Resistance genes in wild accessions of Triticeae--inoculation test and STS marker analyses

Sequence tagged site (STS) markers have been developed recently to identify resistance genes in wheat. A number of wild relatives have been used to transfer resistance genes into wheat cultivars. Accessions of wild species of Triticeae: Aegilops longissima (4), Ae. speltoides (6), Ae. tauschii (8), Ae. umbellulata (3), Ae. ventricosa (3), Triticum spelta (2), T. timopheevi (3), T. boeoticum (4) and T. monococcum (1), 34 in total, were examined using PCR-STS markers for resistance genes against Puccinia recondita f.sp. tritici (Lr) and Erysiphe graminis (Pm). Additionally, a set of cv. Thatcher near-isogenic lines conferring resistance genes Lr 1, Lr 9, Lr 10, Lr 24, Lr 28, Lr 35 and Lr 37 were examined with the same procedure. Twenty-two accessions were tested using the inoculation test for resistance to Erysiphe graminis, Puccinia recondita, P. striiformis and P. graminis. The most resistant entries were those of Aegilops speltoides and Triticum timopheevi and among T. boeoticum accession #5353. Markers of all mentioned Lr resistance genes were identified in all corresponding cv. Thatcher near-isogenic lines (except Lr 35 gene marker). The following resistance gene markers were identified in wild Triticeae accessions: Lr 1 in two accessions of Ae. tauschii and one accession of Ae. umbellulata, Lr 9 in one accession of Ae. umbellulata, Lr 10 in one accession of T. spelta, Lr 28 in 11 accessions: Ae. speltoides (4), Ae. umbellulata (2), T. spelta (2) and T. timopheevi (3), Lr 37 in 3 accessions of Ae. ventricosa, Pm 1 in all 34 accessions, Pm 2 in 28 accessions, Pm 3 in all 4 accessions of T. boeoticum, 1 accession of T. spelta and 1 of T. timopheevi, and Pm 13 in 5 out of 6 accessions of Ae. speltoides. Reliability and usefulness of STS markers is discussed.     

一些小麦白粉病抗源抗性基因鉴定分析

研究鉴定了我国３７份小麦白粉病抗源的抗性基因,１９份材料不具有任何抗性基因;６份材料具有来自１ＢＬ／１ＲＳ易位系的抗性基因Ｐｍ８;５份材料具有抗性基因Ｐｍ５ａ;３份分别具有对目前欧洲所有生理小种均抗的抗性基因Ｐｍ２１、Ｐｍ１６和Ｐｍ１２;４份材料具有新的抗性基因。     

Resistance of spring wheat cultivars and lines to leaf rust

Spring wheat nursery accessions, including 18 spring wheat lines derived in CIMMYT, Mexico, and 12 spring wheat cultivars bred in Poland, along with cultivars Frontana and Sumai 3 as resistant controls, were examined for resistance to leaf rust under field conditions. Multipathotype tests with 16 different pathogen isolates were performed for postulation of Lr genes in Polish cultivars. Besides, STS markers for resistance genes Lr1, Lr9, Lr10, Lr24, Lr28, Lr37 were analysed in the studied cultivars and lines with Thatcher near-isogenic lines as positive controls. All Polish cultivars appeared to be susceptible to leaf rust. Ten of the CIMMYT nursery lines (IPG-SW: #7, 11, 14, 21, 22, 23, 27, 29, 30, 32) and cv. Frontana were resistant in the same environment and can be sources of resistance genes. Marker for the Lr10 gene was identified in 6 accessions (IPG-SW #14, 22, 23, 29, 30, 32) exhibiting resistance to leaf rust, whereas markers for Lr1 and Lr28 genes were observed in all the examined accessions. STS markers for Lr9, Lr24 and Lr37 genes were not identified in the investigated accessions.     

