Mol Genet Genomics(2015)290:239-255 241 Flower development is controlled by a complex network The Pfam database (http://pfam.sanger.ac.uk/)was of interactions between transcription factors,most of them used to screen the genome assemblies of Prunus per- belonging to the MADS-box family (Airoldi and Davies sica,Arabidopsis lyrata,Capsella rubella,Thellungiella 2012).To get a better picture about the size and phylog- halophila,Solanum tuberosum,Solanum lycopersicum, eny of the MADS-box family in plants,we sorted and Aquilegia coerulea and Volvox carteri.The genome data compared the MADS-box genes from 22 different plant were downloaded from the genome browser phytozome species.To better understand these transcription factors in (http://www.phytozome.net/),and the evolutionary rela- Chinese cabbage,we determined 160 MADS-box genes tionships of these species were determined using the and analysed the phylogenetic relationships,conserved genome browser phytozome and the public database PGDD motifs,retention and ortholog groups between these Chi- (http://chibba.agtec.uga.edu/duplication/)(Lee et al.2013) nese cabbage MADS-box genes and Arabidopsis MADS- box genes.We further studied the chromosomal locations, Phylogenetic analysis gene duplication and tissue-specific expression of BrMADS genes.The expression of all of the BrMIKCC genes was In the phylogenetic tree,the Arabidopsis MADS proteins also investigated under different treatments.which included were used to classify the Chinese cabbage MADS proteins GA.SA.ABA.heat and cold. into different groups.Full-length sequences of MADS pro- teins of Chinese cabbage and Arabidopsis were aligned using the Clustalw2 program with default parameters Materials and methods (Thompson et al.1997).Then,a phylogenetic tree was then constructed by the neighbour-joining method,and boot- Identification of MADS-box gene family in Chinese strap values were calculated with 1,000 replications using cabbage MEGA5.2 (Tamura et al.2011).Additionally,an Arabidop- sis MADS proteins phylogenetic tree was used to detect the All the files that are related to Brassica genome sequence reliability of this method,and to test and verify the classi- data that were used for the identification and annotation of fication,a phylogenetic tree of Chinese cabbage,Arabidop- MADS proteins were downloaded from the Brassica data- sis,rice and grapevine was built. base (BRAD;http://brassicadb.org/brad/)(Wang et al.2011). To estimate the nucleotide divergence between Proteins with SRF-TF domains (PF00319)were retrieved sequences,all nucleotide sequences of Chinese cabbage from the Pfam 27.0 database (http://Pfam.sanger.ac.uk/) MADS-box genes were also analysed by MEGA5.2 using (Punta et al.2012).The hidden Markov model (HMM)was the Jukes-Cantor model.Bootstrap(1,000 replicates)analy- used to identify the putative MADS proteins in Chinese ses were also performed for this estimation. cabbage (Finn et al.2011).To obtain the proteins,first we used the tool hmmsearch,with an expected value (e-value) Identification of conserved motifs and gene structure cut-off 1.0.Then,we verified these sequences using the tool SMART (http://smart.embl-heidelberg.de/)(Letunic et al. To identify the conserved motifs in full-length Chinese cab- 2012),the Pfam database (http://Pfam.sanger.ac.uk/)and the bage and Arabidopsis MADS proteins,the Multiple Expec- NCBI database (http://www.ncbi.nlm.nih.gov/). tation-maximisations for Motif Elicitation (MEME)pro- gram version 4.9.0(Bailey et al.2009)was used with default Sequence retrieval parameters,except for the following parameters:(1)opti- mum motif width was set to =10 and <100;and (2)the max- The Arabidopsis thaliana MADS proteins were retrieved imum number of motifs was set to identify 15 motifs.The from the TAIR database (http://www.arabidopsis.org/) MEME motifs were annotated using the SMART program according to a previous report by Parenicova et al.(2003). (http://smart.embl-heidelberg.de)and the Pfam database. The dataset of predicted Oryza sativa MADS proteins The coding domain sequences(CDS)and DNA sequences was retrieved from previous analyses by Arora et al. of Chinese cabbage MADS-box genes were used to reveal (Arora et al.2007).A MADS-box domain was not found the gene structure using the tool GSDS (http://gsds.cbi.pku. in LOC_Os02g01360 (OsMADS60),LOC_Os12g31010 edu.cn/). (OsMADS67),and LOC_Os08g20460 (OsMADS69).The MADS proteins of Populus trichocarpa,Medicago trun- Ortholog groups of MADS-box genes in Brassica catula,Glycine max,Cucumls sativus,Citrus sinensis,Cit- and Arabidopsis genome rus clementine,Vitis vinifra,Sorghum bicolor,Zea mays, Selaginella moellendorffi and Physcomitrella paters were The program OrthoMCL (http://www.orthomcl.org/cgi- retrieved from a previous report. bin/OrthoMclWeb.cgi)(Li et al.2003)was used to identify ②SpringerMol Genet Genomics (2015) 