12 crop breeding for salt tolerance in the era of molecular markers and marker assisted selection

Plant Breeding 132, 10–20 (2013) © 2012 Blackwell Verlag GmbH doi:10.1111/pbr.12000 Review Crop breeding for salt tolerance in the era of molecular markers and marker-assisted selection M U H A M M A D A S H R A F and M A J I D R F O O L A D 2, Department of Botany, University of Agriculture, Faisalabad, Pakistan; 2Department of Plant Science and The Intercollege Graduate Degree Programs in Plant Biology and Genetics, The Pennsylvania State University, University Park, PA 16802, USA; Corresponding author, E-mail: mrf5@psu.edu With tables Received March 2, 2012/Accepted July 10, 2012 Communicated by R Tuberosa Abstract Crop salt tolerance (ST) is a complex trait affected by numerous genetic and non-genetic factors, and its improvement via conventional breeding has been slow Recent advancements in biotechnology have led to the development of more efficient selection tools to substitute phenotypebased selection systems Molecular markers associated with genes or quantitative trait loci (QTLs) affecting important traits are identified, which could be used as indirect selection criteria to improve breeding efficiency via marker-assisted selection (MAS) While the use of MAS for manipulating simple traits has been streamlined in many plant breeding programmes, MAS for improving complex traits seems to be at infancy stage Numerous QTLs have been reported for ST in different crop species; however, few commercial cultivars or breeding lines with improved ST have been developed via MAS We review genes and QTLs identified with positive effects on ST in different plant species and discuss the prospects for developing crop ST via MAS With the current advances in marker technology and a better handling of genotype by environment interaction effects, the utility of MAS for breeding for ST will gain momentum Key words: abiotic stress — breeding for stress resistance — molecular breeding — quantitative trait loci — salinity — stress tolerance In arid and semi-arid regions of the world, with insufficient annual precipitation, agriculture depends mainly on irrigation water Irrigation agriculture poses a serious problem, that of accumulation of high concentrations of soluble salts in the soil where the plant roots normally grow High salinity in the root zone severely impedes normal plant growth and development This impairment could be due to (i) water stress arising from the more negative water potential of the rooting medium, (ii) adverse specific ion effects (toxicity), usually associated with either excessive sodium or chloride intake that may disturb membrane integrity and function, and (iii) nutrient ion imbalance, when the excess of sodium or chloride leads to either a diminished uptake of potassium, nitrate or phosphate, or impaired internal distribution of one or more of these ions (Shannon 1984, Gorham et al 1985, Ashraf et al 2008, Lenis et al 2011) Irrespective of the cause, plant productivity may be reduced partially or completely depending on the intensity of the salt stress The various strategies proposed to overcome the threat of salinity stress can be deciphered from a number of comprehensive reviews published on plant salt tolerance (ST; Epstein et al 1980, Flowers 2004, Chinnusamy et al 2005, Yamaguchi and Blumwald 2005, Munns et al 2006, Ashraf et al 2008, Ashraf and Akram 2009, Rai et al 2011) However, generally, there are two major approaches/strategies to minimize the deleterious effects of high soil or water salinity and both must be applied to achieve sustainable crop production in the presence of excessive salts (Epstein et al 1980) One is a technological approach, that is, implementing large engineering schemes for reclamation, drainage and irrigation with high-quality water Although these practices have had continuing success in some areas, the associated costs are high and often provide only a temporary solution to the problem The second approach, that is a complement to technological approach, entails biological strategies focused upon the exploitation or development of plants capable of tolerating excessive levels of salts This approach includes (i) diversifying cropping systems to include crops that are known to be salt tolerant (e.g by crop substitution); (ii) exploiting wild or feral species that are adapted to saline environments (e.g by domestication); and/or (iii) genetically modifying domesticated crops by breeding and selection to develop cultivars with enhanced ST Breeding for salt-tolerant genotypes that can grow more efficiently than the conventional varieties under high salinity stress is a fundamental approach which is considered economically feasible (Blum 1988, Ashraf et al 2008) The possibility and desirability of selection and breeding for salt-tolerant plants was first discussed by Lyon (1941), Dewey (1962) and Epstein (1963) Dewey (1962) was actually the first to conduct a systematic study of the ST of 60 strains of Agropyron desertorum and to outline a breeding programme for improving plant ST Further reports (Epstein and Jefferies 1964) also emphasized the importance of breeding for salt-tolerant crops In the early 1970s, genetic investigations were sporadically introduced into applied research on salinity for the first time (Epstein 1976), and in 1980, Epstein et al (1980) advocated the development of crops tolerant to salinity as a strategy to overcome this enduring problem Since then, there have been numerous reports and reviews dealing with the development of salt-tolerant crops (Epstein 1983, Richards 1983, Shannon and Qualset 1984, Staple and Toennissen 1984, Shannon 1985, Foolad 1999a, 2005, 2007, Munns et al 2006, Ashraf et al 2008, Ashraf and Akram 2009, Azzedine et al 2011, Rai et al 2011) Development of a breeding programme for improved ST requires (i) efficient screening techniques