Phytopathological and molecular genetic identification of brown rust resistance genes in common wheat accessions with alien genetic material

Brown rust resistance genes were sought in 23 resistant common wheat accessions with alien genetic material of Aegilops speltoides, Ae. triuncialis, and Triticum kiharae from the Arsenal collection. The genes were identified by common phytopathological tests and PCR analysis with STS markers directed to the known Lr genes. None of the methods identified the resistance genes in two accessions. In the other accessions, the combination of the two methods broadened the spectrum of detectable genes and, in some cases, allowed double verification of the presence of a resistance gene. Most accessions proved to contain several brown rust resistance genes, combining juvenile and adult plant ones. The accessions were found to contain gene combinations that ensured field resistance and immunity under the conditions of the Non-Chernozem region (Lr13 + Lr10 and Lr12 + Lr34). Accessions with alien genetic material contained a unique combination of five or six resistance genes. Since the accessions were rich in brown rust resistance genes, including effective ones, and carried rare combinations of these genes, they were proposed as donors to be universally employed in breeding for immunity in all regions of Russia.     

Resistance of cultivars and breeding lines of spring wheat to Fusarium culmorum and powdery mildew

Twelve Polish spring wheat cultivars and 18 spring wheat accessions from CIMMYT, Mexico, were examined for resistance to a highly pathogenic Fusarium culmorum strain KF846 and powdery mildew in 5-year field experiments. Resistant wheat cultivars (Sumai 3 and Frontana) served as controls. The mean percentage of Fusarium-damaged kernels (% FDK) for 5 years was lower in CIMMYT accessions (16.7%) than in Polish spring cultivars (28.3%). In all Polish spring cultivars, % FDK was higher than in the control cultivars Sumai 3 and Frontana (12-20%). The mean disease score (on a scale of 1-9) for powdery mildew (natural infection) for all examined cultivars and lines ranged from 0 to 7 and in the Polish spring cultivars was significantly lower (0-5). Cultivars Eta, Henika, Ismena, Jasna and Olimpia were found to be the least susceptible to powdery mildew in field experiments. The laboratory host-pathogen tests with Blumeria graminis f. sp. tritici isolates showed that only two cultivars were characterized by identical resistance patterns as the standard differential lines with documented resistance genes. Cultivar Alkora had the gene Pm3d, and Henika had Pm5. The gene Pm3d was identified in cultivars Jasna and Eta in combination with another unknown gene/genes. Cultivars Santa and Torka had the gene Pm5 in combination with another unknown gene/genes. Four cultivars: Banti, Ismena, Olimpia and Sigma, showed resistance to all mildew isolates employed in a laboratory test. The accession IPG-SW-14 was the least susceptible to both pathogens (F. culmorum and powdery mildew) in all 5 years of experiments. This line is the best candidate for deriving new cultivars with improved resistance to fungal diseases.     

54份CIMMYT小麦材料抗白粉病分析

选用来自我国不同地区的20个白粉病菌毒性菌株,对54个CIMMYT小麦品种(系)进行抗病性分析.结果表明:(1)34个品种(系)含有抗病基因,以Pm8基因出现频率最高,有15个品种(系)携带该基因;(2)参试主效基因中,Pm1、Pm3e、Pm5、Pm6和Pm7基因已丧失对我国白粉菌的抗性,Pm16和Pm20基因的抗性最强;(3)50个1B/1R易位系品种(系)中31个含有抗病基因,48%的抗病1B/1R易位系可检测到Pm8基因.根据田间成株期病程曲线下面积(AUDPC)聚类分析结果,可将54份材料分为高抗、中抗、中感和高感4类,7个品种(系)不含任何主效抗病基因而田间表现中到高的抗性,是典型慢病性品种.     

