High-resolution mapping of the leaf rust disease resistance gene <Emphasis Type="Italic">Lr1</Emphasis> in wheat and characterization of BAC clones from the <Emphasis Type="Italic">Lr1</Emphasis> locus

Leaf rust is the most common disease in wheat production. There are more than 45 specific resistance genes described and used in wheat breeding to control epidemics of leaf rust, but none of them has been cloned. The leaf rust disease resistance gene 1 ( Lr1) is a good model gene for isolation by map-based cloning because it is a single, dominant gene which is located in the distal region of chromosome 5DL of wheat. As the first step towards the isolation of this gene we constructed a high-resolution genetic map in the region of the Lr1 locus by saturation mapping of two large segregating F(2) populations (Thatcher Lr1 x Thatcher, Thatcher Lr1 x Frisal). The resistance gene Lr1 was delimited in a 0.16-cM region between the RFLP markers ABC718 and PSR567 (0.12 cM from ABC718 and 0.04 cM from PSR567). A genomic BAC library of Aegilops tauschii (D genome) was screened using the RFLP markers ABC718 and PSR567. Five positive BAC clones were identified by ABC718 and four clones by PSR567. Two NBS-LRR type of resistance gene analogs, which encode proteins highly homologous to the bacterial blight disease resistance protein Xa1 of rice, were identified on BAC clones isolated with PSR567. Polymorphic BAC end probes were isolated from both ends of a 105-kb large BAC clone identified by ABC718. The end probes were mapped at the same locus as ABC718, and no recombination event was found within 105 kb around ABC718 in our analysis of more than 4,000 gametes.     

Physical mapping and identification of a candidate for the leaf rust resistance gene <Emphasis Type="Italic">Lr1</Emphasis> of wheat

Lr1 is a dominant leaf rust resistance gene located on chromosome 5DL of bread wheat and the wild species Aegilops tauschii. In this study, three polymorphic markers (WR001, WR002, and WR003) were developed from resistance gene analogs (RGAs) clustering around the Lr1 locus. Using these and other markers, Lr1 was mapped to a genetic interval of 0.79 cM in Ae. tauschii and 0.075 cM in wheat. The CAPS marker WR003, derived from LR1RGA1, co-segregated with Lr1 in both mapping populations of wheat and Ae. tauschii. For isolation of Lr1, two genomic BAC libraries (from Ae. tauschii and hexaploid wheat) were screened using the tightly flanking marker PSR567F and a set of nested primers derived from the conserved region of the RGA sequences. Approximately 400 kb BAC contig spanning the Lr1 locus was constructed. The LR1RGA1 encoding a CC-NBS-leucine-rich repeat (LRR) type of protein was the only one of the four RGAs at the Lr1 locus, which co-segregated with leaf rust resistance. Therefore, it represents a very good candidate for Lr1. The allelic sequences of LR1RGA1 from resistant and susceptible lines revealed a divergent DNA sequence block of ∼605 bp encoding the LRR repeats 9–15, whereas the rest of the sequences were mostly identical. Within this sequence block, the 48 non-synonymous changes resulted in 44 amino acid differences. This indicates that LR1RGA1 likely evolved through one or more recombination or gene conversion events with unknown genes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Aegilops-Secale</Emphasis> amphiploids: chromosome categorisation,pollen viability and identification of fungal disease resistance genes

