Figure 1
RNA-Seq data expression profiles in C. alismatifolia colour bracts. (A) Phenotypes of ‘Dutch Red’ at three typical development stages and ‘Chocolate’ at the blossomed stage. (B) Numbers of transcripts in the four bract samples. (C) Principal component analysis of the RNA-Seq data. (D) Venn diagram of the RNA-Seq data from the four bract samples. BF, blossomed flowering; HF, half-flowering; FPKM, fragments per kilobase of transcript per million mapped reads.
Table 1
Summary of assembly results for ‘Dutch Red’ at three development stages and ‘Chocolate’ at the blossomed stage.
| Features | SF | HF | BF | YF |
|---|---|---|---|---|
| Total raw reads (Mb) | 45.24 | 48.46 | 52.98 | 45.47 |
| Total clean reads (Mb) | 44.47 | 47.55 | 52.10 | 44.63 |
| Total clean bases (Gb) | 6.59 | 7.05 | 7.75 | 6.61 |
| Clean reads Q20 (%) | 98.27 | 98.23 | 98.34 | 98.40 |
| Clean reads Q30 (%) | 94.63 | 94.56 | 94.83 | 95.01 |
| Clean reads (pair reads) | 22.24 | 23.78 | 26.05 | 22.31 |
| Mapped reads | 16,378,830 | 17,511,187 | 19,200,458 | 16,316,551 |
| Mapped ratio (%) | 73.66 | 73.66 | 73.69 | 73.13 |
| Total number of transcripts | 159,687 | |||
| Total number of unigenes | 69,453 | |||
| Total sequence base | 197,307,177 | |||
| Average length of transcripts | 1,236 | |||
| N50 value of transcripts | 1,830 | |||
| E90N50 value of transcripts | 1,921 | |||
| GC (%) | 42.49 | |||
| TransRate score | 0.30 | |||
| BUSCO score | 60.1% (3.5%) |
[i] Q20: The rate of bases whose quality is greater than 20; Q30: The rate of bases whose quality is greater than 30; N50: A weighted median statistic in which 50% of the total length is contained in unigenes greater than or equal to this value; E90N50: A weighted median statistic in which 50% of the total length is contained in the top 90% unigenes greater than or equal to this value; GC (%): The percentage of G and C bases in all unigenes; TransRate: Score the assembly results with transrate; BUSCO: Score the assembly integrity with BUSCO.
[ii] BF, blossomed flowering; HF, half-flowering.
Figure 2
(A) Distributions and (B) KEGG analysis of DEGs identified from pairwise comparisons between different developmental stages and between varieties. The FDR value is the multiple hypothesis test-corrected p-value. The FDR value is in the range of [0–1]. The closer that number is to 0, the more significant the enrichment. The rich factor refers to the ratio of the number of genes among the DEGs located in a number of pathways to the total number of genes in the pathway entries in all of the annotated genes. The greater the rich factor, the greater the degree of enrichment. BF, blossomed flowering; DEGs, differentially expressed genes; FDR, false-discovery rate; HF, half-flowering; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 3
Comparisons of DEGs. (A) Heatmap comparison of DEGs in the most enriched KEGG pathways during the three development stages. (B) Heatmap comparison of DEGs in the most enriched KEGG pathways between ‘Chocolate’ and ‘Dutch Red’. DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Table 2
Putative structural genes in anthocyanin biosynthesis identified from DEGs.
