Euphytica 119: 335–341, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

335

Analysis of genetic diversity in Agave tequilana var. Azul using RAPD

markers

Katia Gil Vega1 , Mario González Chavira2 , Octavio Martı́nez de la Vega2, June Simpson2 &
George Vandemark∗
1 Departamento de Biotecnologı́a y Bioquı́mica, CINVESTAV del IPN Apdo. Postal 629, Irapuato, Gto., Mexico
36500; 2 Departamento de Ingenerı́a Genética, CINVESTAV del IPN Apdo. Postal 629, Irapuato, Gto. Mexico,
36500; ( ∗ author for correspondence: U.S. Department of Agriculture-Agricultural Research Service, 24106 N.
Bunn Road, Prosser, WA 99350, U.S.A.)

Received 6 April 2000; accepted 11 August 2000

Key words: Agave tequilana, genetic diversity, RAPDs, tequila

Summary
By federal law in Mexico, A. tequilana Weber var. Azul is the only variety of agave permitted for the production
of any tequila. Our objective was to assay levels of genetic variation in field populations of A. tequilana var.
Azul using randomly amplified polymorphic DNA (RAPD) markers. Ten plants were collected from each of four
different fields, with two fields being located in each of two principal regions of Mexico for the cultivation of A.
tequilana var. Azul. The two regions are separated geographically by approximately 100km. Genetic relationships
between A. tequilana var. Azul and two other varieties of A. tequilana Weber, ‘Chato’ and ‘Siguin’, were also
investigated using RAPDs. Among the three varieties, 19 decamer primers produced 130 markers, of which 20
(15.4%) were polymorphic between A. tequilana var. Chato and A. tequilana var. Siguin. The results of RAPD
analysis suggest that A. tequilana var. Siguin is more closely related to A. tequilana var. Azul than is A. tequilana
var. Chato. Among the 40 field selections of A. tequilana var. Azul, only 1 of 124 RAPD products (0.8%) was
polymorphic and 39 of 40 plants were completely isogenic. This is one of the lowest levels of polymorphism
detected to date for the analysis of a crop species, and is proposed to be the result of the promotion of a single
conserved genotype over many years due to an exclusive reliance on vegetative propagation for the production of
new planting materials. The significance of these results is discussed in relation to breeding programs focused on
the improvement of A. tequilana var. Azul.

