Published online 18 May 2006
Published in Crop Sci 46:1467-1470 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY–NOTE
SNP-Based Improvement of a Microsatellite Marker Associated with Karnal Bunt Resistance in Wheat
Steven A. Brooksa,*,
Deven R. Seeb and
Gina Brown-Guedirac
a Dale Bumpers National Rice Research Center, USDA ARS, 2890 Hwy 130 E. (P.O. Box 1090), Stuttgart, AR 72160
b Department of Plant Pathology, Kansas State University, Manhattan, KS 66506
c USDA ARS, Plant Science Research Unit, North Carolina State University, Raleigh, NC 27606
* Corresponding author (ricegenes{at}mac.com)
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ABSTRACT
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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 indica Mitra) 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 improved with gwm538snp since the amplification of competing nontarget fragments is eliminated, and ambiguity is reduced since heterozygous plants are easily identified among backcross progeny.
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INTRODUCTION
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MICROSATELLITE-based 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 marker Xgwm538 with a QTL for KB resistance on the long arm of chromosome 4B, that accounted for 18.3% of disease variation (P value = 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 implementation of Xgwm538 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.
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MATERIALS AND METHODS
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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 KB RIL mapping population HD29(KB resistant) x 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).
PCR Conditions and Fragment Separation
PCR was performed with the Promega Taq DNA polymerase kit (Promega, Madison, WI). A 25-µL reaction was used containing 50 ng of template DNA, 1x concentration reaction buffer, 0.1 mM dNTPs, 1.25 mM MgCl2, 4 pmol of each primer and 1.25 units of Taq DNA Polymerase. MJ Research (Waltham, MA) PTC-200 thermal cycler conditions were as follows: initial denaturing step at 94°C for 3 min followed by 35 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1.5 min. Primer sequences for gwm538 were obtained from Röder et al. (1998): gwm538F 5'-GCATTTCGGGTGAACCC-3' and gwm538R 5'-GTTGCATGTATACGTTAAGCGG-3'. gwm538snpF1 was designed by extending the original forward primer by five nucleotides on the 3' end (5'-GCATTTCGGGTGAACCCATCAT-3'). Ten microliters of each reaction was run on 2.3% (w/v) agarose gels in 0.5x TBE for 3 h at 5 V per centimeter of gel length. Gels were stained with a solution of ethidium bromide in water (0.5 µg/mL) for 30 min on a rotary platform shaker and destained for 5 min with water before visualization with a UV light box.
TA Cloning and Plasmid Preparations
CS derived NT lines N4BT4D and N4DT4B were used to amplify and clone PCR products of gwm538. Excised bands were purified with the QIAGEN MinElute Gel Extraction Kit, reagents and protocol (QIAGEN, Valencia, CA). PCR products were cloned with the TOPO TA Cloning kit, reagents and protocol (Invitrogen, Carlsbad, CA). Amplified products were ligated directly into plasmid vector pCR 2.1-TOPO and transformed into TOP10 One Shot chemically competent E. coli cells (Invitrogen). Plasmids were prepared for sequencing with the QIAprep Spin Miniprep Kit, reagents, and protocol (QIAGEN, Valencia, CA).
Sequencing and Sequence Analysis
Purified plasmids were sequenced directly with primers M13 reverse and T7 by the Kansas State University DNA Sequencing Facility (Manhattan, KS). Three independent clones were sequenced from both directions for each PCR fragment. Vector sequence was trimmed manually and alignment of sequences was performed with AssemblyLIGN version 1.0.9c (Oxford Molecular Ltd., Madison, WI).
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RESULTS
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Two polymorphic alleles of 152 and 171 bp in the resistant (HD29) and susceptible (WL711) parent, respectively, were amplified by gwm538. Two additional monomorphic fragments (137 and 147 bp) also were amplified, and the 147-bp fragment masked the 152-bp fragment in HD29 (Fig. 1). In Lane 2, HD29 appears to have only two fragments, whereas WL711 (Lane 3) clearly has three fragments. What appears as a single upper band in HD29 is actually both the 152- and 147-bp fragments, that can only be resolved on denaturing polyacrylamide sequencing gels (not shown). Furthermore, the monomorphic fragments compete for amplification in the PCR reaction resulting in inconsistent amplification of the target sequence (not shown).
