CsNAM-like protein encodes a nuclear localized protein and responds to varied cues in tea [Camellia sinensis (L.) O. Kuntze]

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Gene 502 (2012) 69–74

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CsNAM-like protein encodes a nuclear localized protein and responds to varied cues in tea [Camellia sinensis (L.) O. Kuntze] Asosii Paul a, 1, Richard Chalo Muoki a, b, 1, 2, Kashmir Singh b, Sanjay Kumar a,⁎ a b

Biotechnology Division, Council of Scientific and Industrial Research-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh-176061, India Biotechnology Department, Panjab University, Chandigarh-160014, Punjab, India

a r t i c l e

i n f o

Article history: Accepted 9 April 2012 Available online 19 April 2012 Keywords: Abiotic stress Drought Gene expression Hydrogen peroxide NAC Subcellular localization

a b s t r a c t Abiotic stress possesses serious threat to plant distribution and production. In response to stress, plants induce the expression of many genes that function to protect the cellular machinery from stress-induced damages. These genes are largely regulated by specific transcription factors (TFs). NAC family proteins are plant specific TFs implicated in diverse processes including development, and biotic and abiotic stress responses. The present work described (i) cloning of CsNAM-like protein gene from a tree crop tea [Camellia sinensis (L.) O. Kuntze], (ii) its cellular localization, and (iii) regulation of the gene by external cues. The gene had an open reading frame of 873 base pairs encoding 291 amino acids with calculated molecular weight of 33.4 kDa and an isoelectric point (pI) of 6.72. Expression characterization showed the gene to be induced by drought, osmoticum, salt, heat and hydrogen peroxide. During the period of active growth, CsNAM-like protein showed ubiquitous expression in all the tissues analyzed, with higher level of transcripts in stem, flower bud and mature leaf as compared to the root, young leaf and fruit. The common response of CsNAM-like protein to various cues suggests its important role in imparting tolerance against abiotic stress. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Plants induce many genes in response to abiotic stress. These genes and their products function to protect the cellular machinery from stress induced damages. Expression of stress-related genes is largely regulated by specific TFs that bind to the cis-acting element in the promoters of respective target genes. Regulatory proteins activated in response to abiotic stress include DREB1/CBF and DREB2, AREB/ABF, MYB/MYC and NAC (Nakashima et al., 2009). NAC family proteins have a consensus sequence known as the NAC domain that is located in the N-terminal region and divided into five subdomains (Ooka et al., 2003). NAC

Abbreviations: TFs, transcription factors; Cs, Camellia sinensis; NAC, NAM and ATAF1/2, and CUC2; NAM, no apical meristem; DREB, dehydration-responsive element binding protein; CBF, C-repeat binding factor; AREB, ABA-responsive element binding protein; ABF, ABA-responsive element binding factor; ESTs, expressed sequence tags; IP, irrigated plant; DS, drought stress; PEG, polyethylene glycol; NaCl, sodium chloride, H2O2, hydrogen peroxide; cDNA, complementary DNA; YL, young leaf; ML, mature leaf; FB, flower bud; RACE, rapid amplification of cDNA ends; ORF, open reading frame; GFP, green fluorescence protein; qPCR, quantitative polymerase chain reaction; Ct, threshold cycle; REST, relative expression software tool; pI, isoelectric point; CaMV, cauliflower mosaic virus; LT, low temperature; ROS, reactive oxygen species; ABA, abscisic acid; ATAF, Arabidopsis thaliana activation factor; NAP, NAC-like, activated by APETALA 3/PISTILLATA; PCR, polymerase chain reaction; SENU5, senescence up-regulated5; TIP, TCV-interacting protein. ⁎ Corresponding author. Tel.: + 91 1894 233339x340; fax: + 91 1894 230433. E-mail addresses: [email protected], [email protected] (S. Kumar). 1 Authors contributed equally. 2 Permanent address: Tea Research Foundation of Kenya, P.O. Box 820, Kericho-20200, Kenya 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.04.017

