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Taking an Integrated Approach for Plant Germplasm Characterization and Utilization

Taking an Integrated Approach for Plant Germplasm Characterization and Utilization. Ming Li Wang Molecular and Biochemical Genetics Laboratory PGRCU. Curators Workshop at Atlanta, February 3, 2010. Our Mission for Plant Germplasm Research. Utilization. Utilization.

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Taking an Integrated Approach for Plant Germplasm Characterization and Utilization

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  1. Taking an Integrated Approach for Plant Germplasm Characterization and Utilization Ming Li Wang Molecular and Biochemical Genetics Laboratory PGRCU Curators Workshop at Atlanta, February 3, 2010

  2. Our Mission for Plant Germplasm Research Utilization Utilization

  3. An Integrated Approachfor Plant Germplasm Research ► Genetic analysis of sweet sorghum germplasm ► Genetic and biochemical analysis of peanut germplasm

  4. Sorghum as Feedstock for Bioethanol Production

  5. Feedstock Pretreatment Fermentation Products Grain sorghum (Starch) Animal feed Ethanol Chemicals Hydrolysis Forage or energy sorghum (Biomass) Glucose Fructose Sucrose C5 sugars Delignification Hydrolysis Ethanol Chemicals Stalk residue Squeeze Sweet sorghum (Sugars) There are about 3,000 sweet sorghum accessions in the U.S. germplasm collection

  6. Selected 96 Sweet Sorghum Accessions for Genotyping

  7. Sweet Sorghum Growing in the Field

  8. Selected SSR Markers for Genotyping

  9. Genotyping Sweet Sorghum Accessions with SSRs SSR marker Xcup1

  10. Statistics Results * Polymorphism information content.

  11. Determination of the Number of Subpopulations • Likelihood plot of the models, • Stability of grouping patterns across ten runs, • Germplasm information.

  12. Analysis of Molecular Variation (AMOVA)

  13. (24/33) Mainly from Africa (26/29) US historic syrup type (10/14) Race durra from Asia (16/20)

  14. Genetic Distances between Sweet Sorghum Groups Note: The top diagonal is Nei’s minimum distance and the bottom diagonal is pairwise Fst.

  15. Genotyped Sweet Sorghum Accessions on the World Map

  16. Principal Component Analysis (PCA)

  17. Geographical and Genetic Distributions of Genotyped Sweet Sorghum Accessions Wang et al., 2009 TAG

  18. Summary of Sweet Sorghum Research Results • Four subpopulations (G1, G2, G3, and G4) have been identified. • Four branching groups (B1, B2, B3, and B4) have been classified. • Results from genetic diversity and population structure analysis were correlated well with the geographical locations where these accessions were curated. • Geographical origin of accessions had significant influence on genetic similarity of sweet sorghum germplasm. • Germplasm accessions curated from different geographical regions should be used for developing sweet sorghum cultivars.

  19. Income Fatty Acid Composition (12 fatty acids) Seed Oil Content (40-60%) Grain Yield of Oilseed Crop (bushel/acre) Biochemical and Genetic Analysis of Peanut Germplasm $

  20. Peanut as Nutritional and Bioenergy Crop 50 gallon oil /acre for soybean 123 gallon oil /acre for peanut

  21. Peanut oil extraction by ether solvent Ankom XT15 Fat Extractor

  22. Measure Oil Content by Nuclear Magnetic Resonance (NMR)

  23. Convert Plant Oil to Fatty Acid Methyl Esters (FAME) Catalyst Plant oil + Methanol Esters + Glycerol

  24. Peanut Fatty Acid Composition 11.0% 48.0% 32.0% 80% (high oleic acid) C18:1 C18:2 C16:0 Oleic acid Linoleic acid Palmitic acid 3.5% 3.2% 1.6% C18:0 C22:0 C20:0 Arachidic acid Stearic acid Behenic acid

  25. GC Analysis of Peanut Fatty Acid Composition

  26. 5 F.A.M.E. Standard 1 = C14:0 2 = C16:0 3 = C16:1 4 = C18:0 5 = C18:1 6 = C18:2 7 = C18:3 8 = C20:0 9 = C20:1 10 = C22:0 11 = C24:0 6 3 2 10 1 4 7 8 9 11 0 2 4 6 8 10 12 Time (min.)