The aim of this study was to assess the potential breeding value of goatgrass-rye amphiploids, which we are using as a “bridge” in a transfer of Aegilops chromatin (containing, e.g. leaf rust resistance genes) into triticale. We analysed the chromosomal constitution (by genomic in situ hybridisation, GISH), fertility (by pollen viability tests) and the presence of leaf rust and eyespot resistance genes (by molecular and endopeptidase assays) in a collection of 6× and 4× amphiploids originating from crosses between five Aegilops species and Secale cereale. In the five hexaploid amphiploids Aegilops kotschyi × Secale cereale (genome UUSSRR), Ae. variabilis × S. cereale (UUSSRR), Ae. biuncialis × S. cereale (UUMMRR; two lines) and Ae. ovata × S. cereale (UUMMRR), 28 Aegilops chromosomes were recognised, while in the Ae. tauschii × S. cereale amphiploid (4×; DDRR), only 14 such chromosomes were identified. In the materials, the number of rye chromosomes varied from 14 to 16. In one line of Ae. ovata × S. cereale, the U-R translocation was found. Pollen viability varied from 24.4 to 75.4%. The leaf rust resistance genes Lr22, Lr39 and Lr41 were identified in Ae. tauschii and the 4× amphiploid Ae. tauschii × S. cereale. For the first time, the leaf rust resistance gene Lr37 was found in Ae. kotschyi, Ae. ovata, Ae. biuncialis and amphiploids derived from those parental species. No eyespot resistance gene Pch1 was found in the amphiploids.     

Microsatellite mapping of adult-plant leaf rust resistance gene <Emphasis Type="Italic">Lr22a</Emphasis> in wheat

This study was conducted to identify microsatellite markers (SSR) linked to the adult-plant leaf rust resistance gene Lr22a and examine their cross-applicability for marker-assisted selection in different genetic backgrounds. Lr22a was previously introgressed from Aegilops tauschii Coss. to wheat (Triticum aestivum L.) and located to chromosome 2DS. Comparing SSR alleles from the donor of Lr22a to two backcross lines and their recurrent parents showed that between two and five SSR markers were co-introgressed with Lr22a and the size range of the Ae. tauschii introgression was 9–20 cM. An F₂ population from the cross of 98B34-T4B × 98B26-N1C01 confirmed linkage between the introgressed markers and Lr22a on chromosome 2DS. The closest marker, GWM296, was 2.9 cM from Lr22a. One hundred and eighteen cultivars and breeding lines of different geographical origins were tested with GWM296. In total 14 alleles were amplified, however, only those lines predicted or known to carry Lr22a had the unique Ae. tauschii allele at GWM296 with fragments of 121 and 131 bp. Thus, GWM296 is useful for selecting Lr22a in diverse genetic backgrounds. Genotypes carrying Lr22a showed strong resistance to leaf rust in the field from 2002 to 2006. Lr22a is an ideal candidate to be included in a stack of leaf rust resistance genes because of its strong adult-plant resistance, low frequency of commercial deployment, and the availability of a unique marker. An erratum to this article can be found at     

<Emphasis Type="Italic">Lr41, Lr39,</Emphasis> and a leaf rust resistance gene from<Emphasis Type="Italic"> Aegilops cylindrica</Emphasis> may be allelic and are located on wheat chromosome 2DS

The leaf rust resistance gene Lr41 in wheat germplasm KS90WGRC10 and a resistance gene in wheat breeding line WX93D246-R-1 were transferred to Triticum aestivum from Aegilops tauschii and Ae. cylindrica, respectively. The leaf rust resistance gene in WX93D246-R-1 was located on wheat chromosome 2D by monosomic analysis. Molecular marker analysis of F₂ plants from non-critical crosses determined that this gene is 11.2 cM distal to marker Xgwm210 on the short arm of 2D. No susceptible plants were detected in a population of 300 F₂ plants from a cross between WX93D246-R-1 and TA 4186 (Lr39), suggesting that the gene in WX93D246-R-1 is the same as, or closely linked to, Lr39. In addition, no susceptible plants were detected in a population of 180 F₂ plants from the cross between KS90WGRC10 and WX93D246-R-1. The resistance gene in KS90WGRC10, Lr41, was previously reported to be located on wheat chromosome 1D. In this study, no genetic association was found between Lr41 and 51 markers located on chromosome 1D. A population of 110 F₃ lines from a cross between KS90WGRC10 and TAM 107 was evaluated with polymorphic SSR markers from chromosome 2D and marker Xgdm35 was found to be 1.9 cM proximal to Lr41. When evaluated with diverse isolates of Puccinia triticina, similar reactions were observed on WX93D246-R-1, KS90WGRC10, and TA 4186. The results of mapping, allelism, and race specificity test indicate that these germplasms likely have the same gene for resistance to leaf rust.Contribution number 03-348-J from the Kansas Agricultural Experimental Station, Manhattan, KansasCommunicated by J. Dvorak     

