Identification of powdery mildew resistance loci in wheat by integrating genome-wide association study(GWAS) and linkage mapping

Wheat powdery mildew(Blumeria graminis f.sp.tritici, Bgt) is a disease of increasing importance globally due to the adoption of high yielding varieties and modern sustainable farming technologies.Growing resistant cultivars is a preferred approach to managing this disease, and novel powdery mildew resistance genes are urgently needed for new cultivar development.A genome-wide association study was performed on a panel of 1292 wheat landraces and historical cultivars using 5011 single nucleotide polymorphism(SNP)markers.The association panel was evaluated for reactions to three Bgt inoculants, OKS(14)-B-3-1, OKS(14)-C-2-1, and Bgt15.Linkage disequilibrum(LD) analysis indicated that genome-wide LD decayed to 0.1 at 23 Mb, and population structure analysis revealed seven subgroups in the panel.Association analysis using a mixed linear model(MLM) identified three loci for powdery mildew resistance on chromosome 2 B, designated QPm.stars-2BL1,QPm.stars-2BL2, and QPm.stars-2BL3.To evaluate the efficacy of GWAS in gene discovery,QPm.stars-2BL2 was validated using F2 and F2:3 populations derived from PI420646 × OK1059060-126135-3.Linkage analysis delimited the powdery mildew resistance gene in PI 420646 to an interval where QPm.stars-2BL2 was located, lending credence to the GWAS results.QPm.stars-2BL1 and QPm.stars-2BL3, which were associated with four SNPs located at 457.7–461.7 Mb and two SNPs located at 696.6–715.9 Mb in the Chinese Spring reference IWGSC RefSeq v1.0, respectively, are likely novel loci for powdery mildew resistance and can be used in wheat breeding to improve powdery mildew resistance.

The Relationship of Methyl Jasmonate Enhanced Powdery Mildew Resistance in Wheat and the Expressions of 9 Disease Resistance Related Genes

[Objective] This study was carried out to determine the induction effect of jasmonic acid（JA）on powdery mildew resistance in wheat,the activation effect on the expressions of plant disease resistance related genes,and to investigate the relationship between the induced resistance and the gene expression patterns.[Method] Three powdery mildew susceptible cultivars of ＂Chinese Spring＂,＂Pumai 9＂ and ＂Zhoumai 18＂ typically representing different phenotypes in the field were employed.The powdery mildew was assessed by detached leaf assay,and real time quantitative RT-PCR was used to determine the expression patterns of 9 disease resistance related genes of PR1（PR1.1）,PR2（β,1-3 glucanase）,PR3（chitinase）,PR4（wheatwin1）,PR5（thaumatin-like protein）,PR9（TaPERO,peroxidase）,PR10,TaGLP2a（germin-like）and Ta-JA2（jasmonate-induced protein）in leaf of the three cultivars.[Result] MeJA application enhanced the powdery mildew resistances of ＂Chinese Spring＂,＂Pumai 9＂ and ＂Zhoumai 18＂.The induced powdery mildew resistance could be detected from 12 h to 96 h after MeJA treatment,and the peak value was at 24 h.Though there were differences between the three cultivars,MeJA significantly effect on the expressions of the 8 disease resistance related genes except TaGLP2a,and the peak values were at 12 h,24 h or 48 h after treatments.The strongest activation of MeJA was on PR9 and PR1 that their expressions could reach more than 100 times of the untreated samples.MeJA strongly activated PR2、PR4、PR5、PR3、PR10 and Ta-JA2,their expression could reach 10 to 70 times,and there was almost no activation effect on TaGLP2a.The induced powdery mildew resistance positively correlated with the induced expressions of the 8 disease related genes.[Conclusion] The induced powdery mildew resistance positively correlated with the induced expressions of the disease related genes.Jasmonate signalling plays a role in defence against Blumeria graminis f.sp.tritici.and future manipulation of this pathway may improve powdery mildew resistance in wheat.

