Botanical Studies (2006) 47: 223-229.
*
Corresponding author: E-mail: hanyaoz@163.com.
INTRODUCTION
China is the world¡¦s largest producer and consumer of
tobacco. Flue-cured tobacco (Nicotiana tabacum L.) is one
of the most important commercial crops in the world. The
results of genetic diversity study provide estimates on the
level of genetic variation among diverse materials that can
be used in germplasm management, varietal protection,
and flue-cured tobacco improvement.
Morphological, karyotypical, and physiological
characters have already been used to study the genetic
diversity of flue-cured tobacco germplasm (Goodspeed,
1945; Zhang, 1994; Lei et al., 1997; Lu, 1997). However,
morphological characters usually vary with environments.
The number of karyotypical characters is limited, and the
study of genotypic diversity based on isozyme variation is
restricted to a few polymorphic enzyme systems encoded
by a small number of loci (Lu, 1997). These methods
have been improved greatly by new molecular marker
techniques such as simple sequence length polymorphism
(SSLP), restriction fragment length polymorphism
(RFLP), randomly amplified polymorphic DNA (RAPD),
sequence characterized amplified regions (SCAR),
and amplified fragment length polymorphism (AFLP)
(Botstein et al., 1980; Jarman and Wells, 1989; Williams
et al., 1990; Williams et al., 1991; Vos et al., 1995).
The AFLP technique allows the identification of a
greater number of polymorphisms than RFLP or RAPD
analysis. This technique is easy to perform compared to
RFLP, reproducible, and requires only small amounts
of DNA. Furthermore, it is a reliable and efficient DNA
marker system that has been extensively used for genetic
diversity study in different plant species (Maughan et
al., 1996; Ellis et al., 1997; Breyne et al., 1999; Erschadi
et al., 2000; De Riek et al., 2001). We employed the
Amplified Fragment Length DNA Polymorphism (AFLP)
technique to clarify the genetic relationships between
51 distinct flue-cured tobacco accessions with desirable
agronomic characteristics from the germplasm collections
of the South China Tobacco Breeding Research Center.
MOLECULAR BIOLOGY
Genetic diversity among flue-cured tobacco (Nicotiana
tabacum L.) revealed by amplified fragment length
polymorphism
Han-Yao ZHANG
1,2,
*, Xiao-Zhen LIU
1
, Tong-Sen LI
1
, and Yu-Ming YANG
1
1
Department of Forestry, Southwest Forestry College, White Dragon Temple, Kunming, Yunnan Province 650224, People's
Republic of China
2
College of Agronomy, South China University of Tropic Agriculture, Dangzhao, Hainan 571737, People¡¦s Republic of
China
(Received August 9, 2004; Accepted January 13, 2005)
ABSTRACT.
Flue-cured tobacco (Nicotiana tabacum L.) is one of the most important commercial
crops in the world. Genetic diversity studies provide estimates on the level of genetic variation among
diverse materials that can be used in germplasm management, varietal protection, and flue-cured tobacco
improvement. Amplified fragment length polymorphism (AFLP) analysis of 51 flue-cured tobacco cultivars
produced a tota l of 1479 unambiguous DNA fragments. Cluster a nalyses using the unweighted pair group
method with arithmetic mean (UPGMA) showed that the cultivars could be grouped into American or
Chinese types, with the Chinese types being further clustered into four subgroups and American ones into
two subgroups. The average pairwise genetic distance was 0.167 and ranged from 0.024 to 0.267. AMOVA
analysis showed that 55.76% of genetic variation came from cultivars having different origins and 44.24%
from cultivars having the same origin. The overall average Fst of the 51 accessions was 0.177. Mean Fst
of each accession against the rest ranged from 0.152 to 0.238. The findings of this study revealed that the
present day commonly grown flue-cured tobacco germplasm has narrow genetic diversity among the cultivars,
necessitating a sustained effort to preserve flue-cured tobacco germplasm resources. Further crosses should be
made only with genetically distant varieties.