  27. Oil Content and Fatty Acid Composition among Different Subspecies A B FA A B HY A B A B A B A A A B A B A A

  28. Oil and FAC among Different Botanical Varieties A A B Hi A Hy B B A B A A B B A B A B A B

  29. Fatty Acid Desaturase (FAD) with Fatty Acid Composition COOH C18:0 Stearic acid Δ9 FAD1 COOH x C18:1 Oleic acid Δ12 FAD2 COOH C18:2 Linoleic acid ω-3 FAD3 COOH C18:3 Linolenic acid

  30. From Gene Mutation to Fatty Acid Composition Change Wild type Mutation on A Mutation on B Mutation on A + B A B A B A B A B Gene mutation for FAD2 FAD2 enzyme activity Normal ½ Normal ½ Normal Abnormal Oleic acid level 64% 64% 80% 48% Middle Middle Low High

  31. Ol2Ol2 ol2ol2 Detection of FAD2 Mutation on B Genome by Real-time PCR Wild type Mutant Ol2ol2 ol2ol2 Heterozygous Ol2ol2 Ol2Ol2 Barkley et al., 2009 Molecular Breeding

  32. a. FAD2 mutation b. Allele-specific PCR amplification prediction Wild type Substitution Insertion SUB + INS

  33. Detection of Mutation in FAD2 by Allele-Specific PCR Substitution Mid oleate Insertion Mid oleate Sub + Ins High oleate Wild type Low oleate Common band Wild type band + + + - Common band Substitution band Common band Substitution band - + - + Common band Insertion band Common band Insertion band - - + + Chen et al., 2010 Plant Molecular Biology Reporter

  34. Substitution on A O/L = 63.6 / 18.5 = 3.4 Substitution + Insertion O/L = 80.0 / 2.9 = 27.4 Insertion on B O/L = 60.2 / 20.95 = 2.9 600 600 600 600 Wild Type O/L = 43.2 / 34.2 = 1.3 300 300 300 300 min 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Oleic acid Low Oleic Acid Type Linoleic acid Mid Oleic Acid Type Similar to Olive oil (64%) Mid Oleic Acid Type High Oleic Acid Type

  35. Summary of Peanut Germplasm Research Results • Significant difference identified on oil content and fatty acid composition among botanical varieties and subspecies. • Real-time PCR assay was developed for detection FAD2 mutation on B genome. • Allele-specific PCR assay was developed for detection FAD2 mutations on both A and B genomes including: Wild type (no mutation), Substitution type (G→A) on A genome, Insertion type (→A) on B genome, Double mutation type (Substitution + Insertion). • Real-time PCR and allele-specific PCR markers developed in our lab can be used for MAS and germplasm screening. • GC analysis identified accessions with different levels (L, M, H) of oleic acid. • The results from Genetic analysis and GC analysis were consistent. • Genetic analysis in combination with biochemical analysis is a powerful approach for germplasm research.

  36. Acknowledgements PGRCUCollaborators Mr. Brandon Tonnis Dr. John Erpelding USDA-ARS, Puerto Rico Dr. Noelle Barkley Dr. Charles Chen USDA-ARS, Dawson Mr. Dave Pinnow Dr. Paul Raymer UGA, Griffin Ms. Sarah Moon Dr. Manjee Chinnan UGA, Food Science Dept. Ms. Jessica Norris Dr. Zhenbang Chen UGA, Crop & Soil Dept. Dr. Corley Holbrook USDA-ARS, Tifton Mr. Ken Manley Dr. Dick Auld Texas-Tech University Ms. Lee-Ann Chalkley Dr. Baozhu Guo USDA-ARS, Tifton Ms. Tiffany Fields Mr. Jerry Davis UGA, Statistics Dept. Ms. Merrelyn Spinks Dr. Tom Stalker NCSU, Crop & Soil Dept. Dr. Gorge Mosjidis Auburn University, All Supporting Staff Dr. Zhanguo Xin USDA-ARS, Lubbock All Curators Dr. Anna Resurrreccion UGA, Food Science Dept. Dr. Gary Pederson Dr. Jianming Yu Kansas State University

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