| Unigene | Annotation | FPKM-SF | FPKM-HF | FPKM-BF | Log2 Ratio (BF/SF) | FPKM-YF | Log2 Ratio (BF/YF) |
|---|---|---|---|---|---|---|---|
| DN17923_c1_g3 | CHS-like protein | 2,593.0 | 1,906.1 | 185.4 | −3.80 | 2,667.3 | −3.8 |
| DN16695_c0_g5 | CHS | 80.4 | 760.5 | 298.4 | 1.9 | 2,187.2 | −2.9 |
| DN10857_c0_g4 | Probable chalcone – flavonone isomerase 3 isoform X1 (CHI) | 501.8 | 344.7 | 38.0 | −3.7 | 561.0 | −3.9 |
| DN15898_c0_g1 | Chalcone – flavonone isomerase (CHI) | 152.8 | 139.5 | 20.0 | −2.9 | 237.0 | −3.6 |
| DN14172_c2_g1 | F3H | 976.4 | 886.8 | 232.7 | −2.1 | 1,440.4 | −2.6 |
| DN22055_c1_g3 | Flavonol synthase/F3H | 5.1 | 7.3 | 11.9 | 1.2 | 1.4 | 3.1 |
| DN22236_c1_g1 | Flavonol synthase/F3H | 296.2 | 125.1 | 3.0 | −6.6 | 257.7 | −6.4 |
| DN16064_c1_g1 | Flavonoid 3′,5′-hydroxylase 1-like (F3′5′H) | 1,422.9 | 1,389.6 | 1,138.9 | −0.3 | 64.0 | 4.2 |
| DN8362_c0_g1 | Flavonoid 3′,5′-hydroxylase 1-like (F3′5′H) | 6.6 | 14.1 | 2.3 | −1.5 | 20.0 | −3.1 |
| DN14193_c0_g5 | DFR | 7.5 | 8.8 | 2.4 | −1.6 | 18.5 | −2.9 |
| DN16691_c4_g1 | DFR | 67.8 | 259.7 | 117.8 | −0.8 | 570.8 | −2.3 |
| DN12990_c1_g6 | Anthocyanidin reductase (ANR) | 64.7 | 331.3 | 90.8 | 0.5 | 323.2 | −1.8 |
| DN13257_c0_g1 | Anthocyanidin reductase (ANR) | 27.8 | 249.5 | 85.3 | 1.6 | 277.8 | −1.7 |
| DN12119_c3_g2 | Anthocyanidin 3-O-glucosyltransferase 7-like flavonoid 3-O-glucosyltransferase (UFGT) | 191.51 | 222.9 | 58.1 | −1.7 | 250.3 | 0.38 |
| DN13065_c1_g1 | Anthocyanin 3′-O-beta-glucosyltransferase-like (UFGT) | 3.74 | 2.6 | 0.24 | −4.0 | 11.2 | −5.54 |
| DN13592_c1_g3 | Anthocyanidin 3-O-glucosyltransferase-like (UFGT) | 155.8 | 42.5 | 3.1 | −5.67 | 196.8 | −6.0 |
[i] BF, blossomed flowering; CHI, chalcone isomerase; CHS, chalcone synthase; DEGs, differentially expressed genes; DFR, dihydroflavonol 4-reductase; F3H, flavanone-3-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; FPKM, fragments per kilobase of transcript per million mapped reads; HF, half flowering.
Figure 4
The anthocyanin biosynthesis process with its core metabolites and enzymes and the expression levels of core enzyme genes. Enzyme names and expression patterns are indicated at the side of each step. Colour boxes from left to right represent unigenes showing lower or higher expression level in the colour bracts of ‘Dutch Red’ of SF, HF, BF and YF, respectively. ANS, anthocyanidin synthase; BF, blossomed flowering; CHI, chalcone isomerase; CHS, chalcone synthase; C4H, cinnamate-4-hydroxylase; DFR, dihydroflavonol 4-reductase; F3H, flavanone-3-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; HF, half-flowering; PAL, phenylalanine ammonia lyase.
Figure 5
Distribution and selection of key differently expressed TFs associated with anthocyanin biosynthesis. (A) Distribution of TF family of unigenes. (B) Clustering heat maps of significantly enriched differently expressed TFs including MYB, bHLH and WD during three development stages and between species ‘Chocolate’ and ‘Dutch Red’. TF, transcription factors.
Table 3
Differently expressed genes in transcription factor families of MYB and WD40.
| Unigene | Annotation | FPKM-SF | FPKM-HF | FPKM-BF | FPKM-YF | Regulation |
|---|---|---|---|---|---|---|
| DN13707_c0_g2 | WD-repeat protein | 306.8 | 283.4 | 244.7 | 226.5 | Whole flowering |
| DN14303_c2_g4 | WD-repeat protein | 90.3 | 97.0 | 97.2 | 101.5 | periods |
| DN17014_c0_g2 | MYB_superfamily | 613.5 | 1,695.1 | 531.6 | 718.1 | |
| DN20674_c0_g1 | WD-repeat protein | 121.4 | 131.1 | 136.2 | 67.3 | |
| DN15014_c1_g1 | MYB_superfamily | 66.3 | 27.2 | 13.4 | 11.8 | Early flowering |
| DN11279_c3_g3 | MYB_superfamily | 105.1 | 64.9 | 0.8 | 38.9 | periods |
| DN19744_c1_g3 | MYB_superfamily | 160.6 | 132.0 | 45.9 | 53.1 | |
| DN12166_c1_g8 | MYB_superfamily | 16.3 | 10.9 | 41.8 | 6.4 | Blossom periods in |
| DN12860_c2_g5 | MYB_superfamily | 4.5 | 5.2 | 22.7 | 2.8 | ‘Dutch Red’ |
| DN15887_c1_g3 | MYB_superfamily | 14.1 | 10.7 | 36.5 | 2.8 | |
| DN22635_c0_g1 | MYB_superfamily | 7.1 | 11.2 | 26.8 | 3.3 | |
| DN12661_c0_g1 | WD-repeat protein | 1.9 | 5.5 | 36.5 | 36.2 | Blossom periods |
| DN21970_c4_g5 | MYB_superfamily | 15.3 | 121.8 | 220.9 | 298.7 |
[i] FPKM, fragments per kilobase of transcript per million mapped reads; HF, half-flowering.
