Impaginato 13 1. Introduction Cotton is an economically important plant grown world-wide as a principal source of staple fiber and vegetable oil. A great deal of effort has been made to improve cotton cultivation and characteristics by breeders. Cotton is one of the major fiber crops in Syria, with a cultivated area of 125,000 ha, a produc- tion of 470,000 t of seed cotton, and lint production at 160,000 t. Yarn spinning capacity is estimated at 180,000 t (USDA, 2011). Salinity tolerance is a com- plex trait that involves physiological, biochemical, cellular, and genetic strategies. At present, out of 1.5 billion ha of cultivated land around the world, about 77 million ha (5%) is affected by excess salt content (Moradi et al., 2011). There is evidence that high salt concentrations cause an imbalance of the cellular ions resulting in ion toxicity and osmotic stress, lead- ing to the generation of reactive oxygen species (ROS) which alter cellular metabolism causing lipid peroxidation, protein denaturing, and DNA mutation (Dat et al., 2000; Davenport et al., 2003; Implay, 2003). Moreover, salt stress causes nuclear deforma- tion and subsequent nuclear degradation (Katsuhara and Kawaski, 1996). Structural changes of nuclei caused by salt stress have been previously reported as well (Werker et al., 1983). At present, there are several methods (physiologi- cal, biochemical, and molecular) available for detect- ing different kinds of DNA damage but with some limitations. Recently, molecular markers have been successfully applied to detect DNA damage induced by different abiotic stresses, particularly salinity. Among others, RAPD technique has been well docu- mented as a sensitive means of detecting DNA dam- age and shows potential as a reliable and repro- ducible assay for the detection of DNA fragmentation and chromosomal mutations (Citterio et al., 2002). The RAPD marker has been extensively applied for salinity tolerance screening in plant breeding pro- grams, such as in date palm (Phoenix dactylifera L.) (Kurup et al., 2009), aquatic plants Hydrilla verticilla- ta and Ceratophyllum demersum (Gupta and Sarin, 2009), in Euplotes vannus (Protozoa, Ciliophora) (Zhou et al., 2011), and in Acacia Senegal (Daffalla et al., 2011); in cotton (Dojan et al., 2012) and in fish full-sib Nile tilapia (Oreochromis niloticus), Blue tilapia (Oreochromis aureus) and their diallel inter- specific hybridization (El-Zaeem, 2012); and recently, also in soybean (Glycine max L.) (Khan et al., 2013). RAPD bands can be scored for genomic template stability (GTS) evaluation to detect various types of DNA damage and mutations (rearrangement, point mutations, small insertions or deletions of DNA and polyploidy changes) which suggests that RAPD bands may potentially form the basis of novel biomarker Adv. Hort. Sci., 2016 30(1): 13-21 DOI: 10.13128/ahs-18697 DNA changes in cotton (Gossypium hirsutum L.) under salt stress as revealed by RAPD marker B. Saleh Department of Molecular Biology and Biotechnology, AECS, P.O. Box 6091, Damascus, Syria. Key words: cotton, genomic template stability, RAPD, salt stress. Abstract: Random amplified polymorphic DNA (RAPD) analysis was applied to evaluate DNA changes among four upland cotton (Gossypium hirsutum L.) varieties [Niab 78 (N78), Deir-Ezzor 22 (DE22), Deltapine 50 (DP50) and Aleppo 118 (A118)] grown under non-saline conditions (control) and salt stress (200 mM NaCl) for seven weeks. Changes in RAPD profiles were measured as genomic template stability (GTS%). The highest estimated