Genome-Wide Identification, Characterization, and Expression Profiling Analysis of SPL Gene Family During The Inflorescence Development in Trifolium Repens

doi:10.21203/rs.3.rs-1430343/v1

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Genome-Wide Identification, Characterization, and Expression Profiling Analysis of SPL Gene Family During The Inflorescence Development in Trifolium Repens

https://doi.org/10.21203/rs.3.rs-1430343/v1

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Trifolium repens is the most widely cultivated perennial legume forage in temperate region around the world. It has rich nutritional value and good palatability, seasonal complementarity with grasses, and can improve the feed intake and digestibility of livestock. However, flowering time and inflorescence development directly affects the quality and yield of T. repens, as well as seed production. Squa promoter binding protein–like (SPL) gene family is a plant specific transcription factor family, which has been proved to play a critical role in regulating plant formation time and development of flowers. In this study, a total of 37 TrSPL genes were identified from the whole genome of T. repens and were divided into 9 clades based on phylogenetic tree. The conserved motif of squamosa promoter binding protein (SBP) contains two zinc finger structures and one NLS structure. Gene structure analysis showed that all TrSPL genes contained SBP domain, while ankyrin repeat region was just distributed in part of genes. 37 TrSPL genes were relatively dispersedly distributed on 16 chromosomes, and 5 pairs of segmental repeat genes were found, which indicated that segmental duplication was the main way of gene expansion. Furthermore, the gene expression profiling showed that TrSPL11, TrSPL13, TrSPL22 and TrSPL26 were highly expressed only in the early stage of inflorescence development, while TrSPL1 and TrSPL6 are highly expressed only in the mature inflorescence. Significantly, the expression of TrSPL4 and TrSPL12 increased gradually with the development of inflorescences. The results of this study will provide valuable clues for candidate gene selection and elucidating the molecular mechanism of T. repens flowering regulation.

Trifolium repens

SPL gene family

Phylogenetics

Flowering regulation

Expression patterns

Trifolium repens is the most important perennial legume forage in temperate regions. It is mainly cultivated in perennial pasture together with other forage, and utilization through directly grazed or mechanically harvested into hay and silage (Birte et al., 2014). T. repens was a kind of high quality forage with rich protein and mineral content, good palatability and high nitrogen fixation ability, which was beneficial to improve grassland quality, complement seasonal growth patterns of commonly forages and promote the intake and digestibility of livestock (Caradus et al., 1995; Abberton et al., 2005). However, flowering always reduces the number of axillary buds growing into branch stolons and leaf production (Gibson et al., 1985), which lead to sharply dropped of the nutritional value and digestibility (Kilcher et al., 1981). Therefore, delaying the flowering time and prolonging the vegetative growth period of T. repens will greatly improve the forage quality and yield (Fiorella et al., 2017). Also, T. repens flowering at the suitable time and inflorescence well developed are not only an important guarantee for seed production, but also increasing the opportunity of resowing in the pasture under adverse conditions and enhancing the persistence (Pederson et al., 2000; Christian et al., 2017). Indeed, it is important to study the flowering time regulation and inflorescence development for furture T. repens genetic improvement and breeding program, nevertheless, the molecular mechanism remain unknow due to the absence of omics data and limited availability of genome resource. The newly published genome article provides a possibility platform for the further study of T. repens (Griffiths et al., 2019).

