Evaluation phenotypes of three Chinese wild thymes and seven European thymes, and construction of F 1 hybrid population
Thymus is a herbaceous perennial or sub-shrub with valuable medicinal and aromatic properties. The types of Thymus are erect-type and creeping-type, which display remarkable differences in morphology. The plant types of 10 different thyme varieties are shown in Fig. 1A (Table 1). Among them, T. rotundifolius (Tr), T. vulgaris ‘Elsbeth’ (Tve), T. thracicus (Tt), and T. vulgaris ‘Fragrantissimus’ (Tvf) are erect-type, and T. serpyllum ‘Aureus’ (Ts), T. guberlinesis ‘Iijin’ (Tg), T. longicaulis (Tl), T. quinquecostatus (Tq), T. quinquecostatus var. przewalskii (Tqp), and T. mongolicus (Tm) are creeping-type. Thyme fertility is shown in Fig. 1B. Tr, Tve, Tg, Tt, Ts, and Tl have only stigma and no pollen (stamen), and they are male-sterile. Tq, Tqp, Tm, and Tvf have both stigma and pollen (stamen), and they are male-fertile (Table 1). The increased genetic diversity of the thymes could be partially attributed to the consistent introgression of Chinese wild thymes into the male-sterility and erect-type European thymes during long cultivation periods and partially to the adaptations of thymes to new environments during spreading.
Table 1
Phenotype survey results of three Chinese wild thymes and seven European thymes.
Species
|
Chemotype
|
Plant type
|
Oil yield (%)
|
Pollen
|
Seed
|
Sterility
|
Flowering phase
|
Thymus quinquecostatus
|
carvacrol
|
creeping
|
0.50
|
yes
|
yes
|
fertile
|
2020.4.20–2020.8.30
|
Thymus quinquecostatus var. przewalskii
|
carvacrol
|
creeping
|
0.50
|
yes
|
yes
|
fertile
|
2020.4.20–2020.8.30
|
Thymus mongolicus
|
thymol
|
creeping
|
0.40
|
yes
|
yes
|
fertile
|
2020.5.12–2020.8.30
|
Thymus rotundifolius
|
thymol
|
erect
|
0.95
|
no
|
no
|
sterile
|
2020.6.20–2020.7.30
|
Thymus vulgaris 'Elsbeth'
|
thymol
|
erect
|
0.75
|
no
|
no
|
sterile
|
2020.6.1–2020.6.20
|
Thymus guberlinesis 'Iijin'
|
thymol
|
creeping
|
0.50
|
no
|
no
|
sterile
|
2020.5.6–2020.5.20
|
Thymus thracicus
|
thymol
|
erect
|
1.20
|
no
|
no
|
sterile
|
2020.5.8–2020.5.30
|
Thymus serpyllum 'Aureus'
|
thymol
|
creeping
|
0.52
|
no
|
no
|
sterile
|
2020.5.22–2020.6.15
|
Thymus longicaulis
|
geraniol
|
creeping
|
0.70
|
no
|
no
|
sterile
|
2020.4.20–2020.5.15
|
Thymus vulgaris 'Fragrantissimus'
|
α–terpineol
|
erect
|
1.25
|
yes
|
yes
|
fertile
|
2020.4.20–2020.5.10
|
Glandular trichomes are specialized hairs found on the surface of about 30% of all vascular plants and are responsible for a significant portion of a plant’s secondary chemistry [35]. Terpenoids are stored and synthesized in glandular trichomes which is an organ that originates from epidermal cells of flowers, leaves, and stems [36]. Glandular trichomes are widely found in lavender, thyme, rosemary, oregano, basil, and other Lamiaceae, with two existing types: peltate and capitate trichomes. The regulation of these trichome-related genes may underlie the regulation of glandular trichomes density to increase the terpenoid content. The adaxial and abaxial planes of thyme leaves are shown in Fig. 1C, E. The most glandular trichomes per unit area (glandular trichomes density) of adaxial were Tve and Tr, followed by Tvf and Tt. The most glandular trichomes per unit area of abaxial (glandular trichomes density) were Tve and Tr. In total, The most glandular trichomes density of leaves are Tve, Tr, Tvf, Tt, and Ts. The EO yield (extraction rate) are shown in Fig. 1D, F (Supplementary Fig. S1). The highest oil yield are Tt (1.20%) and Tvf (1.25%), followed by Tr (0.95%), Tve (0.75%), and Tl (0.70%), respectively. The EO yield of Tq, Tqp, Tm, Tg, and Ts are between 0.40% and 0.50% (Table 1; Supplementary Fig. S1). There was a certain correlation between EO yield and glandular trichomes density (Fig. 1E, F).