Introduction ively produced vegetatively from ramets that emerge

The center of origin of the genus Agave is located
in Mexico (Gentry, 1982), where alcoholic bever-
ages have been produced through the fermentation
of Agave sp. for over 750 years (Ramírez & López,
1985). Tequila, the most popular contemporary bever-
age made from agave, is an important cultural icon
of the nation of Mexico, and is also its most widely
recognized export product. The blue agave, Agave
tequilana Weber var. Azul, is the only variety of agave
permitted by federal law in Mexico to be used for
the production of tequila (Diario Official, 1993). New
planting materials of A. tequilana var. Azul are exclus-
from the end of rhizomes, a practice that has contin-
ued without interruption for the last 200 years (Nobel,
1994; Valenzuela, 1997). Plants begin to flower at
between 7–9 yr of age under conditions of field pro-
duction and the inflorescence is cut off the plant once
it reaches a height of about 1 m. Six months later the
mature crown is harvested for use in the production of
tequila.
In Mexico, approximately 55,000 ha are dedicated
to the cultivation of A. tequilana var. Azul, with the
most of the productive area being located in the state
of Jalisco (Valenzuela, 1997). Federal law restricts the
336
Figure 1. Map of the state of Jalisco showing the locations of the
four field sites surveyed in this analysis. OR = Orendain; TC =
Tequila Cuervo; SD = Santo Domingo and SJ = San Juan.
cultivation of the variety to specific localities within
the country (Diario Official, 1993). Two regions of
Jalisco are each responsible for approximately 25% of
the total annual production (Valenzuela, 1997). The
first region is centered around the town of Tequila, ap-
proximately 50 km to the west of the city of Guadala-
jara, and the second region, collectively known as ‘Los
Altos de Jalisco’, is located approximately 70 km to
the southeast of Guadalajara (Figure 1).
Legislative restrictions, on both the permissible
areas of cultivation of A. tequilana var. Azul and
the exclusivity of this variety for the production of
tequila, have promoted interest in the development of
higher yielding genotypes of the variety. The timely
production of improved genotypes will be largely de-
pendent on the degree of genetic diversity present in
the base population of breeding materials. A common
approach for assessing levels of genetic diversity is
the use of molecular markers such as randomly amp-
lified polymorphic DNA (RAPDs) (Williams et al.,
1990). RAPDs have previously been used to assess
levels of genetic diversity and phylogenetic relation-
ships in a wide range of clonally propagated plant
species including garlic (Allium sativum) (Al-Zahim
et al., 1997), grapevine (Vitis vinifera) (This et al.,
1997), strawberry (Fragaria × ananassa) (Degani et
al., 1998) and sugarcane (Saccharum spp.) (Nair et al.,
1999).
Phylogenetic relationships both between and
within taxa in the family Agavaceae have been previ-
ously examined using different molecular techniques.
These include restriction fragment length polymorph-
isms of chloroplast DNA (Bolger & Simpson, 1995),
sequence variations of internally transcribed spacer
(ITS) regions of nuclear genes coding for ribosomal
DNA (Bolger & Simpson ,1996), variations in the se-
quence of the chloroplast rbcL gene (Eguiarte et al.,
1994), isozyme analysis (Massey & Hamrick, 1998;
Massey & Hamrick, 1999; Martínez-Palacios et al.,
