Rapid phenotyping of adult plant resistance in barley (Hordeum vulgare) to leaf rust under controlled conditions

Breeding for adult plant resistance (APR) is currently impeded by the low frequency of annual field‐based testing and variable environmental conditions. We developed and implemented a greenhouse‐based methodology for the rapid phenotyping of APR to leaf rust in barley to improve the efficacy of gene discovery and cloning. We assessed the effects of temperature (18 and 23°C) and growth stage (1–5 weeks) on the expression of APR in the greenhouse using 28 barley genotypes with both known and uncharacterized APR. All lines were susceptible in week 1, while lines carrying Rph20 and several with uncharacterized resistance expressed resistance as early as week 2. In contrast, lines lacking Rph20 and carrying either Rph23 and/or Rph24 expressed resistance from week 4. Resistant phenotypes were clearest at 18°C. A subset of 16 of the 28 lines were assessed for leaf rust across multiple national and international field sites. The greenhouse screening data reported in this study were highly correlated to most of the field sites, indicating that they provide comparable data on APR phenotypes for screening purposes.     

Identification and characterization of seedling and adult plant resistance to Puccinia hordei in Chinese barley germplasm

Leaf rust of barley, caused by Puccinia hordei, occurs in all barley‐growing regions of Australia causing significant yield losses under epidemic conditions. The development and use of resistant cultivars are the most economical and environmentally sustainable method to control leaf rust which in turn relies on ongoing efforts to identify and characterize new sources of resistance. The aim of this study was to postulate known genes and/or identify new sources of resistance to P. hordei. Fifty‐two genotypes were assessed at the seedling and adult plant growth stages. On the basis of multipathotype tests, 39 genotypes lacked detectable seedling resistance, and nine were postulated to carry the genes Rph2, Rph4, Rph12 and Rph19 singly. Four genotypes carried uncharacterized seedling resistance; however, the gene(s) present in each were ineffective to at least one of the pathotypes used. Field tests at the adult plant growth stage revealed the presence of adult plant resistance (APR) in 12 genotypes. Tests of allelism and marker analysis indicated that resistance genes present in these genotypes were independent of the APR gene Rph20.     

Inheritance and characterization of the new and rare gene Rph25 conferring seedling resistance in Hordeum vulgare against Puccinia hordei

In this study, we characterized and mapped a new and rare resistance gene (RphFT) in the Chinese barley variety ‘Fong Tien’. RphFT, a dominant gene, was mapped to chromosome 5HL at a genetic position of 142.1 cM using DArT‐seq markers. The gene was also confirmed to be present in Australian cultivar ‘Yagan’ based on allelic tests, and likely ‘Lockyer’ based on multipathotype tests. The genetic studies also confirmed the presence of Rph12 in Australian cultivar ‘Baudin’. Rph12 is also located on chromosome 5HL close to RphFT, and the two loci were confirmed to be independent. Gene RphFT is of limited breeding value because it is effective to only one pathotype of P. hordei, 220P+ +Rph13 in Australia; nevertheless, it may play a role in controlling leaf rust if used in combination with other Rph genes. The locus symbol Rph25 is recommended for RphFT in accordance with the rules and numbering system of barley gene nomenclature.     

‘CPAN 1842’: a new source of adult plant leaf rust resistance in bread wheat

There is worldwide interest in adult plant resistance (APR) because of greater durability of APR to the cereal rusts. Peruvian bread wheat genotype ‘CPAN (Coordinated Project Accession Number) 1842’ (LM 50–53) has shown leaf rust resistance in disease screening nurseries since its introduction in 1977. However, it is susceptible at the seedling stage to several Puccinia triticina (Pt) pathotypes including the widely prevalent 77‐5 (121R63‐1) that infects bread wheat. Inheritance studies showed that CPAN 1842 carried a dominant gene for APR to pathotype 77‐5, which was different from Lr12, Lr13, Lr22a, Lr34, Lr35, Lr37, Lr46, Lr48, Lr49 and Lr68, based on the tests of allelism; and from Lr67, based on genotyping with the closely linked SSR marker cfd71. This gene should also be different from Lr22b as the latter is totally ineffective against pathotype 77‐5. CPAN 1842 therefore appears to be a new promising source of leaf rust resistance. Also having resistance to stem rust and stripe rust, this line can contribute to breeding for multiple rust resistances in wheat.     