genes are plant specific, with 117 genes in Arabidopsis thaliana Heynh. (Nuruzzaman et al., 2010), 151 genes in Oryza sativa L. (Nuruzzaman et al., 2010), 101 genes in soybean [Glycine max (L.) Merr.] (Pinheiro et al., 2009) and 163 genes in Populus trichocarpa Torr. & Gray (Hu et al., 2010) genomes. The family has been implicated in diverse processes including development, and biotic and abiotic stress responses (Hu et al., 2006; Nakashima et al., 2009; Olsen et al., 2005). Drought poses serious threat to the sustainable production and distribution of tea [Camellia sinensis (L.) O. Kuntze]. Tea is a major source of revenue for over 40 tea producing countries worldwide with leading producers namely, China, India, Kenya and Sri-Lanka, reporting an estimated gross production value of million USD 5338, 387, 864 and 133, respectively in the year 2009 (http://faostat.fao.org). However, drought impacts tea production to an extent of 14–33%, with about 6–19% plant mortality (Cheruiyot et al., 2010). The consequence of drought is the imposition of heat and salt stress (Carr, 2010; Mittler, 2006). Several reports suggested that response of tea to drought stress involved molecular (Muoki et al., 2011; Rani et al., 2009, 2012; Sharma and Kumar, 2005; Singh et al., 2008, 2009), physiological (Carr, 2010) and biochemical (Upadhyaya and Panda, 2004) mechanisms. Recently, ESTs were reported in tea (Muoki et al., 2011; Sharma and Kumar, 2005). However, most of these putative genes have not been functionally characterized. Several advancements toward identifying potential stress related genes capable of increasing the tolerance of plants to abiotic stress have been reported (Nakashima et al., 2009). TFs act as master regulators of cellular processes and hence are the target candidates to

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understand the gene regulatory network as influenced by various abiotic stress. In an earlier study, a drought responsive cDNA library of tea was constructed and deposited in GenBank at NCBI, wherein a CsNAM-like protein, a member of the NAC family was reported vide accession number JK341660 (Muoki et al., 2011). However, neither the full-length cDNA was reported, nor its cellular localization and regulation of the gene to the external cues was reported. The present work reports on full-length cloning of the gene followed by detailed analyses. The encoded protein was found to be localized in the nucleus strengthening the proposition for encoding a TF. The gene was up-regulated in drought, osmotic (polyethylene glycol; PEG), salt (sodium chloride; NaCl), heat and hydrogen peroxide (H2O2) stresses suggesting its crucial role in abiotic stresses. 2. Materials and methods 2.1. Plant materials and stress imposition For drought experiment, 2 year old vegetatively propagated plants of a high yielding and drought resistant tea clone, UPASI-9 (Camellia sinensis var. assamica) (Balasaravanan et al., 2003), grown in plastic sleeves (24 cm length, 12 cm width and 8 cm diameter) containing

tea garden soil: farm yard manure: sand (1:2:1) were used in the present study (Muoki et al., 2011). Plants were subjected to progressive water stress by withholding irrigation for 30 days. Thereafter, drought was released by irrigating the plants experiencing DS (recovery experiment). Control plants were irrigated every other day. Leaf tissues (4th to 6th position from the apical bud) were harvested on day 10, 20 and 30 of withholding irrigation. Also, at day 30 of withholding irrigation, stem and root tissues were harvested. Any of the tissues, including root tissue, was never rinsed with water to avoid recovery from drought stress. On day 30 of withholding water, soil on the root tissue was dried up and could easily be removed using a soft paper napkin. Care was taken to avoid tissue injury. Similar attempts to harvest root tissue on days 10 and 20 of withholding water resulted in tissue injury since these were not being washed with water to avoid any recovery from drought. For this reason, sampling of root as well as stem was avoided during these two early time points. For irrigated plants (IP, control), roots had to be rinsed with water to separate the attached wet soil. Corresponding control tissues (IP) on the corresponding day were always harvested, immediately frozen in liquid nitrogen and stored at −80 °C for further analyses. In a separate experiment, shoot cuttings containing apical bud and the associated six leaves were collected from clonal bushes of

Fig. 1. Alignment of the deduced CsNAM-like protein sequence (accession number: JQ619837) with homologous NAC of Gossypium hirsutum L. (GhNAC2: ACI15342.1), Cicer arietinum L. (CaNAC5: ACS94038.1), Brassica napus L. (BnNAC5-11: AAP35053.1) and Prunus mume Siebold & Zucc. (PmNAC: BAE48667.1). The locations of the five highly conserved amino acid motifs (I–V) comprising the NAC domain are indicated by lines above the sequences. The putative nuclear localization signal (PRDRKYP) as predicted by PSORTII in the deduced CsNAM-like protein sequence is underlined. Residues varying between sequences are shown with gray background.

