Single nucleotide polymorphism analysis in plastomes of eight Catharanthus roseus cultivars

Figures & data

Figure 1. Photographs of eight varieties of Catharanthus roseus. (a) Patricia White (PW); (b) First Kiss Polka Dot (FKD); (c) First Kiss Peach (FKP); (d) Experimental Rose Pink (ER); (e) Experimental Deep Pink (ED); (f) Cooler Orchid (CO); (g) Victory Red (VR); (h) Blue Pearl (BP). Note: The colourless and coloured flowers are red and blue framed, respectively.

Table 1. Characteristics of eight varieties of Catharanthus roseus.

	No.	Name of variety	Code	Petal colour	Eye colour	TIA content	Accession No.
Colour-less	1	Patricia White	PW	White	White eye with yellow center	Low	PRJNA417292
	2	First Kiss Polka Dot	FKD	Milky white	Red radiating eye with small red center	Low	PRJNA417297
	3	First Kiss Peach	FKP	Whitish pink	White and Pink radiating eye with yellow center	Low	PRJNA417299
Coloured	4	Experimental Rose Pink	ER	Pale pink	Red radiating eye with red center	High	PRJNA417300
	5	Experimental Deep Pink	ED	Pink	Dark pink radiating eye with pink center	High	PRJNA417303
	6	Cooler Orchid	CO	Pinkish red	White radiating eye with yellow center	High	PRJNA417309
	7	Victory Red	VR	Deep red centre	Dark red eye with small red center	High	PRJNA417311
	8	Blue Pearl	BP	Purple blue	White large radiating base with yellow center	High	PRJNA417312

Table 2. Summary of alignment results to the chloroplast reference genome (GenBank No. KC561139).

Variety	Total no. reads	No. mapped reads	Coverage	% mapped reads
PW	73,925,432	12,156,126	7,003.41	16.44
FKD	73,995,072	10,959,007	6,340.78	14.81
FKP	74,686,038	11,831,482	6,847.73	15.84
ER	73,981,268	9,309,744	5,387.75	12.58
ED	75,019,248	11,661,747	6,745.48	15.54
CO	74,722,670	12,998,306	7,491.05	17.39
VR	75,406,360	10,571,820	6,115.36	14.01
BP	73,061,630	12,235,439	7,040.31	16.74

Figure 2. Number of total and unique SNPs detected for each C. roseus variety. Note: More information about variety abbreviations is provided in Table 1.

Figure 3. Locations of SNPs in the chloroplast genome. IGS, intergenic spacer; NS, non-synonymous; S, synonymous; I, intron; tRNA, tRNA-related sequence; rRNA, rRNA-related sequence. Note: More information about variety abbreviations is provided in Table 1.

Table 3. Unique SNPs and their positions in the chloroplast genome.

Variety	Number of unique SNPs	Positions
PW	36	286, 293, 4797, 32316, 32657, 32660, 32673, 32675, 32683, 32714, 37702, 37703, 43547, 54156, 77058, 77059, 86366, 86388, 86492, 86493, 97250, 97254, 97257, 97293, 97298, 111009, 111012, 111353, 111354, 111359,111378,115013, 121734, 121736, 144530, 145946.
FKD	4	86639, 86669, 101836, 131548
FKP	1	145811
ER	2	52027, 131530
ED	1	145952
CO	18	6362, 32324, 32447, 32519, 32534, 32571, 32589, 52142, 69044, 95377, 95393, 95396, 99952, 99970, 121738, 130572, 131771, 154031
VR	13	109364, 109367, 109374, 109390, 109413, 109421, 109431, 109436, 143709, 145320, 145339, 145368, 145385
BP	7	33205, 52132, 60684, 93301, 93308, 93319, 133426

Table 4. Summary of SNPs hotspots analysis results.

No.	Hotspot position (size)	Location	Variety	No. SNPs
1	≈ 6,340 .. 6,400 (60 nt)	Between rps16 and trnQ-UUG genes	CO	7
2	≈ 32,260 .. 32,720 (460 nt)	Downstream, within and upstream of trnT-GGU gene	PW, FKD, FKP, ER, ED, CO and VR	27–37
3	≈ 51,960 .. 52,160 (200 nt)	Between ndhC and trnV-UAC genes	PW, FKP, ER, ED, CO and BP	5–7
4	≈ 86, 340 .. 86,680 (340 nt)	In intron of rpl2 gene	PW, FKD, ER and ED	5–9
5	≈ 99,920 .. 100,000 (80 nt)	In CDS and upstream of rps12 gene	FKP and ER	5
6	≈ 109,360 .. 109,440 (80 nt)	Within trnR-ACG gene	CO and VR	5–11
7	≈ 111,000 .. 111,380 (380 nt)	Within and upstream of ycf1p gene	PW	9
8	≈ 131,520 .. 131,840 (320 nt)	Downstream of rrn5 gene and between rrn5 and rrn4.5 genes	FKD, FKP, ER, CO and VR	6-9
9	≈ 132,920 .. 133,440 (520 nt)	Within rrn23 gene	FKD and BP	7-9
10	≈ 154,020 .. 154,060 (40 nt)	In intron of rpl2 gene	ER, VR and BP	5

Table 5. The total of chloroplast DIPs ordered by their position in the genome.

