Background
In the plant kingdom, dioecious plants are estimated to constitute 4% of all flowering plants and are represented in 75% of plant families (Yakubov et al., 2005). Dioecious plants are highly evolved members of the plant kingdom in terms of sexual differentiation. Mechanisms of sexual differentiation in plants are almost overwhelmingly diverse, therefore, it is impossible to answer sex-related questions during seedling stage. Most models of sex determination in dioecious plants are based annuals such as Silene or Rumex that possess heteromorphic sex chromosomes (Parker, 1989). Although these species represent useful models for understanding the control of gender in a broad sense, the liability of sex determination mechanisms in plants without sex chromosomes suggests that such models may prove to be unsuitable for the majority of dioecious plants.

The application of molecular techniques has advanced the isolation and characterization of many floral meristem identity genes (Ainsworth, 2000). However, it is unclear what direct effect such genes may have on sex determination in monoecious and dioecious species. Furthermore, despite evidence that sex determination is under genetic control in dioecious species with heteromorphic sex chromosomes (e. g., Rumex and Silene), it is doubtful that the same mechanism would control sex in individual flowers in monoecious plants or in dioecious plants without sex chromosomes (Ainsworth et al., 1998). However, the presence of sex chromosome has been claimed for some dioecious angiosperms, but only in few cases have these claims been documented (Singh et al., 2002).

Trichosanthes dioeca, a plant with great medicinal and nutritive value is yet to be exploited to its full extent due to absence of reliable breeding programmes owing to low growth rate of seedlings and well-established dioecism nature of the sex types (Nanda et al., 2013). There is no robust evidence of sex chromosomes in T. dioica, thus there is no method for distinguishing between male and female plantlets prior to flowering in T. dioica. A reliable method for determining the gender of pointed gourd before flowering would facilitate breeding programs. The last decade has witnessed an increasing number of research efforts directed at identifying and characterrizing molecular markers and genes involved in plant dioecy (Yakubov et al., 2005). Previous studies have provided information indicating that use of molecular markers in breeding programs can save time and economic resources in breeding programs. Several researchers have shown that random amplified polymorphic DNA (RAPD) banding patterns are linked to sex in (Esfandiyari et al., 2012).  RAPD is a rapid and inexpensive method for studies on genotypic relationships and selection of traits of interest. Sequence characterized amplified regions (SCARs) are commonly derived from RAPD markers and represent a genetically defined locus, which is amplified from the genomic DNA by specific oligonucleotide primers. As there is only single report on the sex associated molecular markers of pointed gourd (Nanda et al., 2013), this study can also be regarded as the leading report on the use of the SCAR technique for gender determination in pointed gourd.

1 Result
1.1 Identification of sex-specific RAPD markers
Of 104 RAPD primers evaluated, 21 produced clear and reproducible DNA profiles with a total of 971 amplification bands for the bulked sample of female and male plants of which 96 bands were polymorphic. The approximate size range of the RAPD products was 200 bp (22E1) to 2000 bp (OPA-17). Only two markers, OPC-05 and OPK-07, were producing distinguish banding pattern of 1.0 kb (named as OPC-05₁₀₀₀; Figure 1) and 300 bp (named as OPK-07₃₀₀; Figure 2) and were considered as associated with maleness and femaleness, respectively. These amplicons were extremely reproducible under a broad range of amplification temperature without any variation in the results. The marker OPC-05₁₀₀₀ amplified a unique band in bulk DNA of male and 10 individual male DNA, whereas similar fragment is not observed in the bulk and 44 individual females. Similarly, marker OPK-07₃₀₀ amplified a unique fragment only in the bulk and individual females. This amplification was repeated for five times in individuals as well as bulk genomic DNA corresponding to male and female accessions to confirm the reproducibility and consistency. Similar results were obtained in each repetition. The bands were consistently presence/absent in the bulk and male/female individuals. These unique markers indicating that both markers was closely linked to the sex gene and are candidate for SCAR marker development, hence will useful for determining sex in pointed gourd.

