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Creation of new germplasm resources, development of SSR markers, and screening of monoterpene synthases in thyme
BMC Plant Biology volume 23, Article number: 13 (2023)
Thyme derived essential oil and its components have numerous applications in pharmaceutical, food, and cosmetic industries, owing to their antibacterial, antifungal, and antiviral properties. To obtain thyme essential oil with different terpene composition, we developed new germplasm resources using the conventional hybridization approach.
Phenotypic characteristics, including essential oil yield and composition, glandular trichome density, plant type, and fertility, of three wild Chinese and seven European thyme species were evaluated. Male-sterile and male-fertile thyme species were crossed in different combinations, and two F1 populations derived from Thymus longicaulis (Tl) × T. vulgaris ‘Fragrantissimus’ (Tvf) and T. vulgaris ‘Elsbeth’ (Tve) × T. quinquecostatus (Tq) crosses were selected, with essential oil yield and terpene content as the main breeding goals. Simultaneously, simple sequence repeat (SSR) primers were developed based on the whole-genome sequence of T. quinquecostatus to authenticate the F1 hybrids. A total of 300 primer pairs were selected, and polymerase chain reaction (PCR) was carried out on the parents of the two hybrid populations (Tl, Tvf, Tve, and Tq). Based on the chemotype of the parents and their F1 progenies, we examined the expression of genes encoding two γ-terpinene synthases, one α-terpineol synthase, and maybe one geraniol synthase in all genotypes by quantitative real-time PCR (qRT-PCR).
We used hybridization to create new germplasm resources of thyme, developed SSR markers based on the whole-genome sequence of T. quinquecostatus, and screened the expression of monoterpene synthase genes in thyme. The results of this study provide a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, and identification of quantitative trait loci (QTLs), and help gain insight into the mechanism of monoterpenoids biosynthesis in thyme.
Species belonging to the genus Thymus (family Lamiaceae) are commonly used for food, cosmetic, and medicinal purposes [1,2,3]. Thyme (Thymus) species are important aromatic and medicinal plants that have been used as traditional medicine for thousands of years in the Mediterranean basin . These species are highly appreciated for the wide spectrum of pharmacological properties of their essential oils. The antirheumatic, antiseptic, antispasmodic, antimicrobial, anti-inflammatory, carminative, diuretic, and expectorant activities of thyme essential oil have been researched and validated [5,6,7,8]. The main components of thyme essential oil belong to the chemical classes of terpenoids, terpene alcohols, phenolic derivatives, ketones, aldehydes, ethers, and esters . Generally, these oils contain oxygenated monoterpenoids (e.g., thymol, carvacrol, γ-terpinene, p-cymene, 1,8-cineole, linalool, α-terpineol, geraniol, and borneol), sesquiterpenoids (e.g., β-caryophyllene), and oxygenated sesquiterpenoids (e.g., caryophyllene oxide) [4, 8, 9]. Depending on the characteristics and composition of thyme essential oil, the occurrence of different chemotypes have been described within several species of Thymus [2, 9,10,11], including T. vulgaris.
To date, a few studies have been conducted by scientific research institutions and companies on thyme breeding, especially in European countries, and few of the high-performance industrial and horticultural varieties developed in these studies have been cultivated. In the 4th International Symposium on the Breeding of Medicinal Aromatic Plants, a breeding study was reported, in which male-sterile and male-fertile clones were crossed to optimize the terpenoid content and yield of thyme (T. vulgaris) . In this study, 56 new hybrids, which were tested by assessing the homogeneity, dry weight, essential oil yield, winter frost tolerance, and seed production potential of the parents, were obtained from 2000 to 2002. The most dominant hybrid, named ‘Varico 3’, showed 4.9% essential oil yield and thymol-type chemotype . Such hybrid varieties have been entered into the market. Clone T-12, with high phenol content, was selected from among 10 T. vulgaris clones . The global collection of Thymus resources is highly diverse, with more than 300 species, which are native to the Mediterranean basin and are widely distributed in the temperate regions of Europe, North Africa, and Asia . In China, Thymus species are mainly distributed in the northwest, north, and northeast regions based on the Flora of China . Therefore, according to the breeding goals, wild Chinese thyme species could be crossed with European thyme species to develop a series of new varieties for applications in different fields.
With the rapid development of modern molecular biology approaches and genome sequencing technologies, DNA-based molecular markers have become an important tool for cultivar identification, fingerprinting [16, 17], and genetic diversity analysis [18,19,20]. To improve the industrial applications of medicinal and aromatic plants, breeders often select plants with high genetic divergence and essential oil content . Phenotypic variation could be very valuable for molecular breeding approaches such as marker-assisted selection (MAS), which has been very helpful in elucidating the genetic diversity of plant species. A dendrogram, based on cluster analysis, showed that T. daenensis and T. fallax are clearly distinct from the other Thymus species, indicating that T. daenensis shares some genetic similarity with T. fallax . Previously, several studies used randomly amplified polymorphic DNA (RAPD) markers to investigate the genetic diversity and essential oil composition of various Thymus species as well as the phylogenetic relationship among these species [11, 17, 22].
Phenolic monoterpenoids in thyme are synthesized via two pathways: the mevalonate (MVA) pathway and 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway . Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are canonically condensed head-to-tail by trans-prenyltransferases to generate geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Terpene synthase (TPS) converts GPP and FPP into the basic skeleton of monoterpenes (C10) and sesquiterpenes (C15), respectively. TPSs have been characterized as multifunctional enzymes, owing to multiple single amino acid substitutions, which result in altered metabolic profiles [24,25,26]. A wide range of TPSs and other terpene-modifying enzymes have been characterized to date . TPS genes have been identified in several Thymus species, including T. caespititius [28,29,30], T. vulgaris [31,32,33,34], T. serpyllum , T. albicans , and T. citriodorus.
In this study, male-sterile and male-fertile thyme varieties were crossed in two different combinations, T. longicaulis (Tl) × T. vulgaris ‘Fragrantissimus’ (Tvf) and T. vulgaris ‘Elsbeth’ (Tve) × T. quinquecostatus (Tq), to generate two F1 populations. Then, simple sequence repeat (SSR) markers were developed based on the whole-genome sequence of T. quinquecostatus , to authenticate all F1 individuals. In addition, the expression profiles of two γ-terpinene synthase genes (Tq13G005250.1 and Tq02G002290.1), one geraniol synthase gene (Tq04G005190.1), and maybe one α-terpineol synthase gene (Tq03G001560.1) were analyzed by quantitative real-time PCR (qRT-PCR). Overall, this study provides a valuable collection of new thyme varieties, which could be used for MAS and the verification of TPS gene function in future studies.
Phenotypic evaluation of different thyme species and construction of F1 hybrid populations
Thyme is a herbaceous perennial or sub-shrub with valuable medicinal and aromatic properties. Thyme plants are of two types, depending on their growth habit: erect-type and creeping-type. These two plant types display remarkable differences in morphology. The plant types of 10 different thyme species are shown in Fig. 1a; Table 1. Among these species, 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 (Tg), T. longicaulis (Tl), T. quinquecostatus (Tq), T. quinquecostatus var. przewalskii (Tqp), and T. mongolicus (Tm) are creeping-type. The fertility of different thyme species is shown in Fig. 1b. Tr, Tve, Tg, Tt, Ts, and Tl contained only stigma and no pollen (stamen), and were therefore male-sterile. By contrast, Tq, Tqp, Tm, and Tvf possessed both stigma and pollen (stamen), and therefore were categorized as male-fertile (Table 1). The increased genetic diversity of thyme species could be attributed partially to the consistent introgression of wild Chinese thyme germplasm into the male-sterile and erect-type European thyme germplasm during long cultivation periods, and partially to the adaptation of thyme species to new environments in new geographical locations.
Glandular trichomes are specialized hairs that originate from the epidermal cells of flowers, leaves, and stems. These organs exist as two types, peltate and capitate, on the surface of approximately 30% of all vascular plants, including lavender, thyme, rosemary, oregano, basil, and other Lamiaceae species . Glandular trichomes are responsible for a significant portion of the secondary metabolite of a plant , and serve as the storage and synthesis sites of terpenoids . The regulation of glandular trichome formation related genes potentially underlies the regulation of glandular trichome density (number of glandular trichomes per unit area) for increasing the terpenoid content of plants.
Tve and Tr showed the highest glandular trichome density on both the adaxial and abaxial leaf surfaces (Fig. 1c, e). Glandular trichome density on the adaxial leaf surface was the second highest in Tvf and Tt. The glandular trichome density of leaves, overall, decreased in the following order: Tve, Tr, Tvf, Tt, and Ts. The essential oil yield of thyme species is shown in Fig. 1d, f and Supplementary Fig. S1. Tvf showed the highest essential oil yield (1.25 mL·100 g− 1), followed by Tt (1.20 mL·100 g− 1), Tr (0.95 mL·100 g− 1), Tve (0.75 mL·100 g− 1), and Tl (0.70 mL·100 g− 1). The essential oil yield of Tq, Tqp, Tm, Tg, and Ts varied between 0.40 mL·100 g− 1 and 0.50 mL·100 g− 1 (Table 1; Supplementary Fig. S1). There was a certain correlation between essential oil yield and glandular trichome density, the higher the density of glandular trichome, the higher the yield of essential oil (Fig. 1e, f).
The relative contents of essential oil components in 10 different thyme species are shown in Fig. 2a. Only 20 compounds showed relative contents of > 0.3% and were shared by 10 different thyme species (Table 2). The most abundant volatile compounds in Tq essential oil were p-cymene and carvacrol, which accounted for 23.00% and 20.74% (relative content), respectively. Tqp showed the highest content of carvacrol, which accounted for 48.37% of the total content of volatile compounds. In Tm, thymol was the most abundant volatile compound (38.57%), followed by p-cymene (16.40%). Similarly, in Tve, thymol was the most abundant volatile compound (35.43%), followed by p-cymene (18.42%), and γ-terpinene (13.96%). The thymol contents of Tr, Tt, Tg, and Ts were 36.02%, 41.04%, 26.26%, and 28.96%, respectively. In Tl, geranyl acetate (34.81%) was the most predominant volatile compounds, followed by geraniol (28.54%). The most abundant volatile compounds in Tvf were α-terpineol acetate (45.46%) and α-terpineol (30.84%).
Cluster analysis of the 20 main compounds found in the essential oil of 10 different thyme species (Fig. 2b) revealed four clusters. Tq and Tqp, which contained carvacrol as the most abundant compound (carvacrol-type essential oil), with relative contents of 20.74% and 48.37%, respectively, clustered together; Tm, Tve, Tr, Tt, Tg, and Ts grouped together, and contained thymol as the most abundant compound (thymol-type essential oil); and Tl and Tvf clustered separately (geraniol-type and α-terpineol-type essential oil, respectively). Principal component analysis (PCA) analysis was carried out on the main compounds found in the essential oils of all 10 thyme species (Fig. 2c, d). The results showed that the relatively high contents of thymol, carvacrol, p-cymene, and γ-terpinene contributed greatly to the volatile components of the essential oil of all 10 species. Tq and Tqp grouped together in the first quadrant, and their corresponding characteristic volatile substances included carvacrol, p-cymene, and γ-terpinene; Tvf and Tl were distributed in the third quadrant, which corresponded to volatiles geraniol and α-terpineol; Tm, Tve, Tr, Tt, Tg, and Ts were distributed in the fourth quadrant, and the corresponding predominant volatile was thymol (Fig. 2d); these results verified the results of cluster analysis.
Development and application of SSR markers
The chromosome-level genome assembly and annotation using high-fidelity (HiFi) and chromatin conformation capture (Hi-C) technologies revealed 13 chromosomes in T. quinquecostatus, with a total length of 528.66 Mb, 70.61% (373.28 Mb) of which was annotated as highly repetitive . A total of 239,400 tandem repeats were identified, accounting for 48.30 Mb (9.14%) of the genome. Additionally, 191,847 SSR loci were detected, of which 183,536 (95.67%) could be used for primer design. Among the top 10 contigs with the greatest distribution of SSR sites, Contig00377 showed the highest number of SSR sites (4,774), and Contig00721 contained the least number of SSR sites (2,089) (Supplementary Fig. S2a). The density of SSR loci (number of SSR loci per unit Mb), among these 10 contigs, was the highest in Contig00808 (435) and lowest in Contig00630 (264) (Supplementary Fig. S2b). Seven dinucleotide, twelve trinucleotide, and one tetranucleotide repeats were detected among the SSR loci of T. quinquecostatus (Supplementary Fig. S2c). Among the dinucleotide repeats, the CT/AG-type repeat was the most abundant (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%). Among the trinucleotide repeats, ATT/AAT accounted for the highest proportion (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 (0.30%). AAAT/ATTT (0.40%) was the only tetranucleotide repeat type identified in the T. quinquecostatus genome (Supplementary Fig. S2c). The length of SSR loci ranged from 18 to 87 bp in the T. quinquecostatus genome, with 10 bp SSRs being the most abundant (61,865, accounting for 32.00% of all SSR loci) and 25 bp SSR loci being the least abundant (1,356, 0.71%). The number of SSRs gradually decreased with the increase in repeat length (Supplementary Fig. S2d).
Male-sterile (without pollen) and male-fertile (with pollen) thyme species were crossed as female and male parents, respectively, in different combinations. Finally, two F1 hybrid populations were obtained from two crosses: T. longicaulis × T. vulgaris ‘Fragrantissimus’ (Tl × Tvf, 14 lines) and T. vulgaris ‘Elsbeth’ × T. quinquecostatus (Tve × Tq, 11 lines) (Supplementary Table S1). To design SSR markers for the verification of F1 progenies, 300 primer pairs were screened by performing PCR amplification on the parental lines of the two crosses (Supplementary Table S2). Analysis of the PCR products revealed 1–2 polymorphic bands between the two parents of each cross. After many repetitions, the primers showing clear and stable banding patterns were selected (Fig. 3a, d).
