Identification of an active miniature inverted-repeat transposable element mJing in rice

Abstract

Miniature inverted-repeat transposable elements (MITEs) are structurally homogeneous non-autonomous DNA transposons with high copy numbers that play important roles in genome evolution and diversification. Here, we analyzed the rice high-tillering dwarf (htd) mutant in an advanced backcross population between cultivated and wild rice, and identified an active MITE named miniature Jing (mJing). The mJing element belongs to the PIF/Harbinger superfamily. japonica rice var. Nipponbare and indica var. 93-11 harbor 72 and 79 mJing family members, respectively, have undergone multiple rounds of amplification bursts during the evolution of Asian cultivated rice (Oryza sativa L.). A heterologous transposition experiment in Arabidopsis thaliana indicated that the autonomous element Jing is likely to have provides the transposase needed for mJing mobilization. We identified 297 mJing insertion sites and their presence/absence polymorphism among 71 rice samples through targeted high-throughput sequencing. The results showed that the copy number of mJing varies dramatically among Asian cultivated rice (O. sativa), its wild ancestor (O. rufipogon), and African cultivated rice (O. glaberrima) and that some mJing insertions are subject to directional selection. These findings suggest that the amplification and removal of mJing elements have played an important role in rice genome evolution and species diversification.

Keywords: DNA transposon; MITE; amplification; rice; targeted high-throughput sequencing.

Figure 1

Identification of the miniature inverted‐repeat transposable element miniature Jing (mJing). (a) Phenotypes of the recipient parent Teqing (wild‐type, WT) and the high‐tillering dwarf (htd) mutant at the heading stage. Scale bars represemt 10 cm. (b) and (c) Comparison of plant height (b) and tiller number (c) between WT and htd plants at the harvest stage. Values are represented as mean ± SD (n = 20). Two‐tailed Student's t‐tests were performed between WT and htd (**P < 0.01). (d) Mapping of htd and identification of the candidate mutant gene for the high‐tillering and dwarf phenotypes. The htd gene was mapped to a 565 kb interval on chromosome 4. The number of recombinant plants is shown below the corresponding markers. HTD1 (LOC_Os04g46470) is a strong candidate gene due to the presence of a 246‐bp insertion in its coding region in the htd mutant compared with WT. Black boxes and black lines represent exons and introns, respectively. Gray boxes represent the 5′‐untranslated region (UTR) and 3′‐UTR. The red triangle represents the insertion site. (e) Validation of the insertion within HTD1 gene by PCR analysis. (f) Sequences of the 246‐bp insertion and its flanking region. Red lowercase letters and black uppercase letters represent the mJing insertion and the 5′‐ and 3′‐flanking sequences of the mJing insertion in HTD1, respectively. Red underlining and black arrow indicate the target site duplications (TSDs) and terminal inverted repeats (TIRs) of mJing element, respectively.

Figure 2

Excision of mJing within the htd1 mutant allele. (a) Excision frequency of mJing within the htd1 mutant allele in the F₃ through F₆ generations. The number of surveyed families in each generation is shown in the histogram. (b) Frequency comparison of excision with and without perfect TSDs for the 97 independent excision events. (c) Footprint analysis of the excision at the original site of mJing. The mJing insertion within the htd1 mutant allele is shown in the box. Dashes represent the deleted bases after mJing excision. The numbers in parentheses indicate the proportion of corresponding excision events. (d) Phenotypes of chimeric mutants at the tillering stage (left) and the maturity stage (right). Red arrows show tillers with partial phenotypic recovery. Scale bar represemts 10 cm. (e) Two types of precise excision events at the original site of mJing in normal tillers of chimeric plants. Chromatograms show the sequences covering the original sites of mJing in dwarf tillers and taller tillers from chimeric plants. M1 and M2 are two normal tillers from different chimeric plants. Gray regions represent those sharing identical sequences. Back box represents the footprint left by mJing excision. In (c) and (e), red lowercase letters represent the mJing insertion, black uppercase letters represent the 5′‐ and 3′‐flanking sequences of the mJing insertion in HTD1, and red underlining indicates the target site duplications (TSDs) of mJing element.

