Published online 20 May 2008
Published in Crop Sci 48:1124-1128 (2008)
© 2008 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Molecular Mapping of the Leaf Rust Resistance Gene Lr17a in Wheat
Barbara Bremenkamp-Barretta,
Justin D. Farisb and
John P. Fellersc,*
a Dep. of Plant Pathology, Kansas State Univ., Manhattan, KS 66506
b USDA-ARS, Cereal Crops Research Unit, Fargo, ND 58105
c USDA-ARS Plant Science and Entomology Unit, 4008 Throckmorton Hall, Manhattan, KS 66506. Mention of a trademark of a proprietary product does not constitute a guarantee of warranty of the product by the United States Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable
* Corresponding author (john.fellers{at}ars.usda.gov).
 |
ABSTRACT
|
|---|
Wheat leaf rust, caused by Puccinia triticina Eriks., infects millions of acres of wheat (Triticum aestivum L.) and causes leaves to senesce prematurely, which in turn decreases yield by reducing the plant's ability to complete kernel fill. Lr17a, a leaf rust resistance gene in wheat, is present in many wheat varieties available today. The objective of this research was to identify molecular markers linked to Lr17a. Microsatellite markers were used on a mapping population of 161 F2 lines made from a cross of Chinese Spring and Thatcher-Lr17a. Capillary fragment analysis was performed and eight markers were linked to Lr17a, of which Xgwm614 and Xwmc407 flanked Lr17a at genetic distances of 0.7 and 2.5 cM, respectively. An evaluation of cultivars with and without Lr17a, and grown in the midwestern United States, revealed that multiple alleles were present for all markers and little correspondence between alleles of closely linked markers and Lr17a was observed. Therefore, caution must taken when using them for marker-assisted selection.
Abbreviations: CS, Chinese Spring • dNTP, deoxy nucleotide triphosphate • IT, infection type • PCR, polymerase chain reaction
|
INTRODUCTION
|
|---|
ONE OF THE MOST DEVASTATING diseases of wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD genomes) is leaf rust caused by the fungal pathogen Puccinia triticina Eriks. This persistent pest decreases yield by reducing the number of kernels per spike, reducing test weight, and diminishing kernel quality. The amount of damage inflicted by leaf rust varies with the degree of infection and host plant resistance. Susceptible wheat plants can suffer yield losses up to 40% under severe infection (Marasas et al., 2004). Although fungicides can be used to control leaf rust to some degree, the most economical and effective way to control the pathogen is through host plant resistance. Over 50 leaf rust resistance genes have been identified in wheat (McIntosh et al., 1995). Though many have been overcome by the evolution of virulent rust races, some of these "defeated" genes still provide some level of resistance when combined with other resistance genes (McIntosh et al., 1995; Oelke and Kolmer, 2005).
In 1968, Dyck and Samborski described what was then a new leaf rust resistance gene, Lr17. Two lines, Klein Lucero (CI 14047) and Maria Escobar (PI 150604), were identified as having novel leaf rust resistance genes. However, both genes produced similar infection types of 0; to 1+ when challenged by different rust races. Both lines were crossed and the progeny backcrossed to Thatcher (CI 10003) for several generations to develop isogenic lines. Allelism tests, through intercrossing between the parents and the isolines, produced only resistant plants. Therefore, it was determined that the resistance was due to the same gene, which was designated Lr17. Lr17 is partially dominant and gives infection types of 1+ to 2 in the heterozygous condition. Lr17 is located on the short arm of chromosome 2A and is independent of Lr11 (Dyck and Kerber, 1977).
There are two resistance alleles at the Lr17 locus. The Australian cultivar Harrier contained an unknown resistance gene, which was given the temporary designation of LrH (McIntosh et al., 1995). Monosomic analysis determined that LrH was on chromosome 2A and allelism tests with the Thatcher Lr17 isoline could not prove that LrH was different from Lr17. Thus, LrH was redesignated as Lr17b, and the first gene was redesignated Lr17a (Singh et al., 2001). Lr17a and Lr17b exhibit increased resistance at higher temperatures, but can be separated by differential rust races (Singh et al., 2001).
