AFLP marker linked to water-stress-tolerant bulks in barley ( Hordeum vulgare L . )

The amplified fragment length polymorphism (AFLP) assay is an efficient method for the identification of molecular markers, useful in the improvement of numerous crop species. Bulked Segregant Analysis (BSA) was used to identify AFLP markers associated with water-stress tolerance in barley, as this would permit rapid selection of water-stress tolerant genotypes in breeding programs. AFLP markers linked to water-stress tolerance was identified in two DNA pools (tolerant and sensitive), which were established using selected F2 individuals resulting from a cross between water-stress-tolerant and sensitive barley parental genotypes, based on their paraquat (PQ) tolerance, leaf size, and relative water content (RWC). All these three traits were previously shown to be associated with water-stress tolerance in segregating F2 progeny of the barley cross used in a previous study. AFLP analysis was then performed on these DNA pools, using 40 primer pairs to detect AFLP fragments that are present/absent, respectively, in the two pools and their parental lines. One separate AFLP fragment, which was present in the tolerant parent and in the tolerant bulk, but absent in the sensitive parent and in the sensitive bulk, was identified. Polymorphism of the AFLP marker was tested among tolerant and sensitive F2 individuals. The presence of this marker that is associated with water-stress tolerance will greatly enhance selection for paraquat and water-stress tolerant genotypes in future breeding programs.


Introduction
Water stress is one of the greatest yield-reducing factors.It causes various physiological and biochemical effects on plants (Tanaka et al., 1990;Irigoyen et al., 1992;Smirnoff, 1993;Bohnert and Jensen, 1996;Jamaux, 1997;Tabaeizadeh, 1998), and is therefore a serious problem in many parts of the world where barley, wheat, and other small-grained cereals form the staple diets (Quarrie et al., 1999).This is true not only in arid and semiarid regions, but also in places where total precipitation is high, but is not evenly distributed over the growing season (Ribaut et al., 1997).The effects of water stress upon leaf physiology could be mediated by the production and accumulation of toxic reactive oxygen intermediates.Reactive oxygen intermediates generated during water stress may represent a serious challenge to a number of cellular functions.For in-stance, chlorophyll destruction, serious de-organization of chloroplast fine structure, and enzyme inactivation can be due to these toxic oxygen forms (Irigoyen et al., 1992).These effects of water stress on the physiology of the plants is very similar to the stress caused by the peroxidizing herbicide paraquat (PQ), which leads to the production of highly toxic free radicals generated by reaction of molecular oxygen with PQ radicals formed in the chloroplast during photosynthesis (Dodge, 1971).Therefore, a close correlation is expected between the plant's tolerance to stresses imposed by water and PQ.It is also possible that drought-tolerant plants can be selected based on their response to PQ in segregating breeding populations (Altinkut et.al., 2001).Other morphological and physiological traits of interest with respect to water-stress tolerance mechanisms include leaf size and relative water content (RWC) (Teulat et al., 1997 a, b;Grainer and Tardieu, 1999;Gonzalez et al., 1999).In our previous study (Altinkut et al., 2001), we reported that leaf size, RWC and PQ tolerance are associated with enhanced tolerance to water-stress in barley and wheat.
Tolerance to water-stress must be incorporated into breeding material to be grown in regions of low rainfall.However, a major constraint in introducing this trait into otherwise elite genotypes is the lack of meaningful selection criteria.An alternative would be to identify molecular markers associated with water-stress tolerance, and to use these markers to indirectly select for tolerance (Courtois et al., 2000).Molecular marker analysis allows to identify genome segments contributing to the genetic variance of a trait, and thus to select superior genotypes at these loci, without uncertainties regarding the genotype, due to environment interaction and experimental error.
The amplified Fragment Length Polymorphism (AFLP) technique (Vos et al., 1995) is based on the amplification of selected restriction fragments of a total genomic digest by PCR, and separation of labeled amplified products by denaturing polyacrylamide gel electrophoresis (Becker et al., 1995).It has been extensively used for developing polymorphic markers linked to important agronomic traits.High reproducibility, rapid generation, and high frequency of identifiable polymorphisms make AFLP analysis an attractive technique for determining linkages by analyzing individuals from segregating populations (Goodwin et al., 1998;Hartl et al., 1999).In this study, we have used the bulked segregant analysis (BSA) technique (Michelmore et al., 1991) in combination with AFLPs to identify potential molecular marker(s) associated with water-stress tolerance in barley, so that these markers shall be used for selecting water-stress tolerance in segregating populations.

