Stable SNP Allele Associations With High Grain Zinc Content in Polished Rice (Oryza sativa L.) Identified Based on ddRAD Sequencing

Abstract

Polished rice is widely consumed staple food across the globe, however, it contains limited nutrients especially iron (Fe) and zinc (Zn). To identify promising genotypes for grain Zn, a total of 40 genotypes consisting 20 rice landraces, and 20 released high yielding rice varieties were evaluated in three environments (wet seasons 2014, 2015 and 2016) for nine traits including days to 50% flowering (DFF), plant height (PH), panicle length (PL), total number of tillers (TNT), single plant yield (SPY), Fe and Zn in brown (IBR, ZBR) and polished rice (IPR, ZPR). Additive Main Effect and Multiplicative Interaction (AMMI), Genotype and Genotype × Environment Interaction (GGE) analyses identified genotypes G22 (Edavankudi Pokkali), G17 (Taraori Basmati), G27 (Chittimuthyalu) and G26 (Kalanamak) stable for ZPR and G8 (Savitri) stable for SPY across three environments. Significant negative correlation between yield and grain Zn was reaffirmed. Regression analysis indicated the contribution of traits toward ZPR and SPY and also desirable level of grain Zn in brown rice. A total of 39,137 polymorphic single nucleotide polymorphisms (SNPs) were obtained through double digest restriction site associated DNA (dd-RAD) sequencing of 40 genotypes. Association analyses with nine phenotypic traits revealed 188 stable SNPs with six traits across three environments. ZPR was associated with SNPs located in three putative candidate genes (LOC_Os03g47980, LOC_Os07g47950 and LOC_Os07g48050) on chromosomes 3 and 7. The genomic region of chromosome 7 co localized with reported genomic regions (rMQTL₇.₁) and OsNAS3 candidate gene. SPY was found to be associated with 12 stable SNPs located in 11 putative candidate genes on chromosome 1, 6, and 12. Characterization of rice landraces and varieties in terms of stability for their grain Zn and yield identified promising donors and recipients along with genomic regions in the present study to be deployed rice Zn biofortification breeding program.

Keywords: SNP; candidate genes; ddRAD sequencing; grain zinc; rice; stable donors.

FIGURE 1

Box plots for nine traits [DFF, Days to fifty percent flowering; PH, Plant Height (cm); PL, Panicle Length (cm); TNT, Total Number of Tillers per plant; SPY, Single Plant Yield (g); IBR, Fe content in Brown Rice (ppm); ZBR, Zn content in Brown Rice (ppm); IPR, Fe content in Polished Rice (ppm); ZPR, Zn content in Polished Rice (ppm)] across three environments E1 – Kharif 2014, E2 - Kharif 2015, E3 – Kharif 2016 and combined across environments.

FIGURE 2

(A) GGE Biplot for Zn content in polished rice (ZPR). (B) GGE Biplot for Zn content in polished rice (ZPR)-Ranking of Genotypes. (C) GGE Biplot for Zn content in polished rice (ZPR) – Mean v/s Stability. (D) Which Won Where/What plot for Zn content in polished rice (ZPR). (E) AMMI Biplot for Zn content in polished rice (ZPR).

FIGURE 3

(A) GGE Biplot for Single Plant Yield (SPY). (B) GGE Biplot for Single Plant Yield (SPY)-Ranking of Genotypes. (C) GGE Biplot for Single Plant Yield (SPY) – Mean v/s Stability. (D) Which Won Where/What plot for Single Plant Yield (SPY). (E) AMMI Biplot for Single Plant Yield (SPY).

FIGURE 4

Genomic position of distribution of SNPs chromosome wise.

FIGURE 5

(A) Selected Manhattan and QQ plots of associated SNPs across 40 genotypes for Zn content in polished rice (ZPR). (B) Selected Manhattan and QQ plots of associated SNPs across 40 genotypes for Single Plant Yield (SPY).