Powdery mildew resistance in 155 Nordic bread wheat cultivars and landraces

The occurrence and distribution of seedling resistance genes and the presence of adult plant resistance to powdery mildew, was investigated in a collection of 155 Nordic bread wheat landraces and cultivars by inoculation with 11 powdery mildew isolates. Eighty-nine accessions were susceptible in the seedling stage, while 66 accessions showed some resistance. Comparisons of response patterns allowed postulation of combinations of genes Pm1a, Pm2, Pm4b, Pm5, Pm6, Pm8 and Pm9 in 21 lines. Seedling resistance was three times more frequent in spring wheat than in winter wheat. The most commonly postulated genes were Pm1a+Pm2+Pm9 in Sweden, Pm5 in Denmark and Norway, and Pm4b in Finland. Forty-five accessions were postulated to carry only unidentified genes or a combination of identified and unidentified genes that could not be resolved by the 11 isolates. Complete resistance to all 11 isolates was present in 18 cultivars. Adult plant resistance was assessed for 109 accessions after natural infection with a mixture of races. In all, 92% of the accessions developed less than 3-5% pathogen coverage while nine lines showed 10-15% infected leaf surface. The characterization of powdery mildew resistance in Nordic wheat germplasm could facilitate the combination of resistance genes in plant breeding programmes to promote durability of resistance and disease management.     

Phytopathological and molecular genetic identification of leaf rust resistance genes in common wheat accessions with alien genetic material

Leaf rust resistance genes were sought in 23 resistant common wheat accessions with alien genetic material of Aegilops speltoides, Ae. triuncialis, and Triticum kiharae from the Arsenal collection. The genes were identified by common phytopathological tests and PCR analysis with STS markers linked with the known Lr genes. None of the methods identified the resistance genes in two accessions. In the other accessions, the combination of the two methods broadened the spectrum of detectable genes and, in some cases, allowed double verification of the presence of a resistance gene. Most accessions proved to contain several leaf rust resistance genes, combining juvenile and adult plant ones. The accessions were found to contain gene combinations that ensured field resistance (Lr13 + Lr10 and Lr12 + Lr34) and immunity under the conditions of the Non-Chernozem region. Accessions with alien genetic material contained a unique combination of five or six resistance genes. Since the accessions were rich in leaf rust resistance genes, including effective ones, and carried rare combinations of these genes, they were proposed as donors to be universally employed in breeding for immunity in all regions of Russia.     

Leaf rust resistance genes of wheat: identification in cultivars and resistance sources

Thirty-seven wheat cultivars originating from seven European countries were examined by using sequence tagged site (STS) markers for seven Lr (leaf rust = brown rust) resistance genes against the fungal pathogen of wheat Puccinia recondita f. sp. tritici (Lr9, Lr10, Lr19, Lr24, Lr26 and Lr37). Additionally, 22 accessions with various Lr genes from two germplasm collections were tested. A Scar (sequence-characterized amplified region) marker for Lr24 and a CAPS (Cleaved Amplified Polymorphic Sequence) marker for Lr47 were also used to identify those genes in the wheat accessions. Each marker amplified one specific DNA fragment. Three Lr gene markers were identified in wheat cultivars (Lr10, Lr26 and Lr37). Another four markers (Lr9, Lr19, Lr24 and Lr47) were found in breeding lines carrying leaf rust resistance genes. The results were compared with leaf rust resistance gene postulations made in previous studies, based on multipathotype testing. Markers for Lr10, Lr26 and Lr37 may be useful in marker-assisted breeding.     

小麦3个被白粉菌诱导基因表达的分析

含有抗白粉病基因Pm2 1的小麦 簇毛麦 6VS/6AL易位系在接种白粉菌后 ,叶片无任何病症。应用mRNA差异显示技术从小麦 簇毛麦 6VS/ 6AL易位系分离到 3个叶绿体蛋白基因片段 ,它们是TaD5、TaD2 3和TaD33,3个基因片段分别与小麦叶绿体基因rbcL ,拟斯卑尔脱山羊草叶绿体RNA聚合酶α亚基基因rpoA和大麦 1,5 二磷酸核酮糖羧化酶活化酶基因(Rubiscoactivase ,RcaA2 )同源性达 97%、98%和 88%。据此推测TaD5、TaD2 3和TaD33分别是 6VS/ 6AL易位系中的rbcL、rpoA和 1,5 二磷酸核酮糖羧化酶活化酶基因的片断。Northern分析表明这 3个叶绿体基因的表达在白粉菌诱导下得到增强。叶绿体基因组含有胸腺嘧啶重复区是在mRNA差异显示中克隆到叶绿体基因组基因的原因     