Eighteen SSR markers were co-dominant in the Tl × Tvf population (Supplementary Table S3). For example, TqSSR289 amplified band 1 in the female parent T1 and band 2 in the male parent Tvf (Fig. 3a); TqSSR292 amplified band 3 in the female parent T1 and band 4 in the male parent Tvf (Fig. 3a). Similarly, 23 SSR markers were co-dominant in the Tve × Tq population (Supplementary Table S4). For example, TqSSR284 amplified band 1 in the female parent Tve and band 2 in the male parent Tq (Fig. 3d), so as to be used for the identification and verification of the hybrid progenies of the combination, accounting for 7.60% of 300 primer pairs. These co-dominant SSR primers were used to identify F1 individuals in the two hybrid populations. Progenies with complementary parental bands or only paternal-specific bands were true hybrids, and those with only maternal-specific bands were pseudo-hybrids or inbreds. Based on the genotyping results, 14 lines in the Tl × Tvf progeny (Fig. 3b, c), and 11 lines in the Tve × Tq progeny were identified as true hybrids (Fig. 3e, f).
Determination of volatile organic compounds (VOCs) in the leaves of F1 hybrids and their parents
Geraniol (22.75%) and geranyl acetate (41.75%) were the most abundant VOCs in the female parent Tl, and α-terpineol (11.76%) and α-terpineol acetate (61.37%) were the most abundant in the male parent Tvf (Fig. 4a; Supplementary Table S5). Among the 14 F1 lines derived from the Tl × Tvf cross, F1-1, F1-2, F1-8, and F1-9 showed the highest contents of thymol (12.51%, 8.03%, 9.55%, and 8.39%, respectively), carvacrol (13.93%, 9.72%, 12.17%, and 12.92%, respectively), p-cymene (16.52%, 23.22%, 29.39%, and 23.01%, respectively), and γ-terpinene (14.16%, 9.50%, 9.02%, and 11.36%, respectively). Additionally, lines F1-3, F1-4, F1-5, F1-6, F1-10, F1-11, F1-13, and F1-14 showed the highest contents of geraniol (18.17%, 22.75%, 23.79%, 28.98%, 28.84%, 26.91%, 25.44%, and 23.57%, respectively) and geranyl acetate (15.76%, 29.59%, 21.89%, 23.41%, 15.05%, 23.60%, 22.50%, and 24.56%, respectively). Thus, the essential oil compositions of these progenies were biased toward the female parent Tl. VOCs with high contents in line F1-7 were geraniol (4.48%), geranyl acetate (27.75%), α-terpineol (3.00%), and α-terpineol acetate (28.03%), thus representing a good aggregation of the dominant compounds found in the two parents. The most abundant compounds in line F1-12 were α-terpineol (9.07%) and α-terpineol acetate (57.44%), indicating a bias toward the male parent Tvf. Cluster analysis of the 17 main chemical compounds found in T1, Tvf, and their 14 F1 lines showed that lines F1-3, F1-4, F1-5, F1-6, F1-10, F1-11, F1-13, and F1-14, which clustered with the female parent Tl, were geraniol-type; F1-12, which grouped with the male parent Tvf, was α-terpineol-type; lines F1-1, F1-2, F1-8, and F1-9 were thymol and carvacrol polymerization-type; and line F1-7 was geraniol and α-terpineol polymerization-type (Fig. 4b).
Next, we analyzed the parents and progeny of the Tve × Tq combination (Fig. 4c; Supplementary Table S6). Thymol (35.43%), p-cymene (18.42%), and γ-terpinene (13.96%) were the most abundant in the female parents Tve, and carvacrol (20.74%), p-cymene (23.23%), and γ-terpinene (11.45%) were the most abundant in the male parent Tq. Among the 11 Tve × Tq F1 lines, F1-3, F1-4, F1-5, F1-8, and F1-11 showed the highest contents of thymol (23.57%, 24.71%, 46.19%, 46.38%, and 33.26%, respectively), p-cymene (37.42%, 27.03%, 23.16%, 18.56%, and 21.14%, respectively), and γ-terpinene (17.14%, 9.03%, 8.55%, 12.58%, and 6.85%, respectively). The compositions of these F1 lines were biased towards the female parent Tve, and the thymol contents of F1-5 and F1-8 were higher than that of the female parent, accounting for 46.19% (F1-5) and 46.38% (F1-8) of the progeny plants. Lines F1-1, F1-2, F1-6, F1-7, F1-9, and F1-10 showed the highest contents of α-terpineol (16.84%, 20.34%, 11.25%, 29.45%, 20.72%, and 23.98%, respectively) and α-terpineol acetate (75.72%, 71.58%, 80.44%, 60.21%, 72.17%, and 67.11%, respectively), and the sum of the proportions of each of these two compounds (α-terpineol and α-terpineol acetate) in the six above-mentioned F1 lines was more than 90%. Thus, α-terpineol and α-terpineol acetate were the absolute dominant compounds, although these compounds were not dominant in the parents; neither one of the two compounds was detected in the female parent Tve, and only α-terpineol was detected in minor amounts (0.95%) in the male parent Tq. Cluster analysis revealed that, among the 11 progeny lines, F1-3, F1-4, F1-5, F1-8, and F1-11 were thymol-type, while F1-1, F1-2, F1-6, F1-7, F1-9, and F1-10 were α-terpineol-type (Fig. 4d).
Bioinformatics analysis and screening of TPSs in thyme
TPSs convert GPP and FPP into the basic skeleton of monoterpenes (C10) and sesquiterpenes (C15), respectively. We determined the volatile terpenes in the leaves of Tl × Tvf and Tve × Tq populations. The chemical types found in the Tl × Tvf population were geraniol-type, geraniol/α-terpineol-type, thymol/carvacrol-type, and α-terpineol-type, while those found in the Tve × Tq population were carvacrol-type, thymol-type, and α-terpineol-type (Fig. 4b, d). Considering that TPS is the key enzyme involved in plant terpenoid biosynthesis, we aimed to identify the main TPSs, such as γ-terpinene synthase, geraniol synthase, and α-terpineol synthase, that determine the essential oil composition of the two F1 progenies. Based on the results of T. quinquecostatus whole-genome sequencing , 69 TPS sequences were selected according to gene function annotation, of which 17 sequences were removed based on conserved domain analysis. A phylogenetic tree was constructed using the remaining 52 TPS sequences of T. quinquecostatus and the TPS sequences of other species (Fig. 5a; Supplementary Table S7). The results showed that 22 TPS sequences belonged to the TPS-b family, which contains monoterpene synthases. Among these sequences, Tq02G002290.1 and Tq13G005250.1 were predicted to encode γ-terpinene synthases; Tq03G001560.1 encoded α-terpineol synthase; and Tq04G005190.1 maybe encoded geraniol synthase. These genes were compared with their homologs in T. caespititius ; comparison of Tq02G002290.1 and Tq13G005250.1 with the reported γ-terpinene synthase genes TcTPS02.1 and TcTPS02.2 in T. caespititius revealed 96.60% and 89.35% sequence similarities, respectively (Fig. 5b, c), and Tq03G001560.1 showed 95.51% sequence similarity with the α-terpineol synthase genes TcTPS05.1 and TcTPS05.2 in T. caespititius (Fig. 5d). Tq04G005190.1 was compared with the Ocimum basilicum the geraniol synthase gene ObGES gene  (Supplementary Fig. S3), which revealed 68.90% sequence similarity. Alignment of the deduced amino acid sequences of Tq02G002290.1, Tq13G005250.1, and Tq03G001560.1 revealed three characteristic TPS domains, including RRX8W, DDXXD, and NSE/DTE (Fig. 5b–d), while Tq04G005190.1 showed two characteristic TPS domains, including DDXXD and NSE/DTE (Supplementary Fig. S3).
The chemical types of the male parent (Tq) and female parent (Tve) of Tve × Tq were carvacrol- and thymol-type, respectively. Since γ-terpinene is the catalytic precursor of thymol and carvacrol, we verified the γ-terpinene synthase function of Tq02G002290.1 and Tq13G005250.1 by monitoring the expression levels of these genes in Tve, Tq, and their progenies F1-3 and F1-4 by qRT-PCR (Fig. 6a). In lines F1-3 and F1-4, the relative expression levels of Tq02G002290.1 and Tq13G005250.1 were consistent with the relative content of γ-terpinene, suggesting that Tq02G002290.1 and Tq13G005250.1 catalyze γ-terpinene biosynthesis (Fig. 6a). Similarly, to better verify the α-terpineol synthase and geraniol synthase functions of Tq03G001560.1 and Tq04G005190.1, respectively, we examined the expression levels of these two genes in Tl (geraniol-type), Tvf (α-terpineol-type), and their progenies F1-6 and F1-12 by qRT-PCR (Fig. 6a). In lines F1-6 and F1-12, the expression levels of both genes were consistent with the relative contents of α-terpineol and geraniol, indicating that Tq03G001560.1 and Tq04G005190.1 catalyze α-terpineol and geraniol biosynthesis, respectively (Fig. 6a).
Phenotypic evaluation of thyme species and construction of two F1 hybrid populations
In this study, the essential oil yield of erect-type thyme was higher than that of creeping-type thyme, and there was a positive correlation between the density of glandular trichomes and essential oil yield (Fig. 1e, f). Some wild Chinese thyme species showed a long flowering period of up to four months, while some European thyme species showed a short flowering period of one month (Table 1). The tillering capacity of creeping-type species was greater than that of erect-type species. In addition, the harvest time, extraction method, and stem/leaf ratio of thyme species likely affected their essential oil yield. In this study, the type and relative content of VOCs in the essential oil varied significantly among the 10 different thyme species, which further illustrated the diversity of the Thymus genus (Table 2). A previous study reported that the essential oil composition of a given thyme species and the contents of individual VOCs vary among different environments . Additionally, extraction methods affect the composition and content of essential oil . Therefore, the effects of species, region, and extraction method should be considered when studying the content and composition of thyme essential oil (Supplementary Fig. S1; Table 2). A certain correlation was also detected between the color and composition of thyme essential oil. Essential oil extracted from α-terpineol-type thyme was milky white whereas that extracted from geraniol-type thyme was light yellow. Essential oils extracted from thymol-type and carvacrol-type thyme species were golden yellow. The most well-studied volatile compounds of Chinese and European thyme species are thymol and carvacrol, since these two compounds are the two most abundant VOCs in most thyme species [4, 8, 9]. In this study, the chemical types of the seven European thyme species were mainly thymol-, geraniol-, and α-terpineol-type, while those of the three wild Chinese thyme species were carvacrol- and thymol-type.
The phenomenon of male sterility in plants enables cross breeding while avoiding complicated process of emasculation. Darwin reported that thyme plants in southern England produce two kinds of flowers: one with intact male and female organs, and the other (a small flower) with no or completely cut anthers; the latter are completely male-sterile . In different ecological environments, the proportion of female plants in different thyme species is more than 50% . Because thyme flowers are very small in size, emasculation and crossing are difficult. Therefore, if some thyme plants possess female reproductive organs, crossing is very easy. Among the 10 thyme species investigated in this study, Tr, Tve, Tg, Tt, Ts, and Tl were male-sterile, while Tq, Tqp, Tm, and Tvf were male-fertile. Therefore, Tve and T1 were used as female parents, and Tq and Tvf were used as male parents to generate two hybrid thyme populations. Male sterility is jointly determined by genetic and environmental factors. The male-sterile thyme lines used in this study were introduced from Europe; therefore, the phenomenon of male sterility in these lines may be caused by maladjustment to the new environment.
Chinese thyme plants are widely distributed in the northwest, north, and northeast regions in China . Therefore, the research on Chinese thyme species should be intensified, and wild Chinese thyme species should be crossed with European thyme species to breed new species suitable for ornamental, medicinal, and edible applications to promote the development of the thyme planting industry. Hybrid breeding is an important means of germplasm resource innovation . The advantages of both parents can be introgressed into a single genetic background through interspecific hybridization. In this study, new thyme germplasm resources were generated by crossing different chemical types and plant types of thyme.
SSR marker development and application
Hybrid breeding is an important means of germplasm resource innovation. The advantages of parents can be combined in a single genetic background through interspecific hybridization. In this study, new thyme germplasm resources were generated by hybridizing different chemical types and plant types. SSR markers are simple, time-saving, cost-effective, reproducible, and stable, and therefore have been widely used for the identification of the hybrid progenies in different species [16, 43]. Early identification and selection of hybrid progeny is an important link in cross breeding. Therefore, molecular marker technology, combined with morphological observation, can be used to identify hybrid progenies with high accuracy.
The SSR-based genotyping process first involves the screening of co-dominant SSR primers in parents, followed by the identification of progenies . However, there may be errors in SSR marker-based identification of hybrid progenies using a single primer pair. For example, in this study, SSR primers TqSSR119 and TqSSR124 were used to identify F1 progenies derived from the Tl × Tvf cross. Analysis of the same sample using different primer pairs can produce different results, possibly because of the high heterozygosity of thyme, lead to some of the parents can’t dominance or appear some non-parental bands. This may also be caused by the process of meiotic division during gamete formation; exchange of DNA between homologous chromosomes during recombination at marker loci can lead to the disappearance of bands in progenies.
Generally, the phenotype of F1 progenies is intermediate between that of the two parents or biased towards the phenotype of the female or male parent, which also provides the possibility of breeding excellent new varieties . In this study, among the 14 Tl × Tvf F1 lines, eight lines showed the same VOC profiles in leaves as their female parent Tl; one line was similar to the male parent Tvf; and five lines were dissimilar to both parents. This conclusion was supported by the 11 F1 lines derived from the Tve × Tq cross; five of these F1 lines showed the same leaf VOC profiles as their female parent Tve, and the remaining six lines were different from both their parents. However, some F1 progenies also showed some volatile compounds that were superior to the both parents or not found in either parent; for example, thymol and carvacrol were found in the progeny of the Tl × Tvf cross, and α-terpineol and α-terpineol acetate were found in the Tve × Tq population. These results suggest that new varieties with related VOC profiles could be developed in thyme through cross breeding. Heterosis may also be present, and the greater the phenotypic differences between parents, the stronger is the heterosis expected to be in their progeny.
Bioinformatics analysis and screening of TPSs in thyme