for the selection and wileyonlinelibrary.com Crop breeding for salt tolerance in the era of molecular markers evaluation of specified characters, (ii) identification of genetic variability, (iii) knowledge of the inheritance of tolerance trait(s) at specific developmental stages, (iv) knowledge of biological mechanisms underlying tolerance, (v) reliable direct or indirect selection criteria, and (vi) designing the most appropriate breeding methodologies/strategies to transfer the tolerance trait(s) into improved genetic backgrounds Among these, the identification of genetic variability, attainment of genetic knowledge of STrelated traits and development of reliable selection criteria are most crucial for the establishment of a successful breeding programme Accumulating evidence in different crop species has indicated that ST is a developmentally regulated, stage-specific phenomenon Tolerance at one stage of plant development may be poorly correlated with tolerance at other developmental stages (Foolad 1999a, Zhang and Blumwald 2001, Ashraf and Akram 2009, Uddin et al 2011) Specific stages throughout the ontogeny of the plant such as germination and emergence, seedling survival and growth, and vegetative and reproductive growth should be evaluated separately for the assessment of ST and identification of contributing genetic components Formal genetic analysis of ST at specific developmental stages may simplify the underlying genetic components and therein facilitate breeding efforts by introgressing each component trait into superior genetic backgrounds Subsequent integration of differential tolerance at specific stages into a single highly tolerant cultivar might then be accomplished faster than selection strategies based on yield performance, which is the ultimate objective Reducing ST into developmental units may also facilitate the identification of individual genes or quantitative trait loci (QTLs), amenable to manipulation with current molecular genetic techniques Direct selection in field sites for quantitative traits, such as ST, is difficult because uncontrollable environmental factors adversely affect the precision and repeatability of such trials One suggested approach to improve the efficiency of the breeding programmes is adoption of new selection criteria based on the knowledge of the physiological processes limiting crop production under conditions of stress (Ashraf 2004) and of their genetic control (Tal 1985) Some of the physiological responses to high salinity, which may be used as indirect selection criteria for improving crop ST, are tissue water potential, tissue ion content, K+/Na+ ratio, succulence, water-use efficiency, chlorophyll fluorescence, content of chlorophyll, contents of proline, phenolics and sugars, and activities and levels of enzymatic and nonenzymatic antioxidants Whether these physiological responses are correlated with ST or there is variation in these responses in the plant species of interest must be elucidated before the question of genetic control can be addressed A contemporary approach to improving the efficiency of selection and breeding for complex traits such as ST is to discover genetic markers that are associated with the trait(s) of interest and which could be used as indirect selection criteria for the evaluation of large breeding populations A common method for the identification of associated genetic markers is the detection of linkage between genetic markers already mapped on the chromosomes and the loci or gene complexes (e.g QTLs) that control quantitative variation and are inherited as major hereditary factors This technique entails the identification of QTL-linked markers in controlled environment, using appropriate genetic material, and then introgressing marker-linked QTLs into desirable genetic backgrounds by a process known as marker-assisted selection (MAS) Because often several traits and mechanisms are involved in plant ST, pyramiding traits of interest via MAS may be an effective approach to substantial improvement in 11 plant ST Furthermore, MAS may allow early selection for trait (s) of interest, multiple cycles of selection in a year, and pyramiding of tolerance components from different genetic resources These advantages may facilitate the development of crops with improved ST, compared to traditional methods of field evaluation and phenotypic selection (Foolad 2004, Collins et al 2008, Witcombe et al 2008) For example, it has been estimated that breeding rice cultivars for tolerance to salinity stress and phosphorus deficiency via MAS may be accelerated by 3–6 years compared with the conventional breeding, leading to a saving of US$ 50–900 million over a 25-year period (Alpuerto et al 2009) Since the advent of molecular markers and MAS technology, numerous studies have been conducted to identify genes or QTLs affecting ST in different plant species and during different developmental stages These studies have been conducted with the promise of using marker-linked QTLs or genes in MAS breeding for ST However, limited progress has been made in developing salt-tolerant cultivars via the use of MAS technology This is in contrast to the extensive application of MAS for breeding for many simple traits in numerous crop species In this article, we review and summarize the genes and QTLs that have been identified with positive effects on ST in different plant species and discuss their use in MAS breeding for improving plant ST Further, we discuss challenges and opportunities for developing crop ST via the use of MAS technology QTLs Associated with Salt Tolerance It has been known that salt stress imposes two primary effects on plants, osmotic and ionic, and both reduce plant growth and final yield to varying extents in different crop species In addition, salt stress imposes secondary effects such as nutrient imbalances, generation of oxidative stresses and