Transcriptome of Gossypium hirsutum floral and extrafloral nectaries

Comparative RNA-Seq study of expression in Gossypium hirsutum nectaries Seeds for Gossipium hirsutum cv. TM1 were chipped and germinated in 8cm x 8cm x 10 cm pots filled with a soil mixture of 3-parts LC8 soil to 1-part sand. Plants were grown in Environmental Growth Chambers kept at 12h light 26 °C /12h dark 22 °C cycle. Seedlings were transplanted into two-gallon (2A) pots after reaching approximately 30 cm in height. Upon transplanting, 10 g of Osmocote Pro 19-5-8 was mixed into the previously described soil mixture per pot. Plants were water each day and once per week with a 10% fertilizer solution of Scotts Excel 21-5-20 all purpose water soluble fertilizer and Scotts Excel 15-5-15 Cal-Mag water soluble fertilizer. All tissues were collected from plants after the first flower bloomed, approximately ten weeks after sowing, between the times of 1000 and 1500. RNA isolation: For a single biological replicate, approximately five nectaries were transferred with a clean forcep to a 2 ml Lysing matrix A tube (MP Biomedicals, Ref # 6910-500) containing a ceramic bead, resting in a liquid nitrogen bath. The tubes were quickly transferred to a QuickPrep adaptor (containing dry ice) and attached to the FastPrep 24™-5G (MP Biomedicals) benchtop homogenizer. The tissue were subjected to 5 to 6 homogenizing runs of 40 seconds each at 6 m/sec, with each run interjected with a period of immersing the tubes in liquid nitrogen and refilling the adaptor with dry ice. Post-pulverization, 600µl of the RNA lysis buffer of the Quick-RNA™ MiniPrep kit (Zymo Research, Cat# R1054) was quickly added to the Lysing matrix tube and the tubes were vortexed. This was followed by addition of 50µl of the Plant RNA Isolation Aid (Thermo Fisher Scientific Cat#AM9690; erstwhile Ambion) and a quick vortex to aid removal of common plant contaminants such as polyphenolics and polysaccharides. Quick-RNA™ MiniPrep kit directions were followed for RNA isolation. Agarose gel electrophoresis and UV spectrophotometry were used to assess RNA quality for all samples prior to submission to the University of Minnesota Genomics Center for barcoded library creation and Illumina HiSeq 2500 sequencing. All libraries were pooled and sequenced across two and a half lanes, then sequencing was repeated for quality concerns. This generated ≥24 M reads for each sample and the average quality scores were above Q30. Tissue types and stages: RNA was isolated from four types of nectaries (foliar, bracteal, circumbracteal, floral) at two or three different stages for each (pre-secretory, secretory and/or post-secretory). In parallel, RNA was also isolated from control samples from tissues adjacent to each nectary type. A total of 51 RNA samples were isolated as described below. Foliar nectary stages included: pre-secretory (‘FO_pre_x’; lack of visible nectar and midvein length of 5 to 6 cm on main stem leaf; 2 replicates), secretory [‘FO_S_x’; fully mature main stem leaf (midvein length 12 to 15 cm) with visible nectar, 1 replicate], and post-secretory (‘FO_P_x’; lack of visible nectar on fully mature main stem leaf; 3 replicates). In parallel, RNA was also isolated from control samples from tissues adjacent to the foliar nectaries (labeled ‘FO_pre_xC’, ‘FO_S_xC’ and ‘FO_P_xC’ where ‘x’ refers to a sample number). Bracteal nectary stages included: pre-secretory (‘B_pre_x’; matchhead square, 2 replicates) and post-secretory (‘B_post_x’; 19 to 24 days after anthesis; 3 replicates). In parallel, RNA was also isolated from control samples from tissues adjacent to the bracteal nectaries (labeled ‘B_pre_xC’ and ‘FO_post_xC’ where ‘x’ refers to a sample number). Circumbracteal nectary stages included: pre-secretory (‘C_pre_x’; matchhead square; 3 replicates) and post-secretory (‘C_post_x’; 19 to 24 days after anthesis; 3 replicates). In parallel, RNA was also isolated from control samples from tissues adjacent to the circumbracteal nectaries (labeled ‘B_pre_xC’ and ‘FO_post_xC’ where ‘x’ refers to a sample number). Floral nectary stages included: pre-secretory (‘FL_pre_x’; Candle, 24 hours pre-anthesis), secretory (‘FL_sec_x’; anthesis), and post-secretory (‘FL_post_x’; 24 hours post-anthesis). In parallel, RNA was also isolated from control samples from tissues adjacent to the floral nectaries (labeled ‘FL_pre_xC’, ‘FL_S_xC’ and ‘FL_P_xC’ where ‘x’ refers to a sample number.