Identification of Co-Segregating RAPD Marker Linked to Powdery Mildew Resistance Gene Pm18 in Wheat

The Pm18 gene of wheat confers resistance to the powdery mildew which is one of the mostserious diseases in many regions of the world. In this study, bulked segregant analysis(BSA) was used to develop randomly amplified polymorphic DNA (RAPD) markers linked toPm18 gene. Three hundred and twenty decamer primers were screened and one of them wasidentified as RAPD marker (S411600) linked to Pm18. Using the F2 mapping population fromthe cross Pm18Chancellor, the marker S411600 was shown to co-segregate with the genePm18. This marker can be conveniently used for marker-assisted selection in wheatbreeding programs for the identification or pyramiding of Pm18 with other resistancegenes.

Molecular and Physical Mapping of Powdery Mildew Resistance Genes and QTLs in Wheat: A Review

Wheat powdery mildew （Pro） is a major disease of wheat worldwide. During the past years, numerous studies have been published on molecular mapping of Pm resistance gene（s） in wheat. We summarized the relevant findings of 89 major re- sistance gene mapping studies and 25 quantitative trait loci （QTL） mapping studies. Major Pm resistance genes and QTLs were found on all wheat chromosomes, but the Pm resistance genes/QTLs were not randomly distributed on each chromosome of wheat. The summarized data showed that the A or B genome has more major Pm resistance genes than the D genome and chromosomes 1A, 2A, 2B, 5B, 5D, 6B, 7A and 7B harbor more major Pm resistance genes than the other chromosomes. For adult plant resistance （APR） genes/QTLs, B genome of wheat harbors more APR genes than A and D genomes, and chromo- somes 2A, 4A, 5A, 1B, 2B, 3B, 5B, 6B, 7B, 2D, 5D and 7D harbor more Pm resistance QTLs than the other chromosomes, suggesting that A genome except 1A, 3A and 6A, B genome except 4B, D genome except 1D, 3D, 4D, and 6D play an impor- tant role in wheat combating against powdery mildew. Furthermore, Pm resistance genes are derived from wheat and its rela- tives, which suggested that the resistance sources are diverse and Pm resistance genes are diverse and useful in combating against the powdery mildew isolates. In this review, four APR genes, Pm38/Lr34/Yr18/Sr57, Pm46/Lr67/Yr46/Sr55, Pm？/Lr27/Yr30/ SY2 and Pm39/Lr46/Yr29, are not only resistant to powdery mildew but also effective for rust diseases in the field, indicating that such genes are stable and useful in wheat breeding programmes. The summarized data also provide chromosome locations or linked markers for Pm resistance genes/QTLs. Markers linked to these genes can also be utilized to pyramid diverse Pm resis- tance genes/QTLs more efficiently by marker-assisted selection.

IDENTIFICATION OF RAPD MARKER LINKED TO POWDERY MILDEW RESISTANCE GENE Pm12 IN WHEAT

Powdery mildew is one of the most serious diseases of wheat in China. In this paper,bulked segregant analysis (BSA) was used to search for randomly amplified polymorphic DNA (RAPD) markers linked to the Pm12 gene,which confers resistance to the powdery mildew in wheat. 200 decamer primers were screened and one RAPD marker (S107 1900 ) was identified to be linked to Pm12 in coupling phase,and their genetic distance is 11.98± 4.00cM. This marker can be used for marker-assisted selection in wheat breeding for the identification or pyramiding of Pm12 with other resistance genes.