hormonal imbalances (Ashraf et al 2008) To counteract such salt-induced adverse effects, plants respond in different ways, including ion exclusion/accumulation to provide ion homoeostasis, accumulation of organic osmolytes to establish osmoregulation, production of antioxidants to counteract salt-induced generation of reactive oxygen species (ROS), and changes in uptake and accumulation of essential mineral nutrients (Mittler 2002, Byrt et al 2007, Ashraf et al 2008) During the past two decades, numerous studies have been undertaken in different crop species to identify QTLs that directly or indirectly affect the various plant responses to salt stress at different developmental stages, including seed germination, seedling and vegetative growth and reproduction These studies are summarized in Table 1, and in the following paragraphs, we review and discuss some of the major findings QTLs for ion uptake and accumulation The key transport systems involved in ion homoeostasis in plants grown under saline conditions are regulated by the salt overly sensitive (SOS) signal pathway (Hasegawa et al 2000, Zhu 2000) The SOS2, a serine/threonine protein kinase, regulates SOS1 (plasma membrane Na+/H+ antiporter)-mediated Na+ efflux from the cytosol, as well as regulating vacuolar Na+/H+ antiporter-mediated Na+ sequestration into the vacuole (Shi et al 2000, Qiu et al 2003) In addition to this mechanism of ion homoeostasis, the high-affinity K+ transporters (HKT), which mediate Na+-specific transport or Na+/K+ co-transport, play an important role in maintaining Na+ homoeostasis in plants under saline conditions An important locus (Kna1) in hexaploid bread 12 M ASHRAF and M.R FOOLAD Table 1: Identification of quantitative trait loci (QTLs) for salt tolerance (ST) in different plant species Crop plants Wheat (Triticum aestivum L.) Arabidopsis thaliana L Rice (Oryza sativa L.) Barley (Hordeum vulgare) Molecular markers Traits governed ESTs TmHKT7-A2 SSR Kna1 Reduces Na+ concentration in leaf blades by retaining Na+ in the sheaths Controls the selectivity of Na+ and K+ transport from root to shoot and maintains high K+/Na+ ratio SSR Nax1 Both are involved in decreasing Na+ uptake and enhancing K+ loading into the xylem EST Nax2 Not mentioned in the article AFLPs Xglk683–Xcdo460 and Xfbb168–Xbcd147 Not mentioned in the article SSRs RAS1 QTL1, QTL2, QTL3, QTL5 qRL-7, qDWRO-9a and qDWRO-9b qBI-1a and qBI-1b Increase biomass, shoot length, root length, chlorophyll and proline contents at both the germination and seedling stages under saline conditions Functions as a negative regulator of ST during seed germination and early seedling growth by enhancing ABA sensitivity and its loss of function contributes to increased ST Control germination under salt stress Play important roles in root length and root dry weight at seedling stage under saline conditions Affect shoot Na+ and K+ concentrations, respectively ESTs qSNC-7 and qSKC-1 SSRs QNa, QNa:K, SKC1/OsHKT8 Regulate K+ / Na+ homoeostasis AFLPs QNa, QK1, QK2 and QNaK SSRs qDM-3 and qDM-8, qSTR-6 Improve Na+, K+ and discrimination of Na+ or K+ uptake Improve Na+/K+ ratio under saline conditions RFLPs, SSRs, AFLPs and isozymes SSRs qST1 and qST3 Enhance ST in shoots qNAK-2 and qNAK-6 Improve Na+/K+ ratio SSRs Saltol Controls shoot Na+/K+ homoeostasis SSRs Saltol and non-Saltol Control shoot Na+/K+ homoeostasis SSRs QKr1.2 Controls K+ content in root SSRs bPb-1278 and bPb-8437 RFLPs QFv2H, QCh7Ha, Qch2Ha and QWSC2H Associated with tiller number, plant height, spikes per line and spikes per plant (SPP) Enhance ST by improving chlorophyll content, fluorescence and proline content SSR Five QTL for ST were identified on chromosomes 1H, 2H, 5H, 6H and 7H, which accounted for more than 50% of the phenotypic variation A locus HvNax3 on the short arm of chromosome 7H in wild barley (Hordeum vulgare ssp spontaneum) accession CPI71284-48 HvNax4 was fine-mapped on the long arm of barley (Hordeum vulgare) chromosome 1H ORS674, ORS784, ORS235, ORS681 RFLPs Sunflower (Helianthus annuus L.) Locus ESTs, SNPs Enhance vegetative growth under saline stress References Huang et al (2006) Dubcovsky et al (1996), Gorham et al (1990) Huang et al (2006), Lindsay et al (2004) Byrt et al (2007) Ma et al (2007) Ren et al (2010) Galpaz and Reymond (2010) Sabouri and Sabouri (2008) Lin et al (2004), Pushparajan et al (2011) Ren et al (2005) Flowers et al (2000) Sabouri et al (2009) Lee et al (2007) Ming-zhe et al (2005) Thomson et al (2010) Alam et al (2011) Ahmadi and Fotokian (2011) Xue et al (2010) Siahsar and Narouei (2010) Zhou et al (2012) Reduces shoot Na+ content by 10–25% in plants grown under salt stress (150 mM NaCl) Shavrukov et al (2010) Promotes shoot Na+ exclusion up to %59 Rivandi et al (2011) Lexer et al (2003) Enhance ST by increasing Ca2+ uptake, coupled with greater exclusion of Na+ (continued) Crop breeding for salt tolerance in the era of molecular markers 13 Table (continued) Crop plants Tomato (Solanum lycopersicum L.) White clover (Trifolium repens L.) Soybean (Glycine max (L.) Merr.) Molecular markers RFLPs Locus Traits governed RFLPs Five QTLs on chromosomes 1, 3, and Contribute to rapid germination under salt stress (in crosses with Solanum pennellii) Contribute to rapid germination under salt stress (in crosses with Solanum pimpinellifolium Affect ST during vegetative stage RFLPs Five QTLs on chromosomes 1, 3, 5, and 11 Affect ST during vegetative stage RFLPs Several QTLs for ST–related traits Affect ST during reproductive stage SSR and SNPs Several QTLs for ST, some at common locations, but each of low scale Affect ST during vegetative stage RFLP and SSR SSR A major QTL for ST was identified near the Sat091 SSR marker on linkage group (LG) N Eight QTLs for ST were detected Maintains healthy growth under salt stress Maintains growth under salt stress SSR A major QTL for ST was detected Maintains growth