小麦tae-MIR156前体基因的克隆及其靶基因TaSPL17多态性分析

Squamosa-promoter binding protein (SBP)-box基因是植物特有的一类转录因子, 广泛参与植物生长发育, 其部分成员受miR156调控。文章克隆了小麦(Triticum aestivum) tae-MIR156前体基因, 转录后能够形成茎环结构。小麦10个SBP-box基因中, 仅TaSPL3和TaSPL17在编码区存在tae-miR156识别位点。SPL17在普通小麦的A基因组供体种乌拉尔图小麦(Triticum urartu, AA) UR209和B基因组供体种拟斯卑尔脱山羊草(Aegilops speltoides, BB) Y2001中均为多拷贝(SPL17-A1、SPL17-A2和SPL17-A3; SPL17-B1、SPL17-B2和SPL17-B3), 在D基因组供体种粗山羊草(Aegilops tauschii, DD) Ae38中仅检测到一种序列(SPL17-D); SPL17-A2与SPL17-B2, SPL17-A3与SPL17-B3、SPL17-D两两之间序列的一致性程度均大于99%, 且与普通小麦(中国春、衡观35和双丰收)的TaSPL17序列具有较高的一致性, 提示它们可能来源于共同的祖先基因, 并且在进化过程中高度保守。靶基因TaSPL17中的tae-miR156识别位点非常保守, 在根据单株穗数和基因型多样性挑选的SubP1和SubP2群体中均未检测到tae-miR156识别位点存在变异碱基。     

Identification and mapping of a new powdery mildew resistance gene on chromosome 6D of common wheat

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most serious wheat diseases. The rapid evolution of the pathogen's virulence, due to the heavy use of resistance genes, necessitates the expansion of resistance gene diversity. The common wheat line D57 is highly resistant to powdery mildew. A genetic analysis using an F(2) population derived from the cross of D57 with the susceptible cultivar Yangmai 158 and the derived F(2:3) lines indicated that D57 carries two dominant powdery mildew resistance genes. Based on mapping information of polymorphic markers identified by bulk segregant analysis, these two genes were assigned to chromosomes 5DS and 6DS. Using the F(2:3) lines that segregated in a single-gene mode, closely linked PCR-based markers were identified for both genes, and their chromosome assignments were confirmed through linkage mapping. The gene on chromosome 5DS was flanked by Xgwm205 and Xmag6176, with a genetic distance of 8.3 cM and 2.8 cM, respectively. This gene was 3.3 cM from a locus mapped by the STS marker MAG6137, converted from the RFLP marker BCD1871, which was 3.5 cM from Pm2. An evaluation with 15 pathogen isolates indicated that this gene and Pm2 were similar in their resistance spectra. The gene on chromosome 6DS was flanked by co-segregating Xcfd80 and Xmag6139 on one side and Xmag6140 on the other, with a genetic distance of 0.7 cM and 2.7 cM, respectively. This is the first powdery mildew resistance gene identified on chromosome 6DS, and plants that carried this gene were highly resistant to all of the 15 tested pathogen isolates. This gene was designated Pm45. The new resistance gene in D57 could easily be transferred to elite cultivars due to its common wheat origin and the availability of closely linked molecular markers.     

Identification of efficient leaf-rust resistance genes in wheat (Triticum aestivum) using STS markers

Molecular STS markers J13, Gb, and J09 were used for screening wheat (Triticum aestivum L.) accessions previously found to possess leaf-rust resistance genes according to test crosses or phytopathological tests. Specific amplicons were detected in all accessions assumed to possess the Lr9 gene, in nine of ten accessions with the conjectured Lr19 gene, and in 13 of 29 accessions with the conjectured Lr24 gene. Application of STS markers to identification of accessions possessing efficient leaf-rust resistance genes is discussed.     