GTS% value was recorded for the two sensitive varieties, DP50 (79.1%) followed by A118 (58.2%); whereas, the lowest value was recorded for the two other tolerant varieties DE22 (36.7%) followed by N78 (26.4%). Based upon the data presented, RAPD marker could be used as potential tool for early identification of cotton tolerance to salt stress. (*) Corresponding author: ascientific@aec.org.sy Received for publication 28 May 2015 Accepted for publication 20 December 2015 Copyright: © 2016 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Adv. Hort. Sci., 2016 30(1): 13-21 14 assays for the detection of DNA damage and muta- tions in the cells of bacteria, plants, and animals (Savva, 1998; Atienzar et al., 1999; Tanee et al., 2012). It is well documented that genomic template stability ratios (GTS) were calculated. GTS implies qualitative measure reflecting changes in RAPD pro- files. Changes in RAPD and profiles were expressed as reductions in GTS in relation to profiles obtained from control samples (Gupta and Sarin, 2009; Aly, 2012; Dojan et al., 2012; Tanee et al., 2012). Therefore, this investigation aimed to detect DNA changes induced by NaCl application by monitoring the RAPD profiles of control and stressed plants in four upland cotton (Gossypium hirsutum L.) varieties grown in Syria. 2. Materials and Methods Plant materials and growth conditions Two local varieties were selected on the basis of their wide-ranging tolerance towards salinity: Deir- Ezzor 22 (DE22) as salt-tolerant and Aleppo 118 (A118) as salt-sensitive variety (Saleh, 2011). These two varieties were compared with two introduced cotton varieties, Niab 78 (N78) (known as salt-toler- ant) and Deltapine 50 (DP50) (known as salt-sensi- tive) under 0 and 200 mM NaCl for seven weeks. Seeds of upland cotton (G. hirsutum L.) were provid- ed by the General Commission for Scientific Agricultural Research of Syria (GCSAR). Seeds were soaked in distilled water for 24 h and then planted in pots filled with a 1:2 (v/v) mixture of perlite:peatmoss. Germination was carried out in a greenhouse at 18°C, 12 h photoperiod, and relative humidity of 80%. Seedlings were allowed to grow in a greenhouse under controlled conditions (tempera- ture 25°C, 12 h photoperiod, and relative humidity 80%). Seedlings were irrigated with tap water for one week before the initiation of NaCl treatments. Salt stress application was carried out by adding NaCl (200 mM) to the water. Plants were irrigated twice a week with water with or without salt. All solutions were changed twice a week. The same environmen- tal conditions were maintained during the experi- ment. The experiment (five replicates/treatment) was carried out in the greenhouse for seven weeks. Genomic DNA extraction Plant genomic DNA was extracted from young leaves (bulk of five plants/variety) including the con- trol and stressed plants (200 mM NaCl) using CTAB (cetyltrimethylammonium bromide) protocols described by Doyle and Doyle (1987) with minor modifications. Leaf tissue (150 mg) was ground in liquid nitrogen and the powder was transferred to a 2 ml Eppendorf tube, mixed with 900 μl of extraction buffer (100 mM Tris-HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 0.0018 ml β-mercaptoethanol, 2% CTAB), and incubated at 65°C for 20 min. One volume of a chloroform:isoamyl alco- hol mix (24:1, v/v) was added and centrifuged at 12,000 g for 10 min at 4°C. The aqueous phase was transferred to a fresh tube, and the DNA was precipi- tated with an equal volume of cold isopropanol and kept at -20°C