The Copy Number Variation of OsMTD1 Regulates Rice Plant Architecture

Abstract

Copy number variation (CNV) may have phenotypic effects by altering the expression level of the gene(s) or regulatory element(s) contained. It is believed that CNVs play pivotal roles in controlling plant architecture and other traits in plant. However, the effects of CNV contributing to special traits remain largely unknown. Here we report a CNV involved in rice architecture by modulating tiller number and leaf angle. In the genome of Oryza sativa ssp. japonica cv. Nipponbare, we found a locus Loc_Os08g34249 is derived from a 13,002-bp tandem duplication in the nearby region of OsMTD1, a gene regulating tillering in rice. Further survey of 230 rice cultivars showed that the duplication occurred in only 13 japonica rice cultivars. Phenotypic investigation indicated that this CNV region may contribute to tiller number. Moreover, we revealed that OsMTD1 not only influences rice tiller number and leaf angle, but also represses pri-miR156f transcription in the CNV region. Intriguingly, this CNV performs function through both the dosage and position effects on OsMTD1 and pri-miR156f. Thus, our work identified a CNV and revealed a molecular regulatory basis for its effects on plant architecture, implying this CNV may possess importance and application potential in molecular breeding in rice.

Keywords: OsMTD1; copy number variation; plant architecture; pri-miR156f; rice.

FIGURE 1

OsMTD1-located segment involves a CNV. (A) The schematic shows the fragment of OsMTD1 located on chromosome 8 in 93–11 and Nipponbare. Primer67/65, Primer64/66, and Primer64/65: primer pairs using for tandem duplication event analysis; OsMTD1 and OsmiR156f: the two comprising elements in the corresponding DNA sequence of the OsMTD1 located CNV. (B) Validity analysis on the primers detecting polymorphisms between 93–11 and Nipponbare. (C) Partial results from PCR amplification using different rice cultivars. (D) Phylogenetic analysis of CNV corresponding region’s sequences in different rice cultivars. Nipponbare: the first sequence of the two DNA segments in japonica cv. Nipponbare genome; ZH11-rep1, ZH11-rep2: the first and the second sequence of the two DNA segments in japonica cv. ZH11 genome; Shuhui498-rep, 93–11-rep, Minghui 63, RP Bio-226, and Zhanshan97-rep: the corresponding DNA sequence of the OsMTD1 located-CNV in different indica cultivars’ genome.

FIGURE 2

Phenotypic distribution of rice tiller number. (A) The maximum tiller number from 94 indica accessions. (B) The statistical result of the maximum tiller number between indica and japonica accessions, the maximum tiller number between one-copy and two-copy CNV cultivars. Statistical significance was estimated by Student t tests. *P < 0.05 and **P < 0.01. (C) The maximum tiller number in different japonica accessions with one copy of OsMTD1 nearby genome sequence. (D) The maximum tiller number in different japonica accessions with a tandem replication at OsMTD1 nearby genome sequence.

FIGURE 3

Effects of OsMTD1 on rice tillering. (A) Lines with different OsMTD1 expression levels and the tillering ability performed. (B) Comparative analysis of the tiller number in lines with different OsMTD1 expression levels. Statistical significance was estimated by Student t tests, and different letters indicate a significant difference (P < 0.05). (C) The OsMTD1 overexpression lines produce less tillers compared to wild type. (D) The OsMTD1 CRISPR/Cas9 editing lines show different tiller traits. WT: Kitaake; OE-15, OE-20, and OE-27: independent OsMTD1 overexpression line; A-3, A-8, and A-44: Independent OsMTD1 CRISPR/Cas9 editing line.

FIGURE 4

Effects of OsMTD1 on rice leaf angle. (A) Lines with different OsMTD1 expression levels show different flag leaf angles. (B) Comparative analysis the flag leaf angle in lines with different OsMTD1 expression levels. Statistical significance was estimated by Student t tests, and different letters indicate a significant difference (P < 0.05). WT: Kitaake; OE-15, OE-20, and OE-27: independent OsMTD1 overexpression line; A-3, A-8, and A-44: independent OsMTD1 CRISPR/Cas9 editing line.

FIGURE 5

Effects of OsMTD1 on accumulation of miR156f. (A) The osa-miR156 relative levels in different rice lines. WT: Kitaake; A-3, A-8, A-16, A-27, A-42, and A-44: different OsMTD1 CRISPR/Cas9 editing lines; OE-15, OE-16, OE-19, OE-20, OE-26, OE-27: different OsMTD1 overexpression lines. (B) Quantification of miR156 in tobacco leaves expressing the pri-miR156f including OsMTD1 sequence (OsMTD1), or the pri-miR156f in which the OsMTD1 was deleted (Δ-OsMTD1), or in which the OsMTD1 start codon was mutated to ATT (ATT-OsMTD1). The empty vector was used as control. Statistical significance was estimated by Student t tests, and different letters indicate a significant difference (P < 0.05).