Genome sequences and annotation files of G. hirsutism (Wang et al., 2019), G. barbadense (Wang et al., 2019), G. arboretum (Du et al., 2018) and G. raimondii (Udall et al., 2019) were downloaded from COTTONGENE (http://www.cottongen.org). The hidden Markov model of ALOG (PF04852) were obtained from the InterPro database (https://www.ebi.ac.uk/interpro/), which were used to retrieve cotton ALOG proteins by HMMER (Finn et al., 2011). In addition, we performed a sequence similarity search by BLAST (Matsuda et al., 2013; Nowicki et al., 2018) (E value ≤ E⁻¹⁰) with the ALOG amino acid sequences of Arabidopsis thaliana and Oryza sativa as queries (Li et al., 2019). Then HMMER results were combined with the BLAST search results, and NCBI-CDD-Search (Yang et al., 2020) was used for further confirmation. The physical and chemical properties of cotton ALOG proteins were predicted by the software Compute pI/Mw (https://web.expasy.org/compute_pi/).

Multiple sequence alignment of ALOG proteins was carried out by the Clustal X (Larkin et al., 2007). A maximum likelihood tree with a bootstrap value of 1,000 was constructed by MEGA (Hall, 2013). The result tree was then decorated by iTOL (https://itol.embl.de/upload.cgi) (Letunic and Bork, 2021). The online tool MEME (Bailey et al., 2009) (https://meme-suite.org/meme/) was used to analysis conserved motifs, with motif number set to 5. The cis-elements in promoter sequences upstream 1,500 bp of ALOG genes were predicted by PlantCARE (Lescot et al., 2002). The exon-intron organization of cotton ALOG genes was identified by GSDS (Hu et al., 2015) (https://gsds.gao-lab.org/).

The collinearity relationships of ALOG genes were analyzed by MCScanX (Wang et al., 2012), and the results were visualized using Circos (Krzywinski et al., 2009). Ka/Ks ratios between ALOG members was calculated by KaKs_Calculator software (Wang et al., 2010). The divergence time was calculated by the formula T = Ks/2λ, where λ represents the neutral substitution rate, which is set to 1.5 × 10⁻⁸ in this study (Koch et al., 2000).

The transposable elements library was construct by RepeatModeler, and RepeatMasker was used to predict transposable elements (Tarailo-Graovac and Chen, 2009; Tempel, 2012). The transposable elements in 2,000 bp and 10,000 bp upstream and downstream regions of the ALOG genes were identified in this study.

Transcriptome data were downloaded from NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra/). The SRA data for multiple tissues (PRJNA490626), different fiber development stages (PRJNA263926) and long day and short day conditions (PRJNA529417) were converted to fastq format with the SRA Toolkit. The software Trimmomatic (Bolger et al., 2014) was used to remove the adapters and to perform quality control and hisat2 (Kim et al., 2015) was used to map the reads to the genomes. Transcript abundance for ALOG genes was quantified using the fragments per kilobase million (FPKM) metric, which was calculated by Cuffinks software (Ghosh and Chan, 2016). Heatmaps of the expression profile values were generated with pheatmap package of R language.

The interaction network of ALOG proteins was predicted by the online website STRING (https://string-db.org/) (Szklarczyk et al., 2015). Arabidopsis thaliana was selected as the organism to retrieve the protein interaction network map.

In the present study, a total of 43, 42, 23 and 27 ALOG genes were identified from G. hirsutum, G. barbadense, G. arboretum and G. raimondii, respectively (Supplementary Table S1). The numbers of ALOG genes in diploid cotton (G. arboretum and G. raimondii) was found to be comparable to that of Nicotiana species (12–23) (Turchetto et al., 2023), yet it exceeded the number observed in Arabidopsis (10) (Rieu et al., 2024).

In addition, the results showed that the numbers of ALOG genes in diploid cotton species are almost half those of tetraploid cotton species. Therefore, we speculate that the ALOG gene family likely originated in diploid cotton species and expanded during polyploidization. However, some gene losses may have occurred post-polyploidization.

