GYMNOCLADUS

Pod images obtained using Adobe Photoshop CS4 with Silver Fast 6.0 (Plustek OpticPro A320) set at 300 DPI. Length and width (at widest point) were measured with Adobe Photoshop CS4 by converting length in pixels to length in millimeters.

Seed images obtained using Adobe Photoshop CS4 with Silver Fast 6.0 (Plustek OpticPro A320) set at 300 DPI with diameter measurements captured using SmartGrain v.1.1 (Tanabata et al. 2012). Random sample of 10, intact seeds. Measurements in millimeters. Diameter defined as the widest distance through the medial portion of the seed, which is typically from the hilum to the distal portion. In general, Gymnocladus dioicus seeds are near perfectly round, but often slightly longer in length by usually 1 mm or less. Reference: Tanabata T, Yamada T, Shimizu Y, Kanekatsu M, Takano M. 2012. SmartGrain: High-Throughput Phenotyping Software for Measuring Seed Shape through Image Analysis.

Gymnocladus accessions held at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS) located in Ames, IA were shipped to the USDA-ARS National Center for Agricultural Utilization Research Station (Bio-Oils Research Unit) located in Peoria, IL to be analyzed for their total-oil and fatty-acid methyl-ester (FAME) profiles. Approximately 15 seeds from each accession were used for analysis. Total Oil Content was analyzed using magnetic resonance (NMR; Bruker MQ Minispec) with a 0.47 T permanent magnet maintained at 40C and provided hydrogen nuclei with a resonance of 20 MHz. The spectrophotometer was calibrated with known soybean oil standards due to the similarity in fatty acids contents. Seed samples for the analysis were randomly selected and run in 8-10 replications (1 seed/replication). The seeds were placed into an 18mm test tube and preheated to 40C before analysis. The pulsed NMR analytical method was set for 16 scans which reports grams of total oil. Oil content was calculated on a dry weight basis using the average moisture content using AOCS Official Method Ca 2c-25. The fatty acid profile analysis was carried out in three replications (10 seeds/replication) using gas chromatography (GC). The GC of fatty acid methyl esters (FAMEs) was done with a Hewlett-Packard 6890 gas chromatograph (Palo Alto, CA), equipped with a flame-ionization detector (FID) and an auto sampler/injector. Analyses were conducted on a SP 2380 30 m x 0.25 mm i.d. with a 0.2 micro M film thickness (Supelco, Bellefonte, PA). Saturated C8-C30 FAEEs standards (Nu-Check Prep Inc., Elysian, MN) were used to make FAME assignments. The SP 2380 analysis were carried out as follows: column flow 0.7 ml/min with helium head pressure of 15 psi; split ratio 100:1; programmed ramp 165C to 265C at 15C/min with a hold of 5 min at 265C ; injector and detector temperatures set at 265C. Fatty acid methyl esters were made by placing a portion of the seeds into a 4 dram vial. Sodium hydroxide/methanol solution 5 mL (0.25 M) was added to the vial and a 0.2g portion of the seed ground for 20 s with a Modular Homogenizer System (Cole-Parmer Instrument Company, Vernon Hills, IL) fitted with a 10 mm diameter shaft. The vial was then sealed with an aluminum lined cap and placed in a heating block maintained at 65C. After one half hour, the vials were removed, allowed to cool and 5ml of hexane and 5ml of saturated sodium chloride solution were added to the vials. The contents of the vials were mixed thoroughly and after the layers separated, a 0.25 ml aliquot from the top hexane layer containing the methyl esters was removed with a Pasteur pipette and diluted up to 2 ml with hexane in a GC vial. One (1) microliter of the sample was injected on the GC using the parameters described above.

