Genomic marker assisted identification of genetic loci and genes associated with variation of grain zinc concentration in rice

A study was conducted to examine the genetic divergence and to determine the genetic loci and genes associated with natural variation of grain zinc (Zn) concentration among 28 landraces, improved varieties and advanced breeding lines of rice using candidate gene specific primers. Field evaluation of the experimental material was conducted in randomized block design with three replications and Zn content in unpolished grains of the entries was determined by addition of nitric acid and perchloric acid (1:3) following the procedure of diacid digestion method. Statistical analysis revealed the exploitable extent of variability with respect to grain Zn concentration among the entries. Eighteen entries were selected from the two extremes of grain Zn distribution range and subjected to molecular profiling using a panel of 14 candidate genes specific 12 reported and 14 designed primer pairs. Only eight (OsZIP1-1, OsZIP3a, OsZIP4a, OsZIP5-3, OsZIP7-2, OsZIP8b, OsNRAMP7 and OsNAAT1) reported and eight (OsZIP3K, OsZIP4K, OsZIP5K, OsZIP7K, OsNRAMP7K, OsNAAT1K, OsNACK and OsYSL14K) designed primers generated polymorphic amplified products showing sequence length variation due to targeted amplification of candidate genes specific genomic regions. Ample genetic differentiation and divergence were revealed among the entries, which were accommodated into similarity coefficient-based six clusters, remarkably consistent with grain Zn concentration of the entries. Hierarchical classification pattern of entries was almost completely corroborated by principal co-ordinate analysis-based spatial distribution pattern of their genetic profiles. Molecular analysis based on candidate genes specific primers appeared to be an efficient approach for the elucidation of genetic differentiation and divergence in relation to variation of grain Zn concentration among entries. Hence, these markers can be effectively and efficiently utilized for grain Zn concentration related discrimination of rice genotypes and selection of parental genotypes for grain Zn biofortification. Microsatellites were detected within the candidate genes and amplicons, thereby providing a basis to deduce that the repeat sequence length variation in candidate genes may be a role player in the differential grain Zn accumulation in rice varieties. Single marker analysis established the association of OsNACK, OsZIP1-1, OsNRAMP7 and OsNRAMP7K with grain Zn concentration. Thus, these four markers can be effectively used in marker-assisted selection programme for grain Zn biofortification in rice.