Molecular mapping of the leaf rust resistance gene Rph7 in barley

Leaf rust of barley, caused by Puccinia hordei Otth, is an important foliar disease in most temperate regions of the world. Sixteen major leaf rust resistance (Rph) genes have been described from barley, but only a few have been mapped. The leaf rust resistance gene Rph7 was first described from the cultivar ‘Cebada Capa’ and has proven effective in Europe. Previously mapped restriction fragment length polymorphism (RFLP) markers have been used to determine the precise location of this gene in the barley genome. From the genetic analysis of a ‘Bow‐man’/‘Cebada Capa’ cross, Rph7 was mapped to the end of chromosome 3HS, 1.3 recombination units distal to the RFLP marker cMWG691. A codominant cleaved amplified polymorphic site (CAPS) marker was developed by exploiting allele‐specific sequence information of the cMWG691 site and adjacent fragments of genomic DNA. Based on the large amount of polymorphism present in this region, the CAPS marker may be useful for the marker‐assisted selection of Rph7 in most diverse genetic backgrounds.     

Identification and characterization of seedling and adult plant resistance to <Emphasis Type="Italic">Puccinia hordei</Emphasis> in selected African barley germplasm

A collection of 112 African barley accessions were assessed for response to Puccinia hordei in seedling greenhouse tests using 10 pathotypes and in adult plant field tests over three successive field seasons in Australia. One of the 10 pathotypes (viz. 5457P+) used in seedling tests was also used in field tests to allow assessment of the presence of adult plant resistance (APR) in lines that were seedling susceptible to this pathotype. The seedling resistance genes Rph1, Rph2, Rph3, Rph9.am and Rph9.z were postulated in a number of accessions, singly and in various combinations, with Rph2 and Rph9.z being the most common. Twenty-six accessions carried seedling resistance that was either uncharacterized or could not be determined using the 10 P. hordei pathotypes. One accession carried high levels of APR and 11 accessions showed moderate levels of APR, all of which were susceptible to all P. hordei pathotypes at the seedling stage. All barley accessions were genotyped for the presence of marker alleles that are closely linked to the APR genes Rph20 and Rph23 (bPb-0837 and Ebmac0603, respectively). No accession was positive for bPb-0837, suggesting that Rph20 is not frequent in African germplasm. Thirteen accessions were postulated to carry Rph23 based on the presence of the marker allele Ebmac0603 found in Yerong (Rph23), and 10 out of the 11 accessions with moderate APR lacked the bPb-0837 and Ebmac0603 marker alleles, indicating that they likely carry new uncharacterized APR genes. Inheritance studies were performed using populations derived from four of the accessions that carried APR (Clho 9776, Clho 11958, Mecknes Maroc and Sinai) by crossing with the susceptible barley genotype Gus. Chi squared analysis of the phenotypic data from F₃ populations suggested that CIho9776 carried a single APR gene and CIho11958, Mecknes Maroc and Sinai each carried two genes for APR to leaf rust.     