Characterisation of a new stripe rust resistance gene <Emphasis Type="Italic">Yr47</Emphasis> and its genetic association with the leaf rust resistance gene <Emphasis Type="Italic">Lr52</Emphasis>

Two Iranian common wheat landraces AUS28183 and AUS28187 from the Watkins collection showed high levels of seedling resistance against Australian pathotypes of leaf rust and stripe rust pathogens. Chi-squared analyses of rust response segregation among F₃ populations derived from crosses of AUS28183 and AUS28187 with a susceptible genotype AUS27229 revealed monogenic inheritance of leaf rust and stripe rust resistance. As both genotypes produced similar leaf rust and stripe rust infection types, they were assumed to carry the same genes. The genes were temporarily named as LrW1 and YrW1. Molecular mapping placed LrW1 and YrW1 in the short arm of chromosome 5B, about 10 and 15 cM proximal to the SSR marker gwm234, respectively, and the marker cfb309 mapped 8–12 cM proximal to YrW1. LrW1 mapped 3–6 cM distal to YrW1 in two F₃ populations. AUS28183 corresponded to the accession V336 of the Watkins collection which was the original source of Lr52. Based on the genomic location and accession records, LrW1 was concluded to be Lr52. Because no other seedling stripe rust resistance gene has previously been mapped in chromosome 5BS, YrW1 was permanently named as Yr47. A combination of flanking markers gwm234 and cfb309 with phenotypic assays could be used to ascertain the presence of Lr52 and Yr47 in segregating populations. This investigation characterised a valuable source of dual leaf rust and stripe rust resistance for deployment in new wheat cultivars. Transfer of Lr52 and Yr47 into current Australian wheat backgrounds is in progress.     

Fine genetic mapping of greenbug aphid-resistance gene <Emphasis Type="Italic">Gb3</Emphasis> in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

The greenbug, Schizaphis graminum (Rondani), is an important aphid pest of small grain crops especially wheat (Triticum aestivum L., 2n = 6x = 42, genomes AABBDD) in many parts of the world. The greenbug-resistance gene Gb3 originated from Aegilops tauschii Coss. (2n = 2x = 14, genome D^tD^t) has shown consistent and durable resistance against prevailing greenbug biotypes in wheat fields. We previously mapped Gb3 in a recombination-rich, telomeric bin of wheat chromosome arm 7DL. In this study, high-resolution genetic mapping was carried out using an F_2:3 segregating population derived from two Ae. tauschii accessions, the resistant PI 268210 (original donor of Gb3 in the hexaploid wheat germplasm line ‘Largo’) and susceptible AL8/78. Molecular markers were developed by exploring bin-mapped wheat RFLPs, SSRs, ESTs and the Ae. tauschii physical map (BAC contigs). Wheat EST and Ae. tauschii BAC end sequences located in the deletion bin 7DL3-0.82–1.00 were used to design STS (sequence tagged site) or CAPS (Cleaved Amplified Polymorphic Sequence) markers. Forty-five PCR-based markers were developed and mapped to the chromosomal region spanning the Gb3 locus. The greenbug-resistance gene Gb3 now was delimited in an interval of 1.1 cM by two molecular markers (HI067J6-R and HI009B3-R). This localized high-resolution genetic map with markers closely linked to Gb3 lays a solid foundation for map based cloning of Gb3 and marker-assisted selection of this gene in wheat breeding.     

