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Genetic Improvement of Sweet Sorghum for the production of Biofuels & Chemicals in Florida • Terry Felderhoff • • UF Genetics and Genomics Graduate Program Sanyukta Shukla, Alejandra Abril, & Wilfred Vermerris Why sweet sorghum is attractive for Florida • Tall, annual, seep propagated crop – Annual crop offers flexibility to the producer – High solubile sugars in juice (~18%) • Stress tolerant (drought, flood, heat) and limited input requirements compared to sugarcane • Opportunity to cultivate on marginal land – Abandoned citrus groves – Former tobacco land • Minimal competition with food production • Compatible with sugarcane production in South Florida Next-generation sweet sorghums: Sustainable production of fuels and chemicals from juice and bagasse fermentation CO2 fuels pentoses hexoses fermentation chemicals (e.g. D-and L-lactate) pretreatment Sweet sorghum chemicals fuels chemicals lignin nanotubes United States Department of Agriculture heat enhanced plastics Sweet sorghum is a new crop in Florida • Commercial sorghum breeding programs are mostly based in Texas, Kansas and Nebraska • Primary focus has been on grain and forage sorghums • Mediocre performance of commercial sorghum in Florida – Heavy pests and disease pressure – Low water retention capacity of sandy soils – Restrictions on the volume of irrigation water • The UF sorghum breeding program is focusing on developing regionally adapted sweet sorghums that give high yields with limited inputs Sweet sorghum cultivars • Sorghum makes a single reproductive structure (panicle) and is a naturally self-pollinating species • Cultivars are pure lines (inbreds) that can be propagated via self-pollination • Cultivars are ‘easy’ to develop Cultivar breeding strategy: Pedigree method Parents: high-sugar x other trait 400 F2 plants Each year we evaluate 10-20 sweet sorghums from around the world F2: select 5-20 biggest and cleanest plants 5-20 F3 families F3: select 5-10 individuals among and within families for biomass, maturity, disease resistance 5-10 F4 families F4: as F3 plus select for sugar based on destructive sampling of sibs 1-5 F5 families F5: estimate sugar yield; compare performance against checks; look for homogeneity; bulk seed 1-3 F6 families F6: replicated yield trials (two years, three locations) Release top performers Sweet sorghum breeding targets • High sugar yields • Disease resistance • Insect resistance • High biomass yields • Water use efficiency • Bagasse amenable to processing • Use marker-assisted selection for complex traits to expedite line development Anthracnose • Omnipresent in the southeastern USA • Resistance not common in commercial germplasm developed in central USA Mapping anthracnose resistance Bk7 EH X 135 F5 lines Phenotyping Genotyping - - Field trials Disease score GBS (Cornell University) 5,186 SNPs Genetic mapping of resistance • Chromosome 7 – 45.2 Mb • Chromosome 9 – 3.2 Mb • Identified in UF developed cultivars Sweet sorghum cultivars Location Sugar Fresh stalk weight Genotype F4(Honey*BK7)-45-3-1 Gusher 6.7a 84.4a F4(Mer 81-4 x BK7)-28-4-1 Caramelo 6.8a 67.7bc F4(Mer 81-4 x BK7)-20-2-1 Fortuna 6.6a 73.4ab F4(Mer 81-4 x BK7)-15-2-1 F4(Mer 81-4 x BK7)-1-2-1 Sweet Florida Candycane 6.8a 72.0ab 7.2a 76.6ab 5.3b 60.0c M81E Commercial (t/ha) (t/ha) Dry weight yields: 15-20 t/ha Improved UF cultivars Brown midrib sorghum • Generated in the 70’s at Purdue University via chemical mutagenesis – 19 recessive mutants • Nine spontaneous bmr mutants discovered later on • Initially used as forage because of improved intake and palatability Klason lignin (Purdue) 25 Klason lignin (mg/g ) 20 15 Wt bmr 10 5 0 N-bmr 2 N-bmr 3 N6-bmr N-bmr 12 N-bmr 19 Wt-bmr isolines The bmr mutations reduce lignin content by ~15-20% Saballos et al. (2008) Bioenerg. Res. 1: 198 Enzymatic saccharification of bagasse (Purdue) Pretreated Unpretreated (dilute sulfuric acid) Glucose yields of sorghum stover from bmr and wild-type lines without and with pretreatment after 48 h of enzymatic saccharification (60 FPU/g cellulose) Saballos et al. (2008) Bioenerg. Res. 1: 198 Sweet sorghum hybrids • Hybrids are the product of a cross between two different inbred parents; the progeny is planted and harvested • Benefits of hybrids: – Potential for superior yield – Seed production is combine-compatible (maybe….) • Challenge 1: Since sorghum is a self-pollinating species, you need a male-sterile female ‘A-line’ to ensure cross-pollination • Challenge 2: The only way to propagate the A-line is to cross it with a fertile B-line that is otherwise genetically identical • Challenge 3: In order to get fertile hybrid seed (necessary for sugar accumulation!), a male, fertility-restoring ‘R-line’ needs to be available A B R Seed Parent Maintainer Pollinator Sanyukta Shukla Combine-compatible hybrid seed production • If we select the parents carefully, we can get tall offspring from short, parent lines • Four recessive, independent dwarf genes • Both hybrid parents need to be sweet in order to produce sweet offspring • But is height a prerequisite for being sweet? Tall sorghums tend to be sweeter….. Brix as a function of plant height 20 18 But…. 16 14 Brix 12 10 8 6 y = 0.017x + 8.4817 R² = 0.222 4 2 0 0 50 100 150 200 250 300 350 400 Plant height (cm) Data based on 250 F3 families derived from grain x sweet sorghum Ana Saballos and Sanyukta Shukla Short Sweet Sorghum • Tall sorghums tend to be sweeter than short sorghums, but short sorghums can be sweet – Height is not a prerequisite in sweet sorghums • Good news: It is possible to produce short sweet inbred parents for hybrid sweet sorghums • Short, sweet inbred parents for hybrid production under development: – 22 R-lines – 73 B-lines (being converted to A-lines) Sanyukta Shukla dw1 dw2 Dw3 Dw4 Dw1 Dw2 dw3 dw4 Dw1 Dw2 Dw3 Dw4 Conclusions • Sweet sorghum is an excellent bioenergy crop for Florida • The great genetic diversity within sorghum can be exploited to develop improved, regionally adapted bioenergy sorghums – Cultivars developed at UF outperform other sorghum cultivars • Short sweet sorghums can be developed for tall hybrid sweet sorghum production Dr. Ana Saballos Dr. Lonnie Ingram Amelia Dempere Dr. Julene Tong Randy Powell Maury Radin Jared Lindey Steven Smith Dr. John Erickson Dr. Jim Preston Dr. KT Shanmugam Dr. Brad Krohn Dr. Wilfred Vermerris [email protected] United States Department of Agriculture