Keywords: AFLP analysis; AMOVA; Flue-cured tobacco; Genetic diversity; UPGMA.
pg_0002
224
Botanical Studies, Vol. 47, 2006
MATERIALS AND METHODS
Plant materials
Seeds of 51 accessions of tobacco were obtained
from the germplasm collections of the South China
Tobacco Breeding Research Center in Yunnan Province,
in southwestern China. The collection consists of 772
accessions from China and 12 foreign countries. On the
basis of results from field trials conducted at Yuxi, Yunnan,
from 1994 to 1996 (Lei et al., 1997), 51 accessions, were
mainly from China and America, with desirable agronomic
characteristics such as high leaf yield, low nicotine
content, and resistance to various diseases or insects for
this study. These accessions represented genotypes likely
to be used in future flue-cured tobacco breeding efforts
in south China. The names and origin of the cultivars are
shown in Table 1.
Seeds were planted in pots in a greenhouse at
temperatures of 28 to 32¢XC. Twenty days after
germination, shoots were harvested from 40 seedlings of
each accession representing the cultivar.
DNA extraction
DNA was extracted from shoots by the CTAB method
(De Riek et al., 2001). DNA concentration and quality
was estimated spectrophotometrically by measuring
absorbance at 260 nm and by visual comparison with
£fDNA standards of known concentration on ethidium
bromide (EtBr) stained agarose gels. DNA samples were
diluted in TE-buffer and maintained at -20¢XC.
AFLP analysis
The AFLP analysis was performed following the
manufacturer¡¦s protocol (Life Technologies). The DNA
was digested simultaneously with restriction enzymes
EcoRI and MseI. The selective amplifications were
performed using the primer pairs listed in Table 2.
Restricted genomic DNA fragments were ligated to EcoRI
and MseI adapters. Primers with EcoRI set included the
sequence 5.- GAC TGC GTA CCA ATT C and the primers
of MseI set had the sequence 5.-GAT GAG TCC TGA
GTA A. The pre and the selective amplifications were
performed in a 2400 Perkin-Elmer Thermocycler. An
equal volume (2 £gL) of loading dye (95% v/v formamide
and 0.08% w/v bromophenol blue in 20 mM EDTA)
was added to each sample, which was then denatured at
95¢XC for 3 min and placed on ice for 2 min before loading.
Amplification products were analyzed by electrophoresis
in a 6.5% polyacrylamide gel. The electrophoresis
parameters were set to 1500 V, 40.0 mA, 40.0 W,
50¢XC, and the run time was set to 2.0 h. Separated AFLP
products were visualized using silver staining (Fritz et al.,
1999).
Data analysis
Each accession was scored 1 for presence or 0 for the
absence of a band. Only bright, clearly distinguishable
bands were used in the genetic analysis. Pairwise
similarity matrices were generated using Jaccard¡¦s
coefficient of similarity. A dendrogram was generated with
the unweighted pair-group method with arithmetic average
(UPGMA) algorithm as described by Sneath and Sokal
(1973). The distance coefficient used for analysis was
Nei¡¦s coefficient. The procedures above were performed
using NTSYS-pc, Version 2.1. AMOVA and Fst
calculations were performed with the software package
Arlequin (Schneider et al., 2000).
RESULTS AND DISCUSSION
Twenty-two selective primer pairs were screened
against all 51 accessions. Four primer pairs were
not included in the final analysis because either the
amplification profile was consistently too faint to score
accurately (E-AAC/M-CGC) or only monomorphic
amplification products were produced (E-ACG/M-CTG,
E-AGC/M-CTC, E-ACT/M-CTG). The eighteen
informative primer pairs used in the final analysis were
listed in Table 2. Two (E-AAC+M-CTA) to thirty-two
polymophic bands (E-ACA+M-CTA) of variable lengths
were detected (Table 2), and the polymorphic rate ranged
from 2.56% (E-AAC+M-CTA) to 37.10% (E-ACG+M-
CAA). Three primer pairs were smaller than 10%; nine
primer pairs ranged from 10% to 20%; three primer pairs
Figure 1. Dendrogram of the flue-cured tobacco cultivars using
AFLP analysis. Symbols indicate country of origin: ¡» = China;
¡½ = USA; ¡¿ = Brazil. Symbols indicate ancestry of origin: ¡´
= Speight G-28; ¡¹=K326.
pg_0003
ZHANG et al. ¡X AFLP in flue-cured tobacco cultivars
225
Table 1. 51 Flue-cured tobacco cultivars used in AFLP analysis.