Lr17a is present in several varieties adapted to North America and the popularity of these varieties has produced a significant shift in virulence to Lr17a in the leaf rust pathogen. Though the resistance has been overcome, parents containing Lr17a are still being used in breeding programs. The objective of this work was to develop polymerase chain reaction (PCR)-based markers linked to Lr17a and to evaluate allelic variation of the markers in several germplasm accessions and popular varieties.
|
MATERIALS AND METHODS
|
|---|
Plant Material and Leaf Rust Inoculations
Seed of the Thatcher Lr17a (TcLr17a) isoline (RL6008) was obtained from David Long of the Cereal Disease Laboratory (USDA, St. Paul, MN). TcLr17a was crossed with the spring wheat line Chinese Spring (CS) from which a population of 161 F2:3 families was developed and used for linkage analysis. To evaluate allelic diversity of microsatellite markers linked to the Lr17 locus, 16 varieties adapted to the Central and Southern Great Plains region and nine germplasm accessions were selected.
The P. triticina race BBBM was used to inoculate 25 individuals from each of the F3 families. Urediniospores were suspended in Soltrol 170 (Phillips 66, Bartlesville, OK) and sprayed onto seedlings at the two- to three-leaf stage. The plants were then placed in a mist chamber overnight at 18°C. Plants were transferred to a glasshouse and leaf rust ratings were taken 10 d post inoculation using the scale described by McIntosh et al. (1995).
DNA Extraction and Microsatellite Marker Analysis
Genomic DNA was extracted from individual F2 plants for the initial mapping and from pooled tissue from the F2:3 families using a modified CTAB extraction protocol (Hulbert and Bennetzen, 1991). Leaf tissue was collected from seedlings at the three- to four-leaf stage and frozen in liquid nitrogen. The leaf tissue was ground to a powder, resuspended in 2x CTAB buffer, and incubated in a 65°C water bath. Two extractions by chloroform/isoamyl alcohol (50:48:2 v/v/v) were performed on the supernatant. After centrifugation, the DNA was precipitated by the addition of 0.7 vol of 100% isopropanol to the supernatant. The DNA was then washed with 70% ethanol, dried and resuspended in 1x TE (10 mM Tris-HCl, pH 8.0 and 1 mM Na EDTA, pH 8.0). DNA for the 27 varieties and germplasm accessions used for allelic sizing was graciously provided by Drs. Bai and St. Amand of the USDA-ARS Genotyping Laboratory, Manhattan, KS.
Microsatellites previously mapped to the short arm of 2AS (Somers et al., 2004) were used to screen the parents for polymorphisms. Each 25-µL reaction consisted of the following reagents: 200 ng genomic DNA, 1x PCR Buffer (Sigma, St. Louis, MO), 1.0 mM MgCl2, 2.5 mM dNTPs, 1.25 U Taq DNA polymerase (Sigma), 5.0 pmol of reverse primer, 1.0 pmol of M13-tailed forward primer, and 5.0 pmol of M13 primers labeled with one of the following dyes: 6-FAM, NED, PET, or VIC (Applied Biosystems, Foster City, CA) (Somers et al., 2004). An MJ Research (Watertown, MA) PTC-225 thermal cycler was programmed as follows: initial denaturation at 95°C for 5 min followed by 10 cycles of denaturation at 95°C for 45 s, anneal at 68°C for 1 min, and extension at 72°C for 1 min. Each cycle reduced the anneal temperature by 2°C with the final 10 cycles running a 50°C annealing temperature. A final elongation step of 72°C for 5 min completed the reaction.
Capillary Fragment Analysis
Polymerase chain reaction products were analyzed by capillary electrophoresis on an ABI3730s (Applied Biosystems). Samples were prepared by pooling 3 µL of PCR product from four separate primer sets, each with a different dye. The DNA pool was mixed and centrifuged. One microliter of the pooled DNA was added to a mixture of 6 µL of Hi-Di formamide (Applied Biosystems), 0.25 µL of Genescan 500-LIZ size standard (Applied Biosystems), and 3 µL of water. The samples were again mixed well and centrifuged. The 96-well plate was placed on an MJ Research PTC-225 thermal cycler for 5 min at 95°C and then on an ice slurry for 5 min. Raw data files from the ABI 3730 were imported into GeneMarker v1.5 (SoftGenetics, State College, PA) for fragment analysis.