Monoterpenoid biosynthesis begins with GPP, which is the precursor of all monoterpenoids, and yields α-terpinyl cations, which are highly unstable intermediates that can then be converted to specific monoterpenoids by certain monoterpene synthases such as γ-terpinene and α-terpineol . GPP also serves as the synthetic precursor of geraniol, linalool, myrcene, and ocimene, which are formed through catalysis by different monoterpene synthases. In addition, cytochrome P450 monooxygenase 71D (CYP71D) proteins subfamily and short-chain dehydrogenases/reductases (SDRs) are involved in further modification of the γ-terpinene framework to produce p-cymene, thymol, and carvacrol . The synthetic pathway of some monoterpenes is shown in Fig. 6b. Functions of the γ-terpinene synthase gene TcTPS02 and α-terpineol synthase gene TcTPS05 were previously validated in T. caespititius . Similarly, functions of the γ-terpinene synthase gene TvTPS2 and three cytochrome P450 genes (TvCYP71D179, TvCYP71D180, and TvCYP71D507) were validated in T. vulgaris [45, 46].
The results of qRT-PCR analysis showed that the screened TPSs were expressed in hybrid thyme progenies, and these results were consistent with the relative contents of the TPS-catalyzed products in thyme. The expression levels of two γ-terpinene synthase genes Tq02G002290.1 and Tq13G005250.1 were verified in the progeny derived from the cross between Tve and Tq, whose chemotypes were thymol- and carvacrol-type, respectively. Similarly, the expression levels of α-terpineol synthase gene Tq03G001560.1 and geraniol synthase gene Tq04G005190.1 were validated in the progeny of Tl and Tvf, whose chemotypes were α-terpineol- and geraniol-type, respectively. Additionally, the relative expression levels of Tq02G002290.1, Tq13G005250.1, Tq03G001560.1, and Tq04G005190.1 were consistent with the relative contents of catalytic products in some F1 lines. Therefore, this method enables only a preliminary screening of gene function and provides a basis for further gene function verification. The above results lay a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, mapping of quantitative trait loci (QTLs), and MAS, and provide insight into the mechanism of monoterpenoids biosynthesis in thyme.
Thyme is a multi-purpose plant with a wide range of applications in the pharmaceutical, food, and cosmetic industries, owing to its high essential oil content. To obtain thyme essential oil with different terpene compositions, we cross wild Chinese thyme species with European thyme species, and new germplasm resources have developed using the conventional hybridization approach. Two F1 populations were obtained, simultaneously, SSR primers were developed based on the whole-genome sequence of T. quinquecostatus to authenticate the F1 hybrids. Based on the chemotype of the parents and their F1 progenies, we examined the expression of genes encoding two γ-terpinene synthases, one α-terpineol synthase, and maybe one geraniol synthase in all genotypes by qRT-PCR. The results of this study provide a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, and identification of QTLs responsible for the terpene compositions and essential oil. We screened monoterpene synthases in thyme to gain insight into the mechanism of monoterpenoids biosynthesis in thyme.
Three wild Chinese thyme species, including T. quinquecostatus, T. quinquecostatus var. przewalskii, T. mongolicus, and seven European thyme species, including T. vulgaris ‘Fragrantissimus’, T. vulgaris ‘Elsbeth’, T. guberlinesis, T. serpyllum ‘Aureus’, T. thracicus, T. longicaulis, and T. rotundifolius, were used in this study. T. quinquecostatus plants were collected from the Jinhekou village in the Hebei province of China in 2018. T. quinquecostatus var. przewalskii was collected in 2019 from the Small Wutai Jinhekou Scenic Spot in Yu County, Hebei, China. T. mongolicus was collected in 2018 from Chicheng County, Hebei, China. Three specimens (voucher nos. 2,582,781, 2,582,673, and 2,582,686) were obtained from the Chinese National Herbarium, Institute of Botany, Chinese Academy of Sciences. T. vulgaris ‘Fragrantissimus’, T. vulgaris ‘Elsbeth’, T. thracicus, and T. longicaulis were introduced from the Czech Republic (introduction no. 708–2015, 626–2015, 628–2015, and 709–2015, respectively). T. guberlinensis and T. serpyllum ‘Aureus’ were introduced from Hungary (introduction no. 002-2016 and 003-2016). T. rotundifolius was introduced from Germany (introduction no. 797–2016). Plants were grown on an experimental farm at the Institute of Botany, Chinese Academy of Sciences (IB-CAS), Beijing, China.
Hybrid breeding design
Male-sterile (without pollen) thyme species were crossed as the female parent with male-fertile (with pollen) thyme species, with the essential oil composition and yield as the main breeding goals. Different cross combinations were designed, and the F1 hybrid combinations of thyme were constructed by cross breeding in 2020. Finally, two F1 populations derived from T. longicaulis (Tl) × T. vulgaris ‘Fragrantissimus’ (Tvf) and T. vulgaris ‘Elsbeth’ (Tve) × T. quinquecostatus (Tq) crosses were selected for further analysis.
Leaves were collected from the parents and F1 progenies of Tl × Tvf and Tve × Tq crosses, immediately frozen in liquid nitrogen, and stored at -80 °C. DNA was extracted from the frozen leaf samples using the DNA Secure Plant Kit (Tiangen, China). DNA concentration and quality were assessed by 1% agarose gel electrophoresis and with a 2.0 Fluorometer (Life Technologies, CA, USA).
PCR was performed on the PCR system (Bio-Rad, Hercules, CA, USA) in a 10 µl reaction volume containing 2 µl (20 ng/µl) of genomic DNA, 3 µl of 2× Taq PCR Master Mix II (Tiangen, China), 2 µl of forward and reverse primer mixture, and 3 µl of ddH2O. The thermocycling conditions were as follows: 94 °C for 3 min; 6 cycles at 94 °C for 45 s, 55–65 °C for 1 min, and 72 °C for 1 min; 9 cycles of 94 °C for 45 s, 50–58 °C for 1 min, and 72 °C for 1 min; 19 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min; and final extension at 72 °C for 5 min. PCR products were analyzed by electrophoresis on 8.0% (w/v) denaturing polyacrylamide gel in TBE buffer for 1 h on the DYY-6 C electrophoresis apparatus (Beijing Liuyi Instrument Factory, China) at a constant voltage of 220 V. DNA fragments were visualized by silver staining (Silver sequence staining reagents, Promega, Madison, USA) and sized with a 50 bp DNA ladder marker (Tiangen, China) . SSR primer sequences are listed in Supplementary Table S2.
RNA extraction and cDNA synthesis
Leaves of T. longicaulis, T. vulgaris ‘Fragrantissimus’, T. vulgaris ‘Elsbeth’, T. quinquecostatus, and their F1 lines were frozen in liquid nitrogen and stored at -80 ℃. Total RNA was extracted from the frozen leaves using the Easy Spin RNA extraction kit (Sangon Biotech, Shanghai, China). The isolated total RNA was treated with DNase I, and then purified with the RNA clean kit (Promega, Madison, WI, USA). The concentration of each RNA sample was determined using NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., USA) and 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was analyzed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Then, cDNA was synthesized using the HiScript Reverse Transcriptase Kit (Vazyme, China), according to the manufacturer’s instructions .
Gene expression analysis by qRT-PCR
To analyze the expression patterns of γ-terpinene, α-terpineol, and geraniol synthase genes, the total RNA of T. vulgaris ‘Elsbeth’, T. quinquecostatus, T. longicaulis, T. vulgaris ‘Fragrantissimus’, and their F1 lines was isolated and reverse transcribed, as described above. To amplify four TPS genes (encoding two γ-terpinene synthases, one α-terpineol synthase, and one geraniol synthase), primers were designed using Primer3 (http://primer3.ut.ee) (Supplementary Table S8). Then, qRT-PCR was carried out on the CFX96 instrument (Bio-Rad, USA) with Ssofast EvaGreen Supermix Kit in a 20 µl reaction volume containing 0.8 µl of each primer, 1 µl of template cDNA, 10 µl of 2× T5 Fast qRT-PCR Mix (TSINGKE, China), 7 µl of ddH2O, and 0.4 µl of 50× ROX Reference Dye II (TaKaRa, China). The qRT-PCR protocol included 40 cycles of 95 ℃ for 30 s, 95 ℃ for 10 s, and 58 ℃ for 30 s, along with melting curve analysis. Primer sequence, amplicon length, amplification efficiency (%), and linear correlation coefficient (R2) corresponding to two internal reference genes (18 S rRNA and β-actin) are shown in Supplementary Table S8. Finally, the relative quantification of four TPS transcripts was performed using the internal reference gene 18 S rRNA. Relative expression levels were calculated using the 2−ΔΔCt method after transcript data normalization. All analyses were performed in triplicate .
Essential oil extraction
The essential oil of 10 thyme species was isolated by steam distillation at 180–200 ℃ for 90 min. Essential oil yield (%) was calculated as volume (ml) of the isolated oil per 100 g of dry plant material. The isolated essential oil was dried using anhydrous sodium sulfate, and stored at 4 ℃ until needed for further analysis .
Analysis of essential oil composition
The essential oil composition of 10 thyme species was analyzed by gas chromatography-mass spectrometry (GC-MS) using Agilent 7890 A-7000B gas chromatograph (Agilent, USA), equipped with Agilent 5975 C MS detector (Agilent, USA). Using the HP-5MS (30 m, 250 m ID, 0.25 μm film thickness) capillary column, volatiles were separated using the following temperature program: 5 min at 60 °C; increased to 220 °C at the rate of 4 °C/min; increased to 250 °C at the rate of 60 °C/min; hold at 250 °C for 5 min. The following parameters were used: injector and detector temperature, 250 °C; carrier gas, He; flow rate, 1 m/min; split ratio, 1:10; acquisition range, 50–500 m/z in electron-impact mode; ionization voltage, 70 eV; and injected sample volume, 1 µl. The determination of the content of each compound (%) was based on the normalization of GC peak areas. The identification of essential oil components was based on the comparison of retention indices (RIs), relative to a homologous series of n-alkanes (C7–C40), and mass spectra (MS) from the NIST (v14.0) library and data from scientific literature . RIs were based on the equation:
where RT(x), RT(z), and RT(z + 1) for the composition, and the number of carbons Z and Z + 1 for the retention time of the normal alkane.
Analysis of leaf VOC profiles of thyme
The leaf VOC profiles of T. vulgaris ‘Elsbeth’, T. quinquecostatus, T. longicaulis, T. vulgaris ‘Fragrantissimus’, and their F1 lines were detected via headspace solid-phase microextraction (HS-SPME). Briefly, 0.25 g of fresh leaf powder was weighed and immediately placed into a 20 ml headspace vial (Aligent, Palo Alto, CA, USA) containing 20 µl of internal standard solution (1 mg/ml, 3-Octanol, Cas#589-98-o, Aladdin, Shanghai, China). The vials were sealed using crimp-top caps with TFE-silicone headspace septa (Agilent, Palo Alto, CA, USA). Subsequently, each vial was immediately incubated at 40℃ for 30 min. Then, to absorb the volatiles, the headspace of each vial was exposed to 100 μm coating fiber polydimethylsiloxane (Supelco, Inc., Bellefonte, PA, USA) for 30 min. All VOCs on the coating fiber were analyzed by GC-MS using Model 7890 A GC instrument and 7000B mass spectrometer (Agilent, Palo Alto, CA, USA) .
The GC-MS conditions were as follows: injector temperature, 250 ℃; transfer line temperature, 250 ℃, respectively; column temperature, initially maintained at 50 ℃ for 3 min, gradually increased to 150 ℃ at 4 ℃/min for 2 min, and finally raised to 250 ℃ at 8 ℃/min for 5 min; carrier gas (helium) flow rate, 1 ml/min; injection, splitless mode; ionization voltage, 70 eV; source temperature, 250 ℃; and MS range, 35–500 m/z. Agilent MassHunter 5.0 was used to analyze the chromatograms and MS. VOCs were identified by comparing the retention times of individual peaks with those of authentic standards, and MS were determined based on the NIST v14.0 MS database and the data reported previously . RIs were calculated using the following equation:
where RT(x), RT(z) and RT(z + 1) for the composition, and the number of carbons Z and Z + 1 for the retention time of the normal alkane.
Density of glandular trichomes
Glandular trichomes were visualized using a stereomicroscope (Leica DVM6, Germany). The number of glandular trichomes within a certain leaf area was counted using the ImageJ software. The average glandular trichome density was calculated based on three plants.
Data were expressed as the mean ± standard deviation of three biological replicates. Statistical analysis, including variance analysis, hierarchical clustering analysis, and correlation analysis, was performed using IBM SPSS Statistics for Windows, version 19.0 (Armonk, USA). Significant differences among the different genotypes were tested using a one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test at 5% probability level (p ≤ 0.05).
Availability of data and materials
The raw sequence data and gene sequence information of T. quinquecostatus were deposited in NCBI under the project accession number PRJNA690675.
Gas chromatography-mass spectrometry
Chromatin conformation capture
Headspace solid-phase microextraction
Institute of Botany, Chinese Academy of Sciences
Principal component analysis
Polymerase chain reaction
Quantitative real-time PCR
Quantitative trait locus
Simple sequence repeat
T. guberlinesis Iijin
T. quinquecostatus var. przewalskii
T. serpyllum ‘Aureus’
T. vulgaris ‘Elsbeth’
T. vulgaris ‘Fragrantissimus’
Volatile organic compound
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We thank Yan Zhu and Lu Wang (Plant Science Facility of the Institute of Botany, Chinese Academy of Sciences) for their excellent technical assistance in the GC-MS and HS-SPME analysis of essential oil and leaf volatile organic compounds.
This study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA23080603).
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Plant data were collected with permission from the related institution, and complied with national or international guidelines and legislation.
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The authors declare that they have no conflict of interest.
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Supplementary Fig. S1. Images of the essential oils of 10 different thyme species.
Supplementary Fig. S2. Detection of simple sequence repeat (SSR) loci in the Thymus quinquecostatus genome.
Supplementary Table S1. Phenotypic characteristics of the parents of two hybrid thyme populations.
Supplementary Table S2. Primers used for the development of SSR markers.
SSR primers used for the identification of F1 hybrids in the Tl × Tvf population.
SSR primers used for the identification of F1 hybrids in the Tve × Tq population.
Relative contents of volatile organic compounds (VOCs) in the leaves of Tl, Tvf, and their F1 progeny.
Relative contents of VOCs in the leaves of Tve, Tq, and their F1 progeny.
Reported amino acid sequences used to construct the TPS phylogenetic tree.
Alignment of the deduced amino acid sequence of Tq04G005190.1 with the reported amino acid sequence of geraniol synthase ObGES.
Primer sequence, amplification length, amplification efficiency (%), and linear correlation coefficient (R2) of four TPS genes and two reference genes (18 S rRNA and β-actin).
gels and blots.
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Cite this article
Sun, M., Zhu, L., Zhang, Y. et al. Creation of new germplasm resources, development of SSR markers, and screening of monoterpene synthases in thyme. BMC Plant Biol 23, 13 (2023). https://doi.org/10.1186/s12870-022-04029-2

