AFLP-Based Genetic Diversity Assessment of Commercially Important Tea Germplasm in India

AFLP-Based Genetic Diversity Assessmentof Commercially Important Tea Germplasm in India

R. K. Sharma • M. S. Negi • S. Sharma •

P. Bhardwaj • R. Kumar • E. Bhattachrya •

S. B. Tripathi • D. Vijayan • A. R. Baruah •

S. C. Das • B. Bera • R. Rajkumar • J. Thomas •

R. K. Sud • N. Muraleedharan • M. Hazarika •

M. Lakshmikumaran • S. N. Raina • P. S. Ahuja

Received: 12 August 2009 / Accepted: 19 November 2009 / Published online: 14 April 2010

� Springer Science+Business Media, LLC 2010

Abstract India has a large repository of important tea accessions and, therefore,

plays a major role in improving production and quality of tea across the world.

Using seven AFLP primer combinations, we analyzed 123 commercially important

tea accessions representing major populations in India. The overall genetic simi-

larity recorded was 51%. No significant differences were recorded in average

genetic similarity among tea populations cultivated in various geographic regions

(northwest 0.60, northeast and south both 0.59). UPGMA cluster analysis grouped

the tea accessions according to geographic locations, with a bias toward China or

Assam/Cambod types. Cluster analysis results were congruent with principal

component analysis. Further, analysis of molecular variance detected a high level of

R. K. Sharma (&) � P. Bhardwaj � R. Kumar � P. S. Ahuja (&)

Biotechnology Division, Institute of Himalayan Bioresource Technology, CSIR,

P.O. Box 6, Palampur 176061, Himachal Pradesh, India

e-mail: [email protected]

P. S. Ahuja

e-mail: [email protected]

M. S. Negi � S. B. Tripathi � M. Lakshmikumaran

Plant Molecular Biology Division, The Energy Resources Institute, Darbari Seth Block,

IHC Complex, Lodhi Road, New Delhi 110003, India

S. Sharma � E. Bhattachrya � S. N. Raina

Department of Botany, University of Delhi, Delhi 110007, India

D. Vijayan � R. Rajkumar � J. Thomas � N. Muraleedharan

UPASI Tea Research Institute, Valparai, Coimbatore 642127, India

A. R. Baruah � S. C. Das � B. Bera � M. Hazarika

Tea Research Association, Tocklai Experimental Station, Jorhat 785008, Assam, India

R. K. Sud

HATS Division, Institute of Himalayan Bioresource Technology, CSIR,

P.O. Box 6, Palampur 176061, Himachal Pradesh, India

123

Biochem Genet (2010) 48:549–564

DOI 10.1007/s10528-010-9338-z

genetic variation (85%) within and limited genetic variation (15%) among the

populations, suggesting their origin from a similar genetic pool.

Keywords AFLP � Genetic diversity � Tea accessions � Institute of Himalayan

Bioresource Technology (IHBT) � Tea Research Association � UPASI Tea Research

Foundation

Introduction

Tea is one of the oldest, most widely consumed, and least expensive natural

beverages, known the world over for its heritage brew, with various flavors and

antioxidant properties. Because of specific soil and climate requirements, tea

production is confined to particular regions of Asia (India, China, Sri Lanka, Japan,

Vietnam, and Indonesia), Africa (Kenya, Uganda, and Malawi), and to a small

extent Latin America (e.g., Argentina). It is one of the most important agro-based,

eco-friendly, labor-intensive, employment-generating, and export-oriented indus-

tries in all of the countries in which it is grown.

The genus Camellia has been described as containing 82 species (Sealy 1958),

but other taxonomic classification systems have also been proposed in the recent

past. For example, Chang (1981, 1998) recognized 284 species, and Ming (2000)

identified 119 species. Of these plants, only three have contributed significantly to

the entire genetic pool of cultivated tea worldwide: the China type, Camelliasinensis (L.) O. Kuntze; the Assam type, C. assamica ssp. assamica (Masters); and

the Cambod type, C. assamica ssp. lasiocalyx (Planch MS). Because of their

compatibility across species, these plants breed with other wild Camellia species,

such as C. flava, C. petelotii (Wight 1962), and C. lutescens (Sharma and

Venkataramani 1974), and these wild species have also contributed to the tea gene

pool, in addition to C. irrawadiensis and C. taliensis, which have morphological

proximity to tea (Banerjee 1992). Therefore, tea cultivated at the national level is

highly heterogeneous, having several intergrades, introgressants, and putative

hybrids. These can be categorized from China types through intermediate to Assam

types, and hence the existence of true China, Assam, or Cambod tea is doubtful

(Visser 1969).