Squa Promoter binding protein–Like (SPL) proteins are plant specific transcription factor family and operate on the characteristic genes of flower meristem, participate in the formation and later development of flowers. The most leading feature of SPL proteins is that SBP-box (squamosa promoter binding protein) encodes a conserved protein domain with 76 amino acids (Cardon et al., 1999). The conserved SBP domain contains two zinc finger structures and one NLS structure, which are the structural basis for sequence-specific gene binding (Yamasaki et al., 2004; Birkenbihl et al., 2005). Although SPL gene family had been widely studied in lots of species and the function of this kind of genes was also confirmed, there is no systematic exploration in T. repens. In Snapdragon (Antirrhinum majus), SBP1 and SBP2 acted on the promoter region of flower meristem characteristic gene SQUAMOSA to control early flower development (Klein et al., 1996). A total of 16 AtSPL genes were identified in Arabidopsis thaliana, and AtSPL3 directly activates LEAF, FRUITFULL and APETALA1 to control the formation time of flowers (Ayako et al., 2009). Besides, overexpressing AtSPL10 showed an early flowering phenotype, and the triple loss-of-function mutants with its homologous genes AtSPL2 and AtSPL11 showed flowering later than wild type (Tao et al., 2019). In terms of inflorescence development, AtSPL8 showed significant functions in controlling pollen sac development and it is necessary for the normal development of spore tissue (Unte et al., 2003). The loss of AtSPL9 and AtSPL15 function can lead to the change of inflorescence structure (Schwarz et al., 2008). In rice (Oryza sativa), 19 OsSPL genes were identified (Xie et al., 2006), with function of promoting the grain development to increase yield at the optimal expression level, and preventing the reversal of rice inflorescence during flowering (Lei et al., 2017; Francesca et al., 2021). There were 56 TaSPL genes in wheat (Triticum aestivum), and most of which regulated the development of inflorescence and spike (Ting et al., 2020). In addition, 17 putative DgSPL genes were identified in orchardgrass (Dactylis glomerata), a common companion species of T. repens mixed grassland, which were mainly involved in vegetative to reproductive growth transition, flower development and flowering regulation (Guangyan et al., 2021). In Tribulus alfalfa (Medicago truncatula), a total of 23 MtSPL genes were identified which are involved in the development of seed pods, especially the formation of thorns on pods (Wang et al., 2019).

It is critical to explore the white clover flowering time regulation and inflorescence development mechanism for its genetic improvement and utilization. Genome-wide identification, characterization, and expression profiling analysis of SPL gene family in T. repens will be an important basic work for providing valuable clues of candidate gene selection and breeding program. In this study, TrSPL gene family were identified and comprehensively analyzed, including SBP conserved domain comparation, conserved motif composition, gene structure annotation and chromosome location distribution. The evolution of TrSPL genes were preliminarily predicted by studying the intraspecific replication events, phylogenetic analysis and collinearity analysis with other plant species. Also, an expression profiling analysis of TrSPL genes at different flower development stages were constructed.

2.1 Genome-wide identification of TrSPL genes

The T. repens genome resource information came from previous study (Griffiths et al., 2019) and all files were provided by Stig Uggerhøj Andersen from Aarhus University. The hidden markov model (HMM) file of SBP domain (pf03110) was download from Pfam database (Finn et al., 2014) (pfam.xfam.org/). Take the downloaded HMM file as the query sequence to search the protein sequence data of T. repens by using HMMER3.0. The obtained proteins were aligned by ClustaW (E-value < 1^e − 20) and the SBP HMM file was rebuilt by hmmbuild in HMMER 3.0. Finally, SBP HMM of T. repens was used to identify SPL protein in T. repens genome, and the cut-off value was setted to 0.01(Finn et al., 2011). In order to ensure that all candidate genes contain SBP domain, the NCBI Conserved Domain Search website was used for further confirmation (Conserved Domains Database (CDD) and Resources (nih.gov)). The isoelectric point and relative molecular weight data were obtained using the Expasy website (Compute pI/MW - SIB Swiss Institute of Bioinformatics | Expasy). The subcellular localization information of TrSPL genes were analyzed and predicted online by BUSCA tool (BUSCA - Bologna Biocomputing Group (unibo.it)). Cis acting element analysis was predicted and visualized by using online tools PlantCARE and TBtools software (http://bioinformatics. psb.ugent.be/webtools/plantcare/html/) (Chen et al., 2020).

2.2. Phylogenetic analyses and classification of TrSPL genes

Genes and protein sequences of Arabidopsis thaliana, Medicago truncatula, Trifolium pratense and Glycine max SPL gene family are downloaded from TAIR (https://www.arabidopsis.org/down load/index-auto.jsp?dir=%2Fdownload_files%2FSequences%2FAraport11_blastsets), the Medicago truncatula Genome Database (http://www.medicagogenome.org/), Ensembl website (https://plants. ensembl. org/ index.html) and the Glycine max Genome Database (https://www. soybase.org/), respectively. Multiple sequence alignment of SPL protein sequences was performed by Log-Expectation (MUSCLE) in MEGA v6.0 (Tamura et al., 2013). Eventually, the phylogenetic tree was constructed by the neighbor-joining method of MEGA v6.0 software and the bootstrap replications value was 1000.0.