Characterization and mapping of gene Rph19 conferring resistance to Puccinia hordei in the cultivar 'Reka 1' and several Australian barleys

Previous studies established that the Australian barley cultivar ‘Prior’ possessed resistance to Puccinia hordei (RphP), displaying the same specificity as an uncharacterized resistance in the differential cultivar ‘Reka 1’ (also possessing Rph2). Multipathotype tests confirmed the presence RphP in nine additional barley cultivars and indicated that RphP differed in specificity to the genes Rph1 to Rph15 and Rph18, plus the gene RphX present in the barley cultivar ‘Shyri’. RphP was inherited as a single dominant gene. Mapping studies using a doubled haploid population derived from ‘Chebec’/‘Harrington’ located RphP to the long arm of chromosome 7H, and demonstrated linkage with an restriction fragment length polymorphism marker (pTAG732), a resistance gene analogue marker (RLch4(Nc)), and two microsatellite markers (HVM11 and HVM49) at genetic distances of about 4‐10 cM. RphP showed linkage of 28 ± 4.3 cM with Rph3. RphP was designated Rph19, with the allele designation Rph19.ah. Previous studies have established that virulence for Rph19 occurs in many barley growing regions of the world.     

Identification of recombinants carrying stripe rust resistance gene Yr57 and adult plant stem rust resistance gene Sr2 through marker‐assisted selection

Many stem rust resistance genes have been formally named in wheat. Adult plant stem rust resistance gene Sr2 was mapped in the short‐arm of chromosome 3B. Stripe rust resistance gene Yr57, identified in Aus91463, was mapped about 5 cM away from Sr2 based on its linkage with Sr2‐linked marker gwm533. The objective of this study was to combine Sr2 and Yr57 in a single genotype. A mapping population containing 107 recombinant inbred lines was developed from a cross between Aus91463‐Yr57 and Hartog‐Sr2. This population was tested at the seedling stage in the glasshouse for variation in stripe rust response, and high temperature induced Sr2‐linked seedling chlorosis. The RIL population was screened for Sr2‐linked pseudo black chaff phenotype at the adult plant stage in field. Five recombinants carrying Sr2 and Yr57 in coupling were detected using phenotypic and marker data. Four recombinants also carried leaf rust resistance gene Lr23 from Aus91463. These recombinants are being used as triple rust resistance source in the Australian Cereal Rust Control Program.     

Postulation of leaf-rust resistance genes in Czech and Slovak barley cultivars and breeding lines

Leaf‐rust resistance (Rph) genes in 61 Czech and Slovak barley cultivars and 32 breeding lines from registration trials of the Czech Republic were postulated based on their reaction to 12 isolates of Puccinia hordei with different combinations of virulence genes. Five known Rph genes (Rph2, Rph3, Rph4, Rph7, and Rph12) and one unknown Rph gene were postulated to be present in this germplasm. To corroborate this result, the pedigree of the barley accessions was analysed. Gene Rph2, as well as Rph4, originated from old European cultivars. The donor of Rph3, which has been mainly used by Czech and Slovak breeders, is ‘Ribari’ (‘Baladi 16’). Rph12 originates from barley cultivars developed in the former East Germany. Rph7 in the registered cultivar ‘Heris’ originates from ‘Forrajera’. A combination of two genes was found in 10 cultivars. Nine heterogeneous cultivars were identified; they were composed of one component with an identified Rph gene and a second component without any resistance gene. No gene for leaf rust resistance was found in 17 of the accessions tested. This study demonstrates the utility of using selected pathotypes of P. hordei for postulating Rph genes in barley.     

Molecular genetics of race non-specific rust resistance in wheat

Over 150 resistance genes that confer resistance to either leaf rust, stripe rust or stem rust have been catalogued in wheat or introgressed into wheat from related species. A few of these genes from the ‘slow-rusting’ adult plant resistance (APR) class confer partial resistance in a race non-specific manner to one or multiple rust diseases. The recent cloning of two of these genes, Lr34/Yr18, a dual APR for leaf rust and stripe rust, and Yr36, a stripe rust APR gene, showed that they differ from other classes of plant resistance genes. Currently, seven Lr34/Yr18 haplotypes have been identified from sequencing the encoding ATP Binding Cassette transporter gene from diverse wheat germplasm of which one haplotype is commonly associated with the resistance phenotype. The paucity of well characterised APR genes, particularly for stem rust, calls for a focused effort in developing critical genetic stocks to delineate quantitative trait loci, construct specific BAC libraries for targeted APR genes to facilitate robust marker development for breeding applications, and the eventual cloning of the encoding genes.     