The relative contents of EOs compositions of 10 different thymes are shown in Fig. 2A. In this study, the 20 main compositions (relative content > 0.3%) shared by 10 different thymes were counted and showed in Table 2. The most volatile compositions of Tq essential oil are p-cymene and carvacrol, which are account for 23.00% and 20.74%, respectively. The volatile composition with the most Tqp content is carvacrol, which is accounts for 48.37%. The volatile compositions with the most Tm content are thymol (38.57%) and p-cymene (16.40%). The volatile compositions with the most Tve content are thymol (35.43%), p-cymene (18.42%), and γ-terpinene (13.96%). The volatile composition with the highest content of Tr, Tt, Tg, and Ts is thymol, and their contents are 36.02%, 41.04%, 26.26%, and 28.96%, respectively. The volatile compositions with the most Tl content are geraniol (28.54%) and geranyl acetate (34.81%). The volatile compositions with the most Tvf content are α-terpineol (30.84%) and α-terpineol acetate (45.46%).
Table 2
Relative contents of volatile terpenoid compositions in 10 thyme essential oils.
No.
|
Composition
|
RI Cal
|
RI Lit
|
Relative content(%)
|
Tq
|
Tqp
|
Tm
|
Tve
|
Tr
|
Tt
|
Tg
|
Ts
|
Tl
|
Tvf
|
1
|
γ-Terpinene
|
1059
|
1060
|
11.45 ± 0.66
|
10.77 ± 0.71
|
8.19 ± 0.61
|
13.96 ± 0.50
|
13.90 ± 0.51
|
12.61 ± 0.55
|
11.18 ± 1.02
|
10.59 ± 0.32
|
0.69 ± 0.01
|
0.48 ± 0.03
|
2
|
p-Cymene
|
1026
|
1025
|
23.00 ± 1.02
|
15.34 ± 0.52
|
16.40 ± 0.72
|
18.42 ± 1.01
|
18.08 ± 0.20
|
17.98 ± 0.97
|
16.50 ± 1.03
|
12.03 ± 0.54
|
-
|
0.62 ± 0.02
|
3
|
Thymol
|
1292
|
1291
|
2.12 ± 0.25
|
7.59 ± 0.02
|
38.57 ± 2.01
|
35.43 ± 3.02
|
36.02 ± 3.04
|
41.04 ± 4.02
|
26.26 ± 2.02
|
28.96 ± 2.03
|
0.57 ± 0.05
|
0.59 ± 0.06
|
4
|
Carvacrol
|
1304
|
1299
|
20.74 ± 1.02
|
48.37 ± 4.05
|
6.87 ± 0.56
|
2.27 ± 0.23
|
2.48 ± 0.22
|
6.45 ± 0.72
|
1.95 ± 0.05
|
3.78 ± 0.05
|
-
|
-