Development of wheat lines carrying stem rust resistance gene <Emphasis Type="Italic">Sr39</Emphasis> with reduced <Emphasis Type="Italic">Aegilops speltoides</Emphasis> chromatin and simple PCR markers for marker-assisted selection

The use of major resistance genes is a cost-effective strategy for preventing stem rust epidemics in wheat crops. The stem rust resistance gene Sr39 provides resistance to all currently known pathotypes of Puccinia graminis f. sp. tritici (Pgt) including Ug99 (TTKSK) and was introgressed together with leaf rust resistance gene Lr35 conferring adult plant resistance to P. triticina (Pt), into wheat from Aegilops speltoides. It has not been used extensively in wheat breeding because of the presumed but as yet undocumented negative agronomic effects associated with Ae. speltoides chromatin. This investigation reports the production of a set of recombinants with shortened Ae. speltoides segments through induction of homoeologous recombination between the wheat and the Ae. speltoides chromosome. Simple PCR-based DNA markers were developed for resistant and susceptible genotypes (Sr39#22r and Sr39#50s) and validated across a set of recombinant lines and wheat cultivars. These markers will facilitate the pyramiding of ameliorated sources of Sr39 with other stem rust resistance genes that are effective against the Pgt pathotype TTKSK and its variants.     

New slow-rusting leaf rust and stripe rust resistance genes <Emphasis Type="Italic">Lr67</Emphasis> and <Emphasis Type="Italic">Yr46</Emphasis> in wheat are pleiotropic or closely linked

The common wheat genotype ‘RL6077’ was believed to carry the gene Lr34/Yr18 that confers slow-rusting adult plant resistance (APR) to leaf rust and stripe rust but located to a different chromosome through inter-chromosomal reciprocal translocation. However, haplotyping using the cloned Lr34/Yr18 diagnostic marker and the complete sequencing of the gene indicated Lr34/Yr18 is absent in RL6077. We crossed RL6077 with the susceptible parent ‘Avocet’ and developed F₃, F₄ and F₆ populations from photoperiod-insensitive F₃ lines that were segregating for resistance to leaf rust and stripe rust. The populations were characterized for leaf rust resistance at two Mexican sites, Cd. Obregon during the 2008–2009 and 2009–2010 crop seasons, and El Batan during 2009, and for stripe rust resistance at Toluca, a third Mexican site, during 2009. The F₃ population was also evaluated for stripe rust resistance at Cobbitty, Australia, during 2009. Most lines had correlated responses to leaf rust and stripe rust, indicating that either the same gene, or closely linked genes, confers resistance to both diseases. Molecular mapping using microsatellites led to the identification of five markers (Xgwm165, Xgwm192, Xcfd71, Xbarc98 and Xcfd23) on chromosome 4DL that are associated with this gene(s), with the closest markers being located at 0.4 cM. In a parallel study in Canada using a Thatcher × RL6077 F₃ population, the same leaf rust resistance gene was designated as Lr67 and mapped to the same chromosomal region. The pleiotropic, or closely linked, gene derived from RL6077 that conferred stripe rust resistance in this study was designated as Yr46. The slow-rusting gene(s) Lr67/Yr46 can be utilized in combination with other slow-rusting genes to develop high levels of durable APR to leaf rust and stripe rust in wheat.     

Fine scale genetic and physical mapping using interstitial deletion mutants of <Emphasis Type="Italic">Lr34</Emphasis><Emphasis Type="Italic">/Yr18</Emphasis>: a disease resistance locus effective against multiple pathogens in wheat

