Location: Grain, Forage, and Bioenergy Research2009 Annual Report
1a. Objectives (from AD-416)
The long-term objectives of this project are the development of sorghum (Sorghum bicolor) germplasm lines with improved bioenergy, feed, and food value, and the elucidation of genetic, biochemical, and biological factors impacting these characters. Over the next five years, the following specific objectives will be addressed: 1) Identify and evaluate genes to improve sorghum for bioenergy, food, and feed traits, 2) Develop a better understanding of genes and fundamental mechanisms controlling cell wall formation and energy availability, and 3) Develop molecular and other technologies for monitoring sorghum fungal pathogens and determine the effects of sorghum genetic modification for bioenergy on pathogen populations.
1b. Approach (from AD-416)
The project utilizes a diverse set of technologies and approaches that are best delineated by objective: Objective 1 will be addressed primarily using traditional whole-plant plant breeding techniques, animal feeding trials, and established laboratory methods for assessment of feed and bioenergy value. Objective 2 will be addressed using current molecular and biochemistry technologies including PCR, RT-PCR, HPLC, microarrays, GC-MS. Objective 3 incorporates both field-based experiments and current molecular biology technologies. All experiments will utilize sound statistical designs to allow hypothesis testing at established levels of probability.
3. Progress Report
1.a. Manuscripts delineating the effects on bmr-6, bmr-12-and stacked bmr-6+12 on chemical makeup, ethanol conversion, and dairy nutrition were submitted in collaboration with USDA-ARS Peoria and the WH Miner Institute. New brown midrib lines from USDA-ARS Lubbock were crossed to testers and classified for allelism with known bmr genes in sorghum. Brown midrib genes were identified that were not allelic to genes at known loci. 1.b. Yield trials of waxy B and R-lines were completed. One generation converting waxy B-lines into A3 male sterile cytoplasm has been completed, and nurseries to accomplish the second backcross are established in the field. The first year of a field trial comparing GBSS+ and GBSS- near isolines was planted and is in progress. 1.c. Backcrosses of high starch and high energy lines F1s with elite lines were completed. Selfing is underway. 2.a. Expressed the Bmr6 protein in E. coli and in planta to examine a unique enzymatic feature, histine 57. Expressed Bmr12 (COMT) in E. coli and developed an enzyme assay. In collaboration with the National Genome Resource Center, Santa Fe, NM, sequenced 192 million reads to characterize the difference in the stalk transcriptome between wild-type and bmr mutant lines. 2.b. Identified potential transcription factors which activate lignin biosynthesis based on amino acid sequence identity. 3.a. Established semi-selective media for screening for Curvularia spp. from grain. Established use of sequence analysis of translation elongation factor (TEF) gene to genotype Fusarium spp. Established methodologies for identifying Alternaria isolates to species. Substantial progress in screening near isogenic plant color lines for Alternaria, Fusarium and Curvularia spp. 3.b. Screened of grain from waxy accessions for infections by Alternaria, Fusarium and Curvularia. 3.c. Established stalk rot infection assay for Fusarium thapsinum. Have begun bioassays comparing brown midrib6 (bmr), bmr12, bmr6 and bmr12 double mutants and near isogenic RTx430 and Wheatland wild-type lines for response to stalk infection by F. thapsinum. Collaborated with Louis Prom to conduct field study of bmr6, bmr12, bmr6 and bmr12 double mutants and near isogenic RTx430 lines for infection by anthracnose (Colletotrichum sublineolum). 3.d. Growth chamber bioassays completed. Second paper describing microbial numbers and biochemical and genetic characterization of fluorescent Pseudomonas associated with sorghum submitted. Further molecular genetic characterization of select isolates with genes for antibiotic production on-going.
1. Distribution of Sorghum Brown Midrib Germplasm. The USDA-ARS Sorghum Project at Lincoln, NE is a world supplier of brown midrib germplasm. Brown midrib lines developed by the project have been distributed to public institutions and private sector companies in Argentina, Australia, Brazil, Bolivia, Botswana, Canada, Eretria, France, Greece, Hungary, India, Japan, Mexico, Paraguay, Uruguay, and the twenty three companies, universities, and institutions in the USA. These genetic resources are having impact ranging from direct use as parental lines in hybrid production by industry, to being utilized in fundamental scientific research on cell wall and lignin, and testing of new conversion technologies for bioenergy production.
2. More Complete Understanding of the bmr-6 gene. The USDA-ARS Sorghum Project at Lincoln, NE has sequenced the bmr-6 gene which reduces lignin content in cell walls by reducing activity of a key lignin synthesis enzyme, cinnamylaldehyde dehydrogenase (CAD). The mutation was determined to be a point mutation due to a change in a single amino acid. Furthermore, the sequence coding for the wild-type Bmr-6 was shown to be a highly conserved region in the genetic code of vascular plants ranging including mosses, rice, corn, tobacco, poplar, and pines. This gene has strong preference for several lignin substrates. Both bmr-6 and bmr-12, which reduces caffeic acid O-methyl transferase (COMT), affect total lignin content, lignin composition, and are affected by the genetic background in which they are placed. Stacking the two brown midrib genes was shown to reduce lignin even further than either gene alone. In actual trials converting lines with these genes to ethanol, the lowered lignin was associated with both higher ethanol yields and higher ethanol conversion efficiency. These research results are of direct importance to the development of sorghum as a bioenergy crop, and are also important as a model system for the development of other more complex crops, such as Switchgrass, for bioenergy.
3. Brown Midrib Genes Affect Fungal Pathogens. The USDA-ARS Sorghum Project at Lincoln, NE has demonstrated that the brown midrib genes bmr-6 and bmr-12 are associated with reductions in some fungal pathogens. When bmr6 grain or bmr12 grain were plated onto culture mediums there were significantly fewer isolations of Fusarium members from the Gibberella fujikuroi species complex than from wild-type grain. One Fusarium genotype was the second most commonly recovered Fusarium from wild-type grain, but was not detected in bmr-12 grain. This indicates that the presence or absence of one or more factors in bmr-12 plants excludes F. bullatum from colonizing grain. It is likely that lignin and lignin precursors function in plant defense by limiting or inhibiting fungal growth, either as preformed molecules or as a result of an induced response. This is uniquely different from resistance genes that interact specifically with microorganisms.
Funnell-Harris, D.L., Pedersen, J.F. 2008. Inoculation strategies to assess biological interactions between fusarium and alternaria species infecting sorghum. Canadian Journal of Plant Pathology 30: 404-413.