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The Journal of Agricultural Science
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© Cambridge University Press 2011
(Continued on inside back cover)
Early determination of sex in jojoba plant by
A. G. INCE1AND M. KARACA2*
1Department of Horticulture, Faculty of Agriculture, Akdeniz University, 07059 Antalya, Turkey
2Department of Field Crops, Faculty of Agriculture, Akdeniz University, 07059 Antalya, Turkey
(Revised MS received 7 August 2010; Accepted 31 August 2010; First published online 19 November 2010)
Jojoba [Simmondsia chinensis (Link) Schneider] is a dioecious plant grown for its seeds, which are the
source of liquid wax or jojoba oil. The sex of jojoba plants cannot be determined with morphological
characters until the plants reach reproductive maturity at 3 or more years old. This difficulty of early
sex determination imposes severe constraints in breeding studies and in the sex allocation of seedlings
in seed orchard establishment, and importantly in a priori mating designs to produce superior jojoba
individuals. This study reports three new cleavage-amplified polymorphic sequence (CAPS) assays,
which identify male and female individuals distinctly. One of the assays could also identify
hermaphrodite jojoba plants existing in nature or obtained using mutagenesis studies.
Jojoba [Simmondsia chinensis (Link) Schneider] plant,
a perennial and native of Arizona, southern California
and northern Mexico, is of immense industrial value.
It is sexually dioecious, bearing male and female
flowers on different plants, and mature male and
female plants differ in their vegetative structure and
floral morphology. Male flowers occur in axillary
inflorescences which may contain 3–20 flowers. Each
male flower consists of four to six sepals and 8–16
large, erect anthers. Female flowers also occur in
axillary positions but usually contain a single flower.
Each female flower consists of one to four large bracts,
four to six sepals and three to four long styles. The
jojoba fruit is a green ovoid-shaped capsule enclosing
one peanut-sized, dark-brown seed (Gentry 1958;
In jojoba, sex is genetically determined and there
are no reports of plants changing sex, indicating that
environment does not play a role in sex expression.
Since neither male nor female flowers of jojoba plants
have nectaries or petals, bees generally do not visit
them. Jojoba is a wind-pollinated plant and pollen is
known to travel distances of c. 33m in relatively mild
breezes (Yermenos 1979). Flowering period can vary
from 30 to 45 days depending on ambient tempera-
tures, which have a large effect on stigmatic receptiv-
ity. Depending on the ambient temperatures, female
flowers are receptive for a period of 3–6 days. There
are also dramatic differences in jojoba pollen viability
depending on the ambient temperatures and humidity
(Coates & Ayerza 2008).
Commercial production of jojoba has spread in
recent times to South and Central America, southern
Africa, Australia and even the Middle East. The
product of primary interest is the seed oil, which is a
unique liquid wax consisting of esters formed from
acids and alcohols with chain lengths of 20 or 22
carbon atoms (Wisniak 1987). As many as 300
products containing jojoba oil have appeared on the
market in recent years and the use of jojoba products
is expected to increase in the future (Benzioni &
Vaknin 2002; Benzioni et al. 2005).
The objectives of jojoba breeding studies involve
the selection of superior genotypes producing large
seeds, flowers at every node, more than one seed per
node in clusters, early flowering, consistent high
production from year to year, production of large
quantities of highly viable pollen, co-ordinated bloom
periods for male and female plants in the plantation,
upright growth habit and high oil content with the
desired wax properties such as viscosity, boiling point
and thermal stability, which are important for future
* To whom all correspondence should be addressed.
Journal of Agricultural Science (2011), 149, 327–336.
© Cambridge University Press 2010327
requirements of the jojoba industry. However, the
most common method for jojoba improvement has
been the selection of plants with desirable character-
istics and propagating them asexually, since the
development of cultivars having large seed, fruit at
every node and seeds in clusters can be accomplished
through vegetative propagation in a relatively short
period of time (Yermenos 1979; Vaknin et al. 2003;
Tobares et al. 2004; Benzioni et al. 2005). However,
the use of clones or cuttings in commercial plantations
faces problems of low seed yield and seed wax content
(Dunstone et al. 1985). Several studies have investi-
gated ways in which to improve jojoba seed yield and
wax yield; however, no significant correlation has
been found between the seed yield and seed weight, or
between seed yield and seed wax content (Tobares
et al. 2004). In order to increase jojoba seed yield, the
application of supplemental pollen has been proposed
and indications of increased seed yield by supplemen-
tal pollination were reported by Coates et al. (2006).
