Expression analysis of candidate genes validates digital gene expression

Two types of qRT-PCR experiments were conducted. Experiment 1 included the validation of the digital gene expression results. Therefore selected contigs have been amplified and their expression pattern has been investigated in different tissue. In total of the six selected candidate genes for Experiment 1 (Candidate 1) four candidate genes could be amplified successful during the qRT-PCR

101 Experiment (tdn_85889, tdn_69411, k65_9861 and tdn_146439) (table 24). For the second experiment four candidate genes have been selected and one of them could be amplified successful: tdn_112851 (table 24). The contig tdn_76356 shows up two times, as two different primer pairs were tried out to amplify the contig, but none of them did work. The contig k65_5754 was used as a housekeeping gene, as its expression profile based on the normalized read counts was equal throughout the all libraries (e Appendix: TpT_10_TPM). The second housekeeping gene RC31500 was used as a housekeeping gene in a previous study from Yates et al. (2014). It could not be amplified during my study.

Table 24 Success of qRT-PCR experiments, in addition the contig name is listed and during which qRT-PCR experiment the contig was used.

ID transcript Pattern qRT-PCR

1 k65_5754 Housekeeping success

2 RC31500(Yates et al. 2014) Housekeeping failed

3 tdn_146439 Candidate1 success 4 k65_9861 Candidate1 success 5 tdn_69411 Candidate1 success 6 tdn_85889 Candidate1 success 7 tgg_76356_b Candidate1 failed 8 tdn_125117 Candidat1e failed 9 tdn_76635 Candidate 2 failed

10 tdn_76356_a Candidate 2 failed

11 tdn_103259 Candidate 2 failed

12 tdn_112851 Candidate 2 success

For the validation of the qRT-PCR results the expression profile of four randomly picked contigs were investigated. The expression profile of those four contigs was examined via qRT-PCR in different tissues (axial meristem and leaves) of mown and not mown plants and 12 different transcriptome libraries of mown and not mown plants. Afterwards the expression profile was compared with the expression profile of the results of the digital gene expression (table 25). Each contig showed a very unique expression pattern, indicating the complexity of expression studies.

102 Figure 28 Expression profiling of selected T. pratense genes: A) tdn69411 B) tdn85889 C) tdn146439 D) tdnK65-9861 E) tdn_112861 examined by quantitative real-time PCR (qRT-PCR) in different T. pratense tissues; axial meristem, leaves, Transcriptome library: TPM2, TPM1, TPGHM. In the “mown” treatment compared to the “not mown” treatment. Gene expression levels were normalized to the internal control k65_5754. Graph shows log2fold changes. Data bars represent the mean +/- SD level of relative transcript abundance of three replicates. Given is the contig name, classification in functional group and A. thaliana gene/protein name. Figure was edited using Inkscape Albert et al. (2014) (V. 0.48; available at: https://inkscape.org/de/). Data visualization was aided by Daniel’s XL Toolbox addin for Excel, version 7.2.12, by Daniel Kraus, Würzburg, Germany (www.xltoolbox.net) (Kraus 2014).

For tdn_85889 (homologue Tair ID and corresponding gene name AT4G33220, PME44, figure 28B) it was found, that the expression level is higher in leaves of mown plants (log2fold 0.6±0.5) compared to control plants. In the axial meristem tissue the expression was decreased in the mown plants (log2fold -1.2±0.8). The expression level of tdn_85889 was higher in the mown transcriptomes FaM (TPM2), GM (TPMGH) ,and FbM (TPM1) compared to their not mown counterparts (log2fold 1.6±1.6;

