Genome-wide identification and characterization of R2R3-MYB genes in Medicago truncatula

Abstract

MYB is a large family of plant transcription factors. Its function has been identified in several plants, while there are few reports in Medicago truncatula. In this study, we used RNA-seq data to analyze and identify R2R3-MYB genes in the genome of Medicago truncatula. Phylogenetic analysis classified 150 MtMYB genes into 21 subfamilies with homologs. Out of the 150 MtMYB genes, 139 were distributed among 8 chromosomes, with tandem duplications (TD) and segment duplications (SD). Microarray data were used for functional analysis of the MtMYB genes during growth and developmental processes providing evidence for a role in tissues differentiation, seed development processes, and especially the nodulation process. Furthermore, we investigated the expression of MtMYB genes in response to abiotic stresses using RNA-seq data, which confirmed the critical roles in signal transduction and regulation processes under abiotic stress. We used quantitative real-time PCR (qRT-PCR) to validate expression profiles. The expression pattern of M. truncatula MYB genes under different abiotic stress conditions suggest that some may play a major role in cross-talk among different signal transduction pathways in response to abiotic stresses. Our study will serve as a foundation for future research into the molecular function of M. truncatula R2R3-MYB genes.

Figure 1. Phylogenetic tree analysis of the R2R3-MYB transcription factors in Medicago truncatula. Red circle: subgroup 1; red square: subgroup 2; red triangle: subgroup 3; red diamond: subgroup 4; blue circle: subgroup five; blue square subgroup six; blue indicates subgroup seven; blue diamond subgroup nine; green circular subgroup ten; green square subgroup 11; green triangle: subgroup 13; green diamond: subgroup 14; pink circle: subgroup 16; pink square: subgroup 18; pink triangle: subgroup 19; pink diamond: subgroup 20; cyan circle: subgroup 21; cyan square: subgroup 22; cyan triangle: subgroup 23; cyan diamond: subgroup 24; yellow circle: subgroup 25.

Figure 2. Chromosome distribution and expansion analysis of R2R3-MYB transcription factors in Medicago truncatula. The genome locations of R2R3-MYB transcription factors were retrieved from Medicago genome website, and the duplications between R2R3-MYB genes identified using software PGDD and BLAST analysis.

Figure 3. Microarray expression data of R2R3-MYB transcription factors in Medicago truncatula. The heatmap was generated using R gplots package. The expressional values of 71 R2R3-MYB genes were retrieved from MtGEA, and they were normalized and used as input, red represents high expressional levels, while blue represents low expressional level.

Figure 4. RNA-seq analysis of R2R3-MYB transcription factors in Medicago truncatula. The heatmap was generated using R gplots package, and the FPKM values of Medicago truncatula genes were evaluated and normalized based RNA-seq data from NCBI SRA database. The plot data included expressional profiles of 67 R2R3-MYB genes in six tissues, and red represents high expressional levels, while blue represents low expressional level.

Figure 5. Expression profile analysis of R2R3-MYB transcription factors in response to abiotic stresses in Medicago truncatula. The heatmap was generated using R gplots package, and the FPKM values of Medicago truncatula genes were evaluated and normalized based RNA-seq data from NCBI SRA database. The plot data included expressional profiles of 64 R2R3-MYB genes in response to abiotic stress, and red represents high expressional levels, while blue represents low expressional level.

Figure 6. qRT-PCR validation of R2R3-MYB transcription factors in Medicago truncatula. All expressional levels of each R2R3-MYB genes were normalized with control expression value (group A) set as one. The groups B (cold stress), C (freezing stress), D (drought stress), E (salt stress), and F (ABA treatment) were compared with control group, and fold-changes were calculated. The fold-change data was used to create the plot, and the blue indicates RNA-seq results, while pink indicates qRT-PCR analysis results.