{"paper_id":"3f879618-14ef-412d-92ce-e99b40f717da","body_text":"Insights into the caleosin family in Cyperus esculentus, an oil-rich tuber plant in Cyperaceae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Insights into the caleosin family in Cyperus esculentus, an oil-rich tuber plant in Cyperaceae Zhi Zou, Yuliang Chen, Huijian Huang, Chunqiang Li, Xiangdong Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8567486/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Caleosins (CLOs), a class of structural proteins of lipid droplets (LDs), widely function in LD formation, stabilization, and degradation as well as in plant development and stress responses. However, their characterization in tigernut ( Cyperus esculentus L., Cyperaceae), a rare example accumulating significant amounts of oil in underground tubers, is still in the infancy. Results In this study, we present a first genome-wide analysis of the caleosin family in tigernut. A number of eight members, which represent two previously defined clades (i.e., H and L), were identified from the tigernut genome, in stark contrast to only two present in the basal angiosperm Amborella trichopoda . Comparison of 193 caleosin genes from 31 representative plant species reveals lineage-specific expansion and functional diversification. Eight CeCLO genes belong to eight out of 11 orthogroups identified in this study, and extensive expansion in this species was contributed by whole-genome duplication (WGD), tandem, and dispersed duplications. 1:1 orthologous relationships observed between tigernut and its close relative Cyperus rotundus suggest that gene copies are not the contributor of high tuber oil accumulation in tigernut. Instead, most CeCLO genes was shown to express more than their orthologs in C. rotundus , implying species-specific activation in oil-bearing tigernut tubers. Correspondingly, expression of CeCLO1 , -2 , -3 , and − 7 was shown to positively correlate with oil accumulation during tuber development. Structure and expression divergence of paralogous pairs were also observed, and good examples are 1) CeCLO7 and − 8 that have gained one additional intron and 2) CeCLO1 that has become the predominant isoform in oil-rich tubers. Conclusions These findings provide insights into the evolution, expression, and structural variation of CeCLO genes, which improve our knowledge on the mechanism of high oil accumulation in tigernut tubers. Vegetative tissue Underground tuber Phylogenomics Orthologous analysis Synteny analysis Gene expansion Expression divergence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Plant seeds store triacylglycerols (TAGs) in discrete spherical organelles called oil bodies or lipid droplets (LDs), which are coated by a monolayer of phospholipids embedded with abundant oleosins (OLEs) and several minor structural proteins [ 1 ]. Caleosins (CLOs) are a class of minor LD proteins that are capable of binding to calcium ion (Ca 2+ ) [ 2 , 3 , 4 ]. Since the first caleosin gene (i.e., responsive to desiccation 20 ( RD20 )) that was identified in arabidopsis ( Arabidopsis thaliana ) [ 5 , 6 ], its homologs have been widely found in land plants as well as in some fungi and green algae [ 2 , 7 , 8 , 9 , 10 , 11 , 12 ]. Caleosins exhibit some similarities to oleosins, which feature a long central hydrophobic hairpin of about 72 residues (X 30 PX 5 SPX 3 PX 30 ) [ 1 , 13 , 14 ]. Like oleosins, caleosins are composed of three characteristic structural domains, i.e., an N-terminal hydrophilic EF-hand calcium binding domain, a central hydrophobic anchoring domain, and a C-terminal hydrophilic phosphorylation domain [ 6 , 7 ]. The central hydrophobic domain, which is responsible for anchorage in LDs, comprises an amphipathic α-helix followed by a pair of anti-parallel β-strands that are connected by a proline-knot motif (PX 3 PSX 3 P) [ 7 , 15 , 16 ]. Both N- and C-terminals are more likely to expose to the cytoplasm and are involved in environmental adaptation [ 7 , 8 ]. Though caleosins were first recognized as minor proteins functioning in LD stabilization and degradation in seeds, they were later proven to have catalytic functions as peroxygenases (PXGs, EC 1.11.2.3), whose activity is dependent on a heme group coordinated by the two invariant His residues located in N- and C-terminal regions [ 3 , 4 , 7 , 17 , 18 , 19 ]. Hence, they are also called peroxygenases [ 4 , 11 , 20 , 21 ]. Unlike oleosins, caleosins have additional intracellular locations apart from LDs, e.g., the envelope of chloroplasts, endoplasmic reticulum (ER), plasmalemma, vacuole, and nucleus, and they were also found to be abundant in a range of non-seed plant tissues such as callus, shoot, hypocotyl, root, cotyledon, leaf, megagametophyte, male flower, pollen, stamen, ovule, silique, fruit, and phloem sap [ 8 , 20 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. In land plants, caleosins comprise a small family with up to 10 members [ 9 , 10 , 11 , 15 , 16 , 29 , 30 , 31 , 32 ]. Generally, there are two major types present in each species, i.e., high (H) and low (L) molecular weights (MW), where the H-forms contain an extra N-terminal insertion of about 30 residues relative to L-forms [ 15 , 16 ]. In contrast to only two members present in castor bean ( Ricinus communis , RcCLO-1 and − 2 ) and flax ( Linum usitatissimum , LuCLO-1 and − 2 ) [ 30 ], a number of eight caleosin genes were described in arabidopsis, including four H-forms (i.e., AtCLO1 , - 2 , - 3 , and − 8 ) and four L-forms (i.e., AtCLO4 , - 5 , - 6 , and − 7 ), and the family expansion was shown to be contributed by both local and segmental duplications [ 15 , 29 ]. Expression analyses showed that RcCLO-2 and LuCLO-2 are preferentially expressed in seeds, whereas RcCLO-1 and LuCLO-1 are constitutively expressed, including seeds, cotyledons, and young leaves [ 29 , 30 ]. Similar expression profiles were also observed for AtCLO genes, where AtCLO3 and − 4 are expressed in various organs and others seem to be organ-specific, e.g., AtCLO1 and − 2 in seeds and AtCLO5 in buds [ 15 ]. Functional analyses revealed that caleosins are not only involved in LD formation, stabilization, and degradation, but also are associated with various plant development and stress responses [ 2 , 7 , 8 , 12 , 15 , 25 , 30 , 31 , 32 ]. In rice ( Oryza sativa ), OsCLO5 was initially named OsEFA27 (EF-hand, ABA responsive, 27 kilodalton (kDa)), which could be induced by ABA treatment, salt and drought stresses [ 2 ]. In arabidopsis, four seed-expressed caleosin genes (i.e., AtCLO1 , - 2 , - 4 , and − 6 ) were proven to redundantly function in oil accumulation and embryo development [ 34 ]. Interestingly, a high number of caleosin genes were shown to be regulated by various abiotic and biotic stresses, e.g., OsCLO5 / EFA27 in rice [ 2 ], AtCLO3 / PXG3 / RD20 in arabidopsis [ 8 ], Sopl in sesame ( Sesamum indicum ) [ 7 ], HvCLO1 and − 2 in barley ( Hordeum vulgare ) [ 33 ], BnCLO1-1 in rapeseed ( Brassica napus ) [ 35 ], and OeCLO3 in olive ( Olea europaea ) [ 25 ]. Tigernut ( Cyperus esculentus L. var. sativus Boeckeler), which is also known as chufa and yellow nutsedge, is an unconventional oil crop uniquely accumulating significant amounts of TAGs in its underground tubers [ 36 , 37 ]. It resides in the Cyperaceae family, which is native to Africa [ 38 ]. Tigernut was initially domesticated in ancient Egypt for the nutrient-rich tubers, e.g., 25–45% starch, 20–35% oil, 15–20% sugars, 5–10% protein, 8–10% dietary fiber, 0.08–0.14 g/kg vitamin C and E, high levels of minerals (e.g., calcium (Ca), phosphorus (P), potassium (K), and magnesium (Mg)), and various active substances (e.g., flavone, alkaloid, tannin, terpenoid, saponin, and phytosterol) [ 39 , 40 ]. Recently, much attention has been attracted for its unique feature of high oil accumulation in the non-seed storage organ [ 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 ], and the disclosure of the mechanisms behind shall facilitate oil production in plant vegetative tissues via genetic engineering [ 50 ]. According to a previous proteomic analysis, like oilseeds, the LDs of tigernut tubers are mainly coated by oleosins and caleosins [ 51 ]. Thus far, genome-wide characterization of the oleosin family was reported, and six members present in tigernut are equal to those found in other Cyperaceae plants such as purple nutsedge ( C. rotundus ) and Rhynchospora breviuscula [ 45 ]. Interestingly, the majority of CeOLE genes were shown to exhibit a tuber-preferential expression pattern with mRNA/protein abundances positively correlating with oil accumulation during tuber development [ 45 ]. Transcriptome-based identification of caleosin genes was also reported [ 52 ], however, 21 homologs described in that study are considerably more than eight detected from our full-length transcriptome, which was constructed via single-molecule real-time (SMRT) sequencing of various tissues such as shoot apex, leaf, sheath, root, rhizome, tuber, flower, and floral stem [ 43 ]. Considering the inherent drawback of incorrect de novo assembly from short reads, a genome-scale identification may provide a more precise view of the caleosin family in this special species. In this study, a genome-wide analysis of the CeCLO family was conducted, which include sequence characteristics, gene structures, chromosome localizations, evolutionary relationships, and expression profiles. Significantly, comparison of 193 caleosin genes identified from 31 representative plant species reveals lineage-specific expansion and functional diversification in tigernut. In contrast to only two members present in the basal angiosperm Amborella trichopoda , a high number of eight CeCLO family genes were shown to arise from whole-genome duplication (WGD), tandem, and dispersed duplications, whereas tuber-specific activation of certain members appears to be species-specific. These findings highlight the evolution patterns of the CeCLO family, which provide valuable information for further functional analysis. Results Identification, gene localization, and duplication event analysis of eight caleosin genes in tigernut As shown in Table 1 , HMMER-based homologue search resulted in eight caleosin genes from the tigernut genome [ 53 ]. Though the overall sequence similarity of their deduced peptides varies from 44.32% to 78.57% ( Additional file 1 ), all of them were shown to possess a single caleosin domain (under the Pfam accession number of PF05042) that is specific to this family (Table 1 ). Notably, the amounts of the CeCLO family are somewhat bigger than six members as described for the CeOLE family [ 45 ]. Compared with CeOLEs with an average of approximately 150 residues [ 45 ], the peptide length of CeCLOs is relatively longer, varying from 198 (i.e., CeCLO6) to 261 (i.e., CeCLO5) residues with an average of about 234 residues, which result in the higher MW of 21.82–29.93 kDa, i.e., 26.42 vs 15.52 kDa (Table 1 ). Interestingly, different patterns of isoelectric point (pI) and grand average of hydropathicity (GRAVY) were also observed between CeCLOs and CeOLEs as described in papaya ( Carica papaya ) [ 29 ]. Whereas only 62.50% of CeCLOs (i.e., CeCLO4–8) exhibit the alkaline feature like CeOLEs, others (i.e., CeCLO1–3) harbor the pI values of less than 7, varying from 5.91 (i.e., CeCLO2) to 6.63 (i.e., CeCLO3) (Table 1 ). In contrast to the highly hydrophobic feature of CeOLEs [ 45 ], all CeCLOs have the GRAVY value of less than 0, varying from − 0.593 to -0.117 (Table 1 ). The overall hydrophilic feature of CeCLOs was also supported by the ProtScale analysis. As shown in Fig. 1 a, similar hydropathicity scales were observed for all eight CeCLOs. Whereas the central domain is hydrophobic, N- and C-terminal regions are usually hydrophilic (Fig. 1 a). Except for CeCLO6, one to two transmembrane helixes (TMH) were identified (Table 1 ). Table 1 Caleosin genes identified in C. esculentus . ( AA amino acid, Ce C. esculentus , CLO caleosin, GRAVY grand average of hydropathicity, kDa kilodalton, MW molecular weight, pI isoelectric point, Scf scaffold, TMH transmembrane helix) Gene name Locus ID Position AA MW (kDa) pI GRAVY TMH Caleosin location CeCLO1 CESC_00080 Scf30:3622141..3623943(-) 251 28.96 6.50 -0.366 104..123 74..240 CeCLO2 CESC_16572 Scf47:433600..435126(-) 242 27.87 5.91 -0.410 94..117 63..231 CeCLO3 CESC_16571 Scf47:436090..438271(-) 239 27.38 6.63 -0.228 88..107,184–201 59..225 CeCLO4 CESC_18483 Scf13:1911499..1913058(-) 244 27.63 9.04 -0.293 88..110,184..201 59..226 CeCLO5 CESC_18484 Scf13:1915073..1916809(-) 261 29.93 8.78 -0.324 97..116 68..235 CeCLO6 CESC_02525 Scf21:760234..762404(-) 198 21.82 7.81 -0.593 - 21..188 CeCLO7 CESC_02526 Scf21:770229..771732(-) 217 23.57 8.79 -0.363 7..24,70..92 38..206 CeCLO8 CESC_02527 Scf21:775539..780932(-) 220 24.24 9.03 -0.117 6..25 43..210 The amino acid (AA) composition of CeCLOs was further calculated and compared with that of CeOLEs. Except for the absence of the Cys in CeCLO6–8, other members contain all 20 residues (Fig. 1 b), usually rich in Leu, Gly, and Ala as observed in CeOLEs ( Additional file 2a ). Interestingly, the cluster analysis revealed distinct patterns of AA composition between CeCLOs and CeOLEs, where proteins were grouped in family. Compared with CeOLEs, CeCLOs contain more hydrophilic but less hydrophobic residues ( Additional file 2a ), which contribute to their hydrophilic feature. The secondary structure of CeCLOs was also shown to differ from that of CeOLEs, where CeCLOs harbor more random coils but less alpha helixes and were clustered together (Fig. 1 c and Additional file 2b ). Sequence alignment of CeCLOs is shown in Fig. 1 d. Except for the conserved caleosin domain, N- and C-terminals are highly diverse. Though the usual proline-knot pattern PX 3 PSX 3 P was observed in CeCLO2–6, the PX 4 S/QSX 3 P variant was identified in CeCLO7 and − 8. Moreover, the PX 3 LNX 3 P variant was found in CeCLO1. Prior to the proline-knot are the amphipathic α-helix and the Ca 2+ binding motif. Two His residues, which are essential for the PXG activity [ 4 ], were found in all CeCLOs, residing in front of the Ca 2+ binding motif and somewhat behind the proline-knot motif, respectively (Fig. 1 d). Compared with CeCLO6–8, CeCLO1–5 harbor an H-form insertion as observed in arabidopsis [ 15 ], which contributes to the longer peptide size and higher MW (Table 1 and Fig. 1 d). As shown in Additional file 3 , a high number of phosphorylation sites were identified for each sequence. Among them, four are shared by most sequences, i.e., one for Tyr kinase and three for casein kinase II (CK II) (Fig. 1 d), implying their posttranslational regulation. Interestingly, despite few Cys residues present in CeCLOs, one is shared by CeCLO1–5 (Fig. 1 d), which was also described for Sopl in sesame [ 7 ]. In contrast to the distribution of six CeOLE genes over six scaffolds (Scfs) [ 45 ], gene localization showed that eight CeCLO genes are unevenly spread across four scaffolds, and three hotspots for tandem duplication were detected, i.e., CeCLO2 /- 3 on Scf47, CeCLO4 /- 5 on Scf13, and CeCLO6 /- 7 /- 8 on Scf21, respectively. Further synteny analysis revealed that CeCLO1 and − 2 are located within syntenic blocks, implying their derivation from WGD. On the contrary, CeCLO2 /- 4 and CeCLO4 /- 6 were characterized as dispersed repeats (Fig. 1 e). Phylogenetic and comparative analyses revealed species-specific expansion of the caleosin family among tigernut, rice, and arabidopsis To uncover the evolutionary relationships of CeCLO genes, homologs were also identified from arabidopsis and rice, two model plants for dicots and monocots, respectively. In accordance with previous studies [ 15 , 27 ], eight family members were obtained from updated genomes of both species ( Additional file 4 ). Interestingly, despite harboring the same family amounts, phylogenetic analysis of caleosins in tigernut, rice, and arabidopsis revealed species-specific expansion. As shown in Fig. 2 a, these proteins were clustered into two main clades, corresponding to H and L as described in arabidopsis [ 15 ]. Moreover, these two clades could be divided into two and three groups, respectively. Among them, Group Ia includes CeCLO1–3, OsCLO4, OsCLO5, AtCLO1–3, and AtCLO8; Group Ib includes CeCLO4, CeCLO5 and OsCLO3; Group IIa is specific to arabidopsis, including AtCLO4–7; Group IIb is specific to tigernut, including CeCLO6–8; and Group IIc is specific to rice, including OsCLO1–2, and OsCLO6–8. The results are largely in accordance with sequence similarities and duplication event analyses. As shown in Additional files 1 and 5 , OsCLO1 /- 2 , OsCLO4 /- 5 , and OsCLO6 /- 7 , which share 72.65%, 67.48%, and 95.56% sequence similarities at the protein level, were defined as proximal repeats; OsCLO7 /- 8 (53.41%), AtCLO4 /- 6 (85.71%), and AtCLO5 /- 7 (62.66%) were characterized as tandem repeats; OsCLO1 /- 6 (57.33%), AtCLO1 /- 2 (87.76%), and AtCLO5 /- 6 (66.67%) were characterized as WGD repeats, respectively; OsCLO3 /- 4 (54.10%), OsCLO4 /- 6 (39.74%), AtCLO1 /- 3 (72.47%), and AtCLO1 /- 4 (47.35%) were characterized as dispersed repeats. To learn more about structural variation, exon-intron structures and conserved motifs were further compared. As shown in Fig. 2 b, the most majority of caleosin genes present in three species feature five introns with the conserved phase pattern of 1, 1, 0, 2, and 2. Nevertheless, gain or loss of certain introns was observed in CeCLO7 , CeCLO8 , AtCLO7 , and AtCLO8 . In contrast to the loss of the fifth intron in AtCLO8 , CeCLO7 and CeCLO8 were shown to have gained one more intron (in phase 2) at the 5' end, which is different from the phases of 2, 0, 0, 0, 2, and 2 as observed in AtCLO7 , implying independent origin. Ten motifs identified using MEME are shown in Fig. 2 c. Whereas Motifs 1–5 are widely present, others appear to be sequence-specific. Motifs 1, 2, and 4 were characterized as the conserved caleosin domain; Motif 3 was characterized as the H-form insertion; and Motif 5 was characterized as the C-terminal phosphorylation sites (Fig. 2 d). Notably, Motifs 2 and 4 are absent from AtCLO7, and Motif 1 is absent from AtCLO7 and − 8, implying their divergence. Whereas Motif 3 was found in all H-forms, Motif 10, which was also characterized as the C-terminal of the H-form insertion, are limited to CeCLO1, CeCLO2, OsCLO4, and AtCLO2. Motifs 6 and 7, which are confined to OsCLO6 and − 7, are located at the N- and C-terminals, respectively. Motif 8, which is confined to AtCLO1 and − 2, is located at the C-terminal. Motif 9, which is confined to AtCLO5 and − 7, is located at the N-terminal (Fig. 2 c). Characterization of caleosin genes from representative plant species and insights into lineage-specific family evolution in Poales Above phylogenetic analysis suggests the divergence of Clades H and L