QTL Mapping For Adult-plant Resistance To Stripe Rust In Common Wheat And Candidate Gene Analysis Of Yr26 Based On BSR-Seq And SNP Array

Global wheat production is constantly attacked by various pathogens,and stripe rust caused by Puccinia striiformis f.sp.tritici（Pst）is one of the most devastating diseases.The most effective control strategy is breeding resistant varieties,as it has no additional cost to farmers and is environmentally desirable.Stripe rust resistance is usually categorised as all-stage resistance（ASR）and adult-plant resistance（APR）.ASR is more vulnerable to pathogen evolution when deployed singly in cultivars.In contrast,APR usually provides protection against a broader range of races and tends to be more durable.Therefore,combining APR genes with race specific ASR genes is a preferred strategy for wheat breeding as it may prolong the life of the ASR genes and will significantly reduce losses if virulent races do develop.This highlights the significance of identifying and cloning resistance genes.Molecular mapping has been massively used for identifcation of R genes and it can provide the starting point for gene cloning and marker-assisted selection（MAS）in wheat breeding.In addition,quantitative trait loci（QTL）mapping is the currently preferred method of dissecting the genetic components of disease resistance.However,the conventional markers are sometimes ineffective for mapping genes in chromosomal regions with low gene density or poor recombination.More recently,the rapid development of next-generation sequencing（NGS）technologies have ignited a revolution in the amount of data available for gene identification projects.NGS such as bulked segregant analysis RNA-Seq（BSR-Seq）enables the efficiency and throughput discovery of DNA variants in Triticum and single nucleotide polymorphisms（SNPs）are currently the more appropriate choice for genetic analysis based on sequencing.Current SNP assay platforms including Illumina Bead ChipTM,Affymetrix Gene ChipTMand Kompetitive allele-specific PCR（KASPTM）have been massively used in mapping and MAS studies.Common wheat line 92R137 with Yr26 has been widely used in wheat breeding programs in China.Although Yr26-virulent races have emerged in recent years,Yr26 is still effective against currently predominant races of Pst in China.Gene pyramiding is a possible way to prolong the useful lifetime of R genes in agriculture.Cloning Yr26 can contribute to the understanding of molecular mechanisms of stripe rust resistance and provide basis forwisely using the gene and other genes to achieve long lasting and high level resistances to stripe rust.However,the gene was located in near the centromere region causing that the actual physical distance is more than 10 Mb and the existing markers are far from enough to screen BACs for construction physical map.Hence,additional efficient markers need for further fine mapping and map-based cloning of Yr26.In addition,we screened a diverse panel of over 1,000 wheat germplasms for resistance to stripe rust in field nurseries and identified a number of common wheat genotypes with resistance to prevalent Chinese Pst races.Wheat cultivars or lines Napo 63,03031-1-5 H62,P10057 and Friedrichswerther showed typic adult plant resistance to stripe rust.The objectives of the study were to: fine map and eventually clone Yr26 by BSR-Seq and SNP arrays;identify the major QTL for APR to stripe rust in common wheat Napo 63,03031-1-5 H62,P10057 and Friedrichswerther using SNP arrays following BSA;identify the candidate resistance genes;develop and verify the applicability of KASP markers to enable marker-assisted selection in wheat breeding programs.The follow main results were obtained:1.Saturation mapping of stripe rust resistance gene Yr26 and associated candidate genes analysis via a combination of next-generation sequencing and bulked segregant analysis in hexaploid wheat1)The RNA resistant bulk（R-bulk）and susceptible bulk（S-bulk）were constructed for BSR-Seq in the population of 273 F8 recombinant inbred lines（RILs）derived from the cross Yangmai5/92R137.In addition,three pairs super DNA “R-bulk vs S-bulk” were performed from three populations,i.e.Yangmai5/92R137,Avocet S/92R137 and NIL-S/NIL-R for 90 K and 660 K SNP arrays analysis.A total of 530,252 and 1745 SNPs associated with Yr26 were discovered by BSR-Seq,90 K and 660 K SNP arrays,respectively.2)The sequences of all SNP were used to blast version 1.0 or 0.4 of the assembled Chinese Spring survey sequence to determine their physical positions.Based on the density of SNPs on 1B,most of SNPs were located within the interval 300-350 Mb and the SNPs in this region were selected for conversion to KASP markers.After analyzing polymorphism between parents and bulks,they were used to genotype the entire population for fine mapping Yr26.Finally the Yr26 locus was narrowed down to a 0.03 cM interval flanked by KASP markers WRS303 and WRS351.3)The target region contained a receptor-like kinase gene and partial Sr35 that are possibly involved in plant disease resistance and therefore can be regarded as a potential candidate gene for Yr26.4)Combining BSA with NGS enables the identification of genetic markers for breedingand map-based cloning.KASP genotyping technology provides a high throughput platform at low cost.We identified WRS467 and CM1135 as co-segregation markers suitable for selection of Yr26 when they were used together.2.Development of Yr26 mutant