Conversion of an RFLP marker associated with malting quality<p> to a PCR-based marker
Conversion of an RFLP marker associated with malting quality
to a PCR-based marker

F. Han, J.A. Clancy and S.E. Ullrich
Department of Crop and Soil Sciences, Washington State University
Pullman, WA99164-6420

The North American Barley Genome Mapping Project (NABGMP) has developed a high density molecular marker linkage map (Kleinhofs et al., 1993). A major application of linkage maps is to map quantitative trait loci (QTL) for important agronomic and quality traits, and then to tag these QTL via tightly linked molecular markers. The QTL for a number of traits have been detected based on the Steptoe/Morex map (Hayes et al., 1993; Ullrich et al., 1993; Han and Ullrich, 1994; Han et al., 1994; Hayes et al., 1994). An important region flanked by the markers, Amy2 and Brz, on chromosome 1 has been identified as containing major QTL for malting quality traits such as malt extract percentage, alpha-amylase activity, diastatic power, malt ß-glucan content, and ß-glucanase activity. Amy2 is a locus for the low pI (4.8 - 5.0) alpha-amylase I gene, and Brz is a glucosyl transferase gene. Therefore, breeding for malting quality could be facilitated by manipulating this region via linked molecular markers (Amy2 and Brz). For the purpose of molecular marker assisted selection (MMAS), PCR-based markers are preferable to RFLP-based markers in terms of relatively simple techniques and no radioactive materials involved. Reported herein are results of converting an Amy2 RFLP to a PCR-based marker.

Materials and Methods

Primer design: Based on alpha-amylase 2 mRNA sequence information in GeneBank (accession no. M17128), primers were designed using program Primer (Lincoln et al., 1991, unpublished). The forward primer is: 5'-ACATGATGCTGGGAAAGGTC-3' with Tm of 59.9°C, and the reverse primer is: 5'-CGTAGATGCAGTAGATGCCG-3' with Tm of 59.5°C. The PCR product length is 497 bp. The primers were synthesized by the Laboratory of Biotechnology and Bioanalyses at Washington State University, Pullman.

DNA extraction: Barley DNA was extracted from freeze-dried 4-5 week-old leaf tissues by the CTAB procedure (Ausubel et al., 1987).

PCR reaction: The PCR was carried out in a final volume 50 uL which contains 5 uL 10x Taq buffer, 5 uL 1mM dNTP, 3 uL 25 mM MgCl2, 2 uL each of forward and reverse primers (10 pmol), 5 uL genomic DNA (50 ng/uL), and 0.5 uL Taq polymerase. Thermal cycling was performed on the PTC-100 Programmable Thermal Controller (MJ Research, Inc, Watertown, MA, USA): initial denaturation at 94°C for 3 min, 30 cycles (denaturation at 94°C for 1 min, annealing at 55 °C for 1 min, and extension at 72°C for 1 min), final extension at 72°C for 7 min, and holding at 14°C.

Restriction enzyme digestion: After PCR amplification, the restriction enzyme digestion of the PCR product was performed as follows: in a total of 15 uL volume containing 10 uL PCR-amplified DNA, 0.25 uL BstX I (10 u/uL), 1.5 uL 10x enzyme buffer, and 3.25 uL deionized H2O. The reaction was incubated at 55°C for 2 hrs, stopped with 4 uL 5x loading buffer and separated on 1% agarose in TBE buffer.

Results and Discussion

PCR amplification: The PCR product was checked on a 1% agarose gel, and a strong amplification of expected fragment size was observed (Fig. 1, lanes 2-3). Several non-specific bands were also observed, but were much weaker than the specific band. Elimination of the non-specific amplifications by increasing reaction stringency was not attempted, since they had no effect on subsequently achieving polymorphism. In this study, the primers were chosen from two exon regions to amplify the intron. This consideration has two significant aspects: i) there is high probability of getting polymorphism in terms of more variable intron regions; and ii) exon regions are much conserved, so the primers chosen from exon regions of a target DNA sequence may be used in related species.