Annual world tea production grew by more than 3% in 2006, to reach an estimated

3.6 million metric tons (FAO 2008). An increase was also recorded in India, the

second-largest producer and consumer, where harvests were also 3% higher, totaling

(945,000 metric tons) in the same year. Nonetheless, India ranks first as a producer,

consumer, and exporter of black tea. Tea-producing areas in India are confined to

three major geographic regions: the northeast, represented by Assam, West Bengal,

Bihar, Tripura, Sikkim, Manipur, Nagaland, Meghalaya, Arunachal Pradesh, and

Mizoram; the south, represented by Kerala, Karnataka, and Tamil Nadu; and the

northwest, represented by the hills of Himachal Pradesh and Uttarakhand.

The introduction of tea germplasm from China in the year 1836 and its subsequent

spontaneous large-scale hybridization with indigenous tea accessions and wild

relatives from a wide range of geographic distribution in India led to hybrid swarms

550 Biochem Genet (2010) 48:549–564

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with tremendous genetic variability. In order to maintain a heterogeneous gene pool

at the national level, regional germplasm collection centers were established in

predominant tea-growing areas. Tea germplasm is now maintained at the national

level in northeastern India at the Tea Research Association, Tocklai Experimental

Station, Jorhat, Assam; in southern India at the United Planters’ Association of

Southern India (UPASI), Valparai, Coimbatore; and in northwestern India at the

Institute of Himalayan Bioresource Technology (IHBT), Palampur. It is estimated

that presently more than 2532 accessions are held by Tocklai Experimental Station

(Singh 1999). Large numbers of commercially important accessions are also

available at UPASI and IHBT. There is, therefore, an immediate need for assessing

the highly heterogeneous tea germplasm representing major populations in India at

the DNA sequence level, not only for the future benefits in identification, inventory,

utilization, management, and conservation of the tea germplasm but also for the

development of a strategy for future genetic improvement programs. In the past

decade or so, many efforts have been made to assess the genetic diversity of Indian

tea germplasm at the phenotypic (Barua 1963; Banerjee 1992; Das 2001),

cytogenetic (Bezbaruah 1971), biochemical (Saravanan et al. 2005), and molecular

levels (Paul et al. 1997; Mondal 2002; Wachira et al. 2001; Balasaravanan et al.

2003; Singh and Ahuja 2006; Karthigeyan et al. 2008; Sharma et al. 2009).

Numerous genetic diversity studies have also been conducted on tea germplasm in

other regions of the world, using various molecular marker techniques (Kaundun

et al. 2000; Kaundun and Matsumoto 2002; Chen et al. 2005; Chen and Yamaguchi

2005; Matsumoto et al. 2004; Ohsako et al. 2008; Yao et al. 2008). Most of these

investigations carried out nationally or internationally, however, suffer from an

important lacuna: either small sample size or accessions restricted to a particular

region.

In this study, for the first time, a large number of samples covering the total

spectrum of variation of the tea in India were used to make a comprehensive study

of polymorphism at the DNA sequence level. This analysis provides evidence of

existing genetic relatedness and molecular diversity of the commercial tea

accessions at the national level and thus should be helpful in developing a strategy

for the genetic improvement program in tea. In a coordinated effort, we have

characterized 123 commercially important tea accessions representing major

populations in different geographic regions of India by AFLP markers.

Materials and Methods

Plant Materials

Of the 123 tea accessions, 57 were from northeastern India, 44 from southern India,

and 14 from northwestern India. In addition, four released accessions from the Tea

Research Institute, Sri Lanka, and a single accession from the National Research

Institute of Vegetables, Ornamental Plants, and Tea (NIVOT), Japan were also

included in the study, along with three wild relatives of ornamental C. japonica for

comparison. All of these accessions were either collected from commercial estates

Biochem Genet (2010) 48:549–564 551

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or abandoned tea gardens or developed by open pollination and control crosses

through the breeding program at various centers, and they are regionally maintained

at the Tocklai Experimental Station in the northeast, UPASI in the south, and IHBT

in the northwest (Table 1). The northeastern accessions (at Tocklai) consisted of

collections from Darjeeling in West Bengal and Cachar in Assam. The southern

collections (at UPASI) included accessions from Nilgiris, Anamallais, and Munnar

and released accessions of Tata Tea Limited. The northwestern collections

(at IHBT) consisted of accessions from Himachal Pradesh and Uttarakhand states.

