Characterization of selected south Indian tea (Camellia spp.) germplasm using morphological traits and RAPD markers

· Articles
Authors

DEEPU VIJAYAN*, JIBU THOMAS, K.M. MARIA JOHN, P.K. PIUS & N. MURALEEDHARAN

UPASI Tea Research Foundation, Tea Research Institute, Nirar Dam BPO, Valparai – 642127, Tamil Nadu, India
*Corresponding author present address: Botanical Survey of India, Eastern Regional Centre, Shillong – 793003, India;
email: deepundd@gmail.com

Abstract

The genetic variation and relationship among the selected south Indian tea (Camellia spp.) germplasm was evaluated using morphological traits and RAPD markers. Ninety four mother bushes were selected for germplasm characterization. Morphological characterization of selected mother bushes identified significant divergence among them.The dendrogram constructed using UPGMA method based on RAPD fingerprinting divides the selected mother bushes into two major groups. The present investigation confirms the ability of RAPD markers to characterize the high variation exists within and between the selected accessions of tea germplasm. The results of the present study indicate that there is no correlation between molecular and morphological (both quantitative and qualitative) relationships.

Keywords Camellia spp., genetic diversity, RAPD markers, morphological markers

Introduction

Tea, made from the tender shoots of tea plant (Camellia sinensis (L.) O. Kuntze), is the most popular beverage in the world. South Indian tea accessions are highly genetically variable due to free natural pollination (Balasaravanan et al., 2003). The elevated level of genetic heterogeneity in tea is presumably due to the frequent outcrossing as a result of self-incompatibility (Neog et al., 2004). Germplasm conservation and evaluating their degree of genetic variation are important in order to use representative accessions from the accessible tea population for genetic improvement programme (Bera et al., 2002). The extensive cultivation of clonal tea can reduce the genetic diversity if care is not taken to use clones of disparate origin (Magoma et al., 2000). This is important because old seedlings in tea plantations are being massively replanted with high yielding clones to increase production and hence the seedling population will be lost eternally (Rajkumar et al., 2010). The overdependence on few highly selected and specially chosen “accessions” (clones) has led to a reduction in land dedicated to seedling cultivars of high genetic variability. The success in tea germplasm collection, preservation, exploitation, utilization and long-term breeding programmes of tea depend largely on the understanding of genetic background, diversity, relationship and identification (Sui et al., 2008).

Morphological parameters such as leaf architecture, growth habits and floral biology are important criteria used by tea taxonomists (Mohanan & Sharma, 1981; Banerjee, 1992). The visible variation among the selected germplasm is necessary for easy selection and utilization for the conventional breeding approach (Rajkumar et al., 2010). In spite of several disadvantages, morphological characterization is the most widely adopted method used by tea breeders’ worldwide (Mondal et al., 2004; Tripathi & Negi, 2006). Large variation in the phenotypic characters in existing tea germplasm is helpful in breeding programme and exploitation of such plant variability by careful selection can be utilized for developing improved planting material in this perennial crop.

Molecular markers have several advantages over the traditional phenotypic markers that were previously available to plant breeders. In the recent years, few attempts have been made to assess the genetic diversity in tea populations, growing in certain specific geographical regions using Random Amplified Polymorphic DNA (RAPD) markers (Wachira et al., 1995; Kaundun et al., 2000; Lai et al., 2001; Chen et al., 2005; Goonetilleke et al., 2009). Molecular markers are useful tool to understand the diversity in tea germplasm and also for tea breeding programmes (Chen and Yamaguchi, 2005). The present study attempted to distinguish the selected accessions using morphological traits and RAPD markers. The study would pave the way to improve current understanding on the status of diversity among tea germplasm which can form the baseline information for future tea breeding programme.

Materials and Methods

Mother bush selection/Plant material

Ninety four (94) seedling populations maintained in the germplasm collection centre of United Planters’ Association of Southern India (UPASI) Tea Research Institute, Valparai, Tamil Nadu, India were selected for the present investigation. The bushes were properly tagged and given code numbers for the experimental convenience. The mother bushes were numbered as UP/62/1 to UP/62/94; where UP represents UPASI, 62 symbolize 1962 area of experimental farm and 1 to 94 stands for individual mother bushes.