Figure 6
qRT-PCR validations of 14 putative genes involved in anthocyanin biosynthesis and regulations. The histograms represent expression determined by qRT-PCR (left y-axis), while lines represent expression by RNA-Seq in FPKM values (right y-axis). The x-axis in each chart represents the SF, HF, BF and YF, respectively. For qRTPCR assays, the mean was calculated from three biological replicates. For RNA-Seq, each point is the mean of three biological replicates. BF, blossomed flowering; HF, half-flowering.
Table S1
RNA sequencing data and corresponding quality control.
| Sample | Raw reads | Raw bases | Clean reads | Clean bases | Error rate (%) | Q20 (%) | Q30 (%) | GC content (%) | Accession number |
|---|---|---|---|---|---|---|---|---|---|
| BF_1 | 54,170,030 | 8,179,674,530 | 53,271,940 | 7,920,660,194 | 0.0243 | 98.33 | 94.81 | 48.94 | SRR17783056 |
| BF_2 | 55,251,576 | 8,342,987,976 | 54,405,312 | 8,096,016,364 | 0.0241 | 98.41 | 94.99 | 48.87 | SRR17783055 |
| BF_3 | 49,533,034 | 7,479,488,134 | 48,632,006 | 7,241,216,477 | 0.0244 | 98.29 | 94.7 | 49.33 | SRR17783054 |
| HF_1 | 47,378,590 | 7,154,167,090 | 46,432,354 | 6,880,585,840 | 0.0248 | 98.14 | 94.35 | 50.69 | SRR17783059 |
| HF_2 | 47,325,446 | 7,146,142,346 | 46,485,250 | 6,893,639,691 | 0.0244 | 98.27 | 94.67 | 49.13 | SRR17783058 |
| HF_3 | 50,665,694 | 7,650,519,794 | 49,743,012 | 7,381,231,384 | 0.0244 | 98.28 | 94.67 | 48.55 | SRR17783057 |
| SF_1 | 43,599,698 | 6,583,554,398 | 42,859,044 | 6,355,814,159 | 0.0247 | 98.16 | 94.36 | 48.96 | SRR17783064 |
| SF_2 | 44,423,394 | 6,707,932,494 | 43,751,414 | 6,485,935,396 | 0.024 | 98.48 | 95.15 | 48.33 | SRR17783063 |
| SF_3 | 47,696,422 | 7,202,159,722 | 46,810,850 | 6,927,255,232 | 0.0247 | 98.16 | 94.38 | 48.97 | SRR17783060 |
| YF_1 | 45,005,912 | 6,795,892,712 | 44,227,366 | 6,543,730,785 | 0.0241 | 98.4 | 95 | 49.34 | SRR17783053 |
| YF_2 | 47,249,416 | 7,134,661,816 | 46,353,918 | 6,861,606,053 | 0.0241 | 98.41 | 95.05 | 50.28 | SRR17783062 |
| YF_3 | 44,148,530 | 6,666,428,030 | 43,297,006 | 6,412,165,282 | 0.0241 | 98.39 | 94.99 | 49.7 | SRR17783061 |
[i] Read Number: Pair-end Reads number in Clean Data; Base Number: Total base number in Clean Data; GC Content: The percentages of G and C in Clean Data constitute the total base; Q20/30: The percentage of bases in which Clean Data quality is greater than or equal to 20 or 30; Accession number: Accession number of all raw data in the SRA Sequence Data base in the NCBI.
[ii] BF, blossomed flowering; HF, half-flowering; NCBI, National Center for Biotechnology Information; SRA, short read archive.