Inheritance of seedling and adult plant resistance to leaf rust of selected Australian spring and English winter wheat varieties

Genetic studies were conducted to gain an understanding of the inheritance of adult plant resistance (APR) to leaf rust in six common wheat varieties. The Australian varieties ‘Cranbrook’ and ‘Harrier’ each carry two genes for APR to leaf rust. These genes are genetically independent of the seedling resistance genes Lr23 and Lrl7b, carried by the respective varieties. Adult plant resistance in ‘Suneca’ was conferred by at least two genes, in addition to the seedling genes Lr1 and Lrli. It is likely that the APRs in ‘Cranbrook’, ‘Harrier’ and ‘Suneca’ are conferred by uncharacterized gene(s). Tests of allelism confirmed that seedling resistances in the varieties ‘Avocet R’, ‘Hereward’, ‘Moulin’ and ‘Pastiche’ are conferred by Lrli. Adult plant resistance in the variety ‘Hereward’ was inherited monogenically, whereas varieties ‘Moulin’ and ‘Pastiche’ each carried two dominant genes. On the basis of rust specificity and pedigree analysis, it would seem likely that the APR genes in ‘Hereward’, ‘Moulin’ and ‘Pastiche’ are also currently uncharacterized.     

Evaluation of seedling and adult plant resistance to <Emphasis Type="Italic">Puccinia hordei</Emphasis> in barley

The responses of 92 barley genotypes to selected Puccinia hordei pathotypes were assessed in greenhouse tests at seedling growth stages and in the field at adult plant growth stages to identify known and/or unknown resistances. On the basis of multipathotype tests, 35 genotypes were postulated to carry Rph1, Rph2, Rph4, Rph5, Rph12, RphCantala, alone or in combination (Rph2 + Rph4 and Rph1 + Rph2), whereas 52 genotypes lacked seedling resistance to P. hordei to the pathotypes used. Five genotypes carried seedling resistance that was effective to all pathotypes, of which four were believed to carry uncharacterised resistance based on pedigree information. Field tests at adult plant growth stages indicated that while 28 genotypes were susceptible, 57 carried uncharacterised APR to P. hordei. Pedigree analysis indicated that APR in the test genotypes could have been derived from three different sources. The resistant responses of seven cultivars at adult plant growth stages were believed to be due to the presence of seedling resistance effective against the field pathotypes.     

Evaluation of seedling and adult plant resistance to leaf rust in European wheat cultivars

Summary A set of 105 European wheat cultivars, comprising 68 cultivars with known seedling resistance genes and 37 cultivars that had not been tested previously, was tested for resistance to selected Australian pathotypes of P. triticina in seedling greenhouse tests and adult plant field tests. Only 4% of the cultivars were susceptible at all growth stages. Twelve cultivars lacked detectable seedling resistance to leaf rust, and among the remaining cultivars, 10 designated genes were present either singly or in combination. Lr13 was the most frequently detected gene, present in 67 cultivars, followed by the rye-derived gene Lr26, present in 19 cultivars. Other genes present were Lr1, Lr3a, Lr3ka, Lr10, Lr14a, Lr17b, Lr20 and Lr37. There was evidence for unidentified seedling resistance in addition to known resistance genes in 11 cultivars. Field tests with known pathotypes of P. triticina demonstrated that 57% of the cultivars carried adult plant resistance (APR) to P. triticina. The genetic identity of the APR is largely unknown. Genetic studies on selected cultivars with unidentified seedling resistances as well as all of those identified to carry APR are required to determine the number and inheritance of the genes involved, to determine their relationships with previously designated rust resistance genes, and to assess their potential value in breeding for resistance to leaf rust.     

