Genomewide Identiﬁcation and Characterization of the Genes Involved in the Flowering of Cotton

: Flowering is a prerequisite for ﬂowering plants to complete reproduction, and ﬂowering time has an important effect on the high and stable yields of crops. However, there are limited reports on ﬂowering-related genes at the genomic level in cotton. In this study, genomewide analysis of the evolutionary relationship of ﬂowering-related genes in different cotton species shows that the numbers of ﬂowering-related genes in the genomes of tetraploid cotton species Gossypium hirsutum and Gossypium barbadense were similar, and that these numbers were approximately twice as much as the number in diploid cotton species Gossypium arboretum . The classiﬁcation of ﬂowering-related genes shows that most of them belong to the photoperiod and circadian clock ﬂowering pathway. The distribution of ﬂowering-related genes on the chromosomes of the At and Dt subgenomes was similar, with no subgenomic preference detected. In addition, most of the ﬂowering-related core genes in Arabidopsis thaliana had homologs in the cotton genome, but the copy numbers and expression patterns were disparate; moreover, ﬂowering-related genes underwent purifying selection throughout the evolutionary and selection processes. Although the differentiation and reorganization of many key genes of the cotton ﬂowering regulatory network occurred throughout the evolutionary and selection processes, most of them, especially those involved in the important ﬂowering regulatory networks, have been relatively conserved and preferentially selected.

In the photoperiod pathway, day length is perceived by the leaves and induces a systemic signal called florigen that moves through the phloem sieve elements to the shoot apical meristem (SAM); the FLOWERING LOCUST (FT) protein is a major component of florigen [4,30] in different species, such as Arabidopsis, rice, and temperate cereals [8]. At the SAM of various plant species, such as Arabidopsis, rice, wheat, temperate crops, and cereals [1,2,4,30], the FT-FLOWERING LOCUS D (FD) complex is involved in the network associated with the SAM, and causes changes in the expression of floral meristem-identity other plant specises such as wheat [64], and temperate fruit trees including apple [65], Prunus mume (mei) [66], sweet cherries [67], and kiwifruit [68].
Cotton plants, which are members of the Malvaceae family and Gossypium genus, are considered to be a model species for the study of plant polyploidy [69]. Since most of the planted varieties are allotetraploids, the cotton genome is more complex, coupled with complex traits and a long growth period, and studies on flowering are key to finding a way to regulate flowering time to breed early-maturity cotton. Cotton flowering is positively correlated with the differentiation of flower primordia. However, cotton flower primordia differentiation is driven by axillary bud primordia, which is governed by SAM differentiation. Axillary bud primordia differentiate into leaf primordia during early growth and development; however, they differentiate into flower primordia during later growth and differentiation, marking the beginning of the cotton transition from vegetative growth to reproductive development.
Previous genomewide association study (GWAS) results showed that flowering time, the length of the period from the first flower bloom to the first boll opening, the length of the whole growth period, the yield percentage before frost, and the plant height and height of the first branch node were early-maturity traits, and some important floweringrelated alleles have been identified, such as GhSPY, GhZTL, GhELF6, GhSVP, GhELF4, GhGA2OX6, and GhPHYA [69]. Furthermore, the GhCAL protein can form heterodimers with GhAP1 and GhAGL6 to regulate these two genes' expression and facilitate cotton flowering [70]. Coincidentally, these flowering-related genes are highly homologous to those in A. thaliana, suggesting that the key flowering mechanism was relatively conserved during the evolution and domestication of plant species. However, how strong selection pressure alters the genome of cotton, particularly the genetic components underlying the adaptation to environmental changes and improved early maturity, remains unknown.
In comparison with extensive studies of the flowering mechanisms in model plant species A. thaliana, the flowering mechanisms in cotton remain unknown. In this study, to better understand the flowering mechanism of cotton, flowering-related candidate genes were identified with genomewide identification-based techniques. A detailed analysis of the flowering-related genes, including their functional classification, chromosomal locations, phylogenetic relationships, conserved motifs, and expression patterns, was performed. Moreover, the nonsynonymous substitution rate (Ka), synonymous substitution rate (Ks), and ω (Ka/Ks) ratio were calculated to determine the divergence time and selection pressure of homologous gene pairs. This systematic analysis of flowering-related genes in cotton provides a basis for further studies on the flowering mechanism of cotton.

