Agricultural and Marine Sciences, 12:35-42 (2007) ©2007 Sultan Qaboos University 35 Evaluation of Potato Cultivars Grown in Saudi Arabia by Morphological Characters and RAPD Markers Abd-Rahman Mohamed M. Al-Moshileh*1, Mohamed Zaky El-Shinawy2 and Mohamed Ibrahim Motawei1 1Department of Plant Production and Protection, College of Agriculture and Veterinary Medicine, Al-Qassim University, Saudi Arabia 2Horticulture Department, Faculty of Agriculture, Ain Shams University, Shobra El-Khima, Cairo, Egypt ABSTRACT: Genetic diversity of 10 potato cultivars was investigated at the DNA level with the random amplified polymorphic DNA (RAPD) procedure and at the phenotypic level using morphological characters. The results indicated that there were considerable variations among the different studied cultivars. Cultivars Victoria, Frizia and Safaren had the highest chlorophyll content compared to the other cultivars in both seasons. The largest leaf area and leaf dry weight were measured in cultivar Safaren and Mondial in both seasons. Cultivars Aboulx and Mondia produced the highest tuber yield in both seasons. On the other hand, cultivars Victoria and Edward were characterized by their considerably lower yield than other studied cultivars. Specific gravity varied considerably among the different potato cultivars. Thirteen random decamer primers were used to amplify DNA via the polymerase chain reaction and 75 RAPDs were generated. The RAPD profiles obtained were successfully used to differentiate potato cultivars. Based on the pair-wise comparison of amplification products, genetic similarity was estimated. The genetic similarity among all potato cultivars ranged from 50 to 92 %. Cultivars Victoria and *Corresponding author. E-mail: amoshileh@hotmail.com العربیة السعودیة بالمملكة الوسطى المنطقة في النامیة البطاطس بعض أصناف تقویم (RAPD) الدالئل الجزیئیة و المورفولوجیة الـصفات باستخدام مطاوع ومحمد إبراھیم الشناوي زكى محمد ، المشیلح بن محمد الرحمن عبد طریقة باستخدام وذلك (DNA) على مستوى الحمض النووي البطاطس الوراثیة لعشرة أصناف من درست االختالفات الخالصة: اختالفات وجود النتائج أظھرت المورفولوجیة. الصفات باستخدام المظھري الشكل مستوى على كذلك و (RAPD) الجزیئیة الدالئل الكلوروفیل من محتوى أعلى Safaren و ,Frizia و Victoria أعطت األصناف لقد الدراسة. البطاطس تحت بین أصناف وراثیة كال للورقة في جاف وزن مساحة و أعلى Mondial و Safarenالصنفان أعطى و الموسمین. كال األصناف في بباقي مقارنة من أعلى كانت أنھا إال السابقین بالصنفین مقارنًة اقل Kawalicو Victoria للصنفین الورقیة المساحة كانت بینما الموسمین. بینما الموسمین. كال في للدرنات محصول أعلى Mondial و Aboulx الصنفین من كل أنتج و .Aboulx Edward و الصنفین المدروسة. البطاطس بین أصناف الكثافة النوعیة للدرنات. واختلفت اقل محصول Edward و Victoria الصنفان أنتج النووي الحامض لقطع العشوائي التكبیر طریقة واستخدمت 75 حزمة. وأعطت المتسلسل البلمرة تفاعل في جزیئیا بادئا 13 استخدم من زوجین كل بین المقارنة باستخدام الوراثي التشابھ حساب تم البطاطس. أصناف بین للتفریق بنجاح (RAPD) ریبوز الدیوكسي Etfadoal و Victoria الصنفان 92%. وكان إلى من 50 البطاطس بین أصناف التشابھ الوراثي نسب تراوحت و الناتجة. الحزم أبعد Etfadoal وكان الصنف وراثي. تشابھ درجة Etfadoal أعلى و Victoria الصنفان أعطى تشابھ وراثي بینما درجة أقل أساس على مجموعات ثالث إلي البطاطس أصناف وقسمت األخرى. بقیة األصناف عن الوراثي حیث درجة التشابھ من األصناف تحدید في وكذلك المختلفة البطاطس أصناف على التعرف في الطریقة ھذه استخدام یقترح لذلك .