Evaluation of seedling and adult plant resistance to stem rust in European wheat cultivars

A total of 105 European wheat cultivars were assessed for seedling and adult plant resistance (APR) to stem rust using an array of Australian isolates of Puccinia graminis f. sp. tritici. Twenty-seven cultivars were susceptible at both seedling and adult plant growth stages. Twelve catalogued seedling stem rust resistance genes (Sr7b, Sr8a, Sr8b, Sr9b, Sr9g, Sr11, Sr15, Sr17, Sr29, Sr31, Sr36 and Sr38) were detected in the remaining cultivars, and 13 cultivars carried additional seedling resistance genes that could not be postulated with the isolates used. Low levels of APR to stem rust were found in the cultivars Artaban, Forno, Mec, Mercia, Pandas and Vlada. Although the genetic identity of this APR was not determined, it was clear that the only designated stem rust APR gene Sr2 was not present in any of the cultivars tested based on the absence of the linked traits seedling chlorosis and pseudo black chaff. One of these cultivars, Forno, is believed to carry the leaf rust APR gene Lr34, previously reported to be associated with improved resistance to stem rust. A detailed genetic characterisation of the APRs in these cultivars will be needed to understand their modes of inheritance and relationships with catalogued stem rust resistance genes. Such knowledge may help in developing cultivars with effective gene combinations that confer higher levels of protection.     

Development of a SCAR marker linked with a MYMV resistance gene in mungbean (Vigna radiata L. Wilczek)

Yellow mosaic disease (YMD) caused by mungbean yellow mosaic virus (MYMV) is the most important disease of mungbean, causing great yield loss. The present investigation was carried out to study the inheritance and identify molecular markers linked with MYMV resistance gene by using F₁, F₂ and 167 F_2 : 8 recombinant inbred lines (RILs) developed from the cross ‘TM‐99‐37’ (resistant) × Mulmarada (susceptible). The F₁ was susceptible, F₂ segregated in 3S:1R phenotypic ratio and RILs segregated in 1S:1R ratio in the field screening indicating that the MYMV resistance gene is governed by a single recessive gene. Of the 140 RAPD primers, 45 primers showing polymorphism in parents were screened using bulked segregant analysis. Three primers amplified specific polymorphic fragments viz. OPB‐07_600, OPC‐06₁₇₅₀ and OPB‐12₈₂₀. The marker OPB‐07₆₀₀ was more closely linked (6.8 cM) with a MYMV resistance gene as compared to OPC‐06₁₇₅₀ (22.8 cM) and OPB‐12₈₂₀ (25.2 cM). The resistance‐specific fragment OPB‐07₆₀₀ was cloned, sequenced and converted into a sequence‐characterized amplified region (SCAR) marker and validated in twenty genotypes with different genetic backgrounds.     

Alleles on the two dwarfing loci on 4B and 4D are main drivers of FHB‐related traits in the Canadian winter wheat population “Vienna” × “25R47”

Fusarium head blight (FHB), leaf rust and stem rust are among the most destructive wheat diseases. High‐yielding, native disease resistance sources are available in North America. The objective of this study was to map loci associated with FHB traits, leaf rust, stem rust and plant height in a “Vienna”/”25R47” population. DArT markers were used to generate a genetic map, and quantitative trait loci (QTL) analysis was performed by evaluating 113 doubled haploid lines across three environments in Ontario, Canada. FHB resistance QTL were identified on chromosomes 4D, 4B, 2D and 7A, while a QTL for leaf and stem rust resistance was identified on chromosome 1B. The dwarfing alleles of both Rht‐B1 and Rht‐D1 were associated with increased FHB index and DON content.     