Identification of effective leaf-rust resistance genes in wheat (Triticum aestivum) using STS markers

Molecular STS markers J13, Gb, and J09 were used for screening wheat (Triticum aestivum L.) accessions previously found to possess leaf-rust resistance genes according to test crosses or phytopathological tests. Specific amplification products were detected in all accessions assumed to possess the Lr9 gene, in nine of ten accessions with the conjectured Lr19 gene, and in 13 of 29 accessions with the conjectured Lr24 gene. Application of STS markers to identification of accessions possessing effective leaf-rust resistance genes is discussed.     

PstIAFLP based markers for leaf rust resistance genes in common wheat

The aim of the present study was to detect candidate DNA markers for selected leaf rust resistance genes. A total number of 286 loci in the 'Thatcher' near-isogenic lines carrying resistance gene Lr1, Lr9, Lr10, Lr13, Lr19, Lr21, Lr24, Lr26, Lr28, Lr35, and Lr37 were screened for DNA polymorphism by the PstIAFLP method. A survey with 33 selective primers yielded 16 candidate markers. Further validation studies on cultivars characterized for the presence and absence of selected resistance genes confirmed specificity of markers for Lr24, Lr26 and Lr37. The AFLP-based marker P42-530 was successfully converted into an STS marker. The new marker was linked with the Lr37-specific marker (CslVrga13) at the distance of 1.7 cM. The PstIAFLP method was found to be effective in the identification of DNA changes induced in hexaploid wheat by translocations from Agropyron elongatum, Secale cereale and Aegilops ventricosa.     

Allelic series of four powdery mildew resistance genes at the Pm3 locus in hexaploid bread wheat

At the Pm3 locus in hexaploid wheat (Triticum aestivum), 10 alleles conferring race-specific resistance to powdery mildew (Blumeria graminis f. sp. tritici) are known. A cluster of genes encoding coiled-coil-nucleotide-binding site-leucine-rich repeat proteins spans the Pm3 locus on wheat chromosome 1A, and one member of this gene family has recently been identified as the Pm3b resistance gene. Using molecular markers closely linked to Pm3b, we performed haplotype analysis of 10 lines carrying different Pm3 alleles. All these lines have a conserved genomic region delimited by markers cosegregating with Pm3b and including a structurally conserved Pm3b-like gene. A polymerase chain reaction-based strategy allowed the amplification of one Pm3b-like sequence from lines carrying Pm3a, Pm3d, and Pm3f alleles. These candidate genes for Pm3a, Pm3d, and Pm3f conferred AvrPm3a-, AvrPm3d-, and AvrPm3f-dependent resistance, respectively, to wheat powdery mildew in a single cell transient transformation assay. A high level of amino acid similarity (97.8%) was found between the PM3A, PM3B, PM3D, and PM3F proteins. The coiled-coil domain was 100% conserved, whereas, in the nucleotide binding site region, sequence exchange was detected, indicating intragenic recombination or gene conversion between alleles. All these results indicate that Pm3a, Pm3b, Pm3d, and Pm3f form a true allelic series. The low level of sequence divergence between the four characterized alleles as well as the finding of a conserved Pm3 haplotype are in agreement with the hypothesis of a recent evolution of Pm3-based resistance, suggesting that some or most of the diversity found at the Pm3 locus in modern wheat has evolved after wheat domestication.     

Powdery mildew resistance gene <Emphasis Type="Italic">Pm22</Emphasis> in cultivar Virest is a member of the complex <Emphasis Type="Italic">Pm1</Emphasis> locus in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L. em Thell.)