for 10 min. It was then centrifuged at 12,000 g for 10 min at 4°C, the supernatant was dis- carded, and DNA was spooled out and washed with 1 M ammonium acetate and 100% ethanol. The cleaned DNA pellet was air dried and dissolved in 100 μl of 0.1X TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). Finally 5 μl of RNase (10 mg ml-1) were added and incubation for 30 min at 37°C was applied. DNA concentration was quantified by DNA Spectrophoto- meter at 260/280 nm and adjusted to final concen- tration of 10 ng μl-1. DNA was stored at -80°C until needed. RAPD marker Twenty-three RAPD primers from Operon Technologies Inc. (USA) and three primers from the University of British Columbia were tested to detect DNA changes in stressed plants, and their respective controls, for four cotton varieties. RAPD marker was performed as described by Williams et al. (1990) with a minor modification. PCR amplification reaction was carried out in 25 μl reac- tion volume containing 1xPCR buffer, 2 mM MgCl2, 0.25 mM dNTPs, 25 pmol primer, 1.5 U of Taq DNA polymerase and 30 ng template DNA. PCR amplifica- tion was performed in a T-gradient thermal cycler (Bio-Rad; T Gradient) programmed to fulfill 42 cycles after an initial denaturation cycle for 4 min at 94°C. Each cycle consisted of a denaturation step (1 min at 94°C), an annealing step (2 min at 35°C), and an extension step (for 2 min at 72°C). A final extension cycle was performed for 7 min at 72°C. The PCR prod- ucts were separated on a 1.5% ethidium bromide- stained agarose gel (Bio-Rad) in 0.5xTBE buffer. Electrophoresis was performed for 3 h at 85V and visualized with a UV transilluminator. Band sizes were determined by comparison with a 1 kb DNA Ladder Mix, ready for use (Fermentas). Saleh - DNA changes in cotton under salt stress 15 RAPD data analysis and Genomic Template Stability (GTS) estimations DNA changes induced in treated plants compared to their respective controls were screened by RAPD assay. The polymorphism was calculated in relation to the appearance of new bands and disappearance of bands in treated plants, compared to control band patterns. Genomic template stability (GTS%) was calculated as follows: GST% = (1- a/n) x 100 where (a) is the average number of changes in the DNA profile and (n) the number of total bands in the control. Polymorphism observed in RAPD profiles included disappearance of a normal band and appearance of a new band in comparison to the cotrol RAPD profiles (Atienzar et al., 2002). 3. Results A set of 26 random10-mer primers was used to detect the DNA changes among four cotton varieties under salt stress application compared to their respective controls. RAPD fragment sizes ranged from 200 to 3000 bp. The generated band character- istics for the four varieties (including control and stressed plants) are summarized in Table 1. The total number of characteristic bands (common observed bands in control and stressed plants for the four examined varieties) was 29. The amplification prod- ucts produced from 26 RAPD primers are listed in Table 2 in terms of loss or appearance of new bands (number and size) under salt stress compared to their respective controls for each variety separately. The RAPD analysis carried out on the four cotton varieties produced a number of distinct fragments which varied according to each tested primer. Twelve of the 26 RAPD primers (OPA02, OPB05, OPC08, OPD08, OPD20, OPJ07, OPK13, OPK17, OPR12, OPY10, UBC132 and UBC159) produced polymorphic bands under saline conditions for the four tested varieties (Table 2). Figure 1 shows the amplification products using OPA02, OPB17 and OPY10 RAPD primers with tem- plate DNA from the four varieties under control and saline conditions (200 mM NaCl). Changes in DNA pattern induced by NaCl treat- ment in the four tested cotton varieties were detect- ed based on estimated genomic template stability (GTS%) (Table 3). In this respect, it was found that the highest GTS% was recorded in salt-sensitive cot- ton, whereas the lowest was found among salt-toler- ant varieties (Table 3). 