|
5
|
Geraniol
|
1259
|
1255
|
-
|
-
|
-
|
-
|
-
|
0.49 ± 0.02
|
-
|
0.69 ± 0.06
|
28.54 ± 1.05
|
-
|
6
|
Geraniol acetate
|
1388
|
1382
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
1.50 ± 0.12
|
34.81 ± 2.07
|
-
|
7
|
α-Terpineol
|
1193
|
1189
|
0.95 ± 0.02
|
-
|
1.11 ± 0.08
|
-
|
-
|
-
|
0.75 ± 0.06
|
0.50 ± 0.05
|
0.85 ± 0.03
|
30.84 ± 2.02
|
8
|
α-Terpineol acetate
|
1355
|
1350
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
1.17 ± 0.09
|
45.46 ± 5.05
|
9
|
Eucalyptol
|
1030
|
1032
|
5.78 ± 0.32
|
0.93 ± 0.05
|
7.02 ± 0.23
|
0.81 ± 0.06
|
0.84 ± 0.03
|
0.64 ± 0.07
|
1.71 ± 0.02
|
-
|
0.76 ± 0.07
|
0.67 ± 0.12
|
10
|
endo-Borneol
|
1165
|
1167
|
7.89 ± 0.66
|
1.37 ± 0.22
|
4.96 ± 0.44
|
0.55 ± 0.02
|
1.23 ± 0.02
|
0.56 ± 0.01
|
9.02 ± 0.54
|
4.13 ± 0.33
|
8.17 ± 0.55
|
1.26 ± 0.06
|
11
|
β-Caryophyllen
|
1421
|
1419
|
1.74 ± 0.24
|
1.56 ± 0.34
|
1.75 ± 0.03
|
3.37 ± 0.21
|
4.01 ± 0.24
|
3.11 ± 0.16
|
3.79 ± 0.22
|
3.07 ± 0.03
|
4.19 ± 0.02
|
2.60 ± 0.04
|
12
|
Caryophyllen oxide
|
1585
|
1581
|
1.14 ± 0.02
|
-
|
0.51 ± 0.01
|
0.47 ± 0.02
|
0.74 ± 0.05
|
-
|
0.93 ± 0.12
|
0.34 ± 0.01
|
0.48 ± 0.01
|
-
|
13
|
α-Thujene
|
926
|
929
|
0.91 ± 0.02
|
1.97 ± 0.01
|
1.21 ± 0.02
|
2.09 ± 0.26
|
1.27 ± 0.04
|
1.53 ± 0.05
|
1.07 ± 0.11
|
1.31 ± 0.02
|
-
|
-
|
14
|
Sabinene
|
972
|
974
|
0.81 ± 0.04
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
2.18 ± 0.03
|
15
|
1-Octen-3-ol
|
978
|
980
|
0.64 ± 0.14
|
1.12 ± 0.05
|
2.07 ± 0.33
|
1.20 ± 0.02
|
1.30 ± 0.14
|
0.92 ± 0.01
|
0.88 ± 0.04
|
1.39 ± 0.07
|
0.69 ± 0.03
|
-
|
16
|
β-Myrcene
|
991
|
991
|
0.96 ± 0.02
|
1.09 ± 0.04
|
1.15 ± 0.05
|
1.74 ± 0.13
|
1.37 ± 0.02
|
1.86 ± 0.15
|
0.51 ± 0.01
|
1.12 ± 0.01
|
0.38 ± 0.01
|
1.62 ± 0.25
|
17
|
D-Limonene
|
1028
|
1032
|
0.90 ± 0.05
|
-
|
0.70 ± 0.12
|
0.90 ± 0.01
|
0.79 ± 0.04
|
0.81 ± 0.06
|
1.09 ± 0.25
|
0.77 ± 0.03
|
-
|
6.04 ± 0.72
|
Continued Table 2
No.