sometime before monocot-eudicot split. However, several issues need to be further resolved: 1) the tandem duplications identified in tigernut are species or lineage-specific; 2) the exact time of the WGD that gave birth to CeCLO2 ; 3) the exact origin of CeCLO4 and − 6 ; 4) the exact time of gain of the additional intron as observed in CeCLO7 and − 8 . For the purposes, homologs were also identified from representative plant species, which include the basal angiosperm A. trichopoda (an Amborellaceae plant in Amborellales) and three early diverged monocots that didn't experience the τ WGD shared by core monocots, i.e., American sweet flag ( Acorus americanus , an Acoraceae plant in Acorales), eelgrass ( Zostera marina , a Zosteraceae plant in Alismatales), and duckweed ( Spirodela polyrhiza , an Araceae plant in Alismatales). After discarding pseudogenes and gene fragments, as shown in Additional file 4 , a total of 169 members were further identified. Notably, a single member for each clade was not only found in two out of three tested core eudicots, i.e., grapevine ( Vitis vinifera ) and castor bean, but also in A. trichopoda and eelgrass, supporting early divergence of Clades H and L in the angiosperm ancestor. By contrast, equal amounts of eight members were identified in purple nutsedge, a close relative of tigernut ( Additional file 4 ). To infer lineage-specific gene expansion and contraction, species-specific duplication events were further examined and orthologs across different species were identified using Orthofinder. As shown in Fig. 3 , a total of 11 orthogroups were obtained and eight CeCLO genes belong to H1, H2, H3, H4, H5, L1, L2, and L3. Among them, H1 and L1 are shared by all species examined in this study, whereas others appear to be lineage-specific: H4 is widely present in core monocots, including garden asparagus ( Asparagus officinalis ), apostasia ( Dendrobium catenatum ), and greater yam ( Dioscorea alata ), implying its early origin that may arise from the τ WGD; H2 is present in most Poales plants and is usually located within syntenic blocks with H1, implying its possible origin along with the Poales-specific σ WGD; H3, which is limited to Cyperaceae and Juncaceae species, was characterized as the tandem repeat of H2; H5 is confined to three out of four examined Cyperus species, i.e., tigernut, purple nutsedge, and C. iria , where further expansion via WGD was also observed in the latter; L2, which is widely present in Cyperaceae plants with the exception of C. littledalei , was characterized as the tandem repeat of L1; L3, which is also Cyperaceae-specific, was characterized as the tandem repeat of L2 in tigernut, purple nutsedge, C. iria , C. fuscus , and R. breviuscula , but the tandem repeat of L1 in C. littledalei ; L4 and L5, which is Poaceae-specific, was characterized as the tandem and WGD (ρ) repeats of L1, respectively; L6, which is also Poaceae-specific, was characterized as the tandem repeat of L5 (Fig. 3 and Additional file 4 ). Inter-specific syntenic analyses were further conducted and results showed that CeCLO1 , CeCLO2/ - 3 , CeCLO4/ - 5 , CeCLO6/-7/-8 have syntelogs in at least one of examined species. Significantly, as shown in Fig. 4 a, 1 :1 and 2:2 relationships were observed between tigernut and purple nutsedge/ R. breviuscula / C. littledalei , implying that the WGD event (i.e., CeCLO1 /- 2 ) detected in tigernut is shared by these species. Moreover, 1:1, 1:2, and 2:4 relationships were observed between tigernut and C. iria , supporting species-specific expansion via WGD in C. iria , followed by group-specific gene loss in H3 and L3. Actually, their gene fragments could be detected in the C. iria genome. In accordance with orthologous analysis, no syntelog was identified for CeCLO4/ - 5 in C. littledalei , implying species-specific contraction. By contrast, their syntelogs were not only identified in pineapple ( Ananas comosus ), Joinvillea ascendens (Fig. 4 b), but also in oil palm ( Elaeis guineensis ), greater yam, duckweed (Fig. 4 c), and rice (Fig. 4 d). Notably, CeCLO4/ - 5 , CeCLO6/-7/-8 , EgCLO2 , and EgCLO3/-4/-5/-6 were shown to locate within syntenic blocks (Fig. 4 c). Since EgCLO3 was characterized as a recent repeat of EgCLO2 from the Arecaceae-specific p WGD ( Additional file 4 ), WGD-derivation of CeCLO4/ - 6 followed by lineage-specific transposition could be speculated. Collectively, these results imply early diversification of the caleosin family before the radiation of angiosperms followed by lineage-specific expansion even in early diverged angiosperms. The sequence identities and evolutionary rates of Ce/CrCLO genes as well as duplicate pairs identified in tigernut are summarized in Table 2 . As expected, high sequence identities of 89.30–98.50% and small Ks (synonymous substitution rate) values of 0.1048–0.1810 were observed between orthologs of tigernut and purple nutsedge. By contrast, only 43.90–68.40% sequence identities were found between seven duplicate pairs identified in tigernut. Moreover, except for high sequence divergence between CeCLO4 and − 6 , the Ks values of other pairs were shown to vary from 0.9012 ( CeCLO4 and − 5 ) to 3.7504 ( CeCLO6 and − 7 ) (Table 2 ), implying distinct time of their birth. Given that the Ka (nonsynonymous substitution rate)/Ks values of all duplicate pairs identified in tigernut are less than one (Table 2 ), a role of purifying selection during their evolution could be speculated. Table 2 Sequence identities and evolutionary rates of duplicate pairs identified in tigernut as well as orthologs between C. esculentus and C. rotundus . ( Ce C. esculentus , CLO caleosin, Cr C. rotundus , Ka nonsynonymous substitution rate, Ks synonymous substitution rate) Species Gene 1 Gene 2 Identity (%) Ka Ks Ka/Ks C. esculentus- C. esculentus CeCLO1 CeCLO2 66.50 0.2346 2.4592 0.0944 CeCLO2 CeCLO3 62.90 0.3019 2.4057 0.1255 CeCLO2 CeCLO5 54.10 0.5105 2.1566 0.2367 CeCLO4 CeCLO5 68.40 0.2376 0.9012 0.2637 CeCLO4 CeCLO6 43.90 0.5190 - - CeCLO6 CeCLO7 58.50 0.3064 3.7504 0.0817 CeCLO7 CeCLO8 62.30 0.3924 1.1637 0.3372 C. esculentus- C. rotundus CeCLO1 CrCLO1 94.70 0.0226 0.1810 0.1246 CeCLO2 CrCLO2 98.50 0.0127 0.1723 0.0736 CeCLO3 CrCLO3 96.80 0.0073 0.1225 0.0598 CeCLO4 CrCLO4 93.70 0.0419 0.1522 0.2751 CeCLO5 CrCLO5 89.30 0.0169 0.1696 0.0995 CeCLO6 CrCLO6 95.80 0.0132 0.1407 0.0938 CeCLO7 CrCLO7 95.30 0.0266 0.1048 0.2536 CeCLO8 CrCLO8 95.80 0.0160 0.1203 0.1333 Caleosin genes in tigernut exhibited distinct expression patterns To provide a global view of expression profiles of CeCOL genes, RNA-seq data of various tissues were first examined, i.e., young leaf, mature leaf, sheath, shoot apex, root, rhizome, young tuber (collected at 40 days after sowing (DAS)), middle tuber (collected at 85 DAS), and mature tuber (collected at 120 DAS). As shown in Fig. 5 , FPKM (fragments per kilobase of exon per million fragments mapped) values exceeding 1 in at least one tested samples were observed for five out of eight CeCOL genes, i.e., CeCLO1 – 3 , -6 , and − 7 . Among them, CeCLO3 , -6 , and − 7 were constitutively expressed, whereas CeCLO1 and − 2 were predominantly expressed in tubers, especially in middle and mature tubers. In accordance with the high oil accumulation, total CeCLO transcripts were most abundant in middle and mature tubers, which were clustered together and whose transcripts were 12.41 and 13.90 folds more than those in young tubers. Followed were roots, young tubers, shoot apexes, sheaths, and rhizomes, whose transcripts were 2.20 to 5.98 folds more than those in mature and young leaves (Fig. 5 a). When CeOLE genes were included for comparison, three tuber stages were clustered together; two most abundant genes CeOLE2 and CeOLE5 formed one group; CeCLO1 was clustered with CeOLE4 , CeOLE6 , and CeOLE1 ; CeCLO2 was clustered with CeOLE3 ; and CeCLO3 / -6 / -7 and CeCLO4 / -5 / -8 formed two independent groups ( Additional file 6a ). Correspondingly, correlation analysis showed that CeCLO1 transcripts were positively related with those of CeOLE genes, with the r value varying from 0.458 (i.e., CeOLE3 ) to 0.913 (i.e., CeOLE2 ). Expression of CeCLO1 was also positively correlated with CeCLO3 ( r = 0.917), which exhibited the r values of 0.390 (i.e., CeOLE1 )–0.843 (i.e., CeOLE2 ) ( Additional file 6b ). Comparison revealed apparent expression divergence of caleosin genes between tigernut and purple nutsedge Our previous study showed that oleosin genes between tigernut and purple nutsedge underwent apparent expression divergence during tuber development, and species-specific activation is in accordance with high oil accumulation in tigernut tubers [ 45 ]. To address whether similar cases are also present for CeCLO genes, their expression profiles over three representative tuber stages were compared, i.e., 20, 50, and 90 days after tuber initiation (DAI). As shown in Fig. 5 b, expression was detected for five CeCLO and four CrCLO genes, i.e., CeCLO1 , CeCLO2 , CeCLO3 , CeCLO6 , CeCLO7 , CrCLO1 , CrCLO3 , CrCLO6 , and CrCLO7 . Among them, CeCLO1 was mostly expressed, followed by CeCLO3 and CrCLO6 , whereas CrCLO3 was lowly expressed. Notably, though most CeCLO genes were expressed more than their orthologs in purple nutsedge, CrCLO6 represents the single one that was expressed more than its ortholog CeCLO6 (Fig. 5 b). The result implies that CeCLO1 , -2 , -3 , and − 7 may contribute to tuber oil accumulation in tigernut. CeCLO transcripts were positively correlated with TAG accumulation during tuber development According to our previous study, the TAG content of Reyan3 gradually increased along with tuber development, from 5.09% at 5 DAI to 34.29% at 35 DAI when tubers have completely matured [ 45 ]. To uncover the correlation between gene expression and TAG accumulation during tuber development, five tuber-expressed CeCLO genes (i.e., CeCLO1 , -2 , - 3 , -6 , and − 7 ) and five representative stages were selected for qRT-PCR analysis, i.e., S1 (5 DAI), S2 (10 DAI), S3 (20 DAI), S4 (25 DAI), and S5 (35 DAI). As shown in Fig. 6 , gradual promotion of transcripts during tuber development was observed for CeCLO1 , -2 , - 3 , and − 7 , whereas CeCLO6 was highly expressed in three early stages, followed by significant downregulation at two latter stages. Further correlation analysis showed that the transcripts of CeCLO1 , -2 , - 3 , and − 7 were positively correlated with TAG accumulation, with the r value varying from 0.837 (i.e., CeCLO1 ) to 0.964 (i.e., CeCLO2 ), whereas a negative correlation with r = 0.933 was observed for CeCLO6 (Fig. 6 ), supporting the result obtained from above transcriptional profiling. Discussion As a rare example accumulating high levels of oil in the nutrient storage tubers, tigernut is emerging as an ideal model to study lipid regulation mechanisms beyond oilseeds [ 36 , 42 , 45 , 46 , 47 , 48 , 49 ]. Like oleosins, caleosins also feature the 12-residue proline-knot motif, a key component of the family-specific caleosin domain [ 11 , 29 , 54 ]. A crucial role of caleosins in LD formation and oil accumulation prompted us to study this special gene family in tigernut. In the current study, the first genome-wide identification and characterization of the CeCLO gene family was reported. A number of eight family members identified from the tigernut genome are considerably less than 21 transcripts obtained from a de novo transcriptome assembly [ 52 ], implying the necessity of a genome-based identification. Interestingly, the family amounts present in this species are equal to those described in two model plants arabidopsis and rice [ 15 , 27 ], but considerably more than two found in A. trichopoda , castor bean, flax, and cucumber ( Cucumis sativus ) [ 11 , 29 , 30 ], implying lineage and/or species-specific family expansion during angiosperm radiation. Further phylogenetic analysis assigned eight CeCLO genes into two evolutionary groups as defined in arabidopsis [ 15 ], i.e., H (5) and L (3), where the H clade features a high MW with the so-called H insertion at the N-terminal (Fig. 1 d). Interestingly, the group composition observed in tigernut is highly distinct from rice (H, 3; L, 5) and arabidopsis (H, 4; L, 4), in contrast to the basal angiosperm A. trichopoda harboring a single member for each group [ 29 ]. Extensive expansion in tigernut was shown to be resulted from WGD, tandem, and dispersed duplications (Fig. 1 e). By contrast, expansion in rice was contributed by WGD, tandem, proximal, and dispersed duplications, whereas in arabidopsis, expansion was contributed by WGD, tandem, transposed, and dispersed duplications ( Additional file 5 ). The pattern is also different from the CeOLE family, whose expansion was shown to be contributed by WGD and dispersed duplication [ 45 ]. The results reflect the occurrence of more than one WGD in these species after monocot-eudicot divergence. According to comparative genomics analyses, arabidopsis in the eudicot clade experienced three additional WGDs known as γ, β, and α in sequence [ 55 ]; similarly, rice in the monocot clade experienced three additional WGDs known as τ, σ, and ρ, where τ and σ WGDs are shared by tigernut [ 48 , 56 , 57 , 58 ]. Correspondingly, four WGD repeats identified in arabidopsis, i.e., AtCLO1 / -2 and AtCLO5 / -6 , were shown to arise from the α WGD that is specific to the Brassicaceae family [ 15 , 29 ], whereas OsCLO1 and − 6 were derived from the ρ WGD that is specific to the Poaceae family ( Additional file 5 ). By contrast, considering the absence of a recent WGD in tigernut, two identified WGD repeats, i.e., CeCLO1 and − 2 , are more likely to arise from the σ WGD that is shared by all Poales plants [ 56 ]. To confirm the hypothesis and gain insights into the origin and evolution of CeCLO genes, approximately 200 homologs from 31 representative plant species were included for comparison, which resulted in 11 orthogroups named H1, H2, H3, H4, H5, L1, L2, L3, L4, L5, and L6. These species cover a wide range of 17 plant families, i.e., Amborellaceae ( A. trichopoda ), Vitaceae (grapevine), Euphorbiaceae (castor bean), Brassicaceae (arabidopsis), Acoraceae (American sweet flag), Zosteraceae (eelgrass), Araceae (duckweed), Asparagaceae (garden asparagus), Orchidaceae (apostasia), Dioscoreaceae (greater yam), Arecaceae (oil palm), Bromeliaceae (pineapple), Typhaceae ( Sparganium stoloniferum ), Cyperaceae (tigernut, purple nutsedge, C. iria , C. fuscus , Bolboschoenus planiculmis , Schoenoplectus tabernaemontani , C. littledalei , R. breviuscula , and Eleocharis parvula ), Juncaceae ( Juncus effusus and J. inflexus ), Joinvilleaceae ( J. ascendens ), and Poaceae ( Pharus latifolius , rice, barley, Brachypodium distachyon , Sorghum bicolor , and Setaria italica ). In accordance with previous studies [ 11 , 29 ], our data suggest that the caleosin family has diverged into Clades H and L in the last common ancestor of angiosperms, which is reserved as a single copy for each clade in some eudicots such as grapevine and castor bean. By contrast, extensive expansion via both local and large-scale duplications was frequently observed in Poales, and CeCLO genes belong to eight orthogroups, i.e., H1–5 and L1–3. Based on the results from orthologous, syntenic, and evolutionary rate analyses, a possible evolution route in tigernut is proposed as follows: the wide present H4 (e.g., CeCLO4 ) first gave birth to H2 (e.g., CeCLO2 ) via dispersed duplication sometime before monocot radiation, which may be along with the τ WGD followed by gene transposition; then, H2 produced H1 (e.g., CeCLO1 ) via the σ WGD in Poales, though H1 is most conserved; the last common ancestor of Cyperaceae and Juncaceae generated H3 (e.g., CeCLO3 ) via tandem duplication (unequal crossover) sometime after the split with other families within Poales; in Cyperaceae, L1 (e.g., CeCLO6 ) first brought forth L2 (e.g., CeCLO7 ) via tandem duplication sometime after the split with Juncaceae, which further gained an additional intron; subsequently, L2 produced L3 (e.g., CeCLO8 ) via tandem duplication; in Cyperus , the last common ancestor of tigernut, purple nutsedge, and C. iria generated H5 (e.g., CeCLO5 ) via tandem duplication sometime after the split with C. fuscus . Interestingly, despite a very close genetic relationship of tigernut and purple nutsedge, purple nutsedge only accumulates less than 2.5% of oil in its tubers, in contrast to up to 35% in tigernut [ 36 , 42 , 45 ]. 