1999; Colunga-GarcíaMarín et al., 1999), and RAPDs
(Trame et al., 1995).
Our objective was to use RAPDs for evaluating ge-
netic diversity among plants of A. tequilana var. Azul
collected from several different commercial fields loc-
ated in the Mexican state of Jalisco. RAPDs were
also employed to investigate intraspecific relationships
between A. tequilana var. Azul and two other vari-
eties of A. tequilana; A. tequilana var. Chato and A.
tequilana var. Siguin.
Materials and methods
Plant material
Ten plants of A. tequilana var. Azul were collected
at random from each of four production fields. Two
of the fields, referred to in this text as ‘San Juan
(SJ)’ and ‘Santo Domingo (SD)’, were located to the
southeast of Guadalajara, in the region known as ‘Los
Altos de Jalisco’. The two other fields, referred to
as ‘Tequila Cuervo (TC)’ and ‘Orendain (OR)’, were
both located near the town of Tequila, to the west of
Guadalajara (Figure 1). These 4 fields represent two
different commercial producers of tequila with Tequila
Cuervo S.A. de C.V. being the owners of fields SD,
SJ, and TC, while Orendain S.A. de C.V. are the own-
ers of field OR. Single plants were also obtained of
A. tequilana var. Chato and A. tequilana var. Siguin.
These 42 plants were used as samples for the analysis
of intra-specific relationships.
DNA extractions
Leaf tissue was excised and surface sterilized for 5
min with a 5% solution of commercial bleach. Tis-
sues were then rinsed in sterile dd H 2 0 and blotted dry
prior to being frozen in liquid N 2 . DNA was extracted
by the method of Doyle & Doyle (1989). RNA was
eliminated by the addition of 5U RNAase (RNAase+,
5 → 3 , Inc., Boulder, CO) per sample. DNA was
resuspended in TE (10 mm Tris-HCl, pH 8.0, 1 mM
 337

EDTA) and the sample concentrations were determ-

ined spectrophotometrically. Samples were diluted in
TE to achieve a final concentration of 20 ng/µl.

RAPDs
A total of 19 different random 10-mer primers (Op-
eron Technologies, Alameda, CA) were used for
RAPD analysis. Polymerase chain reaction (PCR) was
conducted with 25 µl reactions containing 40 ng gen-
omic DNA, 15 ng primer, 200 µm of each dNTP, and
2.5 U of Amplitaq Gold DNA polymerase (Perkin-
Elmer, Norwalk, CT) in buffer (50 mm KCl, 10 mm
Tris- HCl, pH 8.3, 1.5 mm MgCl 2 , and 0.001% (w/v)
gelatin). Amplifications were done using a GeneAmp
PCR System 2400 thermocycler (Perkin-Elmer, Nor-
walk, CT). Each reaction was performed using an Figure 2. Results of gel electrophoresis of RAPD products
initial ‘hot start’ of 94 ◦ C for 9 min, followed by 35 amplified with the random decamer primers OPC 4
cycles with the following temperature profile: 1 min at (5 -CCGCATCTAC-3 ) and OPC 7 (5 -GTCCCGACGA-3 ),
Forty ng of genomic DNA was amplified with the following
94 ◦ C, 1 min at 37 ◦ C, and 1 min at 72 ◦ C. The reac- thermocycling profile: 94 ◦ C for 9 min, followed by 35 cycles
tions were terminated with a final extension at 72 ◦ C of 1 min at 94 ◦ C, 1 min at 37 ◦ C, and 1 min at 72 ◦ C. The
for 7 min. Amplification products were resolved on reactions were terminated with a final extension at 72 ◦ C for 7 min.
Amplification products were resolved on 1X TBE gels containing
1X TBE gels containing 1.4% agarose, stained with
1.4% agarose.
ethidium bromide and visualized with a source of UV
light. Each primer-plant sample combination was re-
peated at least twice, and negative control reactions
lacking DNA were included for each primer.

