Molecular cloning of Sdr4, a regulator involved in seed dormancy and domestication of rice

Abstract

Seed dormancy provides a strategy for flowering plants to survive adverse natural conditions. It is also an important agronomic trait affecting grain yield, quality, and processing performance. We cloned a rice quantitative trait locus, Sdr4, which contributes substantially to differences in seed dormancy between japonica (Nipponbare) and indica (Kasalath) cultivars. Sdr4 expression is positively regulated by OsVP1, a global regulator of seed maturation, and in turn positively regulates potential regulators of seed dormancy and represses the expression of postgerminative genes, suggesting that Sdr4 acts as an intermediate regulator of dormancy in the seed maturation program. Japonica cultivars have only the Nipponbare allele (Sdr4-n), which endows reduced dormancy, whereas both the Kasalath allele (Srd4-k) and Sdr4-n are widely distributed in the indica group, indicating prevalent introgression. Srd4-k also is found in the wild ancestor Oryza rufipogon, whereas Sdr4-n appears to have been produced through at least two mutation events from the closest O. rufipogon allele among the accessions examined. These results are discussed with respect to possible selection of the allele during the domestication process.

Fig. 1.

Genetic effects, delimitation of candidate genomic region, and genetic complementation of Sdr4. (A) Germination of Kasalath (Kas), Nipponbare (Npb), and NIL[Sdr4] (NIL). (B) Germination rates of seeds from panicles sampled 6 weeks after heading. (C) Delimitation of Sdr4. Graphical genotypes of the Sdr4 region in eight plants in which recombination occurred between RM1365 and SNP5 (Fig. S1) are shown at the left. Black and white regions represent chromosomal segments homozygous for the Kas and Npb alleles, respectively. The bar graph at the right shows the germination rates of recombinants. Preharvest sprouting resistance is denoted by “R,” and sensitive is denoted by “S.” Genes predicted by RAP-DB (http://rapdb.dna.affrc.go.jp/) (52) in the Npb genomic sequence around the Sdr4 candidate region are indicated by closed arrows and closed boxes. Two genes were annotated in the region. The 8.7-kb Sdr4 candidate region was defined by linkage analysis. The 3.3-kb fragment was used for complementation analysis. The 1.6-kb region with FNP is indicated by a hatched box. (D) Bar graph showing germination rates of T₃ seeds harboring the Kas fragment (3.3 kb) or empty vector (Vc). (E) RNA gel blot analysis of Os07g0585700 gene expression in knockdown (KD) and Vc lines. Knockdown transgenic lines were generated from Npb and NIL. rRNA stained with methylene blue shows equal loading of RNAs (rRNA). Germination rates 7 days after imbibition are shown as averages of three biological repeats with standard deviation.

Fig. 2.

Subcellular localization of Sdr4. The 35S promoter–driven dimer of sGFP and the Sdr4-k–sGFP fusion were transiently expressed in oc cells, and the localizations were investigated under a confocal laser scanning microscope (Left). Differential interference contrast images (Right) and merged images (Middle) are shown.

Fig. 3.

InDels and SNPs between Sdr4-n and Sdr4-k, and amino acid sequence comparison among Sdr4 and its homologs. (A) Haplotypes derived from 11 SNPs and two InDels of the Sdr4 coding region. (B) Sequences of two alleles around the SNP cluster, indicated by the asterisk in A. The 18-nt direct repeats are indicated by arrows. (C) Sdr4 homologs were searched by BLAST. Aligned sequences by Genetyx version 9 (Genetyx) are shown by unrooted phylogenetic tree using MEGA4 (53).

Fig. 4.

Morphological and physiological analysis of sdr4 mutant. (A) GUS staining of sliced seed of Nipponbare (Npb) with Sdr4-n:GUS. The radicle is indicated by an arrowhead. For tissue-specific expression of Sdr4 mRNA, in situ hybridization used Sdr4-specific antisense and sense probes on sections of Npb seed. The radicle is indicated by an arrowhead. (B) Temporal changes in level of Sdr4 mRNA during the ripening period, based on semiquantitative RT-PCR, at 0, 3, 7, 14, 28, and 42 days after heading. The UBQ2 was used as a control to show equal loading. (C) Longitudinal slices of the seeds of Npb and the sdr4 mutant were stained by toluidine blue. (D) Embryos were harvested (n = 50) and weighed, with three repeats. (E) Germination rates of Npb, NIL[Sdr4] (NIL), two sdr4 mutants, and Kasalath (Kas) at various time points after heading were determined. (F) Germination rates just after harvest (6 WAH) of fresh seeds treated with different concentrations of ABA. (G) Embryoless half-seeds were treated with 30 μM ABA or left untreated for 24 h; expression levels of Osem1 were determined by real-time PCR. (H) AK063726 expression levels in the embryoless half-seeds were measured.

Fig. 5.

Expression analysis of dormancy and germination related genes in sdr4 and Osvp1 mutants. (A) Temporal changes (DAF) in mRNA levels of OsDOG1-like genes, gibberellin biosynthesis gene (OsGA20ox-1), aquaporin gene (PIP1;3), and expansin gene (OsEXPB3) in embryos were monitored by real-time PCR in Nipponbare (Npb), the sdr4 mutant (M100), and NIL[Sdr4] (NIL), with three biological repeats. Expression levels are shown as ratios to Actin-1 gene expression. (B) Sdr4 gene expression in wild-type (Npb), Osvp1-1 (M125), and Osvp1-2 (M101) at 28 DAF was determined.

Fig. 6.

Natural variations in the Sdr4 coding region. (A) SNPs and germination rates at 6 WAH in cultivars in the world rice core collection. Cultivars, ordered according to the genetic distance based on RFLPs, are shown. Germination rates at 6 WAH are indicated in mauve-blue or sky-blue bars (cultivars with Sdr4-k or Sdr4-k′, respectively) or green bars (cultivars with Sdr4-n); the 100% scale is indicated by the gray bars. (B) The frequency of each haplotype in japonica and indica (In-1 and In-2) lines is summarized, and wild relatives showing the same haplotypes as the cultivars are shown.