|
Composition
|
RI Cal
|
RI Lit
|
Relative content(%)
|
Tq
|
Tqp
|
Tm
|
Tve
|
Tr
|
Tt
|
Tg
|
Ts
|
Tl
|
Tvf
|
18
|
α-Terpinolen
|
1087
|
1088
|
1.65±0.13
|
2.10±0.18
|
1.58±0.04
|
3.24±0.32
|
-
|
2.76±0.27
|
0.66±0.08
|
2.83±0.12
|
-
|
-
|
19
|
Linalool
|
1099
|
1099
|
1.17±0.05
|
1.52±0.05
|
1.33±0.15
|
1.06±0.01
|
1.21±0.03
|
5.33±0.75
|
0.72±0.42
|
0.38±0.01
|
4.12±0.69
|
1.81±0.26
|
20
|
Camphene
|
949
|
952
|
2.60±0.56
|
-
|
0.92±0.02
|
-
|
-
|
-
|
1.84±0.08
|
1.04±0.04
|
2.15±0.17
|
-
|
(Notes: Tq, T. quinquecostatus; Tqp, T. quinquecostatus var. przewalskii; Tm, T. mongolicus; Tve, T. vulgaris ‘Elsbeth’; Tr, T. rotundifolius; Tt, T. thracicus; Tg, T. guberlinesis ‘Iijin’; Ts, T. serpyllum ‘Aureus’; Tl, T. longicaulis; Tvf, T. vulgaris ‘Fragrantissimus’. RI Cal, calculated according to C7–C40; RI Lit, obtained by searching the mass spectrum database NIST14.0)
Based on the cluster analysis of the 20 kinds main common compositions in 10 different thymes are shown in Fig. 2B, all thymes are clustered into four categories. The one most abundant composition of Tq and Tqp is carvacrol, and their relative contents are respectively 20.74% and 48.37%, so its chemical type is carvacrol-type; Tm, Tve, Tr, Tt, Tg, and Ts are grouped together, and the most content of them is thymol, so its chemical type is thymol-type; Tl is divided to one group which is geraniol-type, Tvf is divided to other type which is α-terpineol-type, respectively. Principal component analysis (PCA) analysis was carried out on the main compositions common to these 10 different thymes, as shown in Fig. 2C, D. It can be seen that among these 10 thymes, and they contribute to the volatile compositions of their EOs. The most compositions are thymol, carvacrol, p-cymene, and γ-terpinene. It can be seen in Fig. 2D that Tq and Tqp are distributed in the first quadrant, and their corresponding characteristic volatile substances are carvacrol, p-cymene, and γ-terpinene; Tvf and Tl are distributed in the third quadrant, which correspond to the characteristic volatile substances of are geraniol and α-terpineol; Tm, Tve, Tr, Tt, Tg, and Ts are distributed in the fourth quadrant, and the corresponding characteristic volatile substance is thymol, which can be used to verify cluster analysis the result.
Use male sterile (without pollen) thyme as the female parent, and male fertile (with pollen) thyme as the male parent, the EO compositions and yield were taken as the main breeding goals, and other traits were referenced. Different cross combinations were designed, and the F1 hybrid populations of thyme were constructed by means of cross breeding. Finally, two hybrid populations were obtained: T. longicaulis × T. vulgaris 'Fragrantissimus' (Tl × Tvf, 14 lines) and T. vulgaris 'Elsbeth' × T. quinquecostatus (Tve× Tq, 11 lines) (Supplementary Table S1).
Ssr Development And Its Application In F1 Hybrid Progenies
Through the assembly and annotation of T. quinquecostatus via high-fidelity (HiFi) and chromatin conformation capture (Hi-C) technologies, its genome was revealed at the chromosome level, which contained 13 chromosomes at a total length of 528.66 megabases (Mb) (Data unpublished). The T. quinquecostatus genome was highly repetitive with a total of 373.28 Mb of repetitive sequences annotated, accounting for 70.61% of the genome (Data unpublished). Finally, a total of 239,400 tandem repeats were identified, accounting for 48.30 Mb (9.14%) of the genome. A total of 191,847 SSR