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INVESTIGATOR: Ponniah, S.

PERFORMING INSTITUTION:
UNIV OF ARKANSAS
PINE BLUFF, ARKANSAS 71601

GENE EDITING IN RICE GENOME TOWARDS DEVELOPING IMPROVED RICE VARIETIES

NON-TECHNICAL SUMMARY: Chalkiness in rice is a highly undesirable trait as it contributes to milling loss due to grain breakage, in addition to negatively impacting grain appearance, cooking quality, and palatability. The presence of chalk in the milled rice lowers the value of the rice in most domestic and international markets. Reducing the chalk, therefore, would benefit farmers, millers, traders and consumers, by increasing the yield of marketable rice, thereby contributing to food security and potential income. The overall goal of this project is to carry out gene editing in Chalk5, a major genetic determinant of grain chalkiness, in elite Arkansas rice varieties. The immediate objective of this proposal is to delete cis-elements, associated with the elevated Chalk5 expression in early grain filling stages, using CRISPR/Cas9 mediated targeted mutagenesis. The project will lead to: 1) reduced chalkiness in rice, 2) improved rice yield, 3) trained undergraduate/ graduate minority students in biotechnology, 4) establishment of collaboration between UAPB and University of Arkansas, and 4) strengthened research and teaching capacity of UAPB.

OBJECTIVES: Grain quality dictates value of rice in the market, and impacts the yield of the processed 'white' rice (also known as 'head rice yield'). The translucent full-length (head) rice commands higher value in the market compared to 'chalky' rice. Chalk is the opaque area in rice endosperm that reduces overall yield by increasing the fraction of broken rice and/or opaque grains. Chalkiness is a complex trait manifested by small, loosely packed starch granules and increased amount of long chain amylopectin (Lisle et al., 2000; Wang et al., 2013). The natural variation in chalkiness has long been documented, but its genetic basis has been masked by the complex source-sink interaction (Fitzgerald et al. 2009). Chalk content is negatively correlated with head rice yield. With every incremental increase in grain chalk content, there is a concomitant decrease in head rice yield (Lyman et al., 2013; Zhao and Fitzgerald, 2013). Chalkiness is found to be elevated in the rice grown in warmer seasons, therefore, unless chalk is reduced in the high yielding rice varieties, a significant food security issue remains unsolved in the years ahead when earth atmosphere is projected to become warmer (Fitzgerald et al., 2009). Significant efforts in determining chalk QTLs have been made in the recent time as rice biologists recognize the relation of chalky trait with food security (Zhao et al., 2016). However, breeding for low chalk has been very difficult due to the genetic distance from low chalk varieties, instability in multiple environments, and the association of high chalk with yield QTLs.Many studies have explored the correlation of starch biosynthesis genes with grain quality including chalk content. Starch biosynthesis is regulated by complex pathway involving multiple genes. The lossof function mutants in starch biosynthesis genes show drastically reduced starch content and rice yield. Polymorphism discovery efforts in 18 starch biosynthesis genes in 233 Australian rice breeding lines found very limited variation--no polymorphism or significant association with the quality traits in 12 of 18 starch related genes Therefore, breeding for quality traits would require new alleles either by introducing new germplasm or by creating these alleles in the breeding lines by gene editing. Manychalk QTLs have been described. Recently, Chalk5 as amajor genetic determinant of grain chalkiness has been identified. This indica Chalk5 allele, associated with high chalkiness, is tightlylinked to two yield QTLs explaining the linkage of high yield and high chalkiness in indica varieties. Chalk5 QTL has also been reported in more than one population: H94 (low chalk indica) x Z97 (high chalk indica) and LMT (low chalk japonica) x TQ (high chalk indica). Discovery of Chalk5 in different environments and populations derived from indica and japonica indicates its importance in diverse lines and stability in multiple environments. The goal of this proposal is to reduce chalkiness through Chalk5 gene editing in the elite rice varieties by deleting cis-elements, associated with the elevated Chalk5 expression in early grain filling stages, using CRISPR/Cas9 mediated targeted mutagenesis approach.