Introduction

Tea is the oldest and the most popular non-alcoholic beverage all over the world (Wambulwa et al. 2016). Tea plant is originated from China and has been grown in a geographical range from 18°N to 37°N in latitude and from 95°E to 122°E in longitude of this country (Chen et al. 2012; Long et al. 2012). A great number of tea plant germplasms, including the tea plant, allied species and cultivars in the section Theaceae genus Camellia, have been collected and conserved in China, Japan, India, Kenya as well as a number of other countries (Fang et al. 2012). Wild type tea plants are defined as the general name for wild woody evergreen plants belonging to Camellia, Theaceae, with significant value in science and application as they have been naturally selected and evolved during a long period of time. They possess some important characters such as the stubborn resistance to stressful conditions and unique quality etc. that the cultivars don’t have. Consequently, they are the precious materials for development and cultivation of excellent tea varieties. Plants belonging to Camellia from each province in China were listed by Chang HT.A (Chang 1981): a total of 66, 55, and 50 varieties have been found in Guangxi, Yunnan and Guangdong provinces, respectively. The adjacent areas of Guangxi, Yunnan, and Guizhou were considered one of the secondary original centers of tea plants based on the geographic distribution of the wild type tea plants (Yao et al. 2005). The extraordinarily abundant wild tea plants have been growing in Guangxi owning to its unique and favorable geographical conditions.

Types and contents of biochemical components were the essential criteria for evaluation of tea plant quality, depending on the environmental conditions and related genes (Wei et al. 2011). Vital components of tea beneficial to human health are mainly the secondary metabolites of tea plants such as tea polyphenols, amino acids, caffeine etc., which are related to both resistance to stresses and tea quality (Anand et al. 2015; Kottur et al. 2010; Ma et al. 2014). Therefore, identification of the key biochemical components will pave the essential scientific basis for investigation, screening of high quality resources and application of wild type tea plants. To this end, molecular markers are the biomarkers at the DNA level. Random amplified polymorphic DNA (RAPD) is a highly effective tool to investigate the genetic diversity of living organisms and to differentiate tea germplasms at the inter-species level (Chen et al. 2005; Dhakshanamoorthy et al. 2015), represents dominant marker type known for easy handling, moderate cost of the technique, and the highly polymorphic nature (Bali et al. 2015).

There are rich resources of tea plants in China. However, previous researches on wild type tea plant germplasm resource were mainly focused on those growing in Yunnan and Guizhou provinces (Chen et al. 2005; Liu et al. 2010; Qi et al. 2013), scarce studies were done on those growing in Guangxi province (Huang et al. 2014; Zhu and Yahui 2015). However, there are plentiful wild type tea plant resources existing in the region of Daoyao Mountain, Guangxi, due to its unique geology, diverse climates and plentiful rainfall. The important ecological barrier of Dayao Mountain runing from northeast to southwest in the middle of Guangxi, formed in Permian of Paleozoic. Temple Mountain is its major peak with 1979 meters in height. Tea plant has to cross the Dayao Mountain from Yunnan-Guizhou Plateau, Southwest China, leading to the Southeast China.

In this study, we conducted a systemic analyses on the biochemical characteristic of 29 wild type tea plants collected from Liuxiang, Bainiu and Gonghe populations of Dayao Mountain. Liuxiang population mostly grows under the shade of primeval forest, Bainiu population partly grows under the tall trees; Gonghe population grows with scrubby shrubs. In addition, some special tea resources containing high contents of epigallocatechin gallate (EGCG), caffeine, and amino acid etc., have been discovered. This study may also contribute to development and utilization of wild type tea plants and scientific research on tea breeding and processing of high-quality tea.

Materials and methods

Samples and reagents

Sources of tea plants are described in Fig. 1. DYS1-13, DYS14-23 and DYS24-29 are selected from Liuxiang, Bainiu and Gonghe, respectively.

Fig. 1
figure 1

Tea plant (Camellia sinensis (L.) Kuntze ) sources in Dayao Mountain, South China

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All of the following standard chemicals were purchased from Sigma-Aldrich (Co. LLC): Caffeine, theobromine, theophylline, catechins including (+)-gallocatechin (GC), catechin (C), (−)-epigallocatechin (EGC), (−)-epicatechin (EC), (+)-gallocatechin gallate (GCG), (−)-epicatechin gallate (ECG), catechin-3-gallate (CG), (−)-epigallocatechin gallate (EGCG), and amino acids (Siama, St. Luis, MO, USA). Methanol (HPLC), sodium carbonate (Analytical grade, AR), folin-ciocalteu (AR), galllic acid (AR), oxalic acid (AR), citric acid (AR), disodium hydrogen phosphate (AR), potassium dihydrogen phosphate (AR), ninhydrin (AR), stannous chloride (AR), sodium bicarbonate (AR), trifluoroacetic acid (AR), and aluminium chloride (AR) were obtained from Tianjin, China.

