1. Identification of molecular markers for aluminium tolerance in diploid oat through comparative mapping and QTL analysis 1 Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa, ON K1A 0C6, Canada 2 Lacombe Research Centre, Agriculture and Agri-Food Canada, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada (in memorium) Charlene P. Wight 1, Solomon Kibite 2, Nicholas A. Tinker 1, and Stephen J. Molnar 1. Free aluminium present in acid soils affects the growth and yield of oat and other cereal crops worldwide. Since genetic variability for tolerance exists, developing aluminium-tolerant cultivars may be an effective strategy for increasing the productivity of crops on acid soils. A survey of 3500 oat accessions from the USDA World Oat Collection identified the Avena strigosa Schreb. line CIav 9011 as having the highest tolerance to aluminium (Kibite, unpublished). A number of molecular marker maps exist to facilitate genetic analysis in oat, including one for the diploid oat cross A. atlantica Baum et Fedak x A. hirtula Lag. (AH) (O’Donoughue et al. 1992; Van Deyzne et al. 1995) and one for the hexaploid oat cross A. byzantina C. Koch ‘Kanota’ x A. sativa L. ‘Ogle’ (KO) (Wight et al. 2003). Both of these maps are integrated with the international grass databases Gramene (http://www.gramene.org; Ware et al. 2002) and Graingenes (http://wheat.pw.usda.gov/GG2/index.shtml), where queries and tools are available to facilitate comparative mapping among related species and genera. The objectives of this study were to identify genomic regions in diploid oat suspected to contain genes and QTL that are orthologous to those affecting aluminium tolerance in other grass species, and to use this information to identify genetic markers linked to QTL affecting aluminium tolerance in diploid oat. Introductio n Materials and Methods References Results and Discussion Figure 1. Example of comparative mapping study results illustrating the relationships between AH group F and aluminium tolerance-related genes and/or QTL regions located on wheat, barley, rye, maize, and rice chromosomes. The QTL located on rice chromosome 12 in three different populations were related to AH group F through the use of a bridging map; the maize chromosome 10 and rice chromosome 3 maps are composites. References for maps taken directly from the literature include: Miftahudin, et al. (2002) Theor Appl Genet 104:626- 631; Nguyen VT, et al. (2002) Molec Genet Genom 267:772-780; Nguyen VT, et al. (2001) Theor Appl Genet 102:1002-1010; Raman H, et al. (2003) Aus J Agric Res 54:1315-1321; Raman H, et al. (2002) Theor Appl Genet 105:458-464; Riede CR and Anderson JA (1996) Crop Sci 36:905-909; Tang Y, et al. (2000) Crop Sci 40:778-782; and Wu P, et al. (2000) Theor Appl Genet 100:1295-1303. Figure 2. Molecular marker map of the LAG-211 A. strigosa population with aluminium-tolerance QTL positions highlighted. The percentages given represent the percent phenotype explained by each QTL individually. mwKO CIav 2921 CIav 9011 mw K O CIav 2921 CIav 9011 B A calretB1_3SCA08 Acknowledgements This research was made possible by generous financial support from the Quaker Oats Company (USA), Quaker Tropicana Gatorade (Canada), and the Agriculture and Agri-Food Canada Matching Investment Initiative. Our thanks for excellent technical assistance go to Don Beauchesne, Linda Vandermaar, Stefan Halisky, Zachary Fouchard, and Ana Beatriz Locatelli. Figure 3. Presence of SCAR marker bands in hexaploid oats (panel A=calretB1_3, panel B=SCA08). The bands linked to aluminium tolerance are highlighted. K and O contain DNA from the hexaploid oat varieties Kanota and Ogle. CIav2921 is the aluminium sensitive A. strigosa parent, and CIav9011 is the tolerant