Genome-Wide Analysis of the Rad21/REC8 Gene Family in Cotton (Gossypium spp.)

Cohesin is a ring-shaped protein complex and plays a critical role in sister chromosome cohesion, which is a key event during mitosis and meiosis. Meiotic recombination protein REC8 is one of the subunits of the cohesion complex. Although REC8 genes have been characterized in some plant species, little is known about them in Gossypium. In this study, 89 REC8 genes were identified and analyzed in 16 plant species (including 4 Gossypium species); 12 REC8 genes were identified in Gossypium. hirsutum, 11 in Gossypium. barbadense, 7 in Gossypium. raimondii, and 5 in Gossypium. arboreum. In a phylogenetic analysis, the 89 RCE8 genes clustered into 6 subfamilies (I–VI). The chromosome location, exon-intron structure, and motifs of the REC8 genes in the Gossypium species were also analyzed. Expression patterns of GhREC8 genes in various tissues and under abiotic stress treatments were analyzed based on public RNA-seq data, which indicated that GhREC8 genes might have different functions in growth and development. Additionally, qRT-PCR analysis showed that MeJA, GA, SA, and ABA treatments could induce the expression of GhREC8 genes. In general, the genes of the REC8 gene family of cotton were systematically analyzed, and their potential function in cotton mitosis, meiosis, and in response to abiotic stress and hormones were preliminary predicted, which provided an important basis for further research on cotton development and resistance to abiotic stress.


Introduction
Cohesion, a muti-subunit protein complex, is essential for sister chromatid cohesion and chromatin loop structure in mitosis and meiosis [1]. The loss and mutation of cohesion can cause abnormal chromosomal separation or alloploidy gamete production. Genes involved in the regulation of meiosis have been extensively studied in Arabidopsis, rice, maize and many other plants [2][3][4]. Meiotic recombination protein REC8, one of the subunits of the meiosis-specific cohesion protein complex, is conserved in functional evolution from yeast to humans. Previous studies have shown that the REC8 gene is required for correct meiosis. During meiosis in Saccharomyces cerevisiae and Schizosaccharomyces pombe, REC8 was found in the cohesion complex, which has homology to SCC 1/Mcd 1/Rad 21 proteins [5][6][7]. REC8 mutations in S. cerevisiae and S. pombe disrupt the cohesion of sister chromosome arms and centromeres, resulting in reduced recombination, affecting the formation of the synaptonemal complex and causing premature separation of sister chromatids during the first meiotic division [5,[7][8][9]. Subsequently, proteins homologous to yeast REC8 have been found in other species, including mice, humans [6,10], Caenorhabditis elegans [11], Arabidopsis thaliana [12,13], Zea mays [14], Drosophila [15], Xenopus laevis [16], Oryza sativa [17], and Citrullus lanatus [18]. In C. elegans, RNAi of REC8 leads to univalent formation and chromosomal fragmentation at diakinesis [11]. In Arabidopsis, syn1/dif1 tree was then decorated using iTOL (https://itol.embl.de/upload.cgi (accessed on 26 October 2022)).

Chromosomal Locations and Collinearity Analysis of Cotton REC8 Genes
The chromosome location information of four cotton species (G. arboretum, G. raimondii, G. hirsutum, and G. barbadense) was obtained from the reference cotton database. The distribution of REC8 genes on the four cotton chromosomes was drawn using TBtools [29]. The multiple collinearity scan (MCS-scanX) in TBtools was used to analyze the collinearity of REC8 genes. The collinearity map was drawn by using Basic CIRCOS in TBtools (v1.108).

Calculation of Selection Pressure for Duplicated Gene Pairs
CDS sequences of cotton REC8 genes were obtained from the reference cotton database, and then the non-synonymous (Ka) and synonymous substitution rates (Ks) were calculated using TBtools software (v1.108). A Ka/Ka ratio > 1 means positive selection, a Ka/Ka ratio = 1 indicates neutral selection, and a Ka/Ka ratio < 1 represents negative selection.