The Lr34/Yr18 locus has contributed to durable, non-race specific resistance against leaf rust (Puccinia triticina) and stripe rust (P. striiformis f. sp. tritici) in wheat (Triticum aestivum). Lr34/Yr18 also cosegregates with resistance to powdery mildew (Pm38) and a leaf tip necrosis phenotype (Ltn1). Using a high resolution mapping family from a cross between near-isogenic lines in the “Thatcher” background we demonstrated that Lr34/Yr18 also cosegregated with stem rust resistance in the field. Lr34/Yr18 probably interacts with unlinked genes to provide enhanced stem rust resistance in “Thatcher”. In view of the relatively low levels of DNA polymorphism reported in the Lr34/Yr18 region, gamma irradiation of the single chromosome substitution line, Lalbahadur(Parula7D) that carries Lr34/Yr18 was used to generate several mutant lines. Characterisation of the mutants revealed a range of highly informative genotypes, which included variable size deletions and an overlapping set of interstitial deletions. The mutants enabled a large number of wheat EST derived markers to be mapped and define a relatively small physical region on chromosome 7DS that carried Lr34/Yr18. Fine scale genetic mapping confirmed the physical mapping and identified a genetic interval of less than 0.5 cM, which contained Lr34/Yr18. Both rice and Brachypodium genome sequences provided useful information for fine mapping of ESTs in wheat. Gene order was more conserved between wheat and Brachypodium than with rice but these smaller grass genomes did not reveal sequence information that could be used to identify a candidate gene for rust resistance in wheat. We predict that Lr34/Yr18 is located within a large insertion in wheat not found at syntenic positions in Brachypodium and rice. W. Spielmeyer and R. P. Singh contributed equally to the study through the “Thatcher” and “Lalbahadur” genetic stocks, respectively.     

Identification of microsatellite markers linked to leaf rust resistance gene <Emphasis Type="Italic">Lr25</Emphasis> in wheat

The leaf rust resistance gene Lr25, transferred from Secale cereale L. into wheat and located on chromosome 4B, imparts resistance to all pathotypes of leaf rust in South-East Asia. In an F₂-derived F₃ population, created by crossing TcLr25 that carries the gene Lr25 for leaf rust resistance with leaf rust-susceptible parent Agra Local, three microsatellite markers located on the long arm of chromosome 4B were found to be linked to the Lr25 locus. The donor parent TcLr25 is a near-isogenic line derived from the variety Thatcher. The most virulent pathotype of leaf rust in the South-East Asian region, designated 77–5 (121R63-1), was used for challenging the population under artificially controlled conditions. The marker Xgwm251 behaved as a co-dominant marker placed 3.8 cM away from the Lr25 locus on 4BL. Two null allele markers, Xgwm538 and Xgwm6, in the same linkage group were located at a distance of 3.8 cM and 16.2 cM from the Lr25 locus, respectively. The genetic sequence of Xgwm251, Lr25, Xgwm538, and Xgwm6 covered a total length of 20 cM on 4BL. The markers were validated for their specificity to Lr25 resistance in a set of 43 wheat genetic stocks representing 43 other Lr genes.     

Characterization and mapping of cryptic alien introgression from <Emphasis Type="Italic">Aegilops geniculata</Emphasis> with new leaf rust and stripe rust resistance genes <Emphasis Type="Italic">Lr57</Emphasis> and <Emphasis Type="Italic">Yr40</Emphasis> in wheat

Leaf rust and stripe rust are important foliar diseases of wheat worldwide. Leaf rust and stripe rust resistant introgression lines were developed by induced homoeologous chromosome pairing between wheat chromosome 5D and 5M^g of Aegilops geniculata (U^gM^g). Characterization of rust resistant BC₂F₅ and BC₃F₆ homozygous progenies using genomic in situ hybridization with Aegilops comosa (M) DNA as probe identified three different types of introgressions; two cytologically visible and one invisible (termed cryptic alien introgression). All three types of introgression lines showed similar and complete resistance to the most prevalent pathotypes of leaf rust and stripe rust in Kansas (USA) and Punjab (India). Diagnostic polymorphisms between the alien segment and recipient parent were identified using physically mapped RFLP probes. Molecular mapping revealed that cryptic alien introgression conferring resistance to leaf rust and stripe rust comprised less than 5% of the 5DS arm and was designated T5DL·5DS-5M^gS(0.95). Genetic mapping with an F₂ population of Wichita × T5DL·5DS-5M^gS(0.95) demonstrated the monogenic and dominant inheritance of resistance to both diseases. Two diagnostic RFLP markers, previously mapped on chromosome arm 5DS, co-segregated with the rust resistance in the F₂ population. The unique map location of the resistant introgression on chromosome T5DL·5DS-5M^gS(0.95) suggested that the leaf rust and stripe rust resistance genes were new and were designated Lr57 and Yr40. This is the first documentation of a successful transfer and characterization of cryptic alien introgression from Ae. geniculata conferring resistance to both leaf rust and stripe rust in wheat.     