1:1 orthologous relationships of caleosin genes observed between these two species suggest that all duplicate pairs identified in tigernut are lineage-specific and gene copies are not the contributor of high oil accumulation in tigernut tubers. Similar results were also reported for the oleosin family, which is composed of six members in both tigernut and purple nutsedge [ 45 ]. Compared with tuber-preferential expression of most CeOLE genes [ 45 ], eight CeCLO genes was shown to exhibit distinct expression patterns with constitutive expression of CeCLO3 , -6 , and − 7 , tuber-predominant expression of CeCLO1 and − 2 , and rare expression of CeCLO4 , -5 , and − 8 (Fig. 5 a). Moreover, though the CeCLO family contains two more members than the CeOLE family, their total transcripts in tubers were shown to be considerably less abundant ( Additional file 6a ). Nevertheless, the transcript level of CeCLO1 ranks only after CeOLE5 and CeOLE2 , two most abundant CeOLE genes in tigernut tubers ( Additional file 6a ). A putative role of CeCLO1 and − 3 in tuber oil accumulation was first inferred from Pearson's correlation analysis, which was shown to exhibit the high r value up to 0.9 with CeOLE2 ( Additional file 6b ). Correspondingly, our qRT-PCR analysis showed that transcription of CeCLO1 , -2 , -3 , and − 7 in five representative tuber stages was positively correlated with oil accumulation during tuber development (Fig. 6 ). Last but most important, our comparative transcriptome analysis revealed that CeCLO1 , -2 , -3 , and − 7 were expressed considerably more than their orthologs in purple nutsedge (Fig. 5 b), implying species but not tuber-specific activation for oil accumulation. As discussed above, unlike the oleosin family, tandem duplication was shown to play a crucial in the expansion of the caleosin family [ 15 , 27 , 29 ]. Though this duplication mode has been proven to play a key role in environmental adaptation of plants [ 59 – 61 ], an amazing result is that most tandem repeats identified in tigernut, e.g., CeCLO4 , -5 , and − 8 , were not or rarely expressed in all tissues examined in this study. Similar results were also reported for plasma membrane intrinsic protein (PIP), stearoyl-acyl carrier protein desaturase (SAD), and fatty acid desaturase 2 (FAD2) (sub)families [ 47 , 48 , 62 ]. Thereby, further clarifying their biological significance in tigernut is of particular interest. Conclusions This study presents the first genome-wide analysis of the caleosin family in tigernut, a unique oil-rich tuber plant in Cyperaceae. Eight family members representing two clades identified from the tigernut genome are considerably more than two present in the basal angiosperm A. trichopoda . Comparison of 193 members from 31 representative plant species reveals lineage-specific family expansion and functional diversification in tigernut, whose expansion was contributed by WGD, tandem, and dispersed duplications. Comparison between tigernut and purple nutsedge suggests that species-specific activation of certain members but not gene copies is the main contributor of high oil accumulation in tigernut tubers. These findings provide valuable information for further functional analysis and genetic improvement in tigernut and species beyond. Materials and methods Plant materials Tigernut plants of Cyperus esculentus var. sativus Boeckeler (NCBI:txid1504802) variety Reyan3, which was mainly bred by Zhi Zou, were cultivated in sandy soil as described before [ 57 ]. Tubers of various developmental stages were collected at 5, 10, 20, 25, and 35 DAI, and three biological replicates were harvested for each stage. All samples were first freezed with liquid nitrogen and then stored at 80℃for further uses. Datasets and identification of caleosin family genes Genomic and transcriptome data of C. esculentus and representative plant species were accessed from CNGBdb ( https://db.cngb.org/ ), RGAP7 ( http://rice.plantbiology.msu.edu/ ), TAIR11 ( https://www.arabidopsis.org/ ), Phytozome v13 ( https://phytozome.jgi.doe.gov/pz/portal.html ), and NCBI ( https://www.ncbi.nlm.nih.gov/ ): C. esculentus (v1; Cyperaceae, Poales), C. rotundus (v1; Cyperaceae, Poales), C. iria (v1; Cyperaceae, Poales), C. fuscus (v1; Cyperaceae, Poales), B. planiculmis (v1; Cyperaceae, Poales),, S. tabernaemontani (v1; Cyperaceae, Poales), C. littledalei (v1; Cyperaceae, Poales), R. breviuscula (v1; Cyperaceae, Poales), E. parvula (v1; Cyperaceae, Poales), J. effusus (v1; Juncaceae, Poales), J. inflexus (v1; Juncaceae, Poales), Sparganium stoloniferum (v1; Typhaceae, Poales), A. comosus (v3; Bromeliaceae, Poales), J. ascendens (v1.1; Joinvilleaceae, Poales), P. latifolius (v1; Poaceae, Poales), O. sativa (RGAP7; Poaceae, Poales), B. distachyon (v3.2; Poaceae, Poales), H. vulgare (v1; Poaceae, Poales), S. bicolor (5.1; Poaceae, Poales), S. italica (v2.2; Poaceae, Poales), E. guineensis (EG5; Arecaceae, Arecales), D. alata (v2.1; Dioscoreaceae, Dioscoreales), A. officinalis (v1.1; Asparagaceae, Asparagales), D. catenatum (v1; Orchidaceae, Asparagales), Spirodela polyrhiza (v2; Lemnaceae, Alismatales), Z. marina (v3.1; Zosteraceae, Alismatales), A. americanus (v1; Acoraceae, Acorales), A. thaliana (Araport11; Brassicaceae, Brassicales), R. communis (WT05; Euphorbiaceae, Malpighiales), V. vinifera (T2T; Vitaceae , Vitales ), and A. trichopoda (v2.1; Amborellaceae, Amborellales). Caleosin homologs were identified via HMMER (v3.3, http://hmmer.janelia.org/ ) searching with the Pfam profile PF05042 (v35.0, https://pfam.xfam.org/ ). Gene models of all candidates were curated with available mRNAs as previously described [ 63 ], and gene structure display was performed using GSDS2.0 [ 64 ]. Presence of the conserved caleosin domain in deduced proteins was confirmed using Pfam Search. Sequence alignment and phylogenetic analysis Multiple sequence alignment was conducted using Muscle (v5) [ 65 ], and sequence alignment display was carried out using SMS (v1, http://www.bioinformatics.org/sms/ ). Phylogenetic tree construction was performed using RAxML (v8) [ 65 ] with the maximum likelihood method and bootstrap of 1000 replicates. Systematic names of caleosin family genes were assigned with two italic letters denoting the source organism and a progressive number based on sequence similarity. To distinguish with caleosin genes in arabidopsis, three italic letters were used in A. trichopoda , i.e., AtrCLO1 and − 2 . Protein properties and conserved motif analysis Physical and chemical parameters (e.g., MW, pI, and GRAVY) of deduced proteins were calculated using ProtParam (v1, http://web.expasy.org/protparam/ ), whereas hydropathicity scales were determined using ProtScale (v1, https://web.expasy.org/protscale/ ). Protein secondary structures were determined using SOPMA (v1, https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html ), while conserved motifs were identified using MEME (v5.4.1, https://meme-suite.org/tools/meme ) with the parameters as follows: any number of repetitions; the maximum number of motifs, 10; and, the optimum width of each motif, between 5 and 200 residues. Chromosomal localization, synteny analysis, definition of orthogroups, and calculation of evolutionary rate Synteny analysis was conducted as previously described [ 67 ], where duplicate pairs were identified using the all-to-all BLASTp method with the E -value cutoff of 1e-10 and syntenic blocks were inferred using MCScanX [ 68 ] with at least five BLAST hits. Orthologs between different species were identified using OrthoFinder (v2.3.8) [ 69 ], whereas different modes of gene duplication were identified using the DupGen_finder pipeline [ 61 ]. Chromosomal localization and calculation of Ks and Ka values were performed using TBtools-II (v2.390) [ 70 ]. Gene expression analysis based on RNA-seq Global expression profiles of caleosin and oleosin genes in tigernut and yellow nutsedge were investigated by using transcriptome datasets that are under NCBI accession numbers of PRJNA703731 and PRJNA671562. The data are 150 bp paired-end reads with three biological replicates, which were derived from shoot apexes, young leaves, mature leaves, leaf sheaths, roots, rhizomes, and tubers of 40 DAS, 85 DAS, 120 DAS, 20 DAI, 50 DAI, and 90 DAI. Raw reads in the FASTQ format were obtained using fastq-dump (v1) [ 71 ], and quality control was carried out using Trimmomatic (v1) [ 72 ]. Read mapping was performed using HISAT (v2) [ 73 ], and relative gene expression level was presented as FPKM [ 74 ]. Gene expression analysis based on qRT-PCR Total RNA extraction, the integrity and concentration detection, synthesis of the first-strand cDNA, and qRT-PCR analysis were conducted as previously described [ 57 , 63 ]. Primers used are shown in Additional file 7 , where CeTIP41 and CeUCE2 are two reference genes as described before [ 37 ]. All qRT-PCR assays were conducted in triplicate for each biological sample, and relative gene abundance was estimated with the 2 −ΔΔCt method and statistical analysis was performed using SPSS Statistics 20 as described before [ 63 ]. Declarations Acknowledgements The authors appreciate those contributors who make the related genome and transcriptome data accessible in public databases. Authors’ contributions The study was conceived and directed by ZZ. All the experiments and analyses were directed by ZZ and carried out by ZZ, XC, HH, CL, XY, JH, and ZY. ZZ, JH, and ZY wrote the paper. All the authors read and approved the final manuscript. Funding This work was supported by the Hainan Province Science and Technology Special Fund (ZDYF2024XDNY171 and ZDYF2024XDNY156) and the National Natural Science Foundation of China (32460342 and 31971688). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials The datasets analyzed during the current study are available in the NCBI SRA repository (https://www.ncbi.nlm.nih.gov/sra/) under accession numbers of PRJNA703731 and PRJNA671562. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Huang AHC. Plant lipid droplets and their associated proteins: Potential for rapid advances. Plant Physiol. 2018;176(3):1894–918. doi:10.1104/pp.17.01677. Frandsen G, Müller-Uri F, Nielsen M, Mundy J, Skriver K. Novel plant Ca 2+ -binding protein expressed in response to abscisic acid and osmotic stress. J Biol Chem. 1996;271(1):343–8. doi:10.1074/jbc.271.1.343. Chen EC, Tai SS, Peng CC, Tzen JT. Identification of three novel unique proteins in seed oil bodies of sesame. Plant Cell Physiol. 1998;39(9):935–41. doi: 10.1093/oxfordjournals.pcp.a029457. 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Supplementary Files Supplementarymaterial.zip Supplementary Information The online version contains supplementary material available at https://doi.org/. Additional file 1 Percent similarity within and between CeCLOs, OsCLOs, and AtCLOs. ( At A. thaliana , Ce C. esculentus , CLO caleosin, Os O. sativa ) Additional file 2 Comparison of amino acid composition (a) and secondary structure (b) between CeCLOs and CeOLEs. Amino acid composition was calculated using ProtParam (v1), whereas secondary structure was determined using the SOPMA method. The heatmap was generated using the R package with both rows and columns clustered. Color scale represents log 2 transformed percentage, where red indicates high percentage and blue indicates low percentage. ( Ce C. esculentus , CLO caleosin, OLE oleosin) Additional file 3 Predicted phosphorylation sites of CeCLOs. Potential phosphorylation sites were determined using NetPhos (v3.1). ( Ce C. esculentus , CLO caleosin) Additional file 4 Detailed information of caleosin genes identified in this study. Additional file 5 Gene localization and duplication events detected in O. sativa and A. thaliana . Gene localization was conducted using TBtools-II (v2.210), whereas different modes of gene duplication were identified using the DupGen_finder pipeline. Serial numbers are indicated at the top of each chromosome, and the scale is in Mb. Duplicate pairs identified in this study are connected using lines in different colors, i.e., tandem (green), proximal (brown), transposed (blue grey), dispersed (purple), and WGD (gold). ( At A. thaliana , CLO caleosin, Mb megabase, Os O. sativa , WGD Whole-genome duplication) Additional file 6 Tissue-specific expression profiling and correlation analysis of CeCLO and CeOLE genes. a Tissue-specific expression profiles of CeCLO and CeOLE genes. Young, middle, and mature represent tubers collected at 40, 85, and 120 DAS. The heatmaps were generated using the R package (v3.5.0), where color scale represents FPKM normalized log 2 transformed counts. Blue and red indicate low and high expression, respectively. b Pearson's correlation coefficient of CeCLO and CeOLE genes. ( Ce C. esculentus , CLO caleosin, Cr C. rotundus , DAS days after sowing, FPKM fragments per kilobase of exon per million fragments mapped, OLE oleosin) Additional file 7 Primers used in this study. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 28 Apr, 2026 Reviews received at journal 09 Mar, 2026 Reviewers agreed at journal 20 Feb, 2026 Reviewers invited by journal 11 Feb, 2026 Editor assigned by journal 21 Jan, 2026 Editor invited by journal 19 Jan, 2026 Submission checks completed at journal 17 Jan, 2026 First submitted to journal 17 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8567486\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":590184109,\"identity\":\"c829b602-d6a9-448b-aef8-641137f5025b\",\"order_by\":0,\"name\":\"Zhi 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University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jiaquan\",\"middleName\":\"\",\"lastName\":\"Huang\",\"suffix\":\"\"},{\"id\":590184137,\"identity\":\"d6b0b7c0-5e8a-43fe-b6ac-615e706122e9\",\"order_by\":6,\"name\":\"Yongguo Zhao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"College of Biology and Food Engineering, Guangdong University of Petrochemical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yongguo\",\"middleName\":\"\",\"lastName\":\"Zhao\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-01-10 10:08:38\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-8567486/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8567486/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":102779827,\"identity\":\"17be3d64-e5cb-4301-96ee-820171ea716a\",\"added_by\":\"auto\",\"created_at\":\"2026-02-16 14:52:34\",\"extension\":\"jpeg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":601732,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSequence and duplication event analysis of \\u003cem\\u003ecaleosin\\u003c/em\\u003e family genes in \\u003cem\\u003eC. esculentus\\u003c/em\\u003e. \\u003cstrong\\u003ea \\u003c/strong\\u003eKyte-Doolittle hydrophobicity plots of CeCLOs analyzed using ProtScale (v1).\\u003cstrong\\u003e b\\u003c/strong\\u003e Amino acid composition of CeCLOs calculated using ProtParam (v1). \\u003cstrong\\u003ec\\u003c/strong\\u003e Predicted secondary structure of CeCLOs using the SOPMA method. \\u003cstrong\\u003ed\\u003c/strong\\u003e Multiple sequence alignment of CeCLOs using MUSCLE (v5.1). Potential phosphorylation sites were determined using NetPhos (v3.1). Broken lines in the sequences represent gaps introduced for best alignment, whereas identical and similar amino acids are highlighted in black or dark grey, respectively. The SeqLogo of the caleosin domain generated using WebLogo (v3) is shown above the alignment, and the positions of the H-form insertion, an EF-hand calcium binding motif, the amphipathic α-helix, and the proline knot-like motif (PX\\u003csub\\u003e3-4\\u003c/sub\\u003ePSX\\u003csub\\u003e3\\u003c/sub\\u003eP) are boxed in black and are indicated at the bottom of the sequences. Two invariable His residues for heme-binding and four phosphorylation sites (one for Tyr kinase and three for CK II) are underlined and pointed by arrows. Potential phosphorylated residues in the C-terminal regions are boxed in red, whereas a conserved Cys residue prior to the last CK II phosphorylation site is boxed in purple.\\u003cstrong\\u003e e\\u003c/strong\\u003e Gene localization and duplication events detected in \\u003cem\\u003eC. esculentus\\u003c/em\\u003e. Gene localization was conducted using TBtools-II (v2.210), whereas different modes of gene duplication were identified using the DupGen_finder pipeline. Serial numbers are indicated at the top of each scaffold, and the scale is in Mb. Duplicate pairs identified in this study are connected using lines in different colors, i.e., tandem (green), dispersed (purple), and WGD (gold). (\\u003cem\\u003eCe\\u003c/em\\u003e \\u003cem\\u003eC. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCK II\\u003c/em\\u003e casein kinase II, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eMb\\u003c/em\\u003e megabase, \\u003cem\\u003eWGD\\u003c/em\\u003e Whole-genome duplication)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8567486/v1/925ed05a8f260da3428f8dbb.jpeg\"},{\"id\":102962799,\"identity\":\"01f5772a-d1b3-4c09-80f5-e3e830480c03\",\"added_by\":\"auto\",\"created_at\":\"2026-02-19 04:11:19\",\"extension\":\"jpeg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":338239,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eStructural and phylogenetic analysis of \\u003cem\\u003ecaleosin\\u003c/em\\u003e family genes in \\u003cem\\u003eC. esculentus\\u003c/em\\u003e, \\u003cem\\u003eO. sativa\\u003c/em\\u003e, and \\u003cem\\u003eA. thaliana\\u003c/em\\u003e.\\u003cstrong\\u003e a\\u003c/strong\\u003e Shown is an unrooted phylogenetic tree resulting from full-length caleosins with RAxML (maximum likelihood method and bootstrap of 1000 replicates), where the distance scale denotes the number of amino acid substitutions per site and the name of each clade (i.e., H and L) is indicated next to the corresponding group. \\u003cstrong\\u003eb\\u003c/strong\\u003e Shown are the exon-intron structures. “0”, “1”, and “2” indicate that an intron is located between codons, the first and second bases of a codon, and the second and third bases of a codon, respectively. \\u003cstrong\\u003ec\\u003c/strong\\u003e Shown is the distribution of conserved motifs identified using MEME, where different motifs are represented by different color blocks as indicated and the same color block in different proteins indicates a certain motif. \\u003cstrong\\u003ed\\u003c/strong\\u003e Shown is the detailed information of ten conserved motifs identified in this study. (\\u003cem\\u003eAt A. thaliana\\u003c/em\\u003e,\\u003cem\\u003e Ce C. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCLO \\u003c/em\\u003ecaleosin, \\u003cem\\u003eLD\\u003c/em\\u003e lipid droplet, \\u003cem\\u003ePXG \\u003c/em\\u003eperoxygenase, \\u003cem\\u003eOs\\u003c/em\\u003e \\u003cem\\u003eO. sativa\\u003c/em\\u003e)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8567486/v1/c3b0e232e20ca51ba354ec16.jpeg\"},{\"id\":103049608,\"identity\":\"7181263e-82c1-4674-953a-ea8c762f6f6a\",\"added_by\":\"auto\",\"created_at\":\"2026-02-20 07:43:42\",\"extension\":\"jpeg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":286947,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSpecies-specific distribution of 11 caleosinorthogroups in 31 representative plant species. The species tree is referred to NCBI Taxonomy (https://www.ncbi.nlm.nih.gov/taxonomy) and well-established recent WGDs are marked, which include the γ WGT shared by all core eudicots, the β WGD shared by the majority of Brassicales plants, the α WGD specific to Brassicaceae plants, the τ WGD shared by all core monocots, the σ WGD specific to Poales plants, the ρ WGD specific to Poaceae plants, and the p WGD specific to Arecaceae plants. Names of tested plant families are indicated next to the corresponding branches. (\\u003cem\\u003eH\\u003c/em\\u003ehigh molecular weight, \\u003cem\\u003eL\\u003c/em\\u003e low molecular weight, \\u003cem\\u003eWGD\\u003c/em\\u003e whole-genome duplication, \\u003cem\\u003eWGT\\u003c/em\\u003e whole-genome triplication)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8567486/v1/f1c4682507af4f3b64c5456b.jpeg\"},{\"id\":102962793,\"identity\":\"6613b0d3-c4db-4f45-914f-c301f76880ca\",\"added_by\":\"auto\",\"created_at\":\"2026-02-19 04:11:16\",\"extension\":\"jpeg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":568747,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSynteny analyses within and between \\u003cem\\u003eC. esculentus\\u003c/em\\u003e and representative plant species.