Linkage Analysis
The leaf rust reaction types were evaluated on 25 individuals from each of the F3 families and then classified as homozygous resistant, segregating (heterozygous), or homozygous susceptible and used to infer the genotypes of the corresponding F2 plants. This was repeated twice. The phenotypic marker (Lr17a), as well as the polymorphic microsatellite markers were used in linkage analysis. The linkage map was constructed using the computer program Mapmaker v2.0 (Lander et al., 1987) for Macintosh with an LOD of 3.0 and the Kosambi mapping function (Kosambi, 1944). The correctness of marker order was validated using the "Ripple" command with a LOD threshold of 3.0.
|
RESULTS
|
|---|
TcLr17a displayed a 0; to 1 infection type (IT), whereas CS showed a 3 to 3+ IT when inoculated with the P. triticina race BBBM (Fig. 1a
). Segregating F3 families derived from heterozygous F2 plants displayed three ITs, the two parental ITs plus a 1 to 2C IT (Fig. 1b). This indicates that Lr17a is partially dominant and agrees with previous findings (Dyck and Kerber, 1977). Of the F3 families, 38 were homozygous resistant (Lr17a/Lr17a), 84 were heterozygous (Lr17a/lr17a), and 39 were homozygous susceptible (lr17a/lr17a), which fits the expected 1:2:1 segregation ratio for a single locus (
2 = 0.21, P = 0.85).
View larger version (79K):
[in this window]
[in a new window]
|
Figure 1. (A) Infection types observed in the parents, Thatcher Lr17a (TcLr17a) and Chinese Spring (CS), used to make the mapping population. (B) Examples of infection types observed in the heterozygous, homozygous susceptible, and homozygous resistant, respectively, TcLr17a x CS F2:3 families.
|
|
Using the map previously published by Somers et al. (2004), we selected microsatellite markers located near the end of chromosome arm 2AS. Forty primer sets were tested on the parents. Overall, 19 primer sets revealed polymorphisms, however, only eight microsatellite markers were linked to Lr17a in the population generating a linkage map spanning a genetic distance of 61 cM (Fig. 2
). Lr17a was delineated to a 3.2-cM interval by flanking markers. The microsatellite marker Xgwm614 mapped 0.7 cM distal to Lr17a and Xwmc407 mapped 2.5 cM on the proximal side of Lr17a. Three additional markers, Xgwm636, Xwmc382_235, and Xgwm512, were linked to Lr17a but mapped distal to Xgwm614 at distances of 2.9, 4.9, and 10.3 cM, respectively. The marker Xwmc667 mapped 4.4 cM proximal to the Lr17a. Somers and Isaac (2004) reported that WMC382 amplified three fragments, 159 bp located on 2A, 171 bp located on 2B, and 222 bp located on 2A. With the M13 18 bp extension on the forward primer and the use of capillary electrophoresis, fragments of 173, 185, and 235 bp were observed in CS, but no amplification of any of the fragments occurred in TcLr17a. Each fragment was mapped individually and only the 235-bp fragment was linked (in repulsion) with Lr17a. Deletion mapping placed Xgwm512, Xwmc667, Xwmc407, Xgwm359, and Xwmc177 in the deletion bin 2AS-5 (data not shown) which is the most distal bin of chromosome arm 2AS (Qi et al., 2004)
View larger version (10K):
[in this window]
[in a new window]
|
Figure 2. Microsatellite marker linkage map of the Lr17a locus on chromosome 2A of hexaploid wheat derived from analysis of 161 Thatcher Lr17a x Chinese Spring (TcLr17a x CS) F2:3 families.
|
|
To evaluate allelic diversity, the microsatellite markers linked to the Lr17 locus were evaluated in several varieties and germplasm accessions. Multiple alleles were found for all of the markers, but only three different allele sizes were observed for Xgwm512 (Table 1
). As stated above, the primer set for Xwmc382 amplified three fragments in CS, however, eight different fragment sizes, similar in size to the 235-bp mapped fragment, were found among the various lines. Six different fragment sizes were observed for Xgwm636, and eight were found for Xgwm614 (Table 1). The markers Xwmc407 and Xwmc667 showed five and seven different fragment sizes, respectively.
View this table:
[in this window]
[in a new window]
|
Table 1. Alleles of microsatellite markers mapped to the Lr17a locus that are within a select group of popular varieties and germplasm accessions from the Central and Southern Great Plains of the United States.
|
|
|
DISCUSSION
|
|---|
Previous work showed that Lr17 is on chromosome 2A (Dyck and Kerber, 1977) and resistance is controlled by a single, partially dominant gene (Dyck and Samborski, 1968). The precise chromosomal location of Lr17 was not previously established, but it was inferred to be located distally on the short arm of chromosome 2A due to close repulsion linkage of Lr17 to a translocation from Aegilops ventricosa (Tausch) Ces. that contains the genes Yr17/Lr37/Sr38 contained in the cultivar VPM1 (Bariana and McIntosh, 1993). The data contained herein demonstrates that Lr17a is located in the distal region of the short arm of chromosome 2A. Two of the closest markers, Xgwm636 and Xgwm614, were previously mapped to a terminal chromosome deletion of 2AS (Sourdille et al., 2004).
