242 Mol Genet Genomics (2015)290:239-255 the homologous genes of MADS-box between Chinese completed for this cultivar;thus,this cultivar is a typical cabbage and Arabidopsis.Briefly,the tools BLASTP, cultivar for Chinese cabbage research.Seeds were grown with an e-value <le-10,and orthomclPairs were applied in pots containing a soil:vermiculite mixture (3:1)in the to find orthologs,inparalogs and coorthologs in these two greenhouse of Nanjing Agricultural University,and the species.To link these genes to chromosomes,a tool called controlled-environment growth chamber programmed is Circos (Krzywinski et al.2009)was used.In addition.the light 16 h/25 C,dark 8 h/20 C (Song et al.2013).One Cytoscape software was applied to build the network of month later,seedlings at the five-leaf stage were trans- these relationships (Shannon et al.2003). ferred to growth chambers that were set at 4 or 38 C under identical light intensity and day length as the Chromosome localisation and gene duplications cold and heat treatments.Simultaneously.for acclima- tion,some plants were cultured in 1/2 Hoagland's solu- To determine the physical locations of MADS-box genes,the tion in plastic containers,with the pH at 6.5(Jensen and starting and ending positions of all MADS-box genes on each Bassham 1966).After 5 days of acclimatisation,plants chromosome were obtained from the BRAD database.The were cultured in the following four treatments:(1)Con- Perl in-house program was used to draw the location images of trol:(2)100 uMABA:(3)100 uM GA:(4)100 uM SA. the Chinese cabbage MADS-box genes.The positions of each At 4 and 12 h after treatment,the young leaf samples were Chinese cabbage MADS-box gene on the blocks were verified collected,frozen in liquid nitrogen and stored at-70C by searching for homologous genes between Arabidopsis and for further analysis. three B.rapa subgenomes,including least fractionated (LF), medium fractionated (MFl)and most fractionated (MF2) RNA isolation and quantitative real-time PCR genomes (http://brassicadb.org/brad/searchSynteny.php) (Wang et al.2011;Cheng et al.2013). Total RNA was isolated from 100 mg of frozen tissue To determine the gene duplications,first,the CDS using an RNA kit(RNAsimply Total RNA Kit,Tiangen, sequences of Chinese cabbage MADS-box genes were Beijing,China)according to the manufacturer's instruc- blasted against each other (evalue <le-10,identity >85%), tions.Five micrograms of each sample were reverse and then Ks values were calculated for all pair-wise align- transcribed into cDNA using the PrimeScript RT rea- ments of these genes,which previously obtained by blast, gent Kit (TaKaRa).The specific primers of Chinese using the method of Nei and Gojobori as implemented in cabbage MADS-box genes and the housekeeping actin KaKs_calculator (Zhang et al.2006).Lastly,based on phy- gene(Bra028615)were designed using the Primer Pre- logenies,the nucleotide divergence (Dist <0.1)was used mier 5.0 software (Supplementary Table 11).To verify as the final standard (Lynch and Conery 2000).The pur- the primer specificity,we used the program BLAST ple lines were used to link the duplicate genes on different against the Chinese cabbage genome.The qRT-PCR chromosomes. assays were performed with three biological and three technical replicates.Each reaction was performed in a Chinese cabbage RNA-seg data analysis 20 uL reaction mixture containing a diluted cDNA sam- ple as the template,2x Power SYBR Green PCR Mas- For the expression profiling of Chinese MADS-box genes, ter Mix(Applied Biosystems),and 400 nM each of for- we utilised the Illumina RNA-seq data that were previously ward and reverse gene-specific primers.The reactions generated and analysed by Tong et al.(2013).Six tissues were performed using a MyiQ Single-Color Real-Time of B.rapa accession Chiifu-401-42,including callus,root, PCR Detection System (Bio-Rad,Hercules,CA)with stem,leaf,flower,and silique,were analysed.Two sam- the following cycling profile:94 C for 30 s,followed ples of root and leaf tissues were generated from different by 40 cycles at 94 C for 10 s,and 58 C for 30 s.A batches of plants.The transcript abundance is expressed as melting curve(61 cycles at 65 C for 10 s)was gener- fragments per kilobase of exon model per million mapped ated to verify the specificity of the amplification (Song reads (FPKM)values.Heat maps for Chinese cabbage et al.2013).The relative expression ratio of each gene MADS-box genes were generated,which have positive was calculated using the comparative C,value method FPKM values in at least one or more of the samples. (Heid et al.1996).The MADS-box gene expression cluster from each stress treatment was analysed using Plant material and treatments the Cluster program (http://bonsai.hgc.jp/~mdehoon/ software/cluster/software.htm)(Eisen et al.1998).and The Chinese cabbage cultivar Chiifu-401-42 was used the results were shown using the TreeView software for the experiments.Whole genome sequencing has been (http://jtreeview.sourceforge.net/). Springer242 Mol Genet Genomics (2015) 290:239–255 