However, yield increases were not dramatic and were
not consistent between years.
Selection of suitable male and female genotypes
for plantation and breeding studies is important.
Several studies have indicated that low yields in
commercial jojoba plantations around the world
have resulted from a high female:male plant ratio
(Benzioni & Ventura 1998) and undesirable weather
conditions during flowering and fruit set (Coates et al.
2006). Weather temperature and relative humidity for
jojoba production cannot be directly changed by
humans but the female:male plantation ratio could
be easily manipulated by planting new male plants at
appropriate distances from the existing females.
However, a period of 3–6 years would be required
for the new male plants to produce sufficient pollen to
be effective. Furthermore, the selection of male plants
for plantations requires genetic knowledge about
them, since the effect of the male and female parents
on yield and wax compositions are additive and highly
significant (Benzioni & Vaknin 2002).
Commercial jojoba is sexually dioecious and its sex
type cannot be determined from morphological
characters till the reproductive maturity age of 3 or
more years. This creates a problem for early sex
diagnosis in plantation and breeding studies. In jojoba
improvement programmes, this prompts remarkable
constraints for a priori mating design of superior
individuals in breeding practices and for sex allocation
of seedlings in seed orchard establishment.
Plants can be dioecious, monoecious or hermaph-
rodite (Hamadina et al. 2009). Some species have
hermaphroditic flowers and female flowers on the
same plant, known as gynodioecy or female-hermaph-
rodite, while others have hermaphroditic flowers
and male flowers on the same plant (gynomonoecy
or male-hermaphrodite; Borges 1998; Ordas et al.
2009). Although jojoba is a dioecious species, one
hermaphrodite jojoba plant called ‘Hermaphrodite’
has been identified and used in studies (Vaknin et al.
2003). Furthermore, a naturally occurring female-
hermaphrodite jojoba plant called ‘LaRonna’ has
been identified; it has both male and female repro-
ductive organs, with the female trait being dominant
(http://www.laronnajojoba.com, verified 9 September
2010). However, there is no record on the existence of
In most cases, hermaphrodite plants, for example,
papaya (Carica papaya L.), aredesired for commercial
production (Deputy et al. 2002). Identification of
hermaphroditic jojoba plants could be important for
jojoba production and breeding studies. However, the
presence of the two types of flowers on a single jojoba
plant may influence plant fitness because geitonoga-
my, i.e. fertilization of one flower with pollen from
another flower on the same plant, may generate
inferior offspring through inbreeding depression
(Collin & Shykoff 2003). However, females and
males of dioecious species may not allocate the same
proportions of assimilate
(McGowan et al. 2004; Cisneros-Lopez et al. 2010).
DNA markers offer several advantages in plant
identification and genetic studies in comparison with
morphological markers. In comparison with morpho-
logical markers, DNA markers are variable and
exhibit a high level of allelic variation, neutral to the
environmental factors and developmental stages and,
not influenced by other genes and factors (Tan et al.
2003; Karaca et al. 2004). DNA markers have also
been used in sex identification in many crop species
(Kumar & Arumuganathan 1997; Alstrom-Rapaport
et al. 1998; Ruas et al. 1998; Culley & Wolfe 2000;
Shibu et al. 2000; Kafkas et al. 2001; Khadka et al.
2002; Torjek et al. 2002; Jiang et al. 2003; Xu et al.
2004; Danilova & Karlov 2006; Prakash & van
Staden2006; Chaves-Bedoya &
Gangopadhyay et al. 2007; Jhang et al. 2010; Li
et al. 2010).