The order of markers on our genetic map agreed with other genetic maps of hexaploid wheat. Röder et al. (1998), based on the ITMI Synthetic x Opata population, demonstrated that Xgwm636 and Xgwm614 cosegregated and were distal to Xgwm359. Based on a high density consensus map developed by Somers et al. (2004), the marker order in our population is the same and genetic distances are similar, with two exceptions. Xwmc382 mapped proximal to Xwmc407 on the map by Somers et al. (2004), whereas Xwmc382_235 mapped distal to Xgwm614 on our map. Because the WMC382 primer set detected multiple fragments, it is possible that we mapped a different locus than that of Somers et al. (2004). Another discrepancy between our map and that of Somers et al. (2004) is that Xwmc667 was placed by Somers et al. (2004) distal to Xgwm636, but this marker mapped proximal to Xwmc407 on our map. When compared to the Thatcher recurrent parent, it appears that the segment containing Lr17a is contained within a region between Xgwm614 and Xwmc667.
The survey of varieties and germplasm accessions demonstrates that there is a high degree of variability in the short arm of chromosome 2A. Of the varieties evaluated, only Jagger, Jagalene, Trego, and Jupateco 73 have been postulated to contain Lr17, based on reaction to differentiating rust pathotypes (A. Fritz, Kansas State University, personal communication, 2007). However, it is unknown whether they contain Lr17a or Lr17b. Our results show that TcLr17a, Jupateco, Nuplains, and Trego have the same 144-bp allele detected by Xgwm614. The pedigree of Trego has TcLr17a (RL6008) in its background (Martin et al., 2001) and thus the marker allele evidence indicates that TcLr17a (RL6008) is the source of Lr17a. NuPlains also had the same allele size as TcLr17a for Xwmc407, indicating that this variety is likely to contain the same Lr17a segment as TcLr17a.
For Jagger and Jagalene, none of the alleles observed for the markers closely linked to Lr17a correspond to those observed in TcLr17a. It is still possible that Jagger and Jagalene contain the same gene as TcLr17a, but the segment containing Lr17a is small and not inclusive of the flanking markers. This cannot be confirmed until more tightly linked markers are identified and mapped in high-resolution mapping populations generated from these genotypes, or when the gene is cloned. No other lines share the allele sizes of the closest markers found in TcLr17a. The line containing Lr17b was not available and thus the allele size could not be determined. Also, the fungal isolates that differentiate Lr17a and Lr17b were not available
Though virulence is present in the leaf rust pathogen P. triticina, varieties containing Lr17a are still popular in the Midwest and throughout the world due to yield and baking qualities and are still used as parents in many breeding programs. The work presented here has led to the identification of closely linked markers that flank the resistance gene Lr17a. However, caution must be taken when using the markers in selection schemes, as numerous marker alleles are present at the locus. Marker alleles and the state of Lr17a should be determined before selecting genotypes as sources of Lr17 in variety development programs. With that in mind, the markers can be useful in marker-assisted selection schemes to ensure the Lr17a allele is transferred to progeny and to pyramid multiple leaf rust resistance genes.
|
ACKNOWLEDGMENTS
|
|---|
The authors would like to thank Beth Gillett, Sally Hermann, Steve Brooks, Micah Hoelscher, Katie Gleason, Dehlia Burdan, and Amy Bernardo for their technical assistance during the project. Also, we would like to thank Drs. Craig Webb, Gina Brown-Guidera, and Paul St. Amand for their input during the preparation of the manuscript. This project was funded by the USDA-ARS CRIS project 5430-21000-005-00D.
|
NOTES
|
|---|
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication July 9, 2007.
|
REFERENCES
|
|---|
- Bariana, H.S., and R.A. McIntosh. 1993. Cytogenetic studies in wheat XV: Location of rust resistance genes in VPM1 and their genetic linkage with other disease resistance genes in chromosome 2A. Genome 36:476–482.[Medline]
- Dyck, P.L., and E.R. Kerber. 1977. Inheritance of leaf rust resistance in wheat cultivars Rafaela and EAP 26127 and chromosome location of Lr17. Can. J. Genet. Cytol. 19:355–358.[Web of Science]
- Dyck, P.L., and D.J. Samborski. 1968. Host-parasite interactions involving two genes for leaf rust resistance in wheat. p. 245–250. In E.W. Findlay and D.W. Shepherd (ed.) Proc. of the 3rd Int. Wheat Genet. Symp. Australian Acad. of Sci., Canberra, Australia.