Gene Structure, Motif, and Domain Analysis
The conserved motifs of REC8 proteins were identified using Multiple Em for Motif Elicitation (MEME) (http://memesuite.org/ (accessed on 25 June 2022)) with the p-value of each motif on each protein being lower than 1 × 10 −5 . The exon-intron structure of REC8 genes was analyzed using TBtools. Phylogenetic trees with motif, domain, and gene structure were visualized using TBtools.

Analysis of Cis-Acting Elements in REC8 Promoters
The 2000 bp upstream promoter sequences of REC8 genes were extracted. cis-acting elements were then predicted by PlantCare (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/ (accessed on 28 September 2022)). A phylogenetic tree with identified elements was visualized using TBtools.

Expression Profile of REC8 Genes under Different Tissues and Stress
To analyze the expression pattern of REC8 genes under different tissues and stress (heat, cold, salt, and PEG), the transcriptome data were downloaded from the Cotton Omics Database (http://cotton.zju.edu.cn/10.rnasearch (accessed on 2 July 2022)) [30]. The heat map was drawn using the TBtools software (v1.108) by using fragments per kilo base of exon per million mapped (FPKM).

Plant Materials and Treatments
G. hirsutum cv. Coker 312 (R15) was grown in a chamber (28 • C, 16/8 h day/night). Plants at the third-leaf stage were treated with 100 µM MeJA, 2 mM SA, and 200 µM ABA. The leaves of treated plants were collected at 0 h, 3 h, 6 h, and 12 h and were frozen in liquid nitrogen immediately for further analysis.

RNA Extraction and qRT-PRC Analysis
Total RNA was extracted from the frozen leaves using RNAprep Pure Plant Kit (TianGen, Beijing, China). HiScript III Reverse Transcriptanse (Vazyme, Nanjing, China) was used to convert RNA to cDNA. The qPCR analysis was performed using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on the ABI 7500 Fast Real-Time PCR system (Thermo, Waltham, MA, USA). Three biological replicates were performed in all analyses. GhUBQ7 was used as the internal reference gene, and the data were analyzed using the 2 −∆∆Ct method [31]. The primers are listed in Table S6.

Identification of REC8 Genes in Cotton
To identify the REC8 genes in the 4 cotton species and the other 12 species, we selected candidate genes from a search and comparison using HMMsearch and blastp programs. Then, the obtained sequences were filtered via CDD. Listed in Table S1 are the 12 REC8 genes found in G. hirsutum, 11 in G. barbadense, 7 in G. raimondii, 5 in G. arboretum, 3 in A. comosus, 4 in Arabidopsis, 7 in maize, 4 in rice, 4 in A. trichopoda, 9 in P. trichocarpa, 6 in G. max, 5 in S. bicolor, 7 in T. cacao, 3 in V. vinifera, 1 in V. carteri, and 1 in C. reinhardtii. Twice as many REC8 genes were found in G. hirsutum and G. barbadense as in G. raimondii and G. arboretum, which indicated that the tetraploid cotton is obtained by hybridizing the diploid A genome and the diploid D genome, followed by chromosome doubling. In addition, the characteristics of REC8 proteins in cotton were analyzed, including the protein sequence size, molecular weight (MV), isoelectric point (pI), and instability coefficient ( Table S2). The length of 35 REC8 proteins ranged from 529 aa to 1193 aa; GbREC8-02-D had the fewest amino acids, and GhREC8-03-D and GrREC8-02 had the most. The molecular weight was between 59,052.57 and 130,250.69 kD and was lowest for GbREC8-02-D and highest for GhREC8-03-D. The pI ranged from 4.29 to 7.32; two proteins had a pI less than 7, and 33 had a pI greater than 7, indicating that most of the REC8 proteins were alkaline. The REC8 proteins were predicted to be localized in the nucleus where the REC8 proteins worked probably (Table S2).