Preparation of samples

Samples were prepared according to the method described in Descriptions and Data Standard for Tea (Camellia spp.) (Chen et al. 2005): one bud and two leaves were plucked in spring, fixed in microwave oven (Midea, Guangzhou, China), and dried in oven (DHG-101, Shanghai, China).

Methods for determination of biochemical components of tea

The contents of moisture, aqueous extract, tea polyphenols, and free amino acids were determined according to GB/T 8304-2013, GB/T 8305-2013, GB/T 8313-2008, and GB/T 8314-2013, respectively.

The contents of alkaloids and catechin monomers were determined by high performance liquid chromatography (HPLC) (Agilent 1200, Agilent Technologies, Santa Clara, CA, USA). C18 reverse phase column (HyPersil ODS2, 4.6 mm × 250 mm, 5 μm) (Thermo Fisher Scientific, USA) was used. The determination conditions were as set follows: solvent A was pure methanol and solvent B was 0.05% trifluoroacetic acid in water. Column temperature was set at 30 °C and the flow rate was 0.8 mL/min. UV spectra were measured at 278 nm. Sample size for injection was 20 μL. The gradient elution profile was 2% A at 0 to 2 min, to 8% A at 8 min, to 10% A at 30 min, to 20% A at 35 min, to 25% A at 45 min, to 30% A at 50 min. 30% A was maintained from 50 to 55 min, and then changed to 35% A for 60 min.

The contents of free amino acid monomers were determined with amino acid analyzer (L-8800, Japan). The determination conditions were set as follows: concentration of amino acid standard was 100 μmol/L, sample size for injection was 20 μL, column temperature was 134 °C, running time was 110 min, room temperature was 26 °C, and relative humidity was 76%.

DNA extraction

Two wild type tea plants collected from Liuxiang, Bainiu, and Gonghe population were randomly selected. Tea leaf DNA was extracted with the method described by Chen et al. (2002) with a modification. 1.0 g of fresh leaves was quickly ground into powder with liquid nitrogen and appropriate amount of polyvinylpolypyrrolidone (PVPP) in a mortar. The powder was mixed with 1 mL of cetyl trimethylammonium bromide extract (2% CTAB, 0.1 mmol/L Tris, 20 mmol EDTA, 1.4 mol NaCl, pH = 8.0) containing 1 μL of mercaptoethanol, and kept in water bath at 65 °C for 45 min, shaked every 8 min. Phenol–chloroform-isoamyl alcohol mixture (V1:V2:V3 = 25:24:1) in the same volume was added, mixed well, and centrifuged at 12,000 r/min for 5 min. The supernatant was transferred to a fresh clean tube. The elution process was repeated three times. 1.5 mL of isopropanol was added to the centrifuge tube, kept at −20 °C for 1 h after shaking well, and centrifuged at 12,000 r/min for 5 min. White precipitation was transferred to a new clean tube, cleaned three times with 75% of ethanol, then dissolved in 100 μL of double distilled water with RNase after being dried in the air. DNA solution was kept in water bath at 37 °C for 30 min. A fraction was used for DNA quality test and the rest was reserved in the refrigerator at −20 °C for subsequent analysis.

DNA quality test

1 μL of DNA solution was diluted to 100 μL with double distilled water and its purity was analyzed with the ratio of A260:A280 after detecting by nucleic acid protein detector (Biophotometer plus, Germany). Purified DNA solution was mixed with loading buffer in the ratio of 1:5. The DNA mixture was analyzed by 1.5% agarose gel electrophoresis apparatus (DYY-6C,Beijing, China) at 130 V for 20 min.

RAPD molecular marker

Polymerase chain reaction (PCR) assays were performed with Thermal Cycler (My Cycler Tm Thermal, USA) as described by Wang et al. (2008) with a modification. 18.5 μL of ddH2O, 2.5 μL of 10 × Taq Buffer containing 20 mmol Mg2+, 2 μL of dNTPs (2 mmol/L), 0.5 μL of primer (8 PM/μL), 0.5 μL of Taq DNA polymerase, and 1 μL of DNA template (40–100 ng) were in PCR reaction solution. PCR amplification was run under the following programmed procedures: pre-denaturation at 94 °C for 1 min, followed by 38 cycles of annealing at 33–37 °C for 50 s, and extension at 72 °C for 2 min and finally extended at 72 °C for 10 min. PCR products were separated by 1.5% agarose gel electrophoresis at 130 V for 80 min, stained with ethidium bromide and the stained bands were visualized with UV transilluminator.

