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Research Article

Total Wheat DNA Variation in to Varieties Using Known Primers of the Genes Induced in Dehydration and Salinity Stress

Muhammad Ashraf, Afsari S. Qureshi and Abdul Ghafoor
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Ten genotypes of wheat used characterised with their total DNA variation with special reference to known primers of the genes induced in dehydration and salinity stress. Amplification profiles of DRE primers in identification of polymorphic bands that can be used for finger printing the varieties. Four main groups were observed based on 138 polymorphic bands and variance was related to drought, pedigree or geographic origin. Similar pattern of genetic diversity was also observed when 200 markers were recorded from DRE combinations. Pakistani varieties showed higher degree of 70% dissimilarities, While exotic showed low genetic distance.

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  How to cite this article:

Muhammad Ashraf, Afsari S. Qureshi and Abdul Ghafoor, 2003. Total Wheat DNA Variation in to Varieties Using Known Primers of the Genes Induced in Dehydration and Salinity Stress. Pakistan Journal of Biological Sciences, 6: 437-440.

DOI: 10.3923/pjbs.2003.437.440



Wheat is the most important cereal crop and staple food of the world. It covers about one fifth of the Punjab rainfed tract, Where water deficiency most often severely effects the productivity and thus became one of the important limiting factor. The effects of drought on wheat have generally been studied in field and based on data recorded for agronomic, morphological and physiological plant traits. Such data characterize the varieties, but may not necessarily provide an accurate identification of genes for drought tolerance because of environmental influence (Aspinall, 1984). Bio-chemical markers have also been used; most recently DNA makers are being used for this purpose for assessment of genetic variability for drought/stress tolerance. Among the DNA markers, Random Amplified Polymorphic DNA (RAPD) has been reported as a useful tool for evaluating intra-specific variation (Dos Santos et al., 1994; Thormann et al., 1994) and for varietal characterization (Farooq et al., 1994). RAPD markers are generated by Polymerase Chain Reaction (PCR) with a single short oligo-nocleotides of arbitrary sequences and provide numerous markers that give genetic information at DNA level. RAPD are simple to produce, easy to handle and are rapidly detect variability used for varietal characterization (Farooq et al., 1994 and Linc et al., 1996).

Drought specific primers acts as a motif and called Dehydration responsive elements (DRE) identified by Shinozaki and Shinozaki (1994) are also being used these elements are conserved DNA sequence of about 9 base pair (TACCGACAT), present in the 5 ’ promoter regions of genes that response to dehydration at transcriptional level in many plant species which are related to drought (Skriver and Mundy, 1990).

Materials and Methods

Ten pure-lines/varieties of wheat from diverse origin were used in the present study (Table 1). RAPD analysis was conducted in the Biological Resources Division Lab., (JIRCAS) at Tsukuba, Japan. Twenty seeds of each variety were sown in pots. After 30 days, young leaves were harvested and DNA was extracted using 20% SDS with extraction buffer [1 M Tris HCl (pH 8.0), 0.5 M EDTA (pH 8.0), 5 M NaCl and 10 m M of 2 mercaptoethanol]. The quantity was measured with spectra-photo-meter. Quantify of DNA used for amplification was 10 ng–1μl and also re-checked by mini gel using λ DNA as standard.

The PCR was performed in 0.5 ml reaction tube with 25 Fl of DNA solution containing 3.0 Fl genomic DNA (10 ng–1Fl), 2.5 Fl of primer (10 FM), 2.0 Fl dNTP (2.5 FM), Gene Taq 5.0l–1 Fl (Nippon Gen. Co.) 0.3 Fl, buffer (X 10) 2.5 Fl and glycerol (7.5%) 14.7 Fl. The amplification reaction was carried out in the Mj research PTC 100 machine with 94°C for one min, 40°C for 2 min, and 72°C for 2 min completed 49 cycles. After that 72°C for 5 min was applied for complete annealing prior to storage at 4 °C of the amplification reactor 15 random primers, and another 15 random primers with combination of Micro Satellite and 6 were with DRE primers (Table 2).

The data of polymorphic fragments were recorded. After electrophoresis, the content were loaded on to 13% Poly Acrylamide Gel, [13 ml H2.O, 5 ml Acrylamide (40%), 2.0 ml of 10 X TBE, 30% fresh APS and 10 Fl of TEMED]. The solution was mixed thoroughly and poured in to the glass plates to let the gels polymerised. Before applying the sample, the wells of the gel were thoroughly washed and 7.0 Fl from individual sample was applied to each value. Electrophoresis was conducted at 300 V for 120 min. Lambda (λ) DNA was used as molecular marker.

