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.