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UPASI-9 from the field and first stabilized in deionized water for 24 h before start of experiment. Thereafter, shoot cuttings were transferred to deionized water (control), 10% PEG-8000 (Sigma, USA) for osmotic stress (Paul et al., 2012), 250 mM NaCl for salt stress (Munns et al., 2006) and 0.25% H2O2 (Merck, Germany; Paul et al., 2012), separately. These were housed in a plant growth chamber set at 25± 3 °C [light intensity, 200 μE m − 2 s− 1; RH, 70–80%; Saveer Biotech, India]. For imposing heat stress, cuttings were transferred to a plant growth chamber set at 37± 2 °C. In a separate experiment, apical buds and the associated two leaves (young leaf, YL) at node positions 1st and 2nd, mature leaf (ML, 4th, 5th and 6th leaf combined; leaf position with reference to the apical bud), FB, fruit, stem and root were collected during the period of active growth (Muoki et al., 2011; Paul et al., 2012) from a clonal tea bush in the field. All samples were frozen immediately in liquid nitrogen and stored at −80 °C for further analyses. 2.2. Full-length cloning of CsNAM-like protein RACE (5′- and 3′-RACE) was used to clone full-length cDNA essentially as described by Paul et al., 2012 except that the primer pairs used for primary PCR were: 5′-CAAGGCTCCACACGGACTCAAGTTGTT3′ (for 5′-RACE) and 5′-CAAGGCTCCACACGGACTCCAAACAAACAT-3′ (for 3′-RACE). For secondary PCR, the primer sequences were 5′TCGTGTCGCCAGAATTTACGTGTGAGAG-3′ and 5′-TCGTGTCGCCAGAATTACGTGTGAGAG-3′, for 5′-and 3′-RACE, respectively. RACE yielded separate 5′ and 3′ ends of the gene which allowed cloning of ORF using the designated sequences. For the purpose, total RNA was isolated from leaf tissue of tea plant under DS and cDNA was synthesized using total RNA (2 μg) in the presence of 1 μg oligo (dT)12–18 and 400 U of reverse transcriptase SuperScript III (Invirogen, USA) after digestion with 2 U DNase I (Amplification Grade; Invirogen, USA) as described elsewhere (Muoki et al., 2011). Primer pairs, comprising of a forward primer 5′-ATGGCATCAGAGTTGCAAATGC-3′ and a reverse primer 5′-TCAGAATGGCTTCTGAAGGTACATG-3′, were used to amplify the full-length CsNAM-like protein from UPASI-9. PCR was performed using 1 μl of cDNA template, 0.2 μM each of forward and reverse primers, 0.2 mM of dNTPs, 1 U of Taq DNA polymerase, and 1× PCR buffer [20 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2] in a final volume of 25 μl. PCR was carried out on a programmable thermal cycler (GeneAmp PCR system 9700, Applied Biosystems, USA) using the following cycling conditions: 1 min at 95 °C, 25 cycles each of 30 s at 95 °C, 30 s at 58 °C and 72 °C for 1 min and final extension at 72 °C for 7 min. Cloning, sequencing, homologous nucleic acid and protein sequences search and alignment were performed as described by Paul et al. (2012).

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Paul and Kumar (2011). Expression primers (forward: 5′-AAACCAACTGGATTATGCAC-3′ and reverse: 5′-GAATTTCGCTCAATTTCTTG3′) for qPCR were designed using Primer 3 software (Rozen and Skaletsky, 2000). Gene expression was performed on a Stratagene Mx3000P system (Agilent Technologies, Germany) using 2 × Brilliant III SYBR @ Green qPCR Master Mix (Agilent Technologies, Germany). All qPCRs were run in triplicates with a no-template control to check for contamination. PCR was conducted under the following conditions: 10 min at 95 °C (enzyme activation), 40 cycles each of 30 s at 95 °C, 30 s at 55 °C and 72 °C for 30 s and a final melting curve analysis was performed (55° to 95 °C) to verify the specificity of amplicons. The raw threshold cycle (Ct) values were normalized against a housekeeping gene encoding Actin (Shi et al., 2011) to calculate both the difference in expression between control and respective treatment samples and tissue specific gene abundance using the Relative Expression Software Tool (REST; Pfaffl et al., 2002). Expression values were transformed (log2) to generate expression profiles.

