Hd6, a rice quantitative trait locus involved in photoperiod sensitivity, encodes the alpha subunit of protein kinase CK2

Abstract

Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the alpha subunit of protein kinase CK2 (CK2 alpha) in this region. The Nipponbare allele of CK2 alpha contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2 alpha increases days-to-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype.

Figure 1

Plant materials used in this study. (A) Graphical genotype of BC₃F₃-9-28. Black-and-white regions represent segments of the 12 chromosomes derived from Kasalath and Nipponbare, respectively. The circles indicate statistical positions of each QTL identified in QTL analyses of heading date (refs. –; M.Y., unpublished data). (B) Frequency distribution of days-to-heading in selfed progeny derived from BC₃F₃-9-28. We used CAPS markers derived from R2404 and C1329 for genotyping and discovered plants in which recombination occurred between them. Ninety nonrecombinants were used in this analysis.

Figure 2

Fine-scale linkage and physical maps around the Hd6 locus. (A) A linkage map made with 2,807 segregating plants. The vertical bars represent the positions of RFLP markers (8, 10). Numerals indicate the numbers of plants in which recombination occurred between each pair of adjacent markers. YAC (B) and PAC (C) contigs are also depicted in combination with the linkage map. After physical map construction, new RFLP markers from end-fragments of the YACs or PACs (markers preceded with Y or P, respectively) or from ESTs mapped on the YACs (C60478 and E11893) were used for further linkage analysis. Squares on the clones indicate the end-fragments of the clones. Circles indicate that RFLP markers or the end-fragment is contained in each clone. Sequencing analysis revealed that all of the end-fragments derived from one end of clones Y5081, P0686A11, and P0667B11 lay within the Hd6 gene.

Figure 3

Delimitation of Hd6. The upper rows show the genotype of seven plants in which recombination occurred between E11893 and P704B1T. Black, white, and hatched regions represent chromosomal segments that are homozygous for the Kasalath allele, homozygous for the Nipponbare allele, and heterozygous, respectively. The lower row depicts a part of P0689D01. CAPS markers derived from the PAC sequence revealed that three recombinations occurred between 13B/BglII and 13C/RsaI and one between A5 M7/AluI and 13G/NheI. Progeny testing of selfed progeny of each recombinant narrowed down the Hd6 locus into the region between 13B/BglII and 13G/NheI. An arrow indicates a CK2α gene found in this region [from transcription initiation site to poly(A) tail addition site]. The bar below the CK2α gene represents the DNA fragments used in complementation testing. The BglII site in parentheses is present only in the Kasalath genome.

Figure 4

The deduced amino acid sequence of CK2α. (A) Sequence alignment of CK2α. Dots represent amino acids identical to the rice sequence. We identified amino acids conserved among 13 CK2α genes from 10 organisms other than plants; these are listed on the bottom row (marked “conserved”). Amino acids identical to the conserved one are boxed. Roman numerals indicate the 11 conserved subdomains of protein kinases (16). The arrow indicates the position of the premature stop codon in the Nipponbare allele. The following accession numbers reference the sequences used for the alignments: maize, Y11526; Arabidopsis, D10246. (B) The nucleotide sequence and the deduced amino acid sequence around the Nipponbare premature stop codon. The stop codon is changed to a lysine codon in the Kasalath allele.