Plant material
Eighty F 2 plants from the cross Tokak x ST 5819 and their parental lines were grown in individual pots with a soil mixture containing soil/sand/organic matter at a ratio of 1:1:1.All plants were grown in a growth chamber at 23 °C, 70% humidity, and 16 h light/8 h dark periods.

Screening the F 2 mapping population for water-stress tolerance
The methods used for selecting water-stress tolerance in barley plants were described previously (Altinkut et al., 2001).Briefly, to prepare bulks, leaf chlorophyll content after PQ treatment, leaf size, and RWC from the tolerant and sensitive parents of the barley cross and its F 2 population of 80 individuals were measured.Leaf size and chlorophyll content after PQ treatment were measured on the 2 nd top leaf, taken from the plants during the pre-flowering stage.Leaf extracts were analyzed for determination of chlorophyll content, after treating leaves with 100 µM PQ solution, under a light intensity of 12000 lux for 24 h.RWC were measured on the 6 th day of water stress.

Bulked segregant analysis with AFLPs
Freeze-dried leaf materials from F 2 individuals and parents were extracted for genomic DNA, according to the method described by Lodhi et al. (1994).Bulked segregant analysis with AFLP markers was used to identify markers associated with water-stress tolerance in the population Tokak x ST5819.Aliquots of DNA from seven tolerant and seven sensitive plants were combined to produce bulks.
Four AFLP reactions were performed with each primer pair on the DNA extracted from the following groups of barley plants: (1) the tolerant parent; (2) the sensitive parent; (3) the pooled DNA of the most tolerant F 2 plants; and (4) the pooled DNA of the most sensitive F 2 plants.The AFLP procedure was performed as described by Zabeau and Vos (1993) and Vos et al. (1995), and comprised of three steps, as follows.

Electrophoresis
Amplification products were separated on a 6% polyacrylamide (acrylamide: bisacrylamide = 19:1) / 8 M urea sequencing gel at 1750 V for 3 h in 1X TBE buffer (0.09 M Tris-borate and 0.002 M EDTA), and detected by silver staining according to the manufacturer's instructions (Promega).Band sizes were determined by comparison with a 10 base-pair (bp) DNA ladder size standard from Gibco-BRL (MD, USA).

