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Asian cultivated rice (Oryza sativa) is a staple food that provides three-quarters of the caloric intake of the population of Southeast Asia (Khush, 1997). Grain size is measured in terms of either grain weight or shape (grain length (GL) and grain width (GW)), and is a major component of crop yield and an important trait for appearance quality (Xing & Zhang, 2010). Different grain sizes are favoured by different local cultures and cuisines. Grain size characteristics, which are immediately obvious to consumers, are major factors defining market value (Fitzgerald et al., 2009). Detailed knowledge of the genetic factors controlling grain size enables breeders to design appropriate genotypes for distinct preferences.
In Asian cultivated rice (O. sativa), the two major subpopulations, indica and japonica, can be differentiated on the basis of morphological and genetic differences (Chou, 1948; Caicedo et al., 2007). Genome-wide studies have demonstrated that these differences arose from genetically distinct gene pools in a common ancestor, Oryza rufipogon, by a process of continuous selection for desirable features (Huang et al., 2012b). In addition to being an important domestication trait, grain size acts as a distinguishable character for the two rice subpopulations (Fitzgerald et al., 2009). Thus, dissection of the genetic basis of grain size and isolation of grain size genes will increase our understanding of the origin and evolution of rice.
In recent years, several quantitative trait loci (QTLs) regulating grain size have been fine-mapped (Xie et al., 2006; Liu et al., 2009; Bai et al., 2010; Shao et al., 2010). grain size 3 (GS3), the first grain-length QTL to be cloned, was isolated in different genetic backgrounds in several studies and functions as a negative regulator of grain size (Fan et al., 2006; Takano-Kai et al., 2009). A nonsense mutation in the second exon is associated with large grain size and is widespread in global rice collections (Fan et al., 2009; Wang et al., 2011). Further molecular characterization of GS3 identified four putative domains, with the organ size regulation (OSR) domain being both necessary and sufficient for its function (Mao et al., 2010). A major GW QTL, grain width and weight 2 (GW2), encodes an uncharacterized Really Interesting New Gene (RING) type E3 ubiquitin ligase. Increased GW is associated with a mutation that causes premature truncation of the GW2 protein (Song et al., 2007). QTL for seed width 5 (qSW5) is the most important QTL for GW and grain weight. A 1212-bp deletion in qSW5 was confirmed to be the causal mutation that led to increases in both GW and grain weight. A yeast two-hybrid assay further implied a possible role for this gene in the ubiquitin-proteasome pathway (Shomura et al., 2008; Weng et al., 2008). grain size 5 (GS5) is a minor GW QTL that is 2 Mb distant from qSW5 and functions as a positive regulator of grain size, such that higher expression of this gene is associated with both larger grain size and acceleration of the cell cycle (Li et al., 2011). Together with the newly cloned genes OsSPL16 and GL3.1 (Qi et al., 2012; Wang et al., 2012), GS3, GW2, qSW5 and GS5 are the main determinants of grain size dimensions. However, the extent of natural variation, allele frequencies and functional differences associated with these QTLs in rice germplasm remain unclear. In addition, their relative importance in grain shape determination is still unknown. An exploration of these issues will aid in understanding the comprehensive role of each gene in grain shape formation and provide an efficient way to target and improve grain shape.
The natural population, harbouring plentiful natural variation, provides an excellent opportunity to identify key single nucleotide polymorphisms (SNPs)/insertions/deletions (InDels) associated with gene function (Zhu et al., 2008). Nucleotide diversity can reflect the evolutionary history and geographical distribution of important genes or alleles (Izawa et al., 2009). Functional gene-based association analyses facilitate the further dissection of traits controlled by many genes. For example, polymorphisms in six genes involved in the flowering pathway were sequenced in a rice collection; by combining these results with those of further experiments, the authors demonstrated that variation in Hd1 proteins, Hd3a promoters and Ehd1 expression levels all contribute to the diversity of flowering time in cultivated rice (Takahashi et al., 2009). In addition, comparisons of the nucleotide diversity of four major flowering genes, PhyB, Hd1, Hd3a and Ehd1, between cultivars and wild rice revealed significant selection on these four genes and geographical adaption to differing photoperiods (Huang et al., 2012a).
In this study, GW2, qSW5 and GS5 were sequenced in the germplasms of 127 varieties of cultivated rice (O. sativa) and 10–15 accessions of wild rice (O. rufipogon). The GS3 genotypes of all samples were determined using functional markers (Fan et al., 2009). Allelic frequencies and functional differences in rice grain size were compared, respectively, in indica and japonica subpopulations. We found that genes with signatures of significant selection have long been utilized in rice breeding from rice germplasm. These results provide insight into the evolutionary features of grain size genes as well as information that may be useful for future molecular applications of these genes in rice breeding.