loci were detected, 183,536 SSR loci used for primer design, accounting for 95.67%, and 8,311 SSR loci not used for primer design, accounting for 4.33%. Among the top ten Contig sequences with the largest distribution of SSR sites, Contig00377 has the largest number of SSR sites (4,774), and Contig00721 has the least number of SSR sites (2,089) (Supplementary Fig. S2A). Among these ten Contig sequences, the most frequent occurrence of SSR per Mb is Contig00808 (435), and the least frequent occurrence of SSR is Contig00630 (264) (Supplementary Fig. S2B). The results showed that there were seven dinucleotide repeat types, 12 trinucleotide repeat types and one tetranucleotide repeat type in the SSR sequence of thyme genome (Supplementary Fig. S2C). CT/AG had the highest proportion of dinucleotide repeats (23.70%), followed by TC/GA (20.50%), TA/TA (14.80%), AT/AT (12.00%), TG/CA (3.80%), GT/AC (3.60%), and GC/GC (0.40%). ATT/AAT accounted for the highest proportion of trinucleotide repeats (2.50%), followed by TTA/TAA (2.00%), TTC/GAA (1.60%), ATA/TAT (1.60%), AGA/TCT (1.50%), CTT/AAG (1.20%), GCC/GGC (0.60%), CCG/CGG (0.50%), GAG/CTC (0.50%), CGC/GCG (0.40%), ATC/GAT (0.40%), and GGA/TCC accounted for the least, which was 0.30%. Only AAAT/ATTT tetraponucleotide repeats accounted for 0.40% (Supplementary Fig. S2 C). The length of SSR sequence in T. quinquecostatus genome ranged from 18 bp to 87 bp, and 10 bp SSR locus was the most (61,865), accounting for 32.00% of all SSR loci, while 25 bp SSR locus was the least (1,356), accounting for 0.71% of all SSR loci. The overall trend of changes in SSR motif length was that the number of SSR gradually decreased with the increase of the length (Supplementary Fig. S2D).
The primers of 300 pairs were selected from the designed primers, and PCR amplification was carried out in the parents of the two populations of Tl × Tvf and Tve × Tq (Supplementary Table S2). Through the polymorphism analysis between the parents, the polymorphisms detected in the parents of the primers are 1–2 bands, after many repetitions, the primers with clear and stable bands are screened out (Fig. 3A, D). There are 18 pairs of SSR primers in the hybrid combination Tl × Tvf that show parental co-dominant (Supplementary Table S3). For example, primer TqSSR289 amplifies characteristic band 1 in the female parent T1, and amplifies characteristic band 2 in the male parent Tvf (Fig. 3A). Primer TqSSR292 amplified characteristic band 3 in the female parent T1, and amplified characteristic band 4 in the male parent Tvf (Fig. 3A). There are 23 pairs of SSR primers in the hybrid combination Tve × Tq that are co-dominant with the parents (Supplementary Table S4). For example, the primer TqSSR284 amplifies characteristic band 1 in the female parent Tve, and amplifies characteristic band 2 in the male parent (Fig. 3D), so as to be used for the identification and verification of the hybrid progenies of the population, accounting for 7.60% of the total number of primers. These co-dominant SSR primers were used to identify hybrids in these two populations. In the progeny amplification results, those with parental complementary bands and only paternal-specific bands are true hybrids, and the progeny with only maternal-specific bands are pseudo-hybrids or inbreds. After identification, the 14 F1 lines of the hybrid combination Tl × Tvf are all true hybrids (Fig. 3B, C), and the 11 F1 lines of Tve × Tq are all true hybrids (Fig. 3E, F).