No. Cultivar
Pedigree
Origin
1 Hubei 517
NC2326
China
2 581
Chujingyan
China
3 Yunyan 3
Zhaojie 8 dui¡ÑCoker347
China
4 Zhongyan 15
Danyu 2¡ÑSpeight G-28
China
5 Yunnanduoyeyan Dajingyuan
China
6 Yongding 1
Special 401
China
7 Baofong
(401-2¡ÑG-80)¡ÑG-80
China
8 Yanyan 97
(401-2¡ÑG-80)¡ÑG-80
China
9 82-3041
G-28¡ÑBurley599
China
10 Zhongyan 14
Jingxing6007¡ÑSpeight G-28
China
11 CV85
(CV58¡ÑSpeight G-28)¡Ñ[CV58¡Ñ(G-28¡ÑNC82)F1]
China
12 Yunyan 84
Yunyan 2¡ÑK326
China
13 Yunyan 85
Yunyan 2¡ÑK326
China
14 Bicui 1
401
China
15 Gexin 5
Dahuangjing
China
16 77809-12
(Lingi 1¡ÑVirginia 115)F6
China
17 Yanbianzi
Unknown
China
18 Xiaohuangjing 1025 Xiaohuangjing
China
19 311
Yunyan 4¡ÑK329
China
20 Changbohuang
Unknown
China
21 Baoshangtuanyeyan Unknown
China
22 Jiyan 5
Jingyehuang¡ÑCoker86
China
23 NCTG 55
K326¡ÑCoker 371-Gold
America
24 G-23
Unknown
America
25 Liaoyan 9
(5203¡ÑKy 56)F1¡Ñ(5637¡ÑBeiyu 29)F2
China
2 6
Special 400
Orinoco
America
27 RGH51
Unknown
Brazil
28 PVH08
Unknown
Brazil
29 CU 236
[(MC944¡ÑTI170) ¡ÑMC944]¡ÑK326
America
30 NC 82
6129¡ÑCoker319
America
31 RG11
NC50¡ÑK399
America
32 Oxford 1
[(Florida 301¡ÑVirginia Bright Leaf) ¡ÑVirginia Bright Leaf] ¡ÑVirginia Bright Leaf America
33 Reams44
Coker319¡ÑHicks
America
34 Coker213
Coker319¡ÑCoker139
America
35 K149
[G-28¡ÑCoker254]¡Ñ(CB139¡ÑF-105)¡Ñ(G-28¡ÑCoker254)]¡Ñ McN399
America
36 Mc Nair 373
(Coker319¡ÑCoker139) ¡Ñ Mc Nair30
America
37 Bell 93
Bell 15¡ÑCoker 187-Hicks
America
38 V2
Unknown
America
39 Vesta47
Oxford¡ÑYellow Special
America
40 Coker 371 Gold
[(G-28¡Ñ354)¡Ñ(CB139¡ÑF-105)¡Ñ(G-28¡Ñ354)]¡ÑNC82
America
41 K346
K326¡Ñ80241
America
42 Dixie Bright101 [(TI448A¡Ñ400)F3¡ÑOxford]¡Ñ(Florida301¡Ñ4008)
America
43 Guanghuang 55
Jingxing6007¡ÑDixie Bright 101
China
44 Hicks
White Stem Orinoco
America
45 Yunyan 1
Gold Dollar
China
46 Yunyan 2
Red Flowers Gold Dollar¡ÑSpeight G-28
China
47 Speight G-28
(Oxford 1-181¡ÑCoker 139)F4¡ÑNC95
America
48 CV87
(CV58¡ÑSpeight G-28)¡Ñ[CV58¡Ñ(G-28¡ÑNC82)F1]
China
49 CV73
(CV58¡ÑSpeight G-28)¡Ñ[CV58¡Ñ(G-28¡ÑNC82)F1]
China
50 K326
McNair 225¡Ñ(McNair 30¡ÑNC95)
America
51 Yunyan 86
Yunyan 2¡ÑK326
China
pg_0004
226
Botanical Studies, Vol. 47, 2006
ranged from 20% to 30%; and three primer pairs ranged
from 30% to 40% (Table 2). Of course, the use of primer
pairs selected for reproduction of higher polymorphism
in the target group of genotypes could further increase
the efficiency and the applications of the AFLP approach
while the genetic loci which were invariant are also
important for their potential to detect polymorphism in
other flue-cured tobacco genotypes.
