Development of STS and CAPS markers for variety identification and genetic diversity analysis of tea germplasm in Taiwan

Plant materials and DNA extraction

The DNA was isolated from buds and leaves by using a modification of Doyle and Doyle (1990) described by Hu et al. (2005).

Design of STS markers

To develop cytoplasmic STS markers, primer pairs of two chloroplast and seven mitochondria were designed according to Hu (2004). In addition, 54 primer pairs of nuclear STSs, including four developed by Kaundun and Matsumoto (20032004), and 50 based on the public EST database (dbEST) of NCBI (National Center for Biotechnology Information, USA, http://www.ncbi.nlm.nih.gov/) were designed, using the Primer3 software (Rozen and Skaletsky 2000) (http://frodo.wi.mit.edu/). These primer pairs were prescreened with eight cultivars comprising of the following: TTES-8; TTES-12; TTES-13; TTES-18; TTES-19; TTES-20; Chin-Shin-Oolong and Shy-Jih-Chuen. The amplification was performed in a total volume of 38 μL containing 80 ng genomic DNA, 0.3 μM each primer, 4.7 mM MgCl₂, 0.27 mM dNTPs, and 1 U of Taq DNA polymerase (Invitrogen by Life Technologies). The amplification was done by T-Gradient (Biometra, Germany) with denaturation at 94°C for 4 min; 40 cycles of 94°C for 30 s, 55-60°C (depending on the primer pair) for 30 s, and 72°C for 30 s; and final extension at 72°C for 4 min.

Design of CAPS markers

Nuclear amplicons that amplified two bands with length polymorphisms were directly applied as STS markers. Meanwhile, the DNA bands were sequenced by ABI PRISM 3730 DNA Analyzer (Applied Biosystems, USA) once the PCR products were less than 1 kb. For SNPs (single nucleotide polymorphism) and InDels (insertion/deletion) screening, sequence analyses were conducted with SeqMan Pro v.7.1 software (DNAStar, Inc., Madison, WI, USA). The sequences with SNPs or InDels were converted to CAPS markers by SNP2CAPS software (Thiel et al. 2004). To check restriction patterns, PCR reactions were performed in a final volume of 11.7 μL with 1× Taq buffer, 2 mM MgCl₂, 0.27 mM dNTPs, 0.26 μM each primer, 1 U Taq DNA polymerase (Invitrogen by Life Technologies), and 40 ng DNA. Amplification was done by T-Gradient (Biometra, Germany) with programmed for 5 min preheating at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 55-60°C (depending on the primer pair) and 1 min at 72°C for the denaturation, annealing and extension steps, respectively. There was a final incubation for 10 min at 72°C. Amplification products were analyzed on 2% agarose gels stained with ethidium bromide to check the fragments being amplified. Amplified fragments were digested with restriction enzymes to detect CAPS and the products were resolved by electrophoresis on 2% agarose gels.

Data analysis and variety identification

, where Pi and Pj are the frequencies of the i th and j th alleles, and n is the number of alleles (Botstein et al. 1980).

Based on the identified STS and CAPS markers, the core markers and the flow chart for identifying 12 prevailing cultivars in Taiwan, including Chin-Shin-Oolong, TTES-12, Shy-Jih-Chuen, Chin-Shin-Dahpan, TTES-13, Chin-Shin-Gantzy, TTES-8, TTES-18, TTES-7, TTES-19, TTES-20, and TTES-21, were developed.

Genetic diversity analysis

In this study, the tea germplasm consist of three main groups, including sinensis type (S and SA), assamica type (A and AS) and wild species in Taiwan (F and FY) (shown in Table 1). The genetic diversity of those germplasm was analyzed by Popgene v.1.32 (Yeh and Boyle 1997) to estimate the observed number of alleles (N_A), the effective number of alleles (N_e), the observed heterozygosity (H_O), the Nei’s gene diversity (H), and Shannon’s Information index (I) per group.

