Genetic and flower volatile diversity in two natural populations of Hyssopus officinalis L. in Bulgaria

Figures & data

Figure 1. Developmental stages of Hyssopus officinalis L. single flowers.

Table 1. Nucleotide sequences of forward and reverse primers used for PCR amplification of SRAP markers.

Forward primer		Reverse primer
ME1	5′-GACTGCGTACGAATTAAA-3′	EM1	5′-TGAGTCCAAACCGGAAA-3′
ME2	5′-GACTGCGTACGAATTTGC-3′	EM2	5′-TGAGTCCAAACCGGAGC-3′
ME3	5′-GACTGCGTACGAATTATT-3′	EM3	5′-TGAGTCCAAACCGGTAA-3′
ME4	5′-GACTGCGTACGAATTATA-3′	EM4	5′-TGAGTCCAAACCGGACC-3′
ME5	5′-GACTGCGTACGAATTCAA-3′	EM5	5′-TGAGTCCAAACCGGAAG-3′
ME6	5′-GACTGCGTACGAATTTTT-3′	EM6	5′-TGAGTCCAAACCGGAGA-3′
ME7	5′-GACTGCGTACGAATTAGC-3′	EM7	5′-TGAGTCCAAACCGGTCC-3′
ME8	5′-GACTGCGTACGAATTCAC-3′	EM8	5′-TGAGTCCAAACCGGCTG-3′
ME9	5′-GACTGCGTACGAATTAAT-3′	EM9	5′-TGAGTCCAAACCGGTTC-3′
ME10	5′-GACTGCGTACGAATT TCA-3′	EM10	5′-TGAGTCCAAACCGGTGA-3′

Figure 2. Distribution of data for Nei’s gene diversity index (a) and Shannon’s index (b) among plants from the two analysed populations, Godech and Izvor.

Table 2. Average gene diversity (GD)/polymorphic information content (PIC) for each SRAP marker/primer pair as well as range of variation per the average gene diversity for all SRAP markers.

Marker	Average GD/PIC	SD	SEM
EM1 ME2	0.1869	0.147	0.0121
EM2 ME3	0.1567	0.1309	0.0114
EM3 ME1	0.1548	0.1447	0.0193
EM4 ME1	0.1679	0.1467	0.0131
EM5 ME4	0.1638	0.1351	0.0108
EM6 ME6	0.1544	0.138	0.0112
EM10 ME1	0.175	0.1564	0.0174
EM10 ME10	0.1677	0.131	0.0148
MAX Average GD/PIC	0.1869
MIN Average GD/PIC	0.1544
Average	0.1659

Table 3. Genetic variation within the two analysed populations.

Population	Pop size	Number of loci	Number of polymorphic loci	% polymorphic loci	h* (Mean ± SD)	I* (Mean ± SD)
Godech	28	927	650	70.12	0.1453 ± 0.1506	0.2414 ± 0.2176
Izvor	31	927	744	80.26	0.1648 ± 0.1544	0.2727 ± 0.2161

“h”, average Nei’s gene diversity [20], “I” = Shannon’s information index [21].

Asterisk (*) indicates that the values vary significantly between groups at p ≤ 0.01.

Figure 3. Principal Coordinates Analysis (PCoA) (a) and UPGMA clustering of Jaccard indices (b) of samples from the two analysed populations. Blue diamonds indicate samples from population Godech; red squares indicate samples from population Izvor. Ellipses and semicircles marked with 1 and 2 indicate clusters of samples belonging to population Godech and Izvor, respectively.

Figure 4. Genetic structure of two Hyssopus officinalis L. populations based on SRAP marker data. (a) Estimation of the most probable K using the delta K method by Evano et al. [19]. (b) Bar plot representing the genetic structure at K = 2 for populations Godech and Izvor.

Table 4. Genetic differentiation between the two analysed populations.

Fst	Gst	Nei's original distance	Nei's original identity	Nei's distance after Lynch and Milligan	Nm
0.0487	0.0645	0.0256	0.9747	0.0073	7.2481

“Nm”, gene flow.

Table 5. Summary AMOVA table based on analysis of two natural H. officinalis populations in Bulgaria.

