A reliable gender diagnostic PCR assay for jojoba (Simmondsia chinensis (Link) Schneider)
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- Ince, A.G., Karaca, M. & Onus, A.N. Genet Resour Crop Evol (2010) 57: 773. doi:10.1007/s10722-009-9516-1
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Jojoba (Simmondsia chinensis (Link) Schneider) is an obligate cross-pollinated shrub native to the Sonora desert. The most valuable product of the jojoba seeds is the liquid wax or jojoba oil which is marketed extensively in the cosmetic industry. Differing from the most of the cultivated crop species, jojoba has slow-growing habit, difficulties in the determination of sex at the early seedling stages, a male-biased ratio and low seed yield. In jojoba, the development of molecular strategies for the identification of sexes at early stages has been a priority in plantations and breeding programs. Two previous studies reported two candidate male-specific jojoba DNA markers. However, present study indicated that these markers were not useful in jojoba sex identification. A reliable gender diagnostic marker for jojoba is, therefore, needed. In the present study a novel jojoba male-specific touch-down polymerase chain reaction based DNA marker (JMS900) was reported using a total of 120 individual jojoba plants bulked into 16 samples. This sex specific DNA marker may have considerable theoretical and practical applications in the establishment of jojoba plantation and breeding studies.
KeywordsGender specific DNA markerSex identificationSimmondsia chinensisTouch-down PCR
Jojoba (Simmondsia chinensis (Link) Schneider) is a dioecious, obligate cross-pollinated and evergreen, small multi-stemmed shrub of the family Simmondsiaceae native to the Sonora desert (Gila Desert) which straddles part of the United States and Mexico border (Gentry 1958; Tobares et al. 2004; Benzioni et al. 2007). Although, jojoba grows wild in the desert of south-western United States and north-western Mexico, nowadays it is cultivated in other countries including Israel, Argentina, Peru and Morocco. The economic value of jojoba comes from its seeds which contain about 50–60% of a light yellow, odorless wax ester commonly referred to as jojoba oil. As many as 300 products containing jojoba have appeared in markets in recent years and the use of jojoba products will increase in future (Benzioni and Vaknin 2002; Benzioni et al. 2005).
The jojoba fruit is a green capsule enclosing up to three seeds. During the jojoba seed development, seeds accumulate long-chain esters of monounsaturated fatty acids and alcohols (known as liquid wax) along with some proteins and carbohydrates. The most valuable product of the jojoba seeds is the liquid wax or jojoba oil which is marketed extensively in the cosmetic, lubricant, computer industries, diesel fuel substitute, candles, plasticizers, detergents, fire retardants, transformer oil, and material for the leather industry (Radwan et al. 2007). Jojoba seeds also contain a group of chemicals known as simmondsins which have an anorexic effect and can be extracted and used as a satiating factor. Defatted meal that remains after winning the jojoba oil has not yet found wide commercial application (Purcell et al. 2000; Bellirou et al. 2005; Van Boven et al. 2000; Benzioni et al. 2007).
As it may occur in other industries of crop species, the jojoba industry faces the challenge of finding ways to improve productivity and quality of the products. Differing from most of the cultivated crop species, jojoba has slow-growing habit, difficulties in the determination of sex at the early seedling stages, a male-biased (5:1; male: female) ratio and low seed yield (Benzioni and Vaknin 2002; Agrawal et al. 2007; Sharma et al. 2008). Commercial plantations of jojoba are mainly established using cuttings, or in a few cases grafting, resulting in a narrow genetic diversity that may cause the low seed yield (Dunstone et al. 1985). Coates and Ayerza (2008) reported that commercial plantations of jojoba in the Hyder Valley in Arizona and in Catamarca, Argentina dramatically decreased in size and number, going from 10,000 ha in the early 1980s to about 2,000 ha in Arizona, and from 3,000 ha in Catamarca in the late 1990s to approximately 300 ha in 2008. Coates and Ayerza (2008) argued that the main reasons for this decrease were the low pollination percentage and the use of inferior genetic material.