Polymorphism: After digestion of the PCR product with restriction enzyme BstX I, distinctive polymorphism between Steptoe and Morex was detected (Fig. 1, lanes 4-11). The Morex fragment was about 80 bp shorter than the Steptoe fragment after restriction enzyme digestion. Therefore, this PCR-based marker still maintains a co-dominant nature. A set of 104 doubled haploid lines (DHLs) from Steptoe/ Morex F1s were genotyped with this marker, as well as the RFLP Amy2 probe. The genotypes of the 104 DHLs for the PCR-based and RFLP-based Amy2 markers showed consistency, indicating this PCR-based marker is reliable.

An RFLP marker for Brz, which together with Amy2 brackets the important region containing QTL for malting quality on chromosome 1, is likewise being converted to a PCR-based marker. The coding region of Brz DNA sequence has an extremely high G-C content (about 75%), and it has proved to be very difficult to amplify G-C rich regions by PCR. Therefore, it was decided to amplify the 3' transcribed, nontranslated region of Brz which is not G-C rich. A 307 bp PCR product was obtained by using the forward primer 5'-AGATAGTTTGTCGGGTGTGATC-3' (Tm = 58.1°C) and reverse primer 5'-AATAAATG GGTAGAAGGCAGC-3' (Tm = 58.7°C). Sequencing the Brz PCR products of Steptoe and Morex indicated that the Morex fragment is 3 bp longer than the Steptoe fragment. To date, efforts continue to find a simple way to resolve this difference.

Reference

Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1987. Current protocol in molecular biology. Greene Publishing Associates and Wiley-Interscience, pp2.3.1-2.3.3.

Han, F. and S.E. Ullrich. 1994. Mapping of quantitative trait loci for malting quality traits in barley. Barley Genet. Newslttr. 23:84-97.

Han, H., S.E. Ullrich, B.L. Jones, P.M. Hayes D.M. Wesenberg, A. Kleinhofs, A. Kilian, and the North American Barley Genome Mapping Project. 1994. Mapping of quantitative trait loci for ß-glucan concentration in barley and malt. Agron. Abstr. 86:202.

Hayes, P.M., B.H. Liu, S.J. Knapp, F. Chen, B. Jones, T. Blake, J. Franckowiak, D. Rasmusson, M. Sorrells, S.E. Ullrich, D. Wesenberg, and A. Kleinhofs. 1993. Quantitative trait locus effects and environmental interaction in a sample of North American barley germplasm. Theor. Appl. Genet. 87:392-401.

Hayes, P.M., O. Iyamabo, and the North American Barley Genome Mapping Project. 1994. Summary of QTL effects in the Steptoe x Morex population. Barley Genet. Newslttr. 23:98-143.

Kleinhofs, A., A. Kilian, M.A. Saghai Maroof, R.M. Biyashev, P. Hayes, F.Q. Chen, N. Lapitan, A. Fenwich, T.K. Blake, V. Kanazin, E. Ananiev, L. Dahleen, D. Kudrna, J. Bollinger, S.J. Knapp, B. Liu, M. Sorrells, M. Heun, J.D. Franckowiak, D. Hoffman, R. Skadsen, and B.J. Steffenson. 1993. A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome. Theor. Appl. Genet. 86:705-712.

Ullrich, S.E., P.M. Hayes, W.E. Dyer, T.K. Blake, and J.A. Clancy. 1993. Quantitative trait locus analysis of seed dormancy in "Steptoe" barley. p136-145. In M.K. Walker-Simmons and J.L. Ried (eds). Preharvest Sprouting in Cereals 1992. Amer. Assoc. of Cereal Chemists, St. Paul, MN, USA.



Figure 1. Amy2 PCR products. Lane 1 - 1 Kb DNA ladder; Lanes 2 - 3, uncut (497 bp) Steptoe and Morex, respectively; Lanes 4 - 11, cut with Bst XI; Lane 4 - Steptoe, Lane 5 - Morex, Lanes 6 - 11, doubled haploid lines.