Cluster analysis and principle coordinates analysis

Both the analyses of average genetic distances among three main groups and genetic distances between the pairs of germplasm were based on modified Roger’s distance (MRD) method (Wright 1978) by using TFPGA v.1.3 (Miller 1997). Upon the genetic distances between all pairwise combinations MRD, the cluster analysis and principal coordinate analysis (PCoA) were completed with NTSYSpc v.2.10 (Rohlf 1997). A dendrogram of the genetic relationships was developed by unweighted pair group method with arithmetic mean algorithm (UPGMA) using cluster analysis. The principal coordinate analysis (PCoA) was performed and the first two extracted coordinates extracted were used to derive the PCoA plot.

Polymorphism of STS and CAPS markers

A total of 98 alleles out of 39 polymorphic loci were detected in 55 germplasm. In 10 cytoplasmic CAPS loci, the average number of alleles was 2 and polymorphism information content (PIC) ranged from 0.13 (C02) to 0.35 (M06), with an average of 0.25 per locus. In 29 nuclear STS and CAPS markers, the number of alleles varied from 2 to 7, with an average of 2.7 per locus. The PIC values widely varied from 0.04 (G16) to 0.62 (G22), with an average of 0.34 per locus (Table 2).

Identification of the prevailing tea cultivars in Taiwan

Two STS and 37 CAPS markers developed in this study can be used to distinguish all 55 core germplasm in Taiwan, and their band patterns are shown in Additional file 1: Table S1. For the identification of 12 prevailing tea cultivars in Taiwan, the electrophoresis patterns of cleaved fragments in each STS and CAPS marker are shown in Additional file 1: Figure S2. In order to establish a flow chart for identifying 12 prevailing tea cultivars in Taiwan, five core markers, including M02 (mitochondria), C02 (chloroplast), G01 G03, and G04 (nuclear), were selected by variety-specific marker and PIC value. First, the sinensis type and the assamica type groups were distinguished by using the M02 marker. Secondly, the G03 and C02 can be employed to discriminate four cultivars within assamica type group, and the G03, G01 and G04 were used to separate eight cultivars within sinensis type group (Figure 1). In addition to five core markers, the remaining 34 markers could be used as a supplementary tool if more new varieties need to be identified in the future.

Genetic diversity of tea germplasm in Taiwan

Cluster analysis and principle coordinates analysis of tea germplasm in Taiwan

The genetic distances between all pairwise combinations were listed in Additional file 1: Table S2. The values among 55 surveyed germplasm in Taiwan ranged from 0.08 to 0.69, with an average value of 0.49. Among the germplasm of 47 cultivated tea (C. sinensis), the average values was 0.47. If only 37 sinensis type tea (S and SA) were surveyed, the genetic distances among this group ranged from 0.11 (TTES-14 and TTES-15) to 0.62 (Chin-Shin-Oolong and TTES-17), with an average value of 0.44. As for 14 assamica type tea (A and AS), the genetic distances ranged from 0.08 (TTES-8 and Jaipuri; Shan-1 and Shan-2) to 0.58 (Shan and Manipuri), with an average value of 0.41. However, the genetic distances among eight wild species (F and FY) were relatively small, ranging from 0.11 (Long-Tou wild tea and Le-Ye wild tea; Ming-Hai wild tea and Nan-Fong wild tea) to 0.38 (De-Hua-She wild tea and Yung-Kang wild tea), with an average value of 0.25.

In PCoA based on MRD estimates of all 55 germplasm, the first, second and third principle coordinates (abbreviated to PC1, PC2 and PC3) explained 24.5%, 15.9% and 11.3% of the molecular variance, respectively, while the cumulative contribution was 51.8%. The first two principle coordinates were used to develop the PCoA plot shown in Figure 2. In the PC1, the 55 tea germplasm were divided into two major groups, cultivated tea (C. sinensis) and wild species in Taiwan (C. formosensis). In the PC2, the cultivated tea (C. sinensis) were divided into two major groups, sinensis type (S and SA) and assamica type (A and AS).