(UPGMA) الوراثي التحلیل البطاطس. لعشائر الخرائط الوراثیة الھامة في االقتصادیة الصفات Al-Moshileh, El-Shinawy and Motawei 36 37 Evaluation of potato cultivars by morphological characters and RAPD markers Etfadoal presented the least similarity (0.50) while cultivars Mondial and Citrix had the greatest similarity (0.92). Etfadoal cultivar displayed the greatest genetic diversity of all cultivars. A dendrogram was constructed using UPGMA analysis. On the basis of this analysis, the cultivars were grouped into three clusters. The polymorphism detected suggests that RAPD markers are reliable for identification of potato cultivars and could be exploited in genetic mapping of populations to tag economically important traits. Keywords: Potato cultivars, fingerprint, genetic diversity, RAPD markers, morphological characters. Introduction Potato (Solanum tuberosum L.) is considered as one of the most important vegetable crops all over the world. Qassim region generates about half of the potatoes produced in Saudi Arabia (Zaag, 1991). The total production of potatoes in Saudi Arabia in 2002 was 315,000 tons (Ministry of Agriculture, 2004). Potato production can be affected by several environmental factors, such as moisture, temperature, day length, light intensity, nutrient availability and genetic variation. The catalogue of potato varieties provides an indispensable source for obtaining optimum potato crops. Any new variety should comply with the strict requirements of growers and users. Therefore, information on the performance of varieties allows selection of the most suitable variety for any specific conditions and provides information on the latest developments in the field of potatoes (Coumou, 2000). Identifying plant cultivars by molecular markers procedures is becoming a practical necessity (Smith and Smith, 1992). The relatively narrow range of morphological traits and limited numbers of polymorphic isoenzyme systems are not sufficient to discriminate all the cultivars of any given species. Furthermore, many phenotypic traits are developmentally regulated or influenced by the genotype X environment interaction. However, with the advent of DNA-based genetic markers, such as restriction fragment length polymorphisms (RFLP) and random amplified polymorphic DNAs (RAPD), the fingerprinting of plant cultivars has become more efficient, reliable, and useful (Caetano-Anolles et al., 1991 and Nybom, 1994). RAPD markers have been used successfully to identify many useful polymorphisms quickly and efficiently, and therefore the method has potential for use in cultivar identification (Schneider and Douches, 1996). Also, RAPDs were shown to provide a level of resolution equivalent to RFLPs for determining genetic relationships among some cabbage (Brassica oleracea L.) genotypes (Dos Santos et al., 1994) and among B. napus L. breeding lines (Hallden et al., 1994). Estimates of genetic similarity based on RAPDs have been developed for Lycopersicon Miller (Williams and St. Clair, 1993), Allium cepa L. (Wilkie et al., 1993), and Solanum tuberosum L. (Rocha et al., 2002 and Collares et al., 2004). RAPDs have been used to investigate developmental variation in micropropagated potatoes (Joyce et al., 1999) and also to correlate genetic variation observed in natural wild potato populations in the USA with a series of different ecological, geographical and reproductive variables (del Rio et al., 2001). Farmers and agricultural companies in Saudi Arabia use mainly imported potato seeds from Europe. Therefore, it is important to have evaluation and identification studies for potato cultivars widely grown in Saudi Arabia. The main objective of this study was to investigate genetic diversity among 10 potato cultivars introduced from The Netherlands using morphological markers and RAPD markers. Materials and Methods Plant materials and experimental design This study was conducted at the Experimental Farm of the College of Agriculture and Veterinary Medicine, Al-Qassim University during 2003 and 2004 fall seasons. The soil type of this farm is classified as a sandy soil. Data in Table 1 show specific soil characteristics. The irrigation water has a pH = 7.12 and 990 ppm total soluble salts. Drip irrigation was used and the amount of 12 mm of irrigation water was applied every other day throughout the two growing seasons. Each treatment was represented with three replicates and arranged in a randomized complete block design. The plot area was 3 x 6 m and contained 