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Eid ul Azha Recipes-Dhuan Daar Kabab Recipe. You have tried many types of kabab recipes. But you will never tried this delicious kabab recipe. Dhuan Daar Kabab Recipe is one of the famous Kabab recipes. Dhuan Daar Kabab recipe are very popular, exotic and delicious in Pakistan. It can be served as snacks or appetizer. You can serve this tasty kababs with naan or paratha. You can also make it at home. Little kids like this recipe very much.
The ingredients used in this recipe is boneless beef, black pepper, salt, crusheds red chilli, corn flour, boiled and crushed potatoes, soya sauce, onion, oil, ginger garlic paste and egg. Eid ul Azha Recipes-Dhuan Daar Kabab Recipe.
Boneless Beef ½ kg
Black pepper 1 tea spoon
Salt 1 tea spoon
Corn Flour 2 spoon
Boiled potatoes 2
Soya Sauce 1 spoon
Crushed Red chilli 1 tea spoon
Ginger Garlic Paste 1 spoon
Oil For shallow fry
Firstly, make paste of ½ boneless beef, 1 tea spoon salt, 1 tea spoon black pepper, 1 tea spoon of crush red chilli, 1 spoon of ginger garlic paste and add 1 cup of water for cooking.
Cook it until the beef cooked and water becomes dry.
Now make small pieces of beef and add 2 boiled and crushed potatoes, 1 onion, 2 spoon of corn flour and crushed masala. Mix it well and give smoke of charcoal.
Eid ul Azha Recipes-Dhuan Daar Kabab Recipe
Then make kabab and Shallow fry it.

