Comparative Transcriptome Analyses of a Mangrove Treesonneratiacaseolaris and Its Non-Mangrove

Comparative transcriptome analyses of a mangrove treeSonneratiacaseolaris and its non-mangrove relatives, Trapabispinosa and Duabanga grandiflora

Jianfang Lia, Yuchen Yanga, Shuhuan Yanga, Zhang Zhanga, Sufang Chena, Cairong Zhongb, Renchao Zhoua,*, Suhua Shi a,*

Methods of transcriptome analysis

Quality control and De novo assembly

Before assembly, reads of low quality were filtered outfrom the three datasets of S. caseolaris, Trapabispinosa and Duabanga grandiflora using customized Perl scripts. After that, high-quality reads of each species werede novo assembled using the Trinity program (trinityrnaseq_r20140413) with a minimum k-mer coverage of 2 and a minimum length of 200 bp(Grabherr et al., 2011). Following the method described by Yang et al.,(2015a, 2015b), transcripts of high similarity were clustered using TGICL-2.1(Pertea et al., 2003) and CD-HIT (Fu et al., 2012), and the reassembled results (unigenes) of low coverage and depth were eliminated from each dataset to increase the reliability of assembly.

Functional annotations and Gene Ontology (GO) classification

All the retainedunigenes of the three datasets were functionally annotated against Swiss-Prot database with an e-value cutoff of 1e-6 using AgBase(McCarthy et al., 2011). Then, Gene Ontology (GO) classification were carried out in the online program WEGO (Ye et al., 2006).

Positively selected gene (PSG) identification in Sonneratia caseolaris

As described in Yang et al., (2015a),open reading frames (ORFs) comprising at least 100 amino acid were extracted from each unigenesof the three datasets using TransDecoder(trinityrnaseq_r20140413; Grabherr et al., 2011). Putative orthologs were extractedamong five species, Sonneratia caseolarisS. alba, Trapa bispinosa, Duabanga grandiflora and Eucalyptus grandiusing OrthoMCL (Fischer et al., 2011). From the identified orthologs, putative positively selected genes (PSGs) along the branch of S. caseolaris, which was set as “foreground branch”, were detected using the branch-site model in the codeml module of PAML (Yang, 2007, Yang and Nielsen, 2002, Zhang et al., 2005). Model A1 with and without ω fixed to 1 was compared with that without ω fixed to 1 for PSG determination. the likelihood ratio test (LTR) were employed to assess the reliability of the results with ap-value cutoffof 0.05, and Bonferroni’s multiple testing correction was performed to control the false discovery rate with a threshold of less than 0.05.

Reference for supplementary methods

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