Phylogenetic Analysis of the Cotton REC8 Gene Family
To further study the evolutionary relationship of the REC8 gene family in plants, 89 REC8 protein sequences from 4 cotton species and 12 other species were used to construct the phylogenetic NJ tree using MEGA 7 software. In the phylogenetic tree, the REC8 proteins were clustered into 6 subfamilies (I-VI) ( Figure 1) with 3 members in subfamily I, 16 in II, 8 in III, 22 in IV, 18 in V, and 22 in VI. Except for subfamilies III and VI, the other four subfamilies were composed of REC8 genes from monocotyledon and dicotyledons, indicating that REC8 proteins were highly conserved. Subfamily III comprised REC8 genes from Arabidopsis and the four cotton species, and subfamily VI comprised REC8 genes from T. cacao and the four cotton species. We found that the REC8 genes of cotton were more closely related to those of T. cacao, since the REC8 genes of cotton and cacao were closely clustered in the phylogenetic tree. In subfamilies II and IV, one TcREC8 gene corresponded to five homologous REC8 genes from three Gossypium species: one from G. arboretum, two from G. hirsutum, and two from G. barbadense. In addition, AtREC8-04 has a role in DNA repair [32], and loss of function in AtREC8-01 and AtREC8-03 affects meiosis [12,20,33]. GhREC8-06-A, GhREC8-02-D, GbREC8-02-D, GbREC8-01-A, GrREC8-03, GrREC8-01, and AtREC8-04 in subfamily III were on the same branch, indicating that they may be involved in DNA repair. GhREC8-04-D, GhREC8-05-A, GbREC8-04-D, GbREC8-05-A, and GaREC8-03 in subfamily IV may have similar functions as AtREC8-03.

Chromosomal Distribution and Synthesis Analysis of REC8 Genes in Cotton
Based on the genomes of the four cotton species, we analyzed the location of the REC8 genes on the chromosomes. One GaREC8 gene was on each of three chromosomes (Chr04, Chr05, and Chr08), and two were on Chr13 (Figure 2a). Two GrREC8 genes were mapped on each of two chromosomes (Chr02 and Chr09), and Chr13 had three (Figure 2b). The chromosome distribution of REC8 genes was highly similar between G. hirsutum and G. barbadense (Figure 2c,d). The G. hirsutum genome had 12 GhREC8 genes among 7 chromosomes (1 each on chromosomes D04 and D05; 2 each on A05, A08, A13, D08, and D13). The G. barbadense genome had 11 GbREC8 genes among 7 chromosomes (1 each on D04, D05, and D13; 2 each on A05, A08, A13, and D08).