Identification of <Emphasis Type="Italic">Pm58</Emphasis> from <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Key message

A novel powdery mildew-resistance gene, designated Pm58, was introgressed directly from Aegilops tauschii to hexaploid wheat, mapped to chromosome 2DS, and confirmed to be effective under field conditions. Selectable KASP? markers were developed for MAS.

Abstract

Powdery mildew caused by Blumeria graminis (DC.) f. sp. tritici (Bgt) remains a significant threat to wheat (Triticum aestivum L.) production. The rapid breakdown of race-specific resistance to Bgt reinforces the need to identify novel sources of resistance. The d-genome species, Aegilops tauschii, is an excellent source of disease resistance that is transferrable to T. aestivum. The powdery mildew-resistant Ae. tauschii accession TA1662 (2n?=?2x?=?DD) was crossed directly with the susceptible hard white wheat line KS05HW14 (2n?=?6x?=?AABBDD) followed by backcrossing to develop a population of 96 BC₂F₄ introgression lines (ILs). Genotyping-by-sequencing was used to develop a genome-wide genetic map that was anchored to the Ae. tauschii reference genome. A detached-leaf Bgt assay was used to screen BC₂F_4:6 ILs, and resistance was found to segregate as a single locus (χ?=?2.0, P value?=?0.157). The resistance gene, referred to as Pm58, mapped to chromosome 2DS. Pm58 was evaluated under field conditions in replicated trials in 2015 and 2016. In both years, a single QTL spanning the Pm58 locus was identified that reduced powdery mildew severity and explained 21% of field variation (P value?<?0.01). KASP? assays were developed from closely linked GBS-SNP markers, a refined genetic map was developed, and four markers that cosegregate with Pm58 were identified. This novel source of powdery mildew-resistance and closely linked genetic markers will support efforts to develop wheat varieties with powdery mildew resistance.

     

Production of intergeneric hybrid between dwarfing polish wheat (<Emphasis Type="Italic">Triticum polonicum</Emphasis> L.) and <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Cosson. with reference to wheat origin

Dwarfing polish wheat is a dwarfing accession of Triticum polonicum L. from Xinjiang of China. In the present study, the artificial hybridization between dwarfing polish wheat and two accessions of Aegilops tauschii Cosson. (AS60 and AS65) was carried out, and the F₁ hybrids were obtained successfully without using embryo rescue techniques for the first time. The crossabilities of hybrids T. polonicum × Ae. tauschii (AS60) and T. polonicum × Ae. tauschii (AS65) were 1.67% and 0.60% respectively. Only the hybrids of T. polonicum × Ae. tauschii (AS60) germinated well, and 24 F₁ hybrid plants were obtained. All the F₁ hybrid plants grew vigorously, and the morphological traits were similar to bread wheat. The F₁ plants had some obvious traits inherited from T. polonicum and Ae. tauschii and were completely sterile. Chromosome pairing in the hybrid was characterized by a large number of univalents, with an average of 20.56 and 0.22 bivalents per PMC, and no ring bivalents and multivalents were observed. Furthermore, the potential value of the F1 hybrids between T. polonicum and Ae. tauschii for studying wheat origin and breeding are discussed. The article is published in the original.     