Comparison of Flowering-Related Genes from Arabidopsis thaliana, Gossypium hirsutum, Gossypium barbadense, and Gossypium arboretum
With the development and improvement of the A. thaliana flowering-related genomic network, 306 flowering time-related genes have been found, and information about their evolution and an interactive database, FLOR-ID, can be found at http://www.flor-id. org (accessed on 3 November 2021) [71]. On the basis of the protein sequences of the 306 flowering-related genes in A. thaliana, we investigated the flowering-related homologous genes in G. hirsutum and G. barbadense. In total, 636 and 673 flowering-related genes were identified in G. hirsutum (Table S1) and G. barbadense (Table S2), respectively, and more than half of the A. thaliana flowering-related genes had putative G. hirsutum (176 out of 306 genes) and G. barbadense (174 out of 306 genes) homologs. As in the G. barbadense genome, homologs in the G. hirsutum genome were lacking for 130 genes, and most of these genes (120 of 130 genes) have a single functional effect on flowering (Tables S3 and S4). Furthermore, 350 flowering-related genes in G. arboreum were identified (Table S5), and more than half of the flowering-related genes in A. thaliana had putative homologs in G. arboretum (173 out of 306 genes; Table S5); in addition, 133 genes have been lost (Table S6).
To explore the flowering mechanism among the cultivated cotton species (G. hirsutum, G. arboretum, and G. barbadense), the putative flowering-related genes were classified into the eight following gene sets according to the classification of A. thaliana genes: those involved in the photoperiod pathway, circadian clock, and light signaling (Ph); sugar (Su); vernalization (Ve); the autonomous pathway (Au); hormone signaling and metabolism (Ho); the aging pathway (Ag); flower development and the apical meristem response pathway (Fd); and the ambient temperature pathway (At) [71]. Interestingly, most of the lost (53) genes belonged to the photoperiod pathway in G. hirsutum. There were 320 genes involved in the photoperiod pathway, and there were too many overlapping genes between the photoperiod and vernalization (62 genes), and hormone (39) pathways. The fewest common genes were detected between the photoperiod and ambient temperature pathways (three genes) ( Figure 1A), and similar results were found in G. barbadense ( Figure 1B). meristem response pathway (Fd); and the ambient temperature pathway (At) [71]. Interestingly, most of the lost (53) genes belonged to the photoperiod pathway in G. hirsutum. There were 320 genes involved in the photoperiod pathway, and there were too many overlapping genes between the photoperiod and vernalization (62 genes), and hormone (39) pathways. The fewest common genes were detected between the photoperiod and ambient temperature pathways (three genes) ( Figure 1A), and similar results were found in G. barbadense ( Figure 1B).
The number of flowering-related genes in G. hirsutum (636 genes) and G. barbadense (673 genes) was approximately twice that in G. arboretum (350 genes, Figures 1 and S1). Moreover, there were no significant differences in the number of flowering-related genes between G. hirsutum (636 genes) and G. barbadense (673 genes). By classifying the putative flowering-related genes in G. hirsutum, G. barbadense, and G. arboretum into the eight gene sets according to the A. thaliana dataset, we found that most of the genes participate in the photoperiod pathway ( Figure S1). Additionally, there was no significant difference in the number of G. hirsutum and G. barbadense genes associated with each gene set ( Figure S1).
Additionally, the hormone signaling gene sets (16.07% were lost) or meristem response and development gene sets (20% were lost) were preferentially retained instead of the other pathway genes. However, more than 30% of the light signaling (38.78%), vernalization (30.86%), and aging (47.5%) genes were lost (Tables S3).  The number of flowering-related genes in G. hirsutum (636 genes) and G. barbadense (673 genes) was approximately twice that in G. arboretum (350 genes, Figures 1 and S1). Moreover, there were no significant differences in the number of flowering-related genes between G. hirsutum (636 genes) and G. barbadense (673 genes). By classifying the putative flowering-related genes in G. hirsutum, G. barbadense, and G. arboretum into the eight gene sets according to the A. thaliana dataset, we found that most of the genes participate in the photoperiod pathway ( Figure S1). Additionally, there was no significant difference in the number of G. hirsutum and G. barbadense genes associated with each gene set ( Figure S1).