under saline stress RFLPs Seven QTLs on chromosomes 1, 2, 3, 7, and 12 Seven QTLs on chromosomes 1, 2, 5, 7, and 12 wheat (Triticum aestivum) has been reported to control the selectivity of Na+ and K+ transport from root to shoot, thereby maintaining a high K+/Na+ ratio in leaves (Gorham et al 1987, 1990, Dubcovsky et al 1996, Luo et al 1996) However, Na+ exclusion mechanism in durum wheat (Triticum turgidum L ssp durum Desf.) was found to be linked to Nax1 (Na+ exclusion 1) and Nax2 loci, which most probably relate to the Na+ transporters HKT1;4 (HKT7) and HKT1;5 (HKT8), respectively (Huang et al 2006, 2008, Byrt et al 2007) The Nax1 and Nax2 loci have been reported to effectively decrease Na+ transport from root to shoot, thereby maintaining reasonably low Na+ content as well as high level of K+ in the leaf blades of durum wheat plants by excluding Na+ from, and loading K+ into, the xylem (James et al 2006) Ming-zhe et al (2005) identified two QTLs for root Na+/K+ ratio in rice (Oryza sativa), which were mapped to chromosomes and using an F2 population of a cross between japonica rice cultivar ‘Jiucaiqing’ and indica rice cultivar ‘IR36’ Many other QTLs for ST traits have been identified in rice, including Saltol on chromosome 1, which explains most of the variation for ion uptake under salt stress (Bonilla et al 2002, Gregorio et al 2002), QNa for high Na+ uptake on chromosome (Flowers et al 2000), QNa:K for Na+/K+ discrimination on chromosome (Singh et al 2001), SKC1/OsHKT8 on chromosome 1, which regulates K+/Na+ homoeostasis in salt-tolerant indica variety ‘Nona Bokra’ (Lin et al 2004, Ren et al 2005), several QTLs on all but chromosome for Na+/K+ ratio in the root (Sabouri and Sabouri 2008), three QTLs for ion exchange on chromosomes and 10 (Sabouri and Sabouri 2008), and one QTL each for Na+ and K+ uptake and four QTLs for tissue Na+/K+ ratio on different chromosomes (Lang et al 2001) Furthermore, Ahmadi and Fotokian (2011) identified 14 QTLs for root and shoot Na+, K+ and K+/Na+ ratio on different rice chromosomes Among them, a QTL (QKr1.2) for root K+ content identified on chromosome was found to be most promising as it explained approximately 30% of the variation observed for ST in rice Furthermore, Islam et al (2011) identified two novel QTLs on rice References Foolad et al (1997) Foolad et al (1998) Foolad and Chen (1999) Foolad et al (2001) Breto et al (1994), Monforte et al (1996) Wang et al (2010) Lee et al (2004) Chen et al (2008) Hamwieh et al (2011) chromosomes and 10 based on an F2 population of a cross between a moderately salt-tolerant (BRRI-dhan40) line and a highly salt-tolerant (IR61920-3B-22-2-1) line Similar to that in rice, numerous studies have identified QTLs for ST-related traits in barley (Hordeum vulgare L.; Table 1) For example, Xue et al (2010) identified 30 QTLs for 10 different traits, including shoot Na+, K+ and Na+/K+ ratio, and several growth and yield-related attributes in populations grown under salt-stress and non-stress conditions This research determined that the QTLs for ion-uptake traits observed under saline stress were different from those under non-stress conditions, suggesting that specific genes related to ion uptake and facilitating plant adaptation to salt stress were expressed only under salt-stress conditions The authors suggested that only the QTLs expressed under saline stress were associated with ST, which could potentially be useful for developing salt-tolerant barley genotypes QTL analysis of mineral ion-uptake traits in three species of sunflower (Helianthus sp.), Helianthus paradoxus (from highly saline habitat – salt marshes) and its putative parents Helianthus annuus and Helianthus petiolaris (both usually categorized as salt sensitive), resulted in the identification of 14 QTLs for ion uptake (Lexer et al 2003) The researchers also reported that ST in Helianthus was achieved through enhanced Ca2+ uptake and Na+ exclusion In follow-up studies, using sunflower expressed sequence tags (EST) database and single-nucleotide polymorphism (SNP) mapping strategy, several candidate genes were detected that co-localized with QTLs for some of the vital adaptive traits (Lexer et al 2004, Lai et al 2005) For example, the genes encoding Ca2+ and K+ transporters as well as a calciumdependent protein kinase (CDPK) co-localized with the QTLs for ion uptake and survival under salt stress In a different study using H paradoxus, transgressive expression of genes encoding K+ and Ca2+ transport system was detected, suggesting that these genes play important roles in the adaptation of H paradoxus to high saline conditions It is apparent that many studies have identified QTLs for ion uptake and/or accumulation in different crop species grown 14 under salt stress However, the utility of these QTL information for improving plant ST via MAS has not been verified Additional work is necessary to determine the utility of such QTLs for use in crop breeding for improved ST First, the actual relationship between ion accumulation (or lack thereof) and ST has to be clearly determined in crop species of interest Ion accumulation or exclusion may only be a small component of the overall response of plants to salt stress, and thus, such QTLs may not play a major role in plant ST Second, most studies have identified QTLs for ion uptake/accumulation at one stage of plant development It has been reported that the extent of operation of ion-uptake mechanism varies considerably at different stages of plant development (Ashraf and O’Leary 1994, Chartzoulakis and Klapaki 2000, Qasim and Ashraf 2006) Thus, it needs to be determined whether ion accumulation at any specific stage of plant development would affect the overall plant response to salt stress Third, as for many other agriculturally important traits, the QTLs for ion accumulation are originally described in genetic populations distantly related