In dioecious plants including jojoba, the development of molecular markers for sex identification at early stages has been a priority in breeding programs, in order to increase their economic potential and understand better the basis of sexual dimorphism. Sex identification in many crop species uses DNA markers which offer several advantages in comparison to morphological markers (Tan et al. 2003; Karaca et al. 2004). DNA markers have been used in sex identification in many plants species including Carica papaya L. (Lemos et al. 2002; Chaves-Bedoya and Nunenz 2007; Urasaki et al. 2002), Salix viminalis L. (Alstrom-Rapaport et al. 1998), Atriplex garrettii Rydb. (Ruas et al. 1998), Mercurialis annua L. (Khadka et al. 2002), Ginkgo biloba L. (Jiang et al. 2003), Melandrium album (Miller) Garcke (Kumar and Arumuganathan 1997), Viola pubescens Aiton (Culley and Wolfe 2000), Humulus lupulus L. (Danilova and Karlov 2006), Cycas circinalis L. (Gangopadhyay et al. 2007), Myristica fragrans Houtt. (Ganeshaiah et al. 2000), Pistacia vera L. (Hormaza et al. 1994; Kafkas et al. 2001), Cannabis sativa L. (Mandolino et al. 1999; Torjek et al. 2002), Encephalartos natalensis R.A. Dyer et I. Verd. (Prakash and Van Staden 2006), Eucommia ulmoides Oliv. (Xu et al. 2004), Calamus simplicifolius C. F. Wei (Yang et al. 2005).
Literature search indicated that two candidate male-specific DNA markers were developed for jojoba. One of the two male-specific markers is a random amplified polymorphic DNA (RAPD) marker; OPG-51400 reported in Agrawal et al. (2007) and the other one is an Inter-Simple Sequence Repeat (I-SSR) marker; UBC-807 reported in Sharma et al. (2008). Both markers were used in identification of male jojoba plants in the present study. However, analyses indicated that the use of these two male-specific markers in identification of male jojoba plants is limited. It is therefore, possible to say that a reliable gender diagnostic marker for jojoba is still needed.
In the present study a novel jojoba male-specific touch-down polymerase chain reaction based DNA marker (JMS900) was developed. JMS900 was tested using a total of 120 individual jojoba plants bulked into 16 samples and some individual plants whose genders were not known (blind samples). Results indicated that JMS900 was highly reproducible and reliable male-specific marker. It is assumed that stated sex specific DNA marker may have considerable theoretical and practical applications in the establishment and breeding studies in jojoba. DNA sequence analysis of JMS900 marker may provide additional information for understanding the genetic basis of sex dimorphism in jojoba and other plants.
Materials and methods
Leaves of jojoba plants grown in a plantation located 102 km to the west of Antalya, Turkey, were used as plant materials. This jojoba plantation was established in March 1990 from the seeds brought from California, USA. A total of 120 samples consisting of 60 female and 60 male jojoba plant leaves were collected and numbered individually. After being transported to the laboratory, samples were divided into 8 subgroups each of which consisted of leaves from either male or female plants. Subgroups A, B, C and D contained leaves from the male plants and numbered 1–15, 16–30, 31–45 and 46–60, respectively. Subgroups E, F, G and H contained leaves from the female plants and numbered 61–75, 76–90, 91–115 and 116–120, respectively. Several bulked samples consisting of 4–8 individual leaves were also made for analyses. In addition to these plant materials a total of 20 jojoba leaf samples whose genders were not known were used to test the reliability and reproducibility of male-specific marker developed in the present study.
A total of 10 g leaf samples randomly picked from gender-specific bulks of the subgroups along with individual male and female samples were ground in a wide mortar using liquid nitrogen. For DNA extractions 5 g powdered leaf samples from bulks and individual samples were used and total genomic DNAs were extracted and analyzed according to Karaca et al. (2005). Also DNAs of randomly selected five bulk and individual plant samples were extracted using a commercial DNA extraction kit (Invitrogen). Concentration of the all DNA samples was readjusted so that each sample contained 0.12 μg/μl genomic DNAs after spectrophotometer and agarose gel electrophoresis analyses.