Molecular Detection of Resistance Genes to Stripe Rust and Powdery Mildew in Common Wheat Cultivar Yunmai52

Yunmai52, developed by crossing with common wheat-Haynaldia villosa6AL/6VS translocation line 92R149 as a resistant parent in 1992, was a common wheat cultivar approved and released in 2007 in Yunnan Province, China, which is characterized by high resistance to powdery mildew and stripe rust. In this study,an F_2 population derived from a cross K78S/Yunmai52 was constructed to investigate the resistance genes, where K78 S is a wheat male sterile line susceptible to powdery mildew and stripe rust. Phenotypic identification of the parents, F_1 and F_2 populations and chi-square analyses showed that F_1 population was immune to stripe rust and powdery mildew; the segregation ratio of resistance and susceptibility to powdery mildew（χ~2=1.10χ~2_（1,0.05）=3.84） and stripe rust（χ~2=0.15χ~2_（1,0.05）=3.84） fit to a 3：1 ratio in F_2 population, indicating that Yunmai52 harbors a dominant stripe rust resistance gene and a dominant powdery mildew resistance gene. The individuals were further detected with a marker co-segregated with Pm21（SCAR_（1400）） and two markers closely linked with Yr26（XWe173 and Xbarc181）. The results showed that polymorphic bands could be amplified between the parents and between resistance and susceptibility gene pools at the same locus. Randomly 96 individuals of F_2 population were selected for verification. The results showed that the phenotype was significantly correlated with the genotype. The detection accuracy of markers SCAR_（1400）, XWe173 and Xbarc181 was 100%, 97.91% and 92.70%, respectively.Yunmai52 harbored powdery mildew resistance gene Pm21 and stripe rust resistance gene Yr26, which were both derived from 6AL/6VS translocation line 92R149.In addition, the results also demonstrate that Pm21 and Yr26 are two genes conferring durable resistance to powdery mildew and stripe rust in wheat.

The Potential Role of Powdery Mildew-Resistance Gene Pm40 in Chinese Wheat-Breeding Programs in the Post-Pro21 Era

Powdery mildew, which is caused by Blumeria graminis f. sp. tritici （Bgt）, is an important leaf disease that affects wheat yield. Powdery mildew-resistance （Pro） gene Pro21 was first transferred into wheat in the 1980s, by translocating the Heuchera villosa chromosome arm 6VS to the wheat chromosome arm 6AL （6VS.6AL）. Recently, new Bgt isolates that are virulent to Pm21 have been identified in some wheat fields, indicating that wheat breeders should be aware of the risk of deploying Pm21, although pathological details regarding these virulent isolates still remain to be discovered. Pm40 was identified and mapped on the wheat chromosome arm 7BS from several wheat lines developed from the progenies of a wild cross between wheat and Thinopyrum intermedium. Pm40 offers a broad spectrum of resistance to Bgt, which suggests that it is likely to provide potentially durable resistance. Cytological methods did not detect any large alien chromosomal segment in the wheat lines carrying Pm40. Lines with Pm40 and promising agronomical traits have been released by several wheat-breeding programs in the past several years. Therefore, we believe that Pm40 will play a role in powdery mildew-resistance wheat breeding after Pm21 resistance is overcome by Bgt isolates. In addition, both Prn21 and Pm40 were derived from alien species, suggesting that the resistance genes derived from alien species are potentially more durable or effective than those identified from wheat.

Resistance Analysis of Wheat Cultivars against Powdery Mildew in Anhui Province of China

[ Objective ] The paper was to determine the resistance level of the tried and pre-examination wheat cultivars against powdery mildew in Anhui Province of China. [ Method ] By using artificial inoculation and identification method in fields, the resistance of wheat cultivars was identified in consecutive three years from 2010 to 2012. [ Result] The highly susceptible （HS） cultivar accounted for 30%, 42% and 11% of total tested cultivars in the years of 2010, 2011 and 2012, respectively; moderately susceptible （MS） cultivar accounted for 53% of total tested cuhivars in 2010, which accounted for 47% and 57% in 2011 and 2012, respectively; moderately resistant （MR） cuhivar accounted for 17% of total tested cultivars in 2010, which accounted for 11% and 32% in 2011 and 2012, respectively. [ Conclusion] The paper can guide breeding direction, and also provide scientific basis for variety approval.