Efforts have been made to identify molecular
markers in jojoba linked to sex alleles. Although
male-sex-linked random amplified polymorphic DNA
(RAPD) and inter-simple sequence repeat (I-SSR)
markers have been reported (Agrawal et al. 2007;
Sharma et al. 2008), these markers were not useful in
jojoba sex identification. Ince et al. (2010a) reported a
novel jojoba male-sex-specific touchdown polymerase
chain reaction (Td-PCR)-based
(JMS900). The JMS900DNA marker is reproducible
and distinct and it may not be necessary to convert
it into the advanced markers of cleavage-amplified
polymorphic sequence (CAPS) or PCR-restriction
fragment length polymorphism (PCR-RFLP) and
sequence-characterized amplified region (SCAR).
However, the JMS900 DNA marker is generated
using a single primer only 10 nucleotides long, thus
being very vulnerable to PCR conditions and quality
A. G. INCE AND M. KARACA
of the genomic DNA (Karaca & Ince 2008).
Furthermore, JMS900cannot discriminate hermaph-
rodite jojoba from male and female plants. New sex
diagnostic markers simultaneously identifying female
and male jojoba plants could be very helpful not only
in sex identification but also in jojoba breeding
studies. This study reports three new CAPS markers,
different from JMS900, and discusses their application
in jojoba plantation and breeding studies.
MATERIALS AND METHODS
Jojoba plants were grown in a plantation located
102km from the Mediterranean
Kumluca, Antalya, Turkey (36°N, 30°W, 55m asl).
The average annual precipitation over the last 10 years
ranged from 600 to 1500mm; the highest temperature
was recorded in August (c. 28·0°C), while the average
lowest temperature was recorded in February (c.
7·0°C). The average humidity ranged from c. 65 to
78%. The leaves of 60 female and 60 male jojoba
plants were used as plant materials. Using the leaves
of female and male plants, several male- and female-
bulked samples (sex-specific bulks) were made. In
addition to these, a total of 36 leaf samples collected
individually from 36 different jojoba seedlings whose
sexes were not known during the collection time
were used as control samples to test the reliability
and reproducibility of male-sex-specific and female-
sex-specific markers developed in this study. These
seedlings were obtained from cuttings of mature male
and female jojoba plants and sexes of the control
samples were determined using the records.
Genomic DNA extraction
A sample of leaves (10g) from sex-specific bulks or
2–3 leaves of individual male or female samples were
ground in a wide or small-sized mortar using liquid
nitrogen. Total genomic DNA was extracted and
Concentration of DNA samples was determined
using a spectrophotometer and concentrations were
readjusted so that each sample contained 0·12μg in
8·5μl for PCR analysis. DNA extraction studies from
those control samples used single leaf material.
et al. (2005).
PCRs and agarose gel electrophoresis analyses of
OPG51400, UBC807 and JMS900 markers using the
primers listed in (Table 1) were performed as reported
in Agrawal et al. (2007), Sharma et al. (2008) and Ince
et al. (2010a), respectively. The following steps were
used for amplification of the OPG51400marker: one
cycle consisting of 60s at 94°C, 30s at 36°C and 60s
at 72°C followed by 45 cycles of 5s at 94°C, 15s at
36°C, and 60s at 72°C and a final cycle of 7min at
72°C. UBC807amplification was carried out with a
preliminary cycle of 3min at 94°C, followed by 35
cycles of 20s at 94°C, 60s at 50°C, 90s at 72°C and a
final cycle of 7min at 72°C. Amplification of JMS900
marker was carried out with the following amplifica-
tion profiles: 3min hold at 94°C, followed by a 10
cycle pre-PCR consisting of 1min at 94°C for
denaturation, 50s at 42°C for annealing and 2min
at 72°C for extension. The annealing temperature was
reduced by 0·5°C per cycle for the first 10 cycles
(touchdown cycles). Amplification of the targeted
DNA templates continued for further 30 cycles at
37°C annealing temperature and ended with a final
extension step at 72°C for 10min. The amplification
products were resolved on 1–2% agarose gels in TRIS-
borate ethylenediaminetetra-acetate (EDTA) buffer
(89mM TRIS-borate and 2mM EDTA), and stained
with ethidium bromide.
Cloning and sequencing
Agarose gel electrophoresis studies indicated that
male-specific markers reported in Agrawal et al.