Gymnocladus accessions held at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS) located in Ames, IA were shipped to the USDA-ARS National Center for Agricultural Utilization Research Station (Bio-Oils Research Unit) located in Peoria, IL to be analyzed for their total-oil and fatty-acid methyl-ester (FAME) profiles. Approximately 15 seeds from each accession were used for analysis. Total Oil Content was analyzed using magnetic resonance (NMR; Bruker MQ Minispec) with a 0.47 T permanent magnet maintained at 40C and provided hydrogen nuclei with a resonance of 20 MHz. The spectrophotometer was calibrated with known soybean oil standards due to the similarity in fatty acids contents. Seed samples for the analysis were randomly selected and run in 8-10 replications (1 seed/replication). The seeds were placed into an 18mm test tube and preheated to 40C before analysis. The pulsed NMR analytical method was set for 16 scans which reports grams of total oil. Oil content was calculated on a dry weight basis using the average moisture content using AOCS Official Method Ca 2c-25. The fatty acid profile analysis was carried out in three replications (10 seeds/replication) using gas chromatography (GC). The GC of fatty acid methyl esters (FAMEs) was done with a Hewlett-Packard 6890 gas chromatograph (Palo Alto, CA), equipped with a flame-ionization detector (FID) and an auto sampler/injector. Analyses were conducted on a SP 2380 30 m x 0.25 mm i.d. with a 0.2 micro M film thickness (Supelco, Bellefonte, PA). Saturated C8-C30 FAEEs standards (Nu-Check Prep Inc., Elysian, MN) were used to make FAME assignments. The SP 2380 analysis were carried out as follows: column flow 0.7 ml/min with helium head pressure of 15 psi; split ratio 100:1; programmed ramp 165C to 265C at 15C/min with a hold of 5 min at 265C ; injector and detector temperatures set at 265C. Fatty acid methyl esters were made by placing a portion of the seeds into a 4 dram vial. Sodium hydroxide/methanol solution 5 mL (0.25 M) was added to the vial and a 0.2g portion of the seed ground for 20 s with a Modular Homogenizer System (Cole-Parmer Instrument Company, Vernon Hills, IL) fitted with a 10 mm diameter shaft. The vial was then sealed with an aluminum lined cap and placed in a heating block maintained at 65C. After one half hour, the vials were removed, allowed to cool and 5ml of hexane and 5ml of saturated sodium chloride solution were added to the vials. The contents of the vials were mixed thoroughly and after the layers separated, a 0.25 ml aliquot from the top hexane layer containing the methyl esters was removed with a Pasteur pipette and diluted up to 2 ml with hexane in a GC vial. One (1) microliter of the sample was injected on the GC using the parameters described above.

Gymnocladus accessions held at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS) located in Ames, IA were shipped to the USDA-ARS National Center for Agricultural Utilization Research Station (Bio-Oils Research Unit) located in Peoria, IL to be analyzed for their total-oil and fatty-acid methyl-ester (FAME) profiles. Approximately 15 seeds from each accession were used for analysis. Total Oil Content was analyzed using magnetic resonance (NMR; Bruker MQ Minispec) with a 0.47 T permanent magnet maintained at 40C and provided hydrogen nuclei with a resonance of 20 MHz. The spectrophotometer was calibrated with known soybean oil standards due to the similarity in fatty acids contents. Seed samples for the analysis were randomly selected and run in 8-10 replications (1 seed/replication). The seeds were placed into an 18mm test tube and preheated to 40C before analysis. The pulsed NMR analytical method was set for 16 scans which reports grams of total oil. Oil content was calculated on a dry weight basis using the average moisture content using AOCS Official Method Ca 2c-25. The fatty acid profile analysis was carried out in three replications (10 seeds/replication) using gas chromatography (GC). The GC of fatty acid methyl esters (FAMEs) was done with a Hewlett-Packard 6890 gas chromatograph (Palo Alto, CA), equipped with a flame-ionization detector (FID) and an auto sampler/injector. Analyses were conducted on a SP 2380 30 m x 0.25 mm i.d. with a 0.2 micro M film thickness (Supelco, Bellefonte, PA). Saturated C8-C30 FAEEs standards (Nu-Check Prep Inc., Elysian, MN) were used to make FAME assignments. The SP 2380 analysis were carried out as follows: column flow 0.7 ml/min with helium head pressure of 15 psi; split ratio 100:1; programmed ramp 165C to 265C at 15C/min with a hold of 5 min at 265C ; injector and detector temperatures set at 265C. Fatty acid methyl esters were made by placing a portion of the seeds into a 4 dram vial. Sodium hydroxide/methanol solution 5 mL (0.25 M) was added to the vial and a 0.2g portion of the seed ground for 20 s with a Modular Homogenizer System (Cole-Parmer Instrument Company, Vernon Hills, IL) fitted with a 10 mm diameter shaft. The vial was then sealed with an aluminum lined cap and placed in a heating block maintained at 65C. After one half hour, the vials were removed, allowed to cool and 5ml of hexane and 5ml of saturated sodium chloride solution were added to the vials. The contents of the vials were mixed thoroughly and after the layers separated, a 0.25 ml aliquot from the top hexane layer containing the methyl esters was removed with a Pasteur pipette and diluted up to 2 ml with hexane in a GC vial. One (1) microliter of the sample was injected on the GC using the parameters described above.

Listing of Omernik Level III Ecoregion where germplasm was obtained. Ecoregions determined using Omernik, J.M., 1987, Ecoregions of the conterminous United States (map supplement): Annals of the Association of American Geographers, v. 77, no. 1, p. 118-125, scale 1:7,500,000.

Listing of USDA Cold Hardiness Zone where germplasm was obtained. Cold Hardiness Zones determined using the USDA Plant Hardiness Zone Map, 2012. Agricultural Research Service, U.S. Department of Agriculture.