4 rows 75 cm apart. The planting distance was 30 cm within the row and presprouted tubers of Citrix, Kawalic, Aboulx, Spunta, Mondial, Victoria, Safaren, Edward, Etfadoal and Frizia cultivars were used. Al-Moshileh, El-Shinawy and Motawei 36 37 Evaluation of potato cultivars by morphological characters and RAPD markers These potato cultivars were introduced from HZPC Holland B.V., The Netherlands. The origin of potato cultivars is listed in Table 2. Planting dates of experiments 1 and 2 were 13th of October 2002 and 10th of October 2003, respectively. The plants in experiment 1 and 2 were harvested on 30th of January 2003 and on 1st of February 2004, respectively. A series of growth and yield parameters were measured and the data were statistically analyzed by the analysis of variance using the SAS package. Comparison of treatment means was performed by Duncan’s multiple range test at the P = 0.05 level of significance. Data were analyzed according to Sendecor and Cochran (1980). Measurements Vegetative growth parameters: Three plants were randomly chosen 50 days after the planting date in 2002 and 2003 seasons, respectively, for recording the vegetative growth parameters, which included leaf area of the fifth upper leaf measured at fully expanded mature stage (cm2) using a leaf area meter, model LI- 1300 Portable Area Meter (Stander Technique No.5, USA), and also leaf dry weight (g). Total chlorophyll: Chlorophyll pigment was measured of the fifth upper leaf using a Minolta chlorophyll Meter SPAD –501. Potato tuber yield: The number and weight of marketable tubers as well as total yield were recorded. Only tubers sized 20-55 mm in diameter were considered marketable tubers. Quality of potato tubers: Specific gravity was measured directly after harvesting by choosing 15 marketable tubers randomly, and was calculated as follows: SG = Weight of tuber in air (Weight in air) - (Weight in water) RAPD-PCR amplification Total genomic DNA of potato cultivars was extracted using the method described by Saghai-Maroof et al., (1984) for RAPD analysis. Twenty random primers were obtained from Operon Technology, USA. RAPD analysis was conducted in a thermal cycler (Thermolyne Amplitron). The reaction mixture (25 µL) contained 1x PCR buffer with Mg Cl (50 mM K Cl, 10 mM Tris- HCl (pH 9.0), 2 mM Mg Cl and Triton X-100), 200 µM each of dATP, dCTP, dGTP, and dTTP, 30 ng template DNA, 50 pM of oligonucleotide primer and 1.5 unit of Taq polymerase. The mixtures were subjected to the following conditions: hold at 94˚C for 2 min, followed by 40 cycles of 94˚C for 1 min, 36˚C for 1 min and 72˚C for 2 min, and a final hold at 72˚C for 5 min. PCR products were visualized along with a DNA marker on 2 % agarose gel with 1X TAE buffer and detected by staining with ethidium bromide. Gels were photographed on Polaroid films under UV light. Data handling and cluster analysis Data were scored for computer analysis on the basis of the presence or absence of the amplified products for each random primer. Basically, if a product was present in a genotype, it was designated "1", but if absent it was designated "0" after excluding irreproducible bands. Pair-wise comparisons of cultivars, based on the presence or absence of unique and shared polymorphic products, were used to generate similarity coefficients based on a simple matching. The similarity coefficients were then used to construct a dendogram by UPGMA Table 1. The soil mechanical features at the experi- mental site. Mechanical Analysis Bulk Density (g/cm3) Water Holding Capacity (%) Field Capacity (%) Wilting Point (%)Sand (%) Silt (%) Clay (%) 96.3 1.8 1.9 1.501 17.17 9.6 4.35 Table 2. The origin of potato cultivars. Cultivars Parents Citrix Cardinal X SVP 709 Kawalic Astarte X SVP AM 66-42 Aboulx Ostara X Renska Spunta Bea X USDA X 96-56 Mondial Spunta X SVP VE 66295 Victoria Agria X Ropta J861 Safaren Fanettex X [Maris piper X INRA 