DNA fingerprinting of cotton cultivars collected from farmers of Sindh and Punjab

Cotton (Gossypium hirsutum L.) is an important fiber crop, which has great impact on the economy of Pakistan. Adoption of transgenic cotton enhanced the cotton production in many countries, but Pakistan couldn't harvest the benefit as was expected. Delay in the approval of transgenic cotton cultivation in Pakistan resulted in an increased malpractice of seed companies. This situation increased the spread of unapproved genotypes. To elucidate this assumption of misconduct on scientific basis, DNA fingerprinting is very useful tool. Therefore, DNA profiling was carried out to assess the genetic similarity/dissimilarity between the seed of a particular variety/genotype, sourced from breeder and farmer. Simple sequence repeats (SSR) based polymorphic markers have the potential to identify the level of impurity by comparing the DNA profiles of any sample with reference breeder's seed sample. Therefore present study was designed to collect 450 seed samples from 25 tehsils of Punjab and Sindh provinces. Seed of reference 39 breeder's varieties was collected from respective breeders. Germination percentage of all the 450 cotton samples collected from farmers and 39 varieties collected from breeder germination test was conducted by following ISTA rules. The results showed a range of variation in germination % from 6% to 80%. The maximum 80% germination was recorded in NEELUM-121 and minimum 6% was recorded in BT-197. DNA was extracted from seed of each sample by modified CTAB method. Hundred SSR primers were synthesized on the basis of already reported PIC (polymorphism information content) value in cotton. On the basis of reproducibility and higher PIC values, only 25 primers were selected for DNA fingerprinting of all collected genotypes. The PCR products were run on PAGE to get higher resolution and more precise results. A standard statistical method was used on the basis of presence and absence of bands to calculate the allele frequencies, heterozygosity and effective number of alleles. Moreover, Genetic divergence and cluster analysis was done using NTSYS-PC software. PIC value of all the primers was calculated which ranged from 0 to 0.61 with an average of 0.44. The comparison of DNA profiles generated on the basis of this analysis identified the percent dissimilarities between the farmer seed samples and the breeder seed samples which ranged from 5.099 to 19.131. It was find out that the minimum genetic dissimilarity index was 2.446, and maximum was 5.292 in breeder varieties. Breeder varieties were 95 to 98% similar so, cotton have very narrow genetic base. These results provide evidence against malpractices in the cotton seed chain.