The Lr34/Yr18/Sr57/Pm38/Ltn1 multi-resistance locus has been deployed and remained effective in wheat cultivars for more than 100 years. The durability and pleiotropic nature makes Lr34 a unique and highly valuable resource for rust resistance breeding. Despite its functional annotation as an ABC transporter, the mode of action is unknown. Considering this, we aimed to decipher molecular factors and signaling components essential for Lr34 function using RNA-seq of Chara resistant (Lr34) and Chara mutant (heavy ion irradiation, HII) susceptible wheat lines. Screening of Chara and Chara HII lines with Lr34-specific markers confirmed the integrity of Lr34 in both lines; however, phenotyping confirmed rust and powdery mildew susceptibility in the Chara HII lines. Plants were grown under controlled conditions and infected with Puccinia triticina pathotype 76-1,3,5,7,9,10,12,13+Lr37 at the flag leaf stage. Flag leaves were sampled at 0, 24, 48, 72, 96 and 168 hours post inoculation (hpi) from mock and infected plants. Based on real-time PCR analysis of basal defense genes and the Lr34 gene, we selected 72 hpi for RNA-seq with four biological replicates per condition. The samples were sequenced on an Illumina Hiseq 4000 at the Beijing Genomics Institute, China. A total of 9.0 Gb of sequence (2.25 Gb/library) from 16 libraries for four conditions was obtained. Differential expression analysis was performed using the Tuxedo analysis pipeline with standard parameters. Analysis revealed deletion of DNA fragments with collinear gene order on chromosomes 1A, 2D, 5A, 5B, 5D and 7D of Chara HII mutants. To determine the significance of the deletions we performed bulk segregant analyses on segregating F2 populations of Chara ? Chara HII crosses. Analyses revealed key genomic regions associated with Lr34-functional resistance and we are in the process of validating candidate genes using qPCR.
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A key objective of BGRI is to breed high yielding, stem rust resistant spring wheat germplasm suitable for releases as successful varieties in wheat growing countries of Africa, Middle East, Asia and Latin America. High emphasis was given to select adult plant resistance (APR) to stem rust in achieving this goal that is especially important in East African highlands where various variants belonging to the Ug99 race group and other lineages of stem rust fungus are now known, disease is endemic and present throughout the year on wheat crops. Recent molecular mapping studies show that combinations of partially effective APR gene Sr2 with 3 to 4 additional APR genes such as Sr55, Sr56, Sr57, Sr58 and other undesignated quantitative trait loci confer adequate to high levels of resistance to stem rust. A ‘Mexico-Kenya shuttle breeding scheme’ was initiated in 2008 to select APR to stem rust under high disease pressures at Njoro, Kenya while selecting for resistance to other rusts, yield, agronomic and quality traits in Mexico. This selection scheme, combined with phenotyping of advanced lines for multiple seasons in Kenya has resulted in identifying a small frequency of high yielding lines that possess a high level of resistance with a stable and low stem rust severity performance over seasons/locations under high disease pressures. These near-immune wheat lines are the best candidates for release in East Africa to achieve durable disease control and simultaneously curtail, or reduce, further selection of new virulences. A significantly higher proportion of wheat lines were also developed with moderate levels of resistance that is considered suitable for deployment in wheat growing areas where rust builds up later in the season. The worldwide distribution of the wheat lines derived from Mexico-Kenya shuttle breeding initiated in 2012 through the international yield trials and nurseries from CIMMYT. Potential releases and cultivation of these lines in different countries together with a reduction in area sown to susceptible varieties are expected to reduce the threat from stem rust.
The Lr34/Yr18 gene has been used in agriculture for more than 100 years. In contrast to many other resistance sources against leaf rust and stripe rust, it has remained effective and no virulence has been reported. This makes Lr34 a unique and highly valuable resource for rust resistance breeding. The pleiotropic nature of the gene conferring partial resistance to different pathogen species, the associated leaf tip necrosis and its durability suggest a molecular mechanism that is different from major gene resistance. This is supported by the molecular nature of Lr34 which was recently found to encode an ABC transporter. Interestingly, all tested wheat lines contain an allele of the Lr34 gene on chromosome 7DS. In its susceptible form, the gene does not confer resistance. The difference between the encoded resistant and susceptible LR34 isoforms consists of only two amino acid changes, whereas the rest of the proteins are identical. These two changes must change the biochemical properties of the resistant LR34 transporter in such a way that the plant becomes resistant. We speculate that there is a slight conformational change in the resistant form of the protein, resulting either in modified specificity or kinetics of the transported molecule, or that the binding properties to an unknown second protein interacting with LR34 are changed, resulting in altered function. While the molecular nature of the molecule(s) transported by the LR34 protein remains unclear, it is likely that a physiological change related to Lr34 activity is at the basis of resistance. We are currently establishing transgenic approaches in heterologous grass species to further investigate the molecular activity of Lr34 and to better understand a physiological mechanisms resulting in disease resistance.
Abstract A recombinant inbred line (RIL) population derived from the cross Arina/Forno was field tested for 2 years against Puccinia graminis f. sp. tritici under artificially created epidemic conditions. Both parents showed intermediate adult plant stem rust responses and the RIL population showed continuous variation for this trait. Composite interval mapping identified genomic regions controlling low stem rust response on chromosomes 5B and 7D consistently across all experiments. These genomic regions were named QSr.Sun-5BL and QSr.Sun-7DS and explained on an average 12% and 26% of the phenotypic variation in adult plant stem rust response, respectively. QSr.Sun-5BL mapped close to Xglk0354 and was contributed by Arina. The Lr34-linked markers csLV34 and swm10 were closely associated with QSr.Sun-7DS suggesting the involvement of Lr34 in controlling adult plant stem rust response of cultivar Forno. Additional minor and inconsistent QTLs explaining variation in adult plant stem rust response were identified on chromosome arms 1AS and 7BL. The QTL located on chromosome 7BL corresponded to the stem rust resistance gene Sr17 carried by cultivar Forno. A seedling stem rust resistance gene carried by Arina, SrAn1, was ineffective under field conditions and was mapped on the long arm of chromosome 2A. Genotypes carrying combinations of QSr.Sun-5BL and QSr.Sun-7DS based on positive alleles of the respective closest marker loci Xglk0354 and XcsLV34 or Xswm10 exhibited a lower response than either parent indicating an additive effect of these genes. Transfer of these genes into cultivars carrying Sr2 would provide a more effective and durable resistance against the stem rust pathogen. Markers csLV34 and/or swm10 could be used in marker assisted selection of QSr.Sun-7DS in breeding programs.