The characteristics of these accessions listed in Table 1 are from either published

information (Bezbaruah and Dutta 1977; Barbora et al. 1996) or observations

recorded and provided by the respective institutes.

DNA Extractions and AFLP Analysis

Total DNA was extracted from fresh leaf tissue by the CTAB method (Doyle and

Doyle 1990) with minor modifications. The quality and concentration of the

extracted DNA were estimated on 0.8% agarose gel using diluted uncut kDNA as a

standard. AFLP fingerprints were generated based on the protocol of Vos et al.

(1995) with slight modifications. Genomic DNA (250 ng) was restricted with EcoRI

and MseI and ligated to standard adapters. The adapter-ligated DNA served as a

template for preamplification, with PCR parameters of 20 cycles of 30 s at 94�C,

60 s at 56�C, and 60 s at 72�C. Selective amplification was subsequently carried out

using EcoRI and MseI primers. The EcoRI primer was labeled with c-33P ATP using

T4 polynucleotide kinase. The first selective amplification cycle consisted of 30 s at

94�C, 30 s at 65�C, and 60 s at 72�C. The annealing temperature was lowered by

0.7�C per cycle during the next 12 cycles, followed by 23 cycles at 94�C for 30 s,

56�C for 30 s, and 72�C for 60 s. All PCR reactions were carried out in a PE-9700

thermocycler (Perkin Elmer, USA). AFLP products were resolved on a 5%

denaturing polyacrylamide gel using 19 Tris–borate–EDTA (TBE) electropho-

resis buffer. The gel was dried for 2 h before subjecting it to autoradiography for

1–3 days at -70�C, depending on the signal intensity. The size of the fragments was

estimated using 20 bp size standards (BioWhittaker Molecular Applications, USA).

Molecular Data Analysis

An AFLP fragment was assumed to represent a single locus, and reproducible

polymorphic fragments were scored as present (1) or absent (0). The polymorphism

information content (PIC) of each marker was calculated according to Anderson

et al. (1993). Data were entered into a binary matrix as discrete variables. Jaccard’s

coefficient of similarity was measured, and a dendrogram based on similarity

coefficients was generated using the unweighted pair group method with arithmetic

mean (UPGMA). The computer package NTsys-PC 2.02 (Rohlf 1998) was used for

cluster analysis. Bootstrap analysis was performed to determine significance of

the clusters with Paup version 4.0b10 (Swofford 1993). In order to identify the

most informative primer pair, discrimination rate was calculated as the number of

polymorphic pairs per total number of pairs of accessions. The structure of the

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Table 1 Tea accessions used for AFLP analysis

Serial

no.

Accession Varietal type Sourced Remarkse

1 KangraAsha China hybrid Himachal Pradesh, Krishi Viswavidyalaya,

Palampur

H

2 KangraJat China hybrid IHBT

3 K-15 China hybrid Khilpet, Palampur

4 BS-26 China hybrid IHBT E

5 BS-1 China hybrid IHBT






11 BS-65a China hybrid IHBT

12 BS-68 China hybrid IHBT G

13 KM-9 China hybrid Vijaypur Tea Estate, Kumaon Hills,

Uttarakhand

14 KM-8 China hybrid Gwaldam, Kumaon Hills, Uttarakhand

15 CSIN-303536 China NIVOT, Japan

16 TS-379b Assam Tocklai ES A

17 TS-464a Assam Tocklai ES A

18 TS-449b Assam hybrid Tocklai ES A

19 TG-270/2/13 Assam Tocklai ES

20 Teenali-17/

154

Assam Teenali, Assam

21 RYDAK-1 China Makaibari Tea Estate, Darjeeling

22 AV-2-Ic China Makaibari Tea Estate, Darjeeling

23 TV-1c Assam-China

hybrid

Tocklai ES A

24 TV-2c Assam Tocklai ES C

25 TV-3c Assam Tocklai ES

26 TV-4c Assam Tocklai ES D

27 TV-5c Assam Tocklai ES A


29 TV-7c China hybrid Tocklai ES B


31 TV-9c Cambod Tocklai ES B, O

32 TV-10c Assam Tocklai ES B

33 TV-12c Assam Tocklai ES B


35 TV-14c Assam hybrid Tocklai ES B

36 TV-15c Assam Tocklai ES A

37 TV-16c Assam hybrid Tocklai ES B

38 TV-17c Assam hybrid Tocklai ES A

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Table 1 continued

Serial

no.