Statistical analysis

Maximum, minimum, mean, and Shannon–Weaver index (H′) were calculated with Excel 2013. H′ = −ΣpilnPi (Pi = Ni/N, occurrence frequency of the ith character). Cluster analysis was done according to Ward method after data normalization by SPSS 18.0. RAPD data were analyzed by NTSYS-pc2.10e software, and PIC (polymorphism information content) = 1−ΣP 2i (Pi: occurrence frequency of the ith polymorphic bands). Genetic similarity coefficient (GS) of germphlasm resources was calculated according to Jacard coefficient.

Results

Genetic diversity analysis

The higher the Shannon–Weaver index (H′) is, the more the information of community has. Species is abundant as H′ > 3.0. H′ of aqueous extract, tea polyphenol, free amino acid, flavonoid, and alkaloid were higher than 3.0, showing that the genetic diversity of wild tea plants from Dayao Mountain is abundant (Table 1). H′ of total catechins, ester-catechins, and non-ester catechins were 3.08, 3.11, 3.01, respectively, which were also greater than 3.0, showing a wide range of evolution of wild type tea plants.

Table 1 Genetic diversity analysis according to main biochemical components
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Cluster analysis of wild tea plant germplasms from Dayao Mountain

Tea plant population can be 90–96% correctly classified by the total contents of tea polyphenol, catechin constitutions, the ratio of caffeine to total nitrogen, theanine (Liu and MJ 1991). The contents of C, EC, GC, EGC, CG, ECG, GCG, EGCG, caffine, theobromine and theophylline were analyzed by HPLC. And factor analysis was according to the dates of catechins and alkaloids after standardization. KMO was 0.609 > 0.500 and the significance level of Bartlett’s test of sphericity was 0.000 < 0.005, showing that the result of factor analysis was credible. And the accumulative contribution rate of the first four public factors total variation accounted for 85.535% > 85%, maintaining most of information of 11 characters.

Then cluster analysis was made according to the four major factors by Ward’s method. The result was shown in Fig. 2A. 29 single plants were clustered into three groups. DYS4, DYS5, DYS6, DYS24, DYS25, DYS26, DYS27, DYS28 and DYS29 were in the first group, DYS8, DYS14, DYS15, DYS16, DYS17, DYS18, DYS19, DYS20, DYS21, DYS22 and DYS23 were in the second group, and DYS1, DYS2, DYS3, DYS7, DYS9, DYS10, DYS11, DYS12 and DYS13 were in the third group. On the one hand, all the Gonghe population was in the first group, all the Bainiu population was in the second group, and the third group was from Liuxiang. The result indicated that wild tea plants from Dayao Mountain were of significant geographical uniqueness. On the other hand, the Liuxiang population were distributed all over the three groups, which indicated the variation of Liuxiang population was the most abundant, and the genetic diversity was the most richest. Therefore, the Liuxiang population was the most primitive.

Fig. 2
figure 2

Cluster analysis of wild tea plants from Dayao Mountain. A Was based on cataechins and alkaloids. B Was based on genetic similarity coefficient. C1 Lintou Dancong, C2 Hongbing Dancong, C3 Huangzhixiang, C4 Tieguanyin, C5 Zhenghe large-leaved white tea, C6 Huangjingui, C7 Maoxie, C8 Zijuan, C9 Changye Baihao, C10 Foxiang 3, C11 Yungui, C12 Xiang 1, C13 Xiang 2, C14 Xiang13, C15 Huangjincha

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Diversity analysis of RAPD amplification production

A total of 17 primers with high polymorphism, clarity, and good repeatability were screened out from 120 common primers of tea plant. The polymorphism information was shown in Table 2. A total of 172 polymorphic bands were amplified by 17 primers (PIC > 0.5), of which, 136 were polymorphic and polymorphism proportion was 79.07%. The numbers of polymorphic bands of each primer ranged from 6 to 15 and the polymorphism of prime 11 reached to 93.33%.