Chromosomal Localization and Duplication Analysis of Flowering-Related Genes in G. hirsutum and G. barbadense
The 636 and 673 flowering-related genes in G. hirsutum ( Figure 2 and Table S7) and G. barbadense ( Figure S2 and Table S8), respectively, were mapped onto pseudomolecular chromosomes. These genes exhibited equal genomic distribution, with 324 (G. hirsutum) and 329 (G. barbadense) genes localized on the At subgenome, and 310 (G. hirsutum) and Int. J. Mol. Sci. 2022, 23, 7940 5 of 18 329 (G. barbadense) genes localized on the Dt subgenome. However, the chromosomal distribution of these genes was uneven. For the At subgenome, 37 (G. hirsutum) and 34 (G. barbadense) genes were localized on chromosome A05, with most located on the top half, representing 11.42% (G. hirsutum) and 10.33% (G. barbadense) of the flowering-related genes in the At subgenome (Figures 2A and S2A). Only 12 flowering-related genes were detected on chromosomes A04 (3.70%, G. hirsutum) and A02 (3.65%, G. barbadense), with most distributed at both ends ( Figures 2A and S2A). In addition, 34 (10.97%, G. hirsutum) and 33 (10.03%, G. barbadense) genes were located on chromosome D05, with most located on the top half. Only 11 genes (3.55%) were detected on chromosome D03 of G. hirsutum, and 12 genes (3.65%) were detected on D01 and D04 of G. barbadense, with most located on the bottom half ( Figure 2B and Figure S2B). Interestingly, most of the other genes were distributed near the ends of chromosomes (Figures 2 and S2).

Chromosomal Localization and Duplication Analysis of Flowering-Related Genes in G. hirsutum and G. barbadense
The 636 and 673 flowering-related genes in G. hirsutum ( Figure 2 and Table S7) and G. barbadense ( Figure S2 and Table S8), respectively, were mapped onto pseudomolecular chromosomes. These genes exhibited equal genomic distribution, with 324 (G. hirsutum) and 329 (G. barbadense) genes localized on the At subgenome, and 310 (G. hirsutum) and 329 (G. barbadense) genes localized on the Dt subgenome. However, the chromosomal distribution of these genes was uneven. For the At subgenome, 37 (G. hirsutum) and 34 (G. barbadense) genes were localized on chromosome A05, with most located on the top half, representing 11.42% (G. hirsutum) and 10.33% (G. barbadense) of the flowering-related genes in the At subgenome (Figures 2A and S2A). Only 12 flowering-related genes were detected on chromosomes A04 (3.70%, G. hirsutum) and A02 (3.65%, G. barbadense), with most distributed at both ends (Figures 2A and S2A). In addition, 34 (10.97%, G. hirsutum) and 33 (10.03%, G. barbadense) genes were located on chromosome D05, with most located on the top half. Only 11 genes (3.55%) were detected on chromosome D03 of G. hirsutum, and 12 genes (3.65%) were detected on D01 and D04 of G. barbadense, with most located on the bottom half ( Figures 2B and S2B). Interestingly, most of the other genes were distributed near the ends of chromosomes (Figures 2 and S2).

