Pollen transcript unigene identifier

log2-fold

changeAnnotation (BLAST)

Unigene7226 -11.1 L. longiflorum chloroplast, complete genomeUnigene24307 -10.1 light harvesting protein 1Unigene25484 -9.8 photosystem II oxygen evoling complex protein 1Unigene13730 -9.7 L. longiflorum chloroplast, complete genomeUnigene11792 -8.9 glutamine synthetaseUnigene9929 -8.8 UnknownUnigene11119 -8.8 glutamine synthetaseUnigene16580 -8.6 L. longiflorum chloroplast, complete genomeUnigene2046 -8.3 L. longiflorum chloroplast, complete genomeUnigene2749 -8.2 ATP synthase CF0 subunit, chloroplast

Tab. S4 Top 10 down-regulated transcripts in lily pollen compared to vegetative tissue. The Lilium longiflorum pollen transcripts (access. no. ERP002303) were compared with the vegetative tissue transcriptome of oriental lily (access. no. SRX250152).

Fig.S1 Classification of the lily pollen transcriptome to GO classes. Sequences were classified to groups of the GO (Gene Ontology) data base. The number of identified sequences (number of genes) for each class is given in logarithmic scale.

biological process cellular component molecular function

Fig.S2 Organization of lily pollen sequences into functional categories (bin classes) using the Mercator software.

Fig.S3 Classification of the transcriptome to mayor metabolic pathways by MAPMAN. Each box presents an unigene which was classified into the given pathway categories. A. Lilium pollen transcriptome data. B. Transcriptome data obtained from 6 different Lilium oriental hybrid tissues (Du et al., 2014). Grey dots: BIN classses without any identified unigene or gene C. Up- (green) and down-regulated (red) lily pollen transcripts compared with the transcript numbers of Lilium oriental hybrid tissue. Data from Tab. S3 were used to construct Fig. S3C.

A B

C

PATHWAYS MAPPINGS EXPERIMENTS

Fig. S4 How to visualize your own pollen transcriptome with MAPMAN. The appropriate data and pathways are given as packed files (lilyPollenRNAseq_supplements_Lang_etal_2014.zip) and should be unpacked and copied into the indicated directories of the MAPMAN software.

Fig. S5 Comparison between pollen sequences and sporophytic tissue sequences / Amborella genome. The percentage of identified Arabidopsis pollen genes from Loraine et al. (2013, orange), the percentage of identified unigenes of the present study (Lilium pollen, Lang et al., dark red), the Amborella CDSs (olive green), Lilium oriental hybrid tissue (Du et al., green) and Lilium longiflorum leaf tissue (Shahin et al., 2012, blue-green) were compared. The percentages of identified genes were calculated for each MAPMAN Bin class 1 to 34.

Fig. S6 MA plots of comparison between pollen unigenes and transcripts of the vegetative tissue pool. A. MA plot of Lilium pollen versus Lilium oriental hybrid tissue (L-6Tis). The log2 fold change is plotted against the log2 of the average expression. According to the assumption that under most experimental conditions the bulk of genes of an organism are not responding differentially, the cloud of points centers around a log2 fold change of 0 (black dots). Red dots indicate pollen unigenes with estimated changes in expression level (P < 0.01) . B. and C. The middle panel shows similar plots but pollen and L-6Tis reads were mapped to the respective unigenes (B) , or mapped to the unigenes and length differences were corrected (C). D. and E. Finally, the lower panel shows the plots for the analysis Lilium pollen versus the 454 read dataset of Shahin et al. (2012) using once again their unigenes without correcting for length (D) and with a unigene length correction (E). For (B)-(E) a trendline was added for illustration

A

B C

D E

Fig. S7 Molecular phylogenetic analysis of plasma membrane H+ ATPase sequences from Lilium pollen and sporophytic Lilium tissue using maximum likelihood method. Assembled amino acid sequences with homologies to P-type PM H+ ATPases were aligned with MUSCLE and a phylogenetic tree was generated using Mega 6 software [3]. Sequences from pollen transcripts (L-pollen), leaf transcripts (L-leaf) and from pooled tissue (L-6Tis) were compared. The full-length ORFs of the pollen PM H+ ATPases LilHA1 (AY029190) and LilHA2 (EF397610.2) were included which clustered only with pollen transcripts (red circle).

The following settings were used for generation of the phylogenetic tree: Maximum Likelihood method based on the Poisson correction model [1]. The bootstrap consensus tree inferred from 500 replicates [2] is taken to represent the evolutionary history of the sequences analyzed [2]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The analysis involved 23 amino acid sequences. There were a total of 1042 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [3].1. Zuckerkandl E. and Pauling L. (1965). Evolutionary divergence and convergence in proteins. In Evolving Genes and Proteins, V. Bryson, H.J. Vogel (eds.), pp. 97-166. Academic Press, New York.2. Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791.3. Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729.