2.3. Gene Structure and Motif Analysis of TrSPL genes

Jalview software for SBP conservative sequence alignment of T. repens (http://www.jalview.org/), two zinc finger structures and one NLS structure are marked in the figure. The conserved motifs of T. repens are analyzed by MEME Suite 5.14 (Introduction - MEME Suite (meme-suite.org)) (Bailey et al., 2009), and the details of each conserved motif are also derived from the same website. The maximum number of motifs are set as 10. Use the program Visualize MEME/MAST Motif Pattern of Tbtools software for conservative motif visual analysis. Gene structure analysis uses the gene sequence file of TrSPL genes to analyze and visualize in TBtools software Visualize Gene Structure function, showing the CDS sequence, SBP conserved domain, ANK conserved domain and intron.

2.4. Chromosomal locations and synteny analysis of TrSPL genes

The chromosome location information of TrSPL genes were obtained from the genome annotation file. The Gene Location Visualize From GTF/GFF function of Tbtools software was used for gene chromosome mapping and visual analysis. The intraspecific collinearity circle map of TrSPL genes were analyzed by using the Dual Synergy Plot for McscanX function of TBtools and visualized by the Advanced Circos function. The genome data of Arabidopsis thaliana was downloaded from TAIR (https://www.arabidopsis.org/download/index-auto.jsp?dir=%2Fdownload_files%2FSequences% 2FAraport11_blastsets). The genome data of Medicago truncatula was downloaded from the Medicago truncatula Genome Database (http:// www.medicagogenome.org/). The genome data of Trifolium pratense was downloaded from Ensembl website (https://plants. ensembl. org/ index.html). The genome data of Glycine max was downloaded from the Glycine max Genome Database (https://www.soybase.org/). Drawing with Dual Synteny Ploter function of TBtools.

2.5. Material culture, sampling and qRT-PCR

T. repens Seed Haifa is provided by Beijing MAMMOTH SEED company, with the registration number of 249. The same genotype material Haifa is used for asexual propagation by cutting in pot (The diameter is 16.5cm, the height is 10cm, and the bottom diameter is 12cm). The growth environment of T. repens is a growth chamber with a Photoperiod of 14h at 22 ℃ and a dark period of 10h at 20 ℃. T. repens after flowering, fresh inflorescences in different developmental states are taken and stored in the freezing tube. (T1: Immature inflorescence, T2: Inflorescences in which no floret was open, T3: Inflorescences in which outermost circle of florets were open, T4: Inflorescences in which 50% of florets were open, T5: Mature inflorescence). Three biological replicates per sample. Put the fresh sample into liquid nitrogen immediately and long-term preservation in -80 ℃ refrigerator. Total RNA was extracted using the Hipure HP Plant RNA Mini Kit (Magen). The obtained RNA was reverse transcribed into cDNA using the MonScript™ RTIII ALL-in-One Mix with dsDNase (Monad) kit. Primer design used primer5 software and Tr β- Actin as internal parameter (Jia et al., 2021). All primers and internal paramenter information have been given in the attachment (Table S1). qRT-PCR was performed with Bio-Rad CFX96 instrument, and used SYBR® green real-time PCR master Mix test kit. The qRT-PCR procedure as follow:30s at 95°C, denaturation (95°C for 10s), anneal/extension (55°C for 30s), for 40 cycles, melting curve detection (65°C-95°C). 2^(−ΔΔCt) analysis method was used to calculate the relative expression of 16 TrSPL genes, and finally the expression maps of 16 TrSPL genes in 5 different flower development stages were obtained.

3.1 Genome-wide Identification of T. repens SPL Genes

Based on the T. repens genome resources, putative TrSPL genes were preliminarily identified by performing HMM (hidden Markov model) search (SBP domain, PF03110) from Pfam database. Subsequently, 38 TrSPL genes were identified after removing redundant sequences preliminarily. However, owing to the incomplete of SBP structure in gene chr13.jg763.t1, 37 genes with highly conserved SBP domain were retained (Table. 1). According to the phylogenetic tree of T. repens and Arabidopsis (Fig. 1), 37 TrSPL genes were named as TrSPL1 - TrSPL37 respectively (Table S2). The isoelectric point (PI) of 40.5% of proteins was less than 7, with the lowest PI of TrSPL5 was 5.72, and the highest PI of TrSPL14 and TrSPL33 were 9.41. The protein sequence length (aa) of all TrSPL proteins ranged from 1053 (TrSPL6) to 124 (TrSPL12) amino acids with an average of 474. The relative molecular weight (MW) ranged from 116204.16Da to 14269.05Da and the corresponding gene was consistent with the length of protein sequence. Subcellular localization results showed that 37 SPL genes in T. repens were located in the nucleus (30 genes), chloroplast (2 genes), plasma membrane (2 genes) and endomembrane system (3 genes), respectively. The basic data of TrSPL gene family varied widely, which indicated that diverse function of these genes.