Morphological characterization

Descriptors of seedlings were prepared by observing foliar, floral, fruit and seed characteristics. In tea, the foliar characters were mostly used for the grouping of plants to determine their affinity towards Assam, China or Cambod jats. In the present study, morphological characters of the seedlings were documented in the format following the tea descriptors developed by International Plant Genetic Resources Institute (IPGRI, 1997). To determine the diversity of the selected population, only some prominent leaf/flower/fruit characters were taken into consideration. Internode length (cm), length of mature leaf, width of mature leaf, immature/mature leaf color, leaf shape, leaf upper surface, leaf margin, leaf pubescence, petiole length, filament length (cm), anther length (cm) and fruit diameter for all the accessions were observed. In each case, at least 25 numbers of mature leaves/crop shoots/ flowers/fruits were examined and the mean values were documented.

Molecular characterization

DNA extraction and purification

DNA was extracted following cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1987) with some modifications. The young leaf samples (0.2 g) were ground in liquid nitrogen using mortar and pestle and resuspended in 2ml of pre-warmed DNA extraction buffer [2% CTAB, 1.4M NaCl, 25mM EDTA (pH 8.0), 100mM Tris-HCl (pH 8.0) and 0.2% b-mercaptanoethanol]. Homogenate containing centrifuge tubes were incubated at 65°C for 45 min with occasional tapping. Samples were mixed with equal volume of chloroform/isoamyl alcohol (24:1 v/v) and centrifuged at 12000 rpm for 20 min. The supernatant was transferred to fresh centrifuge tubes and DNA was precipitated by adding equal volume  of ice cold isopropanol. The DNA precipitate was air dried and stored in 500µL TE buffer. The samples were treated with RNase A (10mgmL-1) at 37ºC for 30 min. Equal volume of chloroform/isoamyl alcohol (24:1 v/v) was added to this and mixed thoroughly by several inversions. Aqueous phase was then transferred to a fresh tube after centrifuging at 12000 rpm for 10 min. To the aqueous phase, 0.6-1.0 volume of ice-cold isopropanol was added, mixed well and incubated at room temperature for 30 min. Later it was centrifuged at 5000 rpm for 10 min at 4°C and the pellet obtained was washed with 1 ml of wash buffer (70% ethanol containing 10 mM ammonium acetate). DNA was air dried and dissolved in 200µl of sterile water. Further purification steps are as follows. The total volume of DNA was made up to 500µl with de-ionized water. Equal volume of phenol:chloroform:isoamyl alcohol (25:24:1 v/v) was added and mixed gently with inversion and centrifuged at 5000 rpm for 15 min. The aqueous layer was transferred in to a new eppendorf tube using a blunt/cut tip. The aqueous phase was extracted with equal volume of chloroform isoamyl alcohol. One by tenth volume of 3M sodium acetate (pH 4.8) was added to the aqueous upper layer, followed by 2.5 volumes of absolute alcohol and mixed by inversion. Then it was incubated for half an hour at -20ºC followed by centrifugation (6000 rpm, 10 min, 4ºC). DNA pellet was then washed with 70% ethanol for 1 hour. The DNA pellet was dried at room temperature and dissolved in 200µl of Milli-Q water and left for overnight at room temperature to dissolve completely. Quantification of the purified DNA was done on 0.8% agarose gel by comparing with uncut lambda DNA (100 ng).

RAPD fingerprinting

For each polymerase chain reaction, reaction mixture (25 µl) containing 25 ng of DNA, 1U of Taq DNA polymerase, 1X Taq buffer, 25mM MgCl2, 2.5mM dNTPs, 3µM of 10-mer oligo-nucleotide primers (Operon Technologies Inc., Alameda, CA, USA). Out of 18 primers screened, 10 primers revealed polymorphism (OPA 01, OPA 05, OPA 08, OPA 09, OPA 13, OPA 20, OPC 02, OPC 07, OPC 15 and OPC 20) and selected for analysis. All the reaction chemicals except primers were procured from M/s. Genei, Bangalore, India.

Amplifications were carried out in a DNA thermal cycler (MJ Research PTC-100) using following parameters: 94°C for 5 min; 44 cycles at 94°C for 1 min; 34°C for 1 min and 72°C for 2 min; and a final extension at 72°C for 15 min. Each amplification reaction for the screened primers was replicated two times individually with the same procedure in order to verify that the RAPD markers were reproducible and consistent. PCR products were subjected to electrophoresis on 1.5% (w/v) agarose gel in 1X TBE buffer, along with 1kb DNA ladder as size markers. The gels were visualized with a UV transilluminator and documented using a digital camera.

