MOLECULAR MARKERS ASSOCIATED WITH DROUGHT TOL- ERANCE IN Citrullus colocynthis

erbal remedy drugs production became so important, especially with flooding the pharmaceutical Egyptian market with a number of synthetic drugs of questionable efficacy associated with the increasing cost of such drugs. Therefore, the demand of high-yield and highquality medicinal plants will continue to increase in the future. In addition, a growing concern with the awareness of the side effects of the drugs associated with regular exposure to synthetic chemicals has triggered a “back to nature” idea with an appeal of new discovery natural products to meet primary health care.

1. Dept. of Genetics, Fac. of Agric., Ain Shams University 2. Desert Research Center, Cairo, Egypt erbal remedy drugs production became so important, especially with flooding the pharmaceutical Egyptian market with a number of synthetic drugs of questionable efficacy associated with the increasing cost of such drugs. Therefore, the demand of high-yield and highquality medicinal plants will continue to increase in the future. In addition, a growing concern with the awareness of the side effects of the drugs associated with regular exposure to synthetic chemicals has triggered a "back to nature" idea with an appeal of new discovery natural products to meet primary health care.
More than two thousand species are grown wild in Egypt with no complete inventory of medicinal and aromatic plants in each region and, in general, the list of medicinal plants in Egypt and the Arab countries is inexhaustible (Batanouny, 1999).
Citrullus colocynthis L. (Cucurbitaceae) is a widely grown desert plant with multi-use potential, is one of the native. Plants of the Middle East countries which is used in traditional medicine. It contains active substances such as saponins, alkaloids and glycosides (Abdel-Hassan et al., 2000) and is used in traditional medicine to inhibit the implantation of embryos. Also used to treat (constipation, rheumatism, cancer, oedema, bacterial infections and diabetes). The plant possesses of large amounts of phenolics and flavonoids, which prompted the need to evaluate its antioxidant (Sunil and Mamal, 2008). Moraver, it is also used as antidiabetic and immunostimulant and antioxidant (Bendjeddou et al., 2003). However, there have been some reports of its side effects which can induces infertility in both sexes (Chaturvedi et al., 2003).
Drought stress is a major environmental factor influencing plant growth and development. Citrullus colocynthis is a very drought tolerant cucurbit species with a deep root system (Si et al., 2008), it is a source of drought tolerance genes. Since this species is widely distributed in the desert areas and well adapted to drought stress (Dane et al., 2006). DNA fingerprinting is a technology that has matured and is poised for very widespread practical application, such as the identification of plants in commerce, plant breeding and research. In addition, commercial applications include the pro-H tection of medicinal plant breeder's rights and patents, quality control in plant production, processing, and labeling of plantderived drugs (Soltis et al., 1992).
Molecular markers have been used to determine the association of drought tolerance and shown to be useful for diversity assessment in a number of plant species (Waugh and powell, 1992). These markers, based on the polymerase chain reaction (PCR) technique, are the most commonly used for these purposes, several different PCR-based techniques have been developed during the last decade, each with specific advantages and disadvantages. Inter-simple sequence repeat (ISSR) markers permit detection of polymorphisms in inter-microsatellite loci, using a primer designed from dinucleotide or trinucleotide simple sequence repeats.
The objectives of this study were to: 1. Identify some molecular markers based on ISSR primers associated with drought tolerance in Citrullus colocynthis.
2. Detect of some drought tolerance genes.
3. Sequence the successfully amplified drought tolerance genes related to.

MATERIALS AND METHODS
This study was carried out at the laboratories of Genetics Department, Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima, Cairo, Egypt and Biotechnology Laboratory in North Sinai Station, Desert Research Center, Egypt.

Plant materials
Citrullus colocynthis L. (Cucurbitaceae) plants were collected from four different sites, Red sea coast (Elba Mountain), New Valley area, North Sinai, and Saint Katreen areas by Egyptian Desert Genebank.

1-DNA preparation
Genomic DNA from each site was isolated according to the method of Junhans and Metzlatt (1990).

2-ISSR-PCR analysis
Inter-Simple sequence repeats (ISSRs) has been developed as an anonymous, RAPDs-like approach that accesses variation in the numerous microsatellite regions dispersed throughout the various genomes (particularly the nuclear genome) and circumvents the challenge of characterizing individual loci that other molecular approaches require. Microsatellites are very short (usually 10-20 base pair) stretches of DNA that are "hyper variable", expressed as different variants within populations and among different species.

Polymerase chain reaction
ISSR-PCR reactions were conducted using 14 primers, (

Statistical analysis
Data was subjected to statistical analysis of molecular variance (AMOVA) using performed GENALEX6 Genetic analysis in Excel, (Peakall and Smouse, 2006) to partition the total molecular variance between and within populations.

Detection of some drought tolerance genes
Genomic DNA based analysis of four Citrullus colocynthis plants from Elba Mountain, New Valley, North Sinai, and Saint Katreen areas were amplified by five primers of drought tolerance genes, namely (Dehydrin gene), (UB gene), (P5CS gene), (PEPKS gene) and (ACT gene) ( Table 2) these primers were designed as degenerate primers based on genes conserved sequences and depending on the data bases of NCBI (National Center for Biotechnology Information).

Polymerase chain reaction
PCR reactions were optimized as follows: dNTPs (8 mM  Amplification was carried out in Stratgene Robocycler Gradient 96 which was programmed as follows: Denaturation (one cycle) 94C for 5 minutes, followed by 30 cycles; as follows denaturation 94C for30 second, annealing 50-60C for 60 secs, extention 72C for 30 secs, and finally one cycle extension at 72C for 5 minutes.