domain: food


[Sam Barker] had a boring dingy that he wanted to spice up a bit, so he resolved to 3D print a hydrofoil wing for it so that it could fly across the water. (Video, embedded below.)
With a large wing designed and sliced into several pieces, and a total print time of 200 hours, [Sam] was ready to glue the foil wing together when he realized his scale was way off and the wings were far too large for his boat. With some hacking, [Sam] was able to use a single wing across the bottom of the ship. [Tom Stanton] came over to help with fiberglassing, and they were ready for a test.
As you might have guessed from the title, the test wasn’t particularly successful. Swapping the engine on the boat for a more potent motor gave the lift he needed in the front, but without a back foil, it was a wheelie rather than what [Sam] hoped for. Back at home, they printed a second wing and went back for a second test. The boat would start to lift out the water, but the shaft of the engine lifted out of the water, sending him back down. Unfortunately, a downpour cut the test short.
Not to be defeated entirely, [Sam] connected it to a much larger boat once the weather cleared and pulled his dingy along behind. To [Sam’s] credit, they did get some solid foiling, and the ship did lift out of the water until the wings sheared off from the stress. All in all, an entertaining story of engineering while racing against the weather.
We admire [Sam’s] ambition, and if you’re thinking about building a whole hydrofoil, we suggest starting with a smaller RC model and scaling up from there.