Table 2 Polymorphism information of PCR amplification products
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Cluster analysis of wild tea plants from Dayao Mountain based on GS

GS ranged from 0.65 to 0.88 among tea plant samples, and GS between DYS6 and Huangjingui, Changye Baihao was the minimum one (0.65), indicating that their genetic distance is far away. Genetic distances among tea plants from Liuxiang population and those in other areas were long according to their GS, which were 0.65–0.77. GS between DYS19 and Foxiang 3 was maximum one (0.83). Tieguanyin and Huangjincha were the most similar to DYS28 (0.80).

Cluster analyses of tea plants from five provinces based on GS were shown in Fig. 2B. 21 samples were classified into 2 groups based on their similarity coefficient, which was 0.74. DYS6 and DYS12 from Liuxiang population of Dayao Mountain were clustered into the same group; those from the rest regions of Dayao Mountain, Yunnan, Guangdong, Fujian, and Hunan were clustered in the other group. It suggested that tea resources from Liuxiang population of Dayao Mountain possessed extremely unique characters. 21 samples were classified into 4 groups based on their similarity coefficient, which was 0.76. Tea plants from Liuxiang population were clustered into two groups, those from Bainiu and Gonghe population were in one group, and those from Yunnan, Guangdong, and Hunan were clustered into the same group, further demonstrating that tea plants from Dayao Mountain are extremely diverse and unique.

Special germplasm resource of wild tea plant from Dayao Mountain

Some special germplasm resources of wild type tea plant from Dayao Mountain were discovered according to their biochemical components (Table 3). These special resources will be directly used for production or as breeding parents. Amino acid content in Tea plant of high free amino acid was more than 4% (Yu et al. 1992). Hereby, 9 tea plants were higher than, including 5 samples from Liuxiang population and 4 samples from Gonghe population. Germplasm with over 5% of caffeine is considered as high caffeine resource (Yang et al. 2013). Wild type tea plants from Dayao Mountain were expected to become a special resource with high caffeine content, as their average content of caffeine was 5.77%. Caffeine contents of 23 tea plants were high, including 13 samples from Liuxiang population, 6 samples from Gonghe population, and 4 samples from Bainiu population. Catechins, a majority components of tea polyphenols, account for 70–80% of tea polyphenols content, and tea with catechins higher than 30.40% is considered as resource of high catechins (Yu et al. 1992). DYS3 from Liuxiang population and DYS22 from Bainiu population were discovered, whose catechins contents were 32.10 ± 0.14% and 30.66 ± 0.08%, respectively. Tea plant with EGCG content over 10% is regarded as high EGCG resource (Zheng et al. 2007). EGCG content of samples were 4.18–12.13%, and DYS11 was the highest one (2.13 ± 0.06%). 3 samples from Liuxiang population were the high EGCG resources.

Table 3 Special resources
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Discussion

Plants in Camellia are generally distributed in the southern and southwestern China, and the central parts in Yunnan, Guangxi, Guangdong and near the Tropical regions (Chang 1981). Consequently, Guangxi is of the vital geographical significance during the evolution of plants in Camellia. The most obvious character of original plants are their diverse transitional types ranging from primitive to evolutionary types (Fulian 1986). Previous studies have shown that evolutionary process of tea plant is successional, periodical and irreversible (Chen and Yajun 2013). Primitive type was large-sized leaves, arbor type with single terminal flowers while the evolutionary type has small leave, and is shrub type with fascicled flowers. Wild type tea plants from Liuxiang population had large leaves in the arbor or semi-arbor form, solitary or fascicled flowers, those from Bainiu population were middle leave, arbor form. Those from Gonghe population had small leaves and in shrub type with most fascicled flowers (Huang et al. 2014). Tea plants from Liuxiang population had large leaves, and were clustered in the same group with Yunnan large leaf species, Vienam large leaf species and Assam varieties based on their leaf area. Tea plants from Liuxiang and Bainiu populations had middle and small leaves, respectively (Fig. 3), whose palisade tissue were 1 layer while those from Gonghe population were 2 layers (Fig. 4). All above described features imply that Liuxiang and Bainiu tea population are relatively primitive, and Gonghe tea population is the most evolutionary one. These results are in accordance with cluster analysis based on RAPD markers, indicating that wild type tea plants from Dayao Mountain are consisted of various types from the primitive to more evolutionary one. These wild type tea germplasms are valuable for research on the origin, evolution and exploitation of tea.

