1a. Objectives (from AD-416)
1. In cooperation with breeders and geneticists, identify and facilitate the manipulation of genetic variation of end use quality characteristics in western wheat. Specific end-use quality traits to be studied include: kernel texture (sub-objective 1a), starch and non-starch carbohydrates (sub-objective 1b), polyphenol oxidase activity (sub-objective 1c), and overall milling and baking performance of wheat breeding lines (sub-objective 1d). Sub-Objective 1a. Alter wheat kernel hardness characteristics by identifying and incorporating novel puroindoline gene alleles and texture-related QTLs. Sub-Objective 1b. Determine the degree to which manipulation of polysaccharide content and structure can affect the end-use quality characteristics of wheat flour. Sub-Objective 1c. Determine the degree to which the many polyphenol oxidase genes in wheat impact noodle dough color stability. Sub-Objective 1d. Develop wheat cultivars with superior end-use quality characteristics in collaboration with regional wheat breeders and geneticists. 2. Develop, modify, and evaluate technologies and methodologies for measuring wheat kernel characteristics that impact end use functionality. Specific characteristics to be measured include: Kernel texture (sub-objective 2a), arabinoxylan composition (sub-objective 2b), gluten quality (sub-objective 2c) and polyphenol oxidase activity (sub-objective 2d). Sub-Objective 2a. Determine the reliability and performance of an endosperm brick test developed at the WWQL. Sub-Objective 2b. Develop a diagnostic assay for oxidative gelation of arabinoxylans in flour batters using free-radical-induced/peroxidase-mediated oxidative cross-linking. Sub-Objective 2c. Test a recently developed laser-based prototype instrument for measuring gluten strength via SDS-sedimentation (in collaboration with Dr. Rangan Chinnaswamy, GIPSA) Sub-Objective 2d. Modify our existing PPO L-DOPA assay to identify very low levels of grain PPO. 3. Identify and manipulate the biochemical constituents of wheat to improve the nutritional functionality of grain and flour, specifically dietary fiber and antioxidant content.
1b. Approach (from AD-416)
Determine the molecular and genetic basis of wheat grain texture by assessing puroindoline gene structure. Determine the effect of two different hardness alleles on grain texture, milling performance and baking quality. Assess the molecular genetic basis of discoloration in Asian noodles by identifying and characterizing polyphenol oxidase from wheat. Develop or adapt methods to evaluate wheat end-use quality with an emphasis on early generation testing. Employ gene expression measurement technology to identify genes contributing to desirable Asian food characteristics.
3. Progress Report
This work falls under NP306. Over 1,000 Aegilops tauschii accessions have been obtained; many have been haplotyped for the puroindoline genes (objective 1a). A Supersoft by Mjolner population was grown in the greenhouse (objective 1a). Crosses were made between waxy germplasm and Stephens, MDM and Xerpha; plots were grown at the WSU agronomy farm (objective 1b). A new family of PPO genes expressed in developing wheat kernals was identified and the three members named PPO-A2, PPO-B2, and PPO-D2 (objective 1c). The relative expression levels of these new PPO genes as well as the previously identified PPO-A1 and PPO-D1 genes were measured; the chromosomal location of the new PPO-2 genes has been determined (objective 1c). The 2010 harvest breeder sample evaluations were completed. Endosperm brick specimens were prepared and distributed to collaborating labs (objective 2a). A study on viscosity of batters showed that oxidative cross-linking was an important factor (objective 2b). Our SDS sedimentation instrument was used to analyze wheat samples and showed that the laser-diodes were capturing substantive gluten quality information (objective 2c). Over 1,000 Regional Nursery samples were obtained in furtherance of an AFRI/NIFA grant; milling and baking tests were completed (objective 3).
1. Discovery of three new polyphenol oxidase (PPO) genes. PPO, causes graying and browning of wheat dough and leads to discoloration in the end products. This is considered to be undesirable by consumers, and thus considerable effort is being made to minimize PPO in wheat varieties. ARS scientists in Pullman, WA have identified three additional PPO genes in wheat that were not previously known to be produced in wheat kernels. The new genes actually appear to account for 72% of the PPO RNA levels in developing wheat kernels, making them major contributors to discoloration of wheat dough products. Identification of three new very important targets that must be addressed for improvement of noodle dough color stability.
2. Development of soft kernel durum wheat. Hard kernel texture of durum wheat limits its utilization, thus an alternative product is necessary. ARS scientist in Pullman, Washington, in cooperation with scientists at Univ. of Tuscia, Viterbo, Italy, transferred the puroindolines and some unknown amount of chromatin from hexaploid wheat to durum wheat; stabilized the trait and developed back-cross derived lines showing soft kernel texture provided milling and flour properties consistent with soft wheats. As a result, soft durum grain can be milled using soft wheat milling equipment producing high yields of break flour with low starch damage levels.
