Application of SSR Markers for Testing the Uniformity/Purity Analysis of a Large Wheat ( Triticum Aestivum L .) Tilling Population

Me-hboob-ur-Rahman, et al., Application of SSR Markers for Testing the Uniformity/ Purity Analysis of a Large Wheat ( Triticum Aestivum L .) Tilling Population. Biomed J Sci & Tech Res 38(3)-2021. BJSTR. MS.ID.006165. The objective of the present study was to conduct DNA-based genotyping assay, specifically exploring microsatellite regions, on wheat M2 population for identifying the genuine mutant plants (other than the natural variants) before conducting TILLING experiments. Seed of a wheat variety ‘NN-Gandum-1’ was exposed to two different time exposure and temperatures i.e., 1 and 2 hours and 33°C and 35°C, respectively. It was established that 0.8% EMS when exposed for 2 hours at 35°C was found appropriate for developing a suitable TILLING population. For determining the quality of the mutant population, a purity analysis was carried out using SSR primer pairs with high PIC values. Initially, 77 SSRs were surveyed on wheat genotypes. In total, 20 SSRs showing high PIC values were selected for surveying 3634 M2 plants. Out of these, three SSR primer pairs WMS-46, WMS-249 and WMS-311 detected natural variants in M2 TILLING population with 5.09, 7.23 and 8.29%, respectively. The While, WMS-249 and WMS-311 were located in densely mapped regions (near the centromere) than that of the WMS-46. It was concluded that 20 SSRs with high PIC value are sufficient to identify true-to-type mutants (other than natural variants) in a wheat TILLING population. This study has implications for identifying genes and their function as well as in genetic improvement of wheat. This information can also be translated on other crops.

Introduction different genetic pathways because of its huge and complex genome size and its ploidy level Slade, et al. [2]. TILLING [3][4][5][6], can address the major challenge of linking sequence information to the Biol. function of genes and can also identify novel variation for crop Breeding. Slade, et al. [2].
Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polypoid. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of Res.ers Chen, et al. [7][8][9][10][11][12][13]5]. Before inducing mutations, genetic uniformity of the genetic material is extremely important Sabetta, et al. [14]. Seed purity is undertaken by morphological means. Due to uncertainties in such methods, there is always a risk of incorrectly rejecting or accepting a seed lot Remund, et al. [15]. Morphological traits are growth stage specific, prone to environmental errors and are limited in number. Wheat is a self-pollinating plant but occasionally outcrossing occurs. However, it is impractical to have pollen control by bagging spikes in the greenhouse or in the field. Therefore, rapid and accurate assessment of a large TILLING population through such means is difficult. Thus, as a standard procedure, putative mutants identified are further subjected to microsatellite marker analysis for confirming their authenticity Wu, et al. [16][17][18][19][20]. Simple Sequence Repeats (SSRs) are co-dominant in expression-make them a marker of choice and have been used extensively in genotyping individuals of different plant species Asif, et al. [21].
The propensity of SSRs showing hyper variability has been exploited to study the natural polymorphisms found in TILLING population of different crop species Xin, et al. [22,14] and or identifying contamination (off type plants) in mutant population that usually occurs by several means including mixing at harvesting, threshing, etc. Bora, et al. [19]. We consider this approach as a costeffective way to maintain quality control of the mutant stock. Thus, it is extremely important to test for heterogeneity and or natural variants in TILLING population.

Rohtas-90) at the Plant Genomics & Mol. Breeding Lab, National
Institute for Biotechnol. and Genetic Engineering. NN-Gandum-1 (also called as Gandum-1)' has been evaluated for studying its adaptability throughout the Punjab province in Pakistan.

Mutagenesis and Tilling Population Development
Genetic purity of the variety was ensured by planting 500 single spike rows. Out of these, only one line was selected by observing uniformity in different morphological traits. Then the seed was increased by planting small plot. This seed was used to expose to chemical mutagen EMS. For optimization of dose, 180 seed in three replicates of each line were treated with eight different EMS doses

DNA Isolation
Genomic DNA was isolated from leaves of 21-day-old seedlings of Gandum-1 and each of 74 the M2 plants using the modified cetyl trimethyl ammonium bromide (CTAB) method.
The DNA quantification was carried out using Nano-dropTM1000 spectrophotometer (Thermo scientific, USA) and diluted accordingly. The quality and quantity of the genomic DNA was also

Results
In present study, seed of wheat variety Gandum-1 were exposed to EMS at two exposure times (1 and 2 h) as previously reported Bahar, et al. [23]. It was observed that the germination rate showed gradual depression with the gradual increment of