4. Discussion and Conclusions Detection of DNA changes in cotton via salt stress was assessed using RAPD marker system. As shown in Table 1, characteristic bands ranged between 0 (OPB17, OPD20, OPE15, OPJ01, OPJ07, OPK12, OPK13, OPK17 and UBC132) and 2 (OPC08 and OPT18), whereas the highest number (three) was yielded by OPA04, OPE07, OPG11, OPQ01 and OPQ18 RAPD primers (Table 1). Our findings reveal that nine out of the 26 tested RAPD primers generat- Table 1 - Characteristic bands identified for the four tested cot- ton varieties using 26 RAPD primers Primer name Sequence (5' - 3') Characteristic bands (number and size) OPA02 TGCCGAGCTG (1) 950 OPA04 AATCGGGCTG (3) 250, 550 & 1600 OPB05 TGCGCCCTTC (1) 1500 OPB17 AGGGAACGAG 0 OPC08 TGGACCGGTG (2) 500 & 800 OPC13 AAGCCTCGTC (1) 450 OPD08 GTGTGCCCCA (1) 1200 OPD20 GGTCTACACC 0 OPE07 AGATGCAGCC (3) 550, 650 & 800 OPE15 ACGCACAACC 0 OPG11 TGCCCGTCGT (3) 700, 1200 & 2100 OPJ01 CCCGGCATAA 0 OPJ07 CCTCTCGACA 0 OPK12 TGGCCCTCAC 0 OPK13 GGTTGTACCC 0 OPK17 CCCAGCTGTG 0 OPQ01 GGGACGATGG (3) 450, 600 & 1000 OPQ18 AGGCTGGGTG (3) 650, 750 & 1200 OPR09 TGAGCACGAG (1) 250 OPR12 ACAGGTGCGT (1) 300 OPT18 GATGCCAGAC (2) 1200 & 1350 OPW17 GTCCTGGGTT (1) 2000 OPY10 CAAACGTGGG (1) 650 UBC132 AGGGATCTCC 0 UBC159 GAGCCCGTAG (1) 1600 UBC702 GGGAGAAGGG (1) 400 Total 29 Adv. Hort. Sci., 2016 30(1): 13-21 16 Table 2 - Markers identified by 26 RAPD primers for the four tested cotton varieties under salt stress compared to their respective con- trols. DNA changes induced by NaCl treatment using RAPD marker as described by loss or appearance of new bands (number and size) under salt stress compared to their respective controls for each variety separately Primer name N78 DE22 DP50 A118 Total poly- morphic bandsC T C T C T C T OPA02 9 8 9 9 ₋ (4) 400, 650, 1800 & 2500 (3) 650, 800 & 2500 (2) 1800 & 2500 (2) 1800 & 2500 16 ₊ (2) 600 & 1500 (3) 500, 600 & 900 ND ND OPA04 3 3 3 3 ₋ ND ND ND ND 5 ₊ (3) 400, 500 & 1000 (2) 400 & 500 ND ND OPB05 5 5 4 5 ₋ (4) 450, 650, 1000 & 2000 (3) 450, 1000 & 2000 (1) 900 (1) 2000 14 ₊ (3) 500, 700 & 1100 (2) 500 & 1100 ND ND OPB17 7 9 8 8 ₋ (5) 600, 700, 1100, 1200 & 1800 (4) 350, 800, 1500 & 1800 ND (5) 400, 600, 800, 1000 & 1800 20 ₊ (2) 400 & 650 (2) 700 & 1100 ND (2) 450 & 750 OPC08 4 5 5 4 ₋ (2) 1850 & 1900 (1) 1200 (1) 1850 ND 10 ₊ (2) 300 & 900 (2) 1350 & 1900 ND (2) 1850 & 1900 OPC13 6 6 5 5 ₋ (1) 1000 (2) 500 & 1000 ND ND 12 ₊ (5) 300, 800, 900, 1100 & 1350 (4) 300, 1100, 1350 & 1500 ND ND OPD08 4 3 4 3 ₋ (2) 450 & 700 (2) 450 & 750 (1) 900 (2) 450 & 800 15 ₊ (2) 650 & 1350 (4) 500, 600, 800 & 1350 (1) 800 (1) 600 OPD20 6 4 2 2 ₋ (4) 650, 850, 1100 & 1850 (3) 700, 900 & 1350 (1) 800 (1) 300 13 ₊ (1) 1350 (1) 800 (1) 300 (1) 200 OPE07 8 8 8 8 ₋ (5) 1000, 1300, 1800, 2300 & 3000 (1) 1800 ND ND 7 ₊ (1) 1100 ND ND ND OPE15 5 4 6 6 ₋ (1) 200 (2) 800 & 1800 ND ND 8 ₊ (4) 800,1300, 1500 & 1800 (1)1500 ND ND OPG11 3 3 3 3 ₋ ND ND ND ND 2 ₊ (1) 600 (1) 600 ND ND OPJ01 7 6 7 7 ₋ (5) 400, 600, 850, 1200 & 2000 (3) 400, 600 & 850 