The seeds were derived from introgressing G. soja (PI468916) into G. max (A81-356022). Specifically, the BC5F5 plant P-C609-45-2-2 was heterozygous for the LG I protein QTL introgression from G. soja. These seeds were planted directly into pots containing Bradyrhizobium japonicum-inoculated soil and supplemented with full nutrient fertilizer (Osmocote 14-14-14) in growth chambers at the University of Minnesota. Chambers were set initially to a photoperiod of 14/10 and thermocycle of 22°C/10°C and monitored to mimic Illinois field growing conditions. Relative humidity settings were 50-60%, and light intensity was measured at 550-740 μE m-2 sec-1. All harvests occurred at 1400 hours and consisted of samples pooled from a minimum of three plants [52 (link)]. Samples were harvested from plants in parallel and flash frozen in liquid nitrogen before storage at -80°C. Open flowers and young leaf tissue samples were collected simultaneously. Pods and seeds were harvested by seed weight and pod lengths that correspond to approximated Days After Flowering (DAF) as specified. The one-cm pod was processed intact (approximately 7-DAF), while the four and five cm pods (approximately 10-13 DAF and 14-17 DAF) were divided into seed and pod-shell components. Seed 21-DAF, Seed 25-DAF, Seed 28-DAF and Seed 35-DAF had seed weights between 10 and 25 milligrams, 25 and 50 milligrams, 50 and 100 milligrams, 100 and 200 milligrams, and greater than 200 milligrams, respectively.
Root and nodule tissues were harvested from plants grown in growth chambers set to 16-hr photoperiods with light intensities ranging from 310-380 μE m-2 sec-1. Seeds were imbibed for three days, planted in quartz sand and fertilized with a full nutrient solution. Root tissue was harvested after 12 days. Nodules were harvested at 20-25 days after inoculation; for these samples, plants were fertilized for the first seven days with nutrient solution containing 3.5 mM NO3 and subsequently fertilized every other day with a full nutrient solution lacking nitrogen.
Soybean tissue samples were ground with liquid nitrogen by mortar and pestle. Total RNA was isolated by a modified TRIzol® (Invitrogen) protocol [53 (link)]. DNA was removed by digest with on-column RNase-free DNase (Qiagen), and RNA was purified and concentrated by RNeasy column (Qiagen). RNA quality was evaluated by gel electrophoresis, spectrophotometer and Agilent 2100 bioanalyzer.
Root and nodule tissues were harvested from plants grown in growth chambers set to 16-hr photoperiods with light intensities ranging from 310-380 μE m-2 sec-1. Seeds were imbibed for three days, planted in quartz sand and fertilized with a full nutrient solution. Root tissue was harvested after 12 days. Nodules were harvested at 20-25 days after inoculation; for these samples, plants were fertilized for the first seven days with nutrient solution containing 3.5 mM NO3 and subsequently fertilized every other day with a full nutrient solution lacking nitrogen.
Soybean tissue samples were ground with liquid nitrogen by mortar and pestle. Total RNA was isolated by a modified TRIzol® (Invitrogen) protocol [53 (link)]. DNA was removed by digest with on-column RNase-free DNase (Qiagen), and RNA was purified and concentrated by RNeasy column (Qiagen). RNA quality was evaluated by gel electrophoresis, spectrophotometer and Agilent 2100 bioanalyzer.
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