Floral Pathway Integrators
In flowering, endogenous elements always undergo cross-talk with exogenous factors through certain integrators [4]. The functional classification of flowering-related genes showed that systemic signal FT [1,4,8,28], which is called GhFT or TWIN SISTER OF FT (GhTSF) in G. hirsutum, functions in all eight flowering pathways, namely, Ph, Ho, Ag,
To systematically determine the conserved motifs of the flowering-related genes involved in the Ph pathway, the distribution of conserved motifs of these genes was estimated by using the online MEME server in conjunction with 25 putative conserved motifs ( Figure S5). Results show that the length of the conserved motifs ranged from 19 to 50 amino acids, and the number of conserved motifs within each flowering-related gene involved in the Ph pathway ranged from 0 to 10 ( Figure 3C,D), showing that all GhSVP, GhAP1 and GhSOC1 homologs contained three conserved motifs. Interestingly, the three GhFT homologs contained no conserved motifs with other Ph pathway flowering-related genes ( Figure 3C).

Flowering-Related Genes Involved in the Photoperiod Pathway, Circadian Clock, and Light Signalling
Light is indispensable for plant life, and the perception of the light environment dictates seed germination, photomorphogenesis, phototropism, shade avoidance, and flowering [61]. In this study, 320 genes in G. hirsutum homologous to 174 genes related to the photoperiod pathway, circadian clock, and light signaling in A. thaliana were identified ( Figure 1 and Table S1). To explore the evolution and genomic structure of Ph pathwayrelated genes, the phylogenetic analysis and intron diagrams of Ph pathway floweringrelated genes were generated on the basis of their sequences ( Figure 3A,B). The findings show that GhSPL3, GhGI, and photoreceptors GhFKF1, GhCRY1/2, and GhPHYB are closely genetically related ( Figure 3A). The exon number of each gene widely ranged from 1 to 15 exons. Significantly, 3 GhELF4 genes were identified, and all of them contained only 1 exon, 2 GhSPL3 copies had 10 exons, and 4 GhPHYB copies had 3 to 5 exons. Four GhGIs were identified and contained 14 to 15 exons ( Figure 3B). FKF1 belongs to a family of F-box proteins of which ZTL and LKP2 are members, and all of them comprise light, oxygen, or voltage (LOV) domains [35]. Like in G. barbadense, there are eight copies of each of the genes GhZTL, GhLKP2, and GhFKF1 in G. hirsutum (Table S2). Moreover, seven GhFKF1 genes contained two exons ( Figure 3B). Blue light photoreceptors CRY1 and CRY2, which monitor light signals to regulate plant flowering [34,80], were identified in G. hirsutum (Table S1), and four GhCRY1 and GhCRY2 contained four exons ( Figure 3B).
Furthermore, most GA biosynthesis homologs, such as GA2ox, GA3ox, and GA20ox [81], were retained ( Figure 3); they were simultaneously induced in response to light signaling and contained six conserved motifs in G. hirsutum ( Figure 3C and Table S1). Our conserved motif analysis showed that genes GhELF4, GhLFY, GhSPL3, and GhPHYB had no conserved motifs. However, the five GhTEM2 genes had two conserved motifs, the GhGI genes contained one conserved motif, the GhFKF1 genes had eight conserved motifs, and the GhCRYs had seven conserved motifs ( Figure 3C).