\\u003cstrong\\u003e a\\u003c/strong\\u003e Synteny analysis within and between \\u003cem\\u003eC. esculentus\\u003c/em\\u003e, \\u003cem\\u003eC. rotundus\\u003c/em\\u003e, \\u003cem\\u003eC. iria\\u003c/em\\u003e,\\u003cem\\u003e C. littledalei\\u003c/em\\u003e, and\\u003cem\\u003e R. breviuscula\\u003c/em\\u003e. \\u003cstrong\\u003eb\\u003c/strong\\u003e Synteny analysis within and between \\u003cem\\u003eC. esculentus\\u003c/em\\u003e, \\u003cem\\u003eJ. effusus\\u003c/em\\u003e, \\u003cem\\u003eJ. ascendens\\u003c/em\\u003e, \\u003cem\\u003eA. comosus\\u003c/em\\u003e, and \\u003cem\\u003eS. stoloniferum\\u003c/em\\u003e. \\u003cstrong\\u003ec\\u003c/strong\\u003e Synteny analysis within and between \\u003cem\\u003eC. esculentus\\u003c/em\\u003e, \\u003cem\\u003eE. guineensis\\u003c/em\\u003e, \\u003cem\\u003eD. alata\\u003c/em\\u003e, and \\u003cem\\u003eA. officinalis\\u003c/em\\u003e. \\u003cstrong\\u003ed\\u003c/strong\\u003e Synteny analysis within and between \\u003cem\\u003eC. esculentus\\u003c/em\\u003e, \\u003cem\\u003eO. sativa\\u003c/em\\u003e, \\u003cem\\u003eA. americanus\\u003c/em\\u003e, \\u003cem\\u003eA. thaliana\\u003c/em\\u003e, and \\u003cem\\u003eA. trichopoda\\u003c/em\\u003e. Syntenic blocks were inferred using MCScanX (\\u003cem\\u003eE\\u003c/em\\u003e-value≤1e−10; BLAST hits≥5). Shown are \\u003cem\\u003ecaleosin \\u003c/em\\u003egene\\u003cem\\u003e-\\u003c/em\\u003eencoding chromosomes/scaffolds and only syntenic blocks containing \\u003cem\\u003ecaleosin \\u003c/em\\u003egenes are marked, where red and purple lines indicate intra- and inter-species, respectively. The scale is in Mb. (\\u003cem\\u003eAa A. americanus\\u003c/em\\u003e, \\u003cem\\u003eAc A. comosus\\u003c/em\\u003e, \\u003cem\\u003eAo A. officinalis\\u003c/em\\u003e,\\u003cem\\u003e At A. thaliana\\u003c/em\\u003e, \\u003cem\\u003eAtr A. trichopoda\\u003c/em\\u003e, \\u003cem\\u003eCe C. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCi C. iria\\u003c/em\\u003e,\\u003cem\\u003e Cl C. littledalei\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eCr C. rotundus\\u003c/em\\u003e, \\u003cem\\u003eDa D. alata\\u003c/em\\u003e, \\u003cem\\u003eEg E. guineensis\\u003c/em\\u003e, \\u003cem\\u003eJa J. ascendens\\u003c/em\\u003e, \\u003cem\\u003eJe J. effusus\\u003c/em\\u003e, \\u003cem\\u003eOs O. sativa\\u003c/em\\u003e, \\u003cem\\u003eR. breviuscula\\u003c/em\\u003e, \\u003cem\\u003eSs S. stoloniferum\\u003c/em\\u003e)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8567486/v1/574174a25589720d593feb45.jpeg\"},{\"id\":102962636,\"identity\":\"d3844c33-e5e7-4d39-baeb-5774dc9a4f6e\",\"added_by\":\"auto\",\"created_at\":\"2026-02-19 04:10:13\",\"extension\":\"jpeg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":172627,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExpression profiles of \\u003cem\\u003eCe/CrCLO\\u003c/em\\u003e genes. \\u003cstrong\\u003ea\\u003c/strong\\u003e Tissue-specific expression profiles of \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes. Young, middle, and mature represent tubers collected at 40, 85, and 120 DAS. \\u003cstrong\\u003eb\\u003c/strong\\u003e Expression profiles of \\u003cem\\u003eCe/CrOLE\\u003c/em\\u003e genes in three representative stages of tuber development. The heatmaps were generated using the R package (v3.5.0), where color scale represents FPKM normalized log\\u003csub\\u003e2\\u003c/sub\\u003e transformed counts. Blue and red indicate low and high expression, respectively. (\\u003cem\\u003eCe C. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eCr C. rotundus\\u003c/em\\u003e, \\u003cem\\u003eDAS\\u003c/em\\u003e days after sowing, \\u003cem\\u003eFPKM\\u003c/em\\u003e fragments per kilobase of exon per million fragments mapped)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8567486/v1/430f0b406fbda5cf3c3261fe.jpeg\"},{\"id\":102779829,\"identity\":\"99ee3f50-a341-4480-9651-b5b3ea0db21f\",\"added_by\":\"auto\",\"created_at\":\"2026-02-16 14:52:34\",\"extension\":\"jpeg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":100134,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExpression profiles of five \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes during tuber development. S1–5 represent tubers collected at 5, 10, 20, 25, and 35 DAI. Bars indicate SD (N=3) and lowercase letters indicate difference significance tested following Duncan's one-way multiple-range post hoc ANOVA (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.05). \\u003cem\\u003er\\u003c/em\\u003e represents the Pearson's correlation coefficient. (\\u003cem\\u003eCe C. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eDAI\\u003c/em\\u003e days after tuber initiation)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8567486/v1/c998c9fe7b3fb32d08df4219.jpeg\"},{\"id\":103503843,\"identity\":\"a25700c4-d006-45db-9601-2bb739d3cb70\",\"added_by\":\"auto\",\"created_at\":\"2026-02-26 13:03:07\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3677931,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8567486/v1/471a4825-8180-4358-948a-3ab00d68d8d8.pdf\"},{\"id\":102779831,\"identity\":\"d602d52c-64dd-4f4e-ac6c-007fd8091569\",\"added_by\":\"auto\",\"created_at\":\"2026-02-16 14:52:34\",\"extension\":\"zip\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":2278193,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSupplementary Information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe online version contains supplementary material available at https://doi.org/.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional file 1\\u003c/strong\\u003e Percent similarity within and between CeCLOs, OsCLOs, and AtCLOs. (\\u003cem\\u003eAt\\u003c/em\\u003e \\u003cem\\u003eA. thaliana\\u003c/em\\u003e, \\u003cem\\u003eCe\\u003c/em\\u003e \\u003cem\\u003eC. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eOs O. sativa\\u003c/em\\u003e)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional file 2\\u003c/strong\\u003e Comparison of amino acid composition (a) and secondary structure (b) between CeCLOs and CeOLEs. Amino acid composition was calculated using ProtParam (v1), whereas secondary structure was determined using the SOPMA method. The heatmap was generated using the R package with both rows and columns clustered. Color scale represents log\\u003csub\\u003e2\\u003c/sub\\u003e transformed percentage, where red indicates high percentage and blue indicates low percentage. (\\u003cem\\u003eCe\\u003c/em\\u003e \\u003cem\\u003eC. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eOLE\\u003c/em\\u003e oleosin)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional file 3\\u003c/strong\\u003e Predicted phosphorylation sites of CeCLOs. Potential phosphorylation sites were determined using NetPhos (v3.1). (\\u003cem\\u003eCe\\u003c/em\\u003e \\u003cem\\u003eC. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional file 4\\u003c/strong\\u003e Detailed information of \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes identified in this study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional file 5\\u003c/strong\\u003e Gene localization and duplication events detected in \\u003cem\\u003eO. sativa\\u003c/em\\u003e and \\u003cem\\u003eA. thaliana\\u003c/em\\u003e. Gene localization was conducted using TBtools-II (v2.210), whereas different modes of gene duplication were identified using the DupGen_finder pipeline. Serial numbers are indicated at the top of each chromosome, and the scale is in Mb. Duplicate pairs identified in this study are connected using lines in different colors, i.e., tandem (green), proximal (brown), transposed (blue grey), dispersed (purple), and WGD (gold). (\\u003cem\\u003eAt\\u003c/em\\u003e \\u003cem\\u003eA. thaliana\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eMb\\u003c/em\\u003e megabase, \\u003cem\\u003eOs\\u003c/em\\u003e \\u003cem\\u003eO. sativa\\u003c/em\\u003e, \\u003cem\\u003eWGD\\u003c/em\\u003e Whole-genome duplication)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional file 6\\u003c/strong\\u003e Tissue-specific expression profiling and correlation analysis of \\u003cem\\u003eCeCLO\\u003c/em\\u003e and \\u003cem\\u003eCeOLE\\u003c/em\\u003e genes. \\u003cstrong\\u003ea\\u003c/strong\\u003e Tissue-specific expression profiles of \\u003cem\\u003eCeCLO\\u003c/em\\u003e and \\u003cem\\u003eCeOLE\\u003c/em\\u003e genes. Young, middle, and mature represent tubers collected at 40, 85, and 120 DAS. The heatmaps were generated using the R package (v3.5.0), where color scale represents FPKM normalized log\\u003csub\\u003e2\\u003c/sub\\u003e transformed counts. Blue and red indicate low and high expression, respectively. \\u003cstrong\\u003eb\\u003c/strong\\u003e Pearson's correlation coefficient of \\u003cem\\u003eCeCLO\\u003c/em\\u003e and \\u003cem\\u003eCeOLE\\u003c/em\\u003e genes. (\\u003cem\\u003eCe C. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eCr C. rotundus\\u003c/em\\u003e, \\u003cem\\u003eDAS\\u003c/em\\u003e days after sowing, \\u003cem\\u003eFPKM\\u003c/em\\u003e fragments per kilobase of exon per million fragments mapped, \\u003cem\\u003eOLE\\u003c/em\\u003e oleosin)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional file 7\\u003c/strong\\u003e Primers used in this study.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Supplementarymaterial.zip\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8567486/v1/1a664f1cd6c6f2024361de02.zip\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Insights into the caleosin family in Cyperus esculentus, an oil-rich tuber plant in Cyperaceae\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003ePlant seeds store triacylglycerols (TAGs) in discrete spherical organelles called oil bodies or lipid droplets (LDs), which are coated by a monolayer of phospholipids embedded with abundant oleosins (OLEs) and several minor structural proteins [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. Caleosins (CLOs) are a class of minor LD proteins that are capable of binding to calcium ion (Ca\\u003csup\\u003e2+\\u003c/sup\\u003e) [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Since the first \\u003cem\\u003ecaleosin\\u003c/em\\u003e gene (i.e., \\u003cem\\u003eresponsive to desiccation 20\\u003c/em\\u003e (\\u003cem\\u003eRD20\\u003c/em\\u003e)) that was identified in arabidopsis (\\u003cem\\u003eArabidopsis thaliana\\u003c/em\\u003e) [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], its homologs have been widely found in land plants as well as in some fungi and green algae [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Caleosins exhibit some similarities to oleosins, which feature a long central hydrophobic hairpin of about 72 residues (X\\u003csub\\u003e30\\u003c/sub\\u003ePX\\u003csub\\u003e5\\u003c/sub\\u003eSPX\\u003csub\\u003e3\\u003c/sub\\u003ePX\\u003csub\\u003e30\\u003c/sub\\u003e) [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. Like oleosins, caleosins are composed of three characteristic structural domains, i.e., an N-terminal hydrophilic EF-hand calcium binding domain, a central hydrophobic anchoring domain, and a C-terminal hydrophilic phosphorylation domain [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. The central hydrophobic domain, which is responsible for anchorage in LDs, comprises an amphipathic α-helix followed by a pair of anti-parallel β-strands that are connected by a proline-knot motif (PX\\u003csub\\u003e3\\u003c/sub\\u003ePSX\\u003csub\\u003e3\\u003c/sub\\u003eP) [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. Both N- and C-terminals are more likely to expose to the cytoplasm and are involved in environmental adaptation [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Though caleosins were first recognized as minor proteins functioning in LD stabilization and degradation in seeds, they were later proven to have catalytic functions as peroxygenases (PXGs, EC 1.11.2.3), whose activity is dependent on a heme group coordinated by the two invariant His residues located in N- and C-terminal regions [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Hence, they are also called peroxygenases [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Unlike oleosins, caleosins have additional intracellular locations apart from LDs, e.g., the envelope of chloroplasts, endoplasmic reticulum (ER), plasmalemma, vacuole, and nucleus, and they were also found to be abundant in a range of non-seed plant tissues such as callus, shoot, hypocotyl, root, cotyledon, leaf, megagametophyte, male flower, pollen, stamen, ovule, silique, fruit, and phloem sap [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn land plants, caleosins comprise a small family with up to 10 members [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. Generally, there are two major types present in each species, i.e., high (H) and low (L) molecular weights (MW), where the H-forms contain an extra N-terminal insertion of about 30 residues relative to L-forms [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. In contrast to only two members present in castor bean (\\u003cem\\u003eRicinus communis\\u003c/em\\u003e, \\u003cem\\u003eRcCLO-1\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e2\\u003c/em\\u003e) and flax (\\u003cem\\u003eLinum usitatissimum\\u003c/em\\u003e, \\u003cem\\u003eLuCLO-1\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e2\\u003c/em\\u003e) [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e], a number of eight \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes were described in arabidopsis, including four H-forms (i.e., \\u003cem\\u003eAtCLO1\\u003c/em\\u003e, -\\u003cem\\u003e2\\u003c/em\\u003e, -\\u003cem\\u003e3\\u003c/em\\u003e, and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e8\\u003c/em\\u003e) and four L-forms (i.e., \\u003cem\\u003eAtCLO4\\u003c/em\\u003e, -\\u003cem\\u003e5\\u003c/em\\u003e, -\\u003cem\\u003e6\\u003c/em\\u003e, and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e7\\u003c/em\\u003e), and the family expansion was shown to be contributed by both local and segmental duplications [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. Expression analyses showed that \\u003cem\\u003eRcCLO-2\\u003c/em\\u003e and \\u003cem\\u003eLuCLO-2\\u003c/em\\u003e are preferentially expressed in seeds, whereas \\u003cem\\u003eRcCLO-1\\u003c/em\\u003e and \\u003cem\\u003eLuCLO-1\\u003c/em\\u003e are constitutively expressed, including seeds, cotyledons, and young leaves [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. Similar expression profiles were also observed for \\u003cem\\u003eAtCLO\\u003c/em\\u003e genes, where \\u003cem\\u003eAtCLO3\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e4\\u003c/em\\u003e are expressed in various organs and others seem to be organ-specific, e.g., \\u003cem\\u003eAtCLO1\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e2\\u003c/em\\u003e in seeds and \\u003cem\\u003eAtCLO5\\u003c/em\\u003e in buds [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. Functional analyses revealed that caleosins are not only involved in LD formation, stabilization, and degradation, but also are associated with various plant development and stress responses [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. In rice (\\u003cem\\u003eOryza sativa\\u003c/em\\u003e), \\u003cem\\u003eOsCLO5\\u003c/em\\u003e was initially named \\u003cem\\u003eOsEFA27\\u003c/em\\u003e (EF-hand, ABA responsive, 27 kilodalton (kDa)), which could be induced by ABA treatment, salt and drought stresses [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. In arabidopsis, four seed-expressed \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes (i.e., \\u003cem\\u003eAtCLO1\\u003c/em\\u003e, -\\u003cem\\u003e2\\u003c/em\\u003e, -\\u003cem\\u003e4\\u003c/em\\u003e, and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e6\\u003c/em\\u003e) were proven to redundantly function in oil accumulation and embryo development [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. Interestingly, a high number of \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes were shown to be regulated by various abiotic and biotic stresses, e.g., \\u003cem\\u003eOsCLO5\\u003c/em\\u003e/\\u003cem\\u003eEFA27\\u003c/em\\u003e in rice [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e], \\u003cem\\u003eAtCLO3\\u003c/em\\u003e/\\u003cem\\u003ePXG3\\u003c/em\\u003e/\\u003cem\\u003eRD20\\u003c/em\\u003e in arabidopsis [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], \\u003cem\\u003eSopl\\u003c/em\\u003e in sesame (\\u003cem\\u003eSesamum indicum\\u003c/em\\u003e) [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e], \\u003cem\\u003eHvCLO1\\u003c/em\\u003e and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/em\\u003e in barley (\\u003cem\\u003eHordeum vulgare\\u003c/em\\u003e) [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e], \\u003cem\\u003eBnCLO1-1\\u003c/em\\u003e in rapeseed (\\u003cem\\u003eBrassica napus\\u003c/em\\u003e) [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e], and \\u003cem\\u003eOeCLO3\\u003c/em\\u003e in olive (\\u003cem\\u003eOlea europaea\\u003c/em\\u003e) [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTigernut (\\u003cem\\u003eCyperus esculentus\\u003c/em\\u003e L. \\u003cem\\u003evar. sativus\\u003c/em\\u003e Boeckeler), which is also known as chufa and yellow nutsedge, is an unconventional oil crop uniquely accumulating significant amounts of TAGs in its underground tubers [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. It resides in the Cyperaceae family, which is native to Africa [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. Tigernut was initially domesticated in ancient Egypt for the nutrient-rich tubers, e.g., 25\\u0026ndash;45% starch, 20\\u0026ndash;35% oil, 15\\u0026ndash;20% sugars, 5\\u0026ndash;10% protein, 8\\u0026ndash;10% dietary fiber, 0.08\\u0026ndash;0.14 g/kg vitamin C and E, high levels of minerals (e.g., calcium (Ca), phosphorus (P), potassium (K), and magnesium (Mg)), and various active substances (e.g., flavone, alkaloid, tannin, terpenoid, saponin, and phytosterol) [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. Recently, much attention has been attracted for its unique feature of high oil accumulation in the non-seed storage organ [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e], and the disclosure of the mechanisms behind shall facilitate oil production in plant vegetative tissues via genetic engineering [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. According to a previous proteomic analysis, like oilseeds, the LDs of tigernut tubers are mainly coated by oleosins and caleosins [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. Thus far, genome-wide characterization of the \\u003cem\\u003eoleosin\\u003c/em\\u003e family was reported, and six members present in tigernut are equal to those found in other Cyperaceae plants such as purple nutsedge (\\u003cem\\u003eC. rotundus\\u003c/em\\u003e) and \\u003cem\\u003eRhynchospora breviuscula\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. Interestingly, the majority of \\u003cem\\u003eCeOLE\\u003c/em\\u003e genes were shown to exhibit a tuber-preferential expression pattern with mRNA/protein abundances positively correlating with oil accumulation during tuber development [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. Transcriptome-based identification of \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes was also reported [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e], however, 21 homologs described in that study are considerably more than eight detected from our full-length transcriptome, which was constructed via single-molecule real-time (SMRT) sequencing of various tissues such as shoot apex, leaf, sheath, root, rhizome, tuber, flower, and floral stem [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. Considering the inherent drawback of incorrect \\u003cem\\u003ede novo\\u003c/em\\u003e assembly from short reads, a genome-scale identification may provide a more precise view of the \\u003cem\\u003ecaleosin\\u003c/em\\u003e family in this special species.