Microsatellite tagging of the leaf rust resistance gene <Emphasis Type="Italic">Lr16</Emphasis> on wheat chromosome 2BSc

Leaf rust, caused by Puccinia triticina, is one of the most damaging diseases of wheat worldwide. Lr16 is a widely deployed leaf rust resistance gene effective at the seedling stage. Although virulence to Lr16 exists in the Canadian P. triticina population, Lr16 provides a level of partial resistance in the field. The primary objective of this study was to identify markers linked to Lr16 that are suitable for marker-assisted selection (MAS). Lr16 was tagged with microsatellite markers on the distal end of chromosome 2BS in three mapping populations. Seven microsatellite loci mapped within 10 cM of Lr16, with the map distances varying among populations. Xwmc764 was the closest microsatellite locus to Lr16, and mapped 1, 9, and 3 cM away in the RL4452/AC Domain, BW278/AC Foremost, and HY644/McKenzie mapping populations, respectively. Lr16 was the terminal locus mapped in all three populations. Xwmc764, Xgwm210, and Xwmc661 were the most suitable markers for selection of Lr16 because they had simple PCR profiles, numerous alleles, high polymorphism information content (PIC), and were tightly linked to Lr16. Twenty-eight spring wheat lines were evaluated for leaf rust reaction with the P. triticina virulence phenotypes MBDS, MBRJ, and MGBJ, and analyzed with five microsatellite markers tightly linked to Lr16. There was good agreement between leaf rust infection type (IT) data and the microsatellite allele data. Microsatellite markers were useful for postulating Lr16 in wheat lines with multiple leaf rust resistance genes.     

Introgression of a leaf rust resistance gene from <Emphasis Type="Italic">Aegilops caudata</Emphasis> to bread wheat

Rusts are the most important biotic constraints limiting wheat productivity worldwide. Deployment of cultivars with broad spectrum rust resistance is the only environmentally viable option to combat these diseases. Identification and introgression of novel sources of resistance is a continuous process to combat the ever evolving pathogens. The germplasm of nonprogenitor Aegilops species with substantial amount of variability has been exploited to a limited extent. In the present investigation introgression, inheritance and molecular mapping of a leaf rust resistance gene of Ae. caudata (CC) acc. pau3556 in cultivated wheat were undertaken. An F(2) population derived from the cross of Triticum aestivum cv. WL711 - Ae. caudata introgression line T291-2 with wheat cultivar PBW343 segregated for a single dominant leaf rust resistance gene at the seedling and adult plant stages. Progeny testing in F(3) confirmed the introgression of a single gene for leaf rust resistance. Bulked segregant analysis using polymorphic D-genome-specific SSR markers and the cosegregation of the 5DS anchored markers (Xcfd18, Xcfd78, Xfd81 and Xcfd189) with the rust resistance in the F(2) population mapped the leaf rust resistance gene (LrAC) on the short arm of wheat chromosome 5D. Genetic complementation and the linked molecular markers revealed that LrAC is a novel homoeoallele of an orthologue Lr57 already introgressed from the 5M chromosome of Ae. geniculata on 5DS of wheat.     

Characterization of the gene <Emphasis Type="Italic">Mre11</Emphasis> and evidence of silencing after polyploidization in <Emphasis Type="Italic">Triticum</Emphasis>

The MRE11 protein is a component of the highly conserved MRN complex, along with RAD50 and NBS1. This complex is crucial in the repair of breaks in double stranded DNA, and is involved in many other cell processes. The present paper reports the molecular characterization of Mre11 gene in all three genomes of wheat, making use of the diploid species Triticum monococcum (genome A) and Aegilops Tauschii (genome D), the tetraploid T. turgidum (genomes A and B), and the hexaploid T. aestivum (genomes A, B and D). The genomic sequences characterized ranged from 4,662 to 4,766 bp in length; the cDNA corresponding to the processed mRNA was 2,440–2,510 bp long. In all cases, Mre11 coded for a highly conserved protein of 699 amino acids with a structure involving 22 exons. Mre11 expression was determined by real-time PCR in all the species analysed. The tetraploid species showed an expression similar to that of the diploid Ae. tauschii and lower than that of T. monococcum. Stronger expression was detected in the hexaploid T. aestivum. The SSCP technique was modified by introducing fluorescent labelling to the procedure in order to analyse the expression of the different Mre11 genes (i.e., those belonging to the different genomes) in the polyploid species. In both polyploids, the Mre11 gene belonging to the B genome was the least expressed. This probably reflects a first step in the process of silencing duplicate genes after polyploidization.     