To further characterize the cotton ALOG proteins, molecular weight, amino acid sequence length and isoelectric point value were analyzed. Notably, the sequence of Ghir_D10G021590.1 in G. hirsutum, Gbar_D10G021160.1 in G. barbadense and Ga10G0697 in G. arboretum are significantly longer than other ALOG proteins. The NCBI-CDD database revealed that these three proteins contain not only the ALOG domain but also a LRR domain (NCBI CDD: 443914), while all other proteins only contain the ALOG domain. Proteins containing LRR domain include tyrosine kinase receptors, cell-adhesion molecules, virulence factors, and extracellular matrix-binding glycoproteins, and are involved in a variety of biological processes, including signal transduction, cell adhesion, DNA repair, recombination, transcription, RNA processing, disease resistance, apoptosis, and the immune response (Kobe and Kajava, 2001).

Except for these 3 special ALOG proteins, the sequence length of cotton ALOG proteins range from 142 aa (Ghir_A07G015930.1) to 280 aa (Ga08G2374), and the isoelectric point ranged from 8.36 (Ghir_D13G025500.1) to 10.41 (Ghir_D02G004400.1). According to the results, cotton ALOG proteins have a wide range of sequence length and isoelectric point, however, the statistical results of them in the 4 cotton species are very similar, for example, the average molecular weight is around 23,627 Da, the average number of amino acids is about 215 aa, and the average isoelectric point is around 9.7.

To better understand the origin and diversification of the ALOG gene family, a phylogenetic tree was inferred with ALOG protein sequences of Physcomitrella patens, Oryza sativa, Zea mays, Arabidopsis thaliana, Populus trichocarpa and the 4 cotton species. The ALOG proteins can be divided into five groups (Figure 1), among them, the group B and E contain a larger number of proteins. Noticeably, most Physcomitrella patens, Oryza sativa and Zea mays ALOG proteins were distributed in group A and C, while Arabidopsis thaliana and Populus trichocarpa ALOG proteins were distributed in group B, D and E along with 4 cotton species. This result indicated that ALOG proteins from the same monocot species are clustered into a branch, but those from the same dicot species are dispersed into different branches. Furthermore, Figure 1 shows that many sub-groups contain similar numbers of ALOG family from the 4 cotton species and other dicot species, which suggested that the expansion has occurred before the divergence of dicot species.

A total of 5 motifs were identified from the conserved domains of cotton ALOG proteins. The majority members of ALOG proteins contain motif 1–4, indicating that they are the main motifs that make up the ALOG domain (Figure 1). Motif 5 were mainly present in the group E. Group C could be further divided into 2 sub-groups. One of the sub-groups contain Oryza sativa and Zea maysALOG proteins, and all of them contain motif 1–4, which is similar to the majority members of ALOG proteins. More interestingly, the other sub-group included 1 ALOG proteins from each diploid cotton species and 2 ALOG proteins from each tetraploid cotton species, and all of them only contain motif 1 and motif 4. These results suggested that ALOG members of group C originated very early in evolution, and they were very conserved since the divergence of cotton species.

Studies have shown that the ALOG domain includes two conserved regions: N-terminal DNA-binding region and C-terminal region (Liu et al., 2024). The sequence of N-terminal with a “HxxxC” and “CxC” signature, which is consistent with Motif 2 (Rieu et al., 2024). The C-terminal region is fused to a tyrosine recombinase catalytic region (Rieu et al., 2024), which is consistent with Motif 1 (Figure 1). In contrast, the functions of Motif 3–5 remain unknown.

The exons and introns were analyzed to get better understand the gene structural evolution of cotton ALOG gene family (Roy and Gilbert, 2006). The results showed that 80.7% (109/135) cotton ALOG genes did not contain introns (Supplementary Table S1). The majority of ALOG genes belonging to group B contain introns, and most of them contained only one intron. Additionally, in group E, 1 ALOG gene from G. hirsutum, G. barbadense and G. arboretum contain 3 introns. Taken together, it appears that genes from the same group also have similar motif and gene structure features, so there may be consistency in the protein function.

