Malus domestica cultivar:Granny Smith and Honey crsip Transcriptome or Gene expression

GS-1-16: ‘Granny Smith’ apples were obtained the day of harvest, 13 September, 2016, from a commercial orchard near Wenatchee, WA USA. Boxed fruit was then stored at 1˚C in air. Fruit peel was collected at harvest and after storage from 3 replicates of 6 apples each using a vegetable peeler. Evenly spaced peel sections (n=3 for each fruit) cut from the stem end to calyx end (resultant peels were ~20 cm x 2 cm) and centered at the equator were immediately flash frozen on liquid nitrogen and stored at -80˚C. These samples represent a short time course sampling scheme in the first two weeks of air storage (TC-0 = at harvest, TC-1 = 1 week at 1˚C, TC-2 = 2 weeks at 1˚C, TE-1 = 1 week at 1˚C, then 5 days at 20˚C, T1.1 through T1.5 are a 24 hour time course of the 5 days at 20˚C TE-1 treatment).HC-1-17: ‘Honeycrisp’ apples were obtained in August 2016 from a commercial orchard near Wenatchee, WA USA. Fruit peel and cortical tissue were collected from individual apples after approximately six months of boxed storage at 1 ˚C in air. A 4-mm biopsy punch, to depth of 6 to 8 mm, was used to harvest tissues, where the peel was immediately separated from the cortex using a razor blade and both tissues immediately frozen separately in liquid nitrogen, then stored at -80 ˚C. Four samples, in biological triplicate (a total of 12 observations - where each replicate was a pool of tissues from 10 fruit), represent peel and cortical tissues each from fruit with and without bitter pit lesions (ASP = asymptomatic peel, ASC = asymptomatic cortex, SP = symptomatic peel, SC = symptomatic cortex).RNA was extracted using a CTAB/Chloroform protocol modified specifically for pome fruit tissue (Honaas and Kahn, 2017- DOI 10.1186/s13104-017-2564-2). Extracted RNA was analyzed for quantity and purity using the Nanodrop (ND-1000, Thermo Fisher Scientific, Waltham, MA) and for quantity and integrity on the Agilent Bioanalzyer 2100 (G2938C, Agilent Technologies, Santa Clara, CA) with the Agilent-RNA Pico Kit (cat# 5067-1513). Only RNA that met the following standards was used for downstream analysis: A260/A280  2.0, RNA Integrity Number (RIN) of ≥ 8.0.

GS-1-16：实验材料为‘澳洲青苹（Granny Smith）’苹果，于2016年9月13日收获当日从美国华盛顿州韦纳奇附近的商业果园采收。装箱后的果实被置于1℃空气环境中冷藏。分别在收获时及冷藏后，使用果蔬削皮器采集3组重复样本，每组包含6个苹果的果皮。从每个果实的果柄端至花萼端、以赤道为中心均匀截取间距一致的果皮片段（每果取n=3片，尺寸约20cm×2cm），截取完成后立即置于液氮中快速冷冻，并于-80℃下保存。本系列样本采用冷藏前两周的短期时序采样方案：TC-0为收获时样本，TC-1为1℃冷藏1周样本，TC-2为1℃冷藏2周样本；TE-1为1℃冷藏1周后置于20℃环境5天的样本；T1.1至T1.5则为TE-1处理后20℃环境下5天内的24小时间隔时序样本。
HC-1-17：‘蜜脆（Honeycrisp）’苹果于2016年8月从美国华盛顿州韦纳奇附近的商业果园采收。果实经1℃空气环境装箱冷藏约6个月后，采集果皮与皮层组织。使用直径4mm、穿刺深度6~8mm的活检打孔器获取组织，随后立即用剃须刀片将果皮与皮层分离，两种组织分别置于液氮中快速冷冻后，于-80℃保存。本实验共设置4类样本，每类设置3次生物学重复，总计12份观测样本（每重复为10个果实的组织混合样），分别采集带有苦痘病病斑与无病斑果实的果皮和皮层组织：ASP为无症状果皮（asymptomatic peel），ASC为无症状皮层（asymptomatic cortex），SP为显症果皮（symptomatic peel），SC为显症皮层（symptomatic cortex）。
总RNA提取采用针对梨果类组织优化的CTAB/氯仿法（Honaas与Kahn, 2017, DOI: 10.1186/s13104-017-2564-2）。提取得到的RNA通过超微量紫外分光光度计（Nanodrop ND-1000, Thermo Fisher Scientific, 赛默飞世尔科技，马萨诸塞州沃尔瑟姆市）检测浓度与纯度，并使用安捷伦生物分析仪2100（Agilent Bioanalyzer 2100, 型号G2938C, Agilent Technologies, 安捷伦科技，加利福尼亚州圣克拉拉市）配套安捷伦RNA Pico试剂盒（货号5067-1513）检测浓度与完整性。仅满足以下标准的RNA方可用于后续实验：A260/A280≈2.0，RNA完整性数（RNA Integrity Number, RIN）≥8.0。