by comparing the genome sequences in the public released databases.Gene annotation of Chinese Spring chromosome 2B identified 689 genes in the region between markers CINAU134 and CINAU140.Comparative analysis found 426 of the above genes had their homologous in all the surveyed genomes or subgenomes,and they also showed well conserved collinearity for their location across different Triticeae species,indicating that region without obviously structural variation in evolution,and the possibility of chromosome rearrangment caused recombination inhibition could be ruled out.The chromosome 2G of T.timopheevii was sorted using flow cytometry,and a draft sequence was produced and assembled,which provided the essential information for fine mapping and cloning the candidate gene of Pm6.Comparison of the 2G and 2B sequences,and using the identified SNPs between 2G and 2B by Wheat55K array allowed us to develop more 2G specific markers.A total of 34 chromosome 2G specific markers harboring the Pm6 region were developed,their well conserved position and order on chromosome 2G showed its good collinear relationship with other Triticeae species.（2）Fine mapping of Pm6 by overcoming 2G/2B recombination inhibition using CSph1bCSph1b is a deletion mutant of Ph1b gene in wheat variety Chinese Spring.To increase the recombination rate between 2B and 2G,the introgression line IGV 1-465 was crossed with CSph1b,followed by backcrossing the F1 individuals to CSph1b.The derived BC1F1 progenies were screened to identify individuals heterozygous for Pm6 and homozygous for ph1b,and these individuals were self-crossed to produce BC1F2 or BC1F2 equivalents.The residual heterozygous lines of BC1F2、BC1F2:3、BC1F2:4 and BC1F2:5 could also produce the segregation population to select the recombinants.By using the Pm6 flanking markers CIT02g-14-900bp and CIT02g-19-750bp,a total of 164 recombinants were identified from 820 BC1F2:3 individuals.Genotyping and powdery mildew resistance evaluation using Bgt isolate E26 of the recombinants enabled us to map Pm6 to a 10 Mb physical region,flanked by markers CIT02g-13500bp and CIT02g-18-300bp.The recombination rates of 2B and 2G in CSph1b and normal CS were 20%and 0.54%per million base pairs（RPM）,respectively.The new flanking markers,CIT02g-13₅00bp and CIT02g-18-300bp were used to identify new recombinants from 643 BC1F2:4 individuals,and 18 were identified.Genotyping and powdery mildew resistance evaluation using E26 of recombinants enabled us to map Pm6 to a 0.9 Mb physical region,flanked by markers CIT02g-20-490bp and CIT02g-18-300bp.The recombination rates of 2B and 2G in CSph1b and normal CS were 2.8%and 0.28%RPM,respectively.The new flanking markers CIT02g-20-490bp and CIT02g-18-300bp were used to identify new recombinants from 1,190 BC1F2:5 individuals,and only one recombinant was identified.Genotyping and powdery mildew resistance evaluation using E26 of the recombinants enabled us to map Pm6 to a 218 kb physical region,flanked by markers CInDel02g-3130 and CIT02g-18-300bp.The recombination rates of 2B and 2G in CSph1b and normal CS were 0.084%and 0.093%RPM,respectively.Above results indicated the introduction of ph1b significantly enhanced 2B and 2G recombination frequency.We succeed in narrowing the Pm6 physical region from 57 Mb to 218 kb,which should be feasible for the candidate gene prediction and cloning of Pm6.（3）Candidate gene prediction for Pm6Gene annotation of Chinese Spring chromosome 2B corresponding to the Pm6 region identified four high-confidence genes,TraesCS2B02G504600.2,TraesCS2B02G504700.1,TraesCS2B02G504800.1 and TraesCS2B02G504900.1.In silico transcription analysis using the public transcriptome data of Chinese Spring showed only two genes,TraesCS2B02G504600.2 and TraesCS2B02G504000.1,were up-regulated in response to Bgt inoculation,while the remained two were not responsive.Sequence alignment of above four genes to the genomic sequences of chromosome 2G showed only TraesCS2B02G504600.2,TraesCS2B02G504800.1 and TraesCS2B02G504900.1 could be found in 2G,and they were named as TiSNF2-G,TiUbox-G and TiNLR-G,respectively.qRT-PCR were used to analyze the expression patterns of candidate genes at two-and four-leaf stage in IGV1-465 after Bgt infection.The results