Figure 1 A male-specific RAPD fragment in pointed gourd amplified using primer OPC-05. M indicated size marker. The arrows denote the position of the 1000-bp fragment, and cause gender differences among female and male plants

Figure 2 A female-specific RAPD fragment in pointed gourd amplified using primer OPK-07. M indicated size marker. The arrows denote the position of the 300-bp fragment, and cause gender differences among female and male plants

1.2 Conversion of OPC5₁₀₀₀ and OPK-07₃₀₀ to SCAR markers
As the fidelity and the reproducibility of RAPD marker is often questionable, the current trend is to convert the RAPD sequence into a SCAR marker which is more reliable and reproducible. Based on the sequence of OPC5₁₀₀₀ and OPK-07₃₀₀, five and one pair of specific forward and reverse SCAR markers were designed, respectively (Table 1). The designed SCAR primer pairs were used to amplify genomic DNA of all 54 accessions. Among five primer pair combinations designed from male specific sequence, SCF/R-3 marker discriminated male and female accessions. This SCAR amplification produced a single specific band of predicted size of 628 bp in all the males without any amplification in the females. Reduction of the annealing temperatures did not generate any spurious fragment other than the specific SCAR, confirming the specificity of the SCAR primer for maleness. However, this marker failed to discriminate one accession among 44 female accessions. With this exception, remaining 43 female accessions were clearly discriminated by this marker. In case of female specific OPK-07₃₀₀ amplicon, with small size of sequence, Primer3 could design only one pair of primer. But this marker was failed to discriminate male and female accessions.

Table 1 Sequence of SCAR primer used for male and female identification

1.3 BLAST analysis of male sex specific RAPD amplicons
The OPC-05₁₀₀₀ with 38% G+C content (A:371; C:211; G:212; T:323) did not possess any open reading frames in either orientations. BLAST results revealed that the sequence has partial homology (ranging from 54.8% to 59.8% identity over a length of 169-232 bp) with known plant nucleotide sequences at different sequence-similarity levels. BLAST searches were made to determine if the sequenced fragment might identify a known gene or mapped sequence. The Phylogenetic tree obtained based on sequence similarity revealed that Solanum lycopersicum, Populus trichocarpa, Lycopersicon esculuntum and L. pimpinellifolium were similar and grouped together. In the same cluster, Salix chamissonis and Phoenix dactylifera were also present, but Trichosanthes dioica Roxb and Coccidiodes posadassis were not clustered. This reveals that, even though sequences obtained in present studies are similar to the sequences of above mentioned plant species, but they are not identical to them.

2 Discussion
Dioecy prevents intra-individual self-pollination and is one of the most extreme inbreeding avoidance mechanisms (Ainsworth, 2000). Efforts to identify sex type in dioecious plant at seedling stage is important for selecting female or hermaphrodite plants for transfer to the field, to gain time and reduce costs (Chaves-Bedoya and Nunez, 2007). To date, several molecular markers including RAPD have been exploited for sex type discrimination in dioecious plants. RAPD, without prior DNA sequence information, can reveal high degree of polymorphism; easy to perform and provides wider coverage of the genome. However, RAPD might lead to irreproducibility; therefore RAPD marker was converted into a SCAR to distinguish sex type in pointed gourd (Paran and Michelmore, 1993). This would increase the reliability and reproducibility of PCR assays. SCARs are advantageous over RAPD markers because they are co-dominant, detect only single genetically defined loci, identified as distinct bands in agarose gels, are easier to score, less sensitive to reaction conditions and are more reproducible. The SCAR has been successfully developed in the case of Mercurialis annua (Khadka et al., 2002), Carica papaya (Urasaki et al., 2002) and Cannabis sativa (Mondolino et al., 1999).

