1. Molecular Mapping of Seed Tocopherols in Soybean HEINRICH S. WOHLESER 1, YUKIO KAKUDA 2, and ISTVAN RAJCAN 3 1 University of Guelph, Department of Plant Agriculture, Guelph ON, N1G 2W1, Canada, e-mail: wohleseh@uoguelph.ca 1 University of Guelph, Department of Plant Agriculture, Guelph ON, N1G 2W1, Canada, e-mail: wohleseh@uoguelph.ca 2 University of Guelph, Department of Food Science, Guelph ON, N1G 2W1, Canada, e-mail: ykakuda@uoguelph.ca 3 University of Guelph, Department of Plant Agriculture, Guelph ON, N1G 2W1, Canada, e-mail: irajcan@uoguelph.ca INTRODUCTION INTRODUCTION Soybeans contain among many beneficial components considerable amounts of tocopherol (  -,  -,  -,  -tocopherol). Tocopherols are vitamin E active components with major antioxidant properties. Many studies have shown that natural tocopherols play an important role in preventing chronic diseases. Beside its health benefits tocopherols have a high market value and are utilized by the food and pharmaceutical industry for diverse applications. The genetic control of these constituents is not well understood. Therefore an identification of markers closely associated with tocopherol accumulation is the first step to generate an understanding of the genetic control. Furthermore these markers are potentially useful for plant breeders to develop high tocopherol soybean lines through marker assisted selection (MAS). We used simple sequence repeat (SSR) markers to construct a genetic linkage map based on an recombinant inbreed line (RIL) population derived from crossing two high-yielding commercial soybean cultivars with different tocopherol profiles. Therefore, the main objective of this study was to identify major genes or quantitative trait loci (QTLs) controlling tocopherol accumulation. MATERIALS AND METHODS MATERIALS AND METHODS RESULTS RESULTS CONCLUSIONSCONCLUSIONS ACKNOWLEDGMENTSACKNOWLEDGMENTS 1.This is the first report of finding markers and QTL for tocopherol accumulation in soybean. Markers associated with alpha and delta tocopherol have been identified on LG C2 and F, therefore suggesting the location of genes involved in this trait. 2.No QTLs were found for beta and gamma tocopherol, although ANOVA showed evidence towards potential QTLs. Fine mapping would eventually reveal additional QTLs for these constituents. 3.Most QTL were consistent among growing environments confirming the genomic regions where these gene(s) involved in this biosynthetic pathway reside. 4.Enough genetic variation is present in elite germplasm to allow for a rapid development of high tocopherol soybean lines. 5. Beta and gamma tocopherol seems to be more influenced by growing conditions than alpha and delta. AOCS. (1997). Determination of tocopherols in vegetable oils and fats by high performance liquid chromatography Ce 8-89. D. Firestone ed. In Official methods and recommended practices of the American Oil Chemist’s Society. 5 th. Ed. Champaign, Illinois. Cregan et al., 1999. An Integrated Genetic Linkage Map of The Soybean Genome. Crop Science, 39: 1464 – 1490. SAS Institute. (1998). SAS/STAT User’s Guide, Version 8, 2th Edition, Vol. 1. SAS Institute, Inc. Carry, N.C. REFERENCESREFERENCES The authors wish to thank Wade Montminy and the field crew for their great help during the planting and harvesting period. In addition we would like to thank Luidy Rodriguez, Chris Grainger, and Patricia Egea for their skilled help. Finally we want to acknowledge the Ontario Ministry of Agriculture for their generous financial contribution to this research project. Combined ANOVA  Genotypes (G) and locations (L) considered fixed effects; alpha, beta, gamma, and delta tocopherol showed significant G, L, and GxL interactions at  <0.05.  QTL analysis conducted on location basis rather than combining locations. Marker verification  26 linkage groups were determined including 14 unlinked markers.  Chi-square test detected 49 markers with segregation distortion at  <0.05. Marker Trait Association  A total of 11markers were found at St. Pauls, 12 at Woodstock and 11 markers at Elora as significantly associated with alpha tocopherol accumulation (Table 1).  Over all 3 markers were found at St. Pauls, 3 at Woodstock and 4 markers at Elora as significantly associated with delta tocopherol accumulation (Table 1).  Fewer markers were closely associated with beta and gamma tocopherol, only Satt335 was significantly associated with gamma tocopherol across locations (Table 1). Interval Mapping (MAPMAKER/QTL)  St. Pauls: 2 QTLs for alpha tocopherol both on LG C2 (combined R 2 = 22.8%); one QTL for delta tocopherol located on LG F (Figure 1).  Elora: 2 QTLs for alpha tocopherol both on LG C2 (combined R 2 = 34%); one QTL for delta tocopherol located on LG F (Figure 1).  Woodstock: 3 QTLs for alpha tocopherol, two on LG C2 and one on LG F (combined R 2 = 35.7%) (Figure 1); no QTLs were found for delta tocopherol but data suggests (LOD 1.9) a possibility of a QTL at the end of LG F near Satt522.  No QTLs associated with beta and gamma tocopherol were found. Figure 1: Partial linkage map of a cross between OAC Shire and OAC Bayfield showing QTLs associated with tocopherol accumulation in soybean seeds, including LOD-scores and R 2 values. Linkage group (LG) designations are used according to a public consensus map (Cregan et. al. 1999). Numbers on the left of the LGs indicate distances in centimorgans (cm) between marker pairs. Table 1: Marker trait associations based on single factor analysis of variance (ANOVA), including P-values (<=0.01) and R 2, in context with alpha-, beta-, gamma-, and delta-tocopherol accumulation. Bottom shows range of tocopherols in RIL population at 3 locations. Tocopherol Analyses: According to AOCS method Ce 8-89 (AOCS, 1997); Saponification of soybean meal and extraction of tocopherols using hexane; Extracts were washed with ddH 2 O to increase purity; Tocopherol quantification using HPLC (high-pressure-liquid-chromatography) expressing amounts in ug/g meal. Statistical Data Evaluation: SAS: SAS for Windows, version 8.2 (SAS Institute, Cary NC) with Type-1-error rate (  ) of 0.05; Computation of LSMEANS; ANOVA (Proc GLM) to determined genotype (G), location (L), and GxL effects. Mapmaker/EXP: Linkage map was constructed using a likelihood of odds (LOD) score of 3.0, and a maximum linkage distance of 40 centimorgans (cm). Mapmaker/QTL: Interval mapping to determine QTLs at a threshold of LOD 2. Marker verification: Leave tissue collection and DNA extraction of 93 RILs; Parental screen using 450 SSRs; RILs screened with 120 polymorphic markers; Linkage map construction based on marker data (MAPMAKER/EXP); QTL detection by combining marker and phenotypic data using interval mapping approach (MAPMAKER/QTL).