*4.1. Identification and Sequence Analysis of Trihelix Transcription Factor Family in Rice*

The rice trihelix transcription factors were identified according to a previously described method with minor changes [39]. The Hidden Markov Model of Myb/SANT-LIKE domain (PF13837) was downloaded from the Pfam database (http://pfam.xfam.org/) [40]. The entire rice amino acid, genome, and CDS sequence assembly and corresponding annotation were downloaded from the EnsemblPlants database (http://plants.ensembl.org/index.html) [41]. The candidate proteins were sought by the HMMSEARCH program (https://www.ebi.ac.uk/Tools/hmmer/search/hmmsearch) base on the Bio-Linux system (Dr Tracey Timms-Wilson, Centre for Ecology & Hydrology (CEH), Oxfordshire, UK). The domain sequences of these candidate proteins were extracted and used to build a rice-specific Hidden Markov Model. All rice proteins were detected by the rice-specific Hidden Markov Model. Those with E-value < 0.01 were selected. The trihelix proteins were verified using the Pfam and InterPro databases (http://www.ebi.ac.uk/interpro/) [42]. Proteins obtained by the domain and

database screening confirmation were considered trihelix family members. The corresponding CDS and gene sequences were extracted according to their protein identifications.

The MEME program (http://meme-suite.org/) identified conserved motifs of the trihelix family proteins with the following parameters: Any number of repetitions; minimum seven motifs; maximum 49 motifs; optimum 10–200 amino acids; expected E-value < 1 <sup>×</sup> <sup>10</sup>−48. The trihelix family gene structures were displayed by comparing the coding and genomic sequences with the Gene Structure Display Server tools (http://gsds.cbi.pku.edu.cn/) [43]. The chromosomal locations of the trihelix family genes were mapped onto the rice linkage map with an online tool according to their TIGR numbers [44]. The isoelectric points and molecular weights of trihelix family proteins were estimated with ExPASy (http://expasy.org/) [45].