67.86.37] Edward Primura X Alcmria Etfadoal Monalisa X Hudson Frizia ZPC 69L 160 X AM 66-42 Al-Moshileh, El-Shinawy and Motawei 38 39 Evaluation of potato cultivars by morphological characters and RAPD markers (Unweighted Pair-Group Method with Arithmetical Averages) using NTSYS-PC (Rohlf, 2000). Results and Discussion Morphological characters Total chlorophyll and vegetative growth: It is evident from Table 3 that cultivars Victoria, Frizia, and Safaren resulted in highest chlorophyll content compared to the other cultivars in both seasons. The lowest chlorophyll content was produced by cv. Aboulx in both seasons. The leaf area and leaf dry weight varied considerably among the different potato cultivars (Table 3). The highest leaf area was produced by Safaren and Mondial in both seasons. Cultivars Victoria and Kawalic had a lower leaf area than cultivars Safaren and Mondial and higher than cultivars Edward and Aboulx. Moreover, cultivars Safaren and Mondial had the highest leaf dry weight in both seasons. On the other hand, cultivar Citrix had the lowest leaf dry weight in both seasons. These results could reflect the variation of vegetative growth and development among the different studied potato cultivars. Similar trends of variation in potato plant development and its reflection on potato yield was observed by Tibbits et al. (1992), Al-Moshileh and Motawei (2001) and Al-Moshileh et al. (2003). Tuber yield and specific gravity: Data in Table 4 clearly indicates that cultivars Aboulx and Mondial produced the highest tuber yield in both seasons. These two cultivars were followed by Spunta, Etafadoal, and Frizia in both seasons. On the other hand, cultivars Victoria and Edward were characterized by considerably lower yield production than the other studied cultivars. Table 4 indicates that specific gravity varied greatly among the different potato cultivars. Aboulx, Kawalic, and Mondial cultivars gave the highest specific gravity in both seasons. These cultivars, except cultivar Kawalic, were also characterized by the highest tuber yield. Cultivars Spunta and Frizia gave the lowest specific gravity. In spite of the importance of the photosynthesis process, formation of carbohydrates and translocation to the tubers, tuber yield did not exactly reflect the vegetative growth. This phenomenon could indicate that yield in potatoes in this study may not only Table 3. Chlorophyll content (Spad), leaf area and leaf dry weight of ten potato cultivars in two successive seasons. Cultivars Chlorophyll Content Leaf Area (cm2) Leaf Dry Weight (g) 2002/2003 2003/2004 2002/2003 2003/2004 2002/2003 2003/2004 Citrix 38.53 38.26 44.97 44.26 0.26 0.29 Kawalic 41.56 41.03 53.00 52.56 0.54 0.55 Aboulx 34.10 33.93 41.46 42.83 0.60 0.58 Spunta 37.63 36.43 54.51 51.90 0.35 0.30 Mondial 38.30 37.86 70.94 67.73 0.75 0.61 Victoria 45.16 40.76 53.20 54.96 0.41 0.38 Safaren 42.36 41.40 71.48 74.20 0.62 0.61 Edward 40.20 40.13 39.83 37.40 0.43 0.48 Etfadoal 38.90 39.43 52.55 53.56 0.53 0.59 Frizia 44.36 42.03 60.01 53.56 0.47 0.47 L.S.D at 5% 2.061 1.667 4.717 5.242 0.05 0.04 Table 4. Marketable yield and specific gravity of ten potato cultivars in two successive seasons. Cultivars Marketable Yield (g/m2) Specific Gravity Units (g/cm-3) 2002/ 2003 2003/ 2004 2002/ 2003 2003/ 2004 Citrix 493.96 548.36 1.080 1.080 Kawalic 595.03 660.23 1.120 1.130 Aboulx 790.33 877.73 1.140 1.130 Spunta 687.73 763.30 1.070 1.060 Mondial 762.16 846.30 1.110 1.120 Victoria 384.26 469.50 1.080 1.090 Safaren 656.56 729.33 1.070 1.070 Edward 431.83 476.63 1.091 1.090 Etfadoal 670.30 740.73 1.111 1.110 Frizia 657.33 738.73 1.041 1.040 L.S.D at 5% 15.52 15.48 0.350 0.048 Al-Moshileh, El-Shinawy and Motawei 38 39 Evaluation of potato cultivars by morphological characters and RAPD markers depend on leaf area and vegetative growth, but also on genotype and possibly other environmental factors (Al-Moshileh, 2001). RAPD analysis In