The powdery mildew resistance gene Pm22, identified in the Italian wheat cultivar Virest and originally assigned to wheat chromosome 1D, was mapped to chromosome 7A with the aid of molecular markers. Mapping of common AFLP and SSR markers in two wheat crosses segregating for Pm22 and Pm1c, respectively, indicated that Pm22 is a member of the complex Pm1 locus. Pm22 also showed a pattern of resistance reaction to a differential set of Blumeria graminis f. sp. tritici isolates that was distinguishable from those from other Pm1 alleles in lines Axminster/8*Cc ( Pm1a), MocZlatka ( Pm1b), Weihenstephan Stamm M1N ( Pm1c) and Triticum spelta var. duhamelianum TRI 2258 ( Pm1d). Based on these results, the gene symbol Pm1e is proposed for the powdery mildew resistance gene in cv. Virest.     

Pm23: a new allele of Pm4 located on chromosome 2AL in wheat

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the major diseases of common wheat (Triticum aestivum) worldwide. The powdery mildew resistance gene Pm23, identified in the common wheat Line 81-7241 and originally assigned to wheat chromosome 5A, was relocated on chromosome 2AL with the aid of molecular markers. Mapping of microsatellite markers in two wheat crosses segregating for Pm23 and Pm4b, respectively, in combination with the reported mapping of Pm4a, indicated that the three genes were all linked to the marker Xgwm356 with a distance of 3-5 cM. Allelism between Pm4b and Pm23 was then confirmed, when the progenies of a cross between VPM1 (Pm4b) and Line 81-7241, were shown to be all resistant to a B. graminis isolate avirulent to the both parents. Pm23 is therefore a new allele of the Pm4 locus, and was redesignated as Pm4c.     

Construction and study of leaf rust-resistant common wheat lines with translocations of Aegilops speltoides Tausch

Genotyping was performed for the leaf rust-resistant line 73/00i (Triticum aestivum x Aegilops speltoides). Fluorescence in situ hybridization (FISH) with probes Spelt1 and pSc119.2 in combination with microsatellite analysis were used to determine the locations and sizes of the Ae. speltoides genetic fragments integrated into the line genome. Translocations were identified in the long arms of chromosomes 5B and 6B and in the short arm of chromosome 1B. The Spelt1 and pSc119.2 molecular cytological markers made it possible to rapidly establish lines with single translocation in the long arms of chromosomes 5B and 6B. The line carrying the T5BS x 5BL-5SL translocation was highly resistant to leaf rust, and the lines carrying the T6BS x 6BL-6SL translocation displayed moderate resistance. The translocations differed in chromosomal location from known leaf resistance genes transferred into common wheat from Ae. speltoides. Hence, it was assumed that new genes were introduced into the common wheat genome from Ae. speltoides. The locus that determined high resistance to leaf rust and was transferred into the common wheat genome from the long arm of Ae. speltoides chromosome 5S by the T5BS x 5BL-5SL translocation was preliminarily designated as LrAsp5.     

Transgenic Pm3 multilines of wheat show increased powdery mildew resistance in the field

Resistance (R) genes protect plants very effectively from disease, but many of them are rapidly overcome when present in widely grown cultivars. To overcome this lack of durability, strategies that increase host resistance diversity have been proposed. Among them is the use of multilines composed of near-isogenic lines (NILs) containing different disease resistance genes. In contrast to classical R-gene introgression by recurrent backcrossing, a transgenic approach allows the development of lines with identical genetic background, differing only in a single R gene. We have used alleles of the resistance locus Pm3 in wheat, conferring race-specific resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici), to develop transgenic wheat lines overexpressing Pm3a, Pm3c, Pm3d, Pm3f or Pm3g. In field experiments, all tested transgenic lines were significantly more resistant than their respective nontransformed sister lines. The resistance level of the transgenic Pm3 lines was determined mainly by the frequency of virulence to the particular Pm3 allele in the powdery mildew population, Pm3 expression levels and most likely also allele-specific properties. We created six two-way multilines by mixing seeds of the parental line Bobwhite and transgenic Pm3a, Pm3b and Pm3d lines. The Pm3 multilines were more resistant than their components when tested in the field. This demonstrates that the difference in a single R gene is sufficient to cause host-diversity effects and that multilines of transgenic Pm3 wheat lines represent a promising strategy for an effective and sustainable use of Pm3 alleles.