Figure 3

Characteristics of mJing‐like elements in the rice genome. (a) Distribution of the 72 mJing‐like elements in the japonica var. Nipponbare genome. Different‐colored dashes represent the location of each element belonging to four clades based on phylogenetic analysis of mJing family members, as shown in Figure 4. Red asterisks indicate the three mJing‐like elements with highly similar internal sequences to mJing. (b, c) Consensus sequences of target site duplications (TSDs) (b) and terminal inverted repeats (TIRs) (c) of the 72 mJing‐like elements in the Nipponbare genome. The size of each letter indicates the frequency of the corresponding nucleotide. Black lines and arrows above the letters indicate the TSDs and TIRs in (b) and (c), respectively. (d) Insertion preferences of the 72 mJing‐like elements in the Nipponbare genome. (e) Sliding‐window analysis of GC content within the flanking regions of mJing‐ and mPing‐like elements. Red and blue lines indicate the average GC content in the flanking sequences of mJing‐ and mPing‐like elements, respectively, and the gray line represents that of randomly selected sequences from the Nipponbare genome as a control. The zero on the x‐axis indicates the insertion sites of both mJing‐ and mPing‐like elements.

Figure 4

Sequence variation and amplification of mJing‐like elements in rice. (a) Analysis of sequence similarity among the 72 mJing‐like elements in the Nipponbare genome. Sequence similarity at each site was calculated based on sequence alignment via MUSCLE. Red curve represents the line of best fit for sequence similarity. (b) Phylogenetic analysis of the 72 mJing‐like elements in the Nipponbare genome. The black dotted oval represents the three family members (Nip_mJing2.10, Nip_mJing4.3, and Nip_mJing7.6) closest to mJing. (c) Frequency distribution of pairwise nucleotide diversity among mJing and mPing family members in rice, respectively. Black and red lines indicate mPing‐like and mJing‐like elements in the japonica var. Nipponbare genome, respectively. Blue dashed line indicates mJing‐like elements in the indica var. 93‐11 genome.

Figure 5

Identification of the putative autonomous element, Jing. (a) Sequence comparison of TSDs and TIRs among mJing, NIP_mJing7.6, and Jing elements. Red lines and black arrows above the bases represent TSDs and TIRs, respectively. Dashes represent the internal sequences of each element. (b) Comparison of mJing and Jing elements. Black triangles represent TIRs. Red and green boxes indicate the 5′ and 3′ regions with high sequence similarity between mJing and Jing, and the numbers show nucleotide identity. Yellow boxes represent exons, and N2, N3, and C1 represent putative catalytic domains. Nucleotide sequences of the TSDs and TIRs and alignment of conserved domains with the DDE motifs of rice Jing, Pong, and maize PIF elements are shown. (c) The detection of mJing excision in transgenic Arabidopsis plants carrying both mJing7.6 and Jing via Sanger sequencing. mJing7.6⁻ represents a transgenic plant in which the mJing7.6 element was excised, while mJing7.6⁺ represents a transgenic plant containing an entire mJing7.6 element. Black uppercase letters and red lowercase letters below the chromatogram represent the 5′‐ and 3′‐flanking and internal sequences of mJing7.6, respectively. Red lines above the bases represent TSDs. Black box represents the TSD of Nip‐mJing7.6 and its footprint after excision.

Figure 6

Amplification and selection of mJing‐like elements in wild and cultivated rice. (a) Consensus sequences of target site duplications (TSDs) of the 297 mJing elements in the rice genome. The letter size represents the frequency of the corresponding nucleotide. Black lines above the letters indicate TSDs. (b) Comparison of the number of mJing insertions among indica, japonica, and O. rufipogon. Black dots represent the number of mJing insertions in each sample. Two‐tailed Student's t‐tests were performed. (c) Venn diagrams showing the number of unique and common mJing insertions among O. sativa, O. rufipogon, and O. glaberrima. (d) Comparison of the number of mJing insertions among O. sativa, O. rufipogon, and O. glaberrima. Black dots represent the number of mJing insertions in each sample. Two‐tailed Student's t‐tests were performed. (e) Phylogenetic analysis based on the polymorphism of 297 mJing‐like elements in 71 accessions of wild and cultivated rice. Green represents Asian cultivated rice (O. sativa) varieties, including indica and japonica subspecies. Yellow and blue represent O. rufipogon accessions that are more and less closely related to cultivated rice, respectively. Red represents African cultivated rice (O. glaberrima) varieties.