GrainGenes

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

Department 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.

Primer3 software (Rozen and Skaletsky, 2000) assisted the process of primer design. PCR products were visualized on 1% or 2%, depending on the fragment size, agarose gel under UV light. PCR products were purified with Exonuclease I (Exo-SAP-IT, UBS, Cleveland, OH). Sequencing reactions were performed on an Applied Biosystems 3100 Genetic Analyzer (Perkin Elmer Applied Biosystem Division, Foster City, CA) with the ABI PRISM Big Dye Terminator v3.1 cycle sequencing kit. Products were confirmed by sequencing in both directions. Analysis of sequences to find restriction sites and/or SNPs was done with the tools provided by the San Diego Super Computer Center (SDSC, http://workbench.sdsc.edu).

Results and Discussion

Details of developed PCR markers are listed in Tables 1 (CAPS) and 2 (SNPs). CAPS marker MWG699 yielded small fragments difficult to visualize after digestion with enzyme Taq1, even when 3% UltraPure Agarose-1000 was used to run PCR products. To avoid this problem, cultivar-specific markers were developed taking advantage of SNPs between the parental genotypes. The two SNP sites are different than the TaqI restriction site, for this reason they are considered two different markers for locus MWG699.

Table 1. Summary of developed CAPS markers, their location, primers and restriction enzymes

*   Restriction enzyme (TaqI) previously reported by Tanno et al. (2002) in a different population

** Primers from Choi et al. (TAG 101:350-354)

Table 2.  Cultivar-specific PCR primers developed exploiting nucleotide polymorphisms between Harrington (H) and Baronesse (B) cultivars

*Note: These primers require stringent conditions when running PCR

References

Choi, D.W., M.C. Koag and T.J. Close. 2000. Map locations of barley Dhn genes determined by gene-specific PCR. Theor. Appl. Genet. 101:350-354.

Edwards K., C. Johnstone and C. Thompson. 1991. A simple and rapid method for the preparation  of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19(6):1349.

Li, C., P. Ni, M. Francki, A. Hunter, Y. Zhang, D. Schibeci, H. Li, A. Tarr, J. Wang, M. Cakir, J. Yu, M. Bellgard, R. Lance and R. Appels. 2004. Genes controlling seed dormancy and pre-harvest sprouting in a rice-wheat-barley comparison. Funct. Integr. Genomics 4:84-93.

Schmierer, D.A., N. Kandemir, D.A. Kudrna, B.L. Jones, S.E. Ullrich and A. Kleinhofs.  2004.  Molecular marker-assisted selection for enhanced yield in malting barley.  Mol. Breed. 14:463-473.

Rozen, S. and H.J. Skaletsky. 2000. Primer3 on the SSS for general users and for biologist programmers. In S. Krawetz and S. Misener (ed.) Bioinformatics methods and protocols: Methods in molecular biology. Humana Press, Totowa, NJ, p. 365-386. Available at http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plusAbout.cgi (verified April 2007).

Tanno, K., S. Taketa, K. Takeda and T. Komatsuda. 2002. A DNA marker closely linked to the vrs1 locus (row-type gene) indicates multiple origins of six-rowed cultivated barley (Hordeum vulgare L.). Theor. Appl. Genet. 104:54-60.