ND (2) 600 & 2000 20 ₊ (4) 350, 500, 950 & 1800 (4) 350, 500, 950 & 1800 ND (2) 500 & 800 OPJ07 3 4 2 3 ₋ (3) 500, 800 & 1600 (4) 300, 500, 700 & 1600 (2) 550 & 1350 (1) 1100 21 ₊ (3) 700, 900 & 1200 (3) 550, 1000 & 1200 (2) 450 & 1600 (3) 450, 550 & 650 to be continuedT(-) loss bands, (+) gains bands, (ND) no differences. Saleh - DNA changes in cotton under salt stress 17 Primer name N78 DE22 DP50 A118 Total poly- morphic bands C T C T C T C T OPK12 3 3 2 2 ₋ (3) 700, 900 & 1350 (3) 800, 900 & 1350 ND ND 11 ₊ (3) 850, 950 & 1100 (2) 500 & 1000 ND ND OPK13 4 4 3 3 ₋ (2) 650 & 1100 (2) 350 & 1100 (1) 200 (1) 490 20 ₊ (4) 450, 750, 1200 & 1350 (4) 500, 750, 1200 & 1350 (3) 300, 500 & 1100 (3) 700, 1200 & 1350 OPK17 8 4 4 6 ₋ (4) 400, 1500, 1850 & 2500 (1) 1850 (2) 700 & 1000 (4) 300, 400, 1000 & 1850 19 ₊ (3) 300, 350 & 800 (2) 350 & 1200 (2) 500 & 1500 (1) 1500 OPQ01 9 4 4 4 ₋ (5) 300, 800, 1100, 1200 & 1800 (1) 1100 ND ND 10 ₊ (3) 200, 700 & 900 (1) 300 ND ND OPQ18 7 3 6 6 ₋ (4) 650, 900, 1350 & 1500 ND ND ND 9 ₊ (2) 550 & 1600 (2) 1500 & 1600 ND (1) 1600 OPR09 6 3 4 4 ₋ (4) 400, 550, 1100 & 3000 (1) 600 ND (1) 600 9 ₊ (2) 600 & 900 (1) 1000 ND ND OPR12 11 10 7 7 ₋ (6) 200, 450, 600, 1100, 2100 & 2250 (6) 250, 500, 800, 900, 1350 & 2250 (1) 900 (3) 200, 1200 & 1350 28 ₊ (4) 250, 500, 900 & 1800 (4) 700, 1000, 1100 & 1800 (2) 1350 & 2250 (2) 450 & 1000 OPT18 5 8 10 10 ₋ (1) 650 (2) 1800 & 2000 ND ND 9 ₊ (4) 400, 500, 1000 & 2250 (1) 950 ND (1) 2000 OPW17 5 4 3 3 ₋ (2) 600 & 1200 (3) 300, 900 & 1500 ND ND 17 ₊ (5) 450, 500, 800, 1350 & 1500 (4) 300, 450, 500 & 800 ND (3) 250, 450 & 800 OPY10 4 5 4 4 ₋ (1) 3000 (2) 1500 & 3000 (1) 400 (3) 400, 900 & 2500 12 ₊ (3) 400, 850 & 2500 (2) 400 & 2500 ND ND UBC132 5 5 4 5 ₋ (4) 1000, 1350, 1500 & 3000 (3) 550, 600 & 3000 (1) 1200 (3) 400, 1000 & 1200 17 ₊ (3) 600, 1200 & 1800 (3) 1000, 1350 & 1500 ND ND UBC159 3 6 3 3 ₋ (1) 500 (2) 1350 & 2000 (2) 500 & 700 (2) 650 & 2000 19 ₊ (5) 550, 700, 950, 1100 & 1350 (3) 300, 700 & 900 (2) 450 & 600 (2) 500 & 700 UBC702 3 3 3 3 ₋ (1) 800 (2) 750 & 800 ND ND 7 ₊ (2) 600 & 2500 (1) 2500 ND (1) 2500 Table 2 (continued) Adv. Hort. Sci., 2016 30(1): 13-21 18 ed no characteristic bands for the for tested cotton varieties (Table 1). It is worth noting that primer OPR12 identified more polymorphisms (28) than any other primer tested (ranging between two for primer OPG11 and 21 for primer OPJ07) (Table 2). Whereas, the banding patterns produced by primers OPA04, OPC13, OPE07, OPE15, OPG11, OPK12 and OPQ01 were not polymorphic for varieties DP50 and A118 (Table 2). Another investigation demonstrated varietal vari- ation in salt tolerance among these cotton varieties based on various examined physiological indices (Saleh, 2011). According to the study, the DE22 vari- ety could relatively be classified as salt tolerant vari- ety to other tested varieties. Dojan et al. (2012) reported the potential of RAPD markers for the detection of DNA damage induced by NaCl in cotton. Likewise, the RAPD marker has the potential to be applied in environmental pollution detection, e.g. Gupta and Sarin (2009) applied the same marker to detect pollution by cadmium (Cd) in two aquatic plants. Zhou et al. (2011) also used RAPD bands to indicate DNA damage in Euplotes vannus (Protozoa, Ciliophora) induced by nitrofurazone in marine cili- ates. Previously, Aly (2012) used the same marker for genotoxic effect detection of Cd stress on Egyptian clover and Sudan grass plants. Changes in RAPD profiles were also measured as Genomic Template Stability (GTS) and the data sug- gest noticeable genomic template instability (Table 3). Reduction in GTS values was observed under salt stress, compared to their respective controls for the four tested varieties (Table 3). Similarly, genetic instability induced by NaCl treatment of cotton was reflected by changes in RAPD profiles: disappearance Fig. 1 - RAPD banding profiles generated by OPA02, OPB17 and OPY10 primers in the four tested cotton varieties showing DNA changes induced by NaCl application for seven weeks, C: Control, T: Treated plants. M: 1 kb DNA Ladder Mix, ready for use. Primer name N78 DE22 DP50 A118 Control 200 mM NaCl 200 mM NaCl 200 mM NaCl 200 mM NaCl OPA02 100 33.3 25 77.8 77.8 OPA04 100 0 33.3 100 100 OPB05 100 40 0 75 80 OPB17 100 0 33.3 100 12.5 OPC08 100 0 40 80 50 OPC13 100 0 0 100 100 OPD08 100 0 100 50 0 OPD20 100 16.7 0 0 0 OPE07 100 25 87.5 100 100 OPE15 100 0 25 100 100 OPG11 100 66.7 66.7 100 100 OPJ01 100 28.6 16.7 100 42.9 OPJ07 100 100 75 100 33.3 OPK12 100 100 66.7 100 100 OPK13 100 50 50 33.3 33.3 OPK17 100 12.5 25 0 16.7 OPQ01 100 11.1 50 100 100 OPQ18 100 14.3 33.3 100 83.3 OPR09 100 0 33.3 100 75 OPR12 100 9.1 0 57.2 28.6 OPT18 100 0 62.5 100 90 OPW17 100 40 75 100 0 OPY10 100 0 20 75 50 UBC132 100 40 20 75 40 UBC159 100 100 16.7 33.3 33.3 UBC702 100 0 0 100 66.7 Mean 100 26.4 36.7 79.1 58.2 Table 3 - Genomic Template Stability (GTS%) estimated by 26 RAPD primers for the four tested cotton varieties under salt stress compared to their respective controls Saleh - DNA changes in cotton under salt stresss 19 of bands and appearance of new bands occurred in the profiles in comparison to those of the controls (Fig. 1, Table 3). Our data supports the suggestion by Dojan et al. (2012) that detected DNA changes, induced by NaCl, using RAPD marker could be explained as previously reported by Atienzar et al. (1999). It has been demonstrated that DNA damage levels could be reflected in GTS (Atienzar et al., 1999). The later investigation suggested that the loss of bands may be attributed to genomic rearrangements or to point mutations causing alterations in oligonucleotide priming sites, while appearance of new bands could be related to the presence of oligonucleotide priming sites which become accessible to oligonucleotide primers after structural changes (DNA mutation, dele- tions or homologous recombination). Table 3 reveals that GTS% values decreased with salt application for the four tested varieties. Our data show that the highest estimated GTS value was recorded for the two sensitive varieties, DP5 (79.1%) followed by A118 (58.2%); whereas, the lowest was recorded for the two tolerant varieties, N78 (26.4%) followed by DE22 (36.7%) (Table 3). Gupta and Sarin (2009) reported that the genomic template stability test was significantly affected by heavy metal stress, while Aly (2012) reported that GTS values decreased obviously with an increase in cadmium (Cd) concentration in Egyptian clover and Sudan grass seedlings. On the other hand, Tanee et al. (2012) used GTS to identify the Vanda species (Orchidaceae) of Thailand. Our results are in accor- dance with Dojan et al. (2012) who reported that there is positive correlation between GTS and other parameters (stem and leaf growth and stem length) under NaCl stress in cotton. In this respect, the estimated GTS values in the current investigation were positively correlated with various physiological indices (biomass and leaf K+/Na+ ratio) tested under NaCl application in cotton (Saleh, 2011). Moreover, a positive relationship