Gel electrophoresis
PCR-Products of 15 μl were resolved in 1.5% agarose gel electrophoresis with 1 x TAE running buffer. The run was performed at 80 V for 180 min and the gel was stained with ethidium bromide. A marker of 1 Kb plus DNA Ladder 1 μg/μl (Invitrogen) that contains a total of twenty bands ranging from 12000 to 100 bp was used. Bands were detected on UV-transilluminator and photographed by Gel doc-umentation system Biometra Bio Doc Analyze 2000.

DNA Sequencing
The automated DNA sequencing reactions were conducted for the fragments with specific forward primer using ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (PE applied Biosystems, USA).
Data analysis was conducted using Blast programs from National Center for Biotechnology Information (NCBI), USA http://www.ncbi.nlm.nih. gov/BLAST/

1-Molecular markers associated with drought tolerance
DNA-based molecular markers have proved their utility in field like taxonomy, physiology, embryology and genetics, etc. As the science of plant genetics progressed, researches have tried to explore these molecular techniques for their applications in food crops, horticultural plants, etc. and recently in pharmacognestic characterization of herbal medicine (Nissen et al., 1995).

ISSR-PCR analysis
Inter simple sequence repeats (ISSR) are ideal as markers for molecular genetic studies because of their abundance and the high degree of polymorphism between individuals within a population of closely related genotypes.
ISSR patterns showed a total of 149 DNA bands detected across the fourteen ISSR primers, 99 of them were polymorphic (about 66%), (Table 3 and

Analysis of molecular variance (AMOVA) based on ISSR results
Analysis of molecular variance indicated that 100% of the genetic variation was attributed to differences within samples. No significant genetic variation was detected among samples, this mean that there is no difference among samples per sites. However, the sum squares was found to be 560.000, 1.957 and 0.720, for within samples, among sites and among samples per sites respectively. Detailed results from AMOVA are shown in Table (5). The estimated variance was 0.001, 0.232 and 0.0 for among sites, within samples and among samples per sites.

Dehydrin Gene
Dehydrin gene is responsive to drought stress and caused accumulation of dehydrin-like proteins (Vinod et al., 2006). Dehydrins (DHNs) compose a family of intrinsically unstructured proteins that have high water solubility and accumulate during late seed development, low temperature or water deficit conditions, They are thought to play a protective role in freezing and drought tolerance in plants and change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a water stimulus.
The PCR product using specific primer of Dehydrin drought tolerant gene showed the appearance of one band with fragment size of 190 bp, that might to be apart of Dehydrin genes (Fig. 2). This was in agreement with Hinniger and Victorea (2006), who isolated and characterized three Dehydrins genes, which were expressed during Coffea canephora (Robusta) in various tissues with molecular sizes of 240 bp.

PEPCS (phosphoenolpyruvate carboxylase) gene
PEPCS gene mode of action is the improvement of drought tolerance and increase efficiency of used water by overexpressing PEPC protein under water stress at reproductive stages with a strong impairment of photosynthesis and grain filling (Kazuo and Shinozak, 2005).
PEPC over expressing increase in intrinsic water use efficiency (WUE) under drought conditions. Opposite effects were observed for transgenic plants underexpressing the corresponding proteins.
The PCR product using specific primer of PEPCS drought tolerance gene indicted the appearance of PEPCS gene, with molecular size of 650 bp as shown in (Fig. 3)

UB gene
UB gene (Ubiquitin) is present in all eukaryotic species, it is a multifunctional protein. One of its main known functions is to tag proteins for selective degradation by the proteosome.
The PCR product using specific primer of UB drought tolerance gene indicted the appearance of UB gene, with molecular size of 450 bp, as shown in (Fig. 4). Our results agreed with those of Zhou and Chang (2010) who reported that ubiquitination plays important roles in plant abiotic stress responses and stated the over expression of soybean ubiquitin gene for enhancing drought stress tolerance through modulating abiotic stress in Arabidopsis. This UB gene fragment was with molecular size of 450 bp.

P5CS gene (1-pyrroline-5carboxylate synthetase)
The mode of action of P5CS gene is the transcription of the amino acid proline which considerd one of the most accumulated osmolytes under salinity and water deficit conditions in plants. Transcription of the P5CS genes is differentially regulated by drought, salinity and abscisic acid, suggesting that these genes play specific roles in the control of proline biosynthesis.
The PCR product using specific primer of P5CS drought tolerant gene indicted the appearance of one fragment with size of 600 bp (Fig. 5).
The same results of P5CS gene was obtained by Hayati and Santoso (2001), who identified P5CS gene on sugarcane by PCR using heterologous primer. The product size was 640 bp.

ACT2 Gene
The mode of action of ACT2 gene is the activation of the process of root hair growth and development.
The PCR product using specific primer of ACT2 drought tolerance gene indicted that appearance of one band with a fragment size of 700 bp as shown in (Fig. 6). Similar result of ACT gene was obtained by Devaiah and Athmaram (2007), who identified 2 new ACT genes with 2 full lengths of 825 and 700 bp from drought tolerant peanut, which were up regulated in response to drought stress.

DNA Sequencing and BLAST analysis
The reactions of automated DNA sequencing were conducted for the five fragments with specific forward primer using ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (PE applied Biosystems, USA), in conjunction with ABI PRISM (310 Genetic Analyzer). However, nucleotide sequence homology could be identified for only the one fragment (ACT2) as shown in Fig. (7), but there is no significant similarity for the other four fragments. BLAST analysis was done using National Center for Biotechnology Information (NCBI), USA (http://www. ncbi.nlm.nih.gov/BLAST/).

E-values or Expect value (E) is a
parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size (Pearson and Lipman, 1988).
The ACT fragment was highly similar to act mRNA from Brassica oleracea       -