Table 1. Primer pairs used in this study and related information
MarkerSequences of primers or primer pairs TypeSex References
Agrawal et al. (2007)
Sharma et al. (2008)
Ince et al. (2010a)
CAPSMale Present study
CAPS assays for early determination of sex
(2007) and Sharma et al. (2008) were not co-
segregated with male samples. Therefore, these mar-
kers were not cloned for sequence analysis. However,
the JMS900marker reported in Ince et al. (2010a) was
found to be reproducible and specific to male-jojoba
A total of 12 JMS900 amplicons obtained from
genomic DNA of different male plants were recovered
from agarose gels and purified using purification kits
(QIAgen, Hilden, Germany). Sizes and integrity of the
purified amplicons were confirmed using agarose gel
electrophoresis studies. Amplicons were transformed
to Escherichia coli strain (JM107) using InsTAclone
cloning kit/Transform Aid protocol (MBI Fermentas
Life Sciences, Amherst, NY, USA). Vectors carrying
JMS900amplicons were extracted from bacteria using
a plasmidisolation kit
Sciences). Inserts carrying the amplified products
were confirmed using PCR or restriction enzyme
digestions and gel electrophoresis studies (Ince et al.
2010b). A total of 12 vectors carrying amplicons were
commercially sequenced in duplicates using both
M13F and M13R primers (Macrogen Inc., Seoul,
Analysis of male-specific DNA sequences
Sequences of vectors including the M13F/R primer
pair were removed from the insert sequences and
assembled into contiguous sequences (contigs) using
the Sequencher software (Gene Codes, Ann Arbour,
MI, USA). Contig assembly parameters were set to a
minimum overlap of 100 bases and 0·95 identity
match. Contig analyses of the sequences grouped
into four from which four primer pairs flanking the
sequences were designed using the PRIMER3 soft-
ware (Rozen & Skaletsky 2000). Sequences were also
analysed using the Basic Local Alignment Search
Tool (BLAST) to identify homology within the
Optimization of JMS900-derived primer pairs for
A gradient PCR procedure was used to determine
optimal annealing temperatures of the JMS900-derived
primer pairs. Two different gradient PCR procedures
were used, one with annealing temperatures of 60–
70°C and the other 50–60°C. Among the four primer
pairs, one did not produce single amplicons, produ-
cing instead multiple weak amplicons at a lower
annealing temperature, while the remaining three
primer pairs produced single amplicons at annealing
temperatures between 60 and 65°C. In order to use a
single amplification protocol for all the three primer
pairs, a Td-PCR procedure was developed and used.
Td-PCR was carried out in 25μl reaction volume
containing 120 ng genomic DNA as a template,
0·5μM of each primer pair (Table 1), 12mM tris-
(hydroxymethyl) aminomethane-hydrogen chloride
(TRIS–HCl; pH 8·8), 60mM KCl, 0·34mM Nonident
P-40, 0·28mM of each deoxyribonucleotide triphos-
phate (dNTP), 3mM MgCl2and 2units of Taq DNA
polymerase (MBI Fermentas Life Sciences).
The Td-PCR amplification profile was as follows:
initial denaturation at 94°C for 3min, 10 cycles with
denaturation at 94°C for 30s, annealing at 65°C for
50s in the first cycle, diminishing by 0·5°C each cycle
and extension at 72°C for 1min in a 96-well P×2
thermal cycler (Thermo Hybaid, Miami, USA). An
additional 30 PCR cycles were run using the same
cycling parameters with constant annealing at 60°C.
Denaturation and extension conditions were the same
as indicated above. The amplifications finished with
final extension at 72°C for 10min.
After completion of Td-PCR, 1μl DNA loading
buffer (MBI Fermentas Life Sciences) was added to
5μl amplified products and these mixtures were
loaded on to 2–3% (w/v) high-resolution agarose gels
(Serva, Heidelberg, Germany or Metaphor, FCM
BioProducts, Rockland, ME, USA) containing 0·6μg/
ml ethidium bromide. Amplicons were then electro-
phoresed at 0·5–0·8V/mm at constant voltage for
8–12h in the presence of 1×TRIS–borate EDTA
buffer (89mM TRIS, 89mM Borate and 2mM EDTA,
pH 8·3) and photographed on an ultraviolet (UV)
transilluminator for analysis.
