| Wheat powdery mildew is a fungal disease caused by the fungus Blumeria graminis f.sp.tritici(Bgt).In recent years,it has become increasingly serious in China,causing great economic losses.Breeding excellent resistant varieties is the most economical and effective way to control powdery mildew.Our laboratory found that Ae.longissima Schweinf.&Muschl.(2n=2x=14,Sl Sl)carried a powdery mildew resistance gene Pm13 a on chromosome 3Sl#2,and introduced the gene into common wheat by inducing Chinese Spring-Ae.longissima translocation lines.On this basis,through the combination of molecular marker development,development of small fragment translocation lines,transcriptome sequencing,and other technologies,we conducted fine mapping,candidate gene discovery,and gene function validation of Pm13 a,and a preliminary analysis of the function of the Pm13 a gene.At the same time,through allelic testing,resistance spectrum analysis,and gene sequence analysis,it was determined that Pm13 a and Pm13 belong to the same gene.The main research results are as follows:1.Creation of resistance loss mutants: 15 susceptible Pm13 mutants of M2 strain were screened using EMS mutagenesis method,and then authenticity identification was conducted using molecular markers and chromosome in situ hybridization technology.Finally,11 authentic resistance loss mutants were selected.2.3Sl#2 specific molecular marker development: 26 specific molecular markers located in different segments of the 3Sl#2 chromosome of Ae.longissima were developed based on the transcriptome sequencing of the 3Sl#2/3B disomic substitution line TA3575;Fourteen molecular markers closely linked to Pm13 were developed using the reference genome sequence of the short arm 0-10 Mb region of the 3Sl chromosome of Ae.longissima.A total of 40 specific molecular markers were obtained.3.Pm13 a gene mapping: A 3Sl#2 F2 segregation population with a homozygous genotype background of homoeologous recombination high-frequency inducing gene(ph1b)was constructed.Combining disease resistance identification and molecular marker analysis,26 different types of recombinants were obtained.Pm13 a was located within the range of less than 3.67 Mb between the molecular markers CL10208 and CL56281 at the short arm end of 3Sl#2.Then,a secondary segregation population was constructed by self-pollination of the disease-resistant recombinants with small fragment of 3Sl,and 20 types of recombinant plants were obtained again.Pm13 a was further localized in the physical region of about 124.43 kb between the molecular markers 3Sl-44661 and 3Sl-31 B.4.Pm13 a gene mapping and cloning: Based on the Pm13 a gene mapping results,combined with transcriptome sequencing and the reference genome sequence of Ae.longissima,a total of 6 candidate genes were mined.Among them,there are three resistance genes,namely NLR gene,CRK gene,and MLKL-ATPK gene.By analyzing the expression and sequence of these genes,it was confirmed that the MLKL-ATPK gene(tentatively named Ael MLKL)was the best candidate gene for Pm13 a.After functional validation such as full length gene sequencing of the resistance loss mutants,BSMV-VIGS induced gene silencing,and wheat transgenic expression,it was determined that Ael MLKL is Pm13.The Pm13 gene is 6413 bp long and contains 7 exons,with a coding sequence(CDS)of 1431 bp.After protein sequence prediction,the gene encodes a 476 amino acid protein that contains an N-terminal MLKL-NTD domain and a functional kinase(STPK)domain.This disease resistance protein encoded by Pm13 has not been reported in any other plant disease resistance genes.5.Homology analysis of Pm13: Resistance spectrum analysis,gene allele analysis,and g DNA sequence analysis were performed on Pm13 a,Pm13b,and Pm13 from different sources of Aegilops longissima,confirming that Pm13,Pm13 a,and Pm13 b are the same gene.6.Preliminary analysis of the function of Pm13 gene: Take 3Sl#2/3B disomic substitution line TA3575 at different stages before and after inoculation,and measure the expression of Pm13 gene in different tissues through real-time fluorescence quantitative analysis.It was found that the expression of Pm13 gene increased after inoculation,reached the highest level at 18 hours,and then decreased slowly;Pm13 is expressed in all tissues,but the expression in leaves is much higher than that in other parts.In addition,after inoculation with wheat powdery mildew,the expression of all PR genes in TA3575 leaves was significantly higher than that in CS,indicating that Pm13 can trigger the defense pathway against powdery mildew;The green fluorescent protein gene(GFP)expression vector was constructed using the Pm13 coding sequence(CDS)and transferred into wheat protoplasts for subcellular localization.It was found that Pm13 was expressed in both the nuclear and endometrial systems.7.Pm13 functional marker development and development of resistant recombinants with tiny 3Sl fragments: Two functional markers,Ael MLKL-1 and Ael MLKL-8,have been developed based on the Pm13 gene sequence.These two markers can be used as diagnostic functional markers for molecular marker assisted selection of Pm13.In addition,a small fragment translocation line of Pm13 has been created,in which the 3Sl#2 chromosome fragment of W12-3 is less than 2.8 Mb,accounting for only 0.35% of the total length of the 3Sl chromosome of Ae.longissima,which can effectively reduce the possible linkage drags of Pm13 and has great application value in wheat powdery mildew resistance breeding. |