Pointed gourd has also been studied and found to have a RAPD marker associated with females (Singh et al., 2002; Kumar et al., 2012). Since pointed gourd is an  importantdiocious vegetable crop, the SCARs developed in present study will be useful for its improvement. To identify the sex associated genome segment, sufficient number of RAPD primers were tested before generating the SCAR marker. This is in agreement with Jiang and Sink (1997) where 760 primers were used in Asparagus officinalis in order to find a SCAR marker associated with the M (male-determining) locus. The chances of any RAPD markers being linked to a gene or a genomic region of interest is mainly dependent on genome size, type of gene or genomic region and on the type of population used for marker analysis (Kumar et al., 2008). For example, (Hormaza et al., 1994) found only one female-specific RAPD marker in pistachio after testing 700 primers. On the other hand, Kumar et al. (2012) reported five bands from 41 random decamer primers were associated with sex expression. In present study, the polymorphic fragments from OPC05 appear to have the potential for development of a molecular marker based technique for early identification of pointed gourd sex expression. This indicates that sex determination in T. dioica is under a simple genetic control, possibly by a single gene or few tightly-linked genes. Similarly Sakamoto et al. (1995) screened only 15 decamer primers and found two markers, viz., Pri-08500 and Pri-11730 that appeared to be tightly linked to the male sex. If the sex expression is governed by heteromorphic chromosomes, a large number of sex-specific bands should be appeared (Jiang and Sink., 1997). Similar to present result, Kumar et al. (2008) also reported a male sex associated RAPD fragment of 1000 bp from OPC-05 in pointed gourd. Cloning and sequencing of OPC-05₁₀₀₀ and subsequent BLAST analysis revealed that this sequence shared 93% identity with Salix Chamissonis.

SCAR primer SCF/R-3 proved to be highly effective for the discrimination of male and female accessions. This result suggests that the SCAR marker is located in a region of the chromosome responsible for maleness as it is present only in male. However, this marker failed to discriminate one accession among 44 female accessions. This may be due to crossing over of male linked gene from Y chromosome with X chromosome or amplification of unspecific product in female may also occur due to presence of some sequence similarity. The same pattern of result was obtained by Chaves-Bedoya and Nunez (2007) in their study to discriminate male and female plants in papaya. In case of female specific OPK-07300 amplicon, the marker was failed to discriminate male and female accessions. This may be due to either small size of the fragment obtained during initial characterization or a single base differences or single base mutation at the primer. The result is in agreement with Niroshini et al. (2008) in papaya. However, the SCAR marker SCF/R-01 was monomorphic and did not discriminate female from male plants. This loss of polymorphism during RAPD to SCAR conversion was most likely due to site mutation at the original RAPD primer binding site (Paran and Michelmore, 1993). This is the first report on the development of SCAR marker in pointed gourd that can be used to discriminate male plants from female plants at early stages of development to support breeding programs. The results presented in this study show a possible new SCAR marker that can be used. This reduces the time required differentiating male and female plants which depends on the evaluation of morphological characters. The marker can also be used to discriminate male and female plants produced at commercial scales in tissue culture laboratories at seedling stage.

3 Materials and methods
3.1 Plant material and DNA extraction
Plant materials used in this study included 10 male and 44 female genotypes, currently being cultivated in Gujarat, were sampled from the different area of Gujarat state. Genomic DNA from three weeks old, field grown plants (raised from stem cuttings) was extracted using a modified cetyltrimethyl ammonium bromide (CTAB) protocol described by Singh et al. (2002). The genomic DNA quality and quantity were assessed through Nano drop V2.02 spectrophotometric measurement and uncut lambda DNA.