was also determined between GTS values and recent findings (Saleh, 2013) based on physiological indices (relative water content, osmotic potential and salt tolerance index) among the same tested varieties (Dojan et al., 2012). Overall, the lowest estimated GTS values com- bined with the highest polymorphism level recorded for the tolerant varieties (N78 and DE22) (where, % polymorphic level was 68.5, 60.9, 21.3 and 36.4% for N78, DE22, DP50 and A118, respectively exposed to 200 mM NaCl for 7 weeks, using the same marker) could explain their salinity tolerance compared to the other tested varieties. However, the lowest estimat- ed GTS value recorded for N78 and DE22 varieties could be attributed to genetic variation, inducing new protein in relation to salinity tolerance. It has been successfully demonstrated that envi- ronmental constraint induced variation in DNA methylation pattern as a developed epigenetic mech- anism after exposure to abiotic stress (Zhong and Wang, 2007; Peng and Zhang, 2009). Our findings could be supported by the data provided in Zhong and Wang (2007), where genotyping variation in wheat (Triticum aestivum L.) cultivar salinity toler- ance was reported. In this respect, the study men- tioned that the salt-sensitive wheat variety had a lower methylation rate compared to salt-tolerant ones. Recently, Saleh (2013) reported that N78 and DE22 varieties showed a better protection mecha- nism against salinity damage than the other tested varieties, demonstrating variation in salt tolerance among cotton varieties based on physiological indices. Likewise, in the same investigation compar- ing the protein profiles between control plants and those salts treated using SDS-PAGE showed protein changes under salt treatment compared to their respective control. In this respect, the expression of ~19, ~21 and ~26 kDa for N78 and ~21 kDa protein for DE22, was highly increased by salt treatment, indicating that it could play a role in salt stress response. On the other hand, newly synthesized pro- tein of ~30 kDa was recorded for both DE22 and N78 varieties under saline treatment which was not observed in their respective controls. The other two tested varieties (DP50 and A118) showed decreases in the same protein bands (~19, 26 and 34 kDa) under saline conditions, with respect to their respec- tive controls, reflecting their sensitivity to salt stress. Salinity promotes the synthesis of salt stress-specific proteins; many of these proteins were suggested to protect the cell against the adverse effect of salt stress. Accumulation of these proteins is a common response to salt stress (Kong-Ngern et al., 2005; Metwali et al., 2011). It is worth noting that identified bands in DE22 and N78 (salt-tolerant varieties), which were not amplified in salt-sensitive varieties (DP50 and A118), could be related to gene(s) involved in salinity toler- ance. These findings are in accordance with Kurup et al. (2009). Adv. Hort. Sci., 2016 30(1): 13-21 20 DNA variation could be exploited in plant breed- ing programs to improve salinity tolerance in germplasm. Overall, the lowest estimated GTS values for NB78 and DE22 varieties (known as salt-tolerant) reflect their highest polymorphic values. Based on the current results, the RAPD marker was useful to establish specific DNA markers associated with NaCl stress. Therefore, the RAPD marker could be used as useful tool in plant breeding programs for early iden- tification of cotton tolerance to salt stress. 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