3.2 Phylogenetic Analyses and Classification of the TrSPL Gene Family

For the classification of the TrSPL gene family, a Neighbor-joining (NJ) tree (with 1,000 bootstraps) of 16 Arabidopsis SPL genes and 37 identified TrSPL genes was constructd (Fig. 1). The results showed that 53 genes were divided into 9 clades, and all 16 SPL genes in Arabidopsis thaliana were distributed in 8 main SPL evolution clades named as Ⅰ to Ⅷ (Salinas et al., 2012; Preston et al., 2013). There were two TrSPL genes grouped into T. repens-specific clade named in Ⅸ, which indicated that potentially emerged after the divergence between two species. Moreover, in order to explore the evolutionary relationship of SPL gene families among the related species of T. repens, 24 SPL genes of red clover (Trifolium pratense) and 23 SPL genes of tribulus alfalfa (Medicago truncatula) were together analzed with white clover and Arabidopsis (Fig. 2). A total of 100 SPL genes are divided into 7 clades (Ⅰ-Ⅶ), and each evolutionary clade containd all 4 species. The results showed that SPL gene distance between three legumes species were closer, while farther than Arabidopsis. Multi species phylogenetic tree revealed that SPL gene family was relatively conservative in evolutionary direction.

Cis-acting elements are important in regulating gene expression. Analysis cis acting elements of 37 TrSPL genes (upstream 2000 bp) showed that TrSPL genes contained a large number of action elements in response to light (light response element, circadian rhythm regulation element, phytochrome down-regulation response element) and hormones (abscisic acid response element, gibberellin response element, plant auxin response element, salicylic acid response element, methyl jasmonate response element). In addition, there were some other elements in response to external stress, such as low temperature response element, defense and stress response element, plant trauma response element. (Table S2; Fig S1).

3.3 Sequence feature and gene structure of TrSPL genes

The full-length protein sequence of T. repens SPL genes were used for sequence alignment. The SBP domain was highly conserved in the TrSPL gene family (Fig. 3). All SBP domains contained two zinc finger structures and a nuclear localization signal (NLS), along with absence of some small fragments from TrSPL12, TrSPL 20, TrSPL 34 and TrSPL 35. Besides, the motif of first TrSPL gene zinc finger for clade Ⅰ (Cys-Cys-Cys-Cys) was different from that in the other clades (Cys-Cys-Cys-His, which was consistent with Tribulus alfalfa and other species (Li et al., 2019; Wang et al., 2019; Tong et al., 2020).

In order to analyze the diversity and similarity of TrSPL gene structure, 10 kinds of motifs were identified in the MEME website (Fig. 4). Among them, motif1 and motif2 contained a complete SBP domain and the length of motif ranges from 21 to 50. TrSPL12 and TrSPL35 only contained one motif, whereas TrSPL6 contained 11 motifs. The relatively conserved parts were motif1, motif2 and motif10 in all TrSPL genes. Motif 4–7 were only appeared in the yellow clade which reveal that these motifs were the main factors for the evolution and even functional conservation of this branch. TrSPL genes in same branch have similar conserved motifs, indicating they may be having similar function. Sequence information for each motif is provided in Table S3. In the analysis of gene structure, all the genes in the yellow clade have Ankyrin repeat regions (Ank-2 and Ank-2 superfamily), which could be involved in protein-protein interaction (Riese, Höhmann et al. 2007). All TrSPL genes had at least one intron and TrSPL34 had the most introns (with 20 introns).

3.4 Chromosomal locations and synteny analysis of TrSPL genes

37 TrSPL genes were accurately mapped onto T. repens chromosomes (Fig. 5, Table S4). TrSPL genes were relatively evenly distributed on all 16 chromosomes, and the number of TrSPL gene on each chromosome ranged from one (Chr6O, chr8O, chr5P and chr7P) to four (chr3O, chr2P and chr3P).

Gene duplication event is an important way to produce new genes with similar or different functions. We visualized the intraspecific replication events of TrSPL genes in Fig. 6. A total of 5 pairs of segmental duplication genes were found, while there was no tandem duplication in the TrSPL gene family (Table S5), indicating that segmental duplication was the main way of TrSPL gene family expansion.