39 TV-18a Cambod Tocklai ES B

40 TV-19a Cambod Tocklai ES A

41 TV-20c Cambod Tocklai ES A

42 TV-21 Assam Tocklai ES D


44 TV-23a Cambod Tocklai ES A

45 TV-24c Cambod/ssp.

hybrid

Tocklai ES B



48 TV-27c Cambod Tocklai ES B

49 TV-28c Cambod Tocklai ES B

50 TV-29a Cambod, triploid Tocklai ES B


52 S-3a/1 Assam Tocklai ES

53 KP-6/25a China-Assam

hybrid

Tocklai ES

54 CB-38c Assam-China

hybrid

Tocklai ES

55 LG-26c China-Assam

hybrid

Cachar, Assam B

56 LG-17a China-Assam

hybrid

Cachar, Assam B

57 P-126 China Tocklai ES

58 P-463 China-Cambod

hybrid

Tocklai ES

59 P-492 China-Cambod

hybrid

Tocklai ES

60 BJ-02b China-Assam

hybrid

Tocklai ES

61 Nokrai-89 Assam Tocklai ES

62 HK-22/14c China-Assam

hybrid

Tocklai ES C

63 HK-23/19c China-Assam

hybrid

Tocklai ES C

64 HV-39a China Darjeeling, West Bengal

65 SS-40Ac China-Assam

hybrid

Tocklai ES B

66 CP-01a China Darjeeling, West Bengal

67 NJ-6/24a China-Assam

hybrid

Tocklai ES B

68 T-78c China Darjeeling, West Bengal B

69 RR-17/144c China Darjeeling, West Bengal

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Table 1 continued

Serial

no.


70 Teesta-

Valley-1cChina Darjeeling, West Bengal

71 S-3 Assam Tocklai ES

72 T-3E/3 Assam Tocklai ES

73 UPASI-1 Assam Brookland A, J, M

74 UPASI-2a Assam Brookland A, J

75 UPASI-3a Assam Brookland D, H, J

76 UPASI-4 Assam Brookland

77 UPASI-5 Cambod Brookland J

78 UPASI-6 Assam Brookland A, J

79 UPASI-7 Assam Brookland A, H, J

80 UPASI-8 Cambod Brookland D, J

81 UPASI-9a Assam Brookland G

82 UPASI-10 China Brookland A, I, M

83 UPASI-11 Assam Brookland J

84 UPASI-12 Cambod Brookland


86 UPASI-14 Cambod Singara Estate, The Nilgiris J

87 UPASI-15 China Springfield Estate, The Nilgiris A, J


89 UPASI-17a Cambod Brookland D, J

90 UPASI-18 Cambod Brookland A, J

91 UPASI-19 Cambod Springfield Estate, The Nilgiris A, E, K

92 UPASI-20 China Brookland H, J


94 UPASI-22c Assam Brookland A


96 UPASI-24 Cambod Brookland A, J

97 UPASI-25c Cambod Anamallais, Valparai, Coimbatore J

98 UPASI-26a Assam Davershola, Gudalur, The Nilgiris A, J, L,

M

99 UPASI-27a Assam Anamallais, Valparai, Coimbatore A, J, L

100 TRI-2026a Assam TRI, Sri Lanka H

101 TRI-2024a Assam TRI, Sri Lanka A, N

102 TRI-2025 Assam TRI, Sri Lanka

103 TRI-2043 Assam TRI, Sri Lanka F

104 ATK-1 Assam Attikunna, The Nilgiri-Wayanad A, J

105 AK-1 China Akkamalai, Anamallais

106 CR-6017 Cambod Craigmore, The Niligiris D

107 SA-6 Assam UPASI E

108 BSS-1 Assam UPASI A, K

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genetic diversity in tea accessions belonging to different geographic regions was

tested by analysis of molecular variance (AMOVA). Principal coordinate analysis

was also carried out to show differentiation of tea accessions in multidimensional

space. The computer program Genalex 6 (Genetic Analysis in Excel 6.0; Peakall

and Smouse 2006) was used for AMOVA and coordinate analysis.

