Tome. In the expression profiling treated with GA for 0 h and

Tome. In the expression profiling treated with GA for 0 h and 2 h, 1379592 the values of RPKM (Reads Per Kilobase of exon model per Million mapped reads) were 720 and 665, respectively (data unpublished). As both FPKM and RPKM were over 300, we considered the GACN01029707 can be used as a housekeeping gene for normalization control. AJ763915 was assigned for the double confirmation. Total RNA from stage 1 to stage 6 was extracted using identical methods as those mentioned before. cDNA was synthesized with 2.5 mg of RNA with oligo(dT) and reverse transcriptase following the protocol for a 50 ml system (Takara). PCR was performed with a 1 ml template for the PCR reaction at 94uC for 3 min, then 94uC for 30 s, 60uC for 20 s, and 72uC for 40 s with 25 to 30cycles in a 20 ml system.Supporting InformationTable S1 Match results of published genes cloned in ourCriteria for Screening Candidate Genes during Pathway 1485-00-3 site AnalysisTo obtain information regarding the metabolism and signal transduction of the phytohormones, we roughly filtered the transcripts that are involved in these pathways by the corresponding annotation of KEGG and KO ID in the KEGG maps (http:// www.genome.jp/dbget/). However, the annotations of one transcript from the Nr, Swiss-Prot and KEGG databases were not always consistent with one another. Therefore, more stringent screening criteria were set up to ensure that the annotations were consistent in at least two different databases. Some sequences with ambiguous annotations were confirmed online by Nr comparison using BLASTX (http://blast.ncbi.nlm.nih.gov/Blast.cgi). To compare the homologs with other species in the Asteraceae, we implemented screening parameters as e-value #1e25 using local BLASTX (version 2.2.23). The multiple alignment analysis of the DELLAs was performed in G. hybrida and Arabidopsis thaliana to construct the phylogram tree using ClustalW2 (http://www.ebi.ac. uk/Tools/MedChemExpress DprE1-IN-2 phylogeny/clustalw2_phylogeny/).laboratory compared to the transcriptome. (XLSX)Table S2 Specific genes identified and involved in GA biosynthesis and signal transduction. (XLSX) Table S3 Statistics of signal transduction genes in otherhormones. (XLSX)Table S4 Specific genes identified and involved in signal transduction in other phytohormones. (XLSX) Table S5 List of primers used for RT-PCR.(XLSX)AcknowledgmentsThe Illumina HiSeqTM 2000 sequencing was performed at the Beijing Genomics Institute (BGI), Shenzheng. The authors would like to thank Huaijun Xiang, Xueying Zhang, Tong Xu and Yuwen Zhou for helping with bioinformatic analysis, the reviewers for providing instructive comments to improve the manuscript and Dr Shan Liang in South China Normal University for discussion.Semi-quantitative RT-PCR Analysis of the Filtered GenesAs the transcriptome was the pooled data with the mixed libraries, semi-quantitative RT-PCR was chosen to detect the expression of the selected genes expression in distinct stages. The candidate genes with lengths greater than 500 bp were initially screened for being designed the primers. Then, in the selected genes, conserved areas were identified using local BLASTN and DNAMAN software to avoid locating primers in there. The sequences with high identities (greater than 90 ) to others were excluded for being designed effective primers. The primer pairs in the selected sequences were designed by Primer Primier 6 software and listed at Table S5. The internal reference sequences used twoAuthor ContributionsConducted the bioinformati.Tome. In the expression profiling treated with GA for 0 h and 2 h, 1379592 the values of RPKM (Reads Per Kilobase of exon model per Million mapped reads) were 720 and 665, respectively (data unpublished). As both FPKM and RPKM were over 300, we considered the GACN01029707 can be used as a housekeeping gene for normalization control. AJ763915 was assigned for the double confirmation. Total RNA from stage 1 to stage 6 was extracted using identical methods as those mentioned before. cDNA was synthesized with 2.5 mg of RNA with oligo(dT) and reverse transcriptase following the protocol for a 50 ml system (Takara). PCR was performed with a 1 ml template for the PCR reaction at 94uC for 3 min, then 94uC for 30 s, 60uC for 20 s, and 72uC for 40 s with 25 to 30cycles in a 20 ml system.Supporting InformationTable S1 Match results of published genes cloned in ourCriteria for Screening Candidate Genes during Pathway AnalysisTo obtain information regarding the metabolism and signal transduction of the phytohormones, we roughly filtered the transcripts that are involved in these pathways by the corresponding annotation of KEGG and KO ID in the KEGG maps (http:// www.genome.jp/dbget/). However, the annotations of one transcript from the Nr, Swiss-Prot and KEGG databases were not always consistent with one another. Therefore, more stringent screening criteria were set up to ensure that the annotations were consistent in at least two different databases. Some sequences with ambiguous annotations were confirmed online by Nr comparison using BLASTX (http://blast.ncbi.nlm.nih.gov/Blast.cgi). To compare the homologs with other species in the Asteraceae, we implemented screening parameters as e-value #1e25 using local BLASTX (version 2.2.23). The multiple alignment analysis of the DELLAs was performed in G. hybrida and Arabidopsis thaliana to construct the phylogram tree using ClustalW2 (http://www.ebi.ac. uk/Tools/phylogeny/clustalw2_phylogeny/).laboratory compared to the transcriptome. (XLSX)Table S2 Specific genes identified and involved in GA biosynthesis and signal transduction. (XLSX) Table S3 Statistics of signal transduction genes in otherhormones. (XLSX)Table S4 Specific genes identified and involved in signal transduction in other phytohormones. (XLSX) Table S5 List of primers used for RT-PCR.(XLSX)AcknowledgmentsThe Illumina HiSeqTM 2000 sequencing was performed at the Beijing Genomics Institute (BGI), Shenzheng. The authors would like to thank Huaijun Xiang, Xueying Zhang, Tong Xu and Yuwen Zhou for helping with bioinformatic analysis, the reviewers for providing instructive comments to improve the manuscript and Dr Shan Liang in South China Normal University for discussion.Semi-quantitative RT-PCR Analysis of the Filtered GenesAs the transcriptome was the pooled data with the mixed libraries, semi-quantitative RT-PCR was chosen to detect the expression of the selected genes expression in distinct stages. The candidate genes with lengths greater than 500 bp were initially screened for being designed the primers. Then, in the selected genes, conserved areas were identified using local BLASTN and DNAMAN software to avoid locating primers in there. The sequences with high identities (greater than 90 ) to others were excluded for being designed effective primers. The primer pairs in the selected sequences were designed by Primer Primier 6 software and listed at Table S5. The internal reference sequences used twoAuthor ContributionsConducted the bioinformati.