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Fig. 1. Panel A; Lanes 1– 6. Fragments generated when the microsatellite gwm538 associated with Karnal bunt resistance was used to evaluate genotypes HD29 (resistant parent), WL711 (susceptible parent) and KB RILs #31 and #134 (heterozygotes). Panel B; Lanes 7– 12. Fragment profiles for HD29(R), WL711(S) and KB RILs #31 and #134 (H) generated with the SNP marker. Panel C; Lanes 13– 18. Physical mapping of microsatellite gwm538 fragments in Chinese Spring (control), N4AT4D (nullisomic for 4A), N4BT4D (nullisomic for 4B) and N4DT4B (nullisomic for 4D). Size standard HyperLadder IV (Bioline, Randolph, MA), with 100- and 200-bp markers indicated in Lanes 1, 7, and 13. Negative (no template DNA) controls in Lanes 6, 12, and 18.
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Using the Chinese Spring (CS) derived nullisomic–tetrasomic lines for group 4 chromosomes, the three fragments amplified by gwm538 were physically mapped, cloned, and sequenced. Use of NT lines allowed easy separation of fragments on 2.3% agarose gels and excision of bands without cross contamination. Two fragments (174 and 147 bp) were located on chromosome 4B and the 137-bp fragment mapped to chromosome 4D (Fig. 1). The same three fragments (174, 147, and 137 bp) were amplified in the nullisomic 4A line (N4AT4D; Lane 15) as in its "parent" line CS (Lane 14), indicating that none of these fragments were located on chromosome 4A. Only the 137-bp fragment was amplified in the nullisomic 4B line (N4BT4D; Lane 16), physically mapping the two uppermost fragments to chromosome 4B. For nullisomic 4D, (N4DT4B; Lane 17) the 137-bp fragment was absent placing it on chromosome 4D.
Alignment of sequence data of the 174-, 147-, and 137-bp fragments amplified by gwm538 in the CS NT lines revealed four SNPs in the regions flanking the SSR (Fig. 2, in bold) from which fragment specific primers were made. Only one SNP was unique to the 174-bp fragment of interest and was used for development of the selective marker (Fig. 2, Position 22). By extending the gwm538F primer an additional five bases on the 3' end, we incorporated a single mismatch base at Position 18 (A in place of G) and a selective base at Position 22 (T). This new primer (gwm538snpF1) was used with the original gwm538R primer at a 60°C annealing temperature to selectively amplify target fragments of 152, 171, and 174 bp in HD29 (Fig. 1, Lane 8), WL711 (Fig. 1, Lane 9) and CS (not shown), respectively. This primer combination also successfully identified genotypes heterozygous for the 152- and 171-bp fragments (KB RILs #31 and #134, Fig. 1, Lanes 10 and 11). In contrast the fragment profiles of the heterozygotes are identical to the susceptible parent when amplified with gwm538 (Fig. 1, Lanes 4 and 5). In the heterozygous lines the 152-bp fragment comigrated with the 147-bp monomorphic fragment when amplification products were separated on 2.3% agarose gels.
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Fig. 2. AssemblyLIGN sequence alignment of the three fragments amplified from Chinese Spring derived nullisomic-tetrasomic lines (N4BT4D and N4DT4B) using gwm538F and gwm538R primers. Fragments are labeled by size in nucleotides. Nucleotide positions are indicated at top of the figure. Primer sequences for gwm538F, gwm538R and gwm538snpF1 are included for comparison. Black arrows indicate SNPs (in bold text) occurring outside the microsatellite region. The SNP indicated at Position 18 was designed as a mismatch to all three fragments (see results) and does not ‘naturally’ occur there. Period ‘.’ characters are used as pads to allow justification for the alignment of different size fragments.
