African Journal of Biotechnology Vol. 10 (4), pp. 470-474, 24 January, 2011
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Sex determination of jojoba (Simmondsia chinensis cv.
Arizona) by random amplified polymorphic DNA (RAPD)
F. S. Hosseini1, H. Shahsavand Hassani2, M. J. Arvin2, A. Baghizadeh3 and G. Mohammadi-
1Graduated Ms Student, Shahid Bahonar University of Kerman, Kerman, Iran.
2Horticultural Research Institute, Shahid Bahonar University of Kerman, Kerman, Iran.
3International Center for Science, High Technology and Environmental Science, Kerman, Iran.
4Horticultural Research Institute, Shahid Bahonar University of Kerman, Kerman P.O.B 76169-133 Iran
Accepted 19 November, 2010
Jojoba (Simmondsia chinensis (Link) Schneider) is a dioecious shrub that produces fruits in female
plants. Its seeds stores liquid wax which is used in cosmetic, pharmaceutical and plastic industries.
This species is generally propagated by seed. The sex of seedlings is not distinguishable by cytological
and seed cultivation methods. This investigation was carried out to study the sex-specific random
amplified polymorphic DNA (RAPD) markers in thirteen 4-year-old jojoba plants from two provinces of
Iran and DNAs of those populations were extracted by CTAB method. Out of the 20 tested primers, two
primers, namely F1 and F10, produced 460 and 680 bp fragments, respectively and were importantly
recognized to distinguish between female and male plants, accordingly. Also, the results of the ratio
difference test showed that, more efficient sex determination of jojoba seedlings is done using both F1
and F10 primers due to gene cooperation between them. The preliminary results of this study for sex
determination would help the recognition of potential fruit-bearing seedlings for having high yield per
hectare in horticultural systems. Furthermore, the findings would help saving time and economic
resources in jojoba breeding programs.
Key words: Jojoba, sex determination, dioecy, random amplified polymorphic DNA (RAPD) molecular marker.
Sexual reproduction is a prominent feature of the life
cycle in most animals and plants (Ming et al., 2007). The
pistil and stamen are the parts of the flower sheltering
ovules and producing pollen grain, respectively. In most
typical dioecious plants, pistil and stamen develop as
separate individuals, which are distinguished as
“pistillate” and “staminate” plants (Xu et al., 2004). The
genetic basis of sex determination in dioecious plants
*Corresponding author. E-mail: G.Mohamadinejad@yahoo.com.
Tel: +98-341-320-2639. Fax: +98-341-322-2043.
Abbreviations: RAPD, Random amplified polymorphic DNA;
CTAB, cetyl trimethyl ammonium bromide; SCAR, sequence
characterized amplified region.
may be extremely diverse. Some dioecious species have
heteromorphic sex chromosomes (for example, silene
latifolia), whereas in other species, sex is determined by
one or several autosomal nuclear loci, possibly influence
by cytoplasmic genes (Alstrom-rapaport et al., 1998).
Jojoba (simmondsia chinensis) a dioecious species, is an
important crop shrub that produces commercially valu-
able seeds in female plants. It is now the only species
belonging to the genus Simmondsia in family of
Simmondsiaceae. Its seeds stores liquid wax which is
used in cosmetic, pharmaceutical and plastic industries
(Tyagi and Prakash, 2004). It has promising physical
properties, such as high viscosity index, high flash and
fire points, high dielectric constant and high stability and
freezing point that can be used in various industries
(Agrawal et al., 2007). Due to its potential to make
canopy and eliminating windy erosion in desert regions in
Iran, its adaptation was initiated by Shahsavand Hasani
et al. (2006).
Jojoba sex chromosomes are not distinguishable.
Therefore, sex type of jojoba seedlings cannot be deter-
mined by cytological methods (Parasnis et al., 2000).
Also, sex type of jojoba seedlings can not be determined
either by embryo shape or morphology at the juvenile
developmental stage. Propagation of jojoba is mainly
through seeds (Singh et al., 2008). Therefore, three to
four years are required for this shrub to reach the flowe-
ring stage of its life cycle and it is a slow growing and the
ratio of male to female plants in the field is around 5:1
(Agrawal et al., 2007). In general, male plants are not
useful commercially, therefore, the farmers eliminate a
considerable number of male plants and this increases
The last decade has witnessed an increasing number
of research efforts directed at identifying and characte-
rizing molecular markers and genes involved in plant
dioecy (Kafkas et al., 2001). Random amplified polymor-
phic DNA (RAPD) is a simple identifier of polymorphism
and has been used for identification of dioecious cultivars
in phylogenetic studies (Xu et al., 2004), selection of
traits of interest (Yakubou et al., 2005) and classification
of plants at the genus and species levels (Wong et al.,
2004; Bouza et al., 2006). The objective of this study is to
develop a sex-specific RAPD marker linked to sex
determination in jojoba seedlings.
