Identification of Elemental Processes Controlling Genetic Variation in Soybean Seed Composition

1. Identification of Elemental Processes Controlling Genetic Variation in Soybean Seed Composition José L. Rotundo, Silvia Cianzio & Mark Westgate Iowa State University, 1301 Agronomy Hall, Ames, IA; rotundo@iastate.edu Abstract: Dissecting quantitative seed traits into more elemental processes may facilitate identifying genes governing those traits. For example, final protein concentration (SPC) is determined predominately by the relative accumulation of protein, oil and carbohydrate (mg seed-1). Likewise, individual component accumulation is determined by their rate and duration of net synthesis. As such, similar values for SPC can result from a variety of developmental and metabolic strategies. We have identified two such strategies to achieve high SPC within a population of F2 families segregating for SPC. One set of ‘high SPC’ lines increased protein accumulation per se (Strategy 1). A second subset of ‘high SPC’ lines maintained protein content fairly constant but decreased accumulation of other seed components (Strategy 2). These lines were screened with ‘high protein’ SSR markers on LG A1 and I to identify genomic regions associated with these two unique strategies. The marker analysis revealed a large impact of parental alleles on the strategies used to achieve highSPC. A conventional SSR marker analysis on seed protein concentration alone would not have revealed these genomic-trait associations. • Results • Two strategies were detected for increasing protein concentration: greater protein synthesis per se; similar protein concentration with reduced oil and residual content. • Genomic regions on LG AI between 27.95 and 28.95 cM were strongly associated with protein and residual contents. Families receiving Evans alleles in this region utilized Strategy 1 (greater protein content) to achieve high SPC, while families receiving PI153296 allele in this region utilized Strategy 2 (lesser residual content). • Materials and Methods: • Population development --100 F2:3 lines • - Low Protein Parent : Evans [SPC: 360 g kg-1; Protein content 48 mg seed-1] • - High Protein Parent: PI153296 [SPC: 450 g kg-1 ; Protein content 72 mg seed-1] • Field evaluation • Three environments: planting on 16 May 2007, 8 June 8 2007, and 15 July 2008; One meter row plots; Two replications in 2007, three in 2008; • Seed sampling every 5-6 days to estimate rate and duration of seed filling; • Mature seeds analyzed by combustion for nitrogen concentration and extracted with hexane for oil determination. • Molecular marker analysis • 24 SSR markers from Linkage Group A1 and I previously associated with high seed protein concentration were assayed for polymorphic alleles in the parents. A subset of F3 families was selected for selective genotyping with 10 of the polymorphic markers (red). Figure 2. Comparison of seed protein concentration and seed protein content of F2 derived lines displaying different strategies to attain high seed protein concentration. Empty and filled circles represent selected families having >40% of protein and contrasting levels of protein content. Introduction: A number of studies have identified genomic regions (quantitative trait loci: QTLs) associated with high seed protein concentration. Brummer et al. (1997) identified a number of QTLs for high protein in diverse populations, but QTL stability varied across environments. Nichols et al. (2006) recently fine mapped a region on Linkage Group I previously associated with increased protein concentration (Chung et al., 2003). Lee et al. (1996) indentified four independent markers stable across locations associated with increased seed protein concentration. No QTLs for variation in seed protein have been associated with an underlying physiological basis for the trait, or even the potential for genetic variation in the elemental processes determining that trait. A logical first step towards identifying marker-associated genes that control a complex trait is to resolve the primary physiological process conditioned by the marker(s). For example, if an increase in final protein concentration is based on increments in seed protein content, the associated gene(s) activity might determine assimilate supply to the seed. Alternatively, if protein concentration increases due to a reduction in oil and/or carbohydrate accumulation, gene activity determining this variation might be related to metabolic partitioning within the seed itself. Despite the vast literature identifying and mapping QTLs associated with seed protein concentration, it is not clear which developmental/physiological/biochemical strategy leading to high SPC can be targeted for genetic improvement of this trait. Objective: To evaluate genotypic variation and genetic basis of different physiological strategies to produce seeds with increased protein concentration. Hypothesis: Different physiological strategies exists for increasing SPC. One is to increase protein content per se (mg protein seed-1). An alternative is to maintain protein content (mg seed-1) while reducing other seed components (oil + carbohydrates). • Results: • Substantial variation was observed among progeny lines for component concentration and content (Figure1). Figure 1. Frequency distribution for protein, oil and residual concentration and content in F2 derived families grown in three environments. (a) to (f): 2006 (Environment 1); (g) to (l): 2007 (Environment 2); (m) to (r): 2007 (Environment 3). Values included for each line are the average of two replicate samples in 2006 and three replicate samples in 2007. The gray arrow indicates values for Evans, the low protein parent. The black arrow indicates values for PI153296, the high protein parent. Table 1. Association between allelic variants for ten polymorphic SSR markers on linkage groups A1 and I and protein concentration and protein, oil and residual contents evaluated in three environments. Marker-trait association was considered significant at P<0.05. R2 represents the proportion of the total variation explained solely by the marker. • Conclusions: • By analyzing these traits in a segregating population we observed substantial genotypic variation for the elemental processes that determine SPC. • The parent with low seed protein content, Evans, provided alleles that contributed to greater protein content in the seed of its progeny. These alleles may regulate assimilate supply per seed, which has been shown in related studies to determine seed protein content. • The markers for protein content in this genomic region probably would not be informative for high SPC in a conventional QTL analysis for protein concentration since some high SPC families have Evans alleles while others have PI153296 alleles. Dissecting SPC into its components , however, exposed this genomic region as important in determining protein accumulation. References Brummer, E.C., G.L. Graef, J. Orf, J.R. Wilcox, and R.C. Shoemaker. 1997. 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