Touch-down polymerase chain reactions
In the present study a touch-down polymerase chain reaction (Td-PCR) approach was used to amplify jojoba DNA samples. One hundred and twenty nanograms genomic DNAs were amplified using a total of 62 primers, some of which generated from minisatellite regions of plant and animal genomic DNAs (Karaca et al. 2008) and some others were randomly designed. In each Td-PCR, a total of 0.12 μg of DNA, and 2.4 μM primer were used in a 25 μl reaction buffer containing 80 mM Tris–HCl (pH 8.8), 19 mM (NH4)2SO4, 0.009% Tween-20 (w/v), 0.28 mM each dNTP, 3 mM MgCl2 and 2 units of Taq DNA polymerase (Bioron). Touch-down polymerase chain reactions were carried out in a Thermo Hybrid Px2 thermal cycler with the following amplification profiles: 3 min hold at 94°C, followed by a 10 cycle pre-PCR consisting of 1 min at 94°C for denaturation, 50s at 50 or 42°C for annealing and 2 min at 72°C for extension. The annealing temperature was reduced by 0.5°C per cycle for the first 10 cycles (touch-down cycles). Amplification of the targeted DNA templates continued for a further 30 cycles at 37 or 42°C annealing temperature and ended with a final extension step at 72°C for 10 min. After Td-PCR completed, samples were stored at −20°C until agarose gel electrophoresis studies. In the present study polymerase chain reactions for candidate male-specific OPG-51400 and UBC-807 were performed according to Agrawal et al. (2007) and Sharma et al. (2008).
Agarose gel electrophoresis
Fifteen microliters of amplified Td-PCR products were mixed with 5 μl loading buffer containing 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF, 40% (w/v) sucrose. Samples were loaded into the wells of 2% agarose gels. Agarose gels were prepared with Tris–borate EDTA buffer consisting of 89 mM Tris–borate, 2 mM EDTA (pH 8.0) and 0.5 μg/ml ethidium bromide. DNA samples were then electrophoresed at 4 V/cm at constant current for 8–16 h in the presence of Tris–borate EDTA buffer. After agarose gel electrophoreses completed DNA samples on the gels were visualized and photographed on an UV transilluminator for analysis.
Gender (sex) specific DNA marker
In the present study a gender or sex specific DNA marker was defined based on the following criteria: a sex specific DNA marker should distinctly differentiate female and male plants, being present or absent between female and male jojoba genomic DNAs and should be reproducible within and between polymerase chain reactions and between different DNA extractions.
In order to represent all the morphological and genetic differences within the plantation the present study used bulked and individual samples obtained from 120 plant samples for analyses. A high level of polymorphic amplicons (DNA markers) between individual male and female samples was obtained using a total of 62 primers some of which consisted of minisatellite regions. The sequence information of the minisatellite primers were obtained from enriched minisatellite regions of several organisms. In addition to minisatellite primers, a total of 40 primers were also used (Karaca et al. 2002; Ince et al. 2009a). Since lengths, sequence and annealing temperatures of primers used in the present study were quite different the value selected for the annealing temperatures and other variables including the concentrations of Mg2+, H+, dNTPs, and template were determined empirically. In the present study several primers were used in the amplification of genomic DNA extracted using a method described in Karaca et al. (2005) and commercial kits (PureLink Genomic DNA Kits, Invitrogen). Several DNA templates were amplified within the same PCR runs and between different PCR runs. The optimized Td-PCR always produced repetitive results within and between PCRs and between templates obtained from different DNA extractions. Reproducible results obtained from the Td-PCRs indicated that amplification of the nonspecific sequences, which are usually present in RAPD technique due to nonspecific primer binding, was avoided (Karaca and Ince 2008).
Among the 27 candidate DNA markers, some of which co-segregated with female or male genotypes, 26 were found to be non gender-specific markers (Fig. 1a). The level of polymorphism at the candidate gender-specific alleles decreased from 27 to 1 when bulked samples were used. In order to determine whether this decrease of polymorphism was due to allelic deletions, which may occur during the PCR amplification of bulked samples, individuals of several bulked sample DNAs were re-amplified using the same Td-PCRs. Analyses indicated that candidate female- and male-specific alleles segregated within male and female samples, indicating that they were not gender-specific markers. This also indicated that decreased level of polymorphism between male and female bulked samples in comparison to individual male and female samples were not due to the allelic deletions (Ince et al. 2009b).