Chromosomal Distribution and Synthesis Analysis of REC8 Genes in Cotton
Based on the genomes of the four cotton species, we analyzed the location of the REC8 genes on the chromosomes. One GaREC8 gene was on each of three chromosomes (Chr04, Chr05, and Chr08), and two were on Chr13 ( Figure 2a). Two GrREC8 genes were mapped on each of two chromosomes (Chr02 and Chr09), and Chr13 had three ( Figure 2b). The chromosome distribution of REC8 genes was highly similar between G. hirsutum and G. barbadense (Figure 2c,d). The G. hirsutum genome had 12 GhREC8 genes among 7 chromosomes (1 each on chromosomes D04 and D05; 2 each on A05, A08, A13, D08, and D13). The G. barbadense genome had 11 GbREC8 genes among 7 chromosomes (1 each on D04, D05, and D13; 2 each on A05, A08, A13, and D08).
To elucidate the relationship of REC8 orthologs in cotton, we performed a synthetic analysis on four cotton species. A total of 35 REC8 genes of cotton have synthetic relationships, including 12 from G. hirsutum, 11 from G. barbadense, 7 from G. raimondii, and 5 from G. arboretum ( Figure 3, Table S3). There were 47 orthologous/paralogous gene pairs: 4 gene pairs between G. hirsutum and G. hirsutum, 20 pairs between G. hirsutum and G. barbadense, 3 between G. hirsutum and G. raimondii, 7 between G. hirsutum and G. arboretum, 4 between G. barbadense and G. barbadense, 6 between G. barbadense and G. arboretum, 2 between G. barbadense and G. raimondii, and 1 between G. arboretum and G. raimondii. These results indicated that REC8 genes in G. hirsutum and G. barbadense had a close evolutionary relationship. Then, we analyzed the collinear relationship of REC8 genes between G. hirsutum and two species (A. thaliana and T. cacao). The results showed that the number of orthologous gene pairs between G. hirsutum and A. thaliana and T. cacao was 2 and 5, respectively ( Figure S1, Table S4). Very few collinear gene pairs of G. hirsutum and the two species were anchored to the highly conserved synthetic blocks.  To elucidate the relationship of REC8 orthologs in cotton, we performed a synthetic analysis on four cotton species. A total of 35 REC8 genes of cotton have synthetic relationships, including 12 from G. hirsutum, 11 from G. barbadense, 7 from G. raimondii, and 5 from G. arboretum (Figure 3, Table S3). There were 47 orthologous/paralogous gene pairs: 4 gene pairs between G. hirsutum and G. hirsutum, 20 pairs between G. hirsutum and G. barbadense, 3 between G. hirsutum and G. raimondii, 7 between G. hirsutum and G. arboretum, 4 between G. barbadense and G. barbadense, 6 between G. barbadense and G. arboretum, 2 between G. barbadense and G. raimondii, and 1 between G. arboretum and G. raimondii. These results indicated that REC8 genes in G. hirsutum and G. barbadense had a close evolutionary relationship. Then, we analyzed the collinear relationship of REC8 genes between G. hirsutum and two species (A. thaliana and T. cacao). The results showed that the number of orthologous gene pairs between G. hirsutum and A. thaliana and T. cacao was 2 and 5, respectively ( Figure S1, Table S4). Very few collinear gene pairs of G. hirsutum and the two species were anchored to the highly conserved synthetic blocks.   Table S5). The results showed that the Ka/Ks ratios from 2 gene pairs (GhREC8-04-D/GbREC8-04-D and GhREC8-06-D/GbREC8-03-D) were larger than 1, indicating that these gene pairs experienced rapid evolution. A total of 23 gene pairs had a Ka/Ks ratio less than 0.5, and 22 gene pairs were between 0.5 and 1, leading us to hypothesize that these gene pairs have undergone strong purifying selection.

Structure Analysis of Cotton REC8 Genes
Structural differences in genes play an important role in the gene evolution of a gene family. To better understand the structure of REC8 genes in cotton, we analyzed the sequence structure, motif, and conserved domains of cotton REC8 genes based on the phylogenetic tree (Figure 4a). Ten motifs (named motifs 1 to 10) were predicted in the REC8 proteins by using MEME. Common motifs and similar motif arrangements were found in most REC8 proteins within the same subfamily, suggesting that the structure was conserved in these proteins. Eleven motifs were found in the proteins of subfamily V and IV except for GrREC8-04 and GrREC8-07. Motif 2 and motif 4 were absent in GhREC8-06-A, GhREC8-02-D, and GbREC8-02-D in subgroup III (Figure 4b). Whether these specific motifs have an effect on the function of REC8 proteins remains to be studied. Most cotton REC8 proteins had a similar domain to Arabidopsis REC8 except that GrREC8-04, GrREC8-07, GhREC8-06-A, GhREC8-02-D, and GbREC8-06-A lacked the N-segment Rad21_REC8 domain. AtREC8-02 in subgroup IV and AtREC8-04 in subgroup III had the MDN1 domain, which was not observed in REC8 proteins from cotton in the same subgroup. In addition, PTZ00449 and N_Asn amidohyd domains were found in GaREC8-04 and GaREC8-03, respectively, indicating that these domains might confer protein different biological functions (Figure 4c). Motifs 1, 2, 3, and 4 were in the N-terminal domain; motifs 9 and 10 were in the C-terminal domain.

