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RAPD Markers Associated with Drought Tolerance in Bread Wheat (Triticum aestivum L.)



H. Pakniyat and E. Tavakol
 
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ABSTRACT

Randomly Amplified Polymorphic DNAs (RAPDs) were used to search genetic diversity and markers associated with drought tolerance in 20 bread wheat cultivars. These cultivars are extensively being used by farmers in Iran, 6 of them are known as drought tolerant. Initial screens involved growing 10 cultivars at seedling stage under drought conditions (-5 and -8 bar) exerted by PEG 6000 in a hydroponic experiment. These tests confirmed the tolerance of the 6 above mentioned cultivars. Thirty 10-mer RAPD primers were used for fingerprinting of the cultivars of which primers P6 (TCGGCGGTTC) and P7 (CTGCATCGTG) produced respectively a 920 and a 750 bp band present in drought tolerant (absent in others) cultivars. These bands may be associated with drought tolerance in bread wheat.

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  How to cite this article:

H. Pakniyat and E. Tavakol , 2007. RAPD Markers Associated with Drought Tolerance in Bread Wheat (Triticum aestivum L.). Pakistan Journal of Biological Sciences, 10: 3237-3239.

DOI: 10.3923/pjbs.2007.3237.3239

URL: https://scialert.net/abstract/?doi=pjbs.2007.3237.3239

INTRODUCTION

Water deficit stress is a major limiting factor for plant growth and productivity resulting large economic losses in many regions of the world (Ashraf and Harris, 2005). The exploitation of tolerant crops is one approach to the problem. Wheat is widely grown as a rain-fed crop in semi-arid areas, where large fluctuations occur in the amount and frequency of rainfall events. The development of tolerant cultivars, however, is hampered by low heritability for drought tolerance and a lack of effective strategies (Kirigwi et al., 2004; Bajji et al., 2001).

To differentiate drought tolerant genotypes, several selection indices have been suggested on the basis of a mathematical relationships between favorable and stress conditions (Clark et al., 1984; Huang, 2000). Tolerance (TOL) (McCaig and Clark, 1982; Clarke et al., 1992), Mean Productivity (MP) (McCaig and Clarke, 1982), Stress Susceptibility Index (SSI) (Fischer and Maurer, 1978), Geometric Mean Productivity (GMP) and Stress Tolerance Index (STI) (Fernandez, 1992) have all been employed under various conditions. Measuring these indices are tedious and time consuming, therefore, selection by aids of genetic markers may be very helpful for this purpose. A wide range of genetic markers is available for study in wheat. Earlier work with barley indicated that RAPD and AFLP markers are associated with salt tolerance (Pakniyat et al., 2004; Pakniyat et al. 1997). Here we use RAPDs as a quick and easy method to search for DNA markers associated with drought tolerance in selected lines of bread wheat contrasting for tolerance.

MATERIALS AND METHODS

DNA extraction: A total of 20 bread wheat cultivars including tolerant, semi-tolerant and non-tolerant were used (Table 1). Six of the cultivars were drought tolerant and extensively are planted by local farmers under drought conditions.

DNA was extracted from 10 days old seedlings using CTAB method (Murry and Thompson, 1980) with some modifications.

Polymerase chain reaction conditions: PCR was carried out essentially as described by Williams et al., 1990. Thirty 10-mer oligonucleotide primers were used for DNA amplification (Table 2). Amplifications were performed in a Technogene Co. thermocycler first 4 min at 94°C followed by 10 cycles of: 1 min at 94°C, 1 min at 36°C and 1 min at 72°C. After that, for next 35 cycles, 0.2°C was added to annealing temperature. After the final cycle, samples were incubated at 72°C for 15 min and then held at 4°C prior to analysis. Amplification products were separated by electrophoresis in 1.5% agarose gels and detected using ethidium bromide and UV light.


Table 1: Bread wheat genotypes and their drought tolerance status, used in the experiment
*Reaction to drought reported by research centers which seeds collected

Table 2: Primer nucleotide sequence used to amplify DNA

Each amplification was performed using a single primer and gels scored for the presence and absence of products.

RESULTS AND DISCUSSION

Preliminary hydroponic experiment performed with a subset (10 cultivars) of bread wheat cultivars including the resistant ones under water deficit stress (-5 and -8 bars) exerted by PEG 6000, confirmed drought tolerance of the 6 tolerant cultivars shown in Table 1 (Tavakol, 2005). Twenty eight primers produced scorable amplification products. Two primers (P15 and P26) produced vague un-scorable bands. From 378 amplified band 156 (41%) were polymorphic and primers P3, P11 and P25 produced more polymorphic bands. Band sizes were between 300 to 1900 bp. Primers P6 (TCGGCGGTTC) and P7 (CTGCATCGTG) produced a 920 and 750 bp bands present in drought tolerant cultivars (Azar 2, Gahar, Koohdasht, Bow, Zagros and Sardari). These bands may be associated with drought tolerance in bread wheat (Fig. 1 and 2) and they may be used in selection of tolerant genotypes in breeding plans.

The results indicate that RAPD technology is a powerful tool in quickly identifying markers related to drought tolerance in wheat. It is interesting that in this research, two out of 28 primers detected associations to drought tolerance in tolerant cultivars which they may be related to involved QTLs.


Fig. 1: Electrophoretic pattern generated by RAPD primer P7 (CTGCATCGTG). Arrow shows a 750 bp band present in drought tolerant (absent in others) cultivars

It is hoped that the discovery of markers associated with drought tolerance with the aid of the genes involved. One strategy for P6 and P7 products is to sequence the 920 and 750 bp bands and search against stress-related ESTs (expressed sequence tags) in public databases (Boguski et al., 1993) to identify the gene involved.


Fig. 2: Electrophoretic pattern generated by RAPD primer P6 (TCGGCGGTTC). Arrow shows a 920 bp band present in drought tolerant (absent in others) cultivars

Sequence data can also be used to develop more robust PCR primers as diagnostics for drought tolerance. Another strategy is to map the two products to confirm or otherwise their genetic co-location.

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