MOLECULAR CHARACTERIZATION OF WHEAT GENOTYPES ( TRITICUM AESTIVUM L . )

Molecular diversity in thirty wheat genotypes was done. For this the genomic DNA isolation was carried out and which were then subjected to PCR amplification using twenty SSR primers. Out of these twenty SSR primers, eighteen yielded amplifications and showed polymorphism. Total 93 loci were generated by amplification with 18 polymorphic primers, all of which 93 loci were polymorphic i.e. 100%. Among the SSR primers, BARC-170, WMC-44, produced maximum number of 2 loci. The size of amplification products ranged from 102 bp to 805 bp. All SSR primers showed 100 % polymorphism and all primers had more than 0.50 PIC value except one primer. Maximum PIC value 0.17 was observed in WMC-468. The maximum number of bands were observed in NIAW-2721 (28 bands), whereas minimum number of bands were present in NIAW-301 and NIAW-2539 (19 bands). The dice similarity coefficient values ranged from 0.50 to 0.95. Maximum similarity value of 0.95 was noticed between NIAW-2891 and NIAW-2837, while minimum similarity value of 0.50 was observed among NIAW-2595, NIAW-2874, NIAW-2995 and NIAW-2725. The consensus tree software revealed two major clusters.


INTRODUCTION
Wheat is the foremost and strategic cereal crop of the world and is the most important and major staple food of more than thirty six percent of world`s population.Globally, it is the most crucial oldest and edible grain cereals.In 2015-16 world production of wheat was estimated to be 727.2 million tons [3].In India wheat is grown in an area of 30.96 million hectors during 2014-15 [3].
Wheat is highly nutritious and provides almost all the nutrients needed for human.wheat endosperm proteins, is the gluten protein and the vital role of gluten proteins in bread making quality and backing quality in wheat [7].
Molecular markers are relatively new technology in breeding and is widely used by breeders to select variations in genotypes.The main marker technologies RFLP, RAPD, AFLP, SSR, ISSR etc. are becoming important for cultivar identification and diversity analysis [9,14].
Traditional method for estimating genetic diversity and relationship among groups of plants were using morphological characters, agronomic information and biochemical variations.Agronomical data of wheat is essential in applied wheat breeding but, individual genotypes of wheat are well adopted to certain regions.Besides, collection of such data is laborious.Due to these region, DNA/molecular markers are attractive alternative [12].The molecular marker reveals genetic diversity at molecular level [5].DNA marker are unlimited in numbers, selectivity neutral and can be organized in linkage maps [14].
Along with agronomical values the genetic diversity should be considered.New sources of variation give new and improved cultivar.The parents with high diversity will give highly variable progenies.Mahjourimajd et al. [15] showed the genetic basis responsible for grain characteristics and how it influenced with cultural practices like fertilizer application.
The knowledge of diversity also helps to develop strategies to incorporate useful diversity in breeding programs.Characterization of crop by markers reveals similarities (i.e.shared alleles) and diversity (i.e.typical alleles) among cultivars of a crop.This also helps in identifying gene pool or origin of cultivar.For development of elite cultivar, the genetic base has to be enlarged.Email: sharadpawar@hotmail.com resources of a crop in gene bank.DNA profile help in selection of distinct parents for obtaining higher genetic variation.The present investigation was conducted to Characterize thirty three wheat genotypes (Triticum aestivum L.) by molecular methods.

MATERIALS AND METHODS
The study was carried out at State Level Biotechnology Centre, Mahatma Phule Krishi Vidyapeeth, Rahuri (Maharashtra) during year 2016-17.
The plant material for the study comprised of thirty three wheat genotypes, which were collected from Agriculture Research Station Niphad, District Nashik, Maharashtra.The seeds thus obtained were sown in plots inside poly house for genomic DNA isolation.Details of these wheat genotypes along with their pedigree are given in table 1.

Isolation of genomic DNA from leaves
Genomic DNA was isolated from 33 wheat genotypes following CTAB (Cetyl Tri methyl Ammonium Bromide) extraction method with some modifications as described by [4]. 2 µl of all DNA extracts were electrophoresed in 0.8 % (w/v) on agarose gel containing 0.5 µg/ml ethidium bromide.After electrophoresis the band intensity of genomic DNA was visualized on gel documentation unit (Flour Chem.TM Alpha InfoTech, USA) and compared to that of standard Lambda phage DNA.These gels also provided a visual measure of purity of DNA.

Optimization of DNA concentration for PCR
To carry out PCR, required template DNA concentration of 20ng/µl is needed.Optimization was done to base on the intensity of electrophoresed DNA.It was done to bring all DNA concentrations to a relatively equal level (20ng/µl) by appropriate dilutions.Accordingly, dilutions were made with calculated amount with TE Buffer.