parent. Unnamed lanes contain DNA from ten other hexaploid oat varieties or breeding lines. De Koeyer DL, et al. (2004) A molecular linkage map with associated QTLs from a hulless x covered spring oat population. Theor Appl Genet 108:1285-1298 Gallego FJ, et al. (1998) Chromosomal location of PCR fragments as a source of DNA markers linked to aluminium tolerance genes in rye. Theor Appl Genet 96:426-434 Holloway JL and Knapp SJ (1993) G-MENDEL 3.0: Software for the analysis of genetic markers and maps. Oregon State University, Corvallis, pp 1-130 Lander E, et al. (1987) MAPMAKER: An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174-181 O’Donoughue LS, et al. (1992) An RFLP-based linkage map of oats based on a cross between two diploid taxa (Avena atlantica x A. hirtula). Genome 35:765- 771 Tinker NA and Mather DE (1995) MQTL: software for simplified composite interval mapping of QTL in multiple environments. J Agric Genomics 1. Full text available from http://www.cabi-publishing.org/JAG Van Deynze AE, et al.(1995) Comparative mapping in grasses. Oat relationships. Mol Gen Genet 249:349-356 Ware DH, et al. (2002) Gramene, a tool for grass genomics. Plant Physiol 130:1606-1613 Wight CP, et al. (2003) A molecular marker map in Kanota x Ogle hexaploid oat (Avena spp.) enhanced by additional markers and a robust framework. Genome 46:28-47 A cross (LAG-211) was made between the A. strigosa Schreb. lines CIav 2921 (Al-sensitive) and CIav 9011 (Al-tolerant). Eighty-eight recombinant inbred lines were established by single seed descent to the F6 generation. Each RI line was screened for aluminium tolerance using a hydroponic system, with sixteen seedlings grown in the presence of 300 uM aluminium, and sixteen more grown as aluminium-free controls. After 14 days of growth, the root lengths of the ten seedlings with the longest roots from each treatment were averaged and compared. An extensive literature review and database search was conducted to identify regions conferring aluminium tolerance across grass species. Potentially orthologous regions in diploid oat were identified by comparative mapping, using bridging maps when common markers were not available. Large scale DNA purification, restriction digestion, Southern blotting, and RFLP analysis were performed as described in Wight et al. (2003). AFLP analysis was performed as described in De Koeyer et al. (2004). The SCAR (sequence-characterized amplified region) marker SCA08 developed by Gallego et al. (1998) to mark aluminium tolerance in rye was mapped using standard PCR conditions and an annealing temperature of 50C. Amplified fragments were visualized in 1.8% agarose gels. In addition, primers were developed from a consensus calreticulin candidate gene sequence (calretB1L, 5’- GAAAAGGAGCGAAGGGAAAG-3’; calretB3R, 5’- CAATCCATGGGATCTTCCAT-3’). In the case of this SCAR, an annealing temperature of 52C was used. Molecular mapping was performed using the programs “GMendel” (Holloway and Knapp, 1993) and “Mapmaker v. 3.0” (Lander et al. 1987) as described in Wight et al. (2003). QTL were detected by simple interval mapping using the program “MQTL” (Tinker and Mather 1995). The distribution of relative root lengths in the LAG-211 population described an approximately normal curve, suggesting that the aluminium tolerance trait in A. strigosa is controlled by more than one gene and/or is strongly affected by environment. An example of how the AH diploid oat reference map was used for the selection of regions to screen for aluminium tolerance QTL in LAG-211 is shown in Figure 1. In this case, it can be seen that the single genes identified in the Triticeae, as well as QTL regions on rice chromosomes 3 and 12 and maize chromosome 10, all show homology