Restriction enzyme digestion studies
Five microlitres of amplified products were digested
separately using each of Aat II, Hae III, Hind III, Rsa
I, Hin6 I, Hinf I, Hha I, Cla I, Hpa II, EcoR I, Msp I,
Mse I, Vsp I (MBI Fermentas Life Sciences) and Taq I
(Bioron, Ludwigshafen, Germany) restriction en-
zymes in 10μl of reaction volume according to the
recommendations of the manufacturers. Agarose gel
electrophoresis analyses were performed as stated
above after completion of the reactions.
Female- and male-specific DNA markers
A sex-specific CAPS DNA marker was defined based
on the following simple criteria: a male-sex-specific
CAPS DNA marker should distinctly differentiate
males from females, a female-sex-specific CAPS DNA
marker should distinctly differentiate females from
males, and gender-specific CAPS DNA marker
should distinctly differentiate males and females in
male–female mixtures mimicking the hermaphrodites.
A. G. INCE AND M. KARACA
Male-specific OPG51400 and UBC807 markers were
screened on a total of 36 jojoba control samples.
Results indicated that these markers could not be
effectively used in jojoba sex identification studies.
Furthermore, none of the amplicons of OPG51400and
UBC807obtained from bulked female and male plants
produced sex-specific products, and therefore were not
used in further studies.
Agarose gel electrophoresis studies showed that
JMS900marker was not present or not detectable in
female-bulked samples, while all the male samples
contained JMS900 markers (Fig. 1a). Male-specific
JMS900 amplicons were cloned and sequenced.
Sequence analysis indicated that JMS900amplicons
contained at least four different DNA sequences
ranging in size from 888 to 852 base pairs (bp). This
indicated that single amplicons in agarose gels
contained at least four different sequences. Using the
four JMS900 amplicon sequences, a total of four
primer pairs were developed.
Gradient PCR studies indicated that one of the four
primer pairs could not amplify, or produced very
weak, amplicons among female and male genomic
DNA samples. Three primer pairs were used for
screening the genomic DNA of bulked male and
female samples; however, none of the amplicons were
polymorphic among the female and male jojoba
plants. It is possible that female DNA samples might
have contained the JMS900amplicon, but they were
not detected using the standard agarose gel electro-
phoresis studies (Figs 1a–c), or females may have a
mutation around the JMS900sequences and hence no
amplicons were produced.
Amplified products of the three primer pairs derived
from the sequence of JMS900 were digested with
several restriction enzymes to investigate whether
JMS900marker could be converted to CAPS markers.
Analyses indicated that among the restriction enzymes
used three (Cla I, Hind III and Hinf I) produced
polymorphic fragments in male- and female-bulked
samples amplified with J880 (Figs. 1d–f) and J818
primer pairs (Figs 1g–i, Table 1). Although majority
of the other restriction enzymes digested the ampli-
cons, they could not produce polymorphism between
female and male samples. The results also indicated
that the product of the J880 primer pair, although
digested with majority of the restriction enzymes,
could not differentiate between male and female
Development of male-specific CAPS marker
(CAPS J888-Hind III)
A male-specific CAPS DNA marker distinctly differ-
entiating males from females was developed. The
forward and reverse (F/R) primer pair of J888
contained 10 bases of the JMS900 primer (5′-
AGACCCAGAG-3′) at their 5′ ends. The F primer
contained an addition of 10 bases of CACACACAGC
and the R primer contained an addition of 11 bases of
GATGAGGAATG. In order to evaluate the differ-
entiation power of the J888 primer pair-amplified
product, 16 male and female genomic DNA bulks
were used. The results clearly indicated that this
primer pair was able to amplify a single DNA
amplicon in all of the male and female plants
analysed, the size of which agrees well with the target
Analyses indicated that when digested with Hind III
restriction enzyme all female amplicons were cut into
two fragments and all the male amplicons were cut
into three fragments (Fig. 2). The PCR amplification
and restriction enzyme studies were repeated three
Fig. 1. Amplification and restriction enzyme digestion studies. (a) Amplification of the MS900marker showing clear male-
specific amplicon at about 900 bp. (b and c) Amplified products of J888and J818primer pairs showing monomorphic amplicons
in male and female samples, respectively. (d–f) J888primer pair-amplified products digested with Hind III, Hinf I and Cla I
restriction enzymes, respectively. (g–i) J818primer pair-amplified products digested with Hind III, Hinf I and Cla I restriction
enzymes, respectively. M, DNA size markers; BF, bulked female; BM, bulked male jojoba genomic DNA.