The UPGMA dendrogram was constructed to separate the 55 germplasm into three major groups (Figure 3). Based on genetic distance coefficient of 0.57, the first group (GroupI) including C. formosensis could be isolated from the cultivated germplasm (C. sinensis). When the coefficient was reduced to 0.51, the assamica type (A and AS) (GroupII) and the sinensis type (S and SA) (GroupIII) germplasm were distinguished. Group III was divided into three subgroups, namely Group IIIa, Group IIIb, and Group IIIc. Many famous varieties belonged to Group IIIa including Chin-Shin-Oolong, Shy-Jih-Chuen, Bair-Mau-Hour, Wuu-Yi, Horng-Shin-Dahpan, and its derived varieties (TTES-3, TTES-4, and TTES-9). The Group IIIb comprised of Tiee-Guan-In, Hwang-Gan, and its derived varieties (TTES-10, TTES-12, TTES-14, TTES-15, and TTES-19). The Group IIIc, on the other hand, contained Chin-Shin-Gantzy, Chin-Shin-Dahpan and its derived variety (TTES-1), Dah-Yeh-Oolong, and its derived varieties (TTES-2, TTES-11, TTES-16, TTES-17, and TTES-20), Ying-Jy-Horng-Shin its derived variety (TTES-13).

Polymorphism of STS and CAPS markers

In this study, 11 nuclear CAPS markers including G03, G12, G14, G15, G17, G18, G20, G22, G23 and G26 showed multi-allele patterns, while the others had only two alleles (bi-allele) (Table 2). There was only one restriction site within each CAPS locus resulting in the bi-allele markers, and their genotypes were easily scored and interpreted. Otherwise, the multi-allele markers were based on different point mutation positions within the locus that had more than two restriction sites. They yielded more complicated genotypes but may still be considered very useful. For example, the multi-allele CAPS markers could be used widely in pepper breeding for viral resistance (Yeam et al. 2005).

Polymorphism information content (PIC) means different informative levels of a locus and it also implies the genetic variation of a marker. The value larger than 0.5, ranging from 0.25 to 0.5, and smaller than 0.25 suggest that the locus is highly informative, reasonably informative, and slightly informative, respectively (Botstein et al. 1980). Of all the 39 cytoplasmic and nuclear markers examined in this study, the PIC ranged from 0.04 to 0.62, with an average of 0.32. The PIC of 10 cytoplasmic markers was 0.25, and seven of them were reasonably informative. Otherwise, the remaining three were slightly informative (Table 2). The 29 nuclear markers had an averaged PIC of 0.34, in which six were found to be highly informative, 16 were reasonably informative, and the remaining seven were slightly informative. The averaged PIC of the nuclear markers was higher than the cytoplasmic, and the average of the mtDNA markers (0.29) was higher than the cpDNA (0.18) (Table 2). Similar results were also reported by Ishii’s group, in which they found that the nuclear microsatellites (the averaged PIC is 0.89) had higher PIC values than the chloroplast microsatellites (the averaged PIC is 0.38) among A-genome species of rice (Ishii et al. 2001). Because the variation of cytoplasmic markers are lower than nuclear markers, the former could be used to examine relationship among distant-related taxa, and the latter are more suitable for the assessment of genetic diversity of close- related taxa.

In our previous study, the observed number of EST-SSR alleles (N_A) per locus was 5.6 (Hu et al. 2011). However, in this study, the values of STS and CAPS markers derived from cytoplasmic and nuclear were 2.00 and 2.69, respectively. The PIC per locus for EST-SSR (0.62) was higher than those of STS and CAPS from cytoplasmic (0.25) and nuclear (0.34). Because small size difference between polymorphic bands was shown in the EST-SSR markers, there was high resolution of agarose gel, polyacrylamide gel electrophoresis or Genetic Analyzer (Hu et al. 2011). However, large size difference between polymorphic bands was found in STS or CAPS markers, and it suggested that only less expensive agarose gel was needed to obtain accurate data.