Results and Discussion

AFLP Analysis

Seven AFLP primer combinations detected 307 reproducible clear fragments

across the 123 tea accessions. Of these fragments, 269 (87.6%) were found to be

polymorphic, with 38.4 polymorphic fragments per primer combination (Table 2).

Table 1 continued

Serial

no.


109 BSBa Assam UPASI

110 B/5/163 China Brookland

111 CH-1a China IHBT A, M

112 C-17 Assam BBTC, Singampatti

113 K-18 Assam UPASI

114 NLT-17/10a - Munnar, Idukki District, Kerala

115 TRF-1a Cambod UPASI

116 TRF-2 - UPASI

117 SMPa Cambod Munnar, Idukki District, Kerala E

118 TTL-1b Cambod Tata Tea Limited, Munnar

119 TTL-2b Assam Tata Tea Limited, Munnar H

120 TES-34 Assam –

121 CJ-1 C. japonica IHBT

122 CJ-2 C. japonica UPASI

123 CJ-3 C. japonica UPASI

a High-yielding (average quality but high yield, with yield potential of 4000 kg made tea/ha)b Quality (high quality but average yield, with yield potential of 2500–2800 kg made tea/ha)c Standard (average yield and quality, with yield potential of 3000–3500 kg made tea/ha)d All sources are in India, unless otherwise indicated. Source abbreviations: Brookland, Brookland

Estate, The Nilgiris, India; IHBT, Institute of Himalayan Bioresource Technology, Palampur, India;

NIVOT, National Research Institute of Vegetables, Ornamental Plants, and Tea, Japan; Tocklai ES,

Tocklai Experimental Station, Jorhat, Assam, India; TRI, Tea Research Institute; UPASI, United Planters’

Association of Southern India, TRI, Valparai, Tamil Nadu, Indiae Remarks key: A, drought tolerant; B, moderately drought tolerant; C, moderately susceptible to

drought; D, susceptible to drought; E, highly resistant to blister blight; F, tolerant of blister blight; G,

moderately resistant to blister blight; H, susceptible to blister blight; I, excellent rooter; J, good rooter; K,

slow rooter; L, frost resistant; M, suitable for wind-prone areas; N, containing high amount of antho-

cyanin pigment; O, tolerant of waterlogging

556 Biochem Genet (2010) 48:549–564

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The remaining 38 bands (12.3%) were monomorphic in all tested accessions and,

therefore, could be used to understand the pattern of divergence and the phylum-

specific genetic clock (Hill et al. 1996). AFLP analysis detected a large number of

distinct scorable fragments per primer combination (Fig. 1). The number of

fragments generated with each primer combination ranged from 21 (E-ACG/M-

CTGC) to 58 (E-ACG/M-CGAT), with an average of 43.9 fragments per primer

combination. The detection of comparatively fewer fragments per primer combi-

nation than was reported by Paul et al. (1997) in an AFLP analysis of Indian and

Kenyan tea germplasm might be due to the presence of four arbitrary nucleotides at

Table 2 AFLP analysis by primer combination

Primer

combination

Total

fragments

Polymorphic

fragments

% Polymorphism PIC Discrimination

rate

E-ACA/M-CACG 36 33 91.66 0.413 0.99

E-AAG/M-CTGC 51 43 84.31 0.418 0.99

E-ACG/M-CTGC 21 17 80.95 0.322 0.99

E-AAC/M-CTAC 53 52 98.11 0.454 0.99

E-ACG/M-CGAT 58 51 87.93 0.500 0.99

E-AAG/M-CGTG 46 31 67.39 0.404 0.99

E-AAG/M-CAGT 42 42 100.0 0.474 0.99

Total 307 269

Mean 43.85 38.42 87.6 0.426 0.99

Fig. 1 A representative amplification profile generated with AFLP primer combination E-ACA/M-CTGC. Lanes 1–55 represent 55 tea accessions (Table 1); Lane M, 20 bp ladder (Cambrex BioScience Rockland, USA) used as size standard

Biochem Genet (2010) 48:549–564 557

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the 30 end of the MseI primers during selective amplifications in the present study.

