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Estimation of genetic diversity in wheat using Dna markers.

Introduction

Knowledge of genetic diversity among adapted cultivars or elite breeding materials has a considerable impact on the improvement of crop plants. It can be obtained from pedigree analysis, morphological traits or using molecular markers (Mohammadi and Prasanna, 2003). Molecular markers offer the best estimate of genetic diversity since they are independent of the perplexing effects of environmental factors. In recent years, several molecular assays have been applied to assess genetic diversity among wheat cultivars. These molecular methods poles apart in principle, application, type, amount of polymorphism detected and in task and time requirements. Assays based on the polymerase chain reaction (PCR) are considered to meet both the technical and genetic requirements for the characterization of plant and animal genetic resources. Several molecular markers like random amplified polymorphic DNAs (RAPD) and simple sequence repeats (SSRs) are presently available to assess the variability and diversity at molecular level (Palombi and Damiano, 2002).

To deal with the frightening global food scarceness, it is the need of hour to develop new crop cultivars with some desired characters like adaptation to dry weather, and resistance to pathogens. The continued improvement in food crops productivity is highly enviable because of increasing demands of the fast growing human population. Being the most important source of staple food, wheat occupies an important place in the crop husbandry of Pakistan. During the last few years, yield improvement in wheat varieties has not been substantial; the narrow genetic base of the germplasm in use has been considered the main reason. The development of improved varieties/cultivars having broad genetic base can increase wheat production under different stresses and agroclimatic conditions. In cereals, broad genetic base shields against spread of diseases (Zhu et al., 2000).

In Pakistan wheat varieties are in-front of a twin threat of biotic and abiotic stresses. It is well known fact that knowledge of germplasm diversity and genetic relatedness among the elite breeding material is a basic constituent in plant breeding (Mukhtar et al., 2002). It provides better understanding of evolutionary relationships among the accessions is and facilitate plant breeders to incorporate useful diversity in their breeding programs to achieve a level of self-sufficiency and sustainability (Rahman et al., 2007).

DNA marker technology has been extensively exploited in varietal improvement programmes during last few years (Legesse et al., 2007). RAPD markers have been used in assessment of genetic variation in populations, and species (Mukhtar et al., 2002; Asif et al., 2005; Rahman et al., 2007) and phylogenetic relationship (Sakowicz and Cieslikowski, 2006). Similarly SSRs have also been employed for investigation of genetic diversity in number of crops including alfalfa (Flajoulot et al., 2005), white clover (George et al., 2006), rice (Thomson et al., 2007), groundnut (Tang et al., 2007) and maize (Legesse et al., 2007). Like other crops, genetic variation in hexaploid wheat have been reported based on RAPD (Swahla et al., 2008) and SSR (Wang et al., 2007), however, there is paucity of information on diversity analysis of Pakistani wheat. Hence the present study was aimed to produce information on genetic diversity among a set of wheat germplasm by means of RAPD and SSR analysis.

Materials and Methods

Plant Material:

Seeds of four wheat genotypes, Punjab-76, Chakwal-86, Inqlab-91and Bt-2549 were obtained from Ayub Agriculture Research Institute (AARI) Faisalabad, Pakistan. Plants were grown in pots in green house under standard agricultural practices.

DNA extraction:

Total genomic DNA was extracted from 8-10 bulked leaves obtained from 4-5 randomly selected plants of the same genotype by CTAB method (Rahman et al., 2002). The DNA quality and quantity was determined by NanoDrop-1000 3.3.1 spectrophotometer and comparison with standard DNA electrophoresed on a 0.8% agarose gel.

RAPD and SSR analysis:

A total of 70 RAPD and 90 SSR primers were employed for finger printing of wheat genotypes. For RAPD analysis, the 25 [micro]l PCR amplification mixture contained 2.5 [micro]l of 10 x PCR buffer [50 mM of Tris (pH 8.3), 500 mM of KCl]; 1.5 mM of Mg[Cl.sub.2]; 0.2 mM each of dATP, dCTP, dGTP and dTTP (Fermentas, USA); 0.3 mM of a decamer primer (GeneLink, USA); one units of Taq DNA polymerase (Fermentas, USA); and 25 ng of genomic DNA as a template. Temperature cycles for DNA amplification were a first denaturation step of 5 min at 94 [degrees]C followed by 40 cycles of 94 [degrees]C for 1 min, 36 [degrees]C for 1 min and 72 [degrees]C for 2 min. After the completion of 40 cycles, a final extension at 72 [degrees]C was carried out for 7 min.