Data analysis

The gel images were scored using a binary scoring system that recorded the presence and absence of bands as “1” and “0” respectively. The statistical analysis is performed using NTSYSpc version 2.1 (Rohlf, 1998). The data matrix was used to construct a phenetic dendrogram using UPGMA (Unweighted Pair Group Method of Arithmetic averages) in order to cluster the accessions (Sneath and Sokal, 1973).

Results and Discussion

Morphological characterization

Morphological characterization of selected mother bushes identified significant divergence among them (Table 1). All the measured characters for mature leaves/ crop shoots/flowers/fruits showed considerable variations. Certain phenotypic characters were found to be very distinct among the seedlings (Figure 1). Among the characters observed, immature leaf color of the population showed considerable variation (yellowish green to green). Dark green color was absent in mature leaves of 26 bushes. Internode length varied from 1.7 cm to 5.8 cm. Majority of the bushes (61%) showed lanceolate leaf shape confirming their affinity towards Assam type and the rest were elliptic in shape. Upper leaf surface of bushes showed that 53% of them were smooth and the rest were found to be rugose. The visual observations on leaf margin identified that majority of the bushes were having serrulated leaf margin while 21 bushes exhibited biserrated margin. Sixty two per cent of the bushes possessed medium sized leaves and the rest were large. Leaf angle were found to be acute in almost all the cases with some exceptions like UP/62/1, UP/62/2, UP/62/7, UP/62/14, UP/62/52, UP/62/57 and UP/62/59. Yet another character, leaf pubescence showed a mixed response (sparse, intermediate and dense). Among the floral characteristics, filament length showed no distinct variation among the bushes studied. From the observations it was found that there was not much variation in the anther length of flower. Fruit diameter recorded from the individual bushes showed discrete variation (3.0 cm to 8.0cm). These findings corroborate the findings of Rajkumar et al. (2010)

Table 1: Morphological characteristics used to distinguish 94 accessions

Vegetative Characters

Range of Variation

Mean±SD

Data type

Internode length (cm) 1.7 – 5.8

3.9±0.93

Quantitative
Length of mature leaf (cm) 10.0 – 17.9

12.71±1.76

Quantitative
Width of mature leaf (cm) 4.0 – 8.1

5.71±0.94

Quantitative
Petiole length (cm) 0.3 – 0.9

0.63±0.17

Quantitative
Filament length (cm) 0.6 – 2.5

1.45±0.45

Quantitative
Anther length (cm) 0.04 – 0.09

5.72E-02±1.37E-02

Quantitative
Fruit diameter (cm) 3.0 – 8.0

5.53±0.95

Quantitative
Mature leaf colour Light green – Green Multistate
Immature leaf colour Yellow green – Green Multistate
Leaf shape Lanceolate – Elliptic Multistate
Leaf upper surface Smooth – Rugose Binary
Leaf margin Serrulate – Biserrate Binary
Leaf pubescence Sparse – Intermediate Binary

Table 2: Affinity of selected mother bushes based on the morphological characteristics