Identification of Resistance of Powdery Mildew and Stripe Rust of Winter Wheat Strains in Xinjiang

Wheat powdery mildew and stripe rust are the major diseases in wheat producing area in Xinjiang.To obtain wheat germplasm resources and varieties resistant to powdery mildew and rust,36 high-generation stable strains of Xinjiang winter wheat were evaluated using the method of natural inducement from 2018 to 2020.A total of 5 strains with high resistance to powdery mildew,4 strains with slow stripe rust and 1 strain with resistance to powdery mildew and adult plant slow stripe rust were obtained.And the parental combination of disease-resistant varieties was analyzed.These studies will provide theoretical basis for the breeding of resistant wheat varieties in Xinjiang.

Characterization of RAPD Markers,and the RFLP Marker Linked to Powdery Mildew Resistance Gene Derived from Different Accessions of H.villosa

The analysis was carried out on performance of the resistance gene from Haynaldia villosa accession of the former Soviet Union to different isolates of Bluemerie graminis. Polymorphisms were revealed between 6D/6V substitution line Pm930640 and its pedigree parents using five RAPD markers of OPAN031700, OPAI01700, OPAL03750, OPAD07480 and OPAG15580 screened out from 120 random 10-mers primers. Three RAPD markers of OPAN03, OPAI01 and OPAL03 were linked with the resistance gene by analysis of F2 population of Chancellor×Pm930640. Analysis of 29 wheat lines including part of lines conferring the known genes from Pm1 to Pm20 respectively, lines conferring resistance gene from two H. villosa accessions and the related wheat parents, were analyzed and the results showed that these markers not only linked to the gene resistant to powdery mildew from H. villosa, but also detected different genetic backgrounds. OPAL03750 can be used as the marker to distinguish the different resistant lines from two H. villosa accessions because it was only observed in the materials from H. villosa of the former Soviet Union. RFLP analysis also showed the polymorphisms between two H. villosa accessions and their derived resistant lines.

RFLP Tagging of a Gene Pm12 for Powdery Mildew Resistance in Wheat (Triticum aestivum L. )

A dominant gene, Pml2, conferring the resistance to powdery mildew (Erysiphe graminis) has been transferred to wheat from Aegilops speltoides. Sixteen RFLP probes were used to test Line 31 and its parents, showing that Line 31 is a 6B/6S translocation lines. The linkage analysis by five probes indicates that Pm12 is located on the chromosome 6B/6S, and tightly flanked by an RFLP marker α-Amy-1 (1.1 cM), identified by RFLP and isozyme analysis. This approach is equally applicable to the identification of other transferred alien genes and the segments of alien chromosome introgressed into wheat.

Gene expression profiling related to powdery mildew resistance in wheat with the method of suppression subtractive hybridization

'Bainong 3217 × Mardler' BC5F4 wheat line at the initial stage of inoculation with powdery mildew pathogen (Erysiphe graminis DC) was used to construct a suppression subtractive hybridization (SSH) cDNA library. Totally 760 ESTs were obtained through sequencing. Similarity analysis of ESTs based on BLASTn and BLASTx with the sequences in GenBank, in combination with macroarray differential screening, revealed that 199 ESTs of 65 kinds were known to be functionally disease resistance related. Based on the gene expression profiling in the present study, it is postulated that salicylic acid (SA) and MAP-related signal transduction pathways were involved in powdery mildew resistance in wheat. System acquired resistance genes were predominant in terms of kinds and quantity. With the initiation of cell defense reaction, the genes conferring anti-oxidation substances were largely expressed and thus cell protection mechanism was activated. Much evidence revealed that phenylpropanes metabolic pathway was

Genome Analysis in Wheat Breeding for Disease Resistance

A brief review on the development of wheat germplasm with introduced powdery mildew and scab resistance from Haynaldia villosa Sch. and Leymus racemosus Lam., Roegneria ciliaris (Trin.) Nevski as well as R. kamoji C. Koch respectively was made. In the course of germplasm development, genome analysis by means of chromosome banding, genomic in situ hybridization (GISH) or fluorescence in situ hybridization (FISH), molecular markers, particularly restriction fragment length polymorphism (RFLP) coupled with aneuploid analysis was employed for the purpose of improving breeding efficiency. Potential use of such germplasm in wheat breeding practice, basic studies and some related problems were also discussed.