- Hulbert, S.H., and J.L. Bennetzen. 1991. Recombination at the Rp1 locus of maize. Mol. Gen. Genet. 226:377–382.[CrossRef][Web of Science][Medline]
- Kosambi, D.D. 1944. The estimation of map distances from recombination values. Ann. Eugen. 12:172–175.
- Lander, E.S., P. Green, J. Abrahamson, A. Barlow, M.J. Daly, S.E. Lincoln, and L. Newberg. 1987. MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174–181.[CrossRef][Medline]
- Marasas, C.N., M. Smale, and R.P. Singh. 2004. The economic impact in developing countries of leaf rust resistance breeding in CIMMYT-related spring bread wheat. Int. Maize and Wheat Improvement Center, Mexico, D.F.
- Martin, T.J., R.G. Sears, D.L. Seifers, T.L. Harvey, M.D. Witt, A.J. Schlegel, P.J. McCluskey, and J.H. Hatchett. 2001. Registration of ‘Trego’ wheat. Crop Sci. 41:929a–930a.
- McIntosh, R.A., C.R. Wellings, and R.F. Park. 1995. Wheat rusts: An atlas of resistance genes. Kluwer Academic, Boston, MA.
- Oelke, L.M., and J.A. Kolmer. 2005. Genetics of leaf rust resistance in the spring wheat cultivars Alsen and Norm. Phytopathology 95:773–778.[Medline]
- Qi, L.L., B. Echalier, S. Chao, G.R. Lazo, G.E. Butler, O.D. Anderson, E.D. Akhunov, J. Dvorak, A.M. Linkiewicz, A. Ratnasiri, J. Dubcovsky, C.E. Bermudez-Kandianis, R.A. Greene, R. Kantety, C.M. La Rota, J.D. Munkvold, S.F. Sorrells, M.E. Sorrells, M. Dilbirligi, D. Sidhu, M. Erayman, H.S. Randhawa, D. Sandhu, S.N. Bondareva, K.S. Gill, A.A. Mahmoud, X.-F. Ma, Miftahudin, J.P. Gustafson, E.J. Conley, V. Nduati, J.L. Gonzalez-Hernandez, J.A. Anderson, J.H. Peng, N.L.V. Lapitan, K.G. Hossain, V. Kalavacharla, S.F. Kianian, M.S. Pathan, D.S. Zhang, H.T. Nguyen, D.-W. Choi, R.D. Fenton, T.J. Close, P.E. McGuire, C.O. Qualset, and B.S. Gill. 2004. A chromosome bin map of 16,000 expressed sequence tag loci and distribution of genes among the three genomes of polyploid wheat. Genetics 168:701–712.[Abstract/Free Full Text]
- Röder, M.S., V. Korzun, K. Wendehake, J. Plaschke, M. Tixier, P. Leroy, and M.W. Ganal. 1998. A microsatellite map of wheat. Genetics 149:2007–2023.[Abstract/Free Full Text]
- Singh, D., R.F. Park, H.S. Bariana, and R.A. McIntosh. 2001. Cytogenetic studies in wheat XIX: Chromosome location and linkage studies of a gene for leaf rust resistance in the Australian cultivar ‘Harrier’. Plant Breed. 120:7–12.[CrossRef]
- Somers, D.J., and P. Isaac. 2004. SSRs from the Wheat Microsatellite Consortium. Available at wheat.pw.usda.gov/ggpages/SSR/WMC/ (verified 31 Mar. 2008). USDA-ARS Plant Genome Research Program.
- Somers, D.J., P. Isaac, and K. Edwards. 2004. A high-density microsatellite consensus map for bread wheat (Triticum aestivum L.). Theor. Appl. Genet. 109:1105–1114.[CrossRef][Web of Science][Medline]
- Sourdille, P., S. Singh, T. Cadalen, G.L. Brown-Guedira, G. Gay, L. Qi, B.S. Gill, P. Dufour, A. Murigneux, and M. Bernard. 2004. Microsatellite-based deletion bin system for the establishment of genetic-physical map relationships in wheat (Triticum aestivum L.). Funct. Integr. Genomics 4:12–25.[CrossRef][Medline]
TOP
ABSTRACT
INTRODUCTION