All primer combinations tested in our study revealed high discrimination power

(C0.99). Primer combination E-ACG/M-CGAT showed the highest PIC (0.500),

whereas the lowest value was recorded with primer pair E-ACG/M-CTGC (0.322).

The three primer combinations E-AAC/M-CTAC, E-ACG/M-CGAT, and E-AAG/

M-CAGT each recorded a PIC C 0.45 (Table 2); therefore, they can be categorized

as informative primer combinations for characterizing an even larger set of tea

germplasm.

AFLP-Based Genetic Relationships

The 123 tea accessions clustered into two major groups, plus an outgroup consisting

of the three C. japonica ornamentals (Fig. 2). A dendrogram constructed on the

basis of shared fragments largely matched the existing taxonomic classification of

tea (Banerjee 1992). Group I consisted of 23 accessions, which could be further

divided into three internal subgroups. The majority of the northwest selections were

from the natural populations of the Kangra region and hence clustered together in

this group. An accession from Japan, CSIN-303536, and the high-yielding HV-39

from the Darjeeling collection made up one subgroup. Two accessions from the

Kumaon Hills of Uttarakhand, KM-8 and KM-9, also clustered with accessions from

the Darjeeling collection.

The first bifurcation of tea accessions clearly separated a cluster of northeastern

plus southern tea populations, including TRI-series accessions of Sri Lanka, into

group II. Further, group II was composed of two major subgroups (IIa and IIb).

Subgroup IIa was dominated by accessions from northeastern India. This finding is

possibly due either to their derivation from the original seeds of wild Assam tea

plants, collected from the hills of Assam, Nagaland, Mizoram, Manipur, and

neighboring Burma, or to their selection from the highly heterogeneous population

derived from seeds collected from established commercial seed baris (Bezbaruah

and Dutta 1977). The three accessions TS-379, TS-449, and TS-464 were biclonal

seed stocks derived from control crosses of the most popular TV-1 with other high-

quality tea accessions; they therefore clustered together as a minor subgroup. TV-18

and TV-19 were derived from open-pollinated seeds of plants 4 and 14,

respectively, from the common stock 107, and hence grouped together. TV-13,

intermediate between the major subgroups IIa and IIb, was the single accession

collected from the seed nursery of Doomur Dullung Tea Estate. Other TV-series tea

accessions, although derived from the open pollination of common seed stock, were

scattered throughout subgroup IIa, confirming the heterogeneous nature of TV-

series clones reported by Mondal (2002).

All but five of the tea accessions representing major populations in southern India

were clustered into subgroup IIb. The popular TRI-series tea accessions (TRI-2024,

TRI-2025, TRI-2026, and TRI-2043) evolved from a single genetic stock (St 123) of

Tocklai. All but one of the TRI-series accessions clustered in a common internal

subgroup, along with biclonal seed stock BSS-1 and TTL-1. The clustering of

northeastern and southern tea accessions in a common major group confirms their

common ancestry and supports the suggestion that early planting materials of tea

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(China or Assam types) used in southern India and Sri Lanka might have been

derived from a similar gene pool (Singh 1979; Bezbaruah and Dutta 1977).

According to the molecular data, the five tea accessions C-17, NJ-6/24-T, ATK,

BSB, and TTL-2 are the most diverse, being intermediate between the cultivated

and related wild C. japonica. Similar results were reported in earlier AFLP studies

0.35 0.49 0.63 0.76 0.90

KangraAsha KangraJat BS-65 BS-1 BS-26 BS-46 BS-68 BS-14 BS-38 K-15 Teesta-Valley-1 CH-1 Teenali P463 P126 KM8 AV2 KM9 RYDAK1 HV39 CSIN-303536 BS-53 BS-54 TG-270/2/13 TV7HK22/14