3. Atomic force microscopy and other techniques examine the difference between soft and hard wheat endosperm. No one has a thorough understanding on the structural levels of soft and hard wheat at the molecular level. An ARS scientist in Pullman, Washington, in cooperation with a scientist with the Dept. of Chemistry, Washington State Univ., Pullman, used state-of-the-art atomic force field emission scanning electron microscopy, Raman and X-ray photoelectron spectroscopies to examine the difference between hard and soft near-isogenic wheat grain endosperm structure. Polished endosperm and flour of near-isogenic lines were examined using the aforementioned techniques. Soft wheat endosperm was less continuous with less distinct small granule structures, but with a greater number of larger features. Results contribute to a basic understanding of how soft and hard wheat grains differ, and how these difference may affect milling and flour quality.
4. Association of puroindoline b-2 genes with other wheat traits. The association between puroindoline b-2 variant genes (Pinb-B2v) and other wheat traits are unclear. An ARS scientist in Pullman, Washington, in cooperation with Henan Agricultural Univ. and Chinese Academy of Agriculture scientists determined the associations between puroindoline b-2 gene allele variants with other grain and agronomic wheat traits. Allelic variation in puroindoline b-2 variant genes was examined in relationship to grain traits, yield components and flag leaf size in bread wheat varieties of the Yellow and Huai river valleys of China. Within the soft wheat class, the Pinb-b2v3 allele was assiciated with harder grain than v2 allele. Results provide a number of testable hypotheses to see if these associations are cause and effect or simply indicate founder effects of wheat germplasm.
5. Distribution of arabinoxylans in wheat grain flour mill streams. Distribution of various arabinoxylan fractions among wheat flour mill streams is unknown. ARS scientists in Pullman, Washington, in cooperation with a graduate student at Washington State Univ., Pullman, measured the total, water-unextractable and water-extractable arabinoxylans of 10 flour streams from each of 31 genetically pure varietal grain lots. Mill stream (the product resulting from the passage of grist through a mill roll pair and subsequent passage through appropriately dimensioned sieves) was a bigger source of variation for arabinoxylans than was wheat variety. Arabinoxylan content was well correlated with flour ash content and increased with flour extraction rate. Selecting specific flour streams will may be a more effective strategy in increasing or decreasing arabinoxylan content compared to selection of specific varieties.
6. Oxidative cross-linking in wheat grain flour mill streams. Oxidative cross-linking forms gels that affect both batter viscostity and food product quality. The oxidative cross-linking potential of various flour mill streams (products resulting from the passage of grist through a mill roll pair and subsequent passage through appropriately dimensioned sieves) is not known. ARS scientists in Pullman, Washington, in cooperation with a graduate student at Washington State Univ., Pullman, measured the endogenous and enhanced oxidative cross-linking potential of 10 flour streams from each of 31 genetically pure varietal grain lots. Hard wheat flours tended to produce more viscous batters; first break and first second middlings flour streams exhibited the greatest potential for oxidative cross-linking. These results will help food manufacturers and flour millers produce products with more consistent processing properties.
7. Maize oil content. Use of the puroindoline transgene in maize must be approved in order to be used by the food production system. An ARS scientist in Pullman, Washington in cooperation with scientists at Montana State University in Bozeman, determined that constitutive expression of the wheat puroindoline proteins caused increased oil content in the germ. Oil produced from maize (i.e. corn oil) will benefit the food industry as well as producers by increasing the oil yields.
8. Kernel texture protein functionality. Proteins called puroindolines control kernel textures. An ARS scientist in Pullman, Washington in cooperation with scientists at Montana State University in Bozeman, Montana, used saturation mutagenesis to determine which regions of the puroindoline proteins are most important for its function in controlling kernel texture. Several portions of the protein were determined to be essential for its function, and many novel hardness mutants with varying degrees of functionality were produced. Some of these new alleles could potentially be more desired by end users through traits such as improved milling yield.
9. Barley grain quality genes. Mutations in the Vrs1 gene cause barley seed heads to form six rows of kernels instead of the usual two. These changes in head row type have a major effect upon end-use quality. ARS scientists at Pullman, WA, in collaboration with scientists at Montana State University, found that different changes of the Vrs1 gene affected end-use quality in different ways. Specific mutations leading to superior end-use quality were identified in this study. The most advantageous alleles can now be preferentially used by barley breedoing programs.
Morris, C.F., Simeone, M.C., King, G.E., Lafiandra, D. 2010. Transfer of soft kernel texture from Triticum aestivum to durum wheat, Triticum turgidum ssp. durum. Crop Science. 51:114-122.