Genome‑wide identification and expression analysis of the UBC gene family in wheat (Triticum aestivum L.)

Abstract

Background: Ubiquitination is an important regulatory step of selective protein degradation in the plant UPS (ubiquitin-proteasome system), which is involved in various biological processes in eukaryotes. Ubiquitin-conjugating enzymes play an intermediate role in the process of protein ubiquitination reactions and thus play an essential role in regulating plant growth and response to adverse environmental conditions. However, a genome-wide analysis of the UBC gene family in wheat (Triticum aestivum L.) has not yet been performed.

Results: In this study, the number, physiochemical properties, gene structure, collinearity, and phylogenetic relationships of TaUBC family members in wheat were analyzed using bioinformatics methods. The expression pattern of TaUBC genes in different tissues/organs and developmental periods, as well as the transcript levels under abiotic stress treatment, were analyzed using RNA-Seq data and qRT-PCR. Meanwhile, favorable haplotypes of TaUBC25 were investigated based on wheat resequencing data of 681 wheat cultivars from the Wheat Union Database. The analyses identified a total of 93 TaUBC family members containing a UBC domain in wheat genome. These genes were unevenly distributed across 21 chromosomes, and numerous duplication events were observed between gene members. Based on phylogenetic analysis, the TaUBC family was divided into 13 E2 groups and a separate UEV group. We investigated the expression of TaUBC family genes under different tissue/organ and stress conditions by quantitative real-time PCR (qRT-PCR) analysis. The results showed that some TaUBC genes were specifically expressed in certain tissues/organs and that most TaUBC genes responded to NaCl, PEG6000, and ABA treatment with different levels of expression. In addition, we performed association analysis for the two haplotypes based on key agronomic traits such as thousand-kernel weight (TKW), kernel length (KL), kernel weight (KW), and kernel thickness (KT), examining 122 wheat accessions at three environmental sites. The results showed that TaUBC25-Hap II had significantly higher TKW, KL, KW, and KT than TaUBC25-Hap I. The distribution analysis of haplotypes showed that TaUBC25-Hap II was preferred in the natural population of wheat.

Conclusion: Our results identified 93 members of the TaUBC family in wheat, and several genes involved in grain development and abiotic stress response. Based on the SNPs detected in the TaUBC sequence, two haplotypes, TaUBC25-Hap I and TaUBC25-Hap II, were identified among wheat cultivars, and their potential value for wheat breeding was validated by association analysis. The above results provide a theoretical basis for elucidating the evolutionary relationships of the TaUBC gene family and lay the foundation for studying the functions of family members in the future.

Keywords: UBC gene family; Allelic variation; Gene expression; Thousand-kernel weight; Wheat.

Fig. 1

Phylogenetic relationships and conserved motifs of UBC proteins. A Phylogenetic analysis of Arabidopsis (47), rice (42), and wheat (93) generated using MEGA 11 software with default parameter settings. The different colored blocks indicate different subclasses of the UBC family. The TaUBC family was clustered into 14 clades, and each clade contained 3, 2, 3, 21, 5, 6, 3, 6, 2, 6, 4, 5, 22 and 5 TaUBCs, respectively. The name of each branch was indicated next to the corresponding branch. B Conserved motifs of UBC proteins. Distribution of the 10 conserved motifs in the TaUBC proteins after analysis by the MEME tool. The different colored boxes represent different motifs and their position in each protein sequence of TaUBC

Fig. 2

Distribution of TaUBCs segment duplication gene pairs on wheat chromosomes. Duplicated gene pairs are connected by lines with the corresponding color. The different colored blocks indicate different chromosome. The chromosome number is indicated on the inner side of each chromosome. The heatmap in the inner circle represents the gene density on the chromosome

Fig. 3

Expression analysis of the TaUBC genes. A Heatmap of TaUBC genes expression in different growth stages and different tissues of Chinese Spring. The heatmap shows the phylogenetic clustering of 93 TaUBC genes. Color scale: Blue represents low expression and red indicates high expression levels. B Expression profiles of TaUBCs over developmental period of wheat. Tubulin (TraesCS1A01G350200.1) was used as a reference gene. The Y-axis and X-axis indicates the relative expression levels and the different tissues, respectively. Means and standard deviations (SDs) were calculated from three biological and three technical replicates. The error bars indicate the standard deviation

Fig. 4

qRT-PCR analysis of selected TaUBCs under 200 mM NaCl, 20% PEG6000, and 100 uM ABA stress. A Relative expression patterns of TaUBCs in leaves after 200 mM NaCl treatment. B Relative expression patterns of TaUBCs in leaves after 20% PEG6000 treatment. C Relative expression patterns of TaUBCs in leaves after treatment with 100 uM ABA. Relative expression values in the control sample (CK 0 h) were normalized to 1. TaActin1 was used as a reference gene. Each bar value is the average value ± standard deviation based on three biological replicates. The different letters denote a significant difference between means (P < 0.05)

Fig. 5

Association analysis of TKW, KL, KW and KT between two haplotypes of TaUBC25 gene in three environments. A The mean thousand grain weight of TaUBC25 haplotypes in the modern cultivars. B The mean kernel length of TaUBC25 haplotypes in the modern cultivars. C The mean kernel width of TaUBC25 haplotypes in the modern cultivars. D The mean kernel thickness of TaUBC25 haplotypes in the modern cultivars. The X-axis represents different environments (Luoyang, 2002; Luoyang, 2005; Shunyi, 2010). The values show the mean ± SE (n > 50 grains). Significant statistical analysis was carried out by Student’s t-test (*, P < 0.05; **, P < 0.01)

Fig. 6

Analysis of the different cis-element existed in the promoter region, spatial and temporal distribution, and expression levels of two TaUBC25 haplotypes. A The distribution of main cis-regulatory elements contained SNPs site in the promoter region of the two haplotypes of TaUBC25 gene. B Geographic distribution of varieties with TaUBC25 haplotypes in China. The map was downloaded using the Standard Map Service System (http://bzdt.ch.mnr.gov.cn/). C Frequencies of TaUBC25 allelic variation in Chinese wheat breeding programs in different decades. D The expression levels of TaUBC25 and TKW carrying TaUBC25-Hap I or TaUBC25-Hap II haplotypes