Analysis of Cis-Acting Elements of the REC8 Genes in Cotton Species
Gene expression is regulated by upstream promoters, where many cis-acting ments are found. To better understand the regulation and potential function of the ta genes, we extracted the 2000 bp upstream sequence from the predicted translation site of each REC8 gene to analyze the cis-acting elements. Cis-acting elements involve hormone (IAA/auxin, GA (gibberelins), MeJA (methyl jasmonate), SA (salicylic acid), ABA (abscisic acid)) and stress responses (low temperature, drought, and defense) development were predicted using the PlantCARE tool. Promoters of 14 genes conta an IAA-responsive element, 17 contained a GA-responsive element, 16 contained an Then, we analyzed the intron/exon structure of the REC8 genes. The structures were similar for REC8 genes in the same subclade, especially among genes in subfamily IV with 15 exons, indicating that REC8 gene structures are conserved among 4 species.

Analysis of Cis-Acting Elements of the REC8 Genes in Cotton Species
Gene expression is regulated by upstream promoters, where many cis-acting elements are found. To better understand the regulation and potential function of the target genes, we extracted the 2000 bp upstream sequence from the predicted translation start site of each REC8 gene to analyze the cis-acting elements. Cis-acting elements involved in hormone (IAA/auxin, GA (gibberelins), MeJA (methyl jasmonate), SA (salicylic acid), and ABA (abscisic acid)) and stress responses (low temperature, drought, and defense) and development were predicted using the PlantCARE tool. Promoters of 14 genes contained an IAA-responsive element, 17 contained a GA-responsive element, 16 contained an SAresponsive element, 20 contained an ABA-responsive element, and 19 contained a MeJAresponsive element. Among stress elements found in the REC8 promoters, low-temperature response elements were found in promoters of 24 REC8 genes, drought response elements in those of 18, and defense and stress response elements in those of 15. Seven genes (GbREC8-03-A, GbREC8-01-D, GhREC8-01-A, GhREC8-04-A, GhREC8-01-D, GrREC8-04, and GaREC8-01) involved in low-temperature and drought responsiveness were found ( Figure 5). These results showed that REC8 genes played a role in hormone, stress, and defense responses in cotton.

GhREC8 Gene Profiles in Different Tissues of Upland Cotton
To evaluate the potential role of the 12 GhREC8 genes in growth and development, publicly available transcriptome data were used to analyze gene expression patterns in fibers, ovules, anthers, bracts, filaments, leaves, petals, pistils, roots, sepals, stems, and torus ( Figure 6). Six GhREC8 genes (GhREC8-03-D, GhREC8-04-A, GhREC8-02-A, GhREC8-06-D, GhREC8-01-D, GhREC8-03-A, and GhREC8-06-A) were highly expressed in almost all tissues and stages. GhREC8-04-D and GhREC8-05-A were similar in being highly expressed during ovule development in leaves, roots, and stems. There were high levels of GhREC8-05-D expression in anthers, bracts, the filament, pistils, and torus. Compared with fiber development, GhREC8-02-D showed relatively higher expression in other issues and stages. These results indicated that most GhREC8 genes were expressed in vegetative tissues.

Expression Patterns of GhREC8 Genes under Abiotic Stress and Hormone Treatments
To explore the potential function of the REC8 genes in response to abiotic stresses in upland cotton, we used public transcriptome data to analyze the effect of stresses from high temperatures, low temperatures, PEG, and salt and treatment with hormones ( Figure 7). The expression of most GhREC8 genes did not change significantly after abiotic stress treatments. GhREC8-01-A and GhREC8-05-D were upregulated after high-temperature, NaCl, and PEG treatments, especially at 12 h and 24 h after treatments. However, GhREC8-01-A and GhREC8-05-D were downregulated after exposure at 4 • C. GhREC8-05-A was induced by high temperatures, 4 • C, NaCl, and PEG with respective peaks in expression at 1 h, 12 h, 6 h, and 24 h. After MeJA, SA, and ABA treatments, most GhREC8 genes were upregulated, and the expression of three genes (GhREC8-01-D, GhREC8-02-A, and GhREC8-06-D) peaked by 6 h after treatments, and two (GhREC8-01-A and GhREC8-05-D) were downregulated. GhREC8-04-D was upregulated after MeJA and SA treatments, with respective maximum expression at 3 h and 6 h (Figure 8). These results indicated that GhREC8 might have potential functions in response to phytohormone and abiotic stresses.