To further explore the evolution of the TrSPL gene family, 4 comparative syntenic maps consisted of Arabidopsis thaliana, Trifolium pratense, Medicago truncatula, and Glycine max were constructed based on collinearity analysis (Fig. 7). The number of homologous pairs between T. repens and other 4 species was 10 (Arabidopsis), 14 (Red clover), 28 (Tribulus alfalfa) and 42 (soybean). The details of homologous pairs are given in Table S6. The comparison results showed that there are more homologous genes between T. repens and leguminosae species.

3.5 Expression patterns of TrSPL genes in different inflorescence development stage

In order to further forecast the function of TrSPL genes, 16 representative TrSPL genes were selected based on phylogenetic tree (Fig. 1). By constructing expression profiles in 5 different inflorescence development stages, preliminarily predicted function of genes was detected (Fig. 8). TrSPL11, TrSPL13, TrSPL22 and TrSPL26 had high expression only in the first development stage (T1), and and expression decreased in subsequent stages. TrSPL33 was highly expressed at T1 and T2, and decreased sharply at three stages after florets bloom. These results suggested that these genes may play an important role in the early development of T. repens inflorescence. Of course, some genes, such as TrSPL1 and TrSPL6, were highly expressed only at inflorescence maturity (T5). With the development of inflorescence, the expression level of TrSPL4 and TrSPL12 gradually increased and reached the highest at inflorescence maturity (T5). It was worth noting that the relative expression of TrSPL12 was the highest among the 16 genes, which may be closely related to the regulation of inflorescence development. The expression level of TrSPL24 and TrSPL25 increased sharply at T2 stage, and then decreased gradually with the development of inflorescences. The gene expression profile of TrSPL genes provided important information to determine the potential regulatory function of T. repens SPL gene family in inflorescence development.

T. repens is high-quality leguminous forage, and has important economic value in temperate agricultural system (Frame et al., 1998). However, fowering directly affects the quality and yield of T. repens, and inflorescence development directly affects seed production. SPL gene family is a plant-specific transcription factor family containing a highly conserved SBP domain (76 amino acids), which can bind DNA in a sequence-specific manner and regulate transcription. SPL genes can specifically bind related motifs in SQUAMOSA promoter of snapdragon and AP1 promoter of Arabidopsis, which have been proved to play an important role in regulating plant growth and development (Huijser et al., 1992; Mandel et al., 1992; Klein et al., 1996). In this study, 37 TrSPL genes were identified in T. repens, and much more than 16 in Arabidopsis, 19 in rice (Xie, Wu et al. 2006), 14 in barley (Hordeum vulgare) (Tong et al., 2020), and 27 in apple (Malus domestica Borkh.) (Li et al., 2013), but less than 56 in wheat (Ting et al., 2020), 57 in mustard (Brassica juncea) (Li et al., 2020), 48 in walnut (Juglans regia) (Zhou et al., 2019), 77 in euphorbiaceae (Li et al., 2019), and 58 in oilseed rape (Brassica napus) (Cheng et al., 2016). Generally, the number of gene family is partly affected by the genome size of species. Although the genomes of Arabidopsis (125Mb) (Schneeberger et al., 2011), rice (389Mb) (Takuji et al., 2005) and apple (632.4Mb) (Xuewei et al., 2016) are much smaller than T. repens (1174Mb) (Griffiths et al., 2019), the genome of mustard (1056.53Mb) (Li et al., 2020; Kang et al., 2021) and walnut (620Mb) (Annarita et al., 2020) were also smaller than T. repens. Gene replication events are also very important in determining the evolution and expansion of gene family, and species-specific gene replication is an important reason for determining the size of SPL gene family. T. repens is an allotetraploid leguminous forage. It is predicted that heterologous polyploidization event occurred in the last great glacier period (Warren et al., 2012), which may have an essential impact on the size of TrSPL gene family. Additionally, 5 pairs of segmental repeat genes were found while no tandem repeat gene pairs, which indicated that segmental repeat is more conducive to the evolution and population expansion of T. repens SPL gene family.

