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 (NA) 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 NA (observed number of alleles) is a count of the mean number of alleles with nonzero frequency across loci; the Ne (effective number of alleles) is an estimate of the mean number of equally frequent alleles in an ideal population; the Ho (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.