Primers used and optimization of annealing temperature of gene specific primers for PCR amplification
Eighteen SSR primers were used for PCR amplification.The details of primers and their sequences are given in table 2. Gradient PCR amplification for different gene specific primers was carried out to determine the annealing temperature of each primer.The PCR programme was set in thermal cycle.(Eppendorf tube, Master Cycler Gradient, Germany).

Data analysis
The clearly resolved PCR amplified bands of wheat genotypes with different SSR and ISSR primers were scored manually as binary matrix for their presence (1) and absence (0) in the data sheet.The polymorphism information content (PIC) value was calculated as Where, n is the number of band positions analysed in the set of accessions and Pi is the frequency of it pattern.

Cluster analysis
The binary data were analysed under the SIMQUAL module of NTSYSpc 2.0i software [10], by using Dice similarity coefficient [8].SAHN module based on UPGMA unpaired group mean algorithm based clustering method [13] was used to generate a tree (dendrogram).

RESULTS AND DISCUSSION
Aim of this investigation was to study the diversity in the available wheat genotypes.For this purpose, molecular marker was used effectively in the assessment of genetic diversity in wheat.
The genomic DNA was isolated from 33 genotypes and was subjected to PCR amplification using 20 primers.Annealing temperature of each of the primer was optimized by gradient PCR.It was observed that out of these 20 SSR primer pairs, 18 amplified and 18 showed polymorphism.
From the SSR analysis it was observed that a total of 93 bands were generated by amplification with 18 polymorphic primers.93 of them were polymorphic, from which 16 were unique.Average polymorphism was 100%.Each SSR primer pair thus produced on an average 5.16 polymorphic bands.
Maximum scorable bands were observed using the primer BARC-170 (11 loci), followed by WMC-44 (9 bands).The least number of bands were reported in the primer WMC-468 (2 each), followed by BARC-62, GMW-610 and WMC-313(3 bands).Eighteen primers showed 100 percent polymorphism.All the genotypes evaluated for the molecular diversity with 18 SSR primer showed variation in their banding pattern.Total number of polymorphic primers 18 5.
Total numbers of bands amplified 93 6.
Total number of polymorphic bands 93 7.
Total number of monomorphic bands 0 8.
Total number of unique bands 16 9.
Average polymorphic bands produced per primer 5.16 12.
Amplified products size 102-805bp  Thirty-three varieties were used for amplification with 18 SSR primers.From eighteen, eighteen primers showed polymorphic.These 18 primers yielded maximum 11 bands and minimum 2 bands with PIC value ranged from 0.17 to 0.806.A total 16 unique bands were observed in 16 genotypes which could be useful in DNA fingerprinting, molecular characterization and varietal identification purpose.
In the present investigation out of 20 primers used 18 primers were able to amplify the genomic DNA of wheat and 2 primers were not able to amplify the genomic DNA.The total of 95 bands was resolved by 18 SSR primers out of which 95 bands were polymorphic.The PIC values of primers ranged from 0.17 to 0.806.Further, it was observed that there was no correlation between per cent polymorphism and PIC values as SSR primers Wmc-468 showed minimum PIC values but was 100 per cent polymorphic.There are previous reports assessment of molecular characterization between 12 wheat genotypes by using 4 SSR markers [17].
Christiansen et al. [2] Assessed genetic diversity in wheat using 47 microsatellite primer pairs were assessed in 75 Nordic spring wheat cultivars bred during the 20th century.Huang et al., [6], assessed genetic diversity using 24 wheat microsatellite markers for 998 accessions of hexaploid bread wheat.
Zala et al., [16] assessed molecular diversity in wheat using18 RAPD and eight SSR markers amplified 5554 fragments with 66.83 % polymorphism and 343 fragments with 90.32% polymorphism respectively.A Jaccard's similarity coefficient of all genotypes derived from RAPD data ranged from 0.65 to 0.90 and that of SSR ranged from 0.45 to 0.94.The PIC value for RAPD and SSR markers ranged from 0.91 to 0.96 and 0.57 to 0.76 respectively, with marker indexes of 11.35 and 5.35, demonstrating its utility in genetic diversity analysis.
Singh et al., [11] screened thirty advance lines of hexaploid wheat (Triticum aestvium L.) cultivars by applying microsatellite markers.Seven primers out of thirty five SSR primers showed polymorphism in banding pattern.A total of 15 alleles were detected.The number of alleles per locus ranged from 1-3 with an average of 2.14 alleles per locus.The overall size of PCR products amplified ranged from 100-300 bp.indicating wide genetic diversity and it may be used in wheat hybridization programme for improving grain yield.
Results from the present study support the potential utility of molecular markers in characterization of the wheat genotypes.The occurrence of unique alleles or rare SSR alleles provides an immense opportunity for characterization and identification of specific wheat genotypes.

Fig. 1 :
Fig. 1: Consensus tree showing clustering of thirty three wheat genotypes using SSR markers