CACTA-like transposable element in ZmCCT attenuated photoperiod sensitivity and accelerated the postdomestication spread of maize

Abstract

The postdomestication adaptation of maize to longer days required reduced photoperiod sensitivity to optimize flowering time. We performed a genome-wide association study and confirmed that ZmCCT, encoding a CCT domain-containing protein, is associated with the photoperiod response. In early-flowering maize we detected a CACTA-like transposable element (TE) within the ZmCCT promoter that dramatically reduced flowering time. TE insertion likely occurred after domestication and was selected as maize adapted to temperate zones. This process resulted in a strong selective sweep within the TE-related block of linkage disequilibrium. Functional validations indicated that the TE represses ZmCCT expression to reduce photoperiod sensitivity, thus accelerating maize spread to long-day environments.

Fig. 1.

ZmCCT-based association mapping and LD analysis of 180 diverse maize lines. (A) Associations between polymorphic sites within the ZmCCT locus (MAF ≥0.05) and APR. Each dot represents a polymorphic site, and the color reflects the level of LD (r²) with the TE (except for the TE, which is red). (B) Structure of the ZmCCT locus. Black rectangles represent exons, and white rectangles represent UTRs. The transcription start site (TSS) is indicated. (C) The pattern of LD for all polymorphic sites within the ZmCCT locus. All polymorphic sites (MAF ≥0.05) excluding the TE were used.

Fig. 2.

Genetic effects and relatedness of ZmCCT-promoter haplotypes. (A) Estimated effects of haplotypes with MAF ≥0.01 on APR in the CAM508 panel after correcting for population structure and kinship. When a string of polymorphic sites are in complete LD, only one is shown. Insensitive alleles are in bold text. ^#The number of lines for each haplotype (N) and the P values are indicated. S1–S7 correspond to sites at −1,983, −1,884, −1,875, −1,722, −1,518, −1,341, and −1,206 bp, respectively. (B) Proposed relatedness of the seven haplotypes. Each circle represents a haplotype, and the size of the circle is proportional to the number of lines within the haplotype: smallest circles <20, small circles 20–30, large circles 30–300, and largest circles ≥300 lines. Green and yellow represent temperate and tropical germplasms, respectively.

Fig. 3.

Sequence diversity of the ZmCCT locus between maize and teosinte. (A) Nucleotide diversity revealed by comparisons between 143 maize lines and 32 teosinte entries across the ZmCCT locus. Nucleotide diversity (π) for teosinte (blue), TE-positive maize (green), and TE-negative maize (red) was calculated using a 100-bp sliding window with a 25-bp step. Results from the Tajima’s D test and three π ratios, TE-positive maize (π₊) to TE-negative maize (π₋), π₊ to teosinte (π_T), and π₋ to π_T, are shown. *P < 0.05; ***P < 0.0001. (B) A minimum-spanning tree for the ZmCCT promoter region including 481 diverse maize sequences and 93 diverse teosinte sequences. Each haplotype group is represented by a circle, and circle sizes represent the number of lines within the haplotype, as in Fig. 2. Green, yellow, and brown represent temperate, tropical, and teosinte germplasms, respectively. The circles with grids indicate haplotypes that have TE insertions; the red stars near the haplotypes show that their TE genotypes are heterozygous.

Fig. 4.

Functional validation of ZmCCT via transformation. (A) The DNA fragment used for the ZmCCT complementation test is shown. (B) The greenhouse performance of T₀ transgenic plants. Red arrows indicate transgenic plants. (C) The greenhouse performance of T₂ transgenic plants. Transgenic plants and sibling controls are indicated by (+) and (−), respectively. (D) Comparison between transgenic lines and controls in T₀ and T₂ generations under long-day greenhouse conditions. DTA, days to anthesis (for T₀ plants, from the date of transplantation to anthesis); EH, ear height; PH, plant height; TLN, total leaf number. Data are shown as mean ± SE.

Fig. 5.

Expression and subcellular localization of ZmCCT and its encoded protein. (A–D) A CaMV35S:ZmCCT-GFP construct was used to assess protein localization. CaMV35S:GFP was used as a control. (A) Nuclear localization of ZmCCT-GFP in maize protoplast. (Scale bar, 10 μm.) (B) GFP localization in maize protoplast. (Scale bar, 10 μm.) (C) Nuclear localization of ZmCCT-GFP in onion epidermal cells. (Scale bar, 20 μm.) (D) GFP localization in onion epidermal cells. (Scale bar, 20 μm.) (E and F) Diurnal rhythms of expression for ZmCCT in NIL1 (TE-negative, blue) and NIL2 (TE-positive, orange) in long-day (E) and short-day (F) environments. Expression was normalized to GADPH. Black bars represent dark periods, and white bars represent light periods. Data are shown as mean ± SE. (G) Allele-specific ZmCCT expression in transgenic and nontransgenic plants (T₂). RT-PCR analysis of transgenic-plant samples yielded a 235-bp band (underlined in red), whereas nontransgenic siblings yielded a 241-bp band (underlined in blue). Phenotypic data associated with each plant are indicated. Transgenic plants and sibling controls are indicated by (+) and (−), respectively. GADPH was used as the control.