from or irrelevant to a given breeder’s germplasm and/or in different agricultural environments from a given breeder’s target climatic region Such information may not be easily transferable to populations used in practical plant breeding programmes Thus, before these issues are addressed, the value of the identified QTLs for improving plant ST cannot be easily determined QTLs for oxidative defence system Under saline stress, similar to that under a variety of other stress conditions, reduction of oxygen (O2) frequently takes place in plants, which leads to the production of different types of ROS, including superoxide (OÁÀ ), hydrogen peroxide (H2O2) and hydroxyl radical (˙OH) ROS are known to adversely interact with a number of metabolites, which may result in the impairment of normal functioning of the cell (Mittler 2002, Ashraf 2009, Mittler et al 2011) However, plants have the ability to generate a variety of enzymatic and non-enzymatic compounds that act as antioxidants to detoxify the ROS The type of antioxidants depends on plant species Some key enzymatic antioxidants include superoxide dismutase, catalase, ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR; Mittler 2002, Ashraf 2009) Important non-enzymatic antioxidants include glutathione (GSH), ascorbate (AsA), carotenoids, tocopherols, phenolics and flavonoids (Mittler 2002, Mateo et al 2004, Gupta et al 2005, Ashraf 2009) Overexpression of these antioxidants has been reported to be associated with ST in many plant species (Lopez et al 1996, Shalata et al 2001, Ashraf 2009, Zhou et al 2011) Furthermore, up-regulation of the genes for different antioxidants has been reported in plants under stress conditions (Zhu et al 2005) Thus, attempts have been made to identify QTLs underlying the expression of various antioxidants in different plant species For example, Frary et al (2010) reported that Solanum pennellii accession LA716 (a salt-tolerant wild accession of tomato) had considerably more accumulation of antioxidants, such as total phenolics, flavonoids and some other key antioxidant enzymes, than the salt-sensitive cultivated species, Solanum lycopersicum In this research, a total of 125 QTLs for antioxidants were identified under saline and nonsaline conditions Among these, some antioxidant QTLs were identified under both saline and non-saline conditions, while M ASHRAF and M.R FOOLAD other QTLs were more specific to one or the other condition However, the authors contemplated that the identification of QTLs for enhanced synthesis of antioxidants under salt stress would be beneficial to breeding programme for developing salt-tolerant cultivars of tomato Because very few reports on the identification of QTLs related to oxidative defence system are available, it is difficult to draw any conclusion as to whether the detection of QTLs for such traits would be useful for improving plant ST via MAS In fact, the regulation of oxidative defence mechanism in plants is considered to be one of the secondary responses of plants to saline stress (Ashraf 2009), so it needs to be determined how relevant QTLs for antioxidants and their overexpression would be in relation to plant ST QTLs for organic osmolytes and osmoprotectants When subjected to stress conditions, including salt stress, most plants accumulate a variety of organic osmolytes (osmoprotectants), including sugar alcohols, proline and quaternary ammonium compounds such as glycine betaine Such organic solutes not only contribute to osmoregulation in stressed plants, but also provide protection to very many enzymes that are active in the cytosol (Bohnert et al 1995, Ashraf and Foolad 2007) There are great variations within and among plant species in the accumulation of organic osmolytes in response to saline conditions In general, plants accumulating higher levels of these osmolytes are more salt tolerant than those accumulating lower amounts (Ashraf and Harris 2004) However, limited research has been conducted to identify putative QTLs underlying various osmoprotectants, besides a few reports related to proline For example, two putative proline QTLs were identified in barley on chromosomes and (http://www.lw23.com/lunwen_492434767/), and recently, Siahsar and Narouei (2010) identified 29 QTLs for a number of physiological traits including proline content under saline conditions in barley Unlike the QTL approach, transgenic approaches have been used frequently to engineer plants with overproduction of different types of osmoprotectants, including glycine betaine, proline and trehalose (Hussain et al 2011) For example, Ziaf et al (2011) transferred an early responsive-todehydration gene (SpERD15) from a drought- and salt-tolerant wild tomato S pennellii to tobacco (Nicotiana tobacum L.) The resultant transgenic tobacco line showed improved drought and ST because of high accumulation of proline and soluble sugars, which occurred because of the overexpression of their respective genes, P5CS and sucrose synthase Similarly, a transgenic line of potato (Solanum tuberosum L.), recently developed by the insertion of a bacterial mannitol 1-phosphate dehydrogenase (mtlD) gene, showed enhanced ST that was associated with increased accumulation of mannitol in both shoot and root (Rahnama et al 2011) The above-mentioned studies clearly demonstrate that ST can be improved by engineering gene(s) controlling enhanced osmolyte synthesis and that the overaccumulation of a specific organic osmolyte depends on the species used For example, in some plant species, high proline synthesis is a useful characteristic related to ST, whereas in others, it may not be The same is true for other osmolytes Because different osmolytes play important roles in conferring ST, it is prudent to conduct research to discern the genetic control of osmolyte production in different plant species Such knowledge may facilitate the development of plants