The hurricane season in 2017 was one of the most devastating seasons in recent years. Here were some of the worst storms from the year, and the areas they affected.
1. Sep 11, 2017
2. Sep 10, 2017
Honey Bees Could Combat Antibiotic-Resistant Bacteria
3. Sep 10, 2017
Apple’s Expensive OLED Handset, iPhone 8, Will Probably Ship After The LCD-Featured Models
4. Sep 08, 2017
Galaxy Note 8 Goes Through The Durability Test: A Recent Video Unveils The whole Fact


text:
Statistics from Altmetric.com
This supplement is focused on the methods, results and recommendations from the research project “Methods of Assessing Response to Quality Improvement Strategies” (MARQuIS). MARQuIS was funded by the Scientific Support to Policies component of the European Union Sixth Framework Research Programme (Contract SP21-CT-2004-513712), and it lasted from January 2004 to June 2008.
MARQuIS was coordinated by R Suñol, assisted by P Vallejo, from the Avedis Donabedian University Institute (Autonomous University of Barcelona) in Spain. Some of the European leading organisations in the healthcare quality field participated in this study, with two different roles:
Eight organisations (partners) were involved in research and dissemination activities: the European Hospital and Healthcare Federation (Belgium), the European Society for Quality in Healthcare (Ireland), the Centre for Research and Advanced Training (Italy), the Department of Social Medicine, Academic Medical Centre/University of Amsterdam (The Netherlands), the Dutch Institute for Healthcare Improvement (The Netherlands), the Manchester Centre for Public Policy and Management (UK), the National Centre for Quality Assessment in Health Care (Poland) and the Avedis Donabedian University Institute (Spain).
A country coordinator was involved in the coordination and development of the MARQuIS field test in each of the eight involved countries (except for Belgium, which had two coordinators): École de Santé Publique, Université Libre de Bruxelles and Katholieke Universiteit Leuven (Belgium), Spojena akreditacni komise Ceske Republiky (Czech Republic), Agence Nationale d’Accréditation et d’Evaluation en Santé (France), National Centre for Quality Assessment in Health Care (Poland), Fundación para la Acreditación y el Desarrollo Asistencial (Spain), Nederland’s Instituut voor Accreditatie …
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

domain: medical


– Primary Air Units
– Air Handling Units
– Terminal Units
– Large scale custom solutions & analytics
– Significant time savings for facility maintenance
– Better function for improved occupant comfort, energy efficiency and money savings
– Continuously improved analytics field verified by facility staff
We’ve deployed SkySpark in approximately 30 million square feet of casinos in Macau including two of the largest buildings in the world!
BIG BUILDING DATA
Connecting the Macau casinos to SkySpark in the cloud was not an option, so BuildingFit created a custom, on-premise functional diagnostic tool (FDT).
SkySpark is deployed on a stand-alone laptop that is connected to the Honeywell building automation system (BAS).
Data is pulled from the BAS through a custom connector (also powered by BuildingFit).
In addition to our standard analytical package, BuildingFit developed custom analytics for each facility specifically designed to test their unique sequences of operations.
A report is generated that identifies Sparks and KPIs.
Operators review the report and create work orders to fix issues.
Using the FDT has streamlined operation and maintenance for casino operators. It has saved countless hours of facility maintenance time while allowing them to optimize system operation to save energy, money, and improve occupant comfort.


text:
The Commissioner of Cruise has called for a Pop-Up Cruise this evening, Friday, May 1, 2020.
The cruise is planned for a meet up at Northridge Plaza, in Pierre, any time after 6pm.
Just like the olden days, Euclid Avenue will be the ultimate destination starting at 7pm.
Top off that fuel tank with cheap gas and plan to spend the evening cruising!!
The disclaimer part:
Social distancing/No group gathering is required.
Safe driving practices are strongly advised.
Bring the fun, be safe, and enjoy the ride!!

domain: travel


- Levels Taught:
Experienced piano teacher of 6 years available for eager students of all experience levels and ages! I structure my lessons so students learn what they want to learn, while also building a strong foundation of piano playing skills. I specialize in classical piano music.
- Teaches students:
- Ages 7 to 18
- Teaching since:
- May 2009
Subjects tought in person: Viola, Violin and Piano
Education: Aug 2006----May 2013 University of Missouri-Kansas City (Doctor of Musical Arts and Performance)
Sept. 2004----May 2006 Mannes School of Music (Performance Diploma)
Sept.2002-----May 2004 Mannes School of Music (Master of Music)
About: I have been a lover of Classical music since I was very young. I was fortunate enough to have my major in Viola performance for my entire study. My educational background has prepared me for the role of Musical Performance teacher. In particular, my studies of Viola Performance and Musical Education have given me a solid background to instruct students in all aspects of musical training. I am eager to contribute my enthusiasm and up-to-date skills to students of all ages in the instruments of my specialty.
*** Lesson Details ***
I am an easy going person with a great passion for music, that I would like to pass on to my students. I hope my Students will be able to learn new things in every lesson, and play their instrument with confidence and beautiful sound.
*** Studio Equipment ***
Music stand and piano .
*** Travel Equipment ***
I expect students to have their own instruments and musical stands.
*** Specialties ***
I could teach all kinds of materials.
The MTNA was founded in 1876 with the goal of advancing the value of music study and music-making to society while supporting the careers and professionalism of teachers of music. With nearly 22,000 members in 50 states—and more than 500 local affiliates—the MTNA is the preeminent source for music teacher support, where members embody like-minded values and commitment to their students, colleagues and society as a whole, while reaping the rewards of collaboration, continuity and connection throughout the lifetime of their careers.Show More
Instructor is encouraging and patient, also pays attention to details. My kid is very happy with the first lesson as the instructor helped her correct and improve finger positions, and taught her how to use muscles properly.
My daughter enjoys Yuan-Yu's class so much that she couldn't stop talking about what she learned from her first class. Great start!


text:
Ghaziabad: A local court here is likely to pronounce its order on framing of charges in the Aarushi-Hemraj twin murder case on Thursday after lawyers for CBI and accused dentist couple Rajesh and Nupur Talwar concluded their submissions Wednesday.
Arguing on behalf of Talwars, defence counsel refuted the prosecution case that their clients could have killed 14-year-old Aarushi and their domestic help Hemraj after having seen them in an objectionable position.
The counsel contended that Rajesh and Nupur Talwar were practicing doctors, had an intercaste marriage and belonged to the elite class of the society and could not have stooped to a level of provocation so as to kill their only daughter.
The defence also refuted the CBI`s case that since there were four people in the house, the onus was on Nupur and Rajesh to explain what happened on the fateful night of May 15-16, 2008.
It said the Talwars had gone to sleep and Hemraj had access to the entire house and could have allowed entry of outsiders who could have carried out the killings.
CBI prosecutor R K Saini, while summing up the arguments, said the defence was trying to examine the evidence which could not be allowed at the stage of framing of charges.
Aarushi was found murdered on May 16, 2008 in her bedroom in her Noida house and the body of Hemraj was found the next day from the terrace.

domain: law


crackerized = Mike Carter aka crackers who owns carter's archery www.cartersarchery.com
Basicly crackers is the Smokey Yunik of bow tuning:wink:
alot of folks ship there bows to him to get them "crackerized" ie "super tuned"
He has a few tricks up his sleeve
A forum community dedicated to bow and crossbow owners and archery enthusiasts. Come join the discussion about optics, hunting, performance, troubleshooting, styles, reviews, accessories, classifieds, and more!


