Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS

Abstract

A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of approximately 12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions.

Figure 1.

A Fine-Scale, High-Resolution Genetic and Physical Map of the Hd1 Region on Chromosome 6. (A) Genetic linkage map showing the relative position of Hd1 with RFLP markers on chromosome 6. Numbers under the horizontal line are numbers of plants with a recombinant chromosome in the adjacent marker intervals. (B) and (C) Yeast artificial chromosome (B) and PAC clones (C) spanning the Hd1 region. A circle indicates the existence of a sequence corresponding to the RFLP markers. Entire insert sequencing was performed on PAC clone P0038C5. (D) Detailed genetic and physical map showing the relative positions of the candidate regions of Hd1 and CAPS markers developed based on sequence data. Rec., approximate positions of recombination events that occurred near Hd1.

Figure 2.

Scheme of the Structural Differences in the Candidate Region of Hd1 in Nipponbare and Kasalath and the Corresponding Genomic Region of se1 Mutants HS66 and HS110 and Their Original Variety, Ginbouzu. (A) Comparison of genomic sequences of Nipponbare and Kasalath Hd1 alleles. Boxes show the predicted open reading frames based on the Genscan software maize model. Vertical lines without labels represent single-base substitutions between Nipponbare and Kasalath. Small rectangular boxes and arrowheads represent deletions and insertions, respectively. (B) A 7.1-kb ApaI genomic fragment containing the entire Hd1 candidate sequence used in the complementation analysis. This fragment does not contain another predicted gene, encoding peroxidase, which was found in the candidate genomic region of Hd1.

Figure 3.

Deduced Amino Acid Sequence of Hd1 Protein, Amino Acid Alignment with Arabidopsis CO and B. napus BnCOA1, and Comparison of Amino Acid Sequences of C-Terminal Regions of the Zinc Finger Domain between Nipponbare and Ginbouzu Hd1. (A) Comparison of Nipponbare Hd1, CO, and BnCOA1 alleles. Boxes in N and C termini are conserved domains of the zinc finger motif (N terminus) and nuclear localization signals (C terminus). Arrowheads indicate cysteine residues in the zinc finger domain. Boldface letters represent identical amino acid residues among the three proteins. (B) Comparison of the C-terminal zinc finger domain of Nipponbare Hd1 and Ginbouzu Se1 (Hd1) proteins. Amino acid substitutions and deletions are indicated by underlines.

Figure 4.

Frequency Distribution for Days to Heading in Self-Pollinated Progeny (T₁) of One 7.1-kb Transformant. All plants were cultivated under SD conditions (10.0 hr) in a controlled growth chamber. Bars indicate plants with (black) and without (hatched) the candidate genomic fragment. NIL(Hd1/Hd2) is the recipient line for transformation, and NIL(Hd2), the nearly isogenic line of Hd2, can be a control of transformation when the functional Hd1 is complemented in NIL(Hd1/Hd2). Means and ranges of days to heading of Nipponbare, NIL(Hd1/Hd2), and NIL(Hd2) are indicated by arrowheads and horizontal bars, respectively.

Figure 5.

Detection of mRNA in the Varieties and Lines Used in This Study by RT-PCR Assay. All plants were raised in LD conditions (16.0 hr) and then subjected to the following treatments: L, additional 10 days of treatment in LD conditions; S0, no additional treatment; S5, additional 5 days of treatment in SD conditions (10.0 hr); S10, additional 10 days of treatment in SD conditions. The actin control is shown at the bottom of figure. Lane M indicates DNA size markers. Lane gDNA is a PCR product of genomic DNA of Nipponbare as a template.