with improved ST via breeding for osmolyte accumulation Crop breeding for salt tolerance in the era of molecular markers QTLs for growth-related traits under salt stress Quantitative trait loci have been identified for growth-related traits as well as physiological and biochemical attributes related to ST during different plant development stages, including seed germination, early and late seedling growth and vegetative growth and reproduction (Foolad and Jones 1993, Foolad et al 1997, 1998, Mano and Takeda 1997, Foolad and Chen 1998, 1999, Foolad 2004; Table 1) For example, QTLs for ST during the germination stage have been identified in different plant species, including tomato (Foolad and Jones 1993, Foolad et al 1997, 1998), rice (Prasad et al 2000, Cheng et al 2008), Arabidopsis (Ren et al 2010, Vallejo et al 2010), barley (Mano and Takeda 1997) and wheat (Ma et al 2007) Similarly, QTLs for ST at the seedling stage (i.e vegetative growth under salt stress) have been detected in a number of plant species, including rice (Ming-zhe et al 2005, Yao et al 2005, Lee et al 2007, Sabouri and Sabouri 2008, Thomson et al 2010, Ahmadi and Fotokian 2011, Alam et al 2011), Arabidopsis (Ren et al 2010), barley (Mano and Takeda 1997, Ellis et al 2002, Zhou et al 2012), soybean (Lee et al 2004, Chen et al 2008, Hamwieh et al 2011), white clover (Wang et al 2010), tomato (Bolarin et al 1991, Asins et al 1993, Foolad and Chen 1999, Foolad et al 2001) and wheat (Ma et al 2007) Furthermore, QTLs for ST during reproductive stage (e.g grain or fruit yield under salt stress) have been identified in different plant species, including rice (Takehisa et al 2004, Manneh et al 2007), barley (Ellis et al 2002, Xue et al 2010) and tomato (Breto et al 1994, Monforte et al 1999, Villalta et al 2007) Many of these studies have indicated the complexity of the genetics of ST across plant species However, most studies have also suggested that this complexity could be simplified by looking at tolerance at individual developmental stages Specific ontogenetic stages, including seed germination and emergence, seedling survival and growth, and vegetative growth and reproduction, may have to be evaluated separately for the assessment of tolerance and the identification and characterization of useful genetic components Partitioning of the tolerance into its component traits related to ontogenic stages would facilitate a better understanding of the genetic basis of tolerance and the development of salt-tolerant genotypes QTL Expression at Different Growth Stages and under Different Conditions Although many QTLs for ST at different phases of plant growth have been identified and mapped in different plant species, a major concern is that the ability to withstand salt stress is a developmentally regulated, stage-specific phenomenon, so that tolerance at one stage of plant development is not necessarily correlated with tolerance at other developmental stages (Kumar et al 1983, Caro et al 1991, Johnson et al 1992, Foolad and Lin 1997, Pearen et al 1997, Shannon 1997, Almansouri et al 2001, Foolad 2004) Thus, often ST QTLs identified at one stage of plant development are different from those identified at other developmental stages (Mano and Takeda 1997, Foolad 1999a, Foolad et al 1999, Zhang et al 2003) For example, Mano and Takeda (1997) reported that in barley, the QTLs identified for ST at the germination stage were different from those contributing to ST at the seedling stage, suggesting that different genes or physiological mechanisms control ST at the two stages Similar observations have been reported in tomato (Foolad and Lin 1997, Foolad 1999a, 2004, Zhang et al 2003) and other plant 15 species (Walia et al 2007, Khan 2011) Similar results have been reported for other abiotic stresses, including cold tolerance (Foolad and Lin 2000, 2001) The overall conclusion from these studies is that to gain a better understanding of the genetic control of stress tolerance (including ST) and to improve plant tolerance, specific ontogenetic stages throughout the plant life cycle should be evaluated separately for the assessment of tolerance and identification, characterization and utilization of useful genetic components This approach is expected to simplify the complexity of stress tolerance and facilitate the development of plants with improved tolerance throughout the plant ontogeny For example, once genetic components of ST at individual developmental stages are tagged with molecular markers, the MAS technology can facilitate transferring of such genetic components to desirable genetic backgrounds leading to the development of plants with enhanced ST throughout the plant life cycle In addition to be stage specific, QTLs have been reported to be environmental and population specific For example, a QTL (fwTG48-TG180) that accounted for 58% of the variation in fruit weight of tomato under non-saline conditions could explain only 14% of the variation under saline stress (Monforte et al 1997) And when the same QTL studied in a different tomato population, it accounted for 17% of the variation under control conditions and 8% of the variation under saline conditions (Monforte et al 1997) These observations suggest the presence of significant genotype genotype and genotype environment interactions when it comes to the expression of genes/QTLs affecting plant ST (Tsuruta et al 2002) Realizing considerable effects of environment on the expression of QTLs, Collins et al (2008) suggested the presence of two types of QTLs, constitutive, which appear in all environments, and adaptive, which normally are detected only in specific environments or show varying expressions with the change in environmental conditions Similar observations and classifications have been made when QTLs were compared under different stress (e.g salt, cold and drought) and non-stress conditions in tomato (Foolad 