MATERIALS AND METHODS
Plant materials consisted of leaves from 11 different jojoba geno-
types (cv. Arizona) collected from plants after the complete
expression of the sexual phenotype in two locations. The first group
is made up of 6 plants from 4 year old jojoba in Kerman University,
Iran, including 3 male and 3 female plants. The second group of
jojoba plants (cv. Arizona) was obtained from agriculture research
centre in Fars province, Iran, consisting of 7 plants including two
male and five female plants. The leaf samples of these plants were
stored in an ice box or liquid nitrogen before transporting to the
laboratory for further processing and then all specimens stored at -
80° C before extracting DNA for screening sex linked DNA marker
by RAPD analysis.
Genomic DNA isolation
Total genomic DNA was isolated from 1 g of leaf tissues of female
and male jojoba plants using the standard CTAB method with minor
modifications (Saghari-maroof et al., 1984). The plant tissues were
ground to a fine powder in liquid nitrogen, transferred to eppendrof
tubes and 700 ml of warm extraction buffer was added. The mixture
was incubated at 65° C for 30 min. Chloroform: octanol (24:1) was
added and the solution was mixed by inversion and centrifuged at
10000 rpm for 10 min at room temperature. The chloroform: octanol
step was repeated. The aqueous phase was transferred to another
tube. DNA precipitated with equal volume of isopropanol, mixed
and incubated at -24° C for 20 min then centrifuged at 10000 for 10
min. The isopropanol was removed and the DNA pellet was rinsed
Hosseini et al. 471
with 75% ethanol. Then, chilled ethanol (-24° C) was added to DNA
precipitation and centrifuged at 10000 rpm for 5 min. Finally, the
precipitated DNA was air-dried and dissolved in TE buffer.
DNA concentration was determined in a Biowave S2100 spectro-
photometer. Also, the purity of DNA was checked by OD260/280
and through gel electrophoresis with 0.8% agarose gel. In all cases,
the extracted DNA was diluted to a final concentration of 50 ng/?l
and then was used for polymerase chain reaction amplification
RAPD marker analysis
Twenty primers from Cinagene Company (Iran) were used for
RAPD analysis. The PCR was performed in a reaction volume of 25
?l using the eppendrof thermal cycler. The reaction mixture con-
tained one unit of Smar Taq polymerase (Cinagene Company), 50
ng of male or female genomic DNA, 0.4 ?M of RAPD primer, 2.5 ?l
of 10 × PCR reaction buffer (500 mM KCl, 100 mM Tris HCl) and
1.5 mM MgCl2, 0.2 mM of each dNTP. The RAPD-PCR reactions
were carried out at 94°C for 5 min, followed by 40 cycles at 94°C for
45 s, different annealing temperature with various type of primer for
45 s, 72°C for 1 min, and final extension at 72°C for 7 min. Amplifi-
cation products were electrophoresed in 1% agarose gel and
visualized by ethidium bromide staining in 0.5 x TBE for 3 h at 80 V.
Standard molecular weight markers were also used in the
The results of primers polymorphism information content
(PIC) of 13 jojoba plants showed that PIC ranged from
0.32 to 0.89 (Table 1). The highest and lowest amounts
were observed in primer 396 and 391, respectively.
Having the Higher amount of PIC value is related to the
power of primer for detection of Genetic variability in the
population, (Mohammadi-nejad et al 2008). Hence,
primer 396 (5'-GAATGCGGAG-3') was identified as the
best primer for genetic diversity in jojoba plants. The
results of RAPD primers indicated 79 bands for sex-
specific DNA markers. 21 monomorphic bands were
identified in male and female plants. Furthermore, 58
polymorphic bands were determined out of them and 56
bands had no role in sex determination, since they were
found in both male and female plants. Two other bands
where detected by F1 (5'-AGGAGTCGGA-3') and F10
(5'- GGGCCACTCA-3') primers, these bands showed
different sex patterns in male and female plants. 460bp
fragment (F1 primer) was present in individual female but
absent in individual male plants (Figure 1). This band was
called “female sex-specific band”.