The genome chromosomal distribution results indicated that ALOG genes were unevenly distributed on different chromosomes, and most chromosomes contain 1–2 ALOG genes (Figure 2). In addition, Ga14G0329 of G. arboretum were located on scaffolds. We refer to the description of Holub that two or more genes of the same family within 200 kb on the same chromosomal is a tandem duplication event (Holub, 2001). There were 2 ALOG genes (Ga10G0696, Ga10G0697) clustered into one tandem duplication event regions on G. arboretum chromosomes Chr10, but no tandemly duplicated genes were found in G. hirsutum, G. barbadense and G. raimondii.

Segmental duplicate gene pairs were searched by MCScanX. The results indicated that 40, 41, 18, and 17 genes formed 110, 140, 27, and 28 segmental duplication pairs in G. hirsutum, G. barbadense, G. arboretum and G. raimondii, respectively, accounting for 93.02%, 97.62%, 78.26%, and 62.96% of the ALOG gene family. G. hirsutum and G. barbadense (AADD) are typical allotetraploid from its diploid ancestors G. arboreum (AA) and G. raimondii (DD).

Segmental duplication relationships between the subgenome and the corresponding ancestral diploid genomes were analyzed to understand the evolutionary mechanism of cotton ALOG gene family. In G. hirsutum, 39 ALOG genes had orthologs in the G. arboreum, 40 genes had orthologs in the G. raimondii, while only 3 genes (Ghir_D10G021590.1, Ghir_D05G010820.1 and Ghir_A10G011070.3) had no ortholog. In G. barbadense, similarly, 40 ALOG genes had orthologs in the G. arboreum, 41 genes had orthologs in the G. raimondii, while only 1 gene (Gbar_D10G021160.1) had no ortholog (Figure 2).

In order to further the understanding of the evolutionary constraints of the ALOG gene family, an analysis was conducted to determine the Ka/Ks ratio. The Ka/Ks ratios of ALOG segmental duplication gene pairs between same or different species are all around 0.08 (Figure 3A). These results suggested that cotton ALOG genes were under strong purifying selection (Hurst, 2002). Furthermore, we estimated the divergence time of cotton ALOG gene family by Ks values. The results indicate that the divergence time of ALOG segmental duplication gene pairs concentrated around 15 MYA (million years ago) and 110 MYA (Figure 3B). Previous studies have estimated that the divergence of cotton species began around 10 MYA (Chen et al., 2016; Chen et al., 2017). Based on these results, we speculate that most of the ALOG duplications took place before the speciation of cotton species. In addition, researches have shown that a major polyploidy event occurred within the eudicots around 117 MYA (Jiao et al., 2012), which suggests that the event has an important impact on the expansion of ALOG gene family.

Transposable elements are widely distributed in the genome, especially in plants, which are important for genome expansion and evolution. We identified the transposable elements located 2,000 bp upstream and downstream of the ALOG genes. Our results indicate that 18.60% (8/43), 19.05% (8/42), 13.04% (3/23), and 25.93% (7/27) of ALOG genes close to transposable elements in G. hirsutum, G. barbadense, G. arboretum and G. raimondii, respectively. Of these transposable elements, most of them are DNA transposon. When the scanning region broadened to 10,000 bp upstream and downstream, 74.72% (32/43), 69.05% (29/42), 60.87% (14/23), and 66.67% (18/27) genes were found near the transposable elements in G. hirsutum, G. barbadense, G. arboretum and G. raimondii, respectively. In addition to DNA transposons, many LTR retrotransposons were identified (Supplementary Table S2). Therefore, our results indicate that cotton ALOG gene family has expansion due to the activity of transposable elements.