Data analysis
All gels were scored for both polymorphic and mono-
morphic bands. It was assumed that bands of the
same molecular weight in different individuals were
identical. Band presence was indicated by a (1) and
absence by a (0). Genetic distances (D) were calcu-
lated using the S-Plus (Version 4.0) software package
according to the method of Skroch et al. (1992). The
genetic distance (D) between two samples, A and B,
is calculated by the
method of Skroch et al (1992)
as follows: DAB = [ i |Ai – Bi |]/N, where Ai and Bi
are the assigned scores (1 or 0) for the i t h band of
A and B respectively, and N = total number of bands
present among all the samples. Cluster analysis was
based on distance matrices using the unweighted pair
Figure 3. Results of gel electrophoresis of RAPD products
group method arithmetic average (UPGMA) (Avise, amplified with the random decamer primer OPC 3
1994) and relationships between samples were graph- (5 -GGGGGTCTTT-3 ). Forty ng of genomic DNA was
ically presented as dendrograms. Confidence limits on amplfied with the following thermocycling profile: 94 ◦ C for 9 min,
relationships were determined by bootstrap analysis followed by 35 cycles of 1 min at 94 ◦ C, 1 min at 37 ◦ C, and 1 min
at 72 ◦ C. The reactions were terminated with a final extension at
(B=1500 replicates) (Felsenstein, 1985). 72 ◦ C for 7 min. Amplification products were resolved on 1X TBE
gels containing 1.4% agarose.
338
Results
Genetic distances among field selections of A.
tequilana var. Azul
All 19 RAPD primers tested in this analysis amplified
DNA from all 40 plants of A. tequilana var. Azul. An
example is presented in Figure 2 with two different
decamer primers from plants of A. tequilana var. Azul
collected from different field sites (Figure 2). These
results (Figure 2) show a complete lack of detectable
polymorphisms among the different plants. The 19
RAPD primers produced a total of 124 bands (an av-
erage of 6.5 bands per primer) of which only 1 (0.8%)
was polymorphic (Figure 3). Plant OR2 is missing a
single band that was present in all other plants of A.
tequilana var. Azul (Figure 3).
The RAPD data indicate that 39 of 40 plants of A.
tequilana var. Azul were isogenic based on the ana-
lysis of 124 amplification products. The dendrogram
demonstrates the virtual absence of genetic diversity
encountered among the sampled plants of A. tequilana
var. Azul (Figure 4). The genetic distance (D) between
the the one variant plant encountered in the analysis,
plant OR2, and the block consisting of 39 isogenic
plants of A. tequilana var. Azul, was 0.007.
Genetic distances among different varieties of A.
tequilana
The 19 RAPD primers amplified a total of 130 markers
among a sample consisting of 40 plants of A. tequilana
var. Azul and one plant each of A. tequilana var. Chato
and A. tequilana var. Siguin. Between A. tequilana
var. Chato and A. tequilana var. Siguin, 20 markers
(15.4%) were polymorphic. Between A. tequilana var.
Siguin and the conserved block of 39 isogenic plants
of A. tequilana var. Azul there were eight (6.2%)
polymorphic markers, and 16 (12.3%) markers were
polymorphic between A. tequilana var. Chato and the
conserved block of 39 isogenic plants. A clear poly-
morphism between A. tequilana var. Chato and the
other plants included in the analysis can be seen in
Figure 3.
The genetic distance between A. tequilana var.
Chato and A. tequilana var. Siguin was 0.15. The
distance between A. tequilana var. Chato and the con-
served block of 39 isogenic plants of A. tequilana var.
Azul was 0.12, and the distance between A. tequilana
var. Siguin and the conserved block of 39 isogenic
plants was 0.06. These relationships are graphically
presented in the dendrogram (Figure 4). The results
of 1500 bootstrap replicates indicate that confidence
limits exceeded 95% for all relationships.
Discussion
The topology of the dendrogram (Figure 4) suggests
that A. tequilana var. Siguin is more closely related
to A. tequilana var. Azul than is A. tequilana var.
Chato. Only one plant each of both A. tequilana var.
Chato and A. tequilana var. Siguin was sampled be-
cause of the paucity of these plants in production
fields of A. tequilana var.Azul. These two varieties
are routinely culled from the fields as soon as they
are encountered due to federal regulations prohibit-
ing their use for tequila production (Diario Official,
1993). We are reluctant to make conclusions regard-
ing phylogenetic relationships based on single plant
samples for these two other varieties, but will note that
relationships inferred from our analysis of molecular
markers are in concordance with those proposed based
on morphology (Gentry, 1982).
A. tequilana var. Siguin is considered to be the
variety of A. tequilana most morphologically similar
to A. tequilana var. Azul (Gentry, 1982; Valenzuela,
1997). A. tequilana var. Chato, however, has many
characteristics that distinguish it from A. tequilana var.
Azul, including a longer life cycle, larger and thicker
leaves that have a pale-green color as compared to
the blue-green color characteristic of A. tequilana var.
Azul (the Spanish word for blue is ‘azul’), and larger
spines (Gentry, 1982; Valenzuela, 1997).
The RAPD results obtained from the forty plants
of A. tequilana var. Azul represent one of the lowest
levels of polymorphism within a plant species repor-
ted to date. Recently, an analysis of 29 individual
plants of Limonium cavanillensi L. with 11 different
RAPD primers failed to detect a single polymorphism
among 131 markers (Palacios & Gonzalez-Candelas,
1997). Although our sample size was limited, we
consider our results to accurately reflect the level of
polymorphism present in the general population of A.
tequilana var. Azul cultivated in the state of Jalisco.
Our samples were obtained from two different produ-
cers of tequila and from four separate fields, two of
each located in one of two geographic regions sep-
arated by over 100 km (Figure 1). Genetic variation
was virtually nonexistent between plants within fields,
between fields, and between producers (Cuervo and
Orendain).
 339