ATAF

NAC

NAP

2.3. Subcellular localization analysis The coding region of CsNAM-like protein was amplified with the primer pair comprising of a forward primer 5′-GCAGATCTAATGGCATCAGAGTTGCAAATGC-3′ and a reverse primer 5′-GCACTAGTGAATGGCTTCTGAAGGTACATG-3′ (restriction sites are underlined), and inserted into the BglII and SpeI sites of the binary vector pCAMBIA1302 (http://www.cambia.org.au) to generate the CsNAM-like protein::GFP in-frame fusion protein. The fusion construct and control GFP vector (pCAMBIA1302) were introduced into the onion epidermal cells through Agrobacterium tumefaciens GV3101 (Peng et al., 2009). The onion epidermal cells treated with bacterial liquid were incubated on MS medium at 25 ± 2 °C for 24 h in the dark. GFP signal was observed under an AxioImager M1 fluorescence microscope with AxioCam HRc (Carl Zeiss, Germany). 2.4. Expression analysis Total RNA was isolated essentially as described previously (Muoki et al., 2011). Double stranded cDNA was prepared as described by

NAM

TIP SENU5

Fig. 2. Phylogenetic tree of CsNAM-like protein along with the previously characterized NACs. The Neighbor-Joining tree was constructed using MEGA 5.05. Accession numbers of deduced protein sequences, mentioned in parentheses, were as follows: CarNAC5 (FJ477886), AtNAM (AF123311), CUC1 (AB049069), CUC2 (AB002560), CUC3 (AF543194), NAC1 (AF198054), TIP (AF281062), OsNAC4 (AB028183), OsNAC5 (AB028184), OsNAC6 (AB028185), OsNAC19 (AY596808), NAM (X92205), BnNAC1-1(AY245879), BnNAC5-1 (AY245881), BnNAC5-8 (AY245883), BnNAC5-11 (AY245884), BnNAC14 (AY245886), GRAB1 (AJ010829), GRAB2 (AJ010830), NAMB1 (DQ869673), TaNAC2 (AY625683), AmNAC1 (EU031047), CaNAC1 (AY714222), HaNAC-1 (AY730866), HvNAC6 (AM500854), PmNAC (BAE48667), SlNAC1 (AAR88435) and StNAC (AJ401151). The genetic distances are indicated by the horizontal bar.

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A. Paul et al. / Gene 502 (2012) 69–74 Bright field

Fluorescence

GFP

CsNAM-like protein::GFP

Fig. 3. Localization of CsNAM-like protein in epidermal cells of onion peel. GFP (top row) and CsNAM-like protein::GFP fusion proteins (bottom row) were transiently expressed in onion epidermal cells and analyzed by florescence microscopy. The photographs were taken under bright field to record morphology of the cell, and in the field to record green fluorescence. The nucleus is indicated by an arrow.

3. Results and discussion 3.1. Cloning and sequence analysis of CsNAM-like protein CsNAM-like protein (accession number: JQ619837) had an ORF of 873 base pairs encoding 291 amino acids (Supplementary Fig. 1) with calculated molecular weight of 33.4 kDa and an isoelectric point (pI) of 6.72. BLAST search and multiple sequence alignment

indicated that the deduced amino acids sequence shared 74.5, 72.7, 71.3, and 69.9% sequence similarity with NACs from Gossypium hirsutum L. (Meng et al., 2009), Cicer arietinum L. (Peng et al., 2009), Brassica napus L. (Hegedus et al., 2003) and Prunus mume Siebold & Zucc. (Mita et al., 2006), respectively (Fig. 1). These results suggested that the cloned cDNA belonged to the NAC gene family and was designated as CsNAM-like protein. The predicted protein sequence of CsNAM-like protein had a NAC domain (amino acid 7–161) at the