Results and Discussion
The main objective of this study was to determine whether different polymorphic AFLP markers could be identified in barley bulks made up based on traits associated with water-stress tolerance.Since water-stress tolerance is a quantitatively inherited trait, controlled by several genetic loci, which are difficult to measure (Forster et al., 2000), identification of molecular markers associated with a major locus contributing to water-stress tolerance would be useful for indirect selection for water-stress tolerance (Altinkut and Gozukirmizi, 2003).However, in most instances, identifying genetic markers associated with important genes or traits requires screening of a relatively large number of individuals in the population (Lawson et al., 1994).For this reason we chose to use BSA, since comparing bulk samples is more convenient than evaluating many individuals in different populations (Sweeney and Danneberger, 1994).
To constitute bulks, individual barley F 2 plants from cross between water-stress tolerant (Hordeum vulgare L. cv.'Tokak' -a known water-stress tolerant cultivar) and water-stress sensitive (H.vulgare L. 'ST 5819' -a highly water-stress sensitive line) barley plants were used to prepare the water-stress tolerant and sensitive bulks.Each water-stress tolerant and sensitive bulk included 7 selected F 2 plants.The methods used for selecting water-stress tolerance in these barley plants were described previously (Altinkut et al., 2001).Briefly, the parental barley genotypes used here differed in their leaf chlorophyll content following PQ treatment, leaf size, and RWC.These traits were scored in the barley cross F 2 population of 80 individuals (Table 1).To prepare the bulks, leaf chlorophyll content after PQ treatment was measured from the tolerant and sensitive parent of the barley cross and its individual segregating F 2 plants.Seven F 2 plants that fell in the extreme ends of the distribution for leaf chlorophyll content were then selected to prepare the tolerant and sensitive bulks.In addition, RWC and leaf size were measured in F 2 plants and their parental genotypes.As shown in  higher RWC values.The correlation coefficient calculated for PQ tolerance and leaf size was significant (r: -0.423, p > 0.05).We then examined the correlation between PQ tolerance and RWC, and the correlation coefficient calculated was highly significant (r: 0.830, p > 0.05).Finally, the association between RWC and leaf size also showed a significant correlation (r: -0.593, p > 0.05).
AFLP fingerprinting was then used to identify markers potentially associated with water-stress tolerance.Combinations of ten MseI primers and four PstI primers, each with three selective bases, on DNA from pooled samples, were used in barley DNA.The DNA pools that we used in this study included F 2 individuals selected for their morphological and physiological traits associated with water-stress tolerance.Approximately 12000 AFLP fragments amplified from barley using a total of 40 primer combinations were analyzed by running on polyacrylamide gels.A majority of the 40 primer pair combinations produced amplification patterns, which were not different between the tolerant and sensitive bulks and their parents.However, one primer pair produced an amplification product that was present in the tolerant parent and in the tolerant bulk, but not in the sensitive one.The primer pair moo+CAT / poo+TGG amplified a DNA fragment of 172 bp, which was present in the tolerant parent and in the tolerant bulk, but not in the sensitive one.The segregation of this marker in the 14 individuals, which constituted the bulks was tested.The 172 bp fragment was present in six out of seven individuals in the tolerant bulk, and was absent in six out of seven individuals in the sensitive bulk.Only 1 sensitive plant generated the polymorphic band (Figure 1).It was significant that the single F 2 plant in the sensitive bulk which generated the 172 bp fragment, and the single F 2 plant in the tolerant bulk which did not generate the 172 bp fragment were two of the misclassified individuals, possibly due to selection error, and that, despite this, BSA was successfully used to identify the fragment.The reproducible amplification of this fragment in repeated amplifications suggests that it is associated with a major gene(s) that contributes to enhanced tolerance to water-stress-associated traits.The results also demonstrate that a BSA strategy may be useful even for the identification of markers for quantitative traits, such as water-stress tolerance.Recently, similar approaches have been used to identify markers associated with other quantitatively inherited traits, namely anther culture response in potato (Boluarte-Medina and Veilleux, 2002), economically important traits in Pinus (Lerceteau et al., 2000), and disease resistance in tomato (Peleman, 1999).However, to determine the transportability of the marker identified in this study to other genotypes, other crosses derived from different parental genotypes should be evaluated.In this regard, the present study only focussed on the barley genotype that is most commonly used in breeding programs in Turkey as a source of drought tolerance.
The marker identified in this study, once verified in other barley genotypes, would allow implementation of marker-assisted selection.We expect that this marker may be even more useful when converted into a simplesequence PCR-based marker that can be used for largescale water-stress tolerance screening of segregating populations.

Table 1 ,
plants with higher chlorophyll content also had smaller leaves and AFLP polymorphism in Hordeum vulgare L. 79

Table 1 -
Morphological and physiological data used for making the water stress tolerant and sensitive barley and wheat bulks in both parental lines and their selected F 2 individuals.Measurements of leaf size and chlorophyll content after PQ treatment were conducted on the 2 nd top leaf taken from the plants during the preflowering stage.Leaf extracts were analysed for determination of chlorophyll content after treating leaves with 100 µM PQ solution under a light intensity of 12000 lux for 24 h.
A: Chlorophyll Content in untreated leaves mg/chlorophyll/fresh weight.B: Chlorophyll Content in PQ treated leaves mg/chlorophyll/fresh weight.T : tolerant F 2 individuals selected for bulk.S : sensitive F 2 individuals selected to constitute the bulks.

Table 2 -
Morphological and physiological data of parental lines* and their F 2 individuals, used to make up the water-stress tolerant and sensitive barley bulks.