Clarke FR

Clarke JM

Fetch T

Knox RE

Pozniak CJ

Reimer S

Singh AK

Somers D

English

Sydney eScholarship

 1Association mapping of UG99 resistance in a diverse durum wheat population  Pozniak CJ1, Reimer S1, Fetch T 2, Clarke JM 3, Clarke FR 3, Somers D2, Knox RE 3, and Singh AK 3 1 Crop Development Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N 5A8. 2Agriculture and Agri-Food Canada, Cereal Research Centre, Winnipeg, Manitoba, Canada, R3T-2M9. 3Agriculture and Agri-Food Canada, Semi-arid Prairie Agriculture Research Centre, Swift Current, Saskatchewan, Canada, S9H 3X2  INTRODUCTION Association mapping (AM) is a complementary strategy to bi-parental mapping for describing associations between genotype and phenotype in crop plants (Somers et al. 2007).  An attractive feature of AM is the ability to perform marker trait associations in well phenotyped breeding populations and locally adapted varieties.  Association mapping has proven to be an effective strategy to identify marker-trait associations for important traits in wheat (Breseghello and Sorrells, 2006), including resistance to diseases (Crossa et al. 2007; Tommasini et al. 2007).    Stem or black rust, caused by Puccinia graminis f. sp. tritici Eriks. & Hennis has caused severe losses to hexaploid wheat (Triticum aestivum L.) and durum wheat (T. turgidum var durum) production.  Stem rust has been controlled effectively through the use of genetic resistance.  However, the identification of stem rust race TTKS (UG99) is of great concern because this race has broad virulence to most Sr genes (Jin et al. 2007) and threatens global wheat production.  Studies of the virulence of TTKS on hexaploid wheat varieties are increasing but data on the virulence of TTKS on durum wheat and the availability of molecular markers for selection for resistance is lacking.  The objectives of this study were to survey virulence of TTKS on a collection of global durum wheat varieties and to perform association mapping to identify genomic regions that could be the target of marker-assisted selection and further genetic dissection in relevant genetic mapping populations.   MATERIALS AND METHODS AM Population and Molecular Analysis– Ninety-six diverse durum wheat cultivars and breeding lines collected from breeding programs in Canada, Argentina, Australia, France, Italy, Germany, Mexico, Morrocco, United States, New Zealand, Russia, Iran and Spain  formed the AM population.  Genotyping was performed on an ABI 3100 capillary electrophoresis using M13-labeled microsatellites.  A total of 241 microsatellite (SSR) markers were used to amplify 245 loci (Somers et al. 2007).   Trait Analysis and AM Mapping –Resistance to stem rust race TTKS was assessed in a field trial at Njoro, Kenya in 2007. UG99 seedling tests using race TTKS were also performed in replicated greenhouse trials.  Prior to AM analysis, population structure was assessed using the program STRUCTURE v.2 (Pritchard et al, 2000).  Structure parameter settings were:  linkage model, allele frequencies correlated, burn-in length 10,000, and 10,000 repetitions.  The highest likelihood of the data was observed for K=5 and a Q matrix was estimated as the average of five runs for K=5. Using field severity and incidence ratings and seedling infection types, marker-trait associations were determined using a general linear model in TASSEL version 2.0.1 (Yu et al. 2006) with the Q-matrix as covariates.  For structure and marker-trait associations, rare alleles (frequency <5%) were either combined into a single genotypic class if their combined frequency was greater than 5%, or scored as missing data.  Significance of associations between loci and disease data was based on an F-test, at a significance level of P≤0.01, corrected for by performing 10,000 permutations. RESULTS AND DISCUSSION In the field trial in Kenya, infection responses ranged from susceptible to resistant with a high proportion of lines rated as resistant (26%) or moderately resistant (18%) (Table 1).  The majority of durum varieties derived from Spanish breeding programs were rated moderately susceptible to stem rust.  Varieties and breeding lines from Canadian and USA breeding programs were moderately resistant or resistant (Table 1).    In greenhouse seedling tests, 23% of lines were highly susceptible to TTKS, with the majority being from Italy (Table 1).  There was a poor relationship between field and seedling reactions, with many lines rated susceptible at the seedling stage displaying good field resistance to stem rust.  Likewise, many breeding lines and varieties susceptible in field screening displayed good resistance to TTKS in greenhouse testing (Table 1).  This suggests the presence of independent seedling and adult plant resistance genes in this population.  Alternatively, it is known that other stem rust races were present in the Njoro field trial, making it difficult to correlate field and greenhouse reactions.   Association mapping was performed on both field and seedling data, and several chromosome regions were identified that were significantly associated with either field severity and infection response, or seedling infection type (Table 2).   2 Table 1.  Seedling infection type (IT) and field severity (Sev) and infection responses (IR) on adult plants in a field nursery at Njoro, Kenya.    Four genomic regions significant for both field and seedling data were identified on 1B, 2A, 6A, and 7A (Table 2), and these chromosomes house known Sr genes.  Two regions were identified on chromosome 7A, one distal to the centromere, and a second at gwm276 (Table 2).  