For SSR analysis, a reaction volume of 20 [micro]l was set up containing 2.0 ul of 10 x PCR buffer [50 mM of Tris (pH 8.3), 500 mM of KCl]; 1.5 mM of Mg[Cl.sub.2]; 0.2 mM each of dATP, dCTP, dGTP and dTTP (Fermentas, USA); 0.3 mM each of reverse and forward primer (GeneLink, USA); one units of Taq DNA polymerase (Fermentas, USA); and 25 ng of genomic DNA as a template. The temperature cycles were a first denaturation step of 5 min at 94 [degrees]C followed by 35 cycles of 94 [degrees]C for 30 s, 55 [degrees]C for 30 s and 72 [degrees]C for 1 min followed by a final extension at 72 [degrees]C for 7 min. All PCR reactions were performed in Mastercycler (Eppendorf, Germany).The completed reactions were then held at 4 [degrees]C until electrophoresis. RAPD and SSR products were run on 1.2 and 2.5% agarose gels, respectively, visualized by ethedium bromide staining under UV light and photographed using Gene Snap Syngene gel documentation system.

Data Analysis:

For genetic diversity analysis each band was considered as a single locus/allele. Only score able bands were considered for genetic diversity analysis. Genetic similarity matrices were generated on the basis of Nei and Li's (1979) coefficients. NTSyspc 2.0 software package was used for dendrograms construction using unweighted pair group method of arithmetic means (UPGMA).

Results and Discussion

DNA markers are important tools to generate information on genetic divergence among germplasm which is useful for improvement of crop productivity. The present study was employed to investigate genetic diversity among three classical wheat cultivars, Punjab-76, Chakwal-86, Inqlab-91, and one reportedly leaf rust resistant promising strain, Bt-2549 using 160 RAPD and SSR primers. RAPD analysis with 70 primers generated a total of 730 DNA, with an average of 10.4 bands per primer. Amplified fragments ranged 250-1500 bp in size. As for as individual RAPD profile of the cultivars/genotype is concerned, 14 fragments were polymorphic in Punjab-76, 22 in Bt-2549, 16 in Chakwal-86 and only 4 were polymorphic in inqlab-91. The highest percentage of polymorphism was observed using RAPD primers GLD D-14 (25%), GLD D-13(25%), GLD C-9 (25%) and GLD C-6 (18.75%). Maximum numbers 192 bands were amplified by the genotype INQLAB91, while minimum number of bands (174) were produced from the genotype Bt-2549.

For SSR analysis, a total of 386 DNA fragments were generated by the 90 primer pairs used, with an average of about 4.3 bands per primer. In SSR amplification, number of polymorphic bands remained 15, 14, 13 and 13 in Inqlab-91, Punjab-76, Bt-2549, and Chakwal-86, respectively. The highest percentage of polymorphism was observed using SSR primers XGWM-374 (50%), XGWM-304 (50%), XGWM-136 (50%), XGWM-155 (50%) and XGWM-186 (37.5%).

Results from similarity matrices generated using Nei and Li's (1979) coefficients indicated high to moderate level of similarity among the cultivars/genotype studied (Table 1). Average similarity based on SSR data was relatively higher with value of 84% compared to 93.5% from RAPD analysis. This high level of polymorphism, associated with SSR markers, is expected because of the unique mechanism responsible for generating SSR allelic diversity by replication slippage. RAPD polymorphisms occurs due to base substitutions at the primer binding sites which prevents stable association with the primer or structural rearrangements within the amplified sequence such as insertions, deletions and inversions (Welsh and McClelland, 1990). Replication slippage is considered to occur more frequently than single nucleotide mutations and insertion\deletion events on RAPD primer annealing sites (Powell et al., 1996). The highest levels of polymorphism for SSRs system compare to other systems also reported in previous studies (Maguire et al., 2002; Belaj et al., 2003; Rajora and Rahman, 2003; Ferreria, et al., 2004; Ullah 2009).

For pair wise comparison, RAPD based similarity matrix revealed that Bt-2549 is 95% related with Punjab-76 whereas 12% dissimilar with Inqlab-91. Similarity matrix generated through SSR data produced almost same results as obtained from RAPD analysis. Bt-2549 showed high level of similarity (86%) with Punjab-76 and significant dissimilarity (21%) with Inqlab-91. These results suggest that RAPD and SSR techniques are equally informative in genetic diversity studies provided PCR conditions are fully optimized although level of polymorphism may depend upon the species under study (Belaj et al., 2003).