Source of variation	df	SS	MS	Est. Var.	% of total variation
Among Populations	1	291.793	291.793	7.38	9%
Within Populations	57	4254.445	74.639	74.639	91%
Total	58	4546.237		82.02	100%

SS, Sum of Squares; MS, Mean Squares; Est. Var., Estimate of Variance; df, degree of freedom.

Table 6. Mean percentage and range of variation for all identified compounds in the two analysed populations and the two identified metabolite clusters as seen in Figure 4. Compounds shown in bold are part of ISO 9841:2007[23].

Compounds			Godech			Izvor			Cl. A			Cl. B
	RI	RT	Mean	MIN	MAX	Mean	MIN	MAX	Mean	MIN	MAX	Mean	MIN	MAX
α-Thujene**	921	8.89	0.14 ± 0.09	0.07	0.37	0.13 ± 0.06	0.08	0.30	0.23 ± 0.07	0.15	0.37	0.09 ± 0.01	0.07	0.11
α-Pinene**	928	9.11	2.26 ± 0.53	1.26	3.07	1.99 ± 0.72	0.70	2.91	1.25 ± 0.44	0.70	1.93	2.45 ± 0.31	1.71	3.07
Camphene**	945	9.62	0.05 ± 0.04	0.02	0.14	0.04 ± 0.03	0.02	0.12	0.09 ± 0.03	0.05	0.14	0.03 ± 0	0.02	0.04
Sabinene	970	10.40	2.3 ± 0.58	0.96	3.29	2.39 ± 0.46	1.30	3.25	2.13 ± 0.26	1.44	2.54	2.48 ± 0.52	0.96	3.29
β-Pinene**	975	10.54	9.23 ± 4.66	5.62	20.04	7.93 ± 2.33	5.61	14.72	12.76 ± 4.25	6.44	19.57	6.74 ± 0.85	5.61	9.05
β-Myrcene	989	10.95	0.92 ± 0.3	0.00	1.29	1.04 ± 0.14	0.75	1.30	0.96 ± 0.1	0.75	1.13	1 ± 0.27	0.00	1.30
Eucalyptol**	1035	12.19	38.61 ± 16.43	1.75	56.88	35.93 ± 13.27	1.30	49.65	21.29 ± 12.69	1.98	40.07	44.44 ± 6.4	26.11	56.88
cis-Ocimene^#	1039	12.49	11.56 ± 3.34	7.71	19.43	14.37 ± 5.81	5.90	27.31	10.05 ± 2.39	5.90	15.85	14.23 ± 5.12	7.71	27.31
Sabinene hydrate	1073	13.55	t	0.00	0.14	x	x	x	t	0.00	0.14	x	x	x
Terpinolene	1087	13.97	0.05 ± 0.12	0.00	0.47	0.16 ± 0.18	0.00	0.58	0.1 ± 0.09	0.00	0.23	0.12 ± 0.18	0.00	0.58
cis-Thujone**	1109	14.63	0.02 ± 0.04	0.00	0.11	0.03 ± 0.05	0.00	0.14	0.09 ± 0.03	0.03	0.14	x	x	x
Unidentified compound**	1157	16.12	0.51 ± 1.06	0.00	3.55	0.45 ± 0.76	0.00	2.52	1.61 ± 1.1	0.00	3.55	0.03 ± 0.19	0.00	1.27
Pinocamphone **	1162	16.27	0.05 ± 0.12	0.00	0.42	0.03 ± 0.06	0.00	0.27	0.09 ± 0.15	0.00	0.42	x	x	x
Pinocarvone**	1164	16.33	0.68 ± 1.77	0.00	8.42	0.62 ± 1.39	0.00	5.71	2.53 ± 2.53	0.00	8.42	x	x	x
iso-Pinocamphone **	1178	16.75	6.97 ± 12.42	0.00	35.97	7.84 ± 13.83	0.00	45.39	23.72 ± 8.32	13.53	37.67	0.04 ± 0.26	0.00	1.72
δ-Elemene	1333	21.11	0.45 ± 0.25	0.00	1.23	0.35 ± 0.18	0.00	0.91	0.44 ± 0.15	0.14	0.70	0.38 ± 0.24	0.00	1.23
α-Copaene	1375	22.29	0 ± 0.03	0.00	0.14	0 ± 0.01	0.00	0.06	x	x	x	t	0.00	0.14