The cis-element present in the promoters of cotton ALOG genes were identified using PlantCARE. The result indicates that TATA-box and CAAT-box were the most abundant cis-elements, in addition, there were also cis-elements related to stress responses (MYB, MYC, STRE), light responsiveness (Box 4, GT1-motif) and so on (Figure 4). Furthermore, we found that the proportions of these cis-elements are similar across different species and groups which indicated the cis-elements of ALOG genes are very conserved after the divergence of cotton species.

We analyzed the transcriptomic data of leaf, root, stem, filament, anther, pistil sepal, pental, torus and bract in G. hirsutum and G. barbadense. The results indicated that most ALOGs showed tissue-specific expression patterns, As shown in Figure 5, Ghir_A09G024400, Ghir_D09G023550, Ghir_A11G026830 of G. hirsutum and Gbar_A09G024590, Gbar_A11G026170, Gbar_D09G024260 of G. barbadense exhibited significantly higher expression in stem than other tissues. Similarly, Ghir_A08G019950, Ghir_D08G020820, Ghir_A02G004060, Ghir_D02G004400 of G. hirsutum and Gbar_A02G003920, Gbar_D02G004510, Gbar_A08G020490, Gbar_D08G021420 of G. barbadense were mainly expressed in filament but barely expressed in other tissues. Therefore, it perhaps that ALOG gene mainly play its role in a specific tissue. In addition, some ALOG genes expressed in the same tissue were segmental duplicate gene pairs, for example, Ghir_A09G024400, Ghir_D09G023550, Gbar_A09G024590 and Gbar_D09G024260, but not all genes were like this, for example, Ghir_A11G026830 and Gbar_A11G026170 were both highly expressed in the stem, but they were not segmental duplicate gene pairs. Furthermore, members sharing closer phylogenetic relationships displayed similar expression patterns, for example, the six ALOG genes highly expressed in stem were all belong to Group B.

To explore the potential role of ALOG in fiber development, we investigated their expression at different fiber development stages (10–28 DPA) of G. barbadense by RNA-sequencing. The results indicated that the expression pattern of ALOG genes can be grouped into 3 clusters (Figure 6). Thirty G. barbadense ALOG genes showed upregulated expression during fiber development, whereas eight exhibited downregulation. In addition, the expression of Gbar_D08G022570, Gbar_D05G011080, Gbar_A08G025590, Gbar_D03G015070 exhibited differences at different stages of fiber development. Based on the above RNA-seq data analysis result, we believe that the ALOG genes play a significant role in the fiber development.

To investigate the potential function of ALOG family in long day and short day conditions, the transcriptome data from leaf and mristem of G. hirsutum, G. barbadense, G. arboretum and G. raimondii was used to calculate the expression of ALOG genes. The expression ratio for the long day conditions/short day conditions was calculated for the expression pattern of each ALOG genes. In the calculation, we only keep the data with FPKM>1 in both conditions. The results showed that 64.5% of the family members had ratios less than 0.8 or more than 1.2, of which 23.5% is less than 0.8% and 41.0% is more than 1.2 (Supplementary Table S3).

First of all, an interaction network was constructed between cotton ALOG proteins, and the results showed that they do not interact with each other. Furthermore, we predict the possible regulatory mechanism of Ghir_D09G023550.1 based on LSH4 (Light-dependent Short Hypocotyls 4), the protein with the highest homology to Ghir_D09G023550.1 in Arabidopsis (Figure 7). LSH4 belongs to the ALOG family which may act as a developmental regulator by promoting cell growth in response to light and suppress organ differentiation in the boundary region. LSH4 is an important component of the Arabidopsis development network (Rieu et al., 2024), and mainly interacted with five types of biological process, including secondary shoot formation (GO:0010223), meristem development (GO:0048507), anatomical structure formation involved in morphogenesis (GO:0048646), formation of plant organ boundary (GO:0090691), and plant organ formation (GO:1905393).

The interaction study of cotton ALOG family provides important clues for further study of its function.