1999b, 2000, Foolad et al 1999, 2003, 2007) In these studies, it was determined that some QTLs were expressed under both stress and non-stress conditions, whereas other QTLs were detected only under stress conditions Additionally, there were QTLs that were expressed under various stress conditions, whereas others were more stress specific, that is, they were expressed only under certain stress conditions However, depending on breeding goals and target environment(s), constitutive and adaptive QTLs may have different values Improvement in Crop Salt Tolerance through Marker-assisted Selection As described earlier, numerous studies have identified QTLs contributing to ST at different developmental stages and for different ST attributes (Table 1) The challenge, however, has been the utilization of the marker-QTL information in breeding programmes for improving crop ST In general, unlike the situation with simple/qualitative traits where marker information has been frequently and successfully utilized in breeding programmes, for complex/quantitative traits, including ST, which are often controlled by more than one gene (QTL) and exhibit low heritability and strong G E interactions, such information is underutilized The limited use of molecular markers for complex traits is because of various reasons, including QTLs being unreliable or population and environment specific, QTLs not strong enough in terms of linkage to warrant their use for marker-assisted 16 M ASHRAF and M.R FOOLAD breeding, lack of marker validation or marker polymorphism in breeding populations, and problems associated with linkage drag In the case of ST, however, a few studies can be deciphered from the literature where gene or QTL information has been used to develop lines or cultivars with improved ST through marker-assisted breeding (Table 2) One example is the recent development of a highly salt-tolerant cultivar of durum wheat by R Munns and her research team at CSIRO, Australia (Munns et al 2012) Durum wheat is generally known for its more sensitivity to salt stress compared with the common bread wheat, and this has been attributed to its inherent lower ability of excluding Na+ from the leaf blade (Gorham et al 1990) In an earlier study, Munns et al (2000) had identified a durum line (known as Line 149) with Na+ exclusion ability similar to that of bread wheat In a genetic analysis of a cross between Line 149 and a durum wheat accession (Tamaroi) with normal Na+ exclusion ability, two genes, Nax1 and Nax2, were identified for controlling Na+ exclusion (Munns et al 2003) Nax1 was mapped to the distal region of chromosome 2AL (Lindsay et al 2004), and Nax2 was mapped to chromosome 5AL (Byrt et al 2007) In subsequent studies, these two genes were introduced into various durum wheat lines through marker-assisted breeding The newly developed durum wheat lines were tested under natural saline fields in northern New South Wales, Australia, and the lines possessing particularly Nax2 were able to produce approximately 25% more yield than the control durum wheat lines under such saline conditions (Munns et al 2012) The findings of this research are encouraging and provide confidence to the use of MAS for breeding plants for improved ST In a similar study in rice, scientists at the International Rice Research Institute (IRRI) identified a single major QTL (Saltol) on the short arm of chromosome 1, which explained much of the variation for ST in a segregating rice population (Bonilla et al 2002) In subsequent studies conducted at the Bangladesh Rice Research Institute (BRRI), attempts have been made to transfer Saltol to two highyielding popular commercial rice varieties, BR11 and BR28 (Rahman et al., http://www.pbtlabdu.net/abs_rahman_et_al.pdf) In this study, various markers closely linked to Saltol were identified and used to transfer the QTL to the two commercial varieties via marker-assisted backcrossing In tomato, MAS was employed to develop salt-tolerant breeding lines using an F2 population of a cross between the cultivated tomato (salt-sensitive) and a salt-tolerant accession of the tomato wild species Solanum pimpinellifolium (Monforte et al 1996) In this study, a combination of phenotypic selection and MAS was used to develop lines with improved ST However, despite the development of advanced filial generations, which contained the QTLs for ST, there is no report of the evaluation of these lines under field conditions to verify their ST behaviour or agricultural value In summary, despite the extensive efforts made to identify QTLs contributing to ST in different crop species, limited attempts have been made to use such QTL information for marker-assisted breeding for improved ST This situation is similar to that for most other complex traits across crop species However, the limited use of markers for improving crop ST is attributable to various reasons, including (i) generally limited efforts that have been made by breeders to develop plants with improved ST, compared to efforts devoted to other economically important traits such as disease resistance and improved quality and quantity of yield, (ii) limited familiarity of many plant breeders with the marker technology, (iii) insufficient reliability of the identified QTLs or lack of QTL confirmation and (iv) population specificity of the reported QTLs and their associated markers However, with the new advancement in marker technology and trait phenotyping and the greater need in crop plants with improved ST, it is expected that more progress will be made in developing new cultivars with improved ST in particular via using marker-assisted breeding approaches Furthermore, once QTLs have been verified for use in MAS, efforts must be made to clone and characterize important QTLs Cloning of QTLs conferring ST will not only enhance the functional understanding of the tolerance and the underlying genes and