Genetic Diversity Assessment In Aromatic Rice Using Microsatellite Markers
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Date
2014
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Rajendra Agricultural University, Pusa (Samastipur)
Abstract
A study was conducted to examine the genetic diversity in eighteen entries, landraces and advanced breeding lines from aromatic rice germplasm in order to characterize them on the basis of simple sequence length polymorphism and to determine the nature and extent of differentiation and divergence among them using eighteen microsatellite based primer pairs. The materials were grown in petriplates for extraction of genomic DNA from the young seedlings and then targeted amplification of the genomic DNA using a panel of eighteen microsatellite based primer pairs covering six chromosomes in the genome of rice. All molecular studies were conducted in the Molecular Biology Laboratory at Pusa. The statistical methods and parameters used for deriving inference were polymorphism information content, similarity coefficient and numerical taxonomic analysis of divergence.
The amplification was successfully achieved with all the microsatellite primers used in the present study. Appearance of bands at different positions on the gel revealed differential migration of amplified products due to differences in overall size of the products generated from targeted amplification of specific region of genome. The polymorphism among the varieties was recognized on the basis of presence or absence of bands, in addition to variation in respect of number and position of bands. Altogether 180 allelic variants were detected among the eighteen rice entries with an average of 7.2 alleles per locus. The number of alleles per locus ranged from six in the cases of RM 256 and RM 284 to nineteen in the case of RM 42. The primer pairs RM 42, RM 44, RM 223, RM 225, RM 252, RM 330 and RM 505 generated amplified products due to amplification of more than one locus. A total of 89 shared and 91 unique allelic variants were generated in the form of amplified products by polymerase chain reaction using eighteen primer pairs. The number of shared alleles per locus ranged from two out of eleven alleles in the case of RM 444 to nine out of thirteen alleles in RM 44. Similarly, the number of unique alleles per locus ranged from two out of five in the case of RM 16, two out of nine in RM 225, two out of six in RM 284 and two out of eight alleles in the case of RM 426 to fourteen out of nineteen alleles in the case of RM 42. The primer pairs RM 444, RM 42, RM 72, RM 80, RM 13, RM 330, RM 223, RM 505, RM 252 and RM 256 generated considerably greater percentage of unique alleles in descending order of magnitude. Among the primers tested, RM 42, RM 72, RM 80, RM 223, RM 252 and RM 444 appeared to be more informative primers.
A direct relationship was observed between the repeat number involved in the microsatellite based simple sequence repeat locus and the number of identified alleles. In general, the larger the repeat number involved in the di-nucleotide microsatellite locus, the larger was the number of identified alleles. The microsatellite based SSR locus associated with RM 13, RM 80, RM 337, RM 339, RM 426, RM 444 and RM 505 exhibited null alleles ranging from one to three in the entries under evaluation. Occurrence of null alleles for a particular repeat locus was noticed reflecting failure of locus specific microsatellite based primer directed generation of amplified products. The primer pairs RM 42, RM 44, RM 223, RM 225, RM 252, RM 330 and RM 505 generated amplified products due to amplification of more than one locus. Appearance of more than one band in the same genotype was noticed revealing the existence of the duplicated region in the genome of rice.
Considerably greater extent of variation existed at the molecular level with maximum similarity between Champaran basmati and Sanwal basmati among the aromatic rice entries under evaluation in the present study. The microsatellite primer based analysis revealed unique or variety specific allele which could be useful as DNA fingerprints in the identification and preservation of rice entries. The use of eighteen microsatellite markers in the analysis exhibited a remarkably higher level of genetic polymorphism, which allowed unique and unambiguous genotyping of eighteen aromatic rice genotypes included in the analysis.
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