Fig. 4. Expression of CsNAM-like protein in response to various abiotic stresses in tea. Panel ‘a’ shows gene expression in response to drought stress (DS) and upon re-irrigation on 2 year old plants grown in plastic sleeves at different stress levels as mentioned in the ‘Materials and methods’ section. For recovery (Rec) experiment, drought-stressed plants (irrigation withheld) were re-irrigated on day 30 of the imposition of drought and the data was collected on day 10 of drought release. Panel ‘b’ represents gene expression on day 30 for IP and DS root, stem and leaf samples. Only one time-point was selected in the study due to the destructive sampling method involved and the problem of removing wet soil attached to root in the early period of drought as detailed in the ‘Materials and methods’ section. Panel “c” shows the effect of osmoticum (10% polyethylene glycol, PEG8000), salt (250 mM NaCl), heat (37 °C) and hydrogen peroxide (0.25% H2O2) on gene expression of cut shoots (comprising of apical buds and the associated six leaves). ‘Materials and methods’ section details normalization of data using housekeeping gene. Expression values were relative to respective controls where DS was compared with IP, while osmotic, heat and salt were compared to the cut shoots placed in deionized water. Panel ‘d’ represents the relative gene expression in different part of well-watered tea bush maintained in the field. YL, young leaf (apical bud and the associated two leaves); ML (mature leaf, pooled tissue at node positions 4th, 5th, and 6th with reference to the apical bud); FB, flower bud.

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N-terminal region which could be divided into five subdomains (I–V) according to Ooka et al. (2003), whereas the C-terminal region had a more variable region, showing no significant similarity to that of any other members of NAC family (Fig. 1). Further analysis of the CsNAM-like protein primary sequence using PSORTII (http://mwww. bioweb.pasteur.fr/seqanal/interfaces/psort2.html) identified a monopartite nuclear localization signal (PRDRKYP) in the third motif of the NAC domain (Fig. 1, underlined). The predicted secondary structure by SOPMA indicated that CsNAM-like protein had 16.84% αhelix, 64.26% random coil, 4.2% extended strand, and 14.78% β-turn, (Supplementary Fig. 2). Phylogeny tree analysis of CsNAM-like protein with previously characterized NAC revealed that CsNAM-like protein, belonged to ATAF-type (Ooka et al., 2003), showing homology to BnNAC5-11 and GhNAC2 (Fig. 2). The NAC family members in subgroup ATAF were implicated to share a conserved role in the response to stress stimuli (Ooka et al., 2003). BnNAC5-11 and GhNAC2 were shown to be highly induced by exposure to abiotic and biotic stress in B. napus and G. hirsutum, respectively (Hegedus et al., 2003; Meng et al., 2009). 3.2. Localization of CsNAM-like protein Numerous NACs have been reported to be localized in the cell nucleus (Hu et al., 2006; Olsen et al., 2005; Peng et al., 2009). In this study, CsNAM-like protein was expected to be localized in the nucleus as predicted by the analyses using PSORTII and ProComp v8.0 (http://linux1.softberry.com/berry). To confirm the localization, the ORF of CsNAM-like protein was fused to GFP and transiently expressed in onion epidermal cells under the control of the CaMV 35S promoter. Cells expressing GFP alone displayed diffused cytoplasmic and nuclear localization (Fig. 3), whereas CsNAM-like protein::GFP fusion proteins were localized exclusively in the nucleus (Fig. 3) suggesting CsNAM-like protein to encode for a nuclear-localized protein. 3.3. Expression of CsNAM-like protein in response to drought, osmoticum, salt, temperature and H2O2 The pattern of CsNAM-like protein expression was examined in DS samples over a period of time (Fig. 4a). The gene was highly induced by drought and consequently repressed when the plants were reirrigated and allowed to recover (Fig. 4a) suggesting its specific role in DS. Phylogenetic tree (Fig. 2) suggested a close similarity of CsNAM-like protein to GhNAC2 that is also induced by dehydration stress, LT and ABA (Meng et al., 2009). BnNAC5-11 and CsNAM-like protein also showed close similarity to each other on the phylogenetic tree (Fig. 2). BnNAC5-11 was reported to be highly induced upon exposure to LT, mechanical wounding, flea beetles feeding and infection with Sclerotinia sclerotiorum (Hegedus et al., 2003). Various NAC transcription factors have been implicated in imparting tolerance to drought in rice (Oryza sativa) and Arabidopsis (Hu et al., 2006; Nakashima et al., 2009, Olsen et al., 2005). Such similarities with the other NACs, which have a role in abiotic stress response, imply a role of CsNAM-like protein under drought stress in tea. To study gene expression in different plant tissues under DS, day 30 IP and DS root, stem and leaf samples were used. One time-point was selected due to the destructive sampling method involved and the problems of removing wet soil attached to root in the early period of drought. The patterns of gene expression showed up-regulation in stem and leaf tissues as compared to root tissue where the gene was down-regulated (Fig. 4b). These results suggested possible involvement of the gene in imparting protection to the aboveground organs under drought stress. Notably, the gene was highly induced in leaf as compared to the stem tissue. In rice, over-expression of SNAC1 was predominant in guard cells and was associated with enhanced tolerance to drought (Hu et al., 2006).

