1a.Objectives (from AD-416):
The ultimate goal of this project is to provide the means to increase the protein quality of sorghum, a key factor determining the end-use quality of sorghum. This goal will be achieved by accomplishing the following specific objectives:. 1)screening of a genetically diverse sorghum population (n~300) to identify germplasm with intrinsically high protein digestibility levels, and. 2)determine the factors that govern digestibility in wild type sorghum lines.
1b.Approach (from AD-416):
Experimental Design: Objective 1
To provide the necessary variability in grain properties needed for this research, a genetically diverse population of sorghum samples (Casa et al. 2008) (hereafter referred to as the “sorghum diversity panel”) will be used as the initial sample population. The sorghum diversity panel was created to “span the genetic diversity of sorghum” and contains ~300 samples. Samples of the diversity panel grown in two locations will be used. The diversity panel will be screened for digestibility using the method of Mertz et al. (1984). Tannin containing samples will not be evaluated as tannins reduce digestibility via binding to kafirin proteins and inactivating digestive enzymes (the diversity panel has been screened for the presence of tannins; approximately 25% of the samples were found to contain tannins). Thus the presence of tannin containing samples would confound the results of this project.
Based on the initial screening of the diversity panel, 30-40 unique samples that span the range of digestibility values will be selected for additional research to determine what factors govern protein digestibility in “normal” sorghum lines.
Protein composition of the selected samples will be analyzed using multiple techniques. Overall protein composition will be measured using an “Osborne” type fractionation. All samples from the above “Osborne” fractionation scheme will be separated by size exclusion chromatography (SEC) to determine the amount of protein present in each class. Composition of the kafirin extracts will also be measured using reversed-phase high performance liquid chromatography (RP-HPLC) and high performance capillary electrophoresis (HPCE). Data from the RP-HPLC, HPCE, and SEC separations will be correlated (using simple correlation statistics) to protein digestibility to identify which proteins or protein complexes are key in determining digestibility. In addition to the above protein analysis, several other factors that have been proposed to influence protein digestibility in sorghum will be evaluated. Physical grain traits will be measured using the single kernel characterization system (SKCS) with optimum settings for measuring grain hardness, weight, and diameter. The Folin-Ciocalteau assay will be employed to determine the total phenolic content of the selected samples to determine if the levels of phenolic compounds in sorghum influences protein digestibility. If significant correlations between phenolic content and digestibility are found, then the composition of the phenolic compounds will be determined using HPLC. Phytate levels will be measured a commercially available colorimetric assay.
Protein content and in-vitro pepsin digestibility was measured on over 500 samples. A subset of 15 samples that ranged in digestibility from roughly 50 to 80% were selected for further study. Proteins from this subset were extracted and analyzed by HPLC to determine how protein composition might be related to digestibility. In addition, this subset of samples is being grown by collaborators at Kansas State University for a multi-location trial to investigate the effects of genetics x environment interactions in determining protein digestibility in sorghum.