CAPS assays for early determination of sex
times and all the analyses results were consistent,
indicating reliability of the male-specific CAPS mar-
ker. Further analyses were performed on the genomic
DNA of 36 control jojoba samples to test the
reliability and reproducibility of this CAPS marker.
Results clearly indicated that amplified products of
the J888 F/R primer pair and Hind III restriction
enzyme digestion could be used in male jojoba
plant identification. This marker was called (CAPS
Development of male-specific CAPS marker
(CAPS J818-Hinf I)
J818F/R primer pair was developed based on the 818
bp in JMS900sequence. The F primer consisted of
5′-AGGGGATAAATGAGCCGAAT-3′ and the R
ATG-3′ nucleotides. In order to evaluate differen-
tiation power of the J818F/R primer pair a total of 36
male and female genomic DNA was used. Agarose gel
electrophoresis studies clearly indicated that this
primer pair was able to amplify a single DNA
amplicon in all the male and female plants analysed.
Two Hinf I digests (digested amplicons) were
present in all the male samples, while these fragments
were absent in female samples. One of two digestion
products in male samples segregated among male
plants and the other fragment was specific to male
plants. Fragments of higher molecular weight in
the male samples (Fig. 3) segregated as a recessive
allele (being present in eight samples out of 40) as
judged from 40 male jojoba genomic DNA, while the
lower fragment segregated as the dominant allele
(being present in 32 samples out of 40, data not
shown). One of the two amplicons was present in all
the male samples without segregations. Further
analyses were also performed on the genomic DNA
of 36 control jojoba samples to test the reliability and
reproducibility of this CAPS marker. Analyses clearly
indicated that amplified products of the J818 F/R
primer pair and Hinf I restriction enzyme digestion
could be used in male jojoba plant identification. This
marker was called (CAPS J818-Hinf I).
Development of gender-specific CAPS marker
(CAPS J888-Cla I)
Amplified products of the J888 primer pair were
digested with Cla I restriction enzyme. Cla I digestion
resulted in several fragments (Fig. 4). The sizes of
gender-specific markers were between 500 and 300bp,
IF IMIFIF IM
Fig. 2. CAPS J888-Hind III marker. (a) Amplified products of J888primer pair showing clear single amplicon at about 888 bp.
(b) Amplified products digested with Hind III restriction enzyme. At about 252 bp products were always present in individual
and bulked male samples, while these products were not seen in individual and bulked female samples. M, DNA size markers;
IF, individual female; IM, individual male; BM, bulked male; BF, bulked female jojoba genomic DNA.
IF IMIF IMIF
IF IMIF IM
Fig. 3. CAPS J818-Hinf I marker. (a) Amplified products of J818primer pair showing clear single amplicon at about 818 bp. (b)
Amplified products digested with Hinf I restriction enzyme. Two distinct products at about 260–280 bp products were always
present in individual and bulked male samples, while these products were not seen in individual and bulked female samples.
M, DNA size markers; IF, individual female; IM, individual male; BM, bulked male; BF: bulked female jojoba genomic
A. G. INCE AND M. KARACA
with female plants containing two and male plants
containing three fragments of these sizes. In order to
evaluate this CAPS marker, PCR amplification and
restriction enzymes studies were repeated thrice using
the genomic DNA of individual male and female
samples (Fig. 5). The results clearly indicated that
amplified products of the J888F/R primer pair and
Cla I restriction enzyme digestion could be used in
male and female jojoba plant identification.
