Identification and Analysis of the GASR Gene Family in Common Wheat (Triticum aestivum L.) and Characterization of TaGASR34, a Gene Associated With Seed Dormancy and Germination

Abstract

Seed dormancy and germination are important agronomic traits in wheat (Triticum aestivum L.) because they determine pre-harvest sprouting (PHS) resistance and thus affect grain production. These processes are regulated by Gibberellic Acid-Stimulated Regulator (GASR) genes. In this study, we identified 37 GASR genes in common wheat, which were designated TaGASR1-37. Moreover, we identified 40 pairs of paralogous genes, of which only one had a Ka/Ks value greater than 1, indicating that most TaGASR genes have undergone negative selection. Chromosomal location and duplication analysis revealed 25 pairs of segmentally duplicated genes and seven pairs of tandemly duplicated genes, suggesting that large-scale duplication events may have contributed to the expansion of TaGASR gene family. Microarray analysis of the expression of 18 TaGASR genes indicated that these genes play diverse roles in different biological processes. Using wheat varieties with contrasting seed dormancy phenotypes, we investigated the expression patterns of TaGASR genes and the corresponding seed germination index phenotypes in response to water imbibition, exogenous ABA and GA treatment, and low- and high-temperature treatment. Based on these data, we identified the TaGASR34 gene as potentially associated with seed dormancy and germination. Further, we used a SNP mutation of the TaGASR34 promoter (-16) to develop the CAPS marker GS34-7B, which was then used to validate the association of TaGASR34 with seed dormancy and germination by evaluating two natural populations across environments. Notably, the frequency of the high-dormancy GS34-7Bb allele was significantly lower than that of the low-dormancy GS34-7Ba allele, implying that the favorable GS34-7Bb allele has not previously been used in wheat breeding. These results provide valuable information for further functional analysis of TaGASR genes and present a useful gene and marker combination for future improvement of PHS resistance in wheat.

Keywords: ABA; GA; GASR; common wheat; seed dormancy.

Figure 1

Phylogeny of GASRs from wheat, rice and Arabidopsis. The 37 TaGASR genes, 11 OsGASR genes, and 15 AtGASR genes are clustered into three subfamilies. Details of GASR genes from Arabidopsis and rice are listed in Table S6. The tree was generated using ClustalX version 2.11 using the neighbor-joining (NJ) method.

Figure 2

Evolutionary and gene structure analysis of TaGASR genes. (A) Phylogenetic relationships and gene structures of TaGASR genes. Exons, introns and untranslated regions (UTRs) are indicated by yellow rectangles, gray lines, and green rectangles, respectively. Colored boxes indicate the subfamily based on the phylogenetic analysis. (B) Schematic representation of 20 conserved motifs in TaGASR genes. Conserved motifs in TaGASR genes were identified using MEME. Different colored boxes represent different motifs. Box lengths in the figure do not represent actual relative motif sizes. (C) Multiple sequence alignment of TaGASR proteins. Sequences were aligned using DNAMAN software. The GASA motif is clearly highly conserved.

Figure 3

Cis-acting element analysis of the promoter regions of TaGASR genes. Based on functional annotation data, cis-acting elements were classified into two major classes: phytohormone responsive elements (i.e. those responsive to ABA, auxin, GA, MeJA, and/or SA) and abiotic stress response cis-acting elements (e.g. those involved in plant defense, drought stress response, and/or low temperature stress response).

Figure 4

Expression profiles of TaGASR genes in different tissues and at different developmental stages. Heatmap shows hierarchical clustering of the 18 TaGASR genes among different tissues. Abbreviations represent specific developmental stages: GSC, germinating seed, coleoptile; GSR, germinating seed, root; GSE, germinating seed, embryo; SR, seedling, root; SC, seedling, crown; SL, seedling, leaf; II, immature inflorescence; FBA, floral bracts, before anthesis; PBA, pistil, before anthesis; Aba, anthers, before anthesis; 3–5 DAP C, 3–5 DAP caryopsis; 22 DAP EM , 22 DAP embryo; 22 DAP EN, 22 DAP endosperm.

Figure 5

Chromosomal localization and gene duplication events of TaGASR genes. Respective chromosome numbers are indicated above each bar. Duplicated paralogous pairs of GASR genes in tandem duplication blocks are indicated by small boxes of the same color.

Figure 6

Microsynteny related to TaGASR family in wheat. Wheat chromosomes are shown in different colors. Each chromosome box indicates sequence length in megabases. Different color lines represent syntenic relationships between TaGASR regions, whereas thick red lines represent paralogous TaGASR genes.

Figure 7

Expression patterns of 37 TaGASR genes during seed imbibition in six wheat varieties. Y-axis: relative expression; Error bars indicate 6 ± SE. *Statistical analysis of materials and methods has been labeled.

Figure 8

Expression in response to ABA, GA, High Temperature (HT), and Low Temperature (LT) treatments in wheat varieties Jing411 (J411) and Hongmangchun 21 (HMC21). (A) The levels of endogenous GA₃/ABA in J411 and HMC21 (B) Expression patterns of five TaGASR genes in response to ABA, GA, High Temperature (HT), and Low Temperature (LT) treatments in wheat varieties Jing411 (J411) and Hongmangchun 21 (HMC21). Y-axis: relative expression; Error bars, 6 ± SE.

Figure 9

Different genotypes identified by the functional marker GS34-7B in different wheat varieties. Shown are: Suiningtuotuo (SNTT), Yangxiaomai (YXM), Yangnong 24 (YN24), Jing 411 (J411), Zhongmai 895 (ZM895), Yangmai 16 (YM16), Shimai12 (SM12), Zhongmai 18 (ZM18), Jimai 20 (JM20), Zhongyou 9507 (ZY9507), and Hongmangchun 21 (HMC21).