GhREC8 Gene Profiles in Different Tissues of Upland Cotton
To evaluate the potential role of the 12 GhREC8 genes in growth and development, publicly available transcriptome data were used to analyze gene expression patterns in fibers, ovules, anthers, bracts, filaments, leaves, petals, pistils, roots, sepals, stems, and torus ( Figure 6). Six GhREC8 genes (GhREC8-03-D, GhREC8-04-A, GhREC8-02-A, GhREC8-06-D, GhREC8-01-D, GhREC8-03-A, and GhREC8-06-A) were highly expressed in almost all tissues and stages. GhREC8-04-D and GhREC8-05-A were similar in being highly expressed during ovule development in leaves, roots, and stems. There were high levels of GhREC8-05-D expression in anthers, bracts, the filament, pistils, and torus. Compared with fiber development, GhREC8-02-D showed relatively higher expression in other issues and stages. These results indicated that most GhREC8 genes were expressed in vegetative tissues.

Expression Patterns of GhREC8 Genes under Abiotic Stress and Hormone Treatments
To explore the potential function of the REC8 genes in response to abiotic stresses in upland cotton, we used public transcriptome data to analyze the effect of stresses from high temperatures, low temperatures, PEG, and salt and treatment with hormones ( Figure  7). The expression of most GhREC8 genes did not change significantly after abiotic stress treatments. GhREC8-01-A and GhREC8-05-D were upregulated after high-temperature, NaCl, and PEG treatments, especially at 12 h and 24 h after treatments. However, GhREC8-01-A and GhREC8-05-D were downregulated after exposure at 4 °C . GhREC8-05-A was induced by high temperatures, 4 °C , NaCl, and PEG with respective peaks in expression induced by high temperatures, 4 °C , NaCl, and PEG with respective peaks in expression at 1 h, 12 h, 6 h, and 24 h. After MeJA, SA, and ABA treatments, most GhREC8 genes were upregulated, and the expression of three genes (GhREC8-01-D, GhREC8-02-A, and GhREC8-06-D) peaked by 6 h after treatments, and two (GhREC8-01-A and GhREC8-05-D) were downregulated. GhREC8-04-D was upregulated after MeJA and SA treatments, with respective maximum expression at 3 h and 6 h ( Figure 8). These results indicated that GhREC8 might have potential functions in response to phytohormone and abiotic stresses.

Discussion
Cohesin is a ring-shaped protein complex that is required for sister chromatid cohesion and DBS repair [34,35]. Centromeric sister chromatid cohesion is essential during meiosis. Previous studies have shown that REC8 is crucial for meiosis. The four members (SYN1-SYN4) belonging to the Rad21 gene family in A. thaliana have been identified. SYN2/4 are involved in mitosis, while SYN1 is essential for meiosis [13], and SYN3 is essential for megagametogenesis [36]. Mutations of SYN1 (REC8) lead to male and female sterility in A. thaliana [37]. In rice, an Osrec8 mutant had abnormal homologous chromatid pairing and telomere bouquets [38]. The germination of pollen grains declined, and fruits