Table S2
Numbers of transcripts in the 12 sequenced libraries.
| seq_id | FPKM (<0.5) | FPKM (0.5–5) | FPKM (5–100) | FPKM (>100) | SUM (30.5) |
|---|---|---|---|---|---|
| SF_1 | 65,107 | 61,397 | 31,536 | 1,647 | 94,580 |
| SF_2 | 62,631 | 63,656 | 31,774 | 1,626 | 97,056 |
| SF_3 | 64,446 | 62,140 | 31,444 | 1,657 | 95,241 |
| HF_1 | 80,443 | 49,508 | 27,811 | 1,925 | 79,244 |
| HF_2 | 67,574 | 59,459 | 30,864 | 1,790 | 92,113 |
| HF_3 | 65,607 | 60,672 | 31,696 | 1,712 | 94,080 |
| BF_1 | 67,310 | 59,403 | 31,170 | 1,804 | 92,377 |
| BF_2 | 66,614 | 60,394 | 30,896 | 1,783 | 93,073 |
| BF_3 | 76,848 | 51,186 | 29,570 | 2,083 | 82,839 |
| YF_1 | 76,226 | 51,191 | 30,451 | 1,819 | 83,461 |
| YF_2 | 84,464 | 45,418 | 27,806 | 1,999 | 75,223 |
| YF_3 | 82,758 | 46,325 | 28,622 | 1,982 | 76,929 |
[i] BF, blossomed flowering; FPKM, fragments per kilobase of transcript per million mapped reads; HF, half-flowering.
Table S3
Functional annotation of transcripts and unigenes in databases.
| Transcript number (%) | Unigene number (%) | |
|---|---|---|
| NR | 104,303 (0.6532) | 34,189 (0.4923) |
| Swiss-Prot | 82,193 (0.5147) | 26,589 (0.3828) |
| Pfam | 73,262 (0.4588) | 24,101 (0.347) |
| COG | 25,088 (0.1571) | 7,537 (0.1085) |
| GO | 71,758 (0.4494) | 23,229 (0.3345) |
| KEGG | 47,453 (0.2972) | 14,838 (0.2136) |
| Total_anno | 1,05,993 (0.6638) | 35,183 (0.5066) |
| Total | 1,59,687 (1) | 69,453 (1) |
[i] COG, Clusters of Orthologous Groups of Proteins; KEGG, Kyoto Encyclopedia of Genes and Genomes; NR, non-redundant.
Table S4
Detailed annotation of differentially expressed MYBs and WD proteins.
| Unigene | Hit_name | Description | Identity (%) | Similarity (%) | KO | Paths | Swiss-Prot |
|---|---|---|---|---|---|---|---|
| DN13707_c0_g2 | XP_009415403.1 | WD-repeat protein | 88 | 95.3 | – | – | gi|74698589|sp|Q9Y7K5.2|YGI3_SCHPO |
| DN14303_c2_g4 | ONM38806.1 | WD-repeat protein | 76.8 | 85.4 | K10752 | – | gi|22096353|sp|O22607.3|MSI4_ARATH |
| DN17014_c0_g2 | XP_009404081.1 | MYB_superfamily | 88 | 91.6 | – | – | gi|115502385|sp|O80622.2|EXP15_ARATH |
| DN20674_c0_g1 | XP_010931565.1 | WD-repeat protein | 79.7 | 91.5 | – | – | gi|75318693|sp|O80775.2|WDR55_ARATH |
| DN15014_c1_g1 | XP_009385043.1 | MYB_superfamily | 54.9 | 63.7 | K14491 | map04075 | gi|75323583|sp|Q6H805.1|ORR24_ORYSJ |
| DN11279_c3_g3 | XP_017696697.1 | MYB_superfamily | 95.6 | 97.8 | K09422 | – | gi|75317981|sp|O22264.1|MYB12_ARATH |
| DN19744_c1_g3 | XP_009418439.1 | MYB_superfamily | 76.3 | 82.4 | – | – | gi|75330977|sp|Q8S9H7.1|DIV_ANTMA |
| DN12166_c1_g8 | XP_016205675.1 | MYB_superfamily | 86.2 | 92.6 | K09422 | – | gi|56749347|sp|Q8LPH6.1|MYB86_ARATH |
| DN12860_c2_g5 | XP_023531843.1 | MYB_superfamily | 49.8 | 59.8 | K09422 | – | gi|75333993|sp|Q9FKL2.1|MYB36_ARATH |
| DN15887_c1_g3 | XP_009408601.1 | MYB_superfamily | 44.7 | 55 | – | – | gi|75338846|sp|Q9ZQ85.2|EFM_ARATH |
| DN22635_c0_g1 | PSS31516.1 | MYB_superfamily | 66.9 | 74.4 | K09422 | – | gi|75335856|sp|Q9M2Y9.1|RAX3_ARATH |
| DN12661_c0_g1 | XP_009389455.1 | WD-repeat protein | 72.9 | 78.9 | K14963 | – | gi|82232080|sp|Q5M786.1|WDR5_XENTR |
| DN21970_c4_g5 | XP_009405310.1 | MYB_superfamily | 59.5 | 66.4 | – | – | gi|75330977|sp|Q8S9H7.1|DIV_ANTMA |
Table S5
Primers used for qRT-PCR analysis.