HK23/19 LG17 LG26 TV9 T78 CB-38 T-3E/3 S-3A S-3 TV4 Nokrai-89 TV14 TV16 RR-17/144 TS379 TV1 TS449 TS464 TV3 TV12 S-3A TV2 TV5 TV8 TV15 TV6 CR-6017 TV23 TV22 TV19 TV18 TV21 TV26 TV28 TV25 TV29 TV27 TV30 TV24 SA-6 KP-6/25 BJ-2-T TV20 SS-40A TV17 TV10 P-492 TV13 UPASI-9 UPASI11 UPASI10 UPASI13 UPASI12 UPASI16 UPASI21 UPASI-8 UPASI15 UPASI-4 UPASI-5 UPASI-2 UPASI-7 UPASI-6 UPASI17 UPASI26 UPASI-1 UPASI27 UPASI20 UPASI14 UPASI19 SMP TRI2043 UPASI18 UPASI25 UPASI24NLT17/10

UPASI22 B/5/163 UPASI23 TRI2024 TRI2025 TRI2026 BSS-1 TTL1 UPASI-3 TRF1 BSB 1 CP-01 TRF2 AK K18 TES34 C17 NJ6/24-T ATK TTL2

CJ-1 CJ-3 CJ-2

63

97 100

86

97

73

65

80

70 83

64

72

73

63

61

89

69

92

Out group

II

IIb

IIa

I

69

72

72

62

66

67

Fig. 2 Genetic relationships of 123 tea accessions based on seven AFLP primer combinations. The scaleindicates Jaccard’s similarity coefficient. Bootstrap values greater than 60% are indicated at the branches

Biochem Genet (2010) 48:549–564 559

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(Balasaravanan et al. 2003). They are highly heterogeneous, and as such seem to be

potential sources for the genetic improvement of tea. The occurrence of a few highly

heterogeneous tea accessions might be due to hybridization between new

introductions and existing old accessions, including wild taxa of Camellia species,

which often grow side by side in the germplasm block. In general, the affinity of tea

accessions on a regional basis, as detected in this molecular study, might be due to

the occurrence of free hybridization within the available genetic base in a particular

region.

AFLP data in this study grouped three varietal types among cultivated accessions

according to geographic locations, having a bias toward either the China or the

Assam/Cambod type. In general, it was possible to separate two distinct tea

varieties, China and Assam. A few exceptions were expected, however, since the

existing classification is based solely on a few morphological characteristics that are

subject to substantial environmental influences (Wachira et al. 1995; Paul et al.

1997; Mondal 2002). Cultivation of northwestern tea is dominated by the China

type or China hybrids, clustering together in group I, along with other China

accessions from Darjeeling, Assam, and Japan. Tea cultivation in the northeast and

the south, with a few exceptions, is dominated by the Assam or Cambod types or

their hybrids, and most of those accessions clustered together in group II. Most of

the Cambod tea accessions formed minor subgroups in IIa. Traditionally, Cambod is

considered a subgroup of the Assam type or is sometimes referred to as a subspecies

of Assam (C. assamica ssp. lasiocalyx; Wight 1962); our data broadly support this

view. A few hybrids (China–Assam, China–Cambod, and Assam–China), however,

were intermingled in other groups. It is possible that these hybrids of China and

Assam types had morphological characters that predisposed them to identification as

either a China or an Assam type. A few of these hybrids clustered as intermediate

between cultivated and ornamentals, suggesting the possibility of free hybridization

between cultivated varieties of tea (Visser 1969). Another important observation

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C. japonica

NW tea populations; broadly China types

NE tea populations; broadly Assam (39.5 %) & Cambod (27 %) types S tea populations; Assam (51%) & Cambod

(22 %) types

25

Coordinate 1

1816

24

NW

NE

S

Fig. 3 Principal coordinate plot of 123 tea accessions based on AFLP data. Northwest accessions areindicated by diamonds, northeast by squares, and south by triangles. Accession numbers as in Table 1

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made in this study was the inclusion of UPASI-9 in the Assam group, consistent

with the conclusion made by Wachira et al. (1995) and Balasaravanan et al. (2003)

but inconsistent with the report of Mondal (2002), who designated UPASI-9 as a

China type. This study puts the controversy to rest by concluding that UPASI-9 is

more or less an Assam type and not a China type. Clustering of CJ-1, CJ-2, and CJ-3

of C. japonica as outgroups was expected and was validated by the molecular data.

Principal coordinate analysis was based on the similarity matrices generated with

AFLP markers; eigenvalues were extracted for two components and plotted against

each other (Fig. 3). The first two components accounted for more than 50% of the

variation. Major groupings were based on accessions from the northwest, northeast,

and south populations in direct relationships with China, Assam, or Cambod types,

somewhat similar to the UPGMA-based cluster analysis. Related C. japonicaaccessions clearly separated out from the cultivated tea accessions.