Characterization of low-molecular-weight glutenin genes in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

This paper reports the characterization of the low-molecular-weight (LMW) glutenin gene family of Aegilops tauschii (syn. Triticum tauschii), the D-genome donor of hexaploid wheat. By analysis of bacterial artificial chromosome (BAC) clones positive for hybridization with an LMW glutenin probe, seven unique LMW glutenin genes were identified. These genes were sequenced, including their untranslated 3 and 5 flanking regions. The deduced amino acid sequences of the genes revealed four putative active genes and three pseudogenes. All these genes had a very high level of similarity to LMW glutenins characterized in hexaploid wheat. The predicted molecular weights of the mature proteins were between 32.2 kDa and 39.6 kDa, and the predicted isoelectric points of the proteins were between 7.53 and 8.06. All the deduced proteins were of the LMW-m type. The organization of the seven LMW glutenin genes appears to be interspersed over at least several hundred kilo base pairs, as indicated by the presence of only one gene or pseudogene per BAC clone. Southern blot analysis of genomic DNA of Ae. tauschii and the BAC clones containing the seven LMW glutenin genes indicated that the BAC clones contained all LMW glutenin-hybridizing bands present in the genome. Two-dimensional gel electrophoresis of an LMW glutenin extract from Ae. tauschii was conducted and showed the presence of at least 11 distinct proteins. Further analysis indicated that some of the observed proteins were modified gliadins. These results suggest that the actual number of typical LMW glutenins may in fact be much lower than previously thought, with a number of modified gliadins also being present in the polymeric fraction.     

Low molecular weight glutenin subunit gene composition at <Emphasis Type="Italic">Glu</Emphasis>-<Emphasis Type="Italic">D3</Emphasis> loci of <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and common wheat and a further view of wheat evolution

Key message

A comprehensive comparison of LMW-GS genes between Ae. tauschii and its progeny common wheat.

Abstract

Low molecular weight glutenin subunits (LMW-GSs) are determinant of wheat flour processing quality. However, the LMW-GS gene composition in Aegilops tauschii, the wheat D genome progenitor, has not been comprehensively elucidated and the impact of allohexaploidization on the Glu-D3 locus remains elusive. In this work, using the LMW-GS gene molecular marker system and the full-length gene-cloning method, LMW-GS genes at the Glu-D3 loci of 218 Ae. tauschii and 173 common wheat (Triticum aestivum L.) were characterized. Each Ae. tauschii contained 11 LMW-GS genes, and the whole collection was divided into 25 haplotypes (AeH01–AeH25). The Glu-D3 locus in common wheat lacked the LMW-GS genes D3-417, D3-507 and D3-552, but shared eight genes of identical open reading frame (ORF) sequences when compared to that of Ae. tauschii. Therefore, the allohexaploidization induces deletions, but exerts no influence on LMW-GS gene coding sequences at the Glu-D3 locus. 92.17% Ae. tauschii had 7-9 LMW-GSs, more than the six subunits in common wheat. The haplotypes AeH16, AeH20 and AeH23 of Ae. tauschii ssp. strangulate distributed in southeastern Caspian Iran were the main putative D genome donor of common wheat. These results facilitate the utilization of the Ae. tauschii glutenin gene resources and the understanding of wheat evolution.

     

Construction and study of leaf rust-resistant common wheat lines with translocations of <Emphasis Type="Italic">Aegilops speltoides</Emphasis> Tausch. Genetic material

Genotyping was performed for the leaf rust-resistant line 73/00i (Triticum aestivum × 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 · 5BL-5SL translocation was highly resistant to leaf rust, and the lines carrying the T6BS · 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 · 5BL-5SL translocation was preliminarily designated as LrAsp5.