Genome-wide identification and expression analysis of the GhIQD gene family in upland cotton (Gossypium hirsutum L.)


 
 Calmodulin (CaM) is one of the most important Ca2+ signaling receptors because it regulates diverse physiological and biochemical reactions in plants. CaM functions by interacting with CaM-binding proteins (CaMBPs) to modulate Ca2+ signaling. IQ domain (IQD) proteins are plant-specific CaMBPs that bind to CaM by their specific CaM binding sites.
 
 
 In this study, we identified 102 GhIQD genes in the Gossypium hirsutum L. genome. The GhIQD gene family was classified into four clusters (I, II, III, and IV), and we then mapped the GhIQD genes to the G. hirsutum L. chromosomes. Moreover, we found that 100 of the 102 GhIQD genes resulted from segmental duplication events, indicating that segmental duplication is the main force driving GhIQD gene expansion. Gene expression pattern analysis showed that a total of 89 GhIQD genes expressed in the elongation stage and second cell wall biosynthesis stage of the fiber cells, suggesting that GhIQD genes may contribute to fiber cell development in cotton. In addition, we found that 20 selected GhIQD genes were highly expressed in various tissues. Exogenous application of MeJA significantly enhanced the expression levels of GhIQD genes.
 
 
 Our study shows that GhIQD genes are involved in fiber cell development in cotton and are also widely induced by MeJA. Thw results provide bases to systematically characterize the evolution and biological functions of GhIQD genes, as well as clues to breed better cotton varieties in the future.


evolution of GhIQD genes. As shown in Fig. 2, among the 102 GhIQD genes identified in G. hirsutum L., 100 (98.04%) were derived from segmental duplication events, and only two genes (GhIQDA13.3 and GhIQDD13.3) resulted from proximal duplication. In contrast, none of the GhIQD genes was found to have arisen from tandem duplication events (Additional files 2 and 3). These results indicate that segmental duplication is the main driving force in the expansion of the GhIQD genes.

Phylogenetic analysis of GhIQD proteins
To examine the molecular evolutionary relationships among plant IQD proteins, the amino acid sequences of the IQD proteins from Arabidopsis, tomato, soybean, and G. hirsutum L.
were used in a phylogenetic analysis. As shown in Fig. 3, a phylogenetic tree was constructed with the Neighbor-Joining (NJ) method from an alignment of all complete IQD protein sequences. The NJ tree showed that the IQD proteins group into seven clusters (Ia, Ib, Ic, II, IIIa, IIIb and IV).
A Ka/Ks ratio 1> indicates that paralogous gene pairs were produced by positive selection, a ratio <1 indicates that paralogous gene pairs were under purifying selection and a ratio equal to 1 indicates that paralogous gene pairs were not subjected to selection pressure [27]. To explore the type selection pressure experienced by the duplicated GhIQD genes, paralogous GhIQD gene pairs were used to calculate synonymous (Ks) and nonsynonymous (Ka) substitution rates to assess the ratio of non-snonymous to synonymous substitutions. As shown in Fig. 3, 50 paralogous gene pairs were identified. The Ka/Ks ratios of 48 members were <1.0, and the Ka/Ks ratios for the remaning two paralogous gene pairs were >1 (Additional file 4), suggesting that the GhIQD paralogous gene pairs were mainly produced by purifying selection. expressed in the leaf, petal, and hypocotyl (Fig. 5e).

Expression profiling of GhIQD genes in response to MeJA treatment
According to previous studies, the expression of most IQD genes can be induced by MeJA stress in plants [22] In this study, the expression patterns of GhIQD genes in plants exposed to MeJA treatment were examined in an qRT-PCR experiment. The results showed that the expression levels of the 20 randomly-selected GhIQD genes were significantly increased by MeJA treatment (Fig. 6). As the time of treatment increased, the transcript levels for most genes increased significantly. In detail, the expression levels of

Discussion
Calcium is one of the most important cytosolic second messengers, and calcium levels can be induced by intracellular and extracellular stimuli. CaM is one of the most important Ca 2+ signaling receptors that regulates diverse physiological and biochemical reactions. were under purifying selection, which indicates that the segmentally duplicated GhIQD genes were subjected to strong purifying constraints during evolution.
GhIQD genes participate widely in the regulation of growth in G. hirsutum L.
In Arabidopsis, the AtIQD proteins were reported to widely link calcium signaling to microtubules, membrane subdomains, and the nucleus [28]. From the results of the transcriptome analysis, the GhIQD gene expression patterns could be clustered into six groups. In the fiber development process, the 5 dpa ovule stage is primary cell wall synthesis stage of fiber cells; 10 dpa corresponds to the elongation stage of fiber development; and 20-25 dpa is the transition stage of fiber development from elongation to secondary wall synthesis, which is important for fiber strength [29]. PdIQD10 gene was found to involve in the secondary cell wall biosynthesis and biomass formation in Populus [30]. Therefore, the genes in cluster 6 might participate in primary cell wall synthesis, the GhIQD genes in cluster 5 may contribute to fiber elongation, and the GhIQD genes in clusters 1, 3, and 4 may be involved in fiber strength development.
In Arabidopsis, AtIQD genes function as hubs in Ca 2+ signaling to regulate growth and development with tissue-specifically [31]. To further elucidate the possible functions of the GhIQD genes, their expression patterns were investigated in various tissues (Fig. 5).
The results show that some GhIQD genes are predominantly expressed in specific tissues, Methods gene expression patterns.

Plant materials and treatments
The G. hirsutum L. cultivar TM-1 was grown in the field in Anyang, Henan province, China.
Leaves, stems, roots, and hypocotyl tissues were collected at the seedling stage. Stigma, petal, pollen, and calyx samples were collected at the flowering stage. The G. hirsutum L.

Evaluation of psychometric properties
Exploratory Factor Analysis. EFA models of 1-4 factors revealed that a 3-factor model was the best fit for the 19-item scale ( = 228.834, p < .000; CFI = .953; TLI = .931; RMSEA = .069; SRMR = 0.032), but low factor loading (below 0.4)was indicated for item 1 ("Could you suggest to your supervisor ways to change your work to reduce discomfort?") and item 7 ("Could you avoid re-injury?").
After removing two items from the item pool, EFA was conducted for 1 Floor and ceiling effect. No floor or ceiling effects were found for total RTWSE and subscale scores using the criteria of 15%. Regarding communicating needs RTWSE, 12.4% achieved the highest score (10), below the 15% cutoff (table 3).
Reliability. All Cronbach's alphas for the overall scale and subscales were satisfactory.
The Cronbach's alpha for the overall RTWSE-17 was 0.925 and was 0.842 for communicating needs, 0.851 for modifying job tasks, and 0.926 for meeting job demands (table 3).

Construct validity
Significant correlations were found between fear-avoidance beliefs about physical activity