Fingerprinting revealed a total number of 1479
unambiguous DNA fragments with an average of
13.83 polymorphic loci per primer pair. The average
polymorphism rate was 16.84%. Using these data, a
dendrogram showing the relationships among the 51
accessions was created using NTSYS 2.1. Two distinct
clusters were apparent in the dendrogram produced
by cluster analysis (Figure 1). Group A consisted 21
accessions (41.18%), and group B included 29 accessions
(56.86%). Interestingly, accession PVH08, a Brazilian
cultivar, did not cluster with either of the groups.
The genetic variation among the 51 accessions was
estimated using a pair-wise comparison of genetic
distance. The average pairwise genetic distance was 0.167
and ranged from 0.024 to 0.267. 25.65% of them ranged
from 0.10 to 0.15; 56.35% of them ranged from 0.15 to
0.20; and 14.37% of them ranged from 0.20 to 0.25. The
results also showed that most of them (96.37%) ranged
from 0.10 to 0.25, and only about 15.73% of the pair-
wise comparisons among accessions exhibited a genetic
distance greater than 0.20 (Figure 2). The most closely
related cultivars, Yunyan 2, Yunyan 1, Yanyan 97, and
Baofong, shared a genetic distance of 0.024. The least
related, PVH08 and Bicui 1, had a genetic distance of
0.267. When the genetic variation of the cultivars was
partitioned by AMOVA, 55.76% of the variation was
found among the cultivars that had different geographic
origins while 44.24% was found among the ones that had
the same geographic origin. Both of the variation figures,
within and among the origins, were highly significant (P <
0.001) (Table 3).
Mean Fst of each accession against the rest were shown
in Figure 3. The overall average Fst of the 51 accessions
was 0.177. In 29 Chinese cultivars, mean Fst of each
accession against the rest ranged from 0.152 to 0.198,
and the mean was 0.164. Those in American cultivars
were 0.174 (mean), 0.152 (min.), and 0.193 (max.). In
Brazil cultivars, they were 0.208 (mean), 0.178 (min.)
and 0.238 (max.).The means were largest in Brazilian
cultivars and smallest in Chinese cultivars. Low value of
mean Fst revealed narrow genetic diversity, reflecting the
consequence of inbreeding from a limited gene pool.
Table 2. The number of bands and degree of polymorphism
revealed by AFLP primer combinations.
Primer
combinations
Total
bands
Polymorphic
bands
Polymorphic
rate (%)
E-ACG+M-CAA 62
23
37.10
E-AAC+M-CTA
78
2
2.56
E-AGC+M-CAC 85
13
15.29
E-AGG+M-CAG 81
9
11.11
E-ACA+M-CTA 113
32
28.32
E-AAG+M-CTG 77
8
10.39
E-AGC+M-CAA 94
21
22.34
E-AGG+M-CTT 86
14
16.28
E-AGG+M-CTA
57
21
36.84
E-ACT+M-CAC 102
12
11.76
E-AAC+M-CTG
91
20
21.98
E-ACA+M-CAC 86
15
17.44
E-ACA+M-CTT
74
11
14.86
E-AAG+M-CTC 89
3
3.37
E-ACT+M-CTC
67
8
11.94
E-AAG+M-CAG 86
12
13.95
E-ACG+M-CTA
93
4
4.30
E-ACG+M-CAG 58
21
36.21
Total
1479
249
Average
82.17 13.83
16.84
Figu re 2. Distribution of pair-wise comparison of genetic
similarity among tobacco cultivars.