The isoelectric point, relative molecular weight and protein sequence length analysis of TrSPL genes showed that rich variation within this gene family. A large number of cis-acting elements related to light, hormone and stress response were found, which speculated that the functions of this gene family in T. repens are diverse and may play a regulatory role in this physiological process. Furthermore, TrSPL gene showed similar gene structure and conserved motifs in the same clade, but there were significant differences among clades. Ankyrin repeat regions were found in all genes of the yellow clade, indicating that these genes may play an important role in protein-protein interaction. Owing to the ancestor SPL originally formed into two different lineages, named clade Ⅰ and clade Ⅱ (Hua et al., 2019). Based on the phylogenetic trees of SPL gene families of white clover, red clover, tribulus alfalfa and Arabidopsis further reveal the phylogenetic relationship between them. Besides, there were only ten pairs of homologous pairs between T. repens and Arabidopsis, while more homologous pairs were found in leguminous species such as red clover, Tribulus terrestris, alfalfa and soybean, indicating that the evolution of SPL gene in leguminous also had high conservation and homology.

Generally, genes in the same branch of the phylogenetic tree have the similar function. Gene expression patterns can provide crucial information for determining gene function prediction (Zhou et al., 2018). Previous studies have shown that the Arabidopsis SPL gene in clade V (AtSPL3) and clade VI (AtSPL2, AtSPL10 and AtSPL11) could regulate flowering time (Cardon et al., 1997; Tao et al.,2019), and it was speculated that TrSPL19-25 located in the same clade may have similar functions in regulating the flowering time of T. repens. Interestingly, the light response elements were detected in all of these genes. Similarly, the Arabidopsis SPL genes (AtSPL8, AtSPL9 and AtSPL15) in clade III and clade VIII have been proved to affect inflorescences development (Schwarz et al., 2008; Unte et al., 2003), and TrSPL10 to14 and TrSPL32 to 35 (assigned into clade III and clade VIII) were possible relevance to inflorescences development of T. repens. Among these genes, TrSPL11 and TrSPL13 was highly expressed only at T1 stage, and TrSPL33 was highly expressed at T1 and T2 stages, indicating that these genes play an important regulatory role in the early development of T. repens inflorescences. Specially, with the development of T. repens inflorescences, the expression of TrSPL12 gradually increased and peaked at T5, indicating that TrSPL12 may play an important effect with the development of inflorescences. AtSPL genes (AtSPL1, AtSPL12 and AtSPL14) has been proved to be involved in regulating the development and its sensitivity to fumonisin B1 of Arabidopsis. Similarly, Vpsbp5 in the same clade has also been proved to prevent powdery mildew in grapes (Hongmin et al., 2013). In this study, results showed that TrSPL genes in cade Ⅱ may play an important role in enhancing disease resistance during the development of T. repens inflorescences. In brief, TrSPL gene family is such important in T. repens flowering regulation, especially in inflorescence development.

Trifolium repens is the most widely cultivated perennial legume forage in temperate region around the world. However, flowering time and inflorescence development directly affects the quality and yield, as well as seed production. SPL gene family is a plant specific transcription factor family, which has been proved to play a critical role in regulating plant formation time and development of flowers. In this study, a total of 37 TrSPL genes were identified from the whole genome of T. repens and were divided into 9 clades based on phylogenetic tree. The basic information of 37 TrSPL genes was obtained, including isoelectric point (PI), relative molecular weight (MW), protein sequence length (aa) and subcellular localization. The result of cis acting element anlysis showed that a large number of action elements in response to light were identified and potential flowering regulation function was predicted. Besides, 37 TrSPL genes were relatively dispersedly distributed on 16 chromosomes, and 5 pairs of segmental repeat genes were found, which indicated that segmental duplication was the main way of gene expansion. Furthermore, the gene expression profiling showed that TrSPL11, TrSPL13, TrSPL22 and TrSPL26 were highly expressed only in the early stage of inflorescence development, while TrSPL1 and TrSPL6 are highly expressed only in the mature inflorescence. The results of this study will provide valuable clues for candidate gene selection and elucidating the molecular mechanism of T. repens flowering regulation.

SPL

Squa promoter binding protein–like

SBP

squamosa promoter binding protein

CDS

Coding sequence

HMM

Hidden Markov model

Molecular weight

isoelectric point

NLS

nuclear localization signal.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the China Agriculture Research System of MOF and MARA, and the Sichuan Province Breeding Research grant (2021YFYZ0013).