Fig. 3
figure 3

Cluster analysis of Dayao Mountain based on leaf area (Huang et al. 2014)

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Fig. 4
figure 4

Leaf transverse section of Dayao Mountain tea plants (Zhu and Yahui 2015)

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The biochemical components of tea were mainly affected by variety, environment and cultivation conditions as well as plucking standard. Clouds and mist drift through Dayao Mountain, and soil there is rich in organic matters, which are favorable for cultivating abundant tea plants. The majority of catechins in primitive tea plant are non-galloylated catechins, while those of the evolved tea plant are ester-catechins. Ratios of ester catechins to non-ester catechins of 29 tea single plants varied hugely. Furthermore, genetic diversity of ester catechins was greater. The contents of theophylline, theobromine of tea were decreased with the improvement of its evolution level (Shengxiang 2009). Theobromine content of single plants from Liuxiang (\({\bar{\text{x}}}\) = 0.34%) was greater than those from Bainiu \({\bar{\text{x}}}\) = 0.12%) and Gonghe (\({\bar{\text{x}}}\) = 0.03%), indicating that Liuxiang population is relatively primitive. However, catechin composition was just contrary, probably due to the reason that biochemical components of tea plant are affected by both environment and variety. Other studies have shown that variations of catechin contents were larger among locations than among cultivars (Wei et al. 2011). Since Liuxiang tea population grow under the shade of tall trees, this environmental condition contributes to the biosynthesis of non-ester catechin (Hong et al. 2014). In general, wild tea plants from Dayao Mountain are the mixture ranging from the primitive type to the evolutionary type, with abundant genetic diversity and geographical uniqueness according to the previous studies and molecular biology research in this study. Wild type tea plants from Liuxiang were the most primitive ones while those from Bainiu were in the middle-evolution stages, and those from Gonghe were the most evolutionary ones.

Changye Baihao was systematically bred among tea plant populations from Nannuoshan, Menghai county from 1973 to 1985 in Tea Research Institute, Yunnan Academy of Agricultural Sciences. Foxiang 3 was the hybrid from F1 with Fuding large-leaved white tea as male parent, Changye Baihao as female parent in Tea Research Institute, Yunnan Academy of Agricultural Sciences. Therefore, there is a genetic relationship between two varieties, which is consistent with result of genetic similarity coefficient, according to RAPD makers in this study. Besides, Zijuan and Changyebaihao were systematically bred from Yunnan dayezhong group, which is also consistent with result of RAPD makers. The above mentioned evidences suggest that genetic relationships among different varieties analyzed according to genetic similarity coefficient based on RAPD maker are highly reliable. Result of cluster analysis showed that tea plants with similar genetic background are always classified in one group. Cluster analyses according to RAPD markers was accordant with that analyzed based on their biochemical components, which turned out that the diversity of biochemical components reflects DNA-based genetic diversity. It is further illustrated that tea plants from Dayao Mountain are of abundant genetic diversity and geographical uniqueness.

Special tea plant germplasm resource is the important source with higher added-value product, and wild tea plants possess a lot of the extraordinary characteristics due to the unique growth environment. Special tea plant resources with high catechin content are important for industrial production of tea polyphnols and catechins. DYS3 and DYS22 are the catechin at high levels in tea plants. DYS11 with high ester catechin tea plant is significant original material for ester catechin extraction and breed improvement. EGCG is a predominant ingredient of catechins (Vuong et al. 2010). There are broad application prospects for tea with high EGCG content due to its function in human health (Sur et al. 2016; Wei et al. 2016). However, EGCG can’t be artificially synthesized at present, and it can only be extracted from tea. DYS11, DYS12, and DYS3 are the resources containing high EGCG (EGCG ≥ 10%), which is essential for high EGCG tea production. Caffeine has the functions of regulation of blood sugar level, anti-depression, improvement of nerve excitability (Fang et al. 2015; Grosso et al. 2015). The average relative content of caffeine of wild type tea plants from Dayao Mountain is 5.77%, these tea plants are expected to become the resource with higher caffeine. Amino acids are one group of important biochemical components contributing to tea quality They have the intimate connection with taste and fragrance of tea. Besides, main amino acids in tea such as theanine have the physiological functions of tranquilization, depressurization, enhancement of the immunnity. Therefore, tea plant with high theanine content is of important values (Song et al. 2015; Zhao et al. 2013). A total of 26 types of amino acids have been identified in tea, accounting for about 1–4% of the dry weight (Xu et al. 2013). Wild tea plants from Dayao Mountain are rich in free amino acids with the average content of 3.60%, and DYS8 is up to 4.72 ± 0.02%. These special wild type tea germplasm resources can not only be used as the materials for extraction of some valuable natural products, but also can serve as breeding materials for commercial exploitation.