Transcriptome responses of two date palm (Phoenix dactylifera L.) varieties (Khalas and Sultana) exposed to NaCl

Provided are Supplementary tables (Table S4-12) to the manuscript in which transcriptome reponses of two date palm (Phoenix dactylifera L.) varieties (Khalas and Sultana) to NaCl are reported. The experiment (Experiment 3 of tha manuscript) was conducted on ten two-year old plants of micro-propagated plants which were obtained from Date Palm Developments Ltd. (Somerset, U.K.). Upon arrival to the lab, plants were planted in 1.5 L pots filled with a mixture of Compo Sana® Potting Soil for Green Plants and Palms (Compo GmbH, Münster, Germany) and sand (5:2 v/v) and watered to water-holding capacity with double-distilled water. Plants were acclimatised to growth in a controlled environment chamber with 60% humidity, a temperature regime of 25 °C day and 20 °C night, day length of 16 h, and 250 µmol s-1 m-2 light intensity at plant height. During one month of acclimation plants were then watered as needed, after which five plants per variety were watered with 100 mL of either double-distilled water (non-saline, control treatment) or five plants per variety with 150 mM NaCl three times per week for two weeks, followed by 300 mM NaCl two times per week for four weeks (saline treatment). At harvest shoots and roots were separated and roots were thoroughly washed in water; roots and shoots were frozen in liquid nitrogen and kept at -80 °C until RNA extraction, when plant material was first homogenised by grinding manually in liquid nitrogen, then a subsample was taken and further pulverised before extraction. The RNAqueousTM kit for total RNA isolation (Thermo Fisher Scientific, MA, USA) was used according to the manufacturer’s instructions. RNA integrity and concentration were confirmed using a 1% (w/v) agarose gel. RNA was sent to BGI Hong Kong (https://www.bgi.com/global) for sequencing. Single-end sequencing was performed on a DNBseq platform. Library preparation, sequencing, alignment and bioinformatics were performed by BGI according to their standard procedures. Each sample generated an average of 4.33 Gb of sequence and had an average mapping ratio of 84.04% with the reference genome. Genes were mapped with 100% accuracy and 28,027 genes were identified of which 2,355 were novel genes and 17,570 were novel transcripts. After filtering the raw reads using SOAPnuke (version1.5.2), which removed adaptors and unknown bases as well as low-quality reads, clean reads were mapped to the reference genome (NCBI_GCF_009389715.1_palm_55x_up_171113_PBpolish2nd_filt_p) using HISAT2 (version 2.0.4). StringTie (version 1.0.4) (Pertea et al., 2015) was used to predict transcripts and Cuffcompare (Trapnell et al., 2012) to identify new transcripts. Finally, CPC (version 0.9-r2) (Kong et al., 2007) was used to predict the coding potential of the new transcripts and the subsequently selected new transcripts were merged with the reference transcripts. Gene expression was calculated using RSME (version 1.2.5) (Li and Dewey, 2011). Differentially expressed genes were extrapolated by using DEseq2. The expression was given as Transcripts Per Kilobase Million (TPM). A fold change was determined from the average expression in two different groups and DEGs were then filtered for a fold change ≥2 and an adjusted p value ≤0.05. 

Li, B. & Dewey, C.N. (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics, 12, 323