Assam

China

Cambod

UP/62/1

UP/62/48

UP/62/5

UP/62/2

UP/62/3

UP/62/50

UP/62/8

UP/62/6

UP/62/4

UP/62/51

UP/62/17

UP/62/25

UP/62/7

UP/62/53

UP/62/18

UP/62/27

UP/62/9

UP/62/54

UP/62/20

UP/62/31

UP/62/10

UP/62/57

UP/62/21

UP/62/33

UP/62/11

UP/62/59

UP/62/40

UP/62/35

UP/62/12

UP/62/60

UP/62/43

UP/62/38

UP/62/13

UP/62/61

UP/62/52

UP/62/39

UP/62/14

UP/62/62

UP/62/55

UP/62/45

UP/62/15

UP/62/63

UP/62/56

UP/62/47

UP/62/16

UP/62/64

UP/62/58

UP/62/49

UP/62/19

UP/62/65

UP/62/67

UP/62/75

UP/62/22

UP/62/66

UP/62/68

UP/62/85

UP/62/23

UP/62/69

UP/62/70

UP/62/89

UP/62/24

UP/62/72

UP/62/71

UP/62/26

UP/62/73

UP/62/77

UP/62/28

UP/62/74

UP/62/81

UP/62/29

UP/62/76

UP/62/84

UP/62/30

UP/62/78

UP/62/88

UP/62/32

UP/62/79

UP/62/90

UP/62/34

UP/62/80

UP/62/93

UP/62/36

UP/62/82

UP/62/37

UP/62/83

UP/62/41

UP/62/86

UP/62/42

UP/62/87

UP/62/44

UP/62/91

UP/62/46

UP/62/92

UP/62/94

Affinity of the accessions based on morphology

In the present study no bush showed identical morphological characters that can sustain the particular characteristics of “Assam”, “China” or “Cambod” cultivars. Hence, on the basis of the data generated 94 bushes were categorized into Assam, China and Cambod groups particularly based on their morphological similarity and affinity. Even though not all the parameters were in relation to their respective jats, grouping was based on their major characteristics. Among the selections characterized morphologically, 57 selections grouped under Assam category, 22 under China and 15 in Cambod (Table 2). These phenotypic traits could result in the selection of elite mother bushes from the existing tea germplasm. Phenotyping can be coupled with molecular characterization as they can outline the base of modern genomics (de Vicente, 2004).

Molecular characterization

Molecular characterization of selected tea germplasm using RAPD exhibited a very high proportion of DNA diversity and high genetic distances. It was possible to establish a minimum number of primers with great capacity to differentiate all the germplasm. Total number of bands generated by 10 primers was 134, out of which 87 were polymorphic (65% polymorphism). The size of the polymorphic amplified fragments ranged from 0.2 to 3 kb. RAPD data generated by ten primers were subjected to UPGMA cluster analysis. The dendrogram constructed on the basis of shared fragments divides the selected mother bushes into two major clusters (Figure 2)

One of the major contributory factors to the high degree of polymorphism observed in tea indicates towards predominant out-crossing in this species. Forty nine accessions were grouped in cluster I, and 45 accessions in cluster II. The accessions grouped as two major clusters based on RAPD data could not be correlated with grouping based on morphological affinity and similarity. RAPD fingerprinting was more discriminatory and able to differentiate the selected accessions than the morphological descriptors. From the present investigation it is clear that there is no direct association between morphological data and molecular information among the selected seedling population of south India. Thus it may be postulated that RAPD fingerprinting is a powerful tool among the available genetic fingerprinting techniques and can be adopted for any kind of tea breeding programme. Chen et al. (2005) and Chen & Yamaguchi (2005) ascertained the use of RAPD markers in detecting the genetic diversity and relationship among the tea germplasm repository.

In addition, estimation of genetic similarity based on molecular markers may provide more precise information to plant breeders than the pedigree methods, allowing breeders to decide and strategically plan the breeding programme on a long term basis (Barret & Kidwell, 1998; Lima et al., 2002). The background information on these germplasm for important traits and genetic diversity helps the breeder in selecting the right material (Tripathi & Negi, 2006). It is also possible that a large proportion of valuable tea germplasm may have been already lost through the continuous removal of older seed-stocks, especially seedlings for commercial planting of vegetatively propagated plants. To avoid further degradation of germplasm resources, existing population must be preserved and by employing molecular markers can be effectively utilized for conservation and tea breeding activities. Planting of a particular clone to the exclusion of other cultivars is not advisable because such a practice narrows the genetic base. Therefore, planting of diverse cultivars comprising about 20, 30 and 50 per cent of clones, nursery grafts and biclonal seed stocks repectively, is recommended (Satyanarayana et al., 1995). Moreover, planting of diverse cultivars widens the genetic base and minimizes the risk of susceptibility to abiotic and biotic stress factors, both prevailing and unforeseen. Seeing that more old seed stocks are being uprooted and substituted by fewer popular tea clones of narrow genetic base, it is imperative to document the genetic variability of tea germplasm to avoid the loss forever (Rajkumar et al., 2010).

Conclusion

The characterization of tea germplasm is important because of their huge genetic diversity. Introduction of tea germplasm from China and its uncontrolled hybridization with indigenous tea germplasm has led to the formation of a highly divergent population. Therefore, characterization
of highly heterogeneous tea germplasm is important for proper utilization, management and improvement of tea in the country.

Acknowledgement

The authors thankfully acknowledge the financial support of Department of Biotechnology, Ministry of Science and Technology, Government of India.

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