Extent of Genetic Variation

Overall genetic similarity (GS) among the 123 tea accessions ranged from 0.32 to

0.88, averaging 0.51. A slightly higher average (0.52) was recorded among the

released accessions, which ranged from 0.42 (TTL-2 and TV-20) to 0.88 (SS-40A

and TV-20). No significant difference was recorded in the average GS among the

tea populations cultivated in various geographic regions (0.60 in the northwest and

0.59 in both the northeast and south). The clustering pattern, however, suggested

that existing tea populations of northeastern and southern India are derived from a

single regional genetic background (Wood and Barua 1958; Cannel et al. 1977). The

high level of heterozygosity recorded in this study possibly provides the required

genetic plasticity essential for the survival of the crop in diverse climatic conditions

(Singh et al. 2006). Among the populations, the most diverse were BS-54 and BS-53

in the northwest (GS 0.48), TV-13 in the northeast (0.52), and TTL-2 in the south

(0.43). Somewhat similar genetic diversity patterns were also recorded in earlier

studies, irrespective of molecular marker techniques (Wachira et al. 1995, 2001;

Paul et al. 1997).

This study revealed considerable diversity among the tea accessions having

quality and tolerance of or resistance to various biotic (blister blight) and abiotic

stresses (drought, waterlogging, frost). Among the tea accessions associated with

trait attributes, SMP, TRI-2043, UPASI-19, SA-6, and BS-26 (average GS 0.56) are

resistant to blister blight; UPASI-26 and UPASI-27 (0.62) are resistant to frost;

UPASI-1, UPASI-10, UPASI-26, and CH-1 (0.53) are tolerant to wind; TV-9 is

tolerant to waterlogging; and 26 accessions (0.57) are tolerant to drought. Overall,

44% genetic diversity was recorded in these accessions. Since these accessions are

Table 3 Analysis of molecular variance of 123 tea accessions

Source of genetic variation df Sum of squares Variance component P value % variation

Among populations 2 527.515 5.824 \0.010 15

Within populations 120 3926.687 32.452 \0.010 85

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highly heterogeneous and were clustered in different groups and subgroups, they are

potential parental groups for recombination of these traits in quality tea accessions

through controlled crossing in future tea improvement programs.

Partitioning of genetic diversity indicates the existence of large-scale variation

within populations. These findings were also confirmed by AMOVA, which

detected a high level of genetic variation (85%) within populations, as well as

limited genetic variation (15%) among populations (Table 3). A further explanation

for these genetic diversity results could be that tea populations cultivated at the

national level are derived from a similar gene pool and share a common genetic

background. Lack of genetic differentiation among the populations has also been

reported in earlier studies of tea (Wachira et al. 1995, 2001; Paul et al. 1997; Yao

et al. 2008; Ohsako et al. 2008). Exchange of genetic material in cultivated crop

species in most of the cases is dictated more by human intervention than by natural

factors. A similar genetic diversity trend was recorded in earlier studies, irrespective

of the various molecular markers (Wachira et al. 2001; Paul et al. 1997).

In conclusion, this study demonstrates for the first time the extent of genetic

diversity in a large collection of commercially important Indian tea germplasm

representing major populations at the national level. The findings of this study can

be a good source of molecular information for future tea breeders because it

eliminates the limitations and shortcomings of selection methodology based on

morphological, biochemical, and physiological data, which are easily influenced by

environmental conditions and the age of the plant. Detailed information about the

existing genetic diversity among standard, quality, and high-yielding tea accessions,

along with trait attributes, would be useful in proper identification, management,

utilization, and strategizing in future tea breeding programs at the regional and

national levels. A low level of genetic variation among populations indicates an

urgent need for diversification and exchange of tea genetic resources at the

international level.

Acknowledgments A financial grant received from the Department of Biotechnology and the

Council of Scientific and Industrial Research, Government of India, is gratefully acknowledged. We

thank Dr. S. Rajkumar for providing help in bootstrap analysis and AMOVA. This is IHBT Publication 0649.

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AFLP-Based Genetic Diversity Assessment of Commercially Important Tea Germplasm in India

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