Novel gene for adult plant resistance to Puccinia recondita in the wheat cultivar'Arjun'

Inheritance of resistance in the wheat cultivar‘Arjun’(HD 2009) against leaf rust pathotype 77–1 revealed that its durable resistance is attributable to a novel dominant adult plant resistance (APR) gene. Lr13, another gene reported in the cultivar played no role. This new gene is established as different from Lr34, the only effective APR gene from Triticum aestivum known for durability.     

Genetic Diversity for Adult Plant Resistance to Leaf Rust (Puccinia recondita) in Near-Isogenic Lines and in Indian Wheats

Variation for adult plant resistance in near-isogenic wheat lines carrying Lrl4b, Lrl4ab and Lr30 in a ‘Thatcher’ background indicated the possible presence of novel adult plant resistance genes effective against the Indian leaf rust population. Sixty-one wheats released for cultivation in India were grown in isolated nurseries. Each nursery was separately inoculated with one of four leaf rust pathotypes which had been selected to aid identification of resistance effective only in the adult plant stage. Seven distinct response groups were recognised and a minimum of six sources of adult plant resistance were postulated. In a group of 14 wheats, resistance was explained on the basis of the seedling response genes that were identified. Similar results for two years with pathotype 77-1 gave support to the reliability of field tests. Adult plant resistance (APR) sources were either race-specific or effective against all pathotypes used. Seedlings of cultivars with APR showed susceptible reactions. The possible presence of Lr34 in Indian wheats and its role in durable leaf rust resistance are discussed.     

Linkage of Rust Resistance Genes from Wild Barley (Hordeum spontaneum) with Isozyme Markers

Eighty-three third backcross lines which comprise a set of near isogenic lines (NIL's) of the barley cultivar ‘Clipper’ but each carrying a different chromosomal segment from Hordeum spontaneum, marked with a distinct isozyme, were tested for resistance to three races of the barley leaf rust pathogen (Puccmia hordei). Fourteen lines showed resistance to at least one race and three showed resistance to all three races. The resistance in two of these lines was controlled by separate, single partially dominant genes. In one case the resistance gene named Rph1O was on chromosome 3 and linked (r = 0.15 ±0.05) with the isozyme locus Est2. In the second case, the gene (Rph11) was on barley chromosome 6 and linked (r = 0.07±0.02) with the isozyme locus Acp3 and (r = 0.11±0.02) with Dip2.     

QTL mapping of adult‐plant resistance to stripe rust in a ‘Lumai 21 × Jingshuang 16’ wheat population

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a devastating fungal disease in common wheat (Triticum aestivum L.) worldwide. Chinese wheat cultivars ‘Lumai 21’ and ‘Jingshuang 16’ show moderate levels of adult‐plant resistance (APR) to stripe rust in the field, and they showed a mean maximum disease severity (MDS) ranging from 24 to 56.7% and 26 to 59%, respectively, across different environments. The aim of this study was to identify quantitative trait loci (QTL) for resistance to stripe rust in an F₃ population of 199 lines derived from ‘Lumai 21’ × ‘Jingshuang 16’. The F₃ lines were evaluated for MDS in Qingshui, Gansu province, and Chengdu, Sichuan province, in the 2009–2010 and 2010–2011 cropping seasons. Five QTL for APR were detected on chromosomes 2B (2 QTL), 2DS, 4DL and 5DS based on mean MDS in each environment and averaged values from all three environments. These QTL were designated QYr.caas‐2BS.2, QYr.caas‐2BL.2, QYr.caas‐2DS.2, QYr.caas‐4DL.2 and QYr.caas‐5DS, respectively. QYr.caas‐2DS.2 and QYr.caas‐5DS were detected in all three environments, explaining 2.3–18.2% and 5.1–18.0% of the phenotypic variance, respectively. In addition, QYr.caas‐2BS.2 and QYr.caas‐2BL.2 colocated with QTL for powdery mildew resistance reported in a previous study. These APR genes and their linked molecular markers are potentially useful for improving stripe rust and powdery mildew resistances in wheat breeding.