Expression Analysis of Ph Pathway Genes in G. hirsutum
To investigate the divergence in the expression levels of homologous genes, and their participation in the photoperiod and circadian clock pathways, we analyzed the expression patterns of these supposed flowering-related genes in G. hirsutum, including the root, stem, leaf, petal, anther, stigma, ovule 0D (Day), ovule 1D, ovule 3D, ovule 10D, ovule 20D, fiber 5D, fiber 10D, fiber 20D and fiber 25D tissues ( Figure 3D and Table S9). The results showed that most of the expressed genes were in the roots, petals, stems and leaves ( Figure 3D and Table S9). Four GhCRY1/2, four GhGA20ox1, two GhFKF1, two GhPHYB, three GhTEM2 and one GhSPL3 were specifically or preferentially expressed in ovule 1D tissue. Four GhSVP, four GhFKF1, four GhGI, two GhSOC1, two GhGA2ox, two GhELF4, one GhAP1, and one GhFT were specifically or preferentially expressed in the stems and leaves. Eight GhAP1, three GhCRY1, two GhSOC1, two GhPHYB, two GhSPL3, and two GhFT genes were specifically or preferentially expressed in the roots or leaves. Only one copy of GhLFY was found in G. hirsutum, and it was preferentially expressed in the roots ( Figure 3D).

Hormone-Pathway-Related Flowering-Related Genes
In A. thaliana, by controlling the spatial expression of floral regulatory genes throughout the plant independent of light signals, GA signals promote the initiation of floral primordia [27]. 85 genes likely related to the hormone pathway were identified in G. hirsutum genome. GA is perceived by its receptor, GID1, and there are 12 GhGID1s in G. hirsutum ( Figure 4A-C and Table S1). CID1s can bind to bioactive GA through the conformational changes, which then facilitates the interaction between CID1 and DELLA proteins, which are major flowering repressors [82][83][84]. There are five DELLA family genes, namely, GIB-BERELLIC ACID INSENSITIVE (GAI), REPRESSOR OF ga13 (RGA), RGA-LIKE1 (RGL1), RGL2 and RGL3, in the A. thaliana genome [85,86]. We found four GhGAIs, namely, GhRGA, GhRGL1, GhRGL2 and GhRGL3 in G. hirsutum ( Figure 4A-C and Table S1).
To further study the structural features of these genes, the exon-intron structure was analyzed, the results of which show that the 12 GhGID1s possessed 2 exons, and the 4 GhGAI only had 1 exon ( Figure 4A). To visualize the conserved residues, the Ho pathway flowering-related gene motifs were analyzed with 10 putative conserved motifs ( Figure S6), the results of which show that the GhCID1 genes have eight conserved motifs, two GhGAI genes have four conserved motifs, and one GhRGA and one GhRGL have three conserved motifs ( Figure 4B). Phylogenetic analysis revealed that the GhGID1s and GhGAI genes are closely related to their homologs ( Figure 4C). The expression pattern analysis of 15 different cotton tissues showed that GhGAI has divergent expression patterns among their different copies coupled with sequence similarity, five GhGAI genes were highly expressed in petals, and six other genes were expressed in the leaf, stem, and ovule 0D tissues. Two GhGAI genes were highly expressed in ovule 0D; in contrast, two other genes showed nearly no expression in ovule 0D ( Figure 4D and Table S9).
We detected content changes in hormones zeatin riboside (ZR), abscisic acid (ABA), GA, and IAA in the SAM and leaves of two early-maturing cultivars Zhong50 and Zhong74; two late-maturing cultivars Zhong60 and Lu28 at different true-leaf flattening stages (first true-leaf stage 1TLS, second true-leaf stage 2TLS, third true-leaf stage 3TLS, and fourth true-leaf stage 4TLS). Results show that, during the development of the SAMs in cotton, the contents of hormones ZR, ABA, and GA are maximized; conversely, hormone IAA reached its minimal value during the second to third true-leaf stages ( Figure 4E-H). Moreover, the balance between various endogenous hormones peaked at 2 TLS in cotton cultivars Zhong74, and at 3 TLS in Zhong50, Zhong60 and Lu28 ( Figure S7).

