GrainGenes

Towards fine structure mapping and tagging major malting quality QTL in barley
F. Han, S.E. Ullrich, A. Kleinhofs and J.A. Clancy

Quantitative trait locus (QTL) analysis has been revolutionized by the developments in molecular markers, construction of comprehensive linkage maps, and software for linkage and QTL analyses. The most extensive QTL mapping has been done with barley through the North American Barley Genome Mapping Project (Hayes et al., 1993; Oberthur et al., 1995; Han et al., 1995b; Han et al., 1996; Tinkeret al.1996). Based on a molecular marker linkage map developed from the Steptoe x Morex doubled haploid line (DHL) population (Kleinhofs et al., 1993), two important malting quality QTL regions containing putative adjacent overlapping QTLs affecting malt extract, alpha-amylase, diastatic power, malt beta-glucan, malt beta-glucanase, and dormancy in the centromere region of chromosome 1 were identified (Hayes et al., 1993; Han and Ullrich 1994; Oberthur et al., 1995; Han et al., 1995b). The two adjacent overlapping QTL regions are flanked by Brz and ABG011 of 25.8 cM, and ABG011 and Amy2 of 17.7 cM. With the aim of determining whether the two QTL regions are really two or one and whether the overlapping QTLs are due to pleiotropic effects of a single gene or independent effects of several to many loci, we are attempting to fine map this complex chromosome region by breaking down the relatively large region into small segments. Another objective of this study is to facilitate molecular marker assisted selection for the malting quality QTLs in this region by converting linked RFLP-based markers to PCR-based markers.

Creation of isogenic lines

An effective approach for accurate localization of a QTL is to produce isogenic lines by backcrossing lines which carry different chromosome segments only in the region containing the QTL. Different chromosome segments and their regions of overlap are identified using all available genetic markers (Paterson et al., 1991).

1. Strategies

Molecular marker assisted backcrossing has been employed to create isogenic lines for fine structure mapping of QTLs. We developed alternative strategies for obtaining isogenic lines in barley. To facilitate the backcross process, plants were maintained in vegetative growth (12 hr. day and 12 hr. night) during genotyping with selected markers. Flowering was induced by increasing daylength to 16 hr. after desirable genotypes were selected for further backcrossing. To obtain isogenic lines in target regions, recombinants were identified in each backcross population followed by selection for the recurrent parent background. An alternative approach we have employed in creating isogenic lines is that the target region is transferred into the recurrent parent background followed by the generation of recombinants after the last backcrossing. The second approach was preferred since it allowed us to work with smaller populations until the generation of recombinants. At that time, the number of progeny examined was based on the recombinants desired. However, identification of desirable recombinants was somewhat faster with the first approach. To obtain homozygous isogenic lines, one generation of selfing was effective with the appropriate population size calculated based on the assumption that each heterozygous DNA segment behaved as a single heritable unit (i.e. gene).

2. Isogenic lines

Since all favorable alleles (high malt extract, amylase and beta-glucanase activity, low malt beta-glucan content, and low dormancy) are attributed to Morex, it is necessary to select a DHL parent which carries Morex alleles at the target QTL region and a high proportion of Steptoe alleles in all other genome regions to backcross to Steptoe (i.e. placing Morex malting quality alleles at the target QTL region in the Steptoe background). A desirable line, DH73, which carries Morex alleles in the QTL region on chromosome 1 and 65.1% Steptoe alleles in other genome regions, was chosen from the 150 Steptoe x Morex mapping DHLs, and backcrossed to Steptoe (BC1). The BC1F1 was backcrossed to Steptoe again (BC2). The BC2F1s were genotyped by using all available molecular markers in the target region and some markers from other regions which previously had Morex alleles. Thirty-three types of recombinants in the target region were identified. Additional backcrosses between these 33 types of recombinants and Steptoe were made for removing Morex alleles in the other genome regions. In the BC3F1 population, a total of 48 recombinant types were identified. These recombinants have an average Morex segment size of 4 cM in the target region of 43.5 cM. Of them, 24 types of recombinants were backcrossed to Steptoe again (BC4) to further clean-up the background. Before extensively testing all recombinants, we selected 8 priority recombinants which will resolve the QTL(s) into an approximately 10 - 15 cM segment(s) (Figure 1). The homozygosity of the 8 recombinants has been achieved by selfing and a seed increase is currently underway. Recombinant 4414 has an intact Morex segment which contributes high malting quality. This line will be a positive control. Steptoe will be the negative control. Recombinants 2616 and 7713 divide the target region into the two subregions with each subregion containing putative overlapping QTLs. These two recombinants will answer whether there are two independent QTL regions or just one. Recombinants 3408, 3616, 5908, and 6606 contain gradually smaller Morex segments. By comparing the QTL effects among these recombinants, segment(s) of 10 cM or less containing QTL effects should be identified. For example, if recombinants 2616, 3408, 3616, and 3907 have the same QTL effects, then the QTL must be in the overlapping Morex segment, in this case flanked by markers ABG701 and ABG011 with a size of 3.1 cM. Recombinants 6606 and 3907 have a Morex allele and a Steptoe allele at Amy2 locus, respectively. These recombinants will answer if the Amy2 locus is responsible for the alpha-amylase QTL in this complex. It is intended that these 8 recombinants be planted in the field in 1996. Malting quality analysis in the fall of 1996 should reveal the exact segment(s) carrying the QTL(s). Further fine structure mapping and characterization of the QTL region of interest will focus on the smaller segment(s).

