Albino tea cultivars grow white leaves at low temperature which are valuable materials for processing green tea, but they develop green leaves in summer and autumn seasons. It is difficult to discriminate albino tea cuttings from the normal tea cuttings by leaf colour and plant morphological characteristics.Specific RAPD markers for identifying albino tea cultivars &#8216;Qiannianxue&#8217; and &#8216;Xiaoxueya&#8217; were developed in the present paper and they can be used in the authentication of the two albino tea cultivars. An amplified fragment (about 1500 bp) from Primer (S 12 (Sangon Biological Engineering Technology and Services Co., Ltd.) was identified in the albino teas and not from the widely cultivated cultivar; Fudingdabai

Du, YY

Liang, YR

Lin, C

Lu, JL

Shao, SH

Wang, KR

Ye, Q

English

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African Journal of Biotechnology Vol. 9(4), pp. 434-437, 25 January 2010     Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2010 Academic Journals      Full Length Research Paper  Development of specific RAPD markers for identifying albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’  Wang, K. R.1,2, Du, Y. Y.1,3, Shao, S. H.1,3, Lin, C.1,3, Ye Q.1,3, Lu, J. L.1,3 and Liang, Y. R.1,3*  1Zhejiang University Tea Research Institute, Hangzhou 310029, China. 2Ningbo Extension Station of Forestry and Speciality Technology, Ningbo 315012, China. 3The Key Laboratory of Horticultural Plant Growth, Development and Biotechnology of Ministry of Agriculture of China, Hangzhou 310029, China.  Accepted 30 December, 2009  Albino tea cultivars grow white leaves at low temperature which are valuable materials for processing green tea, but they develop green leaves in summer and autumn seasons. It is difficult to discriminate albino tea cuttings from the normal tea cuttings by leaf colour and plant morphological characteristics. Specific RAPD markers for identifying albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’ were developed in the present paper and they can be used in the authentication of the two albino tea cultivars. An amplified fragment (about 1500 bp) from Primer (S 12 (Sangon Biological Engineering Technology and Services Co., Ltd.) was identified in the albino teas and not from the widely cultivated cultivar; Fudingdabai.  Key words: Camellia sinensis, albino tea, RAPD maker, cultivar authentication.   INTRODUCTION  White leaves grown on the albino tea cultivars have high level of amino acids which are important tea components responsible for tea quality (Jang et al., 2008; Liang et al., 2003, 2005, 2008) and healthy functions (Yokogoshi et al., 1995; Du et al., 2006, 2008, 2009; Zhang et al., 2007). The cultivars ‘Qiannianxue’ and ‘Xiaoxueya’ are two of the major albino tea cultivars grown in Zhejiang Province, China. They grow shoots with albino leaves when the environmental temperature was below 20°C during early spring season (Du et al., 2008). However, the leaf color becomes green as is the case with the other cultivars commonly grown in the same areas when the environmental temperature is above 20°C, resulting in difficult to identify the authentity of young tea cuttings by leaf color. The malfeasant counterfeited tea cultivar names as albino tea cultivars and the tea farmers have been deceived and suffered from loss. The tea growers are increasingly paying attention to the authentication of albino tea cultivars. Cultivar ‘Fudingdabai’ is now the widely grown variety in the province. It is important to develop a method to identify the albino  tea  cultivar  from     *Corresponding author. E-mail: yrliang@zju.edu.cn. the ‘Fudingdabai’ in cutting stage.  Various types of DNAbased molecular techniques are utilized to evaluate DNA polymorphism, including random amplified polymorphic DNA (RAPD) (Gupta et al., 2008; Vural and Dageri, 2009). RAPD is a tool widely used to identify genetic variation in ecotypes of crops and in the present paper it was used to develop DNA markers suitable for identifying the albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’ from ‘Fudingdabai’, a widely cultivated tea cultivar in China.   MATERIALS AND METHODS  Materials  First leaf beneath the apical bud on tea shoot of albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’ grown in Deshijia Tea Farm (Yuyao City, Zhejiang Province, China) was sampled on 25th April, 2007 and then were frozen in liquid nitrogen and stored at -20ºC until DNA was extracted. To search for DNA markers of the albino tea cultivars which are distinct from common tea cultivar, ‘Fudingdabai’, the most popular tea cultivar grown in China was used as control in the test. Chemical reagents used in the present paper were purchased from Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Wang et al         435    Table 1. RAPD primers that generated polymorphic bands.  Primer No. Sequence Primer No. Sequence S11 GTAGACCCGT S21 CAGGCCCTTC S12 CCTTGACGCA S22 TGCCGAGCTG S17 AGGGAACGAG S24 AATCGGGCTG S18 CCACAGCAGT S25 AGGGGTCTTG S19 ACCCCCGAAG S27 GAAACGGGTG    Table 2. Distinctive polymorphic bands for Albino cultivars and Fudingdabai  Bands exclusively amplified in Fudingdabai Bands exclusively amplified in Qiannianxue Bands generated exclusively in Xiaoxueya Bands amplified in both albino cultivars S11-1, S11-2, S11-3, S12-1, S17-1, S17-3, S17-4, S17-8, S18-3, S19-1, S19-2, S21-1, S21-2, S22-1, S22-3, S22-4, S24-1,  S27-4 S17-6, S17-9, S24-2, S24-3   S12-3, S22-2, S25-1, S27-1, S17-2, S17-5, S17-7, S27-3, S27-3 S12-2    Genomic DNA extraction  Genome DNA extraction was carried out according to CTAB procedure described by Matasyoh et al. (2008). Concentration of the extracted DNA samples were determined by a GeneQuant Pro RNA/DNA calculator (Biochrom Ltd., Cambridge, England) and diluted to 100 mg ml-1 and then stored at -20ºC until used for PCR.   