3.2 Screening of RAPD
For the initial screening, only two bulk DNA samples (male and female) were prepared by pooling an equal amount of DNA from 10 individual male and female cultivars. These bulks were amplified with 104 RAPD primers. Once polymorphism was detected with a particular primer, then the primer was used to amplify the DNA samples from individual plants for sex-typing. The polymorphic markers were verified for their consistency and reproducibility using DNAs from 10 random male plants and 10 female plants from field. Putative sex-linked markers which differentiated the male and female bulks as well as the individuals of each sex are used for developing the SCAR marker. PCR Amplifications were carried out in a 25 µl reaction volume containing 2 µl DNA (50 ng), 12.5 µl Master Mix (Genei, Bangalore, India), and 1 µl of 10 pmol of primer. Primer concentrations viz., 5, 10 and 20pM were used to optimize the amplification conditions. Based on the results, 10pM concentrations of primers were used for RAPD analysis. Amplification was performed in following steps: initial denaturation at 94°C for 5 min, 40 cycles of denaturation at 94°C for 1 min, primer annealing at 38°C for 1 min, extension at 72°C for 2 min, and final extension at 72°C for 7 min. Electrophoresis was performed in 1.5% agarose gels. Band size was estimated by comparison to 100 bp ladder DNA standard (Fermantas).

Cloning and sequencing of sex specific fragments
To convert the sex-diagnostic RAPD band to a SCAR marker, the bands were excised from 2% Agarose gel. Elution and purification of PCR fragment was performed using Qiagen-gel extraction and purification according to manufactures protocol. The purified PCR product was quantified by ethidium bromide spotting method. The eluted DNA was re-amplified with the corresponding RAPD primers to verify whether the fragment size was amplified consistently. The purified DNA fragments were ligated into a PCR cloning vector pTZ57R/T vector using the TOPO-TA cloning kit (Invitrogen) according to the manufacturer directions. The chimeric plasmid was transformed to E. coli strain DH5ά competent (Sambrook and Russell 2001). Confirmation of the successful cloning was carried out by amplifying the chimeric plasmid DNA with universal M13F/R primers. The total DNA and cloning vector were used as positive and negative controls in the PCR.

PCR reactions for cycle-sequencing were performed using the following profile: 25 cycles of 1 min at 96˚C, 10 s at 96˚C, 5 s at 50˚C and 4 min at 60˚C. Cloned fragments were sequenced by 3130 Genetic Analyzer (Applied Biosystems, USA) using BigDye® Terminator v3.1 Cycle Sequencing FS kit (ABI). The sequence of male and female specificity obtained was trimmed to define PCR end points by using sequence analysis software 5.2.0. The sequences of forward and reverse primers were aligned using BioEdit software. The aligned sequence was subjected to BLAST analysis, at http://www.ncbi.nlm.nih.gov of the National Center for Biotechnology Information, to find out the homology between the sequence obtained in the present study and sequences present at NCBI database.

3.3 Designing and amplifications of SCAR Primers
The nucleotide sequence of each of the cloned RAPD fragment was used to design pairs of SCAR primers. SCAR primer sets were designed using Primer3 clone manager software based on sequence information from polymorphic marker. Care was taken to avoid possible secondary structure or primer dimer generation and false priming, and also to match melting temperatures and to achieve appropriate internal stability while designing SCARs. These primers were synthesized by custom service of Eurofin Genomics Pvt. Ltd, Bangalore, India. These SCAR primers were used for the amplification of DNA obtained from female and male and plants. Amplification of the SCAR marker was performed in 25 µl, using 10ng total DNA, 0.2 µM each SCAR primer, 2.5 mM dNTP, 4mM MgCl₂, and 10x-Taq polymerase (Finnzymes) in 1× Taq buffer. The reproducibility of SCAR amplicon was confirmed by amplifying the marker under a broad range of amplification temperature (51℃ to 57℃). Eventually, cycling condition were as follows: 90℃ for 5 min; 30 cycles at 90℃ for 1 min, 53℃ for 1 min and 60℃ for 4 min, and final extension at 72℃ for 7 min. PCR products were electrophoresed on a 1.5% agarose gel and; presence and absence of the SCAR band was visually scored and compared with samples of each sex type and its pattern recorded on Gel documentation system.

Authors’ contributions
KPS carried out the overall experiment and SK prepared the manuscript. KBK supervised the experiment as Chairman of the advisory committee for the Master degree research work.

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