Fig 1.
Maximum likelihood tree of MADS-box factors in the rubber tree.
A total of 134 MADS-box proteins including 97 from the rubber tree (prefixed with ‘Hb’) and 19 representative sequences of well known clades in Arabidopsis (prefixed with ‘At’) were aligned using ClustalX2.0 [35]. The evolutionary distances were computed using the number of differences method [40]. All positions with less than 80% site coverage were eliminated, and a total of 256 positions in the final dataset were used to create the tree. The evolutionary analyses were conducted in MEGA7 [36]. The accession numbers of the Arabidopsis MADS-box proteins are provided in S4 Table.
Fig 2.
Gene structures of MADS-box transcription factors in the rubber tree.
The graph was prepared with GSDS2.0 [37]. The slashes in introns indicate positions of cutouts to make the sequences fit in page. The lengths of cutouts are respectively 30 kb in intron 2 and 12 kb in intron 5 of HbMADS16, 5 kb in intron 2 of HbMADS14, 10 kb in intron 2 of HbMADS11, 5 kb in intron 2 of HbMADS13, 10 kb in intron 3 of HbMADS19, 10 kb in intron 2 and 4 kb in intron 3 of HbMADS25, 21 kb in intron 2 of HbMADS26, 10 kb in intron 1 of HbMADS30, 5 kb in intron 2 of HbMADS35, 14 kb in intron 2 and 3 kb in intron 9 of HbMADS39, 10 kb in intron 2 of HbMADS4, 1 kb in intron 5 of HbMADS42, 22 kb in intron 1 of HbMADS7, and 5 kb in intron 5 of HbMADS96.
Fig 3.
Conserved motifs of MADS-box factors in the rubber tree.
The motifs were identified with MEME Suite 5.03 [42] and confirmed in InterPro database (http://www.ebi.ac.uk/interpro/). Motifs are shown by different color boxes, while non-conserved sequences are shown in grey line. Length of motifs is present in scale.
Fig 4.
Phylogenetic relationships and expression profile of MIKCc-type MADS-box proteins in the rubber tree.
A, a Neighbor Joining Tree. The evolutionary history was inferred using Neighbor Joining (NJ), Maximum Likelihood (ML) and Minimum Evolution (ME) methods, respectively. A total of 133 MIKCc factor sequences from the rubber tree (prefixed with ‘Hb’), Arabidopsis (prefixed with ‘At’), and rice (prefixed with ‘Os’) are involved in the analysis. The tree is rooted with an ancient MADS-box protein MpMADS1 (GQ334454) from a liverwort species Marchantia polymorpha. All positions with less than 80% site coverage were eliminated and 177 positions were used in the final datasets to create the tree. The analysis were conducted in MEGA7 [36]. B, expression profiles of MIKCc-type MADS factor genes in the rubber tree. C, Real-time quantitative PCR analysis in comparison to RNA-seq results. Rt, root; Bk, bark; Lf, leaf; MF, male flower; FF, female flower; PL, primary laticifer; SL, secondary laticifer. The accession numbers of MADS-box proteins from Arabidopsis and rice are provided in S4 and S5 Tables.
Fig 5.
Phylogenetic relationships and expression profiles of MIKC*-type MADS-box proteins in the rubber tree.
A, a Maximum likelihood tree. The evolutionary history was inferred using NJ, ML, and ME methods. A total of 42 MIKC* MADS-box proteins from rubber tree (prefixed with ‘Hb’), Arabidopsis (prefixed with ‘At’), rice (prefixed with ‘Os’), Zea mays (prefixed with ‘Zm’), Ceratopteris richardii (prefixed with ‘Cr’), Eschscholzia californica (prefixed with ‘Ec’), Funaria hygrometrica (prefixed with ‘Fh’), Sphagnum subsecundum (prefixed with ‘Ss’), Populus trichocarpa (prefixed with ‘Pt’), Hordeum vulgare (prefixed with ‘Hv’), Brachypodium distachyon (prefixed with ‘Bd’), and Selaginella moellendorffii (prefixed with ‘Sm’) are involved in the analysis. The accession numbers of the reference sequences are provided in S6 Table. The tree is rooted with an ancient MADS-box protein MpMADS1 (GQ334454) from a liverwort species Marchantia polymorpha. All positions with less than 80% site coverage were eliminated and a total of 390 positions were used in the final dataset. Bootstrap values of conserved clades as calculated using ML, NJ, and ME methods are provided above or below the branches. Scale bar represent 0.5 substitutions. B, MIKC* expression profile by RNA-seq. C, real-time RT-PCR results of representative genes. Rt, root; Bk, bark; Lf, leaf; MF, male flower; FF, female flower; PL, primary laticifer; SL, secondary laticifer.
Fig 6.
Maximum Likelihood (A) and Neighbor Joining (B) trees of type I MADS-box transcription factor sequences. A total of 127 amino acid sequences were used in the analysis, including all type I MADS from the rubber tree (prefixed with ‘Hb’), Arabidopsis (prefixed with ‘At’), and rice (prefixed with ‘Os’), and one type II (HbMADS85) from rubber tree, and outgroups from distinct species (CAX33869, CAX33874, GQ334454, XP_024027649, XP_021284791, PAN24898, AST36051, CAX33873, and CAX33871). The HbMADS85 is used to confirm that it does not belong to type I subfamily because it is not classified into either MIKCc or MIKC* in Fig 1. The outgroups are represented with a black triangle to simplify the tree. All positions with less than 80% site coverage were eliminated and 153 positions were used in the final datasets to create the trees. The analysis were conducted in MEGA7 [36].
Fig 7.
Expression profile of type I MADS-box genes in the rubber tree.
A, Expression profiles by transcriptome analysis. B, Real-time quantitative RT-PCR results of representative genes as compared with TPM values in transcriptome analysis. Rt, root; Bk, bark; Lf, leaf; MF, male flower; FF, female flower; PL, primary laticifer; SL, secondary laticifer.
Table 1.
Number of annotated MADS-box genes present in genomes.