Polymerase chain reaction (PCR)  The PCR was carried out using S-series of 10-mer RAPD primers from Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Thirty primers; S1 to S30 were tested and those which amplified polymorphic bands (Table 1) were used to evaluate the three cultivars.  The PCR was carried out in 25 L reaction solution that contains ddH2O (17.8 l), 10× buffer (2.5 l), 25 mmol Mg2+ (2.5) l, 10 mmol dNTP  (0.5 l), 0.5  mol l-1 RAPD primer (0.33) l, 100 g ml-1 DNA (1.2 l) and 5 u l-1 Taq polymerase (0.17 l). The thermal amplification was programmed in three phases in a DYADTM PTC-200 PCR Thermal Cycler (MJ Research, MA, USA), that is, the initial denaturation (at 94ºC for 5 min); the 25 cycles of denaturation (at 94ºC for 30 s), annealing (at 36ºC for 1 min) and extension (at 72ºC for 2 min) and the final extension step was 10 min at 72ºC. The PCR product was checked by electrophoresis on 1.5% (W/V) Agarose gel and photographed using the JD-801 ImageQuant Scanner (Jiangsu JEDA Science-Technology Develop-ment Co., Ltd., Nanjing, China).    RESULTS AND DISCUSSION  Among the 30 tested RAPD primers, 10 primers (Table 1) generated polymorphic bands. Compared to control cultivar ‘Fudingdabai’, there were 18 bands which could be generated in cultivar ‘Fudingdabai’ but could not be generated in both albino tea cultivars by the random primers S11, S12, S17, S18, S19, S21, S22, S24 and S27 (Table 2; Figure 1). It suggested that they might be related to some structural genes missed in the albino tea cultivars, compared to the common tea cultivar. These missed genes may be related to the albinism of tea. It offered important information for further studies on the genetic characteristics of albino tea cultivars. Band S12-2 was exclusively generated in the two albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’ by primer S12 (Table 2; Figure 1).  However, polymorphic bands were found between the albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’. Bands S17-6 and S17-9 generated by primer S17 and bands S24-2 and S24-3 by primer S24 were found in ‘Qiannianxue’, but not found in ‘Xiaoxueya’ (Figure 1; Table 2). On the contrary, bands S12-3 generated by primer S12 and band S22-2 by primer S22, band S25-1 by primer S25 and band S27-1 by primer S27 were detected in ‘Xiaoxueya’ but not in ‘Qiannianxue’ (Figure 1; Table 2). Furthermore, bands S17-2, S17-5 and S17-7 generated by primer S17 and bands S27-3 and S27-3 generated by primer S27 were exclusively detected in ‘Xiaoxueya’, which were not amplified in the common tea cultivar ‘Fudingdabai’ and the albino cultivar ‘Qiannianxue’ (Figure 1; Table 2). The polymorphic bands will be useful RAPD markers to discriminate the albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’ from cultivar ‘Fudingdabai’.  This study revealed that there were four groups of bands generated by the ten RAPD primers as shown in Table 1. These were the bands exclusively amplified in Fudingdabai, bands amplified exclusively in Xiaoxueya, bands exclusively amplified in Qianniaxue and bands amplified in both albino cultivars. The bands were gene-rated from genome DNA samples, which were not affected by environmental conditions. Although the albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’ grow green shoots as common tea cultivar at temperature above 20°C, they were identified according to the bands generated  436         Afr. J. Biotechnol.      Figure 1. RAPD markers generated by random primers S12, S17, S19, S22 and S25. L = DNA Ladder; Q = Cultivar ‘Qianianxue’; X = Cultivar ‘Xiaoxueya’; F = Cultivar ‘Fudingdabai’. The bands were numbered by primer number-the polymorphic band number, that is, S11-1 means the largest polymorphic band generated by primer S11.     by the ten primers in Table 1. However, only one widely cultivated cultivar ‘Fudingdabai’ was used as control test in the present study. The result of the present study is suitable for identifying the two albino tea cultivars from ‘Fudingdabai’, instead of all green tea cultivars. Further study should be carried out using more control tea cultivars.    ACKNOWLEDGMENT  The authors wish to thank the Science Foundation of Ningbo City for support of the project (project No. 2007A610069).   REFERENCES  Du YY, Chen H, Zhong WL, Wu LY, Ye JH, Lin C, Zheng XQ, Lu JL, Liang YR (2008). Effect of temperature on accumulation of chlorophylls and leaf ultrastructure of low temperature induced albino tea plant. Afr. J. Biotechnol. 7(12): 1881-1885. Du YY, Liang YR, Wang H, Wang KR, Lu JL, Zhang GH, Lin WP, Li M, Fang QY (2006). A study on the chemical composition of albino tea cultivars. J. Horticult. Sci. Biotechnol. 81(5): 809-812. Du YY, Shin S, Wang KR, Lu JL, Liang YR (2009). Effect of temperature on the expression of genes related to the accumulation of chlorophylls and carotenoids in albino tea. J. Horticult. Sci. 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Development of specific RAPD markers for identifying albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’

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Development of specific RAPD markers for identifying albino tea cultivars ‘Qiannianxue’ and ‘Xiaoxueya’

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