Candidate gene based microsatellite profiling in relation to grain zinc accumulation in rice
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Date
2019
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DRPCAU, Pusa, Samastipur
Abstract
A study was conducted to evaluate the genetic polymorphism using candidate gene based microsatellite specific designed primers and to validate them in relation to grain zinc accumulation in 18 rice genotypes. The experimental materials were grown in aluminum containers for extraction of genomic DNA from the young seedlings and then targeted amplification of the genomic DNA was achieved using a panel of 14 candidate genes based microsatellite specific 22 designed primer pairs covering eight chromosomes in the genome of rice. The statistical methods and parameters used were polymorphism information content, polymorphic percent, similarity coefficient and single marker analysis.
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 regions of genome. The polymorphism among the genotypes was recognized on the basis of presence or absence of bands. Altogether 82 allelic variants were generated with an average of 3.7 alleles per primer. Among the 14 candidate genes under consideration, single microsatellite sequence was investigated in OsZIP3, OsZIP4, OsZIP8, OsYSL2, OsYSL6, OsNRAMP7 and OsVIT1, whereas polymorphism in respect of two microsatellite sequences were surveyed in OsZIP5, OsZIP7, OsYSL14, OsNRAMP6, OsNAAT1 and OsNAC1. Only in the case of candidate
gene OsZIP1, three microsatellites were investigated. Altogether 22 microsatellites were investigated in which 8, 12 and 2 microsatellites had di-nucleotide, tri-nucleotide and tetra-nucleotide repeat motif. Candidate genes based microsatellite specific primers generated polymorphic amplified products. The polymorphic information content of panel of primers ranged from 0.438 to 0.790 with an average value of 0.635.Differential amplification pattern revealing molecular level genetic polymorphism among the entries subjected to molecular characterization provided a basis for deducting that the sequence length variation observed in candidate genes may be a role player in differential grain zinc accumulation in rice. Sizable variation was clearly recognized in the molecular size of the genomic region targeted by the primer pairs. Similarity coefficient data based on proportion of shared alleles showed maximum similarity between Rajendra Kasturi and Rajendra Mahsuri (0.977), whereas minimum similarity between Sanwal Basmati and Jeerabati (0.0) and also between Jeerabati and Swarna SUB1 (0.0). Cluster analysis resulted in distribution of 18 rice genotypes into 5 main genotypic groups.
Wide genetic differentiation and divergence at the molecular level was revealed among the entries. Hierarchical pattern of classification based on similarity coefficient matrix of pair-wise combinations of entries was in extremely good agreement with principal coordinated analysis based spatial distribution pattern of genetic profiles of entries. Hierarchical cluster analysis, as well as principal coordinate analysis using microsatellite-specific markers derived from candidate genes made it possible to differentiate and classify entries with appreciably greater level of consistency in relation to their grain zinc concentration. These markers can therefore be used effectively and efficiently to discriminate the genotypes of rice and to select parental genotypes during genetic improvement in relation to grain zinc biofortification.
Among the 14 candidate genes based 22 designed microsatellite primers utilized for polymorphism survey in relation to natural variation of grain zinc concentration in 18 entries, two primers, namely OsZIP1(A) and OsZIP3 exhibited statistically significant association with grain zinc concentration. These two markers can be used during marker assisted selection for grain zinc bio-fortification programme against hidden hunger.
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