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PCR conditions for gwm538snpF1-gwm538R are sensitive to annealing temperature. With our conditions an annealing temperature less than 59°C amplified all three fragments produced by the original gwm538 primers. A minimum annealing temperature of 59°C was required to selectively amplify the single target fragment.
Marker validation was performed with Xgwm538snp using 132 individuals from the KB RIL mapping population described by Singh et al. (2003). When resolved on 2.3% agarose gels, fragments amplified from Xgwm538snp scored exactly the same as individuals evaluated with Xgwm538 (not shown) with two exceptions. KB RILs #31 and #134 were heterozygous for the 152- and 171-bp fragments when amplified with Xgwm538snp, whereas the 152-bp fragment could not be resolved in these individuals and the fragment profile was identical to the susceptible parent when Xgwm538 was used (see results above, Fig. 1). To resolve this discrepancy, amplification products from all 132 individuals evaluated with the microsatellite and the SNP marker were separated on denaturing polyacrylamide sequencing gels. Fragment profiles generated by both markers were in perfect agreement when the sequencing gel was used since heterozygous RILs #31 and #134 were resolved for Xgwm538 in this system.
To assess the potential value of Xgwm538snp for use marker-assisted breeding, 23 adapted wheat cultivars from the Great Plains were surveyed for polymorphism relative to the resistant donor parent HD29. In seven [Cranbrook (USDA-GRIN accession PI 495816), Heyne (Sears et al., 2001), Jagger (Sears et al., 1997), NuHills (Monsanto Company, Creve Coeur, MO), Overley (Fritz et al., 2004), Recital [USDA-GRIN accession PI 573733; Available: http://www.ars-grin.gov/cgi-bin/npgs/html/csrlist.pl? (verified 15 March 2006)], and Tasman (USDA-GRIN accession PI 591917)] of the 16 cultivars tested, the 171-bp polymorphic fragment was amplified indicating that these cultivars are suitable recurrent parent candidates for incorporating KB resistance through the use of marker-assisted backcrossing.
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DISCUSSION
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MAS is a valuable tool for efficiently introgressing traits with difficult phenotypes into adapted germplasm (Dubcovsky, 2004). Tightly linked markers are available for many desirable traits; however, markers such as gwm538 do not easily transcend basic genetic research into applied plant breeding. For example, the ability to discern heterozygotes is of little concern in an F2:8 RIL mapping population, as was the case for Singh et al. (2003) when mapping the KB resistance QTL, since little heterozygosity is expected to remain within individuals advanced through eight generations of self-pollination. However, the ability to identify heterozygotes among segregating progeny is of great value in early generations of advancement (i.e., BC1, BC2, F2, etc.) when transferring the HD29-derived KB resistance into adapted cultivars, since heterozygotes will be the majority of individuals possessing the trait of interest. To this end, we surveyed 23 wheat cultivars with gwm538snp and found polymorphism between HD29 and seven cultivars.
In wheat, microsatellites detect high levels of polymorphism, however, they often produce multiple bands due to the hexaploid nature of the species (2n = 6x = 42). In cases where paralogous or homoeologous amplification interferes with marker resolution, fragment specific SNPs may be useful for selectively amplifying fragments of interest. The amount of time required to convert a microsatellite marker to a SNP marker is extensive; however, the cost associated with marker development is insignificant compared to the cost of phenotypic evaluation for KB resistance or the costs incurred by screening the original marker on DNA sequencing equipment.
The microsatellite marker Xgwm538 and corresponding primer sequences are protected by U.S. patent (Röder et al., 2004). This patent also covers utility of gwm538snpF1, since it was derived from patented sequence. Use of the markers described herein should be arranged through material transfer agreement with the inventors.
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ACKNOWLEDGMENTS
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We extend our thanks to Dr. Lili Qi, Aaron Jackson and Dr. Sukhwinder Singh for their contributions to this manuscript.
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NOTES
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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 for publication May 11, 2005.
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ABSTRACT
INTRODUCTION
