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Additional file 1: Fig. S1. Length distribution of the assembled transcripts of N. khasiana leaf. Fig. S2. a, E-value distribution of BLASTX hits of N. khasiana leaf transcriptome against the NCBI non-redundant protein database. b, BLASTX similarity score distribution of N. khasiana leaf transcriptome with the NCBI non-redundant protein database. Fig. S3. Top 50 organisms distribution of the assembled transcriptome using BLASTX. Fig. S4. Metabolic pathway mapping of the assembled transcriptome. Fig. S5. Top 10 GO terms in biological processes, cellular components and molecular functions. Fig. S6. Over-represented and under-represented molecular functions in each of the five different parts/zones of the N. khasiana leaf. No enrichment was detected in the lid. Fig. S7. Transcript expression distribution in the five tissue samples. Fig. S8. Assigning Mapman ‘bins’ to the DEGs using the automated annotation software Mercator available online at https://mapman.gabipd.org/app/mercator . Fig. S9. Determining the number of clusters for k-means clustering using the Figures of Merit (FOM) application embedded in the MeV program. The adjusted FOM decreases sharply and levels out after reaching 4 clusters. Fig. S10. Determination of the number of clusters for k-means clustering using the gap statistic algorithm in R. The number of clusters is 6. Fig. S11. N. khasiana plant showing several developing leaves, each attaining distinct stages of development. Stage 5 represents the leaf (L5) showing pitcher expansion with the lid remaining unopened. Transcriptome data of stage 5 was included in the present study. White vertical/horizontal lines specify the dissected regions of each stage. Bar = 6 cm. Fig. S12. Relative abundance of bacterial transcripts against fungal transcripts across the different parts/zones of the N. khasiana leaf. LB: leaf base; T: tendril; D: digestive zone; W: waxy zone; L: lid. Fig. S13. Epidermal nail polish imprints of the abaxial surfaces of four different parts/zones of the N. khasiana leaf. These imprints are then used to estimate stomatal density [1]. Arrow head denotes stomata. Fig. S14. Sample collection for transcriptome sequencing of N. khasiana leaf. a, Nepenthes khasiana plants growing in the natural habitat at Jaraiñ, Jaiñtia Hills District, Meghalaya. b, mature pitcher with fully opened-lid and prominent wings formed along the sides of the pitcher. c-d, preparation of the different parts/zones of the leaf viz. leaf base, tendril, digestive zone, waxy zone and lid for preservation in liquid nitrogen. Note: transition regions represent those regions in the leaf that indicate a shift from one part/zone to another. Fig. S15. Extraction and quality check of total RNA from N. khasiana leaf. a, total RNA extracted from five different parts/zones of the N. khasiana leaf. The full-length gels photos are provided in Fig. S16. below; b, total RNA profile and the corresponding peaks resulting from a quality check of the isolated total RNA (leaf base) using an Agilent Bioanalyzer; c, electropherogram profile of RNA library. Fig. S16. Full-length gels photos of the total RNAs isolated from the five different parts/zones of the N. khasiana leaf. The gel photo on the left shows the extracted RNAs of the digestive zone, tendril and the leaf base (lanes 4, 5 and 6). Since the RNA of the tendril is of poor quality (lane 5), isolation was repeated to yield a better one (lane 8 of middle gel photo). The gel photo on the right shows the extracted RNAs of the lid and the waxy zone (lanes 4 and 5). The RNA gel images of each part/zone were cropped and presented in Fig. S15a. Note S1. Stomatal density and distribution in the model plant Arabidopsis thaliana. Table S1. RNA sequencing read statistics of the different tissue parts of two N. khasiana leaf samples. We performed MD5 CheckSum on the FASTQ files under each category (raw and quality filter passed reads) to ensure data integrity. Table S2. Alignment summary of the individual reads to the reference transcriptome. Table S3. List of qPCR primers used for the validation of RNA-seq derived transcript expression pattern. |