Table 3. AMOVA among and within geographic origins of the cultivars.
Source of variation
d.f. Sum of squares
Variance components Percentage P-value
Among origin places
2
109.687
12.38099
55.76
¡Õ0.001
Within origin places
48
471.50016
9.82292
44.24
¡Õ0.001
pg_0005
ZHANG et al. ¡X AFLP in flue-cured tobacco cultivars
227
Characterization and quantification of genetic diversity
has long been a major goal in breeding. In plant breeding
programs, information on genetic diversity is essential for
a rational use of genetic resources. It is particularly useful
in characterizing individual accessions and cultivars, in
detecting duplications of genetic materials in germplasm
collections, and as a general guide in selecting parents
for hybridisation in breeding programs and in developing
informative mapping populations for genome mapping.
Thus those more polymorphic primer pairs¡Xsuch as
E-ACG+M-CAA, E-AGC+ M-CAA, E-ACA+M-CTA,
E-AGG+M-CTA and E-ACG+M-CAG¡Xwould be very
useful in a flue-cured tobacco breeding program. In a study
of wheat cultivars, Barrett and Kidwell (1998) detected
2-31 polymorphic bands per primer pair, with a mean
polymorphic rate of 11.8%. Studies on rice cytoplasmic
male-sterile (CMS) lines showed 8-23 polymorphic bands
per primer pair, with an average of 16 polymorphisms
(Subudhi et al., 1998). In Texas bluegrass genotypes
Renganayaki et al. (2001) detected 3-116 polymorphisms
per primer pair, with an average polymorphic rate of
64.11%. In this study, 2-32 polymophic bands per primer
pair were detected, with an average polymorphic rate of
16.84%.
AMOVA results showed that most of the variations
(55.76%) were found among the cultivars of different
geographic origins. The dendrogram indicated a pattern
of division among the flue-cured tobacco accessions
based on geographic origin, as seen in some other crops
(Spooner et al., 1996; Paul et al., 1997). The collection of
American accessions was in cluster A, together with one
Brazilian cultivar, RGH 51 and two Chinese cultivars.
Chinese cultivars grouped together with two American
cultivars and formed cluster B. The cultivars originated
from different countries clustered together and shared the
same ancestors. This can well be explained by the fact that
lot of Chinese and Brazil cultivars were used to breed the
American cultivars (Wang and Zhou, 1995). In general,
Chinese cultivars clustered into four sub groups, and
American cultivars clustered into two sub groups, with no
clear pattern of division. Clearly, those shared common
ancestry clustered together. For example, the cultivars
bred by Speight G-28 such as 82-3041, Zhongyan 14,
CV73, CV85 and CV87 clustered together in cluster BIII.
Similarly, the cultivars, Yunyan 84, Yunyan 85 and Yunyan
86, sharing the same crossing parent K326, clustered
together in cluster BIV.
Genetically more diverse genotypes may have good
breeding value. PVH08, a Brazilian cultivar which did
not cluster with any American or Chinese cultivars was
the diverse one among all the genotypes used in the
study. PVH08, V2, K346, and Guanghuang 55 were the
genetically most distinct accessions. The mean Fst of these
accessions was greater than 0.190, and these accessions
would certainly contribute to the breeding program (Figure
3). Genotypes in the same cluster may represent members
of one heterotic group. They displayed a similar DNA
fingerprint. For example, Reams 44 and Coker 213 sharing
common parent Coker 319 were clustered together in the
cluster AII. A closer relationship was detected between
the cultivars Baofong and Yanyan 97. Both cultivars
have similar morphological traits and originated from
the same parents (Lei et al., 1997). The results showed
that AFLP assay could identify the flue-cure tobacco
cultivars possessing similar genetic background. The
availability of large numbers of polymorphic fragments
enables the efficient evaluation of genetic diversity. The
AFLP fingerprinting technique utilized in this paper has
demonstrated that this powerful tool can be valuable in the
breeding program of flue-cured tobacco.
Low level of pairwise genetic distance and mean Fst
suggested the existence of limited genetic variation in
flue-cured tobacco cultivars. The existence of low genetic
diversity within cultivated flue-cured tobacco has been
attributed to self-pollination (Wang and Zhou, 1995).