Sr22 is linked to gwm276, and that gene is effective against TTKS (Jin et al., 2007).  Sr13 resides on 6A and is effective against TTKS and is derived from durum wheat variety “Khapli”.  Marker gwm617 was significantly associated with TTKS resistance (Table 2) and is likely marking Sr13 as they are both located on the distal region of 6AL.  Sr14 is also derived from “Khapli” has been localized distal to the centromere on 1B, and provides intermediate resistance TTKS (Jin et al. 2007).  The cfd48 (Table 2) locus on 1B associated with field and seedling resistance is also located distal to the centromere and is likely detecting variation at Sr14.   Table 2.  Chromosome regions and significance of markers associated with field stem rust severity (Sev), and infection response (IR) and seedling  infection type (IT) determined in greenhouse trials.    Chrom. Position (cM) Marker Sev IR IT 1A 6 gdm133 0.016 0.020 ns1B 40 cfd48 0.019 0.011 0.005 2A 16 wmc382 ns ns 0.017  55 gwm95 ns ns 0.031  60 gwm372 0.031 0.018 0.043  85 cfd168 ns ns 0.014 3B 39 wmc808 ns ns 0.002  150 gwm340 0.014 0.016 ns 4B 38 barc20 0.012 0.010 ns 5A 138 gwm216 0.041 ns 0.000  153 wmc727 0.002 0.006 ns 5B 84 wmc537 0.003 0.002 ns 5B 82 Cdu1 0.007 0.008 ns 6A 95 gwm617 0.031 0.019 0.008 7A 40 wmc283 0.018 0.011 0.035 7A 83 gmw276 0.016 0.012 0.043 7B 143 cfa2040 ns ns 0.000  Barc20 on 4B (Table 2) was significantly associated with variation in disease resistance and is linked to the lipoxygenase gene Lpx-B1.1.  Lipoxygenase is known to play a role in disease resistance and enzyme activity has been reported to increase in wheat treated with a rust fungal elicitor (Bohland et al. 1997).  Gene SrTmp from winter wheat cultivar ‘Triumph 64’ is effective against TTKS (Jin et al., 2007) and that gene is also believed to reside on 4B.  More research is required to determine if barc20 is linked to SrTmp-derived resistance.    Several markers on 2A were significant and Sr genes, Sr21, Sr32, Sr34 and Sr38, are located on that  3chromosome.  Sr32 is effective against TTKS at both the seedling and adult plant stages (Jin et al. 2007).   Only gwm372 was significant for both seedling and adult plant resistance and maybe associated with Sr32, but further testing would be required to validate this hypothesis.      In the Kenya field nursery, “Langdon” was rated as 30MSS (Table 1).  In contrast, its near isogenic line “Langdon[DIC6B]”, which contains a substituted 6B chromosome for T. dicoccoides,  was rated 5RMR.  These results suggest that an effective gene(s) for Sr resistance exists on 6B from T. dicoccoides.  However, no 6B markers were associated with resistance in the AM population (Table 1).     Association mapping proved effective at identifying genomic regions associated with TTKS resistance in durum wheat, but these regions should be validated to determine their effectiveness in marker assisted selection programs.  We are in the process of developing mapping populations from selected lines used in this study that appear to have unique Sr genes based on the haplotypes identified at significant loci (Table 2).   In addition, the poor relationship between seedling and adult plant resistance needs to be further examined to identify those regions which will provide broad-spectrum resistance, particularly at the development stage when plants are most likely to become infected.    ACKNOWLEDGEMENTS We gratefully acknowledge funding of this research by the Western Grains Research Foundation, Agriculture and Agri-Food Canada, and the National Sciences and Engineering Research Council of Canada.    REFERENCES  Bohland C, Balkenhohl T, Loers G, Feussner I, and Grambow H. (1997) Differential Induction of lipoxygenase isoforms in wheat upon treatment with Rust Fungus Elicitor, Chitin Oligosaccharides, Chitosan, and Methyl Jasmonate. Plant Physiol. 114, 679-685. Breseghello F. and Sorrells, M.E. (2006) Association mapping of kernel size and milling quality in wheat (Triticum aestivum L.) cultivars. Genetics 172:1165–1177. Crossa J, Burgueno J, Dreisigacker S, Varga M, Herrera-Foessel SA, Lillemo M, Singh RP, Trethowan R, Warburton M, Franco J, Reynolds M, Crouch J, and Ortiz R.  (2007) Association analysis of historical wheat germplasm using additive genetic covariance of relatives and population structure. Genetics 177: 1889-1913. Jin Y, Singh RP, Ward RW, Wanyera R, Kinyua M, Njau, P, Fetch T, Pretorius Z A, and Yahyaoui, A. (2007) Characterization of seedling infection types and adult plant infectionresponses of monogenic Sr gene lines to race TTKS of Puccinia graminis f. sp. tritici. Plant Dis 91:1096-1099.  Pritchard, J.K., Stephens, M., and Donnelly, P. 2000. Inference of population structure using multilocus genotype data. Genetics 155:945-959. Somers D, Isaac P, and Edwards K. (.2004) A high-density microsatellite consensus map for bread wheat (Triticum aestivum L.). Theor Appl Genet 109, 1105-1114. Somers DJ, Banks T, DePauw RM, Fox S, Clarke JM, Pozniak C, and McCartney C (2007) Genome-wide linkage disequilibrium analysis in bread wheat and durum wheat.  Genome 50:557 – 567. Yu, J., Pressoir, G., Briggs, W.H., Bi, I.V., Yamasaki, M., Doebley, J.F., McMullen, M.D., Gaut, B.S., Nielsen, D.M., Holland, J.B., Kresovich, S., and Buckler, E.S. 2006. A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nature Genetics 38:203-208.   

Association mapping of Ug99 resistance in a diverse durum wheat population

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Association mapping of Ug99 resistance in a diverse durum wheat population

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