Determination of volatile organic compositions (VOC) in the leaves of the population Tl × Tvf and Tve × Tq parents and progenies. In the population Tl × Tvf (Fig. 4A; Supplementary Table S5), the VOC with the most content in the female parent Tl are geraniol (22.75%) and geranyl acetate (41.75%), and the VOC with the most contents in the male parent Tvf are α-terpineol (11.76%) and α-terpineol acetate (61.37%); Among the 14 F1 lines, the most abundant compositions of the progenies numbered F1-1, F1-2, F1-8, and F1-9 are thymol (the contents are 12.51%, 8.03%, 9.55%, and 8.39%, respectively), carvacrol (the contents are 13.93%, 9.72%, 12.17%, and 12.92%, respectively), p-cymene (the contents are 16.52%, 23.22%, 29.39%, and 23.01%, respectively), and γ-terpene (the contents are 14.16%, 9.50%, 9.02%, and 11.36%, respectively); The most abundant compositions of the progenies numbered F1-3, F1-4, F1-5, F1-6, F1-10, F1-11, F1-13, and F1-14 are geraniol (the contents are 18.17%, 22.75%, 23.79%, 28.98%, 28.84%, 26.91%, 25.44%, and 23.57%, respectively) and geranyl acetate (the contents are 15.76%, 29.59%, 21.89%, 23.41%, 15.05%, 23.60%, 22.50%, and 24.56%, respectively), so the compositions of these progenies are biased toward the female parent Tl; The compositions with the most content in the progeny of F1-7 are geraniol (4.48%), geranyl acetate (27.75%), α-terpineol (3.00%), and α-terpineol acetate (28.03%), a good aggregation of the dominant compositions in two parents; The most abundant compositions in the progeny numbered F1-12 is α-terpineol (9.07%) and α-terpineol acetate (57.44%), favoring the male parent Tvf. According to the 17 main chemical compositions, clustering of parents and 14 F1 lines found that F1-3, F1-4, F1-5, F1-6, F1-10, F1-11, F1-13, and F1-14 chemical type is geraniol-type which clustered with the female parent Tl; F1-12 and the male parent Tvf are clustered together, and the chemical type is α-terpineol-type; F1-1, F1-2, F1-8, and F1-9 are one chemical type, they are thymol and carvacrol polymerization-type; F1-7 is geraniol and α-terpineol polymerization-type (Fig. 4B).
In the population Tve × Tq (Fig. 4C; Supplementary Table S6), the compositions with the high content of female parent Tve are thymol (35.43%), p-cymene (18.42%), and γ-terpinene (13.96%); The compositions with the high content of male parent Tq is carvacrol (20.74%), p-cymene (23.23%), and γ-terpinene (11.45%). Among the 11 F1 lines, the most abundant compositions of the progenies numbered F1-3, F1-4, F1-5, F1-8, and F1-11 are thymol (the contents are 23.57%, 24.71%, 46.19%, 46.38%, and 33.26%, respectively), p-cymene (the contents are 37.42%, 27.03%, 23.16%, 18.56%, and 21.14%, respectively) and γ-terpinene (the contents are 17.14%, 9.03%, 8.55%, 12.58%, and 6.85%, respectively). The compositions of these F1 lines are biased towards the female parent Tve, and the contents of F1-5 and F1-8 thymol are higher than the female parent Tve, accounting for 45.45% of the progeny plants; The most compositions contents numbered F1-1, F1-2, F1-6, F1-7, F1-9, and F1-10 are α-terpineol (the contents are 16.84%, 20.34%, 11.25%, 29.45%, 20.72%, and 23.98%, respectively), and α-terpineol acetate (the contents are 75.72%, 71.58%, 80.44%, 60.21%, 72.17%, and 67.11%, respectively), the sum of the proportions of these two compositions is more than 90% in these six F1 lines, Belongs to the absolute dominant compositions, but these two compositions are not dominant in the parents, these two compositions do not appear in the female parent Tve, and only α-terpineol (0.95%) in the male parent Tq. After cluster analysis, among the 11 progeny lines, F1-3, F1-4, F1-5, F1-8, and F1-11 are thymol-type; F1-1, F1-2, F1-6, F1-7, F1-9, and F1-10 are α-terpineol-type (Fig. 4D).
The phenotypes of plant type, fertility, and stem diameter of F1 lines were obviously different from their parents of Tl × Tvf and Tve × Tq hybrid combinations (Supplementary Fig. S3). The stem about parents and F1 lines of two populations display remarkable differences in morphology. The types of Tl is creeping-type, Tvf is erect-type, and their F1 lines are creeping-type or semi-creeping-type (Supplementary Fig. S4). The types of Tve is erect-type, Tq is creeping-type, and their F1 lines are erect-type or semi-erect-type (Supplementary Fig. S5). The creeping-type parent has a soft stem without a main stem, while the erect-type parent has an obvious main stem with a high degree of lignification, and the stem of its progeny is also between the parents. Some of flowers of F1 progenies of above two hybrid combinations have stamens and are fertile males, so the inheritance of fertility has the characteristics of paternal inheritance (Supplementary Fig. S3).