In order to validate OPG-51400 and UBC-807, bulked and individual female and male samples were analyzed using the protocol provided in Agrawal et al. (2007) and Sharma et al. (2008). Analyses of amplified products of candidate male-specific OPG-51400 marker indicated that in some individual and bulked samples, there were weak or strong bands of 1,400 bp in female samples. However, further analyses clearly indicated that OPG-51400 segregated within female and male samples and could not be used in identification of male jojoba plants (Fig. 1b). Analyses also indicated that UBC-807 segregated within male and female jojoba samples. Majority of the plant materials regardless of the genders used in the present study contained UBC-807 (Fig. 1c). Based on these findings the present study indicated that OPG-51400 and UBC-807 could not be reliably used in jojoba gender identification studies.
The results presented here indicate that it is now possible to accurately and rapidly determine the sex of jojoba plants within a single day using a Td-PCR-based technique requiring only a minimal tissue sample taken, for example, from seedlings or mature trees. Since the sex of jojoba plants cannot be determined from morphological characteristics until flowering which takes about 2–3 years development of male-specific JMS900 marker is important for plantation and breeding studies in jojoba.
Sex or gender determination in plants is a fundamental developmental process that is particularly important for economic reasons, because the sexual phenotypes of commercially important crops dictate how they are bred and cultivated. Commercial value of the jojoba has been increased and it is now being cultivated in every part of the country where it is grown for seed production. Sex identification at earlier stages in jojoba is important in plantation for commercial production since the female plants are usually valued for the commercial production. A productive jojoba plantation requires a well balanced female to male plant ratio to obtain sufficient pollination percentages and good agricultural practices. For instance in the subtropical climate, one male plant is considered to be enough to pollinate five female plants (Harsh et al. 1987; Prakash et al. 2003). Knowledge on the gender of the seedlings prior to their transplantation to the field is useful to obtain a desired ratio of male and female plants and this will help to manage resources including planting space, the amount of fertilizers, water and the labor costs.
Sex or gender specific DNA markers in dioecious plant species have been identified based on DNA analyses using polymerase chain reaction. In the present study a reliable jojoba male-specific marker was identified. In jojoba breeding studies the genetic properties of the male and female should be considered since the effect of the male and female parents on yield and wax compositions are additive and highly significant (Benzioni and Vaknin 2002).
Although previous studies reported two different putative male-specific markers in jojoba, the present study could not confirm that these markers (OPG-51400 and UBC-807) were male-specific (Agrawal et al. 2007; Sharma et al. 2008). Inter-simple sequence repeat (I-SSR) marker (UBC-807) is similar to random amplified polymorphic DNA (RAPD) marker (OPG-51400). Although the I-SSR technique is similar to RAPD, I-SSR primers consist of a di or trinucleotide simple sequence repeats with a 5′ or 3′ anchoring sequence of 1–3 nucleotides. Compared with RAPD primers, the I-SSR primer sequence is usually larger, allowing for a higher primer annealing temperature, which results in greater band reproducibility than RAPD markers. Both techniques have been used in many plant and animal species; therefore, they are equally reliable techniques. The reason why these two markers could not reproduced in the present study could be due to the limited number of plants that were tested in as it was reported that UBC-807 was based on eight cultivars (Sharma et al. 2008) and OPG-51400 was based on four cultivars (Agrawal et al. 2007). In the present study we tested OPG-51400 and UBC-807 markers on more than ten cultivars and many genotypes. It is more likely that both OPG-51400 and UBC-807 markers were cultivar specific and apparently co-segregated with the male genotypes.
JMS900 DNA marker is reproducible and distinct; therefore, it may not be necessary to convert JMS900 marker into cleavage amplified polymorphic sequence (CAPS) or PCR-restriction fragment length polymorphism (PCR–RFLP) and sequence characterized amplified region (SCAR) markers. Using the JMS900 marker, male jojoba plants can be rapidly and reliably determined. Recent studies of sex determining mechanisms have demonstrated clearly that several crop plants have evolved a variety of sex-determining mechanisms that involve a number of different genetic and epigenetic factors, from sex chromosomes to plant hormones. A male-biased ratio (5:1; male: female) is an indication of non-sex-chromosome based gender development and DNA marker specific to male was also an indication of existence of non epigenetic factors involve in gender development in jojoba. The cloning and molecular characterization of JMS900 will greatly increase our understanding of the flowering process and of sex determination in jojoba.
This research is supported by the Scientific Research Projects Administration Unit of Akdeniz University and The Scientific and Technological Research Council of Turkey. We also thank Dr. Ozgur Akdesir for providing the plant materials used in this study.