The development of systems, whether high-assurance or mission-critical, faces a perennial problem: We can envision and therefore want to build systems that are much more capable and complex than we can assure with traditional approaches. The result is predictable: System errors resulting in system failures that result in losses—sometimes including loss of life.
Many system errors can be traced to erroneous behavior of critical system software. Software, even more than systems, is prone to escape from the envelope of manageable complexity. Software has no physical extent, no physical weight, and no physical power demand per se. The pressures that constrain the features we want to add to systems don’t constrain software. If we can imagine a feature, we can add the feature to software. The result is again predictable: Software that’s far too complex to assure; that fails; and that causes system failure.
Software assurance has traditionally been gathered through extensive testing. Unfortunately, testing isn’t exhaustive and thus can only reveal the presence—not absence—of errors. But we can do better than just testing. At the system level, we apply analysis as well as testing. For example, when designing an aircraft, aerospace engineers conduct extensive aerodynamic analysis using computational fluid dynamics before going into the wind tunnel. The wind-tunnel tests validate the analytical model, upon which the fundamental assurance is based.
For software, we can apply formal methods. These are techniques based on mathematical representations of software behavior that allow for a truly exhaustive examination of software state to prove the absence of errors. Software testing then validates this comprehensive analysis.
Despite its power, the use of formal methods isn’t a silver bullet. In particular, software engineers must ask the right questions when conducting analysis. Asking the right questions is hard when critical system-level properties haven’t been explicitly traced to software. Software engineers often don’t know which properties need to be proved.
When focusing on SSI, systems engineers identify critical system-level properties and trace those properties through decomposition across system architecture and through translation from one artifact to the next. This attention to the evolution of system-level properties enables systems engineers to effectively communicate to software engineers the critical properties that they should prove. Focusing on SSI enables engineers to take full advantage of formal methods.
As model-based systems engineering has become more popular, tools supporting this approach have included increasingly powerful analyses. These tools go beyond simple checks for latency or space, weight, and power consumption, and they seek to demonstrate functional correctness of the architecture. Effectively, these tools apply formal methods at a higher level of abstraction. To take full advantage of these tools, systems engineers need clearly identified properties that they should prove. Focusing on SSI enables systems engineers to take full advantage of these analysis tools.
Talking to systems and software engineers in industry, we often hear that systems-engineering artifacts like requirements documents and design documents are “tossed over the wall” from one team to the next. This statement is somewhat astonishing. Surely all engineers involved in development—from systems engineers to software engineers—should be a single, integrated team.
Different people have different specialties, and software standards such as DO-178B/C require, at the highest Design Assurance Level, independence between those who develop a component and those who verify it. However, integrity is certain to be lost if there’s not constant and managed communication amongst team members. Focusing on SSI enables systems and software engineers to bridge the walls that separate them, by focusing communication on the critical properties along with the artifacts that serve as the interface between team members.
How Is SSI Supported?
SSI is a trait of high-assurance systems engineering—not a tool or technology. However, to facilitate SSI in systems engineering, tool support is essential. There are four integral parts to this tool support, plus an optional fifth: translation, traceability, analysis, argument, and (optionally) test-case generation.
Translation assists in preserving integrity by automating the representation of properties expressed in one artifact into the next. For example, in the SysML modeling language, a constraint associated with a requirement that’s written in OCL (the Object Constraint Language) and attached to an output port on an Internal Block Diagram might be translated into a synchronous observer in a Simulink model that’s written using Simulink blocks and attached to an output port of a subsystem. Similarly, the constraint might be translated to a postcondition in a programming language like SPARK that supports software contracts.
Translation bridges the gap separating the architecture (in SysML) from the design (the Simulink model) or the implementation (in SPARK). It also reduces the likelihood of error by not requiring that an engineer manually recode the property in the target language. Of course, this requires that we have confidence in the correctness of the translation, but techniques are available (such as tool qualification under DO-178B/C) that can offer this confidence.
Traceability assists in preserving integrity by allowing engineers to follow the evolution of properties through system development. Continuing the example above, the Simulink synchronous observer would be traced to the OCL constraint so that if either was changed, engineers would be notified that the other must be reexamined or regenerated.
Analysis assists in preserving integrity by allowing engineers to prove property satisfaction across decompositions. At each phase of system development, greater abstraction is replaced by greater precision, until the system has been fully and precisely described. Decomposition plays a critical role in this ever-increasing precision, as larger abstract components are broken into multiple, smaller components.
With analysis, engineers can prove that the smaller components, together, satisfy the intended functionality of the larger, abstract component. Continuing the example above, the Simulink subsystem would be broken up into smaller subsystems, each with its own contracts that, together, must satisfy the observer for the top-level subsystem.
Argument assists in preserving integrity by allowing engineers to record their rationale for property continuity when traceability or analysis are insufficient or unable to provide sufficient support. Continuing the example above, one of the subsystems may be COTS and might not have a sufficiently precise contract to enable proof that the observer for the top-level subsystem is satisfied. Engineers may know, however, that the way in which the COTS subsystem is used will, together with the other subsystems, always satisfy the observer for the top-level subsystem. The argument documents this rationale.
Test-case generation assists in confirming integrity by demonstrating that critical properties are satisfied by the system. Continuing the example above, the observer for the subsystem could form the basis of a test case or set of test cases to be included in that phase of integration testing.
This high-level workflow supports SSI.
A key requirement of tools supporting SSI is that they meet systems engineers where they currently are: Rather than ask engineers to adopt entirely new languages and entirely new development methods, these tools should support existing languages and existing development methods. Tools supporting SSI don’t currently exist, but AdaCore is working on building them. When finished, they will support a workflow that might look something like what’s illustrated in the figure.
What About Hardware?
This article has focused on SSI in terms of software: system-to-software integrity. But SSI is equally applicable to hardware. The languages used in the final phases of system development will differ for hardware, but the overall approach can still significantly benefit hardware development as well as software development.
The Bottom Line
Taking an integrated approach to system development and verification—by identifying key properties early and ensuring that they’re preserved as a system progresses throughout its lifecycle from requirements to implementation—can save time and effort. It also prevents mismatches between what a system is supposed to do and what it actually does. With automated support from existing and upcoming technology, SSI can offer the assurance that’s needed for the most critical systems that society depends on.
M. Anthony Aiello is Technology Strategist at AdaCore.


text:
Ema's_glasses.png (96 × 96 pixels, file size: 7 KB, MIME type: image/png)
Picture from Dual Destinies
Appears on these pages
Luminol is a chemical that is known for its chemiluminescence. When it reacts with blood, it...
Some of these are already in progress, some are planned, and others are just suggestions. Feel...


text:
Street Fighter Legends: Cammy
Jim Zub (Author)
Out of Stock. Usually despatches within 3 weeks.
DescriptionIt's Cammy White, the stinging bee of Street Fighter, in her own action-packed adventure! Cammy now leads the all-new special ops team "Delta Blue", as they keep England and the world safe from destructive forces. Whether its art thieves, cyber assaults, or would-be world conquerors, Cammy and Delta Blue are ready to face the enemies of freedom and justice everywhere!
Udon Entertainment Corp
31 October 2017
Earn By Promoting Books
Earn money by sharing your favourite books through our Affiliate programme.Become an Affiliate

domain: literature


text: Menzel’s career has traversed stage, film, television, and music. She was heard as Elsa in Disney’s global box office smash ‘Frozen’, in which she sings the film’s Oscar-winning song ‘Let It Go’. After Menzel’s performance of the multi-platinum song at the 86th annual Academy Awards, she made history as the first person with both a Billboard Top 10 hit and a Tony Award for acting. Menzel earned her first Tony nomination as Maureen in the Pulitzer Prize- winning musical ‘Rent’, and won the award for her performance as Elphaba in ‘Wicked’. Other notable roles include Rachel Berry’s mother, Shelby Corcoran, on the hit television show ‘Glee’ as well as starring opposite Susan Sarandon and Amy Adams in Disney’s ‘Enchanted’. Menzel starred in her own PBS special, ‘Barefoot at the Symphony’, with an accompanying live album of the same name, and her highly successful international concert tour recently included a sold-out performance at Radio City Music Hall. In addition to cast albums, Menzel’s prolific recording career includes the solo albums ‘I Stand’, ‘Here’ and ‘Still I Can’t Be Still’.

domain: artistic


text:
Why does my cat have so many decaying teeth?
Tooth decay in cats can be a painful condition for your pet feline. Tooth decay from feline tooth resorption is a condition in which cellular organisms attach to the teeth.
Can a cat have a tooth knocked out of place?
If your cat has suffered trauma or injury to the mouth, she could experience tooth dislocation or sudden loss of a tooth. When a feline experiences head trauma, a tooth can easily be knocked out of place or removed completely upon impact. A dislocated tooth or sudden loss of a tooth is an urgent condition.
What does it mean when a cat’s tooth tips to the side?
Tooth Luxation or Avulsion in Cats. Vertical luxation is related to the dislocation of the root of the tooth. In lateral lunation, the tooth tips to the side. Lateral luxation usually occurs due to an injury that has pushed the tip of the tooth to one side. A tooth is called avulsed, meaning that it has been torn suddenly from its spot,…
Can a cat have a longer upper tooth?
Sometimes older cats can have a longer tooth from the upper teeth. Once I asked a vet and she told me that a tooth may slip out of its seat and appear longer. It will eventually fall. This is what I was told. My cat has her left upper tooth longer than the right one. Click to expand… So this tooth will fall and new one will come?
How can you tell if your cat has dental problems?
Cats use their mouth for grooming, hunting, and eating. Our felines can spend anywhere from 10-15% of their time grooming themselves. When cats are experiencing tooth pain or dental problems, they will groom less, leading to an unkempt coat.
Why does my cat have so much pain in his teeth?
They will often continue to eat since their survival instinct to eat is so strong. Dental disease can manifest in different forms in the cat. Periodontal disease, tooth resorption, stomatitis, malocclusions, oral tumors and oral trauma are all cat teeth problems that can occur.
Is the tooth in my cat’s mouth loose?
However, for the past few days to possibly a week, the tooth has been poking out constantly. Today I decided to take a more detailed look at her mouth, and found that the fang is indeed loose and protruding horizontally.
Why does my cat have holes in her teeth?
Most of her teeth had tiny holes in them. Not visible by just looking at them, a slight gum redness, but that was it. She had Feline Tooth Resorption, and it was affecting most of her teeth.
What should I do if my cat has lost a tooth?
You may also want to speak with your vet regarding special food, treats or other dental products that may promote oral health. If your cat has had teeth removed as a result of tooth decay, the prognosis remains good. Cats can live full and regular lives even if missing several teeth.
Why is it important to check your cat’s teeth?
But cats can suffer from dental issues that, if unchecked, can lead to serious health problems. So don’t skimp on your cat’s annual checkups. Dental health is an important part of your cat’s annual visit, and your veterinarian will use that time to evaluate your cat’s overall health as well as their oral health.
Can a cat be put under anesthesia for tooth decay?
Placing your cat under anesthesia for treatment does have some risks, but it will ensure a thorough removal of harmful cells and bacteria and allow the vet to treat deep into the gum line. For treatment of severe tooth decay, your veterinarian will likely need to perform oral surgery on your cat.
Is it common for cats to have dental disease?
Diseases of the teeth and gums are common in cats. Studies report that between 50 and 90% of cats older than four years of age suffer from some form of dental disease, but fortunately the most common forms of these diseases are largely preventable or treatable with appropriate preventive dental care and monitoring.
Is it normal for cats to lose their teeth?
Of course, not all tooth loss is caused by dental disease, and not all dental disease results in tooth loss. As with humans, cats accumulate bacterial plaque on the surface of their teeth. If the plaque is not removed quickly, it becomes mineralized to form tartar and calculus.
How can I tell if my cat has tooth decay?
My cat’s back teeth on the top right side seem to have hard, brown growths on them. Her breath has been pretty bad for a while and dental chews aren’t helping. She is eating and drinking normally and there is no sign of bleeding or infection. She doesn’t seem to be in any pain. Her back teeth on both sides are very discoloured.
When do cats get their first permanent teeth?
They are fully grown by 5 to 6 weeks of age and the different types of teeth erupt at different times allowing age to be determined 4 (see below). At 4 months of age the thirty permanent teeth begin to show. These are complete by about 6 months of age.
Can a 17 year old cat have dental disease?
Tara: In the article, she tells the story of a 17-year-old cat with advanced dental disease. The cat’s caretaker was reluctant to put the cat under anesthesia because of concerns about her general health … Thomas: After all, the kitty already had kidney disease and hyperthyroidism…
How old does a cat have to be to have a toothache?
The Cornell University College of Veterinary Medicine website says about 85 percent of cats over 6 years old have tooth decay and that tooth resorption occurs in about 50 percent of cats.
How old do cats have to be to have bad teeth?
Problem Teeth. According to the Cornell University College of Veterinary Medicine, 90 percent of kitties will experience dental issues within their lifetimes and 85 percent of those over 6 years old will experience periodontal disease. Your furry friend cuts her adult teeth at 6 months of age and those pearly white have to last a lifetime.
What happens when a cat’s teeth fall out?
Combined with bacteria, it forms plaque, which in turn becomes tartar. Tartar gets into the gums and becomes inflamed. At this point, the gums can draw back and separate from the teeth. The gums become tender and develop pockets of infection. And then the teeth fall out.
How often should I Check my Cat’s teeth?
It is important to have your cat’s teeth and mouth checked at least twice a year to prevent the formation of tooth decay. Raw meats, small uncooked bones, or hard kibble may help scrape off plaque and keep teeth clean. Maintain your cat’s dental hygiene by brushing its teeth twice weekly with cat-specific toothpaste.