Bioinformatics Analysis And Screen Of Terpene Synthase In Thyme
Terpene synthase (TPS) catalyzes GPP to form the basic skeleton of monoterpenes (C10) and sesquiterpenes (C15), respectively. The leaves volatile terpene compositions of Tl × Tvf and Tve × Tq aforementioned populations were determined. The chemical types of Tl × Tvf population were geraniol-type, geraniol/α-terpineol- type, thymol/carvacrol-type, and α-terpineol-type, while the chemical types of Tve × Tq population were carvacrol-type, thymol-type, and α-terpineol-type (Fig. 5B, D). In view of γ-terpinene was the catalytic precursor of thymol and carvacrol, we want to screen of γ-terpinene synthase, geraniol synthase, and α-terpineol synthase. Based on the results of T. quinquecostatus whole genome sequencing (Data unpublished), 69 TPS sequences were selected according to gene functional annotation, and 17 sequences were removed by conserved domain analysis of terpene synthase. The phylogenetic tree was constructed using remaining 52 TPS sequences and other species reported TPS (Fig. 6A; Supplementary Table S7). There are 22 TPS sequences belonging to the TPS-b family, all members of this family belong to monoterpene synthase, in which the target genes of γ-terpinene synthase are Tq02G002290.1 and Tq13G005250.1, the target genes of α-terpineol synthase are Tq03G001560.1, and the purpose of geraniol synthase gene is Tq04G005190.1. The target genes Tq02G002290.1 and Tq13G005250.1 of γ-terpinene synthase were compared with the reported genes TcTPS02.1 and TcTPS02.2 in T. caespititius [28], and the similarities were 96.60% and 89.35%, respectively (Fig. 6B, C); The target gene Tq03G001560.1 of α-terpineol synthase was compared with the reported genes TcTPS05.1 and TcTPS05.2 in T. caespititius [28], and the similarity is 95.51% (Fig. 6D); The target gene Tq04G005190.1 of geraniol synthase is compared with obGES (Supplementary Fig. S6), which has been reported in Ocimum basilicum [37], and the similarity is 68.90%. Sequence alignment results show that the amino acid sequences of the target genes have the characteristic domain of terpene synthase, Tq02G002290.1, Tq13G005250.1, and Tq03G001560.1 sequences have the characteristic domain of terpene synthase, including RRX8W, DDXXD, and NSE/DTE (Fig. 6B–D), and Tq04G005190.1 sequences has the characteristic domain of terpene synthase, including DDXXD and NSE/DTE (Supplementary Fig. S6).
In population Tve × Tq, the chemical type of the male parent Tq is carvacrol, and the female parent Tve is thymol. Since γ-terpinene is the catalytic precursor of thymol and carvacrol, in order to better verify the γ-terpinene synthase function, Tq02G002290.1 and Tq13G005250.1 were verified in Tve, Tq and their progenies F1-3 and F1-4 (Fig. 6A). The qRT-PCR results showed that the relative expression of Tq02G002290.1 and Tq13G005250.1 were consistent compared with the results of the relative content of γ-terpinene in F1-3 and F1-4 lines. These preliminarily inferred that Tq02G002290.1 and Tq13G005250.1 maybe catalyze γ-terpinene biosynthesis (Fig. 6A). In population Tl × Tvf, the female parent Tl is geraniol-type and the male parent Tvf is α-terpineol-type. Therefore, in order to better verify the functions of α-terpineol synthase Tq03G001560.1 and geraniol synthase Tq04G005190.1, the two TPS genes were subjected to qRT-PCR in Tl, Tvf, and their progenies F1-6 and F1-12 (Fig. 6A). The results showed that the expression levels of Tq03G001560.1 and Tq04G005190.1 genes were were consistent with their relative content of α-terpineol and geraniol in F1-6 and F1-12 lines. These preliminarily inferred that Tq03G001560.1 and Tq04G005190.1 maybe catalyze the α-terpineol and geraniol biosynthesis, respectively (Fig. 6A).