Identification of 12 prevailing tea cultivars in Taiwan

In this study, 12 dominant cultivars were selected for variety identification based on the following criteria: (1) the acreage under cultivation of each variety; (2) the variety suitable for manufacturing unique tea; and (3) the newly bred varieties. According to statistics data from Tea Research and Extension Station in 2011, these 12 cultivars take over 98% acreage of Taiwan. Of these 12 cultivars, Chin-Shin-Oolong, TTES-12, Shy-Jih-Chuen, Chih-Shih-Dahpan, and TTES-13 are the top five cultivars in Taiwan that has been found to be suitable for both Paochong tea and Oolong tea. Shy-Jih-Chuen and Chih-Shih-Dahpan are mainly grown in Nantou County and north-west region of Taiwan, respectively, while others are distributed around Taiwan (estimated by Tea Research and Extension Station in 2011). Besides, Chin-Shin-Gantzy is fitted for green tea, and TTES-18, TTES-8, and TTES-7 are the excellent cultivars for making black tea. Chin-Shin-Gantzy is cultivated in New Taipei City, and the other three cultivars are mainly planted in Nantou County (estimated by Tea Research and Extension Station in 2011). In addition, varieties TTES-19 and TTES-20 were bred for manufacturing Paochong and Oolong tea, having been protected by the “Plant Variety and Plant Seed Act” in Taiwan since 2004 (Tsai et al. 2004a). TTES-21, on the other hand, was designated in 2008 for black tea procession (Chiu et al. 2009). These cultivars are most urgently desirable for variety identification in Taiwan.

Tea commercial products are manufactured through the application ofhigh temperature and the use of fermentation treatments at a panning step. These processes could eventually lead to dramatic DNA degradation. Additionally, tea merchants or farmers often blend the tea with different varieties to increase its flavor or reduce material cost. To solve the above problems, we have reported that DNA markers less than 1 kb are less affected by procession treatments and are useful for variety identification. Moreover, the chloroplast DNA markers with haploid genotypes and maternal inheritance could be effectively applied to identify the mixed-varieties of tea products (Hu et al. 2006). Since most STS and CAPS markers in this study are less than 850 bp, they may have application potential in identifying different varieties or mixed-varieties of processed tea.

Genetic diversity of tea germplasm in Taiwan

The consistent results of germplasm classification were found in the principal coordinate analysis and cluster analysis. A total of 55 germplasm can be divided into three groups: sinensis type (S and SA), assamica type (A and AS) and Taiwan wild species (F and FY). The sinensis type (S and SA) and assamica type (A and AS) are generally called cultivated tea (C. sinensis). The former is a shrub with small leaves and can withstand cold climates; while the latter has tall tree-like structure with large leaves and is suitable for warm tropical climates (Banerjee 1992). Besides, the latter has more flavanols content so it was found to be more suitable for making black tea. Meanwhile, the sinensis type has been found to be suitable for manufacturing green tea or Oolong tea (Takeo 1992). In Taiwan, cultivated tea is mainly distributed in Nantou County (48.4%), Chiayi County (15.5%) and New Taipei City (10.6%) (Council of Agriculture 2012). The assamica type tea retains about 3.9% acreage which is mainly distributed in Nantou County, while the sinensis type is about 96.1% which is widely distributed in Taiwan (estimated by Tea Research and Extension Station in 2011). On the other hand, wild tea species is distributed in the central, southern and eastern regions of Taiwan. Various names have been given to the wild species, and Camellia formosensis is the official name based on the RPB2 (large sub-unit of RNA polymerase) gene of nuclear DNA sequence and morphological analyses (Su et al. 2007; Su et al. 2009). It can be well distinguished from cultivated tea (C. sinensis) by the glabrous ovaries and winter buds (Su et al. 2007). In this study, the results of both principal coordinate analysis and cluster analysis have supported that the wild species (C. formosensis) is monophyletic and independent from the cultivated tea (C. sinensis).