population and identifiation of susceptible mutantsTo identify yr26 susceptible mutants for candidate gene analysis,three wheat EMS induced mutant population were developed using wheat line 92R137 and near-isogenic line NBC6-23,i.e.,2150 M6 lines developed from 92R137,2754 M2 individuals from 92R137 and2697 M2 individuals from NBC6-23.All the mutants were tested with Pst CYR32 in seedling and adult plants and 9 lines derived from 9 individual M1 plants displayed susceptibility.The authenticity of susceptible mutants were validated by SSR markers and 660 K SNP array.3.QTL mapping of adult plant resistence to stripe rust in common wheat1)The population of 224 F2:3 lines and 175 F2:6 recombinant inbred lines（RILs）derived from a cross between Napo 63 and the Pst-susceptible line Avocet S were tested with race CYR32 in Yangling,natural inoculation in Tianshui and Jiangyou.Combined BSA and 90 K SNP arrays placed 275 of 511 polymorphic SNPs on chromosome 2B.Sixty four KASP markers selected from the 275 SNPs and 76 SSR markers on 2B were used to identify a chromosome region associated with rust response.To satuate the region,14 KASP markers more closely linked to the locus were developed following a 660 K SNP array analysis.Finally,a major effect QTL,named Qyrnap.nwafu-2BS,was identified by inclusive composite interval mapping（ICIM）and was mapped to a 0.9 cM interval flanked by KASP markers 660K-AN21 and 660K-AN57 in bin region 2BS-1-0.53.The resistance of Napo 63 was stable across all environments,and as a QTL,explained an average 66.1% and 55.7% of the phenotypic variance in F2:3 lines and in F5:6 RILs,respectively.Candidate gene analysis indicated that gene Traes₂BS₂B483208E is possibly involved in plant disease resistance and therefore can be regarded as a potential candidate gene for Qyrnap.nwafu-2BS.The short genetic interval and ?anking KASP markers developed in the study will facilitate marker-assisted selection,gene pyramiding,and eventual positional cloning of Qyrnap.nwafu-2BS.2)In the mapping population of 167 F2:3 lines developed from the AvS/03031-1-5 H62 cross,the segregation ratios indicated that the resistance was conferred by a single dominant gene,designated as YrH62.We conducted a combination of BSA with wheat 90 K SNP array to identify molecular markers linked to YrH62.And the linkage map was constructed with 15 KASP and 3 SSR markers,and the resistance gene was mapped to a 1.0 cM interval flanked by KASP markers AX-109352427 and AX-109862469 in the centromere-vicinity region ofchromosome 1BS.YrH62 explained 63.8% and 69.3 % of the phenotypic variation in MDS and IT,respectively.Based on marker genotypes,the resistance origins,seedling and adult plant responses to the tested Pst races,and the comparison of relative distance,YrH62 is different from the other reported genes/QTL on 1B and should be a novel adult plant resistance gene.The gene and low cost KASP assays developed as flanking markers will be useful for wheat breeding.3)We evaluated 158 F2:3 lines and 150 F2:6 RILs derived from a cross between Friedrichswerther and the Pst-susceptible landrace Mingxian169.Illumina 90 K iSelect SNP arrays were used to sequence the bulk extreme pools and biparent and 286 of 1135 polymorphic SNPs were identified on chromosome 6B.Sixty two KASP markers selected from the 286 SNPs were used to verify a chromosome region associated with the resistance locus.A linkage map was constructed with 15 KASP-SNP markers and a major effect QTL was identified usingICIM within a 3.5 cM interval flanked by KASP markers IWA1839 and IWB60085 in the region 6BL-3-0.36.The QTL,named Qyrfri.nwafu-6BL,was stable across all environments,and explained an average 53.5 % and 31.2% of the stripe rust severity variation in F2:3 lines and F5:6 RILs.Compared with stripe rust resistance genes previously mapped to chromosome 6B revealed that Qyrfri.nwafu-6BL is likely a new APR QTL for resistance to stripe rust.4)The CIMMYT wheat cultivar P10057 displayed a high level of APR to stripe rust in germplasm evaluation in field environments.To clarify the genetic basis and identify quantitative trait loci（QTL）involved in stripe rust resistance in P10057.Three wheat populations were used,namely,154 F2:6 RILs derived from the cross Mingxian 169 × P10057,and 161 and 140 F2:3 lines from AvS × P10057 and Zhengmai 9023 × P10057,respectively.Genotyping was performed with KASP and SSR markers linked to the resistance loci.Using QTL analysis two genomic regions associated with resistance were found on chromosomes2 BS and 3BS,respectively.These two stable QTL,designated Qyrlov.nwafu-2BS and Qyrlov.nwafu-3BS,were detected across all environments and explained 22.6–24.6% and20.6–43.2% of phenotypic variance,respectively.Qyrlov.nwafu-2BS may be the resistance allele derived from CIMMYT germplasm and Qyrlov.nwafu-3BS likely corresponds to the locus Sr2/Lr27/Yr30/Pbc.The KASP markers 90K-2BMP19 and 90K-2BMP35 linked to QYrlov.nwafu-2BS and 90K-3BSMP4 and 90K-3BSMP7 linked to Qyrlov.nwafu-3BS were reliable for marker-assisted selection（MAS）in the Zhengmai 9023 × P10057 population.These QTL with KASP markers are expected to contribute to improving stripe rust resistance in developing wheat cultivars.In conclusion,validating the SNP array with KASP-SNP assays can lower sequencing costs,and more importantly,it can rapidly isolate a major effect gene/QTL in hexaploid wheat and improve the accuracy of mapping results in the QTL region.