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The response of CsNAM-like protein gene to cues that interact with DS namely osmotic, salt, heat and H2O2 stress was also investigated. The gene was up-regulated by all cues at different time points (Fig. 4c). An initial down-regulation at 1 h, then followed by upregulation with the progress of PEG treatment was observed (Fig. 4c). NaCl stress resulted in gradual up-regulation of the gene (Fig. 4c) confirming the role of NAC genes to salt stress as reported in rice (Hu et al., 2006). Under heat stress, a gradual downregulation for the initial 8 h was noted followed by up-regulation with continued exposure to heat (Fig. 4c), whereas in response to H2O2, a ROS involved in stress signaling response, wavy expression pattern was observed. Interestingly, the gene was induced by all the four stresses at 24 h and 48 h (Fig. 4c) suggesting its role in abiotic stress. Paul and Kumar (2011) reported up-regulation of the CsNAM-like protein EST in response to winter dormancy as influenced by LT and ABA in tea. Thus, the gene appears to respond to varied abiotic stresses and hence might have implication in imparting tolerance to abiotic stress. 3.4. Expression of CsNAM-like protein in various tissues The expression of CsNAM-like protein was ubiquitous in all tissues analyzed, with highest level of transcripts measured in stem, FB and ML, while root, YL and fruit had lower level of transcript (Fig. 4d). High expression of NAC in phloem tissue was attributed to the longdistance transport of CmNACP mRNA from the body of the plant to the shoot apex in pumpkin (Cucurbita maxima Duchesne) (Ruiz-Medrano et al., 1999). This was proposed as a post-transcriptional regulatory mechanism. Elsewhere, Mitsuda et al. (2007) reported the role of NST1 in secondary wall-thickening formation in woody tissues. Several studies have reported the involvement of NAC in flower development and reproduction (Olsen et al., 2005). Mutants of the NAC denoted NAM for ‘no apical meristem’ from petunia, died at the seedling stage and occasional escape shoots displayed aberrant floral development (Souer et al., 1996). To conclude, CsNAM-like protein, a member of the NAC transcription factor was found to encode a nucleus localized protein. The gene responded to all the cues suggesting its important role in regulating varied processes under abiotic stress. Acknowledgment The authors thank the Council of Scientific and Industrial Research (CSIR), India for funding the project through Supra Institutional project entitled “High value products from agroforestry resources from the Himalayan region and improving productivity and quality of product development, including evaluation facility for nutraceutical/ value added products (SIP-003)”. The authors thank the Director, CSIR-Institute of Himalayan Bioresource Technology (IHBT) for provision of necessary facilities for the work. AP thanks CSIR for awarding Junior/Senior Research Fellowships. RCM thanks CSIR and the Academy of Sciences for Developing Countries (TWAS), Italy for the award of Postgraduate Fellowship. Manuscript represents IHBT publication number 2334. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2012.04.017. References Balasaravanan, T., Pius, P.K., Raj Kumar, R., Muraleedharan, N., Shasany, A.K., 2003. Genetic diversity among south Indian tea germplasm (Camellia sinensis, C. assamica and C. assamica spp. lasiocalyx) using AFLP markers. Plant Sci 165, 365–372.

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