Natural variation in rice ascorbate peroxidase gene APX9 is associated with a yield-enhancing QTL cluster

Authors: JEON, YUN-A - Chungnam National University; LEE, HYUN-SOOK - Chungnam National University; KIM, SUN-HA - Chungnam National University; SHIM, KYU-CHAN - Chungnam National University; KANG, JU-WON - Korean Rural Development Administration; KIM, HYUN-JUNG - Lg Chem; Tai, Thomas; AHN, SANG-NAG - Chungnam National University

Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/4/2021
Publication Date: 5/28/2021
Citation: Jeon, Y., Lee, H., Kim, S., Shim, K., Kang, J., Kim, H., Tai, T., Ahn, S. 2021. Natural variation in rice ascorbate peroxidase gene APX9 is associated with a yield-enhancing QTL cluster. Journal of Experimental Botany. 72(12):4254-4268. https://doi.org/10.1093/jxb/erab155.
DOI: https://doi.org/10.1093/jxb/erab155

Interpretive Summary: Oryza rufipogon is a wild ancestor of cultivated rice (Oryza sativa). Using O. rufipogon as a donor of beneficial traits, a cluster of quantitative trait loci (QTL) on chromosome 9 that affects several yield-related traits (e.g., days to heading, plant height, grain size and weight, spikelets per panicle) was identified in the background of the temperate japonica rice variety Hwaseong. The objective of this study was to determine if the effects of this QTL cluster were due to the actions of a single pleitropic gene or multiple tightly-linked genes. Two candidate genes, APX9 and MS5, were identified in the genomic region spanning the QTL cluster. Sequence analysis revealed several DNA polymorphisms in each gene. Analysis of gene expression patterns and the trait evaluation of transgenic plants (T-DNA insertion knockout mutants and overexpressors) resulted in the identification APX9, which encodes a putative L-ascorbate peroxidase, as the causal gene for the QTL cluster. Sequence analysis of APX9 from O. rufipogon and Hwaseong revealed a 3-base pair insertion/deletion polymorphism that results in an additional valine in the protein encoded by the Hwaseong allele. Phylogenetic and nucleotide diversity analyses of the APX9 gene and surrounding genomic region indicated that the 3-bp insertion occurred during the domestication of japonica rice from O. rufipogon before the differentiation of temperate and tropical subgroups of japonica rice and that this region was subsequently introgressed into indica rice. Identification of APX9 provides a foundation for investigating how this gene may interact with other genes to produce transgressive variation in agronomically valuable traits.

Technical Abstract: We previously identified a cluster of yield-related QTL in a near-isogenic line (NIL) from an interspecific cross between Oryza sativa spp. japonica cultivar ‘Hwaseong’ and O. rufipogon. Map-based cloning and transgenic approaches revealed that APX9 (LOC_Os09g36750) encoding an L-ascorbate peroxidase 4 is associated with this cluster. Sequence differences in the APX9 promoter and coding regions were observed between the two parental lines including a 3-bp InDel leading to the addition of a valine in Hwaseong. NIL plants showed higher expression of APX9 in various tissues than Hwaseong. We generated APX9 over-expression (OE) transgenics in the Hwaseong background. APX9-OE plants showed taller stature and delayed heading compared to Hwaseong, whereas T-DNA knock-out mutants showed reduced plant height. These results confirm that APX9 is the causal gene for the QTL cluster. NIL had higher antioxidant ability and APX activity than Hwaseong leading to increased drought tolerance seedlings. Sequence analysis of APX9 from 303 rice accessions revealed that the 3-bp InDel clearly differentiates japonica (APX9HS ) and O. rufipogon (APX9OR) alleles. Indica shared both APX9HS and APX9ORalleles suggesting that APX9HS was introgressed into indica followed by crossing. The finding that O. rufipogon accessions with different origins carry APX9OR strongly suggests that the 3-bp insertion was specifically selected in japonica during domestication. In this study, we demonstrated that a pleiotropic gene APX9 acts as a major regulator of plant development by controlling a valuable suite of agronomically important traits in rice.