| Genes | Sequences (5′-3′) | Product size |
|---|---|---|
| ANR 12990 | CAGCTGAAGCAGATGCAGAG CTCCGGATTCTCCGACATTA | 147 |
| ANR 13257 | TCACATCATCCTCACCGTGT AAGGGGCTGGAGGAGATTTA | 103 |
| CHI 10857 | ACGGAGTTCTTCCAGAGCAA TTTCTGCATCATCTCCACCA | 165 |
| CHI 15898 | GCTCGAACACCTCCTTGAAC CGACAAGTTCACGAGGATCA | 155 |
| CHS 16695 | TGAGGGAGAACCCGAATATG GCAGAAGACGAGGTGTGTGA | 161 |
| CHS 17923 | ATGGCCTGAAGATGGATCAG GCGTCCTCTTCATTCTCGAC | 186 |
| DFR 16691 | TGCCCGTTTAACTCATTTCC ACTGTGGAGAAGGAGCAGGA | 101 |
| DFR 14193 | GTGGTGTTCACCTCCTCGAT CGCTAGCTCTACCCCCTTCT | 189 |
| F3′5′H 8362 | CTCCTCCTCCGCTACCTTCT GGTACATGATGGGGCCATAG | 160 |
| F3′5′H 16064 | ATGTGGAGGTTGCAGAGCTT GCGTACGAAGGACAGGACAT | 80 |
| F3H 14172 | ACGTTGATTGGACCGAACAT CTGCGAGATCTACACGGACA | 186 |
| F3H 22055 | GCCTCTTGCATAGCTTCACC GGTGATCCTGCCAACTCATT | 83 |
| F3H 22236 | AAGGAGAAGTACGCGTCCAA TGTATTCCTCGTTCGCCTTC | 184 |
| MYB 11279 | TTTCTCCCATGCTGCTTTCT TCACAATGCCACGGTTAAGA | 160 |
| MYB 12860 | ACCCTATCGTCGAACCACTG TCGACCAAGAGAGCCAACTT | 174 |
| MYB 15887 | CATCTTCCTCCTTGGAGCTG TCTGGATCTGCGAGGAAAGT | 138 |
| MYB 18449 | TTCCACTTGGACTTGCACTG AAGAGCTTCACGGAGACGAA | 110 |
| MYB 12166 | GCCGTCCAAACACATCTTCT GATTTGGTCGAGCCAGAGAG | 85 |
| MYB 15014 | ATTTCTGCAGATGGCTTGCT ATCAATTGGGCATCGAGAAG | 99 |
| MYB 17014 | CTACGGAGGAGGATGGATGA AAGTTGCCATACCCACAAGC | 93 |
| MYB 19744 | CGAAGAACACTGCACTGGAA TATCCACTGCCTCCTCCATC | 190 |
| MYB 21970 | GCACCGGTAAGTGCGTTAAT TTCGTTTCCATCCATCCATT | 159 |
| MYB 22635 | GTCGCAATAACCGACCATCT ATCATCTGCAGCCTCTTCGT | 125 |
| UFGT 12119 | TCGGCGATCAAGGTAGATTC GGATGAAGCTGAGCAACTC | 137 |
| UFGT 13065 | CGGAGGTAGCCATCAACAAT ATCCCACATGGTGGTCTTGT | 143 |
| UFGT 13592 | CTGGAGCTAAGGAGCAAGGA TACCAGGGGTAATGGGATGA | 118 |
| WD 12661 | CAGTAGCCATGGCAGCTACA ATGGCGGATGAGTACCAAAG | 109 |
| WD 13707 | CCAAAGAAGTCCAGCTCCTG AATTTTTGGCAATGGCAGAG | 91 |
| WD 14303 | CAGGCGATGTGAAGAATTGA GCAACTGATGGGGGTAAAGA | |
| WD 20674 | TTCGTCCCTTTGAGTTTGCT ATGGGCTGTAACACGTAGCC |
[i] CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone-3-hydroxylase.