Figure 4. Dendrogram based on 130 RAPD markers using the genetic distance method of Skroch et al. (1992). Confidence limits noted on the
dendrogram were based on the result of 1500 bootstrap replicates (Felsenstein 1985). Plants beginning with the letters ‘OR’, ‘TC’, ‘SD’, and
‘SJ’ were collected from fields Orendain, Tequila Cuervo, Santo Domingo, and San Juan, respectively.

Several points of evidence argue strongly against
the likelihood that all 39 isogenic plants are the
product of vegetative propagation of a single plant
from the previous generation. Valenzuela (1997), sur-
veyed production fields and determined that 67% of
2 yr old plants only produced between one to three
ramets. Ramets are typically excised only during the
third through fifth years of a mother plant’s life cycle
(Valenzuela, 1997), so the total number of ramets suit-
able for propagation that can be harvested from an
average plant might range from 5–10. Secondly, there
has been no tradition of record keeping among the
agave producers with respect to the origin of plant-
ing materials. Ramets are typically excised during the
months of March-May, and are placed in piles under
the shade of a tree for two to three weeks prior to
planting to harden off the wound site made by ex-
cision (Valenzuela, 1997). The ramets are then placed
in trucks and transported to new field sites, where they
are randomly removed from the truck and planted in
rows. Finally, with respect to our sampling proced-
ure, within a field only one plant was sampled from
any given row and at least four adjacent rows were
then skipped prior to sampling another plant. These
observations suggest that the lack of genetic variation
is the result of a promotion of a predominant geno-
type over the course of many generations of vegetative
propagation.
The apparent paucity of genetic variation detec-
ted in this study contrasts sharply with the results of
previous investigations that used molecular markers
to examine levels of genetic variation present in wild
populations of other Agave sp. Martínez-Palacios et
al. (1999) analyzed 10 populations of A. victoriae-
reginae with ten isozyme loci. An average of 83%
of the loci were polymorphic within each population
and high levels of diversity were encountered between
populations. Similarly, Colunga-GarcíaMarín et al.
(1999) investigated genetic diversity among a wild
population of A. angustifolia and observed 34 different
electrophenotypes for the three isozymes used in the
study. Trame et al. (1995) reported high levels of ge-
netic variation based on analysis of a wild population
of A. schotti using RAPDs. Massey & Hamrick (1998)
used 19 isozyme loci to study genetic diversity in 18
populations of Yucca filamentosa, which belongs to
340
the same taxonomic family (Agavaceae) as do Agave
spp. The mean percentage of polymorphic loci detec-
ted among the different populations was 67.6%, al-
though considerable variation was encountered among
populations (range = 31.6–84.2%).
The lack of genetic diversity observed for cul-
tivated A. tequilana var. Azul is likely a direct
consequence of two cultural practices: 1) The ex-
clusive use of vegetative propagation for producing
new planting materials, and 2) the removal from the
plant of the inflorescence before fertilization and seed
formation occurs. Similar results were observed by
Colunga-GarcíaMarín et al. (1999) for the case of A.
fourcroydes, a plant species cultivated for fiber pro-
duction in the Yucatan Peninsula of Mexico. No ge-
netic variation could be detected within three different
varieties of A. fourcroydes based on the results of an
analysis using three different isozyme loci. Colunga-
GarcíaMarín et al. (1999) concluded that the lack of
genetic variation was a consequence of the practice
of obtaining new planting materials exclusively by
vegetative propagation.
The cultivation of a single, preferred genotype is
very common in commercial agriculture. However,
this practice generally implies that a base of genetic
variation is available for the crop species in ques-
tion. This base of variation can be exploited when
new market demands or biotic pressures dictate the
need to introduce newly desirable traits into the cul-
tivated variety. The condition we have observed for A.
tequilana var. Azul is alarming, as there appears to be
no easily accessible base of genetic variation.
Although approximately 50% of the total produc-
tion of A. tequilana var. Azul is in the state of Jalisco,
the states of Nayarit and Tamaulipas each account for
about 20% of the total production (Valenzuela, 1997).
It would be prudent to survey fields grown in these
states with molecular markers to determine if read-
ily available sources of variation are present. This
approach might be especially worthwhile for plants
grown in the state of Tamaulipas, which is over 500 km
northeast of the state of Jalisco, and would be more
likely to have a distinct cultigen of A. tequilana var.
Azul than would the state of Nayarit, which shares a
border with Jalisco.
Although our results demonstrate an almost com-
plete lack of genetic variation between field selections
of A. tequilana var. Azul, this report did not address
the issue of the amount of heterozygosity present in a
typical plant of this variety. The genome most likely
possesses heterozygous loci that could be used to de-
velop genetically distinct progeny. This would require
that some plants be allowed to reproduce sexually and
the resulting seeds collected and germinated for pro-
ducing variant plants. Valenzuela (1997) has noted that
plants resulting from seed are very heterogeneous and
not suitable for cultivation. However, the observed het-
erogeneity suggests that sexual reproduction can result
in genetically diverse plants that may serve as breeding
materials. Unfortunately, it has also been noted that
plants produced from seed require up to three years
of growth before reaching a size that is suitable for
planting in the field (Valenzuela, 1997).
This report should serve as an alarm for the tequila
industry. Currently the industry is very vulnerable to
the occurence of new and highly destructive pathogens
which could result in a disease epidemic of immense
proportion. Breeding programs should be initiated
promptly, as the long vegetative life cycle (7–9 yr) will
exacerbate any difficulties encountered during the pro-
cess. To the authors’ knowledge, there are currently no
active breeding programs for A. tequilana var. Azul.
Approaches that may facilitate the production of im-
proved genotypes of A. tequilana var. Azul include
the development of tissue culture protocols that could
accelerate the vegetative life cycle and flower produc-
tion so that crosses could be made in vitro. This may
result in the production of segregating populations in a
fraction of the time necessary for a breeding program
that relies on crosses made in the field.
Acknowledgements
The authors wish to thank Tequila Cuervo S.A. de C.V.
for the financial support for this work. K. Gil Vega was
supported by a CONACYT graduate fellowship.
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