Variety	Reference position	Reference	Allele variations	Frequencies	Counts	Coverage	Location
CO	1714	T	-	75	3	4	IGS
FKD	4558	–	-/G	50.0/50.0	3/3	6	IGS
FKD	4560	----	AAAT	50	3	6	IGS
FKD	4662	A	-	100	5	5	IGS
CO	4778	AA	--	83.3	5	6	IGS
CO	4799	T	-/T	60.0/40.0	3/2	5	IGS
BP	4799	T	-	100	5	5	IGS
BP	5830	-----	-----/AATTT	50.0/50.0	2/2	4	I
PW	7160	–	T	100	4	4	IGS
CO	7160	T	-	100	4	4	IGS
FKP	8126	T	T/-	60.0/40.0	3/2	5	IGS
BP	8126	TTT	---	75	3	4	IGS
PW	9926	-----	TATTT	75	3	4	IGS
PW	12946	A	A/-	50.0/50.0	4/4	8	IGS
FKD	12946	A	A/-	50.0/50.0	2/2	4	IGS
ED	12946	A	-	83.3	5	6	IGS
CO	12946	A	A/-	60.0/40.0	3/2	5	IGS
PW	12963	T	-/T	57.1/42.9	4/3	7	IGS
FKD	12963	T	T/-	50.0/50.0	2/2	4	IGS
ED	12963	T	-	83.3	5	6	IGS
BP	12988	TATTTGAA	--------/TATTTGAA	60.0/40.0	3/2	5	IGS
PW	13062	–	-/T	63.6/36.4	7/4	11	IGS
FKD	13062	–	-/T	60.0/40.0	3/2	5	IGS
ED	13062	–	T	71.4	5	7	IGS
CO	13062	–	-/T	50.0/50.0	2/2	4	IGS
BP	14284	-------	-------/TATTTAC	60.0/40.0	3/2	5	IGS
PW	21069	–	-/A	60.0/40.0	3/2	5	NS
PW	28359	-----	TTGTA	80	4	5	IGS
CO	30975	--	CC/--	60.0/40.0	3/2	5	IGS
PW	31078	TGTAC	-----	100	4	4	IGS
CO	33347	A	-	100	4	4	IGS
CO	37707	–	G	100	5	5	IGS
BP	37707	–	G	100	4	4	IGS
FKP	44208	T	T/-	62.5/37.5	5/3	8	I
BP	44882	T	-	100	4	4	I
CO	45972	--------	--------/TAATCGAT	50.0/50.0	2/2	4	IGS
BP	45972	--------	TAATCGAT	80	4	5	IGS
CO	47635	------	ATATAG	75	3	4	IGS
BP	47635	------	------/ATATAG	50.0/50.0	2/2	4	IGS
PW	52934	T	-	100	4	4	IGS
CO	52934	T	-	75	3	4	IGS
BP	54032	T	-	75	3	4	IGS
CO	55424	T	-/T	57.1/42.9	4/3	7	NS
BP	55424	T	-	85.7	6	7	NS
FKD	55429	–	T	66.7	4	6	NS
ED	55429	–	-/T	60.0/40.0	3/2	5	NS
VR	55429	–	-/T	62.5/37.5	5/3	8	NS
FKD	55469	CC	--	77.8	7	9	NS
ER	55469	CC	--	75	3	4	NS
ED	55469	CC	--	66.7	4	6	NS
PW	61304	–	A	80	4	5	IGS
PW	61545	T	-/T	50.0/50.0	2/2	4	IGS
CO	67625	A	-	100	4	4	IGS
BP	69044	C	-	100	4	4	IGS
VR	72136	–	-/T	60.0/40.0	3/2	5	I
PW	79096	T	-	85.7	6	7	NS
FKD	79096	T	-	100	6	6	NS
FKP	79096	T	T/-	64.3/35.7	9/5	14	NS
ER	79096	T	-	76.9	10	13	NS
ED	79096	T	-	90	9	10	NS
CO	79096	T	-	90.9	10	11	NS
PW	79193	T	-	100	7	7	NS
FKD	79193	T	-	86.7	13	15	NS
FKP	79193	T	T/-	60.9/39.1	14/9	23	NS
ER	79193	T	-	80	4	5	NS
ED	79193	T	-	90	9	10	NS
CO	79193	T	-	85.7	6	7	NS
VR	79193	T	T/-	54.5/45.5	6/5	11	NS
BP	79193	T	T/-	60.0/40.0	3/2	5	NS
PW	82536	G	G/-	60.0/40.0	3/2	5	NS
FKP	82536	G	G/-	57.1/42.9	4/3	7	NS
ER	82536	G	G/-	50.0/50.0	3/3	6	NS
ER	82729	–	A	75	3	4	IGS
PW	82752	A	-	75	3	4	IGS
BP	82752	–	A	83.3	5	6	IGS
FKD	113997	T	-/T	50.0/50.0	2/2	4	IGS
BP	115673	T	-	75	3	4	IGS
PW	120668	–	C	66.7	4	6	NS
FKP	120668	–	-/C	50.0/50.0	2/2	4	NS
ER	120668	–	-/C	50.0/50.0	2/2	4	NS
FKD	127365	–	-/T	50.0/50.0	2/2	4	NS

Note: Unique DIPs to a single variety are in bold. IGS = intergenic spacer; NS = nonsynonymous; I = intron.

Figure 4. Neighbour-joining trees with bootstrap for chloroplast SNPs in eight C. roseus varieties. CO, Cooler Orchid; BP, Blue Pearl; FKP, First Kiss Peach; FKD, First Kiss Polka Dot; VR, Victory Red; ER, Experimental Rose Pink; ED, Experimental Deep Pink; RF, Reference plastome (GenBank No. KC561139); PW, Patricia White. Note: Numbers near each node are bootstraps used to derive estimates of standard errors and confidence intervals and the numbers towards the end of each branch represent dNS/dS values (dNS = non-synonymous substitution; dS = synonymous substitution).