\\u003c/p\\u003e \\u003cp\\u003eIn this study, a genome-wide analysis of the \\u003cem\\u003eCeCLO\\u003c/em\\u003e family was conducted, which include sequence characteristics, gene structures, chromosome localizations, evolutionary relationships, and expression profiles. Significantly, comparison of 193 \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes identified from 31 representative plant species reveals lineage-specific expansion and functional diversification in tigernut. In contrast to only two members present in the basal angiosperm \\u003cem\\u003eAmborella trichopoda\\u003c/em\\u003e, a high number of eight \\u003cem\\u003eCeCLO\\u003c/em\\u003e family genes were shown to arise from whole-genome duplication (WGD), tandem, and dispersed duplications, whereas tuber-specific activation of certain members appears to be species-specific. These findings highlight the evolution patterns of the \\u003cem\\u003eCeCLO\\u003c/em\\u003e family, which provide valuable information for further functional analysis.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eIdentification, gene localization, and duplication event analysis of eight\\u003c/b\\u003e \\u003cb\\u003ecaleosin\\u003c/b\\u003e \\u003cb\\u003egenes in tigernut\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eAs shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, HMMER-based homologue search resulted in eight \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes from the tigernut genome [\\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. Though the overall sequence similarity of their deduced peptides varies from 44.32% to 78.57% (\\u003cb\\u003eAdditional file 1\\u003c/b\\u003e), all of them were shown to possess a single caleosin domain (under the Pfam accession number of PF05042) that is specific to this family (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Notably, the amounts of the \\u003cem\\u003eCeCLO\\u003c/em\\u003e family are somewhat bigger than six members as described for the \\u003cem\\u003eCeOLE\\u003c/em\\u003e family [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. Compared with CeOLEs with an average of approximately 150 residues [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e], the peptide length of CeCLOs is relatively longer, varying from 198 (i.e., CeCLO6) to 261 (i.e., CeCLO5) residues with an average of about 234 residues, which result in the higher MW of 21.82\\u0026ndash;29.93 kDa, i.e., 26.42 vs 15.52 kDa (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Interestingly, different patterns of isoelectric point (pI) and grand average of hydropathicity (GRAVY) were also observed between CeCLOs and CeOLEs as described in papaya (\\u003cem\\u003eCarica papaya\\u003c/em\\u003e) [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. Whereas only 62.50% of CeCLOs (i.e., CeCLO4\\u0026ndash;8) exhibit the alkaline feature like CeOLEs, others (i.e., CeCLO1\\u0026ndash;3) harbor the pI values of less than 7, varying from 5.91 (i.e., CeCLO2) to 6.63 (i.e., CeCLO3) (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). In contrast to the highly hydrophobic feature of CeOLEs [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e], all CeCLOs have the GRAVY value of less than 0, varying from \\u0026minus;\\u0026thinsp;0.593 to -0.117 (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The overall hydrophilic feature of CeCLOs was also supported by the ProtScale analysis. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea, similar hydropathicity scales were observed for all eight CeCLOs. Whereas the central domain is hydrophobic, N- and C-terminal regions are usually hydrophilic (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea). Except for CeCLO6, one to two transmembrane helixes (TMH) were identified (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCaleosin\\u003c/em\\u003e genes identified in \\u003cem\\u003eC. esculentus\\u003c/em\\u003e. (\\u003cem\\u003eAA\\u003c/em\\u003e amino acid, \\u003cem\\u003eCe C. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eGRAVY\\u003c/em\\u003e grand average of hydropathicity, \\u003cem\\u003ekDa\\u003c/em\\u003e kilodalton, \\u003cem\\u003eMW\\u003c/em\\u003e molecular weight, \\u003cem\\u003epI\\u003c/em\\u003e isoelectric point, \\u003cem\\u003eScf\\u003c/em\\u003e scaffold, \\u003cem\\u003eTMH\\u003c/em\\u003e transmembrane helix)\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"9\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eGene name\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eLocus ID\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003ePosition\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eAA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eMW (kDa)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003epI\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eGRAVY\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003eTMH\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003eCaleosin location\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO1\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCESC_00080\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eScf30:3622141..3623943(-)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e251\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e28.96\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e6.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-0.366\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e104..123\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e74..240\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO2\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCESC_16572\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eScf47:433600..435126(-)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e242\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e27.87\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e5.91\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-0.410\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e94..117\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e63..231\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO3\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCESC_16571\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eScf47:436090..438271(-)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e239\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e27.38\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e6.63\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-0.228\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e88..107,184\\u0026ndash;201\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e59..225\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO4\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCESC_18483\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eScf13:1911499..1913058(-)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e244\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e27.63\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e9.04\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-0.293\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e88..110,184..201\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e59..226\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO5\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCESC_18484\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eScf13:1915073..1916809(-)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e261\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e29.93\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e8.78\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-0.324\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e97..116\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e68..235\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO6\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCESC_02525\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eScf21:760234..762404(-)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e198\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e21.82\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e7.81\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-0.593\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e21..188\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO7\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCESC_02526\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eScf21:770229..771732(-)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e217\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e23.57\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e8.79\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-0.363\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e7..24,70..92\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e38..206\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO8\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCESC_02527\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eScf21:775539..780932(-)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e220\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e24.24\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e9.03\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-0.117\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e6..25\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e43..210\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe amino acid (AA) composition of CeCLOs was further calculated and compared with that of CeOLEs. Except for the absence of the Cys in CeCLO6\\u0026ndash;8, other members contain all 20 residues (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb), usually rich in Leu, Gly, and Ala as observed in CeOLEs (\\u003cb\\u003eAdditional file 2a\\u003c/b\\u003e). Interestingly, the cluster analysis revealed distinct patterns of AA composition between CeCLOs and CeOLEs, where proteins were grouped in family. Compared with CeOLEs, CeCLOs contain more hydrophilic but less hydrophobic residues (\\u003cb\\u003eAdditional file 2a\\u003c/b\\u003e), which contribute to their hydrophilic feature. The secondary structure of CeCLOs was also shown to differ from that of CeOLEs, where CeCLOs harbor more random coils but less alpha helixes and were clustered together (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec and \\u003cb\\u003eAdditional file 2b\\u003c/b\\u003e).\\u003c/p\\u003e \\u003cp\\u003eSequence alignment of CeCLOs is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed. Except for the conserved caleosin domain, N- and C-terminals are highly diverse. Though the usual proline-knot pattern PX\\u003csub\\u003e3\\u003c/sub\\u003ePSX\\u003csub\\u003e3\\u003c/sub\\u003eP was observed in CeCLO2\\u0026ndash;6, the PX\\u003csub\\u003e4\\u003c/sub\\u003eS/QSX\\u003csub\\u003e3\\u003c/sub\\u003eP variant was identified in CeCLO7 and \\u0026minus;\\u0026thinsp;8. Moreover, the PX\\u003csub\\u003e3\\u003c/sub\\u003eLNX\\u003csub\\u003e3\\u003c/sub\\u003eP variant was found in CeCLO1. Prior to the proline-knot are the amphipathic α-helix and the Ca\\u003csup\\u003e2+\\u003c/sup\\u003e binding motif. Two His residues, which are essential for the PXG activity [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e], were found in all CeCLOs, residing in front of the Ca\\u003csup\\u003e2+\\u003c/sup\\u003e binding motif and somewhat behind the proline-knot motif, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed). Compared with CeCLO6\\u0026ndash;8, CeCLO1\\u0026ndash;5 harbor an H-form insertion as observed in arabidopsis [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e], which contributes to the longer peptide size and higher MW (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed). As shown in \\u003cb\\u003eAdditional file 3\\u003c/b\\u003e, a high number of phosphorylation sites were identified for each sequence. Among them, four are shared by most sequences, i.e., one for Tyr kinase and three for casein kinase II (CK II) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed), implying their posttranslational regulation. Interestingly, despite few Cys residues present in CeCLOs, one is shared by CeCLO1\\u0026ndash;5 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed), which was also described for Sopl in sesame [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn contrast to the distribution of six \\u003cem\\u003eCeOLE\\u003c/em\\u003e genes over six scaffolds (Scfs) [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e], gene localization showed that eight \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes are unevenly spread across four scaffolds, and three hotspots for tandem duplication were detected, i.e., \\u003cem\\u003eCeCLO2\\u003c/em\\u003e/-\\u003cem\\u003e3\\u003c/em\\u003e on Scf47, \\u003cem\\u003eCeCLO4\\u003c/em\\u003e/-\\u003cem\\u003e5\\u003c/em\\u003e on Scf13, and \\u003cem\\u003eCeCLO6\\u003c/em\\u003e/-\\u003cem\\u003e7\\u003c/em\\u003e/-\\u003cem\\u003e8\\u003c/em\\u003e on Scf21, respectively. Further synteny analysis revealed that \\u003cem\\u003eCeCLO1\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e2\\u003c/em\\u003e are located within syntenic blocks, implying their derivation from WGD. On the contrary, \\u003cem\\u003eCeCLO2\\u003c/em\\u003e/-\\u003cem\\u003e4\\u003c/em\\u003e and \\u003cem\\u003eCeCLO4\\u003c/em\\u003e/-\\u003cem\\u003e6\\u003c/em\\u003e were characterized as dispersed repeats (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003ePhylogenetic and comparative analyses revealed species-specific expansion of the\\u003c/b\\u003e \\u003cb\\u003ecaleosin\\u003c/b\\u003e \\u003cb\\u003efamily among tigernut, rice, and arabidopsis\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTo uncover the evolutionary relationships of \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes, homologs were also identified from arabidopsis and rice, two model plants for dicots and monocots, respectively. In accordance with previous studies [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e], eight family members were obtained from updated genomes of both species (\\u003cb\\u003eAdditional file 4\\u003c/b\\u003e). Interestingly, despite harboring the same family amounts, phylogenetic analysis of caleosins in tigernut, rice, and arabidopsis revealed species-specific expansion. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea, these proteins were clustered into two main clades, corresponding to H and L as described in arabidopsis [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. Moreover, these two clades could be divided into two and three groups, respectively. Among them, Group Ia includes CeCLO1\\u0026ndash;3, OsCLO4, OsCLO5, AtCLO1\\u0026ndash;3, and AtCLO8; Group Ib includes CeCLO4, CeCLO5 and OsCLO3; Group IIa is specific to arabidopsis, including AtCLO4\\u0026ndash;7; Group IIb is specific to tigernut, including CeCLO6\\u0026ndash;8; and Group IIc is specific to rice, including OsCLO1\\u0026ndash;2, and OsCLO6\\u0026ndash;8. The results are largely in accordance with sequence similarities and duplication event analyses. As shown in \\u003cb\\u003eAdditional files 1\\u003c/b\\u003e and \\u003cb\\u003e5\\u003c/b\\u003e, \\u003cem\\u003eOsCLO1\\u003c/em\\u003e/-\\u003cem\\u003e2\\u003c/em\\u003e, \\u003cem\\u003eOsCLO4\\u003c/em\\u003e/-\\u003cem\\u003e5\\u003c/em\\u003e, and \\u003cem\\u003eOsCLO6\\u003c/em\\u003e/-\\u003cem\\u003e7\\u003c/em\\u003e, which share 72.65%, 67.48%, and 95.56% sequence similarities at the protein level, were defined as proximal repeats; \\u003cem\\u003eOsCLO7\\u003c/em\\u003e/-\\u003cem\\u003e8\\u003c/em\\u003e (53.41%), \\u003cem\\u003eAtCLO4\\u003c/em\\u003e/-\\u003cem\\u003e6\\u003c/em\\u003e (85.71%), and \\u003cem\\u003eAtCLO5\\u003c/em\\u003e/-\\u003cem\\u003e7\\u003c/em\\u003e (62.66%) were characterized as tandem repeats; \\u003cem\\u003eOsCLO1\\u003c/em\\u003e/-\\u003cem\\u003e6\\u003c/em\\u003e (57.33%), \\u003cem\\u003eAtCLO1\\u003c/em\\u003e/-\\u003cem\\u003e2\\u003c/em\\u003e (87.76%), and \\u003cem\\u003eAtCLO5\\u003c/em\\u003e/-\\u003cem\\u003e6\\u003c/em\\u003e (66.67%) were characterized as WGD repeats, respectively; \\u003cem\\u003eOsCLO3\\u003c/em\\u003e/-\\u003cem\\u003e4\\u003c/em\\u003e (54.10%), \\u003cem\\u003eOsCLO4\\u003c/em\\u003e/-\\u003cem\\u003e6\\u003c/em\\u003e (39.74%), \\u003cem\\u003eAtCLO1\\u003c/em\\u003e/-\\u003cem\\u003e3\\u003c/em\\u003e (72.47%), and \\u003cem\\u003eAtCLO1\\u003c/em\\u003e/-\\u003cem\\u003e4\\u003c/em\\u003e (47.35%) were characterized as dispersed repeats.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo learn more about structural variation, exon-intron structures and conserved motifs were further compared. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb, the most majority of \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes present in three species feature five introns with the conserved phase pattern of 1, 1, 0, 2, and 2. Nevertheless, gain or loss of certain introns was observed in \\u003cem\\u003eCeCLO7\\u003c/em\\u003e, \\u003cem\\u003eCeCLO8\\u003c/em\\u003e, \\u003cem\\u003eAtCLO7\\u003c/em\\u003e, and \\u003cem\\u003eAtCLO8\\u003c/em\\u003e. In contrast to the loss of the fifth intron in \\u003cem\\u003eAtCLO8\\u003c/em\\u003e, \\u003cem\\u003eCeCLO7\\u003c/em\\u003e and \\u003cem\\u003eCeCLO8\\u003c/em\\u003e were shown to have gained one more intron (in phase 2) at the 5' end, which is different from the phases of 2, 0, 0, 0, 2, and 2 as observed in \\u003cem\\u003eAtCLO7\\u003c/em\\u003e, implying independent origin. Ten motifs identified using MEME are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec. Whereas Motifs 1\\u0026ndash;5 are widely present, others appear to be sequence-specific. Motifs 1, 2, and 4 were characterized as the conserved caleosin domain; Motif 3 was characterized as the H-form insertion; and Motif 5 was characterized as the C-terminal phosphorylation sites (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed). Notably, Motifs 2 and 4 are absent from AtCLO7, and Motif 1 is absent from AtCLO7 and \\u0026minus;\\u0026thinsp;8, implying their divergence. Whereas Motif 3 was found in all H-forms, Motif 10, which was also characterized as the C-terminal of the H-form insertion, are limited to CeCLO1, CeCLO2, OsCLO4, and AtCLO2. Motifs 6 and 7, which are confined to OsCLO6 and \\u0026minus;\\u0026thinsp;7, are located at the N- and C-terminals, respectively. Motif 8, which is confined to AtCLO1 and \\u0026minus;\\u0026thinsp;2, is located at the C-terminal. Motif 9, which is confined to AtCLO5 and \\u0026minus;\\u0026thinsp;7, is located at the N-terminal (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eCharacterization of\\u003c/b\\u003e \\u003cb\\u003ecaleosin\\u003c/b\\u003e \\u003cb\\u003egenes from representative plant species and insights into lineage-specific family evolution in Poales\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eAbove phylogenetic analysis suggests the divergence of Clades H and L sometime before monocot-eudicot split. However, several issues need to be further resolved: 1) the tandem duplications identified in tigernut are species or lineage-specific; 2) the exact time of the WGD that gave birth to \\u003cem\\u003eCeCLO2\\u003c/em\\u003e; 3) the exact origin of \\u003cem\\u003eCeCLO4\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e6\\u003c/em\\u003e; 4) the exact time of gain of the additional intron as observed in \\u003cem\\u003eCeCLO7\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e8\\u003c/em\\u003e. For the purposes, homologs were also identified from representative plant species, which include the basal angiosperm \\u003cem\\u003eA. trichopoda\\u003c/em\\u003e (an Amborellaceae plant in Amborellales) and three early diverged monocots that didn't experience the τ WGD shared by core monocots, i.e., American sweet flag (\\u003cem\\u003eAcorus americanus\\u003c/em\\u003e, an Acoraceae plant in Acorales), eelgrass (\\u003cem\\u003eZostera marina\\u003c/em\\u003e, a Zosteraceae plant in Alismatales), and duckweed (\\u003cem\\u003eSpirodela polyrhiza\\u003c/em\\u003e, an Araceae plant in Alismatales). After discarding pseudogenes and gene fragments, as shown in \\u003cb\\u003eAdditional file 4\\u003c/b\\u003e, a total of 169 members were further identified. Notably, a single member for each clade was not only found in two out of three tested core eudicots, i.e., grapevine (\\u003cem\\u003eVitis vinifera\\u003c/em\\u003e) and castor bean, but also in \\u003cem\\u003eA. trichopoda\\u003c/em\\u003e and eelgrass, supporting early divergence of Clades H and L in the angiosperm ancestor. By contrast, equal amounts of eight members were identified in purple nutsedge, a close relative of tigernut (\\u003cb\\u003eAdditional file 4\\u003c/b\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTo infer lineage-specific gene expansion and contraction, species-specific duplication events were further examined and orthologs across different species were identified using Orthofinder. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, a total of 11 orthogroups were obtained and eight \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes belong to H1, H2, H3, H4, H5, L1, L2, and L3. Among them, H1 and L1 are shared by all species examined in this study, whereas others appear to be lineage-specific: H4 is widely present in core monocots, including garden asparagus (\\u003cem\\u003eAsparagus officinalis\\u003c/em\\u003e), apostasia (\\u003cem\\u003eDendrobium catenatum\\u003c/em\\u003e), and greater yam (\\u003cem\\u003eDioscorea alata\\u003c/em\\u003e), implying its early origin that may arise from the τ WGD; H2 is present in most Poales plants and is usually located within syntenic blocks with H1, implying its possible origin along with the Poales-specific σ WGD; H3, which is limited to Cyperaceae and Juncaceae species, was characterized as the tandem repeat of H2; H5 is confined to three out of four examined \\u003cem\\u003eCyperus\\u003c/em\\u003e species, i.e., tigernut, purple nutsedge, and \\u003cem\\u003eC. iria\\u003c/em\\u003e, where further expansion via WGD was also observed in the latter; L2, which is widely present in Cyperaceae plants with the exception of \\u003cem\\u003eC. littledalei\\u003c/em\\u003e, was characterized as the tandem repeat of L1; L3, which is also Cyperaceae-specific, was characterized as the tandem repeat of L2 in tigernut, purple nutsedge, \\u003cem\\u003eC. iria\\u003c/em\\u003e, \\u003cem\\u003eC. fuscus\\u003c/em\\u003e, and \\u003cem\\u003eR. breviuscula\\u003c/em\\u003e, but the tandem repeat of L1 in \\u003cem\\u003eC. littledalei\\u003c/em\\u003e; L4 and L5, which is Poaceae-specific, was characterized as the tandem and WGD (ρ) repeats of L1, respectively; L6, which is also Poaceae-specific, was characterized as the tandem repeat of L5 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e and \\u003cb\\u003eAdditional file 4\\u003c/b\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eInter-specific syntenic analyses were further conducted and results showed that \\u003cem\\u003eCeCLO1\\u003c/em\\u003e, \\u003cem\\u003eCeCLO2/\\u003c/em\\u003e-\\u003cem\\u003e3\\u003c/em\\u003e, \\u003cem\\u003eCeCLO4/\\u003c/em\\u003e-\\u003cem\\u003e5\\u003c/em\\u003e, \\u003cem\\u003eCeCLO6/-7/-8\\u003c/em\\u003e have syntelogs in at least one of examined species. Significantly, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea, \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e:1 and 2:2 relationships were observed between tigernut and purple nutsedge/\\u003cem\\u003eR. breviuscula\\u003c/em\\u003e/\\u003cem\\u003eC. littledalei\\u003c/em\\u003e, implying that the WGD event (i.e., \\u003cem\\u003eCeCLO1\\u003c/em\\u003e/-\\u003cem\\u003e2\\u003c/em\\u003e) detected in tigernut is shared by these species. Moreover, 1:1, 1:2, and 2:4 relationships were observed between tigernut and \\u003cem\\u003eC. iria\\u003c/em\\u003e, supporting species-specific expansion via WGD in \\u003cem\\u003eC. iria\\u003c/em\\u003e, followed by group-specific gene loss in H3 and L3. Actually, their gene fragments could be detected in the \\u003cem\\u003eC. iria\\u003c/em\\u003e genome. In accordance with orthologous analysis, no syntelog was identified for \\u003cem\\u003eCeCLO4/\\u003c/em\\u003e-\\u003cem\\u003e5\\u003c/em\\u003e in \\u003cem\\u003eC. littledalei\\u003c/em\\u003e, implying species-specific contraction. By contrast, their syntelogs were not only identified in pineapple (\\u003cem\\u003eAnanas comosus\\u003c/em\\u003e), \\u003cem\\u003eJoinvillea ascendens\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb), but also in oil palm (\\u003cem\\u003eElaeis guineensis\\u003c/em\\u003e), greater yam, duckweed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec), and rice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed). Notably, \\u003cem\\u003eCeCLO4/\\u003c/em\\u003e-\\u003cem\\u003e5\\u003c/em\\u003e, \\u003cem\\u003eCeCLO6/-7/-8\\u003c/em\\u003e, \\u003cem\\u003eEgCLO2\\u003c/em\\u003e, and \\u003cem\\u003eEgCLO3/-4/-5/-6\\u003c/em\\u003e were shown to locate within syntenic blocks (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec). Since \\u003cem\\u003eEgCLO3\\u003c/em\\u003e was characterized as a recent repeat of \\u003cem\\u003eEgCLO2\\u003c/em\\u003e from the Arecaceae-specific p WGD (\\u003cb\\u003eAdditional file 4\\u003c/b\\u003e), WGD-derivation of \\u003cem\\u003eCeCLO4/\\u003c/em\\u003e-\\u003cem\\u003e6\\u003c/em\\u003e followed by lineage-specific transposition could be speculated. Collectively, these results imply early diversification of the \\u003cem\\u003ecaleosin\\u003c/em\\u003e family before the radiation of angiosperms followed by lineage-specific expansion even in early diverged angiosperms.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe sequence identities and evolutionary rates of \\u003cem\\u003eCe/CrCLO\\u003c/em\\u003e genes as well as duplicate pairs identified in tigernut are summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. As expected, high sequence identities of 89.30\\u0026ndash;98.50% and small Ks (synonymous substitution rate) values of 0.1048\\u0026ndash;0.1810 were observed between orthologs of tigernut and purple nutsedge. By contrast, only 43.90\\u0026ndash;68.40% sequence identities were found between seven duplicate pairs identified in tigernut. Moreover, except for high sequence divergence between \\u003cem\\u003eCeCLO4\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e6\\u003c/em\\u003e, the Ks values of other pairs were shown to vary from 0.9012 (\\u003cem\\u003eCeCLO4\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e5\\u003c/em\\u003e) to 3.7504 (\\u003cem\\u003eCeCLO6\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e7\\u003c/em\\u003e) (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e), implying distinct time of their birth. Given that the Ka (nonsynonymous substitution rate)/Ks values of all duplicate pairs identified in tigernut are less than one (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e), a role of purifying selection during their evolution could be speculated.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSequence identities and evolutionary rates of duplicate pairs identified in tigernut as well as orthologs between \\u003cem\\u003eC. esculentus\\u003c/em\\u003e and \\u003cem\\u003eC. rotundus\\u003c/em\\u003e. (\\u003cem\\u003eCe C. esculentus\\u003c/em\\u003e, \\u003cem\\u003eCLO\\u003c/em\\u003e caleosin, \\u003cem\\u003eCr C. rotundus\\u003c/em\\u003e, \\u003cem\\u003eKa\\u003c/em\\u003e nonsynonymous substitution rate, \\u003cem\\u003eKs\\u003c/em\\u003e synonymous substitution rate)\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"7\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSpecies\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eGene 1\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eGene 2\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eIdentity (%)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eKa\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eKs\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eKa/Ks\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"6\\\" rowspan=\\\"7\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eC. esculentus-\\u003c/em\\u003e\\u003c/p\\u003e \\u003cp\\u003e\\u003cem\\u003eC. esculentus\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO1\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO2\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e66.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.2346\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e2.4592\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.0944\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO2\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO3\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e62.90\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.3019\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e2.4057\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.1255\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO2\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO5\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e54.10\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.5105\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e2.1566\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.2367\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO4\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO5\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e68.40\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.2376\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.9012\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.2637\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO4\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO6\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e43.90\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.5190\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e-\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO6\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO7\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e58.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.3064\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e3.7504\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.0817\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO7\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO8\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e62.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.3924\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1.1637\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.3372\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"7\\\" rowspan=\\\"8\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eC. esculentus-\\u003c/em\\u003e\\u003c/p\\u003e \\u003cp\\u003e\\u003cem\\u003eC. rotundus\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO1\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCrCLO1\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e94.70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.0226\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.1810\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.1246\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO2\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCrCLO2\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e98.50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.0127\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.1723\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.0736\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO3\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCrCLO3\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e96.80\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.0073\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.1225\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.0598\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO4\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCrCLO4\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e93.70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.0419\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.1522\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.2751\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO5\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCrCLO5\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e89.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.0169\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.1696\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.0995\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO6\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCrCLO6\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e95.80\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.0132\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.1407\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.0938\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO7\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCrCLO7\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e95.30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.0266\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.1048\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.2536\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCeCLO8\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cem\\u003eCrCLO8\\u003c/em\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e95.80\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.0160\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.1203\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0.1333\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eCaleosin\\u003c/b\\u003e \\u003cb\\u003egenes in tigernut exhibited distinct expression patterns\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eTo provide a global view of expression profiles of \\u003cem\\u003eCeCOL\\u003c/em\\u003e genes, RNA-seq data of various tissues were first examined, i.e., young leaf, mature leaf, sheath, shoot apex, root, rhizome, young tuber (collected at 40 days after sowing (DAS)), middle tuber (collected at 85 DAS), and mature tuber (collected at 120 DAS). As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e, FPKM (fragments per kilobase of exon per million fragments mapped) values exceeding 1 in at least one tested samples were observed for five out of eight \\u003cem\\u003eCeCOL\\u003c/em\\u003e genes, i.e., \\u003cem\\u003eCeCLO1\\u003c/em\\u003e\\u0026ndash;\\u003cem\\u003e3\\u003c/em\\u003e, \\u003cem\\u003e-6\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e. Among them, \\u003cem\\u003eCeCLO3\\u003c/em\\u003e, \\u003cem\\u003e-6\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e were constitutively expressed, whereas \\u003cem\\u003eCeCLO1\\u003c/em\\u003e and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/em\\u003e were predominantly expressed in tubers, especially in middle and mature tubers. In accordance with the high oil accumulation, total \\u003cem\\u003eCeCLO\\u003c/em\\u003e transcripts were most abundant in middle and mature tubers, which were clustered together and whose transcripts were 12.41 and 13.90 folds more than those in young tubers. Followed were roots, young tubers, shoot apexes, sheaths, and rhizomes, whose transcripts were 2.20 to 5.98 folds more than those in mature and young leaves (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea). When \\u003cem\\u003eCeOLE\\u003c/em\\u003e genes were included for comparison, three tuber stages were clustered together; two most abundant genes \\u003cem\\u003eCeOLE2\\u003c/em\\u003e and \\u003cem\\u003eCeOLE5\\u003c/em\\u003e formed one group; \\u003cem\\u003eCeCLO1\\u003c/em\\u003e was clustered with \\u003cem\\u003eCeOLE4\\u003c/em\\u003e, \\u003cem\\u003eCeOLE6\\u003c/em\\u003e, and \\u003cem\\u003eCeOLE1\\u003c/em\\u003e; \\u003cem\\u003eCeCLO2\\u003c/em\\u003e was clustered with \\u003cem\\u003eCeOLE3\\u003c/em\\u003e; and \\u003cem\\u003eCeCLO3\\u003c/em\\u003e/\\u003cem\\u003e-6\\u003c/em\\u003e/\\u003cem\\u003e-7\\u003c/em\\u003e and \\u003cem\\u003eCeCLO4\\u003c/em\\u003e/\\u003cem\\u003e-5\\u003c/em\\u003e/\\u003cem\\u003e-8\\u003c/em\\u003e formed two independent groups (\\u003cb\\u003eAdditional file 6a\\u003c/b\\u003e). Correspondingly, correlation analysis showed that \\u003cem\\u003eCeCLO1\\u003c/em\\u003e transcripts were positively related with those of \\u003cem\\u003eCeOLE\\u003c/em\\u003e genes, with the \\u003cem\\u003er\\u003c/em\\u003e value varying from 0.458 (i.e., \\u003cem\\u003eCeOLE3\\u003c/em\\u003e) to 0.913 (i.e., \\u003cem\\u003eCeOLE2\\u003c/em\\u003e). Expression of \\u003cem\\u003eCeCLO1\\u003c/em\\u003e was also positively correlated with \\u003cem\\u003eCeCLO3\\u003c/em\\u003e (\\u003cem\\u003er\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.917), which exhibited the \\u003cem\\u003er\\u003c/em\\u003e values of 0.390 (i.e., \\u003cem\\u003eCeOLE1\\u003c/em\\u003e)\\u0026ndash;0.843 (i.e., \\u003cem\\u003eCeOLE2\\u003c/em\\u003e) (\\u003cb\\u003eAdditional file 6b\\u003c/b\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eComparison revealed apparent expression divergence of\\u003c/b\\u003e \\u003cb\\u003ecaleosin\\u003c/b\\u003e \\u003cb\\u003egenes between tigernut and purple nutsedge\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eOur previous study showed that \\u003cem\\u003eoleosin\\u003c/em\\u003e genes between tigernut and purple nutsedge underwent apparent expression divergence during tuber development, and species-specific activation is in accordance with high oil accumulation in tigernut tubers [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. To address whether similar cases are also present for \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes, their expression profiles over three representative tuber stages were compared, i.e., 20, 50, and 90 days after tuber initiation (DAI). As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb, expression was detected for five \\u003cem\\u003eCeCLO\\u003c/em\\u003e and four \\u003cem\\u003eCrCLO\\u003c/em\\u003e genes, i.e., \\u003cem\\u003eCeCLO1\\u003c/em\\u003e, \\u003cem\\u003eCeCLO2\\u003c/em\\u003e, \\u003cem\\u003eCeCLO3\\u003c/em\\u003e, \\u003cem\\u003eCeCLO6\\u003c/em\\u003e, \\u003cem\\u003eCeCLO7\\u003c/em\\u003e, \\u003cem\\u003eCrCLO1\\u003c/em\\u003e, \\u003cem\\u003eCrCLO3\\u003c/em\\u003e, \\u003cem\\u003eCrCLO6\\u003c/em\\u003e, and \\u003cem\\u003eCrCLO7\\u003c/em\\u003e. Among them, \\u003cem\\u003eCeCLO1\\u003c/em\\u003e was mostly expressed, followed by \\u003cem\\u003eCeCLO3\\u003c/em\\u003e and \\u003cem\\u003eCrCLO6\\u003c/em\\u003e, whereas \\u003cem\\u003eCrCLO3\\u003c/em\\u003e was lowly expressed. Notably, though most \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes were expressed more than their orthologs in purple nutsedge, \\u003cem\\u003eCrCLO6\\u003c/em\\u003e represents the single one that was expressed more than its ortholog \\u003cem\\u003eCeCLO6\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). The result implies that \\u003cem\\u003eCeCLO1\\u003c/em\\u003e, \\u003cem\\u003e-2\\u003c/em\\u003e, \\u003cem\\u003e-3\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e may contribute to tuber oil accumulation in tigernut.