mechanisms (Salvi and Tuberosa 2005), but also provide breeders with precise markers for both breeding purposes and exploitation of allelic variations present in germplasm collections The latter is particularly crucial for further identification and characterization of desirable genetic resources and enhancement of crop ST Conclusion and Future Prospects Most modern cultivars of major crop species are highly or moderately sensitive to salt stress and thus no perform well under field saline conditions Fortunately, genetic sources of ST have been identified in most crop species, which could be utilized for breeding purposes However, most of the identified germplasms with ST characteristics have been identified within the related wild or feral species, which could not be utilized in breeding programmes without inherent difficulties The advent of molecular markers and mapping technology has promised opportunities to identify genes or QTLs of interest for complex traits such as ST and transfer them more precisely from unadapted genetic backgrounds into modern cultivars via the process of MAS One of the advantages of MAS when using wild germplasm as genetic resources is to reduce the problems associated with linkage drag With this promise in mind, considerable efforts have been made and many genes or QTLs have been identified Table 2: Improving salinity tolerance in different crop lines/varieties using marker-assisted selection Crop QTL used QTL donor line/cultivar Recipient line/cultivar Line/cultivar developed Durum wheat (Triticum turgidum L.) Nax1 and Nax2 Line 149 Tamaroi Durum wheat Rice (Oryza sativa L.) Saltol FL378 BR11 or BR28 BR11 Tomato (Solanum lycopersicum L.) TG24 Solanum pimpinellifolium (wild tomato) S lycopersicum (cultivated tomato) F3 population of S lycopersicum Traits improved Improved 25% yield by enhancing Na+ exclusion from the leaf blade Higher yield under saline conditions Improved total fruit weight under salinity References Munns et al (2000) Rahman et al (2008) Monforte et al (1996) Crop breeding for salt tolerance in the era of molecular markers contributing to ST in different plant species However, despite such progress, currently, there are few examples of successful development of cultivars or breeding lines with improved ST via the use of molecular markers and MAS technology This is in sharp contrast to the extensive and efficient use of molecular markers for improving simple traits, which are often controlled by one or few genes with independent effects and free of environmental influence The limited use of markers for improving complex traits has been attributable to various reasons as discussed in this article; however, this does not mean that the marker technology will not be useful for developing crops with improved ST Theoretically, it should be possible to use markers for improving complex traits such as ST, assuming that efforts are made to identify reliable QTLs and associated genetic markers However, the development of reliable marker information for quantitative traits necessitates additional efforts, including: (i) Conducting mapping experiments under field production conditions where all factors affecting the expression of the quantitative trait of interest are accounted for Often QTL mapping experiments are conducted under controlled greenhouse conditions, where the environment is different from field conditions For example, most experiments to identify QTLs for ST have been conducted under greenhouse conditions with little or no transpiration Such settings would not impose the kinds of stress that plants may experience under field conditions, which certainly affect gene expression and QTL identification Furthermore, plants respond to various stresses in a coordinated and interactive manner and cross-tolerance exists against such stresses, which make the mechanisms much more complex (Orsini et al 2010) Under field saline conditions, there are always other stresses that plants have to deal with and respond to in order to survive or produce economic yield Thus, the identification of QTLs for ST under confined greenhouse conditions may not be useful for developing plants with improved ST under field conditions At least the QTLs identified under controlled conditions must be re-examined and confirmed under field saline conditions before using them in markerassisted breeding (ii) Repeating screening experiments in multiple environments to minimize the environmental effects on trait expression and maximize the relationship between phenotype and genotype (i.e increasing heritability of the trait) Generally, QTLs identified in multiple environments are more reliable (iii) Minimizing other environmental variation to increase the heritability of the trait, for example by increasing the number of replications in space and using larger population size (iv) Breaking complex traits into their simpler individual components and identifying QTLs and linked markers for such individual components, instead of studying the trait as a whole For example, ST is a developmentally regulated phenomenon, which differs with changes in plant age and developmental stages Tolerance at one stage of plant development may not be correlated with tolerance at other developmental stages or with the overall performance of the plant during its life cycle QTLs must be identified for individual developmental stages as well as for individual physiological parameters contributing to ST Pyramiding of such QTLs may lead to the development of plants with improved ST Although the proposed approach seems challenging, it is certainly doable assuming devoted efforts and is expected to pave 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(2008) Monforte et al (1996) Crop breeding for salt tolerance in the era of molecular markers contributing to ST in different plant species However, despite such progress, currently, there are