Selection Pressure on Flowering Pathway Gene Sets
To investigate the evolution process, the nonsynonymous/synonymous substitution ratio (Ka/Ks), which is related to the evolutionary selection patterns of the corresponding genome [87], of flowering-related gene orthologous pairs between the G. hirsutum and A. thaliana genomes as well as between the G. barbadense and A. thaliana genomes were computed to determine the selection pressure. The calculation of the selection pressure showed that the mean Ka/Ks ratios between A. thaliana and G. hirsutum of different flowering-related gene sets ranged from 0.001 to 0.200 ( Figure 5, Tables S10 and S11). In particular, genes involved in flower development and the apical meristem response pathway appear to have been subjected to less negative selection pressure than the other pathway genes were. Moreover, the range of the selection pressure variation was largest in Ph pathway genes during the evolution of cotton ( Figure 5A,B, Tables S10 and S11).

Selection Pressure on Flowering Pathway Gene Sets
To investigate the evolution process, the nonsynonymous/synonymous substitution ratio (Ka/Ks), which is related to the evolutionary selection patterns of the corresponding genome [87], of flowering-related gene orthologous pairs between the G. hirsutum and A. thaliana genomes as well as between the G. barbadense and A. thaliana genomes were computed to determine the selection pressure. The calculation of the selection pressure showed that the mean Ka/Ks ratios between A. thaliana and G. hirsutum of different flowering-related gene sets ranged from 0.001 to 0.200 ( Figure 5, Tables S10 and S11). In particular, genes involved in flower development and the apical meristem response pathway appear to have been subjected to less negative selection pressure than the other pathway genes were. Moreover, the range of the selection pressure variation was largest in Ph pathway genes during the evolution of cotton ( Figure 5A,B, Tables S10 and S11).

Discussion
Compared with the A. thaliana flowering mechanism, which has been comprehensively described, the exploration of cotton flowering mechanisms is still