103 1.6±0.5, 0.5±0.2) which was in accordance based on the TPM values of the transcriptome analysis data. The contig tdn_146439 (ENGASE85A, figure 28C) was in every tissue and library of mown plants higher expressed compared to the not mown plants. Solely in the axial meristem almost no expression was detectable. Based on the digital expression analysis the contig was found to be upregulated within FaM (TPM2), GM (TPMGH) ,and FbM (TPM1), this goes in accordance with the findings of the TPM values. The contig tdn_K65-9861 (AT2G39800, P5CS_MESCR (figure 28D) is higher expressed in axial meristem of not mown plants compared to mown plants (log2fold -1.9±0.2 in mown T. pratense plants).The contig tdn_69411 (LTP family gene figure 28A) is upregulated in the axial meristem of mown T. pratense plants and showed no expression in leaf. Within the material of the field transcriptomes of the mown T. pratense plants it was found to be upregulated compared to the not mown condition. Within the greenhouse transcriptomes of mown plants it showed a weak expression, but due to the large standard deviation the result was not reliable. Based on the TPM values the contig is expressed in GM and TPM2. The expression pattern of tdn_112861 (ATGA2OX1) revealed that the contig is downregulated in flowers, axial meristem and young buds and leaves of mown plants (figure 28E). In addition it is downregulated within all transcriptome samples.

Figure 29 Expression profiling of selected T. pratense gene. Tdn_112861 examined by quantitative real-time PCR in different T. pratense tissues ;flowers (adult plants), axial mersitem (adult plants), young flowers (adult plants), leaves (adult plants), flowers (NM), axial meristem (NM), young flowers (NM), leaves (NM), axial mersitem (M),young flowers (M), leaves (M), Transcriptome library: TPNM2, TPNM3, TPGHNM, TPM2, TPM,TPGHM, compared to young seedlings. Gene expression levels were normalized to the internal control k65_5754. Graph shows log2fold changes. Data bars represent the mean +/- SD level of relative transcript abundance of three replicates. Figure was edited using Inkscape Albert et al. (2014) (V. 0.48; available at: https://inkscape.org/de/). Data visualization was aided by Daniel’s XL Toolbox addin for Excel, version 7.2.12, by Daniel Kraus, Würzburg, Germany (www.xltoolbox.net) (Kraus 2014).

104 Table 25 TPM values (rounded) for a rough estimation oft he expression strength fort he Candidate genes. a/b refers to the replicas. High expression is highlighted.

TPGH M1a TPGH M1b TPGHN M1a TPGHN M1b TPM 1a TPM 1b TPM 2a TPM 2b TPNM 2a TPNM 2b TPNM 3a TPNM 3b tdn_694 11 27.4 33.6 5.3 3.2 8.7 12 17.0 44.1 10.1 12 4.8 3.3 tdn_858 89 30.9 36.1 9.3 9 47.9 38.5 35.4 28.5 28.8 26 23.8 19.2 tdn_146 439 101.9 131.1 16.5 16.7 45.5 42 76.7 64.9 24.4 24.1 13.2 11.3 k65_986 1 18 18.5 89.5 102.2 17.6 11.9 26.6 6 14.4 9 77.7 58.9 21.1 95.1 tdn_112 851 6.5 6.7 13.1 25.5 7.6 6 7 8.5 15.6 10.9 7.1 8.9 k65_575 4 23.5 27.9 23.1 23.1 42.2 39.6 30.5 28.8 30.2 25.4 35 27.7

As I was interested in which tissue and under which treatment the contig tdn_112861 is further expressed and how strong I compared the expression strength of the contig within young seedling of

T. pratense with other tissue (figure 29). The “young seedling” samples were used as calibrators to

represent the baseline of gene expression to monitor the change in the expression levels for the examined genes in the other tissues. I found that the contig showed a strong expression in flowers and a weaker expression in axial mersitem and young flowers of adult plants compared with young seedlings (log2fold 6.7±2, 1.2±1, 1.5±1). It was weaker expressed in leaves (log2fold -1.3±2). In older plants (adult plus 2-3 weeks) it was still highly expressed in flowers (log2fold 6.6±3) and axial meristem (1.3±2), but was leaves (-2.8±2). Further it was highly expressed in flowers of mown plants (log2fold 4.6±1). But was down regulated in young inflorescences and leaves of mown plants (log2fold -2.7±1, -2.8±1). Compared with young seedlings the contig was downregulated within all transcriptome libraries which consist of leave and shoot tissue.