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eCeCLO\\u003c/b\\u003e \\u003cb\\u003etranscripts were positively correlated with TAG accumulation during tuber development\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eAccording to our previous study, the TAG content of Reyan3 gradually increased along with tuber development, from 5.09% at 5 DAI to 34.29% at 35 DAI when tubers have completely matured [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. To uncover the correlation between gene expression and TAG accumulation during tuber development, five tuber-expressed \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes (i.e., \\u003cem\\u003eCeCLO1\\u003c/em\\u003e, \\u003cem\\u003e-2\\u003c/em\\u003e, -\\u003cem\\u003e3\\u003c/em\\u003e, \\u003cem\\u003e-6\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e) and five representative stages were selected for qRT-PCR analysis, i.e., S1 (5 DAI), S2 (10 DAI), S3 (20 DAI), S4 (25 DAI), and S5 (35 DAI). As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e, gradual promotion of transcripts during tuber development was observed for \\u003cem\\u003eCeCLO1\\u003c/em\\u003e, \\u003cem\\u003e-2\\u003c/em\\u003e, -\\u003cem\\u003e3\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e, whereas \\u003cem\\u003eCeCLO6\\u003c/em\\u003e was highly expressed in three early stages, followed by significant downregulation at two latter stages. Further correlation analysis showed that the transcripts of \\u003cem\\u003eCeCLO1\\u003c/em\\u003e, \\u003cem\\u003e-2\\u003c/em\\u003e, -\\u003cem\\u003e3\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e were positively correlated with TAG accumulation, with the \\u003cem\\u003er\\u003c/em\\u003e value varying from 0.837 (i.e., \\u003cem\\u003eCeCLO1\\u003c/em\\u003e) to 0.964 (i.e., \\u003cem\\u003eCeCLO2\\u003c/em\\u003e), whereas a negative correlation with \\u003cem\\u003er\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.933 was observed for \\u003cem\\u003eCeCLO6\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e), supporting the result obtained from above transcriptional profiling.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eAs a rare example accumulating high levels of oil in the nutrient storage tubers, tigernut is emerging as an ideal model to study lipid regulation mechanisms beyond oilseeds [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e]. Like oleosins, caleosins also feature the 12-residue proline-knot motif, a key component of the family-specific caleosin domain [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e54\\u003c/span\\u003e]. A crucial role of caleosins in LD formation and oil accumulation prompted us to study this special gene family in tigernut.\\u003c/p\\u003e \\u003cp\\u003eIn the current study, the first genome-wide identification and characterization of the \\u003cem\\u003eCeCLO\\u003c/em\\u003e gene family was reported. A number of eight family members identified from the tigernut genome are considerably less than 21 transcripts obtained from a \\u003cem\\u003ede novo\\u003c/em\\u003e transcriptome assembly [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e], implying the necessity of a genome-based identification. Interestingly, the family amounts present in this species are equal to those described in two model plants arabidopsis and rice [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e], but considerably more than two found in \\u003cem\\u003eA. trichopoda\\u003c/em\\u003e, castor bean, flax, and cucumber (\\u003cem\\u003eCucumis sativus\\u003c/em\\u003e) [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e], implying lineage and/or species-specific family expansion during angiosperm radiation.\\u003c/p\\u003e \\u003cp\\u003eFurther phylogenetic analysis assigned eight \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes into two evolutionary groups as defined in arabidopsis [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e], i.e., H (5) and L (3), where the H clade features a high MW with the so-called H insertion at the N-terminal (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed). Interestingly, the group composition observed in tigernut is highly distinct from rice (H, 3; L, 5) and arabidopsis (H, 4; L, 4), in contrast to the basal angiosperm \\u003cem\\u003eA. trichopoda\\u003c/em\\u003e harboring a single member for each group [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. Extensive expansion in tigernut was shown to be resulted from WGD, tandem, and dispersed duplications (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee). By contrast, expansion in rice was contributed by WGD, tandem, proximal, and dispersed duplications, whereas in arabidopsis, expansion was contributed by WGD, tandem, transposed, and dispersed duplications (\\u003cb\\u003eAdditional file 5\\u003c/b\\u003e). The pattern is also different from the \\u003cem\\u003eCeOLE\\u003c/em\\u003e family, whose expansion was shown to be contributed by WGD and dispersed duplication [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. The results reflect the occurrence of more than one WGD in these species after monocot-eudicot divergence. According to comparative genomics analyses, arabidopsis in the eudicot clade experienced three additional WGDs known as γ, β, and α in sequence [\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e]; similarly, rice in the monocot clade experienced three additional WGDs known as τ, σ, and ρ, where τ and σ WGDs are shared by tigernut [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e58\\u003c/span\\u003e]. Correspondingly, four WGD repeats identified in arabidopsis, i.e., \\u003cem\\u003eAtCLO1\\u003c/em\\u003e/\\u003cem\\u003e-2\\u003c/em\\u003e and \\u003cem\\u003eAtCLO5\\u003c/em\\u003e/\\u003cem\\u003e-6\\u003c/em\\u003e, were shown to arise from the α WGD that is specific to the Brassicaceae family [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e], whereas \\u003cem\\u003eOsCLO1\\u003c/em\\u003e and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;6\\u003c/em\\u003e were derived from the ρ WGD that is specific to the Poaceae family (\\u003cb\\u003eAdditional file 5\\u003c/b\\u003e). By contrast, considering the absence of a recent WGD in tigernut, two identified WGD repeats, i.e., \\u003cem\\u003eCeCLO1\\u003c/em\\u003e and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/em\\u003e, are more likely to arise from the σ WGD that is shared by all Poales plants [\\u003cspan citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eTo confirm the hypothesis and gain insights into the origin and evolution of \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes, approximately 200 homologs from 31 representative plant species were included for comparison, which resulted in 11 orthogroups named H1, H2, H3, H4, H5, L1, L2, L3, L4, L5, and L6. These species cover a wide range of 17 plant families, i.e., Amborellaceae (\\u003cem\\u003eA. trichopoda\\u003c/em\\u003e), Vitaceae (grapevine), Euphorbiaceae (castor bean), Brassicaceae (arabidopsis), Acoraceae (American sweet flag), Zosteraceae (eelgrass), Araceae (duckweed), Asparagaceae (garden asparagus), Orchidaceae (apostasia), Dioscoreaceae (greater yam), Arecaceae (oil palm), Bromeliaceae (pineapple), Typhaceae (\\u003cem\\u003eSparganium stoloniferum\\u003c/em\\u003e), Cyperaceae (tigernut, purple nutsedge, \\u003cem\\u003eC. iria\\u003c/em\\u003e, \\u003cem\\u003eC. fuscus\\u003c/em\\u003e, \\u003cem\\u003eBolboschoenus planiculmis\\u003c/em\\u003e, \\u003cem\\u003eSchoenoplectus tabernaemontani\\u003c/em\\u003e, \\u003cem\\u003eC. littledalei\\u003c/em\\u003e, \\u003cem\\u003eR. breviuscula\\u003c/em\\u003e, and \\u003cem\\u003eEleocharis parvula\\u003c/em\\u003e), Juncaceae (\\u003cem\\u003eJuncus effusus\\u003c/em\\u003e and \\u003cem\\u003eJ. inflexus\\u003c/em\\u003e), Joinvilleaceae (\\u003cem\\u003eJ. ascendens\\u003c/em\\u003e), and Poaceae (\\u003cem\\u003ePharus latifolius\\u003c/em\\u003e, rice, barley, \\u003cem\\u003eBrachypodium distachyon\\u003c/em\\u003e, \\u003cem\\u003eSorghum bicolor\\u003c/em\\u003e, and \\u003cem\\u003eSetaria italica\\u003c/em\\u003e). In accordance with previous studies [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e], our data suggest that the \\u003cem\\u003ecaleosin\\u003c/em\\u003e family has diverged into Clades H and L in the last common ancestor of angiosperms, which is reserved as a single copy for each clade in some eudicots such as grapevine and castor bean. By contrast, extensive expansion via both local and large-scale duplications was frequently observed in Poales, and \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes belong to eight orthogroups, i.e., H1\\u0026ndash;5 and L1\\u0026ndash;3. Based on the results from orthologous, syntenic, and evolutionary rate analyses, a possible evolution route in tigernut is proposed as follows: the wide present H4 (e.g., \\u003cem\\u003eCeCLO4\\u003c/em\\u003e) first gave birth to H2 (e.g., \\u003cem\\u003eCeCLO2\\u003c/em\\u003e) via dispersed duplication sometime before monocot radiation, which may be along with the τ WGD followed by gene transposition; then, H2 produced H1 (e.g., \\u003cem\\u003eCeCLO1\\u003c/em\\u003e) via the σ WGD in Poales, though H1 is most conserved; the last common ancestor of Cyperaceae and Juncaceae generated H3 (e.g., \\u003cem\\u003eCeCLO3\\u003c/em\\u003e) via tandem duplication (unequal crossover) sometime after the split with other families within Poales; in Cyperaceae, L1 (e.g., \\u003cem\\u003eCeCLO6\\u003c/em\\u003e) first brought forth L2 (e.g., \\u003cem\\u003eCeCLO7\\u003c/em\\u003e) via tandem duplication sometime after the split with Juncaceae, which further gained an additional intron; subsequently, L2 produced L3 (e.g., \\u003cem\\u003eCeCLO8\\u003c/em\\u003e) via tandem duplication; in \\u003cem\\u003eCyperus\\u003c/em\\u003e, the last common ancestor of tigernut, purple nutsedge, and \\u003cem\\u003eC. iria\\u003c/em\\u003e generated H5 (e.g., \\u003cem\\u003eCeCLO5\\u003c/em\\u003e) via tandem duplication sometime after the split with \\u003cem\\u003eC. fuscus\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cp\\u003eInterestingly, despite a very close genetic relationship of tigernut and purple nutsedge, purple nutsedge only accumulates less than 2.5% of oil in its tubers, in contrast to up to 35% in tigernut [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. 1:1 orthologous relationships of \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes observed between these two species suggest that all duplicate pairs identified in tigernut are lineage-specific and gene copies are not the contributor of high oil accumulation in tigernut tubers. Similar results were also reported for the \\u003cem\\u003eoleosin\\u003c/em\\u003e family, which is composed of six members in both tigernut and purple nutsedge [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. Compared with tuber-preferential expression of most \\u003cem\\u003eCeOLE\\u003c/em\\u003e genes [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e], eight \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes was shown to exhibit distinct expression patterns with constitutive expression of \\u003cem\\u003eCeCLO3\\u003c/em\\u003e, \\u003cem\\u003e-6\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e, tuber-predominant expression of \\u003cem\\u003eCeCLO1\\u003c/em\\u003e and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/em\\u003e, and rare expression of \\u003cem\\u003eCeCLO4\\u003c/em\\u003e, \\u003cem\\u003e-5\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;8\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea). Moreover, though the \\u003cem\\u003eCeCLO\\u003c/em\\u003e family contains two more members than the \\u003cem\\u003eCeOLE\\u003c/em\\u003e family, their total transcripts in tubers were shown to be considerably less abundant (\\u003cb\\u003eAdditional file 6a\\u003c/b\\u003e). Nevertheless, the transcript level of \\u003cem\\u003eCeCLO1\\u003c/em\\u003e ranks only after \\u003cem\\u003eCeOLE5\\u003c/em\\u003e and \\u003cem\\u003eCeOLE2\\u003c/em\\u003e, two most abundant \\u003cem\\u003eCeOLE\\u003c/em\\u003e genes in tigernut tubers (\\u003cb\\u003eAdditional file 6a\\u003c/b\\u003e). A putative role of \\u003cem\\u003eCeCLO1\\u003c/em\\u003e and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/em\\u003e in tuber oil accumulation was first inferred from Pearson's correlation analysis, which was shown to exhibit the high \\u003cem\\u003er\\u003c/em\\u003e value up to 0.9 with \\u003cem\\u003eCeOLE2\\u003c/em\\u003e (\\u003cb\\u003eAdditional file 6b\\u003c/b\\u003e). Correspondingly, our qRT-PCR analysis showed that transcription of \\u003cem\\u003eCeCLO1\\u003c/em\\u003e, \\u003cem\\u003e-2\\u003c/em\\u003e, \\u003cem\\u003e-3\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e in five representative tuber stages was positively correlated with oil accumulation during tuber development (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). Last but most important, our comparative transcriptome analysis revealed that \\u003cem\\u003eCeCLO1\\u003c/em\\u003e, \\u003cem\\u003e-2\\u003c/em\\u003e, \\u003cem\\u003e-3\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e were expressed considerably more than their orthologs in purple nutsedge (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb), implying species but not tuber-specific activation for oil accumulation.\\u003c/p\\u003e \\u003cp\\u003eAs discussed above, unlike the \\u003cem\\u003eoleosin\\u003c/em\\u003e family, tandem duplication was shown to play a crucial in the expansion of the \\u003cem\\u003ecaleosin\\u003c/em\\u003e family [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. Though this duplication mode has been proven to play a key role in environmental adaptation of plants [\\u003cspan additionalcitationids=\\\"CR60\\\" citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e], an amazing result is that most tandem repeats identified in tigernut, e.g., \\u003cem\\u003eCeCLO4\\u003c/em\\u003e, \\u003cem\\u003e-5\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;8\\u003c/em\\u003e, were not or rarely expressed in all tissues examined in this study. Similar results were also reported for plasma membrane intrinsic protein (PIP), stearoyl-acyl carrier protein desaturase (SAD), and fatty acid desaturase 2 (FAD2) (sub)families [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR62\\\" class=\\\"CitationRef\\\"\\u003e62\\u003c/span\\u003e]. Thereby, further clarifying their biological significance in tigernut is of particular interest.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eThis study presents the first genome-wide analysis of the \\u003cem\\u003ecaleosin\\u003c/em\\u003e family in tigernut, a unique oil-rich tuber plant in Cyperaceae. Eight family members representing two clades identified from the tigernut genome are considerably more than two present in the basal angiosperm \\u003cem\\u003eA. trichopoda\\u003c/em\\u003e. Comparison of 193 members from 31 representative plant species reveals lineage-specific family expansion and functional diversification in tigernut, whose expansion was contributed by WGD, tandem, and dispersed duplications. Comparison between tigernut and purple nutsedge suggests that species-specific activation of certain members but not gene copies is the main contributor of high oil accumulation in tigernut tubers. These findings provide valuable information for further functional analysis and genetic improvement in tigernut and species beyond.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePlant materials\\u003c/h2\\u003e \\u003cp\\u003eTigernut plants of \\u003cem\\u003eCyperus esculentus var. sativus\\u003c/em\\u003e Boeckeler (NCBI:txid1504802) variety Reyan3, which was mainly bred by Zhi Zou, were cultivated in sandy soil as described before [\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e]. Tubers of various developmental stages were collected at 5, 10, 20, 25, and 35 DAI, and three biological replicates were harvested for each stage. All samples were first freezed with liquid nitrogen and then stored at 80℃for further uses.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eDatasets and identification of\\u003c/b\\u003e \\u003cb\\u003ecaleosin\\u003c/b\\u003e \\u003cb\\u003efamily genes\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eGenomic and transcriptome data of \\u003cem\\u003eC. esculentus\\u003c/em\\u003e and representative plant species were accessed from CNGBdb (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://db.cngb.org/\\u003c/span\\u003e\\u003cspan address=\\\"https://db.cngb.