Genome-wide identification and characterization of the MADS-box gene family in Salix suchowensis

Abstract

MADS-box genes encode transcription factors that participate in various plant growth and development processes, particularly floral organogenesis. To date, MADS-box genes have been reported in many species, the completion of the sequence of the willow genome provides us with the opportunity to conduct a comprehensive analysis of the willow MADS-box gene family. Here, we identified 60 willow MADS-box genes using bioinformatics-based methods and classified them into 22 M-type (11 Mα, seven Mβ and four Mγ) and 38 MIKC-type (32 MIKCc and six MIKC*) genes based on a phylogenetic analysis. Fifty-six of the 60 SsMADS genes were randomly distributed on 19 putative willow chromosomes. By combining gene structure analysis with evolutionary analysis, we found that the MIKC-type genes were more conserved and played a more important role in willow growth. Further study showed that the MIKC* type was a transition between the M-type and MIKC-type. Additionally, the number of MADS-box genes in gymnosperms was notably lower than that in angiosperms. Finally, the expression profiles of these willow MADS-box genes were analysed in five different tissues (root, stem, leave, bud and bark) and validated by RT-qPCR experiments. This study is the first genome-wide analysis of the willow MADS-box gene family, and the results establish a basis for further functional studies of willow MADS-box genes and serve as a reference for related studies of other woody plants.

Keywords: Expression; Gene family; Genome-wide characterization; MADS-box; Phylogenetic analysis; Willow.

Figure 1. Comparison of the MADS-box domains from the 60 willow MADS-box genes.

The multi-alignment was performed using the ClustalX programme (version 2.1) and coloured using the online tool BoxShade (http://www.ch.embnet.org/software/BOX_form.html). Black indicates a highly conserved region. (A) Mα subgroup. (B) Mβ subgroup. (C) Mγ subgroup. (D) Mδ/MIKC* subgroup. (E) MIKCc subgroup.

Figure 2. Chromosomal localization of the 60 willow MADS-box genes.

The number of each chromosome is given above the lines. The left side of each chromosome is related to the approximate physical location of each MADS-box gene. The four unmapped genes are shown on ChrN. Purple indicates Mα, green indicates Mβ, brown indicates Mγ, yellow indicates MIKC*, and blue indicates MIKCc.

Figure 3. Phylogenetic tree of S. suchowensis and A. thaliana MADS-box proteins.

A total of 60 MADS-box proteins from S. suchowensis and 88 from A. thaliana were used to construct a NJ tree using MEGA 7. Different shapes and colours represent different species and gene categories.

Figure 4. Phylogenetic relationships and gene structures of the willow MADS-box genes.

An unrooted NJ tree was constructed based on the full-length willow MADS-box protein sequences. The exon-intron structures of the willow MADS-box genes were displayed using the online tool GSDS.

Figure 5. Conversed motif distributions of the willow MADS-box proteins.

A total of 15 conserved motifs of the 60 willow MADS-box proteins were identified using MEME. Motifs 1–15 are indicated by different colours.

Figure 6. Expression analysis of the 60 willow MADS-box genes in five tissues (bark, leaf, bud, root and stem).

The colour scale represents RPKM normalized log2-transformed counts. The red blocks indicate high expression, the blue blocks indicate low expression, and the light green blocks indicate no expression in this tissue.

Figure 7. Expression patterns of 6 MIKC genes in five tissues (root, stem, leaf , bud and bark) by RT-qPCR.

SsOTU primers were used as the internal standard for each gene. The mean expression value was calculated from 3 independent replicates. The vertical bars indicate the standard deviation. (A) SsMADS9; (B) SsMADS11; (C) SsMADS15; (D) SsMADS 37; (E) SsMADS44; (F) SsMADS47.