The genetic diversity can be accessed by many parameters. The N_A (observed number of alleles) is a count of the mean number of alleles with nonzero frequency across loci; the N_e (effective number of alleles) is an estimate of the mean number of equally frequent alleles in an ideal population; the H_o (observed heterozygosity) is an estimate proportion of observed heterozygotes at a given locus; the H (Nei’s gene diversity) is estimated proportion of expected heterozygotes under random mating; the I (Shannon information index) is an index as a measure of gene diversity (Yeh and Boyle 1997). According to the genetic diversity analysis, all parameters or indices showed that higher genetic diversity or genetic variation were detected in the sinensis type (S and SA) and the assamica type (A and AS) than wild species (Table 4). One possible explanation is that the cultivated tea (A, AS, S and SA) originated from diverse regions (China, Myanmar, Thailand, India, and so on) and had frequent inter-crossings. However, genetic recombination only occurred in a limited local wild species in Taiwan. This rationalization differs from that of Lai et al. (2001), in which they used RAPD and ISSR markers to evaluate the gene diversity of 37 tea samples in Taiwan. They reported that the native Taiwan wild species had the highest genetic diversity, followed by the sinensis type and the assamica type (Lai et al. 2001). There are two contrarieties that could be raised against this: first, two (Laitou and Shueijing wild tea) of six native Taiwan wild tea samples in Lai et al. (2001) are C. furfuracea instead of C. formosensis authenticated by Su (2007). This would lead to overestimate the diversity of native wild species. Second, all of three assamica varieties surveyed in Lai et al. (2001) merely originated from India, which are not representative of the tea wild species.

The tea industry in Taiwan began in the Jiaqing era of Ching Dynasty (AD 1796 to 1820), and a few tea varieties were introduced from China (Jun 1997). During the Japanese occupation period (AD 1896 to 1945), four landraces including Chin-Shin-Oolong, Dah-Yeh-Oolong, Chin-Shin-Dahpan, and Ying-Jy-Horng-Shin were recommended to the tea farmers. In addition, Hwang-Gan and Horng-Shin-Dahpan were also the prevailing cultivars at that time. Since 1945, the above six varieties have been used as female parents for hybridization breeding (Sanui 2011; Shyu and Juan 1993). According to cluster analysis in this study (Figure 3), Chin-Shin-Oolong and Horng-Shin-Dahpan belonged to Group IIIa, Hwang-Gan was classified in Group IIIb, and the remaining three landraces (Dah-Yeh-Oolong, Chin-Shin-Dahpan, Ying-Jy-Horng-Shin) were categorized in Group IIIc. However, all of these six varieties were introduced from Fukien or Guangdong of China (Sanui 2011).

Genetic vulnerability is a common problem in most of the tea-production countries, because only a few specific varieties are grown in large-scale and not many varieties have been used as the breeding parents (Yao et al. 2008). For example, a famous cultivar Yabukita contributes more than 80% of its tea acreage in Japan for making green tea (Kaundun and Matsumoto 2004). Besides, the other prevailing varieties including Kanayamidori, Sayamakaori, Saemidori, Okumidori, Meiryoku etc. were selected from Yabukita (Tanaka 2012). This could possibly lead some alleles to be eliminated and result in genetic erosion when most cultivars are replaced by a few varieties. Once the dramatically biotic or abiotic stress occurs, it is more likely to cause reduction in the production of the same or close-related cultivars, which could induce a crisis in the tea industry, leading to its possible collapse. In fact, a similar problem also exists in Taiwan. The top three prevailing cultivars in Taiwan take over 84.2% acreage including Chin-Shin-Oolong (57.3%), TTES-12 (13.7%) and Shy-Jih-Chuen (13.2%) (estimated by Tea Research and Extension Station in 2011). According to leaf morphological characters and ISSR DNA markers, a high similarity between Chin-Shin-Oolong and Shy-Jih-Chuen was found previously (Hu 2004). In this study, the genetic distance between these two cultivars (0.21) is far below the average (0.49) (Table 4 and Additional file 1: Table S2), and the alleles of all 10 cytoplasmic markers are identical (Additional file 1: Table S1). It was confirmed that Shy-Jih-Chuen originated from Chin-Shin-Oolong. In addition, these two cultivars accounting for 70.5% of all tea plantations in Taiwan, and Chin-Shin-Oolong is also the male parent of another two new varieties, TTES-19 and TTES-20, which were released in 2004 (Tsai et al. 2004a). In order to avoid the genetic vulnerability and increase the genetic diversity of tea varieties in Taiwan, the new parental lines could be referred to as the dendrogram of cluster analysis in this study (Figure 3). The elite parents from different geographical origins or genetic background could also be chosen.