Identification and molecular characterization of MYB Transcription Factor Superfamily in C4 model plant foxtail millet (Setaria italica L.)

Abstract

MYB proteins represent one of the largest transcription factor families in plants, playing important roles in diverse developmental and stress-responsive processes. Considering its significance, several genome-wide analyses have been conducted in almost all land plants except foxtail millet. Foxtail millet (Setaria italica L.) is a model crop for investigating systems biology of millets and bioenergy grasses. Further, the crop is also known for its potential abiotic stress-tolerance. In this context, a comprehensive genome-wide survey was conducted and 209 MYB protein-encoding genes were identified in foxtail millet. All 209 S. italica MYB (SiMYB) genes were physically mapped onto nine chromosomes of foxtail millet. Gene duplication study showed that segmental- and tandem-duplication have occurred in genome resulting in expansion of this gene family. The protein domain investigation classified SiMYB proteins into three classes according to number of MYB repeats present. The phylogenetic analysis categorized SiMYBs into ten groups (I-X). SiMYB-based comparative mapping revealed a maximum orthology between foxtail millet and sorghum, followed by maize, rice and Brachypodium. Heat map analysis showed tissue-specific expression pattern of predominant SiMYB genes. Expression profiling of candidate MYB genes against abiotic stresses and hormone treatments using qRT-PCR revealed specific and/or overlapping expression patterns of SiMYBs. Taken together, the present study provides a foundation for evolutionary and functional characterization of MYB TFs in foxtail millet to dissect their functions in response to environmental stimuli.

Figure 1. Physical map of 209 SiMYB genes.

SiMYB genes were plotted onto nine chromosomes of foxtail millet. The numbers at left indicates the position of SiMYB genes, and the names of the respective genes are given in right. The ‘MYB-Related’ are given in black, ‘MYB-R2R3’ are in blue and ‘MYB-R1R2R3’ are in red. The SiMYB genes used for expression analysis through qRT-PCR are underlined. Segmentally duplicated gene-pairs are highlighted with different colours. Tandemly duplicated gene-pairs are enclosed within red boxes.

Figure 2. Chromosome-wise distribution of SiMYB genes.

The distribution of three different SiMYB genes on individual chromosomes is shown.

Figure 3. Phylogenetic relationships of foxtail millet MYB transcription factors.

The unrooted phylogenetic tree was constructed by neighbor-joining method with 1000 bootstrap replicates. The bootstrap values are shown at the nodes. The tree was divided into ten phylogenetic cluster designated as I to X.

Figure 4. Gene Ontology distribution for SiMYB proteins.

Blast2GO program defines the gene ontology under three categories. (A) biological processes, (B) molecular functions and (C) cellular component.

Figure 5. Heat map representation of SiMYB genes in four tissues.

The Illumina RNA-seq data were re-analyzed and heat map was generated. Bar at the top represents log₂ transformed values, thereby values 0.0, 2.0 and 8.0 represent low, intermediate and high expression, respectively.

Figure 6. Comparative physical mapping revealed high degree of orthologous relationships of SiMYB genes located on nine chromosomes of foxtail millet.

(A) Sit (foxtail millet) – Sbi (sorghum), (B) Sit – Zma (maize), (C) Sit – Osa (rice), and (D) Sit – Bdi (Brachypodium).

Figure 7. Time of duplication and divergence (MYA) based on synonymous substitution rate (Ks) estimated using duplicated SiMYB gene-pairs of foxtail millet and orthologous MYB gene pairs between foxtail millet- sorghum, -maize, -rice and Brachypodium.

Figure 8. The relative expression ratio of 11 candidate SiMYB genes analysed using qRT-PCR under salinity stress, dehydration stress and hormone treatments for 1 h (early) and 24 h (late).

The relative expression ratio of each gene was calculated relative to its expression in control sample (0 h). Act2 was used as an internal control to normalize the data. Error bars representing standard deviation were calculated based on three technical replicates for each biological duplicate. ABA - abscisic acid, MJ - methyl jasmonate, SA - salicylic acid.