290:239–255 241 1 3 Flower development is controlled by a complex network of interactions between transcription factors, most of them belonging to the MADS-box family (Airoldi and Davies 2012). To get a better picture about the size and phylog￾eny of the MADS-box family in plants, we sorted and compared the MADS-box genes from 22 different plant species. To better understand these transcription factors in Chinese cabbage, we determined 160 MADS-box genes and analysed the phylogenetic relationships, conserved motifs, retention and ortholog groups between these Chi￾nese cabbage MADS-box genes and Arabidopsis MADS￾box genes. We further studied the chromosomal locations, gene duplication and tissue-specific expression of BrMADS genes. The expression of all of the BrMIKCC genes was also investigated under different treatments, which included GA, SA, ABA, heat and cold. Materials and methods Identification of MADS-box gene family in Chinese cabbage All the files that are related to Brassica genome sequence data that were used for the identification and annotation of MADS proteins were downloaded from the Brassica data￾base (BRAD; http://brassicadb.org/brad/) (Wang et al. 2011). Proteins with SRF-TF domains (PF00319) were retrieved from the Pfam 27.0 database (http://Pfam.sanger.ac.uk/) (Punta et al. 2012). The hidden Markov model (HMM) was used to identify the putative MADS proteins in Chinese cabbage (Finn et al. 2011). To obtain the proteins, first we used the tool hmmsearch, with an expected value (e-value) cut-off 1.0. Then, we verified these sequences using the tool SMART (http://smart.embl-heidelberg.de/) (Letunic et al. 2012), the Pfam database (http://Pfam.sanger.ac.uk/) and the NCBI database (http://www.ncbi.nlm.nih.gov/). Sequence retrieval The Arabidopsis thaliana MADS proteins were retrieved from the TAIR database (http://www.arabidopsis.org/) according to a previous report by Parenicova et al. (2003). The dataset of predicted Oryza sativa MADS proteins was retrieved from previous analyses by Arora et al. (Arora et al. 2007). A MADS-box domain was not found in LOC_Os02g01360 (OsMADS60), LOC_Os12g31010 (OsMADS67), and LOC_Os08g20460 (OsMADS69). The MADS proteins of Populus trichocarpa, Medicago trun￾catula, Glycine max, Cucumls sativus, Citrus sinensis, Cit￾rus clementine, Vitis vinifra, Sorghum bicolor, Zea mays, Selaginella moellendorffi and Physcomitrella paters were retrieved from a previous report. The Pfam database (http://pfam.sanger.ac.uk/) was used to screen the genome assemblies of Prunus per￾sica, Arabidopsis lyrata, Capsella rubella, Thellungiella halophila, Solanum tuberosum, Solanum lycopersicum, Aquilegia coerulea and Volvox carteri. The genome data were downloaded from the genome browser phytozome (http://www.phytozome.net/), and the evolutionary rela￾tionships of these species were determined using the genome browser phytozome and the public database PGDD (http://chibba.agtec.uga.edu/duplication/) (Lee et al. 2013). Phylogenetic analysis In the phylogenetic tree, the Arabidopsis MADS proteins were used to classify the Chinese cabbage MADS proteins into different groups. Full-length sequences of MADS pro￾teins of Chinese cabbage and Arabidopsis were aligned using the ClustalW2 program with default parameters (Thompson et al. 1997). Then, a phylogenetic tree was then constructed by the neighbour-joining method, and boot￾strap values were calculated with 1,000 replications using MEGA5.2 (Tamura et al. 2011). Additionally, an Arabidop￾sis MADS proteins phylogenetic tree was used to detect the reliability of this method, and to test and verify the classi￾fication, a phylogenetic tree of Chinese cabbage, Arabidop￾sis, rice and grapevine was built. To estimate the nucleotide divergence between sequences, all nucleotide sequences of Chinese cabbage MADS-box genes were also analysed by MEGA5.2 using the Jukes-Cantor model. Bootstrap (1,000 replicates) analy￾ses were also performed for this estimation. Identification of conserved motifs and gene structure To identify the conserved motifs in full-length Chinese cab￾bage and Arabidopsis MADS proteins, the Multiple Expec￾tation-maximisations for Motif Elicitation (MEME) pro￾gram version 4.9.0 (Bailey et al. 2009) was used with default parameters, except for the following parameters: (1) opti￾mum motif width was set to ≥10 and ≤100; and (2) the max￾imum number of motifs was set to identify 15 motifs. The MEME motifs were annotated using the SMART program (http://smart.embl-heidelberg.de) and the Pfam database. The coding domain sequences (CDS) and DNA sequences of Chinese cabbage MADS-box genes were used to reveal the gene structure using the tool GSDS (http://gsds.cbi.pku. edu.cn/). Ortholog groups of MADS-box genes in Brassica and Arabidopsis genome The program OrthoMCL (http://www.orthomcl.org/cgi￾bin/OrthoMclWeb.cgi) (Li et al. 2003) was used to identify