A low DNA polymorphism level was also reported in
several other self-pollinating plants such as wheat (Joshi
and Nguyen, 1993), pigeonpea (Ratnaparkhe et al., 1995),
tomato (Williams and St. Clair, 1993), and coffee (Steiger
et al., 2002). It is also possible that a large proportion
of valuable flue-cured tobacco germplasm may already
have been lost through the popularity of certain cultivars
in commercial planting and the continuous artificial
selection. To avoid further degradation of germplasm
resources, crosses should be made with genetically distant
varieties or genotypes of diverse origin.
Figure 3. Mean Fst of each cultivar against the rest.
pg_0006
228
Botanical Studies, Vol. 47, 2006
Acknowledgements. The authors thank Ms. M.L. Xu
(South China Tobacco Breeding Research Center) for
providing seeds of the various flue-cured tobacco cultivars
used in this study. We also thank Dr. H.B. Zhang (New
Zealand Royal Institute for Crop and Food Research),
and Dr. C.Z. He,Y.B. Sha and Z.Z. Zhou (Laboratory of
Plant Biotechnology, Institute of Microbiology, Chinese
Academy of Sciences) for their critical review of the
manuscript.
LITERATURE CITED
Barrett, B.A. and K.K. Kidwell. 1998. AF LP-Based genetic
divers ity as ses sm ent am ong whe at culti vars from the
Pacific Northwest. Crop. Sci. 38: 1261-1271.
Botstein, B., R.L. White, M. Skolnick, and R.W. Davis. 1980.
Cons truc tio n of a ge neti c li nkage ma p in m an us in g
restriction fragment length polymorphisms. Am. J. Hum.
Genet. 32: 314-331.
Breyne, P., D. Rombaut, A. Van Gysel, M. Van Montagu, and T.
Greats. 1999. AFLP analysis of genetic diversity within and
between Arabidopsis thaliana ecotypes. Mol. Gen. Genet.
261: 627-634.
De Riek, J., E. Calsyn, I. Everaert, E. Van Bockstaele, and
M. De L oose . 2001. AF LP ba se d a lte rnati ves fo r t he
assessment of distinctness, uniformity and stability of sugar
beet varieties. Theor. Appl. Genet. 103: 1254-1265.
Ellis , R.P., J.W. McNicol, E. Baird, A. Booth, P. Lawrence,
B. Thomas, and W. Powell. 1997. The use of AFLP s to
examine geneti c relatedness in barley. Mol. Breed. 3:
359-369.
Erschadi, S., G. Haberer, M. Schoniger, and R.A. Torres-Ruiz.
2000. Estimating genetic diversity of Arabidopsis thaliana
ecotypes with amplified fragment length polymorphism
(AFLP). Theor. Appl. Genet. 100: 633-640.
Fritz, A.K., S. Caldwell, and W.D. Worall. 1999. Molecular
mapping of Russian Wheat aphid resistance from triticale
accession PI 386156. Crop. Sci. 39: 1707-1710.
Goodspeed, T.H. 1945. Chromosome number and morphology
in Nicotana. VII. Karyotypes of fifty-five species in relation
to a taxonomic revision of the genus. Univ. Calif. Publ. Bot.
18: 345-370.
Jarman, A.P. and R.A. Wells. 1989. Hypervariable minisatellites:
recombinators or innocent bystanders. Trends. Genet. 5:
367-371.
Joshi, C.P. and H.T. Nguyen. 1993. RAPD (random amplified
polymorphic DNA) analysis based on intervarietal genetic
relations hips among hexaploid wheats. P lant. S ci. 93:
95-103.
Lei, Y.H., M.L. Xu, and X.Y. Huang. 1997. Tobacco Collection
in the Yunnan Province. Science and Technology publishing
company of Yunnan, Yunnan, pp. 1-20.
Lu, J .P. 1997. The Applic ati on of PAGE in t he Cul tivars
Identification of Flue-cured Tobacco. Seed 5: 30-32.
Maughan, P.J ., M.A. Saghai Maroof, G.R. Bus s, and G.M.