with a small segment of AH group F. The molecular marker map generated for the LAG-211 population is presented in Figure 2. Of the 70 RFLP clones tested, eighteen identified polymorphisms in this cross, representing nine of eleven potential QTL regions. The AFLP markers were used to provide additional coverage of the genome, and 41 of these loci were mapped across the genome. Both of the SCAR markers, SCA08 and calretB1_3, mapped to LAG-211 group 1. Four QTL were found using simple interval mapping (Figure 2). The QTL with the largest effect was associated with LAG-211 group 1, containing the AH group F-related marker bcd1250 as well as both SCAR markers. This suggests that the AH group F region of diploid oat surveyed contains a gene that is orthologous to the major aluminium tolerance genes found in wheat, rye, barley, and perhaps maize, as well as a minor QTL in rice. It is also intriguing that the calretB1_3 marker is so closely linked to the major QTL in diploid oat, although the current study provides no direct evidence for the involvement of calreticulin in conferring aluminium tolerance in this species. A second QTL was associated with LAG-211 group 10, which contains the AH group A markers cdo393 and cdo1173. The marker cdo1173 is duplicated on AH group A. While it can be associated with the Alm2 gene found on maize chromosome 6 (data not shown), the peak marker for the QTL on LAG-211 group 10 was cdo393, and this marker is located at the opposite end of the chromosome from cdo1173 on maize chromosome 6. It is well known that translocations and other cytogenetic phenomena exist among and within different cereal species, so we can speculate that the QTL associated with LAG-211 group 10 is orthologous to the Alm2 gene, but only with caution. The two remaining QTL were associated with unlinked AFLP markers. Together, the four QTL accounted for 55% of the total phenotypic variation in aluminium tolerance. The tolerant parent, CIav9011, contributed the positive allele in each case. No significant epistasis was detected among these QTL, or between these QTL and other genomic regions. Figure 3 presents the results obtained when the SCAR markers SCA08 and calretB1_3 were tested across hexaploid oat germplasm. The calretB1_3 band associated with aluminium tolerance was present in twelve hexaploid oat lines tested (Fig. 3A, ten lines not shown), while the SCA08 band was missing (Fig.3B). Thus, the SCA08 marker should prove useful for breeders wanting to introgress the major gene for aluminium tolerance from A. strigosa CIav9011 into elite, hexaploid oat germplasm. This work has demonstrated the effectiveness of using different types of information from different species to identify the locations of genes for a complex trait in a diploid oat population previously uncharacterized by molecular mapping. (AH group F) (AH group A) LAG_211_unlinked acc_cat_2 acg_caa_0 aca_caa_6 acc_caa_0c acc_caa_1 acc_cag_1 acc_cag_4 acc_cat_1 acc_cat_3 acc_cat_7 acc_cat_10 acg_caa_4 acg_caa_6 wg110 wg282 cdo358 cdo1445 bcd1095 LAG_211_1 [0] bcd1250 acc_caa_0a aca_caa_1 [4] SCA08 acc_caa_0b acg_caa_1 acc_ctc_0 [5] calretB1_3 acc_cag_5 [7] acc_caa_4 [10] acc_caa_1a LAG_211_2 [0] aca_caa_2b [10] aca_caa_2 [18] acc_cag_2b LAG_211_3 [0] aca_caa_3 [7] cdo1328 [13] acc_ctc_1 LAG_211_4 [0] aca_caa_7 acg_caa_3 [4] cdo1428 LAG_211_5 [0] cdo57b [4] isu1755 [7] acc_cat_4 acg_caa_5 LAG_211_6 [0] acc_caa_5 [7] acc_caa_0d [18] acc_caa_3 LAG_211_7 [0] acc_cat_6 [5] acc_cag_2 LAG_211_8 [0] acc_cat_8 acc_cag_4b [12] cdo1174 [20] bcd115 LAG_211_9 [0] acg_caa_2 [5] aco193 LAG_211_10 [0] cdo393 [8] acc_ctc_2 [10] acc_ctc_5 [15] acc_caa_2 [18] cdo1173 LAG_211_11 [0] cdo669 [10] bcd454 LAG_211_12 [0] cdo783 [4] cdo57a 39% 20% 14% 16%