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quantitative and qualitative disease resistance genes in a doubled haploid population of barley (Hordeum vulgare). Theor. Appl. Genet. 101, 580–589 van Berloo, R., H. Aalbers, A. Werkman, and R. E. Niks, 2001: Resistance QTL confirmed through development of QTL-NILs for barley leaf rust resistance. Mol. Breed. 8, 187-195 Vasu K., S. Sood, H. S. Dhaliwal, P. Chhuneja, B. S. Gill, 2006: Identification and mapping of a tiller inhibition gene (tin3) in wheat. Theor. Appl. Genet. (Published online) Wang, S., C. J. Basten, Z. B. Zeng, 2005: Windows QTL Cartographer 2.5. <strong>Department</strong> of Statistics, North Carolina State University, Raleigh http://www.statgen.ncsu.edu/qtl/crt/WQTL.htm Zang, Z. B., 1994: Precision mapping of quantitative trait loci. Genetics 136, 1457-1468 Zhu, H., L. Gilchrist, P. Hayes, A. Kleinhofs, D. Kudrna, Z. Liu, L. Prom, B. Steffenson, T. Toojinda, and H. Vivar, 1999: Does function follow form? Principal QTLs for Fusarium head blight (FHB) resistance are coincident with QTLs for inflorescence traits and plant height in a doubled-haploid population of barley. Theor. Appl. Genet. 99, 1221–123 20
Barley Genetics Newsletter 37:21-23 (2007) PCR-based markers targeting barley putative grain yield and quality QTLs regions Isabel A. del Blanco*, Deric A. Schmierer, Andris Kleinhofs and Steven E. Ullrich <strong>Department</strong> of Crop and Soil Sciences, Washington State University, Pullman, WA, 99164-6420 *e-mail: mailto:blanco@wsu.edu Abstract Eight restriction fragment length polymorphism (RFLP) and two genes delimiting, or included in, chromosome fragments containing putative QTLs for grain yield and quality were sequenced and converted to PCR-markers. Eight markers were co-dominant between two-rowed barley cultivars Harrington and Baronesse after digestion with restriction enzymes. Three were dominant-recessive after designing specific primers exploiting single nucleotides polymorphisms (SNPs) between those two cultivars. Introduction Two chromosomal regions from Baronesse have been reported as containing putative grain yield QTLs, one on chromosome 2(2HL) (between markers ABG461C and MWG699) and the other on chromosome 3(3HL) (between markers MWG571A and MWG961) (Schmierer et al., 2004). Subsequent analysis of Harrington/Baronesse derived inbred lines suggested other additional regions as candidates for grain yield QTLs. These regions are on chromosome 7(5HL) (between markers ABC717 and ABC718) and on the telomeric region of the short arm of chromosome 2(2HS) (ABG058). Since RFLP methodology needs large amounts of DNA and entails a complex procedure with radioactively labeled probes and Southern blotting, which requires several days to produce results, we converted RFLP targeting those putative QTL regions to PCR-markers: cleaved amplified polymorphic sites (CAPS) and SNPs. The conversion of RFLP clones to PCR-based markers rendered a much simpler technique that facilitated the screening of large numbers of genotypes at the seedling stage, since it requires a small amount of DNA. Materials and Methods Genomic DNA extraction was modified from Edwards et al. (1991); the modification added an extra-step of chloroform-isoamyl alcohol (24:1) extraction. CAPS were developed from RFLP clones. Primers were designed from the cloned sequences and used to amplify genomic DNA from the parental cultivars Harrington and Baronesse. If no fragment length polymorphism was observed, the fragments from both parents were sequenced to discover SNPs. These SNPs were analyzed to identify restriction enzymes that could be used to develop CAPS, or to design cultivar-specific primers. CAPS markers were also developed for gene Dhn1 (Choi et al., 2000) and a candidate gene for seed dormancy and/or pre-harvest sprouting, GA20-oxidase (Li et al., 2004). Reactions for SNPs were set under stringent conditions (annealing temperatures ~ Tm) and short cycles (annealing ≤ 15 s). Extensions were 1min or 2min depending on product size. 21
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Page 37 and 38: References Bhatnagar, V. K., Sharma
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