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. FY13 progress: Several hundred Aegilops tauschii accessions were grown in the greenhouse, nearly all have been haplotyped for the puroindoline genes; a representative botanical specimen was archived; phylogenetic analysis is underway. Selections were made from waxy germplasm and Stephens, MDM and Xerpha; plants were growing in the greenhouse and field for the next round of selection. The 2012-harvest breeder sample evaluations are essentially complete; 2013 samples are being collected. Our SDS sedimentation instrument was successfully evaluated by the Grain Inspection, Packers and Stockyards Administration (GIPSA) and Agriculture and Food Research Initiative (AFRI) invited us to submit a full grant proposal on the instrument; the outcome of the grant application is pending. A complete set of U.S. soft wheat germplasm from the Regional Nurseries were analyzed in furtherance of an AFRI National Institute of Food and Agriculture (NIFA) grant; a paper was published on the results; a post-doc will continue these studies. Objectives 1a, 1b, 1d, 2a, 2b, 2c, and 3 were either substantially or fully met, or exceeded; objective 1c was placed on hold and 2d was not met due to loss of a scientist.
1. Diversity of puroindoline b2 genes. An ARS scientist in Pullman, Washington, in cooperation with Henan Agricultural University studied the variation in puroindoline b2 genes in cultivated and wild diploid wheat. The evolution of the puroindoline genes in wheat is not known. Nine new variant forms of puroindoline b2 genes. Puroindoline b2 genes were sequenced in bread and durum wheat and wild diploid relatives. None of the variants in the diploid species matched those in cultivated wheat. The results provide an evolutionary framework for this gene in wheat and its wild ancestors.
2. Physical location of puroindoline b2 genes in the wheat genome. ARS scientists in Pullman, Washington, with collaborators at Washington State University, and a scientist with the Chinese Academy of Agricultural Sciences, determined the physical location of puroindoline b2 genes in wheat. The location of puroindoline b2 genes in the wheat genome is not known. The locations were determined using special genetic stocks. The location of puroindoline b2 genes was determined to be on the distal end of chromosomes 7AL, 7BL and 7DL.
3. A comprehensive survey of soft wheat grain quality in U.S. germplasm. An ARS scientist in Pullman, Washington, with collaborators at Washington State University conducted a comprehensive survey of soft wheat grain quality in U.S. germplasm. Comparisons of soft wheat from eastern and western regions, including winter and spring, red and white wheats has not been conducted. Elite germplasm from regional nurseries were evaluated for milling and baking end-use quality. In general, soft wheat quality is relatively uniform across America. However, significant variation exists in each class.
4. Segregation analysis of puroindoline b2 variant genes. ARS scientists in Pullman, Washington, in cooperation with Washington State University, and Chinese Academy of Sciences, studied the segregation of puroindoline b2 variants in wheat. The relationship between puroindoline b2 variants 2 and 3 is not known. Segregation analysis among three spring wheat populations was conducted The results indicate that the two forms are allelic. The results provide an evolutionary framework for this gene in wheat and indicate that these two forms share an evolutionary history.
5. Expression of puroindoline b2 genes in wheat. An ARS scientist in Pullman, Washington, in cooperation with Montana State University studied the expression of puroindoline b2 genes in wheat. The level of expression of puroindoline b2 variants is not known. Puroindoline b2 variants 1-4 were measured using mRNA. Expression ranged from near zero to nearly 8% of puroindoline a and b levels. Given the very low level of puroindoline b2 expression, it is unlikely that these genes contribute to grain softness in wheat.
6. Prevalence of puroindoline b2 genes in Pacific Northwest wheat breeding germplasm pools. ARS scientists in Pullman, Washington, with collaborators at Washington State University, and a scientist with the Chinese Academy of Agricultural Sciences surveyed the puroindoline b2 genes in Pacific Northwest soft white, hard red and hard white wheat breeding germplasm. Puroindoline genes may influence kernel properties and end-use quality. The puroindoline genes were determined among 388 wheat varieties and breeding lines, and evaluated against kernel texture. Specific gene variants were present according to germplasm pool, for example, hard red spring versus hard red winter. There was no association of puroindoline b2 genes with kernel texture variation indicating that this gene does not play a direct role in end-use quality.
Chen, F., Shang, X., Morris, C.F., Zhang, F., Dong, Z., Cui, D. 2013. Molecular characterization and diversity of puroindoline b-2 variants in cultivated and wild diploid wheat. Genetic Resources and Crop Evolution. 60:49-58.