indicated that at two-leaf stage of IGV1-465,TiSNF2-G and TiUbox-G were both up-regulated when challenged with Bgt infection,and peaked at 24 hours after inoculation（hai）,while TiNLR-G was not induced at this stage.At four-leaf stage,TiSNF2G and TiUbox-G were both up-regulated in IGV 1-465 when challenged with Bgt,and peaked at 8 hai,and TiNLR-G was also up-regulated but peaked at 24 hai.Because of TiNLR-G encoding a NLR type resistance protein and could be induciable expression after Bgt inoculation,we select TiNLR-G as the candidate gene of Pm6 for further study.2.Cloning and functional analysis of TiNLR-G（1）Gene cloning of TiNLR-GBoth genomic DNA and cDNA of TiNLR-G were cloned from IGV1-465.TiNLR-G has no intron,and the length of cDNA is 2,715 bp which encodes a protein having 904 amino acids.TiNLR-G is a typical NLR gene,containing the CC,NBS and LRR domains.Phylogenetic analysis showed TiNLR-G was most closely related to the homolog in 2B.Comparing with its homologs from other Triticeae species except durum wheat,TiNLR-G has two amino acids insertion within the LRR domain（535-536aa）.（2）Functional analysis of TiNLR-G in wheat powdery mildew resistanceSingle cell transient over-expression assay（TOA）was used by transiently overexpressing TiNLR-G in a powdery mildew susceptible variety Yangmai 158.The overexpression of the target gene significantly decreased the haustorium index（31.11%）of inoculated Bgt isolate E26 in epidermal cells of Yangmai 158,compared with the control（56.77%）.The virus induced gene silencing（VIGS）using barley stripe mosaic virus showed,when silencing the TiNLR-G gene in four-leaf stage plants of IGV1-465,the susceptibility of IGV 1-465 to wheat powdery mildew was obviously increased.Above results indicate that TiNLR-G plays a positive role in wheat resistance to powdery mildew.Powdery mildew susceptible mutants were produced by EMS treatment of IGV 1-465.TiNLR-G was cloned from nine independent susceptible mutants,the gene in one mutant had a G>A SNP variation in its LRR domain.This variation resulted in the amino acid change from Ser to Asn.The 3-D structure prediction showed that this change lead to the conformational change of TiNLR-G.The TiNLR-G expression could not be induced in three independent mutants when infected by Bgt,hinting the important role of this gene in powdery mildew resistance.The over-expression vectors of both wild type TiNLR-G and mutant gene TiNLR-GMu were constructed,respectively.The vectors were transformed into a powdery mildew susceptible wheat variety Fielder by Agro-bacterium mediated transgenic approach.A total of 17 positive transgenic plants of wild type TiNLR-G were obtained.The TiNLR-G expression was increased by 50-375 folds in transgenic plants,compared with that in the receptor Fielder.However,such a high level TiNLR-G expression caused severe retardment of plant growth and development,and all transgenic plant dead.A total of four transgenic plants of TiNLR-GMu was obtained,and the expression level were increased by 18-172 folds compared with that in Fielder.All TiNLR-GMu transgenic plants grew normally,but all were powdery mildew susceptible.This indicated the expression of TiNLR-G affects plant growth as well.We further cloned the promotor of TiNLR-G,pTiNLR-G.The length ofpTiNLR-G is 2.1 kb and contained 14 predicted cis-elements.pTiNLR-G has promoter activity by single cell transient expression assay.The TiNLR-G was transformed into Yangmai 158 using a vector driven by pTiNLR-G through biolistic approach.A total of 512 plants were obtained.Four were positive transgenic plants（T0-96,T0-176,T0-181 and T0-198）,which had improved powdery mildew resistance（Infection type being IT2）at adult stage in the field than the receptor Yangmai158（Infection type being IT6）.These indicated the positive role of TiNLRG in wheat resistance to powdery mildew.In order to investigate whether epigenetic plays role in the developmental stagedependent up-regulation of TiNLR-G,ChIP-QPCR were performed.In response to Bgt infection,the histone H3K4me3 methylation modification was enriched in the promoter,5’UTR and CDS regions.This modification was most enriched in the promotor region,indicating its association with the regulation of TiNLR-G expression.