1a. Objectives (from AD-416):
The long-term objective of this research is to develop genetics and epigenetics of seed chemistry useful in developing high-yielding crops with improved feed, food, and biofuel end-use quality. The main focus will be the genetics/epigenetics of grain phosphorus (P) in barley (Hordeum vulgare L) and maize (Zea mays L.). Since grain total P represents a major pool in the flux of P through agricultural ecosystems, variants for this trait will provide new resources for managing P use in agriculture, thus will contribute to its long-term sustainability. Objective 1: Analyze the physiology and genetics of total phosphorus, phytic acid, and other seed constituents important to end-use quality in barley and maize. [NP301, C3, PS3A] Sub-objective 1.1: Continued genetic analyses of barley High Inorganic P mutants. Sub-objective 1.2: Isolation and study of recessive alleles of barley lpa1 and its maize homolog. Sub-objective 1.3: Forward genetics screens for seed total P mutants in barley and maize. Objective 2: Determine the relative roles of genetic and epigenetic factors in the phytate and related pathways in barley and maize. [NP301, C3, PS3A] Sub-objective 2.1: Epigenetics of maize lpa1. Sub-objective 2.2: Environmental induction of heritable epigenetic change in barley. Sub-objective 2.3: The epigenetic contribution to recurrent selection gain in barley.
1b. Approach (from AD-416):
Sub-objective 1.1: Hypothesis: Barley MAz423 is a novel “high seed total P” mutant with no impact on yield. Analyses of barely MAz423 will define mode of inheritance, confirmation of seed P phenotype and correlation or lack with seed yield. If warranted, mapping and gene identification will proceed. Studies of additional barley lpa mutants will continue. Sub-objective 1.2: Hypotheses: Barley lpa1 encodes a novel seed P transporter important to phytic acid and total P. “Complementation” and “knock-out” will determine if the putative P transporter is the gene perturbed in barley lpa1. New recessive alleles of the transporter in both barley and maize will be isolated using “TILLING”. Analyses of these new alleles will determine if the barley lpa1 phenotype can be engineered in maize. Sub-objective 1.3: Hypothesis: We can isolate alleles that impact seed total P but have little or no impact on maternal plant P. Chemically-mutagenized maize and barley populations will be screened for mutations that perturb seed total P amount and distribution. Genetics and inheritance of selected mutations will be studied in greater detail to identify those that perturb seed P without perturbing maternal plant P. Sub-objective 2.1: Hypothesis: Epigenetic silencing at lpa1 occurs both spontaneously and via paramutation. Analyses will describe the occurrence of these phenomena at maize lpa1, defining rate and critical sequences/modifications. Next-generation sequencing combined with bisulfite sequencing will be used in mapping/sequencing cytosine methylation and sequence changes and additional methods will be used to study histone modification. Sub-objective 2.2: Hypotheses: heritable plant growth phenotype changes and other molecular adaptations, in response to environmental stresses such as P nutrient deficiency, will be observed with barley and can be used to develop lines more productive under nutrient or other stress. Plants will be grown to maturity under various P nutritional or other stresses, and the seed they produce will be used to study inheritance of growth habit and molecular adaptations. The genetic versus epigenetic contribution to heritable adaptive responses will be determined using current methods including high-throughput, low-cost next-generation sequencing. Sub-objective 2.3: Hypotheses: Heritable epigenetic change contributes substantially to gains due to recurrent selection; recurrent selection for yield or stress tolerance can substantially enhance performance of low-phytate lines. Various recurrent selection schemes for performance under field conditions or under experimentally-induced abiotic stress, will be conducted with sets of barley low phytic acid isolines. Gain-due-to-selection for various performance parameters will be quantitated. Current methods of such as mapping-by-sequencing and techniques including bulked-segregant analyses, low-cost high-throughput next-generation sequencing, bisulfite sequencing, and sample multiplexing will be used to quantitate the contribution of genetic versus epigenetics to gain-due-to-selection.
3. Progress Report:
This report documents progress for the parent Project 5366-21000-030-00D, Analysis of the Biochemical Pathway and Genetics of Seed Phytate in Barley which started February 2013 and continues research from Project 5366-21000-025-00D, entitled "Plant and Seed Chemistry Genteics". While several specific lines of research in the prior project’s work addressing the genetics and biology of seed total phosphorus will be continued, a reduced amount of work will be devoted to the genetics of seed phytic acid per se. A substantial amount of the plant materials needed for this new project was accomplished during the prior project period. In the area of seed total phosphorus, the first milestone has already been completed: we have now shown that barley MAz423 represents a “high-yield/high seed total phosphorus” barley line. Substantial progress in the genetic screens in both barley and maize for mutations that alter seed total phosphorus was made. For example, we have advanced progenies segregating for over 100 genetic “events” that alter seed total P in maize. “TILLING” for recessive alleles of transporter gene important to seed total phosphorus is underway. A major focus of the new project will be on the role of epigenetics versus genetics in crop adaptation, response to abiotic stress and in “gain-due-to-selection” in crop breeding. Substantial progress has been made in this second Objective. We have completed genetic screens for the phenomenon of “paramutation” at the maize low-phytic-acid 1 locus, and will be determining the changes in “DNA methylation” that are the result. The first two generations of the experiment to study how plants use epigenetics to respond to and heritably adapt to abiotic stress, in the present case plant nutrient stress, has been completed. We are also at the fourth generation of recurrent selection for yield and drought tolerance in barley low-phytate lines. These last two lines of research are critical to developing enhanced understanding of the potential role of epigenetics in crop improvement.