Phylogentic tree based on RAPD and SSR data grouped four accessions into two clusters (Fig 2). First cluster contained Punjab-76 and Bt-2549 where as cultivars Chakwal-86 and Inqlab-91 were grouped in second cluster. In Punjab, research on variety development in wheat is being carried out mainly at Wheat Research Institute (WRI), Faisalabad, through conventional means. Among the cultivars/genotype studied, Punjab-76, Chakwal-86 and Inqlab-91 has been bred at WRI, Faisalabad. Apparently clustering of cultivars from same centre of origin is surprising. However, the results are quite anticipated if we review wheat cultivar development programme being followed by different research organization in Punjab. Almost all conventional breeding institutes working on wheat get nursery from International Maize and Improvement Centre (CIMMYT), Mexico and develop cultivars either through direct selection from lines adaptive to our eco-climatic conditions or through hybridization of this adaptive material. However, Punjab-76 is among the cultivars developed using local blood (non-Mexican source) released soon after green revolution and is quite different from the material released in eighties, including Chakwal-86 and Inqlab-91, when breeding against rusts got attention of the breeders (K.N.Shah, Personal communication).

Traditional plant breeding utilizes natural variation present in germplasm for crop improvement. However, repeated use of germplasm lines with better yield and quality led to narrowing genetic base of modern cultivars (Ullah, 2008, 2009). During the last few decades the techniques of plant tissue culture have been developed as powerful tool to create genetic variation and crop improvement. Plants regenerated from calli have been referred to as somaclones (Larkin and Scowcroft, 1981). These clones show variation for different parameters like yield, quality, disease resistance, drought tolerance and maturity (Niaz and Quraishi, 2002). The technique is also being used to produce somacolonal rust resistant variants in wheat. Genotype BT-2549 used in this study is an elite line developed exploiting somaclonal variation at Agricultural Biotechnology Research Institute, Faisalabad. High degree of dissimilarity of BT-2549 found with other wheat cultivars suggests that in vitro techniques of tissue culture are reliable source of creating genetic variation.

In conclusion, information obtained here revealed high relatedness among the accessions studied, however, medium level of diversity between resistance source of leaf rust (Bt-2549) and well adaptive and high yielding wheat cultivar (Inqlab-91) suggests their utilization in breeding leaf rust resistance. Moreover, the RAPDs proved equally informative and can be used for genetic diversity studies, especially for resource poor labs of developing countries, provided PCR conditions are fully optimized.

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(1) Muhammad Nawaz, (1) Sayyid Abid Hussain, (2) Ihsan Ullah, (2) Muhammad Younus,

(2) Muhammad Zaffar Iqbal and (1) Shahid Mahboob Rana

(1) Department of Bioinformatics, GC University, Faisalabad.

(2) Agriculture Biotechnology Research Institute, Faisalabad.

Muhammad Nawaz, Sayyid Abid Hussain, Ihsan Ullah, Muhammad Younus, Muhammad Zaffar Iqbal and Shahid Mahboob Rana: Estimation of Genetic Diversity in Wheat Using Dna Markers, Am.-Eurasian J. Sustain. Agric., 3(3): 507-511, 2009

Corresponding Author: Muhammad Nawaz, Department of Bioinformatics, GC University, Faisalabad. E-mail: Ihsan.ullah_tlw@yahoo.com
Table 1: Genetic similarity matrix for wheat cultivars/genotype
Punjab-76, BT2549, Chakwal-86 and Inqlab-91, as assessed by 70 random
amplified polymorphic DNA (Upper diagonal) and 90 simple sequence
repeat (Lower diagonal) primers.

             Punjab-76   BT-2549   Chakwal-86   Inqlab-91

Punjab-76    1.00        0.95      0.92         0.92
BT-2549      0.86        1.00      0.93         0.88
Chakwal-86   0.80        0.83      1.00         0.93
Inqlab-91    0.79        0.82      0.88         1.00
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Title Annotation:Original Article
Author:Nawaz, Muhammad; Hussain, Sayyid Abid; Ullah, Ihsan; Younus, Muhammad; Iqbal, Muhammad Zaffar; Rana,
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Geographic Code:9PAKI
Date:Sep 1, 2009
Words:2836
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