Discussion
Cohesin is a ring-shaped protein complex that is required for sister chromatid cohesion and DBS repair [34,35]. Centromeric sister chromatid cohesion is essential during meiosis. Previous studies have shown that REC8 is crucial for meiosis. The four members (SYN1-SYN4) belonging to the Rad21 gene family in A. thaliana have been identified. SYN2/4 are involved in mitosis, while SYN1 is essential for meiosis [13], and SYN3 is essential for megagametogenesis [36]. Mutations of SYN1 (REC8) lead to male and female sterility in A. thaliana [37]. In rice, an Osrec8 mutant had abnormal homologous chromatid pairing and telomere bouquets [38]. The germination of pollen grains declined, and fruits were seedless after the knockout of ClREC8 in watermelon [18]. It can be seen that REC8 protein plays an important role in the fertility of monocots and dicots. Here, we identified 35 REC8 genes in 4 cotton species, and tetraploid cotton had twice more REC8 genes than diploid cotton, which reflected the fact that tetraploid cotton descended from the hybridization of A-and D-genome ancestors. Phylogenetic tree analysis showed that GhREC8 genes (GhREC8-05-D, GhREC-01-A, GhREC8-06-A, GhREC8-02-D, GhREC8-04-D, and GhREC8-05-A) were in the same clade as SYN1-4 ( Figure 1, Table S1), indicating that these genes may have a similar function in meiosis and mitosis. Other studies have shown that REC8 genes were highly expressed in reproductive tissues [1,13,18]. Similarly, most GhREC8 genes were highly expressed in floral organs ( Figure 6). However, whether it could affect meiosis and miosis needs further research.
Heterosis is widely used in agriculture and has made a great contribution to world food security. The artificial creation of apomiesis may be another effective way to achieve heterosis fixation. The production of cloned gametes is one of the key steps in artificial apomiesis. In previous studies, the REC8 gene was used to produce cloned gametes in rice and Arabidopsis [39,40]. REC8 genes in the same clade as AtREC8-01 (AtREC8) and OsREC8-02(OsREC8) may be used for cloned gamete production in cotton. Rad21/Rec8 proteins from different species share a conserved N-terminal domain and C-terminal domain, which are involved in the interaction with Smc1-Smc3 heterodimer during cohesion establishment [40,41]. Similar N-and C-domains were observed in REC8 proteins from Arabidopsis and four cotton species (Figure 4c). Moreover, different exon/intron structures were observed in cotton REC8 genes (Figure 4d), suggesting differences in gene function and RNA splicing of REC8 gene family members. The promoters of REC8 genes contained cis-acting elements associated with phytohormone, stress, and defense responses ( Figure 5). Then, we analyzed the expression of GhREC8 genes under MeJA, SA, and ABA treatments, and most were upregulated after hormone treatments, especially after SA treatment, indicating these genes might be involved in hormone regulation. ABA could interact with the JA and SA signal pathways [42]. JA acts as a defense hormone and is also involved in male sterility, root growth, leaf senescence, and other processes [43,44]. Because most GhREC8 genes were induced by MeJA, they may be related to fertility (Figure 8). The abiotic stresses tested also had different effects on GhREC8 expression. Most genes were not affected by the abiotic stresses, indicating that these genes did not have an important role in plant responses to these stresses. The expression of GhREC8-01-A, GhREC8-05-A, and GhREC8-05-D, however, was sharply induced by PEG (Figure 7), and they had drought response elements in their promoters, suggesting that these genes might be involved in drought tolerance. These mechanisms still need further research. In conclusion, our results indicated that cotton REC8 genes likely had multiple functions in the hormone and stress responses and development of plants. Our next step is to study the role of the REC8 genes in cotton fertility and responses to stresses, which may contribute to the achievement of apomiesis and a further understanding of the mechanisms of resistance to stresses.

Conclusions
In this study, we identified 89 REC8 family genes in 16 species, and these genes were divided into 6 subfamilies. Tissue-specific expression pattern analysis indicated that GhREC8 genes were expressed variously in cotton, indicating that they played different roles in cotton growth and development. An analysis of promoter cis-elements and expression levels under abiotic stresses and hormone treatments showed that GhREC8 genes responded to different stimuli. This study provides valuable information about the cotton REC8 gene family, which lays a foundation for further revealing the biological roles of REC8 genes in cotton meiosis, mitosis, and stress adaptation.
Author Contributions: Conceptualization, Y.W. and L.Z.; Analysis and experiments, L.Z. and Y.W.; Manuscript preparation, Y.W. and L.Z.; Review and project administration, H.C. and H.G.; All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by the Natural Science Foundation of Hebei Province (grant no. C2022204205) and Nan fan special project, CAAS (grant no: YBXM14).