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The importance of varietal identification has grown dramatically on a global scale, particularly in the context of plant variety protection. To address the same, 100 new hyper-variable mango simple sequence repeats (HMSSRs) markers were screened for polymorphism among 24 mango hybrids bred at ICAR- IARI, New Delhi. Out of which, 89 polymorphic HMSSRs were used for the generation of DNA barcodes of mango hybrids. A total of 1,861 alleles with an amplicon size of 130 to 450 bp have been detected. The average number of alleles was 2.60 per locus, and PIC ranged from 0.04 (HMSSR1382) to 0.72 (HMSSR1289) with an average of 0.39. UPGMA analysis grouped these mango hybrids into two major clusters broadly representing the parental influence in hybrids. Cluster II comprised of 14 hybrids and cluster I had 10 hybrids. Model-based structure analysis revealed two gene pools, and AMOVA indicated that higher molecular variation is due to individuals (82%). The generated allelic variations of these polymorphic markers were translated into DNA barcodes by separating the allele size for each polymorphic HMSSR locus. A total 11 unique and 35 rare alleles from amplified alleles have been observed. Validation of these hybrid-specific alleles was attempted on a set of four tree replicates of the same genotypes grown at different places, of which seven hybrid-specific alleles could be validated in the present study. Overall, the present set of HMSSRs convincingly revealed to be highly informative and useful for future molecular research in mango. Furthermore, identifying hybrid-specific alleles would be highly useful in identifying and protecting mango hybrids.
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