1 3 the homologous genes of MADS-box between Chinese cabbage and Arabidopsis. Briefly, the tools BLASTP, with an e-value ≤1e−10, and orthomclPairs were applied to find orthologs, inparalogs and coorthologs in these two species. To link these genes to chromosomes, a tool called Circos (Krzywinski et al. 2009) was used. In addition, the Cytoscape software was applied to build the network of these relationships (Shannon et al. 2003). Chromosome localisation and gene duplications To determine the physical locations of MADS-box genes, the starting and ending positions of all MADS-box genes on each chromosome were obtained from the BRAD database. The Perl in-house program was used to draw the location images of the Chinese cabbage MADS-box genes. The positions of each Chinese cabbage MADS-box gene on the blocks were verified by searching for homologous genes between Arabidopsis and three B. rapa subgenomes, including least fractionated (LF), medium fractionated (MF1) and most fractionated (MF2) genomes (http://brassicadb.org/brad/searchSynteny.php) (Wang et al. 2011; Cheng et al. 2013). To determine the gene duplications, first, the CDS sequences of Chinese cabbage MADS-box genes were blasted against each other (evalue <1e−10, identity >85 %), and then Ks values were calculated for all pair-wise align￾ments of these genes, which previously obtained by blast, using the method of Nei and Gojobori as implemented in KaKs_calculator (Zhang et al. 2006). Lastly, based on phy￾logenies, the nucleotide divergence (Dist <0.1) was used as the final standard (Lynch and Conery 2000). The pur￾ple lines were used to link the duplicate genes on different chromosomes. Chinese cabbage RNA-seq data analysis For the expression profiling of Chinese MADS-box genes, we utilised the Illumina RNA-seq data that were previously generated and analysed by Tong et al. (2013). Six tissues of B. rapa accession Chiifu-401-42, including callus, root, stem, leaf, flower, and silique, were analysed. Two sam￾ples of root and leaf tissues were generated from different batches of plants. The transcript abundance is expressed as fragments per kilobase of exon model per million mapped reads (FPKM) values. Heat maps for Chinese cabbage MADS-box genes were generated, which have positive FPKM values in at least one or more of the samples. Plant material and treatments The Chinese cabbage cultivar Chiifu-401-42 was used for the experiments. Whole genome sequencing has been completed for this cultivar; thus, this cultivar is a typical cultivar for Chinese cabbage research. Seeds were grown in pots containing a soil: vermiculite mixture (3:1) in the greenhouse of Nanjing Agricultural University, and the controlled-environment growth chamber programmed is light 16 h/25 °C, dark 8 h/20 °C (Song et al. 2013). One month later, seedlings at the five-leaf stage were trans￾ferred to growth chambers that were set at 4 or 38 °C under identical light intensity and day length as the cold and heat treatments. Simultaneously, for acclima￾tion, some plants were cultured in 1/2 Hoagland’s solu￾tion in plastic containers, with the pH at 6.5 (Jensen and Bassham 1966). After 5 days of acclimatisation, plants were cultured in the following four treatments: (1) Con￾trol; (2) 100 μM ABA; (3) 100 μM GA; (4) 100 μM SA. At 4 and 12 h after treatment, the young leaf samples were collected, frozen in liquid nitrogen and stored at −70 °C for further analysis. RNA isolation and quantitative real-time PCR Total RNA was isolated from 100 mg of frozen tissue using an RNA kit (RNAsimply Total RNA Kit, Tiangen, Beijing, China) according to the manufacturer’s instruc￾tions. Five micrograms of each sample were reverse transcribed into cDNA using the PrimeScript RT rea￾gent Kit (TaKaRa). The specific primers of Chinese cabbage MADS-box genes and the housekeeping actin gene (Bra028615) were designed using the Primer Pre￾mier 5.0 software (Supplementary Table 11). To verify the primer specificity, we used the program BLAST against the Chinese cabbage genome. The qRT-PCR assays were performed with three biological and three technical replicates. Each reaction was performed in a 20 μL reaction mixture containing a diluted cDNA sam￾ple as the template, 2× Power SYBR Green PCR Mas￾ter Mix (Applied Biosystems), and 400 nM each of for￾ward and reverse gene-specific primers. The reactions were performed using a MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA) with the following cycling profile: 94 °C for 30 s, followed by 40 cycles at 94 °C for 10 s, and 58 °C for 30 s. A melting curve (61 cycles at 65 °C for 10 s) was gener￾ated to verify the specificity of the amplification (Song et al. 2013). The relative expression ratio of each gene was calculated using the comparative Ct value method (Heid et al. 1996). The MADS-box gene expression cluster from each stress treatment was analysed using the Cluster program (http://bonsai.hgc.jp/~mdehoon/ software/cluster/software.htm) (Eisen et al. 1998), and the results were shown using the TreeView software (http://jtreeview.sourceforge.net/)