Discussion
Compared with the A. thaliana flowering mechanism, which has been comprehensively described, the exploration of cotton flowering mechanisms is still deficient. Cotton species G. hirsutum, G. barbadense, and G. arboretum were sequenced, and the release of genomic data makes it greatly convenient for studying the genes and functions of important agronomic traits [88]. Here, a bioinformatics approach was used to analyze the G. hirsutum and G. barbadense genes that potentially participate in flowering, and implied a possible regulatory model of the multiple feedbacks and inputs during the regulation of cotton flowering in the leaf and meristem ( Figure 6).
In this study, the number of flowering-related genes identified in G. hirsutum was nearly equal to that in G. barbadense and twice that in G. arboretum, which is consistent with the results that G. hirsutum and G. barbadense, both of which are allotetraploid cotton species, were formed by the two closest diploids extant progenitor hybridization, evolution and artificial domestication [88][89][90]. Interestingly, many of the detected flowering-related genes were functionally redundant, suggesting that these genes may play an integrated role during evolution and domestication; moreover, the photoperiod pathway may undergo cross-talk with the vernalization and hormone pathways and play a substantial role in regulating cotton flowering. Inversely, most of the flowering-related homologous that were found in A. thaliana but not in G. hirsutum had a single function, implying that these genes may not participate in the core flowering pathways. Moreover, the genetic localization results show that the flowering mechanism of cotton might be relatively conserved among the allotetraploid species and between the subgenomes. Meanwhile, more than 30% of the light signaling (38.78%), vernalization (30.86%) and aging (47.5%) gene sets were lost. Considering that currently grown cotton varieties evolved and were domesticated from perennial shrub plants in the subtropics [69,89,90], the loss of genes in the photoperiod pathway, vernalization, and aging gene sets may be related to changes in photoperiod and environmental temperature during their northward movement. Notably, all of the floral pathway integrator SOC1 [72,73] and the floral meristem-identity genes AP1 [2,74], LFY [75][76][77][78] and SVP [79] are activated to regulate floral primordium initiation, facilitate the transition from vegetative growth to reproductive growth, and accelerate early flowering in SAM during the transition from vegetative growth to reproductive development in Arabidopsis [4,73], and all of them were identified in G. hirsutum. In addition, a previous study had investigated that GhCAL can form heterodimers with GhAP1/GhAGL6 to regulate their expression and facilitate cotton flowering [70]. GWAS results [69] indicate that, despite the loss of some of the flowering-related genes, the core mode of flowering is evolutionarily conserved in G. hirsutum and A. thaliana to a certain extent, and the basic light signaling-related flowering pathways are likely relatively conserved. These results indicated that the differentiation fate transition from a vegetative SAM to a reproductive SAM might be a pivotal cell biological characteristic affecting the early flowering of cotton. The flowering mechanisms have been relatively conserved during evolution and domestication, and all of these core factors may also play an indispensable role during floral primordium initiation in cotton SAM, which seems to explain why G. hirsutum can flower in temperate areas. Interestingly, combined with the core role of FT in the flowering of Arabidopsis, rice, and temperate crops [1,2,8], the analysis of gene conserved motifs in G. hirsutum, showed that GhFT have no conserved motifs with other flowering-related genes that identified in this study, which indicated that GhFT homologs in cotton likely also play a core part different with other genes during the evolution, and the function of which might can't be reproduced by other genes. Additionally, plants perceive light signaling through their leaves [7] through several photoreceptors, such as FKF1/ZTL/LKP2 [35][36][37]91,92], CRY1/2 [34,42] and PHYA/B/C/D/E [38][39][40], and induce florigen, a systemic signal that consists of primarily FT protein and moves through the phloem to SAM [5,31,93]. At the SAM, via reprograming the expression of floral identity genes, such as AP1, SOC1, LFY and SVP, FT drives SAM differentiation such that floral primordia are produced [2,4]. In this study, the homologs of GhCRY1/2, GhFKF1/ZTL/LKP2 and GhPHYA/B/C/D/E were identified in the G. hirsutum genome. Consistently, the identification of GA biosynthesis-and metabolism-related genes GID1, GA20ox1/2, GA30x1, and GA2ox1/2/3/6/8 [82][83][84] as well as the DELLA family members GAI, RGA, RGL1, RGL2 and RGL3 [85,86], coupled with the changes in hormone contents in the SAM and leaves during the transition of floral primordia in cotton. Furthermore, GA signaling can not only function in leaves by promoting the expression of flowering time integrator genes such as FT and TSF independently of CO and GI [27], but also regulates the expression of SPL genes in both the leaves and the SAM [27]. All of the findings suggest that hormones, especially GA signals and light signals may participate in the initiation of the floral primordia during the transition from vegetative growth to the reproductive stage.
Furthermore, expression pattern analysis indicates that, in cotton, during evolution and domestication, duplicated genes can undergo nonfunctionalization, neofunctionalization, or subfunctionalization. Genes preferentially expressed in stems and leaves may perceive a light signal in the leaves, transport it through the phloem to the SAM, and stimulate the initiation of floral primordia [4,5,31,93]. Remarkably, selective pressure analysis shows that all Ka/Ks ratio values were lower than 0.200, which indicates that the flowering-related genes for cotton underwent strong purifying selection and that the negative selection had acted against extreme polymorphic variants of flowering-related genes in cotton. Additionally, the sequences of flower development and apical meristem response pathway genes exhibit great sequence diversity, with higher Ka/Ks ratios than those of other flowering pathways between not only the G. hirsutum and A. thaliana genomes but also the G. barbadense and A. thaliana genomes, indicating that the cotton flowering-related genes underwent strong purification during evolution, and the same was found between A. thaliana and G. barbadense, and variation in flower development and apical meristem response pathway genes may contribute to the rapid evolutionary capacity to change the thermal requirement to flowering in G. hirsutum.

Data Resources
Details regarding the annotated A. thaliana genome were downloaded from the TAIR10 website (http://www.arabidopsis.org/index.jsp, accessed on 10 December 2021). G. hirsutum, G. barbadense, and G. arboretum genomic data and the fragments per kilobase of transcript per million mapped reads (FPKM) of the flowering-related genes for G. hirsutum were downloaded from the CottonFGD database (https://cottonfgd.org/about/download.html, accessed on 13 January 2022). The genomic, coding, and protein sequences, and the gene expression of flowering-related genes were downloaded from the CottonFGD database (accessed on 25 January 2022).