β-Bourbonene	1383	22.50	0.32 ± 0.15	0.00	0.58	0.2 ± 0.17	0.00	0.73	0.33 ± 0.14	0.00	0.52	0.2 ± 0.14	0.00	0.58
α-Gurjunene	1405	23.10	0 ± 0.01	0.00	0.07	0 ± 0.01	0.00	0.04	x	x	x	t	0.00	0.07
Caryophyllene^#	1418	23.43	0.88 ± 0.94	0.00	3.69	0.96 ± 0.69	0.00	3.62	0.57 ± 0.4	0.00	1.22	1.05 ± 0.9	0.00	3.69
β-Copaene^##	1429	23.69	0.06 ± 0.14	0.00	0.72	0.12 ± 0.09	0.00	0.35	0.05 ± 0.05	0.00	0.15	0.1 ± 0.13	0.00	0.72
Aromadendrene	1442	24.02	0.28 ± 0.17	0.00	0.75	0.25 ± 0.11	0.08	0.63	0.21 ± 0.11	0.07	0.45	0.29 ± 0.15	0.00	0.75
α-Humulene^##	1456	24.34	0.15 ± 0.16	0.00	0.66	0.18 ± 0.1	0.07	0.62	0.11 ± 0.06	0.00	0.22	0.19 ± 0.15	0.00	0.66
Germacrene D	1482	24.99	1.4 ± 0.93	0.15	4.66	1.3 ± 0.65	0.52	2.85	0.81 ± 0.21	0.53	1.22	1.55 ± 0.84	0.15	4.66
Varidiflorene	1492	25.23	0.52 ± 0.21	0.24	1.10	0.46 ± 0.14	0.26	0.87	0.36 ± 0.09	0.24	0.55	0.53 ± 0.18	0.24	1.10
Elixene	1497	25.34	1.52 ± 0.79	0.49	4.04	1.08 ± 0.4	0.54	2.63	1.02 ± 0.4	0.49	2.09	1.4 ± 0.71	0.52	4.04
Pentacosane	2503	44.86	0.19 ± 0.06	0.08	0.37	0.19 ± 0.06	0.09	0.30	0.19 ± 0.07	0.08	0.27	0.19 ± 0.06	0.10	0.37
Hexacosane^#	2600	46.37	0.11 ± 0.04	0.04	0.23	0.1 ± 0.03	0.05	0.17	0.1 ± 0.04	0.04	0.16	0.11 ± 0.04	0.05	0.23
Heptacosane	2702	47.84	0.66 ± 0.22	0.31	1.35	0.57 ± 0.16	0.29	0.88	0.6 ± 0.2	0.29	0.84	0.61 ± 0.2	0.30	1.35
Octacosane^#	2800	49.26	0.23 ± 0.09	0.10	0.55	0.22 ± 0.06	0.12	0.36	0.21 ± 0.07	0.10	0.31	0.23 ± 0.08	0.12	0.55
Nonacosane	2901	50.63	1.32 ± 0.49	0.61	3.15	1.07 ± 0.28	0.61	1.67	1.16 ± 0.4	0.61	1.67	1.2 ± 0.44	0.61	3.15
Triacontane	2999	51.95	0.3 ± 0.11	0.15	0.67	0.27 ± 0.08	0.16	0.45	0.26 ± 0.09	0.15	0.41	0.29 ± 0.11	0.16	0.67
Hentriacontane	3101	53.24	1.31 ± 0.52	0.64	3.37	0.96 ± 0.25	0.52	1.55	1.04 ± 0.37	0.52	1.73	1.15 ± 0.48	0.61	3.37

# and ## indicate that the mean values vary statistically significantly between populations at p ≤ 0.05 and p ≤ 0.01, respectively.

** Indicate that the mean values vary statistically significantly between clusters A and B as shown in Figure 4.

Figure 5. Principal Component Analysis (PCA) of samples from the two analysed populations based on GC/MS data for compound abundances. Red squares indicate samples from population Godech; blue triangles indicate samples from population Izvor. The two ellipses marked with A and B indicate the observed two clusters corresponding to the two different chemotypes.

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