F10 primer detected a 680 bp fragment, only in male
plants (Figure 2). Furthermore, statistical analysis of the
ratio difference showed that, the difference of 460 bp
fragment presence in female plants was significant, since
this band was detected in 7 out of 8 female plants.
Therefore, this band can be referred to as, sex- specific
472 Afr. J. Biotechnol.
Table 1. Number of alleles and polymorphism information content (PIC) value of
rapid markers for 11 jojoba genotypes.
Number of alleles
Frequency of main alleles
Figure 1. RAPD banding patterns of male and female jojoba plants (cv. Arizona)
obtained by the arbitrary primer F1 which indicates a 460 bp sex-linked band in
female plants. (F, female plants; M, male plants; Mw: 100 bp ladder marker).
Hosseini et al. 473
Figure 2. RAPD banding patterns of male and female jojoba plants (cv. Arizona)
obtained by the arbitrary primer F10 which indicates a 680 bp sex-linked band in male
plants. (F, female plants; M, male plants; Mw, 100 bp ladder marker).
band in female plants. The ratio difference test indicated
that, the difference of 680 bp fragment presence in male
plant was not significant and it is referred to as, male sex-
specific band. Since F1 and F10 primers, were able to
differentiate male and female plants from each other, with
460 bp fragment present in seven female plants and ab-
sent in five male plants and 680 bp fragment absent in
seven female plants but present in five male plants; it is
therefore recommended that, both F1 and F10 primers be
used together in the sex determination of jojoba
Due to the absence of genetic information on sex deter-
mination in dioecious plants, the use of molecular mar-
kers for discriminating between staminate and pistillate
genotype is worthwhile (Xu et al., 2004). In recent
decades, the use of molecular markers in sex determi-
nation have been increased, for example amplified
fragment length polymorphism (AFLP) markers were
developed in Asparagus officinallis and Ficus fulva
(Spada et al., 1998; Parrish et al., 2004), DAF marker in
papaya (Somsri et al., 1998) and RAPD markers in
Papaya, Cannabis sativa and Populus tomentosa (Lemos
et al., 2000; Deputy et al., 2002; Mandolino et al., 2002;
Hou et al., 2009). The most common marker for sex
determination is RAPD marker. Agrawal et al. (2007)
introduced a 1400 bp RAPD fragment for sex deter-
mination in jojoba plants but in the present research this
primer (OPG-5) was not able to detect male and female
plants in few individuals of jojoba seedlings. Harvey et al.
(1997) have used the primer F1 for sex identification in
Actinidia chinensis species and was able to detect female
plants from male ones. These results show the possibility
of similar conserved sequences of A. chinensis and S.
chinensis genomes and perhaps also exist in many other
In Humulus lupulus (Polley et al., 1997) and Asparagus
(Jiang, 1997) utilized 1000 and 760 primers for sex
identification, respectively. One primer was only used for
designing sex-specific SCAR primers. Hormaza et al.
(1997) used 400 RAPD primers for sex determination in
pistacia and found one band in female plants. In Salix
viminalis also 350 random decamer primers were tested
and only one band was detected (Alstrom-Rapaport et
al., 1998). Agrawal et al. (2007) worked with 72 markers
474 Afr. J. Biotechnol. Download full-text
in jojoba and found only one sex-specific RAPD marker,
whereas in this study we used 20 RAPD markers in
jojoba and found one sex-specific marker for male and
one for female. Furthermore, in pepper two female-speci-
fic bands was obtained using random decamer primers
(Banerjee et al., 1999). Shirkot et al. (2002) also reported
six female-specific markers and two male-specific
markers in Actinidia deliciosa. Also, in S. viminalis two
female-specific markers were found (Gunter et al., 2003).
The results of this study support that, sex determination
in jojoba may be affected by one or several autosomal
loci, or several loci possibly when an epistatic manner is
involved in sex determination. The preliminary results of
this study for sex determination would help in the
recognition of potential fruit-bearing seedlings for having
high yield per hectare in horticultural systems. Further-
more, the findings would help save time and economic
resources in jojoba breeding programs.
This work was supported by International Center for
Science and High Technology and Environmental
Sciences of Kerman. Also authors express their gratitude
to College of Agriculture and Horticultural research
Institute, Shahid Bahonar University of Kerman, Iran.
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