Huestis. 1996. Amplified fragment polymorphism (AFLP)
in soybean: species diversity, inheritance, and near-isogenic
line analysis. Theor. Appl. Genet. 93: 392-401.
P aul, S ., F.N. Wac hira , W. P owell , a nd R. Waugh. 1997.
Diversity and genetic differentiation among populations of
Indian and Kenyan tea (Camellia sinensis (L.) O. Kuntze)
revealed by AFLP markers. Theor. Appl. Genet. 94:
255-263.
Ratnaparkhe, M.B., V.S. Gupta, M.R. Murthy Ven, and P.K.
Ra nje kar. 1995 . Gene tic fig erpri nti ng of p ige onpea
[Cajanus cajan (L) Millsp.] and its wild relatives using
RAPD markers. Theor. Appl. Genet. 91: 893-898.
Renganayaki, K., J.C. Read, and A.K. F ritz. 2001. Genetic
diversity among Texas bluegrass genotypes (P o a
arachnifera Torr.) revealed by AFLP and RAPD markers.
Theor. Appl. Genet. 102: 1037-1045.
Schneider, S., D. Roessli, and L. Excoffier. 2000. Arlequin ver.
2.000: a Software for Population Genetic Da ta Analy -
s is. Genetics and Biome try Laboratory, Univers ity of
Geneva, Geneva, Switzerland. (Available at URL http://
anthropologie.unige.ch/arlequin/).
Sneath, P.H.A. and R.R. S okal. 1973. Numerical Taxonomy.
Freeman, San Francisco.
Spooner, D.M., J. Tivang, J. Neinhuis, J.T. Miller, D.S. Douches,
and A. Contreras-m. 1996. Comparison of four molecular
markers in measuring relations hips among wild potato
relatives Solanum section etuberosum (subgenus Potatoe).
Theor. Appl. Genet. 92: 532-540.
S tei ger, D.L ., C . Na gai, P.H. Moore , C .W. Morden, R.V.
Osgood, and R. Ming. 2002. AFLP analysis of genetic
diversity within and among Coffea arabica cultivars. Theor.
Appl. Genet. 105: 209-215.
Subudhi, P.K., S. Nandi, C. Casal, S.S. Virmani, and N. Huang.
1998. Classification of rice germplasm: III. High-resolution
fingerprinting of cytoplasmic genetic male-s terile (CMS)
lines with AFLP. Theor. Appl. Genet. 96: 941-949.
Vos, P., R. Hogers, M. Bleeker, M. Reijans , T. van de Lee,
M. Hornes , A. F rij ters , J. Pot, J . P elem an, M. Kuiper,
and M. Zabeau. 1995. AFLP: a new technique for DNA
fingerprinting. Nucl. Acids. Res. 23: 4407-4414.
Williams, C.E. and D.A. St. Clair. 1993. Phenetic relationships
and level of variability detected by restriction fragment
length polymorphism and random amplified polymorphic
DNA a nal ys i s of c ult iv at ed and wil d a cc es s i ons of
Lycopersicon esculentum. Genome 36: 619-630.
Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski, and
S.V. Tingey. 1990. DNA polymorphism s am pli fied by
arbitrary primers are useful as genetic markers. Nucl. Acids.
Res. 18: 6531-6535.
Williams, M.N.V., N. Pande, S. Nair, M. Mohan, and J. Bennett.
1991. Restriction fragment length polymorphism analysis
of polymeras e chain reaction products amplified from
mapped loci of rice (Oryza sativa L.) genomic DNA. Theor.
Appl. Genet. 82: 489-498.
pg_0007
ZHANG et al. ¡X AFLP in flue-cured tobacco cultivars
229
Wang, Y.Y. and J. Zhou. 1995. Blood Relationship Analysis and
Breeding Procedures Comparis on between Chines e and
American Main Tobacco Cultivars. Chinese Tobacco Sin. 2:
11-22.
Zhang, D.M. 1994. Study on S urface S tructure Scanning of
S ome Tobacco S eeds with Electrical-glas s Obs erving.
Chinese Tobacco Sin. 2: 12-15.
pg_0008