the RAPD marker analysis, the selected primers OPA05,OPA07, OPA08, OPA09, OPA11, OPA12, OPA14, OPA15, OPA16, OPA17, OPA18, OPA19, and OPA20, generated 75 amplified products, with fragment numbers varying from 3 (OPA14) to 8 (OPA08 and OPA19) (Table 5), 50 of which were polymorphic. Examples of polymorphism are shown in Figure 1. By means of the 13 RAPD markers, all cultivars were identified and separated. Collares et al. (2004) identified 27 potato genotypes using only four RAPD primers. Demeke et al. (1993), using the RAPD technique with only two primers, identified 36 commercial potato cultivars. According to these authors, specific primers seem to produce more fragments and more DNA amplified polymorphisms and are, therefore, adequate for identifying potato cultivars by the RAPD technique. Ghislain et al. (1999), using 12 primers, obtained 102 polymorphic Table 5. The 12 selected Operon primers and the number of amplified products and polymorphic fragments. Primers Sequence 5’ to 3’ Ampli- fied Products Poly- morphic Fragments OP-A05 AGGGGTCTTG 6 3 OP-A07 GAAACGGGTG 5 4 OP-A08 GTGACGTAGG 8 6 OP-A09 GGGTAACGCC 7 4 OP-A11 CAATCGCCGT 7 6 OP-A12 TCGGCGATAG 6 4 OP-A14 TCTGTGCTGG 3 1 OP-A15 TTCCGAACCC 7 5 OP-A16 AGCCAGCGAA 4 3 OP-A17 GACCGCTTGT 4 2 OP-A18 AGGTGACCGT 4 3 OP-A19 CAAACGTCGG 8 5 OP-A20 GTTGCGATCC 6 4 Figure 1. Polymorphism revealed using primers OP-A08 and OP-A17 to amplify genomic DNA purified from 10 potato cultivars. OP-A08 OP-A17 Al-Moshileh, El-Shinawy and Motawei 40 41 Evaluation of potato cultivars by morphological characters and RAPD markers markers and could discriminate 128 accessions of potato. The similarity analysis based on RAPD markers showed that cultivars Victoria and Etfadoal presented the least similarity (0.50), whereas cultivars Mondial and Citrix had the greatest similarity (0.92) (Table 6). The high value of genetic similarity obtained between Mondial and Spunta (0.81) may be due to the fact that Spunta was one of the parents of cultivar Mondial (Spunta × SVP VE66295). On the other hand, cultivar Etfadoal had the greatest genetic diversity of all cultivars. These data revealed broad variability among potato cultivars. Sun et al. (2003) found genetic similarity coefficients varied from 0.29 to 0.90 among cultivated potato hybrids. Cluster analysis was conducted to generate a dendogram elucidating relationships among potato cultivars. The dendogram constructed with UPGMA analysis revealed three clusters (Fig. 2). Cluster I contained cultivars Victorial and Kawalic. Custer II contained cultivars Safaren, Citrix, Mondial, Spunta, Edward, Aboulx and Frizia. Cluster III contained cultivar Etafadoal. Hosaka et al. (1994), studying the genetic relations of 73 Japanese potato cultivars, reported that the patterns of RAPD banding of closely related cultivars were grouped. Taylor and Ford (1997) were able, by means of RAPD technique, to Figure 2. Dendrogram constructed from similarity coefficients showing the clustering of potato cultivars. Table 6. Simple matching coefficients of similarity determined from analysis using 75 RAPD loci. Cultivars V S K C Ed E A F M Sp Victoria (V) 1.00 Safaren (S) 0.69 1.00 Kawalic (K) 0.69 0.63 1.00 Citrix (C) 0.70 0.81 0.64 1.00 Edward (Ed) 0.69 0.81 0.69 0.75 1.00 Etfadoal (E) 0.50 0.50 0.63 0.52 0.57 1.00 Aboulx (A) 0.69 0.73 0.65 0.77 0.68 0.50 1.00 Frizia (F) 0.67 0.69 0.69 0.70 0.72 0.67 0.69 1.00 Mondial (M) 0.71 0.88 0.62 0.92 0.77 0.50 0.81 0.72 1.00 Spunta (Sp) 0.61 0.81 0.69 0.78 0.75 0.57 0.74 0.79 0.83 1.00 Al-Moshileh, El-Shinawy and Motawei 40 41 Evaluation of potato cultivars by morphological characters and RAPD markers differentiate closely related potato cultivars, which was not possible by morphological techniques. Forapani et al. (1999), Polzrova and Ptacek (2000), and Isenegger et al. 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Saudi Arabia. Received: March 2005 Accepted: April 2007