Availability of data and materials

All data generated or analyzed during this study are included in this article and its attached documents. The Trifolium repens genome resources were downloaded from the EMBL/GenBank data libraries (Bioproject PRJNA523044, https://www.ncbi.nlm. nih.gov/genbank/) (Griffiths et al., 2019). The genome data used for comparative syntenic analysis were obtained from open database. The genome data of Arabidopsis thaliana was downloaded from TAIR (https://www. arabidopsis.org/download/index-auto.jsp?dir=%2Fdownload_files%2FSequences%2FAraport11_blastsets). The genome data of Medicago truncatula was downloaded from the Medicago truncatula Genome Database (http:// www.medicagogenome.org/). The genome data of Trifolium pratense was downloaded from Ensembl website (https://plants. ensembl. org/ index.html). The genome data of Glycine max was downloaded from the Glycine max Genome Database (https://www.soybase.org/).

Authors’ contributions

GN and XZ conceived and designed the experiments. Bioinformatics analysis was performed by JM and ZY. SM and RH carry out planting and management of materials. JM, FW and JF complete the experimental part. JM and GN complete the manuscript. GN and XZ revised the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We gratefully acknowledge Stig Uggerhøj Andersen from Aarhus University for providing Trifolium repens genomic resources.

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Table 1 The basic information and subcellular localization of all identified SPL genes in T. repens

Name	PI	MW (Da)	Length (aa)	Subcellular Localization
TrSPL1	6.12	86752.27	773	nucleus
TrSPL2	6.88	67153.17	591	nucleus
TrSPL3	8.18	109672.44	989	plasma membrane
TrSPL4	5.75	111233.87	1010	endomembrane system
TrSPL5	5.72	111303.94	1014	endomembrane system
TrSPL6	7.01	116204.16	1053	nucleus
TrSPL7	8.51	115211.08	1044	endomembrane system
TrSPL8	5.8	111853.78	1004	nucleus
TrSPL9	5.86	110745.6	995	nucleus
TrSPL10	8.92	37297.64	335	nucleus
TrSPL11	9.14	40922.71	363	nucleus
TrSPL12	6.9	14269.05	124	nucleus
TrSPL13	9.3	20662.94	182	nucleus
TrSPL14	9.41	20574.88	182	nucleus
TrSPL15	5.98	57844.27	527	nucleus
TrSPL16	6.46	55524.04	509	nucleus
TrSPL17	6.31	55948.26	500	nucleus
TrSPL18	8.97	30286.98	267	chloroplast
TrSPL19	8.88	48472.58	439	nucleus
TrSPL20	8.73	44554.3	406	nucleus
TrSPL21	8.17	45859.11	408	nucleus
TrSPL22	8.67	34247.9	313	nucleus
TrSPL23	8.67	34313.96	312	nucleus
TrSPL24	6.1	17499.07	149	nucleus
TrSPL25	6.1	17472.04	149	nucleus
TrSPL26	7.6	41040.79	367	nucleus
TrSPL27	7.61	40962.63	367	nucleus
TrSPL28	8.86	42684.84	383	nucleus
TrSPL29	8.62	42817.9	385	nucleus
TrSPL30	6.62	42612.13	382	nucleus
TrSPL31	6.78	42977.52	387	nucleus
TrSPL32	9.2	37707.11	339	chloroplast
TrSPL33	9.41	37953.47	340	nucleus
TrSPL34	6.99	37614.66	343	plasma membrane
TrSPL35	8.73	36551.32	335	nucleus
TrSPL36	7.04	16415.99	142	nucleus
TrSPL37	7.04	16430.02	142	nucleus

No competing interests reported.

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Genome-Wide Identification, Characterization, and Expression Profiling Analysis of SPL Gene Family During The Inflorescence Development in Trifolium Repens

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Abstract

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1 Introduction

2 Materials And Methods

2.1 Genome-wide identification of TrSPL genes

2.2. Phylogenetic analyses and classification of TrSPL genes

2.3. Gene Structure and Motif Analysis of TrSPL genes

2.4. Chromosomal locations and synteny analysis of TrSPL genes

2.5. Material culture, sampling and qRT-PCR

3 Results

3.1 Genome-wide Identification of T. repens SPL Genes

3.2 Phylogenetic Analyses and Classification of the TrSPL Gene Family

3.3 Sequence feature and gene structure of TrSPL genes

3.4 Chromosomal locations and synteny analysis of TrSPL genes

3.5 Expression patterns of TrSPL genes in different inflorescence development stage

4 Discussion

5 Conclusions

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