Characterization of Three Novel Wheat LThinopyrum intermedium Addition Lines with Novel Storage Protein Subunits and Resistance to Both Powdery Mildew and Stripe Rust

Wide hybridization is an effective approach for enhancing the resistance of bread wheat （Triticum aestivum L.） to biotic and abiotic stresses by introducing favorable alien genes （Sepsi et al., 2008）. Wheatgrass, Thinopyrum intermedium （Host） Barkworth ＆ D.R. Dewey or Agropyron intermedium （Host） Beauvoir （2n = 42; genome formula JJjSjSstst）, is a perennial species in the tribe Triticeae and an important source of wheat improvement for biotic and abiotic stress resistance and quality-related traits, such as high grain protein concentration （Chen et al., 1998; 2001; 2003; Han et al., 2004; Li and Wang, 2009）. In addition, the ready crossing ability of wheatgrass with various Triticum species has made it popular in germ- plasm development.

Introgression of Resistance to Powdery Mildew Conferred by Chromosome 2R by Crossing Wheat Nullisomic 2D with Rye

Using the nulUsomic back-cross procedure, four wheat-rye chromosome substitution 2R （2D） lines with different agronomic performance, designated WR02-145-1, WR01-145-2, WR02-145-3, and WR02-145-4, were produced from a cross between 2D nullisomic wheat （Triticum aestivum L. cv. ＂Xiaoyan 6＂） and rye （Secale cereale L. cv. ＂German White＂）. The chromosomal constitution of 2n=42=21 in WR02-145 lines was confirmed by cytological and molecular cytogenetic methods. Using genomic in situ hybridization on root tip chromosome preparations, a pair of intact rye chromosomes was detected in the WR02-145 lines. PCR using chromosome-specific primers confirmed the presence of 2R chromosomes of rye in these wheat-rye lines, indicating that WR02o145 lines are disomic chromosome substitution lines 2R （2D）. The WR02-145 lines are resistant to the powdery mildew （Erysiphe graminis DC. f. sp. tritici E. Marchal） isolates prevalent in northern China and may possess gene（s） for resistance to powdery mildew, which differ from the previously identified Pm7gene located on chromosome 2RL. The newly developed ＂Xiaoyan 6＂- ＂German White＂ 2R （2D） chromosome substitution lines are genetically stable, show desirable agronomic traits, and are expected to be useful in wheat improvement.

Transcriptome Comparison of Susceptible and Resistant Wheat in Response to Powdery Mildew Infection

Powdery mildew （Pro） caused by the infection of Blumeria graminis f. sp. tritici （Bgt） is a worldwide crop disease resulting in significant loss of wheat yield. To profile the genes and pathways responding to the Bgt infection, here, using Affymetrix wheat microarrays, we compared the leaf transcriptomes before and after Bgt inoculation in two wheat genotypes, a Pm-susceptible cultivar Jingdong 8 （S） and its near-isogenic line （R） carrying a single Pm resistant gene Pm30. Our analysis showed that the original gene expression status in the S and R genotypes of wheat was almost identical before Bgt inoculation, since only 60 genes exhibited differential expression by P = 0.01 cutoff. However, 12 h after Bgt inoculation, 3014 and 2800 genes in the S and R genotype, respectively, responded to infec- tion. A wide range of pathways were involved, including cell wall fortification, flavonoid biosynthesis and metabolic processes. Further- more, for the first time, we show that sense-antisense pair genes might be participants in wheat-powdery mildew interaction. In addition, the results of qRT-PCR analysis on several candidate genes were consistent with the microarray data in their expression patterns. In summary, this study reveals leaf transcriptome changes before and after powdery mildew infection in wheat near-isogenic lines, suggest- ing that powdery mildew resistance is a highly complex systematic response involving a large amount of gene regulation.