domain: pet


text:
Fusion: Trip to India
Fusion: Trip to India is a performance / video / installation created by Leandro Soto.
Video Stills from the first performance
In this improvisational performance piece participant artists originally from Germany, Cuba, France, India and the US play instruments and conduct dance movements with chanting and gestures inspired by South Indian Culture. Fusion stands for integrations of cultures and multidisciplinary art expressions.
Mixing the admiration for Old Culture with their own personal experiences in the New World, the artists lead the audience into an artistic spiritual journey.
Searching for an integration of old values with contemporary life in America, the piece develops its own language of communication to convey the integration of narratives and mythologies through creativity.
Participants in the first presentation:
- Danielle Godfrey: yoga asana movements
- Daniel Hirtz: tabla and percussion's instruments
- Kristopher Rein: tabla, flute, singing and harmonium
- Leandro Soto: narrative movements/dance free improvisation.
Paintings and Drawings from the Trip to India Series
Installation view of paintings in India


text:
Heavily processed foods can make it difficult to maintain steady weight loss, but some of the best foods to help you lose weight are those high in fiber, which many are surprised to learn is a form of a carbohydrate. Fibrous foods are often naturally lower in calories, help keep you satiated after a meal and also regulate blood sugar levels. Research suggests that the more dietary fiber that one incorporates into their daily routine, the more they're able to work against "abdominal fat depots."
Here are some foods that are good for loosing weight:
Peanut butter packs 8 grams of protein and up to 4 grams of fiber per serving, making it an ideal snack to help you fill up and stay satisfied (particularly in stabilizing a glycemic load). Just make sure that you're taking a look at the ingredients label, which should only include peanuts and salt (maybe a little bit of oil, but no high fructose corn syrup or additives necessary!). A published review of research in the Journal of Food Science and Technology highlights the fact that peanut butter can help people feel more satisfied compared to other snacks.
A cup of peas packs 8 grams of protein and tons of key bloat-reducing nutrients. It's got nearly all of what you need daily for vitamin C, plus magnesium, potassium and iron all of which aid in counterbalancing sodium and bringing oxygen to blood cells.
They're filled with plant and fiber based protein as well as immune-boosting antioxidants and bloat-busting minerals. Chickpeas easily go in soups, stews, salads, and side dishes. Plus, chickpea flour is a great baking alternative for a more nutrient-dense and filling end result.
Pumpkin seeds provide tons of immune-boosting zinc, but more importantly, are a significant source of fiber (quite a filling snack!). With about 7 grams of protein per snack-sized serving, pepitas are a great addition to most diets.
Once feared for being high in cholesterol, whole eggs have been making a comeback. Although a high intake of eggs raises the levels of “bad” LDL-cholesterol in some people, they are one of the best foods to eat if you need to lose weight. They are high in protein and fat, and are very satiating.
One study in 30 overweight women showed that eating eggs for breakfast, instead of bagels, increased feelings of fullness (satiety) and made participants eat less for the next 36 hours
Another eight-week study found that eggs for breakfast increased weight loss on a calorie restricted diet, compared to bagels. Eggs are also incredibly nutrient dense and can help you get all the nutrients you need on a calorie-restricted diet. Interestingly, almost all the nutrients are found in the yolks.
Start a meal with a cup of soup, and you may end up eating less. It doesn’t matter if the soup is chunky or pureed, as long as it's broth-based. You want to keep the soup to 100 to 150 calories a serving. So skip the dollops of cream and butter.
Salmon is a rich source of high-quality protein and provides plenty of "good" fats: omega-3 fatty acids. A diet rich in omega-3 fatty acids helped people feel more satisfied when they were watching their calories, per a study in Appetite. Eating salmon can be a delicious and versatile way to get your recommended two weekly servings of heart-healthy fish.
Tuna is another low-calorie, high-protein food. It’s lean fish, meaning it’s low in fat. Tuna is popular among bodybuilders and fitness models who’re on a cut, as it’s a great way to increase protein intake while keeping total calories and fat low. If you’re trying to emphasize protein intake, make sure to choose tuna canned in water, not oil.
All beans are high in fiber, which is your friend when you're trying to lose weight because it helps you feel fuller longer, thus controlling hunger. Eating beans and legumes has also been linked with various other health benefits, including lowering blood pressure, reducing LDL cholesterol and reducing risk of cardiovascular disease. Beans are fairly low in calories and deliver protein as well. Try them in homemade veggie burgers, soups and salads.
Believe it or not, air fried potatoes (yes, really) are an excellent source of potassium which can help manage bloating and counterbalance sodium. They're high in fiber as well, meaning potatoes can be a nutrient-dense food — just as long as they're not served the french-fry way.
Want to enjoy chocolate between meals? Pick a square or two of dark over the milky version. In one study, chocolate lovers who were given dark chocolate ate 15% less pizza a few hours later than those who had eaten milk chocolate.
For a great snack on the run, take a small handful of almonds, peanuts, walnuts, or pecans. Research shows that when people munch on nuts, they automatically eat less at later meals. Nuts are an excellent source of fiber, and they're high in protein. Eating foods with the one-two punch of fiber and protein can help you feel fuller longer-which makes it less tempting to reach for an unhealthy snack between meals.
As long as this popular crunchy treat isn't doused in movie-theater butter, it makes an excellent weight-loss snack. Not only is popcorn high in fiber, it even delivers some protein. A 1-ounce serving of air-popped corn (about 3½ cups) has 4 grams of fiber, almost 4 grams of protein and clocks in at 110 calories. This combination makes it a snack with staying power. Popcorn is filled with air, so you get a pretty large portion without a lot of calories. You can eat 3 whole cups of popcorn for only 100 calories.
Yes, grapefruit really can help you shed pounds, especially if you are at risk for diabetes. Researchers at Scripps Clinic in San Diego found that when obese people ate half a grapefruit before each meal, they dropped an average of 3 ½ pounds over 12 weeks. Drinking grapefruit juice had the same results. But grapefruit juice doesn't have any proven "fat-burning" properties -- it may just have helped people feel full.


Refrigerated fermenter with conical bottom 550 L
Refrigerated Conical Bottom Fermenter 550 L
The fermenter with refrigerated conical bottom 500 L is a resistant container suitable for beer processing, made entirely of AISI 304 stainless steel (18/10 for foodstuffs) with a mirror-polished interior and a flowered exterior. This fermenter with refrigerated bottom has a capacity of 550 litres, a diameter of 80 cm, a height of 190 cm and is equipped with three adjustable feet with a 50 mm diameter floor plate. It is equipped with a cooler pocket, a non-returnable manhole cover (diameter 40 cm, frame height 90 mm) with an arm and a lateral flag opening.
The 3/8" stainless steel ball valve allows the passage of nitrogen, while the 1" pipe and the 65 mm diameter AISI 316 washing ball allow easy sanitisation of the tank. The 500 L fermenter with conical bottom has a stainless steel sample collector, and is equipped with total discharge with DIN32 trough and DN30 DIN 32MG/32DIN FEMM butterfly valve, and Dn 80 bimetallic stainless steel thermometer.
- Material: stainless steel AISI 304
- Capacity: 550 L
- Diameter: 80 cm
- Height: 190 cm
- cooling jacket
- stainless steel AISI 304 anti-retention manhole diameter 400 mm, manhole neck height 90mm, arm closing and side opening + nitrogen tight closing knobs
- nitrogen inlet with stainless steel ball valve 3/8”
- washing tube 1” in the manhole neck and stainless steel butterfly valve DN30 DIN30 DIN 32MG/32 DIN FEMALE
- stainless steel AISI 316 spray ball diameter 65 mm
- plexiglass level gauge with stainless steel protective device and stainless steel tap level holder3/8”
- stainless steel level inlet DN10/external thread 3/8” gas for tube diameter 20 on the washing tube for an easy cleaning
- stainless steel sample-taking tap
- total discharge with stub pipe DIN32 and stainless steel butterfly valve DIN30 DIN32MG/32DIN FEMALE
- partial discharge with stub pipe DIN32 and stainless steel butterfly valve DIN30 DIN32MG/32DIN FEMALE
- stainless steel bimetallic thermometer DN 80, -20 °C/+80 °C with stem 200 mm equipped with thermometer holder
- adjustable stainless steel feet and base plate diameter 50 mm
- liquid entry and exit on the cooling jacket 1"
- Appliance conforms to CE safety standards