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), RGAP7 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://rice.plantbiology.msu.edu/\\u003c/span\\u003e\\u003cspan address=\\\"http://rice.plantbiology.msu.edu/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), TAIR11 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.arabidopsis.org/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.arabidopsis.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), Phytozome v13 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://phytozome.jgi.doe.gov/pz/portal.html\\u003c/span\\u003e\\u003cspan address=\\\"https://phytozome.jgi.doe.gov/pz/portal.html\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), and NCBI (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.ncbi.nlm.nih.gov/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.ncbi.nlm.nih.gov/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e): \\u003cem\\u003eC. esculentus\\u003c/em\\u003e (v1; Cyperaceae, Poales), \\u003cem\\u003eC. rotundus\\u003c/em\\u003e (v1; Cyperaceae, Poales), \\u003cem\\u003eC. iria\\u003c/em\\u003e (v1; Cyperaceae, Poales), \\u003cem\\u003eC. fuscus\\u003c/em\\u003e (v1; Cyperaceae, Poales), \\u003cem\\u003eB. planiculmis\\u003c/em\\u003e (v1; Cyperaceae, Poales),, \\u003cem\\u003eS. tabernaemontani\\u003c/em\\u003e (v1; Cyperaceae, Poales), \\u003cem\\u003eC. littledalei\\u003c/em\\u003e (v1; Cyperaceae, Poales), \\u003cem\\u003eR. breviuscula\\u003c/em\\u003e (v1; Cyperaceae, Poales), \\u003cem\\u003eE. parvula\\u003c/em\\u003e (v1; Cyperaceae, Poales), \\u003cem\\u003eJ. effusus\\u003c/em\\u003e (v1; Juncaceae, Poales), \\u003cem\\u003eJ. inflexus\\u003c/em\\u003e (v1; Juncaceae, Poales), \\u003cem\\u003eSparganium stoloniferum\\u003c/em\\u003e (v1; Typhaceae, Poales), \\u003cem\\u003eA. comosus\\u003c/em\\u003e (v3; Bromeliaceae, Poales), \\u003cem\\u003eJ. ascendens\\u003c/em\\u003e (v1.1; Joinvilleaceae, Poales), \\u003cem\\u003eP. latifolius\\u003c/em\\u003e (v1; Poaceae, Poales), \\u003cem\\u003eO. sativa\\u003c/em\\u003e (RGAP7; Poaceae, Poales), \\u003cem\\u003eB. distachyon\\u003c/em\\u003e (v3.2; Poaceae, Poales), \\u003cem\\u003eH. vulgare\\u003c/em\\u003e (v1; Poaceae, Poales), \\u003cem\\u003eS. bicolor\\u003c/em\\u003e (5.1; Poaceae, Poales), \\u003cem\\u003eS. italica\\u003c/em\\u003e (v2.2; Poaceae, Poales), \\u003cem\\u003eE. guineensis\\u003c/em\\u003e (EG5; Arecaceae, Arecales), \\u003cem\\u003eD. alata\\u003c/em\\u003e (v2.1; Dioscoreaceae, Dioscoreales), \\u003cem\\u003eA. officinalis\\u003c/em\\u003e (v1.1; Asparagaceae, Asparagales), \\u003cem\\u003eD. catenatum\\u003c/em\\u003e (v1; Orchidaceae, Asparagales), \\u003cem\\u003eSpirodela polyrhiza\\u003c/em\\u003e (v2; Lemnaceae, Alismatales), \\u003cem\\u003eZ. marina\\u003c/em\\u003e (v3.1; Zosteraceae, Alismatales), \\u003cem\\u003eA. americanus\\u003c/em\\u003e (v1; Acoraceae, Acorales), \\u003cem\\u003eA. thaliana\\u003c/em\\u003e (Araport11; Brassicaceae, Brassicales), \\u003cem\\u003eR. communis\\u003c/em\\u003e (WT05; Euphorbiaceae, Malpighiales), \\u003cem\\u003eV. vinifera\\u003c/em\\u003e (T2T; \\u003cem\\u003eVitaceae\\u003c/em\\u003e, \\u003cem\\u003eVitales\\u003c/em\\u003e), and \\u003cem\\u003eA. trichopoda\\u003c/em\\u003e (v2.1; Amborellaceae, Amborellales). Caleosin homologs were identified via HMMER (v3.3, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://hmmer.janelia.org/\\u003c/span\\u003e\\u003cspan address=\\\"http://hmmer.janelia.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) searching with the Pfam profile PF05042 (v35.0, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://pfam.xfam.org/\\u003c/span\\u003e\\u003cspan address=\\\"https://pfam.xfam.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Gene models of all candidates were curated with available mRNAs as previously described [\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e], and gene structure display was performed using GSDS2.0 [\\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e64\\u003c/span\\u003e]. Presence of the conserved caleosin domain in deduced proteins was confirmed using Pfam Search.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eSequence alignment and phylogenetic analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eMultiple sequence alignment was conducted using Muscle (v5) [\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e], and sequence alignment display was carried out using SMS (v1, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.bioinformatics.org/sms/\\u003c/span\\u003e\\u003cspan address=\\\"http://www.bioinformatics.org/sms/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Phylogenetic tree construction was performed using RAxML (v8) [\\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e65\\u003c/span\\u003e] with the maximum likelihood method and bootstrap of 1000 replicates. Systematic names of \\u003cem\\u003ecaleosin\\u003c/em\\u003e family genes were assigned with two italic letters denoting the source organism and a progressive number based on sequence similarity. To distinguish with \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes in arabidopsis, three italic letters were used in \\u003cem\\u003eA. trichopoda\\u003c/em\\u003e, i.e., \\u003cem\\u003eAtrCLO1\\u003c/em\\u003e and \\u0026minus;\\u0026thinsp;\\u003cem\\u003e2\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eProtein properties and conserved motif analysis\\u003c/h2\\u003e \\u003cp\\u003ePhysical and chemical parameters (e.g., MW, pI, and GRAVY) of deduced proteins were calculated using ProtParam (v1, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://web.expasy.org/protparam/\\u003c/span\\u003e\\u003cspan address=\\\"http://web.expasy.org/protparam/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), whereas hydropathicity scales were determined using ProtScale (v1, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://web.expasy.org/protscale/\\u003c/span\\u003e\\u003cspan address=\\\"https://web.expasy.org/protscale/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Protein secondary structures were determined using SOPMA (v1, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html\\u003c/span\\u003e\\u003cspan address=\\\"https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), while conserved motifs were identified using MEME (v5.4.1, \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://meme-suite.org/tools/meme\\u003c/span\\u003e\\u003cspan address=\\\"https://meme-suite.org/tools/meme\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) with the parameters as follows: any number of repetitions; the maximum number of motifs, 10; and, the optimum width of each motif, between 5 and 200 residues.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eChromosomal localization, synteny analysis, definition of orthogroups, and calculation of evolutionary rate\\u003c/h3\\u003e\\n\\u003cp\\u003eSynteny analysis was conducted as previously described [\\u003cspan citationid=\\\"CR67\\\" class=\\\"CitationRef\\\"\\u003e67\\u003c/span\\u003e], where duplicate pairs were identified using the all-to-all BLASTp method with the \\u003cem\\u003eE\\u003c/em\\u003e-value cutoff of 1e-10 and syntenic blocks were inferred using MCScanX [\\u003cspan citationid=\\\"CR68\\\" class=\\\"CitationRef\\\"\\u003e68\\u003c/span\\u003e] with at least five BLAST hits. Orthologs between different species were identified using OrthoFinder (v2.3.8) [\\u003cspan citationid=\\\"CR69\\\" class=\\\"CitationRef\\\"\\u003e69\\u003c/span\\u003e], whereas different modes of gene duplication were identified using the DupGen_finder pipeline [\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e]. Chromosomal localization and calculation of Ks and Ka values were performed using TBtools-II (v2.390) [\\u003cspan citationid=\\\"CR70\\\" class=\\\"CitationRef\\\"\\u003e70\\u003c/span\\u003e].\\u003c/p\\u003e\\n\\u003ch3\\u003eGene expression analysis based on RNA-seq\\u003c/h3\\u003e\\n\\u003cp\\u003eGlobal expression profiles of \\u003cem\\u003ecaleosin\\u003c/em\\u003e and \\u003cem\\u003eoleosin\\u003c/em\\u003e genes in tigernut and yellow nutsedge were investigated by using transcriptome datasets that are under NCBI accession numbers of PRJNA703731 and PRJNA671562. The data are 150 bp paired-end reads with three biological replicates, which were derived from shoot apexes, young leaves, mature leaves, leaf sheaths, roots, rhizomes, and tubers of 40 DAS, 85 DAS, 120 DAS, 20 DAI, 50 DAI, and 90 DAI. Raw reads in the FASTQ format were obtained using fastq-dump (v1) [\\u003cspan citationid=\\\"CR71\\\" class=\\\"CitationRef\\\"\\u003e71\\u003c/span\\u003e], and quality control was carried out using Trimmomatic (v1) [\\u003cspan citationid=\\\"CR72\\\" class=\\\"CitationRef\\\"\\u003e72\\u003c/span\\u003e]. Read mapping was performed using HISAT (v2) [\\u003cspan citationid=\\\"CR73\\\" class=\\\"CitationRef\\\"\\u003e73\\u003c/span\\u003e], and relative gene expression level was presented as FPKM [\\u003cspan citationid=\\\"CR74\\\" class=\\\"CitationRef\\\"\\u003e74\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eGene expression analysis based on qRT-PCR\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA extraction, the integrity and concentration detection, synthesis of the first-strand cDNA, and qRT-PCR analysis were conducted as previously described [\\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e57\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e]. Primers used are shown in \\u003cb\\u003eAdditional file 7\\u003c/b\\u003e, where \\u003cem\\u003eCeTIP41\\u003c/em\\u003e and \\u003cem\\u003eCeUCE2\\u003c/em\\u003e are two reference genes as described before [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. All qRT-PCR assays were conducted in triplicate for each biological sample, and relative gene abundance was estimated with the 2\\u003csup\\u003e\\u0026minus;ΔΔCt\\u003c/sup\\u003e method and statistical analysis was performed using SPSS Statistics 20 as described before [\\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e63\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors appreciate those contributors who make the related genome and transcriptome data accessible in public databases.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026rsquo; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe study was conceived and directed by ZZ. All the experiments and analyses were directed by ZZ and carried out by ZZ, XC, HH, CL, XY, JH, and ZY. ZZ, JH, and ZY wrote the paper. All the authors read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the Hainan Province Science and Technology Special Fund (ZDYF2024XDNY171 and ZDYF2024XDNY156) and the National Natural Science Foundation of China (32460342 and 31971688). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets analyzed during the current study are available in the NCBI SRA repository (https://www.ncbi.nlm.nih.gov/sra/) under accession numbers of PRJNA703731 and PRJNA671562.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eHuang AHC. Plant lipid droplets and their associated proteins: Potential for rapid advances. Plant Physiol. 2018;176(3):1894\\u0026ndash;918. doi:10.1104/pp.17.01677.\\u003c/li\\u003e\\n\\u003cli\\u003eFrandsen G, M\\u0026uuml;ller-Uri F, Nielsen M, Mundy J, Skriver K. Novel plant Ca\\u003csup\\u003e2+\\u003c/sup\\u003e-binding protein expressed in response to abscisic acid and osmotic stress. J Biol Chem. 1996;271(1):343\\u0026ndash;8. doi:10.1074/jbc.271.1.343.\\u003c/li\\u003e\\n\\u003cli\\u003eChen EC, Tai SS, Peng CC, Tzen JT. Identification of three novel unique proteins in seed oil bodies of sesame. Plant Cell Physiol. 1998;39(9):935\\u0026ndash;41. doi: 10.1093/oxfordjournals.pcp.a029457.\\u003c/li\\u003e\\n\\u003cli\\u003eHanano A, Burcklen M, Flenet M, Ivancich A, Louwagie M, Garin J, Blee E. Plant seed peroxygenase is an original heme-oxygenase with an EF-hand calcium binding motif. J Biol Chem. 2006;281(44):33140\\u0026ndash;51. doi:10.1074/jbc.M605395200.\\u003c/li\\u003e\\n\\u003cli\\u003eYamaguchi-Shinozaki K, Koizumi M, Urao S, Shinozaki K. Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in \\u003cem\\u003eArabidopsis thaliana\\u003c/em\\u003e: sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein. Plant Cell Physiol. 1992;33:217\\u0026ndash;24.\\u003c/li\\u003e\\n\\u003cli\\u003eTakahashi S, Katagiri T, Yamaguchi-Shinozaki K, Shinozaki K. An \\u003cem\\u003eArabidopsis\\u003c/em\\u003e gene encoding a Ca\\u003csup\\u003e2+\\u003c/sup\\u003e-binding protein is induced by abscisic acid during dehydration. Plant Cell Physiol. 2000;41(7):898\\u0026ndash;903. doi: 10.1093/pcp/pcd010.\\u003c/li\\u003e\\n\\u003cli\\u003eChen JC, Tsai CC, Tzen JT. Cloning and secondary structure analysis of caleosin, a unique calcium-binding protein in oil bodies of plant seeds. Plant Cell Physiol. 1999;40(10):1079\\u0026ndash;86. doi: 10.1093/oxfordjournals.pcp.a029490.\\u003c/li\\u003e\\n\\u003cli\\u003eNaested H, Frandsen GI, Jauh GY, Hernandez-Pinzon I, Nielsen HB, Murphy DJ, Rogers JC, Mundy J. Caleosins: Ca\\u003csup\\u003e2+\\u003c/sup\\u003e-binding proteins associated with lipid bodies. Plant Mol Biol. 2000;44(4):463\\u0026ndash;76. doi:10.1023/a:1026564411918.\\u003c/li\\u003e\\n\\u003cli\\u003eRahman F, Hassan M, Hanano A, Fitzpatrick DA, McCarthy CGP, Murphy DJ. Evolutionary, structural and functional analysis of the caleosin/peroxygenase gene family in the Fungi. BMC Genom. 2018;19(1):976. doi:10.1186/s12864-018-5334-1.\\u003c/li\\u003e\\n\\u003cli\\u003eRahman F, Hassan M, Rosli R, Almousally I, Hanano A, Murphy DJ. Evolutionary and genomic analysis of the caleosin/peroxygenase (CLO/PXG) gene/protein families in the Viridiplantae. 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Nat Methods. 2015;12(4):357\\u0026ndash;60. doi: 10.1038/nmeth.3317.\\u003c/li\\u003e\\n\\u003cli\\u003eMortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat Methods. 2008;5(7):621\\u0026ndash;8. doi: 10.1038/nmeth.1226.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"bmc-plant-biology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"pbio\",\"sideBox\":\"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/pbio/default.aspx\",\"title\":\"BMC Plant Biology\",\"twitterHandle\":\"BMC_series\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Vegetative tissue, Underground tuber, Phylogenomics, Orthologous analysis, Synteny analysis, Gene expansion, Expression divergence\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8567486/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8567486/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eBackground\\u003c/h2\\u003e \\u003cp\\u003eCaleosins (CLOs), a class of structural proteins of lipid droplets (LDs), widely function in LD formation, stabilization, and degradation as well as in plant development and stress responses. However, their characterization in tigernut (\\u003cem\\u003eCyperus esculentus\\u003c/em\\u003e L., Cyperaceae), a rare example accumulating significant amounts of oil in underground tubers, is still in the infancy.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eIn this study, we present a first genome-wide analysis of the \\u003cem\\u003ecaleosin\\u003c/em\\u003e family in tigernut. A number of eight members, which represent two previously defined clades (i.e., H and L), were identified from the tigernut genome, in stark contrast to only two present in the basal angiosperm \\u003cem\\u003eAmborella trichopoda\\u003c/em\\u003e. Comparison of 193 \\u003cem\\u003ecaleosin\\u003c/em\\u003e genes from 31 representative plant species reveals lineage-specific expansion and functional diversification. Eight \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes belong to eight out of 11 orthogroups identified in this study, and extensive expansion in this species was contributed by whole-genome duplication (WGD), tandem, and dispersed duplications. 1:1 orthologous relationships observed between tigernut and its close relative \\u003cem\\u003eCyperus rotundus\\u003c/em\\u003e suggest that gene copies are not the contributor of high tuber oil accumulation in tigernut. Instead, most \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes was shown to express more than their orthologs in \\u003cem\\u003eC. rotundus\\u003c/em\\u003e, implying species-specific activation in oil-bearing tigernut tubers. Correspondingly, expression of \\u003cem\\u003eCeCLO1\\u003c/em\\u003e, \\u003cem\\u003e-2\\u003c/em\\u003e, \\u003cem\\u003e-3\\u003c/em\\u003e, and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;7\\u003c/em\\u003e was shown to positively correlate with oil accumulation during tuber development. Structure and expression divergence of paralogous pairs were also observed, and good examples are 1) \\u003cem\\u003eCeCLO7\\u003c/em\\u003e and \\u003cem\\u003e\\u0026minus;\\u0026thinsp;8\\u003c/em\\u003e that have gained one additional intron and 2) \\u003cem\\u003eCeCLO1\\u003c/em\\u003e that has become the predominant isoform in oil-rich tubers.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e \\u003cp\\u003eThese findings provide insights into the evolution, expression, and structural variation of \\u003cem\\u003eCeCLO\\u003c/em\\u003e genes, which improve our knowledge on the mechanism of high oil accumulation in tigernut tubers.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Insights into the caleosin family in Cyperus esculentus, an oil-rich tuber plant in Cyperaceae\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-02-16 14:52:29\",\"doi\":\"10.21203/rs.3.rs-8567486/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"30741035406498555850372776319728584250\",\"date\":\"2026-04-28T05:01:26+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-03-09T09:53:10+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"112546605419996072095416561691340256815\",\"date\":\"2026-02-20T12:23:28+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-02-11T10:39:09+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-01-21T06:30:17+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2026-01-19T06:15:08+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-01-17T12:46:23+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"BMC Plant Biology\",\"date\":\"2026-01-17T12:37:22+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"bmc-plant-biology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"pbio\",\"sideBox\":\"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/pbio/default.aspx\",\"title\":\"BMC Plant Biology\",\"twitterHandle\":\"BMC_series\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"5015a386-8936-4143-af23-ac05bc95fb88\",\"owner\":[],\"postedDate\":\"February 16th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-02-16T14:52:29+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-02-16 14:52:29\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8567486\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8567486\",\"identity\":\"rs-8567486\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}