SNP-Based Improvement of a Microsatellite Marker Associated with Karnal Bunt Resistance in Wheat

Marker-assisted selection (MAS) has become the technology of choice for introgressing important traits with indistinct phenotypes into agronomically elite cultivars. Karnal bunt (KB, causal agent Tilletia indicaMitra) is an economically important fungal pathogen of wheat (Triticum aestivum L.) which has caused economic losses in the USA since it was first reported in 1996. To protect U.S. wheat from this emerging disease and the losses incurred from export quarantines, genetic sources of resistance are needed by breeders to improve U.S. germplasm. Resistance to KB is difficult to score phenotypically, making MAS an ideal choice for deploying this trait into U.S. wheat. Here we describe the conversion of a codominant microsatellite marker, Xgwm538, associated with a quantitative trait locus (QTL) for KB resistance into a single nucleotide polymorphism (SNP) based marker. The SNP marker was developed to improve gel-based resolution and amplification consistency. The gwm538 primers amplify three fragments in the KB resistant line HD29: 137-, 147-, and a 152-bp fragment that maps to the long arm of chromosome 4B and is linked to the KB QTL. By cloning and sequencing all three fragments, we were able to exploit a SNP and design a new primer to selectively amplify the 152-bp fragment of interest (gwm538snp). Amplification consistency is improvedwith gwm538snp since the amplification of competing nontarget fragments is eliminated, and ambiguity is reduced since heterozygous plants are easily identified among backcross progeny. M markers are commonly used to rapidly survey the genome of cereal crops for association with known genes. In recent years, the development of high-density microsatellite-based genetic linkage maps for wheat has provided genome-wide marker availability (Somers et al., 2004). High polymorphism detection levels, high throughput capability, and low cost compared to other marker systems enhance the utility of microsatellites for mapping genes associated with agronomically important traits and for MAS strategies (reviewed by Dubcovsky, 2004). MAS is especially valuable when selecting for traits that are difficult to screen for, such as resistance to Karnal bunt disease of wheat (http://maswheat.ucdavis.edu; URL verified 21 March 2006; reviewed by Dubcovsky, 2004; Dekkers and Hospital, 2002). KB resistance is identified in the adult plant stage by determining disease incidence as a percentage of bunted kernels following harvest (Singh et al., 2003). Field screening is labor intensive, expensive, and the disease is highly influenced by environmental factors. KB was discovered in the USA in 1996 and poses a serious threat to the wheat export industry because of international quarantine regulations (Ykema et al., 1996). Development of resistant cultivars is an important preemptive measure to prevent the spread of this emerging disease. Since T. indica is a quarantined pathogen in the USA, MAS is the only feasible option for incorporating KB resistance into U.S. wheat germplasm. Singh et al. (2003) reported the association of microsatellite markerXgwm538with a QTL for KB resistance on the long arm of chromosome 4B, that accounted for 18.3% of disease variation (P value 5 0.00) on average in a recombinant inbred line (RIL) mapping population. Xgwm538 was predicted to be valuable for use in MAS to incorporate KB resistance genes into elite germplasm;however,direct implementationofXgwm538 into MAS programs has been difficult because of spurious amplification of target sequences and the masking of the target allele by a paralogous fragment resulting in an inability to identify heterozygotes among segregating progeny. We have found that these problems are not unique to gwm538, and others also have reported difficulties with microsatellite marker resolution (Bryan et al., 1995; Hourihan et al., 2001). Here we described the development of a single nucleotide polymorphism (SNP) based marker from Xgwm538. The SNP strategy was used in a similar fashion as described by Somers et al. (2003) where locus-specific sequence differences were used to distinguish paralogous and homoeologous sequences. Our objective was to selectively amplify the allele associated KB resistance and produce by MAS a marker that has direct utility for cultivar enhancement. MATERIALS AND METHODS Plant Material and Genomic DNA Isolation The following plant materials were provided by the Wheat Genetics Resource Center at Kansas State University: Chinese Spring (CS), CS derived nullisomic–tetrasomic (NT) lines (N4AT4D, N4BT4D, and N4DT4B; Sears, 1954, 1966)), the International Triticeae Mapping Initiative (ITMI) mapping population (TA4040; Nelson et al., 1995) and the KBRILmapping population HD29(KB resistant) 3 WL711(KB susceptible) (TA4325, Singh et al., 2003). One hundred thirty F2:8 RILs were scored for disease incidence for 3 yr at the Punjab Agricultural University, Punjab, India (Singh et al., 2003). Seedling leaf tissue was harvested from all 130 RILs at the three-leaf stage for genomic DNA isolation by a CTAB DNA extraction protocol (Hulburt and Bennetzen, 1991). Steven Brooks, Dale Bumpers National Rice Research Center, USDA ARS, 2890 Hwy 130 E. (P.O. Box 1090), Stuttgart, AR 72160; Deven See, Department of Plant Pathology, Kansas State University, Manhattan, KS 66506; Gina Brown-Guedira, USDA ARS, Plant Science Research Unit, North Carolina State University, Raleigh, NC 27606. Mention of a trademark of a proprietary product does not constitute a guarantee or 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. Received 11 May 2005. *Corresponding author ([email protected]). Published in Crop Sci. 46:1467–1470 (2006). Genomics, Molecular Genetics & Biotechnology–Note doi:10.2135/cropsci2005.05-0065 a Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . 1467 Published online May 18, 2006