66 AJCS 7(1):66-74 (2013) ISSN:1835-2707 Morphological and molecular genetic variation in wheat for salinity tolerance at germination and early seedling stage Munir Ahmad 1 , Armghan Shahzad 2* , Muhammad Iqbal 2 , Muhammad Asif 3 and Arvind H. Hirani 4 1 Pir Mehr Ali Shah Arid Agriculture University, Murree Road 46300 Rawalpindi, Pakistan 2 Plant Biotechnology Program, National Agricultural Research Center, Park Road 45500 Islamabad, Pakistan 3 Agricultural, Food and Nutritional Science, 4-10 Agriculture/Forestry Centre, Univ. of Alberta, Edmonton, AB T6G 2P5 4 Department of Plant Science, University of Manitoba, Winnipeg, Canada * Corresponding author: armghan_shehzad@yahoo.com Abstract Salinity is one of the major constraints to wheat production. Salt affected soils can be better utilized by developing and growing salt tolerant wheat varieties. Genetic diversity for salt tolerance is a prerequisite for developing salt tolerant wheat varieties. Therefore, the present study was conducted to evaluate the level of genetic diversity among 172 (123 Pakistani and 49 exotic) wheat genotypes for salinity tolerance at germination and early seedling stage. All the genotypes were first tested at 200 mM NaCl stress. Based on the results, 34 genotypes were selected and subsequently tested at 250 mM and 300 mM NaCl stress. Genetic variation for salt tolerance existed in the studied wheat genotypes. Plumule growth was affected more than radicle growth at higher salinity levels. Based on salt tolerance index, 18 accessions were identified as salt tolerant at 200 mM NaCl stress. Egyptian accession 11466 was the most salt tolerant at 250 mM NaCl stress, whereas Pakistani accession 11299 and Egyptian accession 11466 were the most salt tolerant at 300 mM NaCl stress. Genetic similarity coefficients based on RAPD marker data ranged from 0.38 to 0.95. RAPD primer OPA 2 produced a unique fragment of 1000 bp, whereas OPF 13 generated two fragments of 1200 bp and 1400 bp only in some tolerant genotypes. Genetic similarity coefficients for SSR markers ranged from 0.45 to 0.95. Both RAPD and SSR markers revealed genetic variation in the studied genotypes. The salt tolerant landraces identified in this study could be used as parents to incorporate salt tolerance in future wheat cultivars. The unique DNA fragments observed in this study should be further investigated in segregating populations to determine their usefulness in Marker assisted selection for salt tolerance in wheat. Keywords: Genetic diversity; germination stage; RAPD; Salinity tolerance; SSR; Triticum aestivum L. Abbreviations RAPD; Random amplified polymorphic DNA; SSR; simple sequence repeats or microsatellite DNA markers: STI; Salt tolerance index: STTI; salt tolerance trait index. Introduction Over 800 million ha of land worldwide (Munns, 2005) and about 6 million ha in Pakistan (Chatrath et al., 2007) is salt affected. Salinity is a major constraint to food production as it limits crop yields and restricts the use of previously cultivated lands. The canal irrigated areas of Pakistan, especially that in Punjab province are the main contributors to crop production in the country. Salinity problem is becoming more severe in the canal irrigated areas of Pakistan (Evans et al., 2012). Soil reclamation and proper drainage may considerably ameliorate salinity problems. However, the area affected by salt is so large that these solutions appear to be unrealistic. One of the possible ways to bring salt affected land under cultivation is by growing salt tolerant crop cultivars. Salt tolerant cultivars are capable of maintaining active water uptake by root cells at high salt levels in the soil solution (Ashraf, 2004). Bread wheat (Triticum aestivum L.) provides more than half of the caloric and protein requirements to one-third of the world’s population (Dhanda et al., 2004). Although wheat is one of the most salt tolerant cereal crops (Badridze et al., 2009), its yield substantially decreases as the soil salinity level rises to 100 mM NaCl (Munns et al., 2006). To date, Kharchia 65 is the only available donor variety of salt tolerance in wheat and has been extensively used in breeding salt tolerant wheat cultivars globally (Chatrath et al., 2007). Therefore, new sources of salt tolerance in wheat need to be identified to broaden the gene pool and to provide donor parents in locally adapted genetic backgrounds. Due to soil heterogeneity, field screening for salt tolerance is difficult. Therefore, most of the studies on salt tolerance of wheat have been carried out in controlled environments. Identification of salt tolerant genotypes at both the germination and seedling stages is particularly useful (Mano and Takeda, 1997). Germination is a crucial stage for plant establishment (Song et al., 2008) and poor germination may lead to poor stand establishment, resulting in lower grain yields. The seedling stage is generally the most sensitive phase of plant development, and studies on salt tolerance in different crop species has mostly included plant assessment at this stage (Song et al., 2008; Tlig et al., 2008; Badridze et al., 2009). Development of salt tolerant wheat varieties requires an efficient system for assaying genetic variation in adapted as well as exotic germplasm. Due to the complex nature of salt tolerance, DNA markers may provide a valuable tool for assessing genetic diversity. Among different marker systems, random amplified polymorphic DNA (RAPD) is a simple technique for diversity analysis (Sun et al., 1997; Fu et al., 2002). However, RAPD analysis is sensitive to reaction conditions