Conversion of linked RFLP markers to PCR-based markers

Tagging QTL via linked molecular markers will provide an alternative approach to manipulating quantitative traits. Further study and manipulation of this region will be facilitated by the flanking markers Brz and Amy2. Brz is a locus for UDPglucose flavonol 3,0 glucosyl transferase (Wiseet al.1990), and Amy2 is a locus for low pI 4.8 - 5.0 alpha-amylase I gene (Knoxet al.1987). Both are RFLP-based markers. For the purpose of molecular marker assisted manipulation of QTL, PCR-based markers are preferable to RFLP-based markers in terms of relatively simple techniques and no radioactive materials involved. We have reported the conversion of Amy2 RFLP marker to PCR-based marker (Hanet al.1995a). Herein, we report the conversion of Brz RFLP marker to PCR marker.

Primers for amplifying Brz segment were designed based on sequence information in the GeneBank (accession no. X15694). Primers were chosen at the 3' transcribed, nontranslated region to avoid a high G-C rich coding region which has proved to be very difficult to amplify. The forward primer is 5'-AGATAGTTTGTCGGGTGTGATC-3'. The reverse primer is 5'-AGAAGGCAGCTTATGCCAAGTC-3'. The PCR product length is 285 bp.

The PCR was carried out in a final volume 50 ul which contains 5 ul 10x Taq buffer, 5ul 1 mM dNTP, 3 ul 25 mM MgCl2, 2 ul each of forward and reverse primers (10 pmol), 5 ul genomic DNA (50ng/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 4 min, 35 cycles (denaturation at 94°C for 1 min, annealing at 50°C for 45 sec, and extension at 72°C for 15 sec), final extension at 72°C for 7 min, and holding at 14°C.

After PCR amplification, the product was separated on 1.5% agrose gel. Steptoe has the 285 bp band while Morex does not. This polymorphism works also on the use of barley plant tissues (leaf, root, anther etc.) as a direct template (Clancy et al., 1996).

This PCR marker, together with Amy2 PCR marker, should facilitate the tagging of major malting quality QTL in breeding programs.

References :

Clancy, J.A., V.A. Jitkov, F. Han and S.E. Ullrich. 1996. Barley tissue as direct template for PCR: a practical breeding tool. Molecular Breeding (in press).

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

Han, F., J.A. Clancy, and S.E. Ullrich.1995a. Conversion of an RFLP marker associated with malting quality to a PCR-based marker. Barley Genetics Newsletter 24:7-9.

Han, F., S.E. Ullrich, S. Chirat, S. Menteur, L. Jestin, A. Sarrafi, P.M. Hayes, B.L. Jones, T.K. Blake, D.M. Wesenberg, A. Kleinhofs, and A. Kilian. 1995b. Mapping of beta-glucan content and beta-glucanase activity loci in barley grain and malt. Theor. Appl. Genet. 91:921-927.

Han, F., S.E. Ullrich, J.A. Clancy, V. Jitkov, A. Kilian and I. Romogosa. 1996. Verification of barley seed dormancy loci via linked molecular markers. Theor. Appl. Genet. 92:87-91.

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Acknowledgments

We are grateful for the financial support of American barley growers, the malting and brewing industry, and USDA-CSREES Special Grant Agreement 94-34213-0030. This work is part of the North American Barley Genome Mapping Project.

Figure 1. Genome constitution of 8 recombinants at target region on chromosome 1