This marker, called CAPS J888-Cla I, will probably
also useful in the identification of hermaphrodite
jojoba plants. Although monoecious or hermaphro-
dite plants are uncommon in jojoba, dioecious,
monoecious or hermaphrodites coexist in some plant
species (Hamadina et al. 2009). When hermaphrodites
are found in natural populations of jojoba, or
mutagenesis-directed hermaphrodite jojoba is ob-
tained by, e.g. targeting induced local lesions in
genomes (TILLING), a DNA marker specific to
only male or female individuals could give contra-
dictory results. In order to test the sensitivity of the
CAPS J888-Cla I marker, the genomic DNA from
male and female samples were mixed to make artificial
hermaphrodite samples by mixing male and female
genomic DNA in different proportions: 0·33 female
and 0·67 male, 0·67 female and 0·33, 0·25 female and
0·75 male and 0·20 female and 0·80 male (Fig. 5). Also
several male and female DNA were equally mixed
(data not shown). In all cases, the CAPS J888-Cla
I marker could identify male and female fragments in
different male and female genomic mixtures.
Sequence analysis of cloned amplicons
Analyses indicated that DNA sequences from which
J888and J818primer pairs were designed were very
similar showing several single nucleotide polymorph-
isms. Different restriction enzyme digestion patterns
on amplicons amplified with J888and J818primer pairs
confirmed that two sequences were not identical. All
the DNA sequences (four in total) were searched using
BLAST for identifying homology within the Gen-
Bank databases. However, none of the sequences had
significant homology within the GenBank databases.
Both sequences from which J888and J818primer
pairs obtained were analysed using the Sequencher
program (Gene Codes, Ann Arbor, MI, USA).
Results indicated that both sequences had recognition
sites for Cla I, Hind III and Hinf I digestions.
However, when genomic DNA was amplified with
the J888and J818primer pairs and digested with these
enzymes, some extra fragments were obtained. This
IF IMIF IM IF
Fig. 4. CAPS J888-Cla I marker. (a) Amplified products of J888primer pair showing clear single amplicon at about 888 bp. (b)
Amplified products digested with Cla I restriction enzyme. There were three restriction enzyme products between 500 and 300
bp. Female plants contained two and male plants contained three fragments. Female-specific fragment sized between the two
female-specific fragments. M, DNA size markers; IF, individual female; IM, individual male; BM, bulked male; BF, bulked
female jojoba genomic DNA.
M SF SM MF MF
Fig. 5. Differentiation power of female- and male-specific
CAPS-Cla I markers. M, DNA size marker; SF, single
female; SM, single male; MF, male and female genomic
DNA were mixed. MF1, 0·33 female and 0·67 male; MF2,
0·67 female and 0·33 male; MF3, 0·25 female and 0·75 male’
MF4, 0·20 female and 0·80 male.
CAPS assays for early determination of sex
was probably due to the fact that J888and J818primer
pairs amplify different regions related with sex loci in
PCR has been proved to be a reliable strategy for
detection of sex-specific markers in dioecious plant
species. Among the PCR-based DNA markers,
RAPD is the cheapest technique, but it has several
drawbacks including false positive/negative and re-
producibility problems. Thus, its use gradually paved
the way for more advanced molecular techniques
(Karaca & Ince 2008) such as SCAR and CAPS
markers, originating from RAPD markers and devel-
oped to identify sexes in many plant species (Urasaki
et al. 2002; Jhang et al. 2010; Li et al. 2010).
The CAPS marker is expected to solve the false-
negative/positive and reproducibility problems associ-
ated with RAPD methods. In comparison with
RAPD, CAPS is more resilient (less sensitive) of
false-positives as the detection of a specific target is
dependent on restriction digestion instead of PCR
amplification, which is usually more vulnerable to
experimental conditions than restriction enzyme di-
gestion. However, CAPS and SCAR systems require
primer pairs that can amplify all the targeted genomic
sequences simultaneously and equally. The CAPS
system, unlike SCAR, also requires selection of
restriction enzymes for the detection of specific
sequences (Ince et al. 2010b).
This study intended to develop SCAR or CAPS
markers from OPG51400, UBC807 and JMS900 pre-
viously reported (Agrawal et al. 2007; Sharma et al.
2008; Ince et al. 2010a). However, in contrast to the
reports by Agrawal et al. (2007) and Sharma et al.
