Genetic purity assessment of scented rice using microsatellite markers.
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Date
2015
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Rajendra Agricultural University, Pusa (Samastipur)
Abstract
A study was undertaken to examine the microsatellite markers based polymorphism for identification of polymorphic and informative markers in order to characterize four aromatic rice varieties, two non-aromatic rice varieties, twelve inter-varietal seed mixtures and to validate the utilization of these markers for assessment of genetic purity of scented rice. The experimental materials were evaluated on the basis of biochemical test to ascertain the presence of aroma. Amplification of the targeted genomic DNA was done using a panel of 24 microsatellite based primer pairs targeting all the chromosomes in the rice genome. All molecular studies were conducted in the Molecular Biology Laboratory at Pusa. The statistical methods and parameters used for deriving inference were polymorphism per cent, polymorphism information content, discrimination coefficient, similarity coefficient and numerical taxonomic analysis of divergence.
The amplification was successfully achieved with all the microsatellite primers used in the present study. Molecular profiling was conducted and amplification pattern based polymorphism using microsatellite primer pairs was recognized on the basis of presence or absence of bands, besides variation in number and position of bands. Altogether 105 allelic variants were detected at 28 microsatellite loci with an average of 3.75 alleles per locus. A total of 42 shared and 63 unique allelic variants were generated. The number of unique alleles per primer pairs ranged from zero out of three alleles in Aro 7 to eight out of ten alleles in the case of RM 7049. Remarkably higher polymorphism per cent was exhibited by the primer pairs RM 42, RM 7049, BAD 2a, RM 85, RM 444, RM 1109, RM 505 and RM 330, since these primer pairs generated considerably greater percentage of unique alleles in descending order of magnitude.
Considerably greater ability to differentiate pair-wise combinations of entries was observed in the cases of primer pairs RM 42, RM 444, RM 7049, BAD 2a, RM 223, RM 252, RM 284, RM 8264, RM 23097, E03_92.0, F05_103.5, and B03_127.8 in decreasing order of magnitude. Contrarily, the primer pairs SCUSSR 1, RM 505, E11_44.5, and BAD 2b exhibited lesser ability to differentiate pair-wise combinations of entries. Considering the number of alleles generated in conjunction with the level of polymorphism detected, the primer pairs RM 42, RM 85, RM 223, RM 252, RM 284, RM 444, RM 1109, RM 23097, Aro 7, BAD 2a, B03_127.8, F05_103.5 and E03_92.0appeared to be highly polymorphic and comparatively more informative for the purpose of molecular characterization of entries under evaluation. These primers generated considerably greater number of allelic and polymorphic variants due to variation in the length of simple sequence repeats.
Appearance of more than one band in the same genotype was noticed revealing most probably the existence of the duplicated region in the genome. The primer pairs RM 330, RM 505, RM 7049, and RM 8264 generated amplified products due to amplification of two loci. However, further investigation under more stringent condition is required to confirm it because the intensity of bands was comparatively lower in some of the cases. Presence of stutter bands indicated the presence of minor products amplified that had lower intensity than the main allele and normally lacked or had extra repeat units. Such bands were observed to be present in the case of primer pairs RM 7049 and RM 8264.
The total repeat count of microsatellite loci did not appear to be associated with the number of alleles generated by the primers. Results did not reflect that the larger the repeat number involved in the microsatellite locus, larger was the number of identified alleles. In general, the microsatellite specific primer pairs flanking di-nucleotide and tri-nucleotide repeat motifs tended to detect lesser number of allelic variants than loci with tetra-nucleotide and complex repeat motifs. Among the microsatellite loci having di-nucleotide repeat motifs, the markers with AG, AT and CT repeat motifs tended to detect greater number of allelic variants than primers targeting microsatellite loci with TA, GA, and TC di-nucleotide repeat motifs.
Analysis of divergence pattern allowed relative assessment of genetic diversity amongst the four aromatic rice varieties and two non-aromatic rice varieties. A combination of only seven microsatellite primer pairs, RM 42, RM 223 and RM 8264 in combination with RM 85, RM 23097, BAD 2a, or E03_92.0 was purposefully effective in differentiation of four aromatic rice varieties, two non-aromatic rice varieties and genetic purity assessment of their seed mixture. Experimental results finally led to validation of three microsatellite primer pairs, which allowed easily recognizable and unambiguous differentiation of scented rice varieties from non-scented rice varieties evaluated in the present study. The molecular profile based on these three microsatellite markers served as distinct molecular tags for distinguishing the genotypes and inter-genotypic mixtures and provided a basis for genetic purity assessment of scented rice genotypes with at least with a single marker allele difference. These microsatellite primer pairs, namely, RM 42, RM 223, and RM 8264 may be further validated and utilized for the purpose of genetic purity assessment of scented rice. Results of the present study also revealed that use of RM 42 along with RM 223 or RM 8264 was equally effective in differentiation of four aromatic rice varieties from two non-aromatic rice varieties and evaluation of genetic purity of scented rice.
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