分子标记辅助育种新尝试——与P_(m2)及P_(m4a)基因紧密连锁RFLP标记在小麦抗白粉病育种中的应用

以当地育成的小麦抗白粉病材料，对国外筛选到的与白粉病抗性基因Ｐｍ２及Ｐｍ４ａ紧密连锁的ＲＦＬＰ标记进行了分析。结果表明，这些ＲＦＬＰ标记即使在遗传背景不同的情况下仍可用于对Ｐｍ２及Ｐｍ４ａ基因的检测及鉴定。研究还表明，利用分子标记辅助小麦抗白粉病育种不仅可行，而且还可对育成的抗病品系所携具体抗性基因进行精确鉴定。

簇毛麦抗白粉病基因的RAPD及RFLP标记

分析了来自前苏联簇毛麦及其抗病衍生系的抗白粉病基因对不同生理小种的抗性反应。用 120 个随机引物对 6D/6V 代换系 Pm930640 进行 RAPD 分析,检测到 5 个引物 OPAN03、OPAI01、OPAL03、OPAD07 和 OPAG15,分别在大约 1 700、700、750、480 和 580 bp 处有区别于小麦亲本的多态性条带。对 Chancellor×Pm930640 F2群体进行 OPAN03、OPAI01 和 OPAL03 等 3 个 RAPD 标记与抗白粉病基因的连锁分析,表明这些标记同簇毛麦的抗白粉病基因是连锁的。对大部分分别含有 Pm1-Pm20 的已知抗病基因、含有簇毛麦抗病基因及其相关亲本的 29 个小麦品系进行RAPD标记分析。结果表明,这些标记不仅可以鉴定簇毛麦的抗病基因,而且可以判断其遗传背景。OPAL03750仅出现在含有前苏联簇毛麦 6VS 染色体的抗病材料中,可作为区别于 Pm21 的分子标记。RFLP 标记的结果也表明两个不同簇毛麦的 6VS 染色体有明显的多态性。

腥掷孢酵母17wy1的分离、鉴定及对小麦白粉病防效初探

采用26S rDNA序列对分离自异常生长小麦白粉菌分生孢子上的菌株17wy1进行鉴定,明确其分类地位;通过孢子萌发抑制试验、室内盆栽和田间小麦白粉病防治试验测定17wy1发酵液对小麦白粉菌分生孢子萌发的影响及对小麦白粉病的生防效果,并通过定量PCR技术分析小麦病程相关蛋白基因PR1、PR2、PR5、TaPAL的表达模式,解析17wy1的拮抗机制,为小麦白粉病的生物防治提供依据。结果表明,菌株17wy1为腥掷孢酵母(Golubevia albescens),其发酵液(5×107个/mL)对小麦白粉菌分生孢子萌发抑制率为85.1%,对室内盆栽麦苗和田间小麦白粉病的防效分别为58.5%和69.8%;喷施酵母17wy1发酵液后,室内显症麦苗体内的病程相关蛋白基因PR1、PR5被诱导显著上调表达。可见,酵母菌17wy1可抑制小麦白粉菌分生孢子萌发,对室内盆栽麦苗和田间小麦白粉病具有较好的防治作用,是小麦白粉病的潜在生防菌。

小麦四个抗白粉病基因的KASP标记检测

为提高Pm13的检测效率,了解Pm21、PmV、Pm12和Pm13共4个小麦抗白粉病基因在山东小麦育种中的利用情况,本研究通过测序和引物设计成功转化了Pm13的KASP标记,并采用4个基因的KASP标记对2022—2023年山东省小麦高产区试组和强筋区试组的140份参试品系和2份区试对照品种进行检测,结果显示140份材料均不含上述4个基因,表明它们在山东小麦育种中应用较少。本研究可为利用KASP标记开展小麦抗白粉病基因的分子标记辅助选择提供参考,促进其育种应用。