(2008), OPG51400and UBC807were not male specific
and therefore were not included in cloning and
sequencing analyses in this study. Several primer
pairs were developed from the sequences of male-
specific JMS900 marker. However, none of these
primer pairs used in SCAR analyses differentiated
male from female samples.
Sequence and restriction enzyme digestion studies
indicated that JMS900amplicon consisted of several
DNA sequences whose primer binding sites were
identical. Two sequences of JMS900amplicon were
very similar, differed only few nucleotide differences
(J818and J888). The other two sequences were different
from each other and from J818, and J888sequences.
Using all the four sequences, no similar sequence was
found by BLAST search within the GenBank data-
bases (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Results
indicated that the frame −2 of J888and J818sequences
contained an open reading frame (ORF) starting 306
bp with an ATG and ending at 428 bp with a TAA,
coding 40 amino acids. Frame −2 sequence also
contained 207 bp length without an ATG start codon.
However, it is not possible to ascertain whether the
sequence is part of the gene(s) for sex determination
in jojoba, and therefore, these sequences were not
submitted to GenBank databases. Further studies
required to identify gene(s) responsible for sex
determination in jojoba.
None of the four primer pairs developed from
four sequences could differentiate male and female
samples; therefore, they could not be converted to
SCAR markers. However, in this study, three different
CAPS assays, based on three different restriction
enzymes and two primer pairs, were determined.
These are the first CAPS markers ever developed, to
our knowledge, for determination of male and female
jojoba plants. They could be used in the plantation of
jojoba with a well-balanced male and female ratio and
The CAPS markers developed in this study could be
used for identifying male and female seedlings
obtained from seeds. Since the male:female ratio of
plants obtained from seeds has a 5:1 male and female
ratio, as reported earlier (Harsh et al. 1987), CAPS
markers could be used in early determination of sexes
in jojoba plantation. Among the CAPS markers
reported in this study, J888-Cla I differed from
JMS900. The use of JMS900 assumes that female
jojoba plants are those which do not have the
JMS900marker. However, the JMS900marker could
not identify individual sexes from mixed male and
female DNA samples, and so it is assumed that it
cannot differentiate naturally occurring hermaphro-
dite jojoba, reported in Vaknin et al. (2003), or
induced hermaphrodite jojoba plants which could be
obtained using reverse-genetic and TILLING exper-
iments (Martin et al. 2009). Therefore, researchers
dealing with TILLING studies or identifying her-
maphrodite from seedlings could use the gender-
specific CAPS marker developed in this study.
The CAPS markers (CAPS J818-Hinf I and CAPS
J888-Hind III) provide
JMS900. The RAPD marker MS900is more prone to
reproducibility problems owing to the fact that it uses
shorter and single primers, unlike CAPS markers
developed in this study, and is more vulnerable to
experimental conditions (Karaca & Ince 2008). The
CAPS system is easy to assay and more informative
than dominant markers such as JMS900.
It is known that the composition of jojoba wax,
simmondsin and its derivatives in the seeds and leaves
is influenced by both male and female genotypes,
along with environmental factors such as climate and
salinity. The use of male jojoba plants with desirable
traits including production of large quantities of
highly viable pollen, and bloom period at the same
time as the female plants in the plantation is critical
in jojoba improvement (Benzioni & Vaknin 2002;
Vaknin et al. 2003; Benzioni et al. 2005). Therefore,
the CAPS markers reported in this study along with
several advantages over
A. G. INCE AND M. KARACA
DNA markers linked to agronomically important
traits will be useful in jojoba breeding studies. For
instance, one of the CAPS markers reported in this
study, CAPS J818-Hinf I, contained another marker
showing segregation in dominant fashion with the
male-specific marker. These kinds of markers could be
used in the selection of superior genotypes with
desired characteristics. Current jojoba improvement
programmes generally use several criteria including
high seed yield, consistency in production, wax
content and quality, and plant habit. Thus, DNA
markers linked to these criteria will be very helpful in
This research was supported by the Scientific
Research Projects Coordination Unit of Akdeniz
University and the Scientific and Technological
Research Council of Turkey. We thank three anony-
mous referees for contributing valuable information
to this manuscript.
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