Table 1:Varieties used for DNA fingerprinting with special reference to drought resistance

Table 2:Primers used for amplification in wheat

After electrophoresis, the gels were stained with silver nitrate. The banding profile appearing on the gel photodocumented and data was recorded and took photographs. The data were recorded as presence (1) or absent (0) and analyzed by UPGMA method (Sokal and Mitchener, 1958, Lance and Williams, 1966; Sneath and Sokal, 1973). Cluster analysis was formed on the basis of total 118 polymorphic fragments and fragments 36, 26, 4, 13, 16, 11 and 12 obtained by P1-DIC, P1-DICKA, P2-DIC, P2-24F, P1-joint and P2-joint primers separately.

Results and Discussion

Fig. 1 shows the amplification profile for overall primers P1-DIC, P1-DICKA, P2-DIC, P2-24F, P1-joint and P2-joint. The variety C 591 exhibited maximum genetic distance when compared with all other varieties (Table 3). Similarly Rawal 87 and F. Kumugi revealed maximum distance. Genetic distance based on 200 polymorphic bands obtained by DRE primers ranged from 4.36 (Janz Vs O. Culm # 380) to 10.54 (F. Kumugi Vs C 591). Genetic distance varied from 9.38 (O. Culm # 380 Vs. F.K.) to 14.53 (Rawal 87 Vs. F. K. and Rawal 87 Vs. O. Culm # 380) as presented in Table 4. The varieties from Pakistan exhibited high range of genetic diversity, which might be due to diverse parentage as most of wheat varieties have been bred from CIMMYT material. Genetic distance for 10 varieties of wheat based on 200 RAPD bands revealed four major groups. Group A represents one variety V 8203 that was a CIMMYT line. Group B represents three varieties viz., Oligo-Culm # 380 (O. Culm # 380), Janz and Fukuho-kumugi (F.K.) that were all exotic and the status for drought were not known. The group C represents five varieties viz., CB 51, Ch 70, V 90R34, Blue Silver and Rawal 87.

Fig. 1:Banding pattern of primers SK-7+OPF 08 (left) and SK-7+OPU 16 in 10 wheat varieties

Out of this group, Blue Silver was short duration and escapes terminal drought; Rawal 87 and V 90R34 were bred for low to medium rainfall, whereas CB 51 and Chenab 70 were susceptible to drought. Group D represents the variety C 591 that is a drought resistant variety and well known for high bread making quality.

Table 6 gave the results of cluster analysis based on 422 RAPD fragments. Cluster IV comprising of two cultivars Rawal 87 and C 591 were drought tolerant. In relation with drought resistance, clustering on the basis of 422 fragments revealed more clear separation for drought stress. It might be due to more number of polymorphic fragments. Group D represents variety C 591 and Rawal 87, which were drought tolerant and these exhibited maximum genetic distance from other groups. Group B comprising of susceptible cultivars except Blue Silver and V 90R34 which bred for low to medium rainfall. Blue Silver has grouped with susceptible group although it is considered drought tolerant. In fact this variety is short duration and due to this feature it escapes drought. Overall analysis based on 422 polymorphic bands showed almost similar pattern except Rawal 87, which was grouped in cluster IV. According to the genetic distance, variety C 591 and Rawal 87 stand apart from all the other varieties and these both varieties were considered drought tolerant (Table 5).

Table 3:Fragment profile and cluster of each variety on the basis of dendrogram in cluster analysis for each primer and overall

Table 4:Genetic distance for 200 RAPD bands in wheat

Table 5:Genetic distance for 422 RAPD bands in wheat

Table 6:Clusters based on 422 fragments in wheat

The RAPD results conquered to the morphological, traits and groups could be placed in to groups based on drought and geographic region. Drought and geographic (rainfall) relationship of the crop plants as discussed by many earlier researchers (Rao et al., 1992; Sharma et al., 1995).

It is evident from the results that RAPD can be used as a tool for the investigation of morphological and geographical relationship of wheat crop. Drought resistance can be identified by the use of RAPD analysis. This technique in combination with plant physiological analysis can efficiently be used to develop drought resistance cultivars to overcome wheat shortage in the country.

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