Identification of Flowering-Related Gene Homologs in Cotton
Homologous genes were detected by using a combination of similarity-and syntenybased approaches. In the similarity-based approach, the flowering-related genes in A. thaliana were used as queries, and the BLASTP program was used to perform searches against G. hirsutum, G. barbadense, and G. arboretum protein sequences with the following conditions: E-value < 1 × 10 −20 , identity > 60%, coverage > 75%, and match length > 70 amino acids.

Chromosomal Localization of Flowering-Related Genes in the Cotton Genome
The locations on the chromosomes or scaffolds indicate the distribution of floweringrelated genes, and detailed information on genome localization for the predicted G. hirsutum, G. barbadense and G. arboretum flowering-related genes was obtained from the cotton genome data. A map was constructed using the software of MapChart (MapChart 2.32, which is supported by dr.ir. RE (Roeland) Voorrips in Wageningen, The Netherlands; https://www.wur.nl/en/show/Mapchart.htm, accessed on 5 February 2022) to visualize the putative flowering-related genes on pseudomolecular chromosomes.

Analysis of Multiple Alignments and Phylogenetic, Gene Structure and Motif Recognition for Flowering-Related Genes
Multiple gene protein sequences were aligned by using ClustalW 1.81 software, which is currently maintained at the Conway Institute UCD Dublin by Des Higgins, Fabian Sievers, David Dineen, and Andreas Wilm (Dublin, Germany). A phylogenetic tree was constructed using the MEGA 6.0 program with the maximum likelihood (ML) method (1000 bootstrap replicates) on the basis of full-length protein sequences.
To visualize the structure of flowering-related genes and illustrate the organization of exons and introns in G. hirsutum, Gene Structure Display Server 2.0 (GSDS 2.0, http: //gads.cbi.pku.edu.cn/index.php, accessed on 16 February 2022) was used.
Multiple Em for Motif Elicitation (MEME Version 4.11.4, http://meme-suite.org/ tools/meme, accessed on 17 February 2022) was used to identify the conserved motifs of the protein sequences of significant flowering-related genes in G. hirsutum with default parameters except for the maximum number of motifs, which was set to 25.

Ka/Ks Ratios of Flowering-Related Gene Pairs between A. thaliana and G. hirsutum/G. barbadense
The Ka/ Ks ratios of homologous gene pairs were calculated to investigate the molecular evolution of homologous gene pairs. In the calculations of Ka/Ks, the full-length amino acid sequences of the G. hirsutum/G. barbadense and A. thaliana flowering-related genes underwent pairwise alignments using MUSCLE. Then, the aligned amino acid sequences were translated into the corresponding nucleotide coding sequences using Perl scripts derived from ParaAT_2.0 software. Lastly, the translated nucleotide coding sequences were used as input files for computing Ka/Ks values by using the Phylogenetic Analysis by Maximum Likelihood (PAML) program (PAML4.9j, http://www.bork.embl.de/pal2nal/index.cgi, accessed on 25 February 2022) with the default parameters. All variable sites of the alignment pairs were used in the calculation of Ka/Ks.

Conclusions
These findings provide genomic evidence and genomewide information for the investigation of the mechanism of early cotton maturation, and the flowering mechanism of cotton might be relatively conserved among the allotetraploid species and between the subgenomes. The core mode of flowering is also evolutionarily conserved between G. hirsutum and A. thaliana, and the basic light and hormone-signaling-related flowering pathways are likely relatively conserved. Additionally, the cotton flowering-related genes underwent strong purification during evolution. On the basis of these results, we provide a possible regulatory model of the multiple feedbacks and inputs during the regulation of cotton flowering in the leaves and SAM, which are further systematic and comprehensive flowering regulatory molecular networks of allotetraploid cotton species.