Whole genome de novo assemblies of three divergent strains of rice,Oryza sativa,document novel gene space of aus and indica

The use of high throughput genome-sequencing technologies has uncovered a large extent of structural variation in eukaryotic genomes that makes important contributions to genomic diversity and phenotypic variation. When the genomes of different strains of a given organism are compared, whole genome resequencing data are typically aligned to an established reference sequence. However, when the reference differs in significant structural ways from the individuals under study, the analysis is often incomplete or inaccurate. Here, we use rice as a model to demonstrate how improvements in sequencing and assembly technology allow rapid and inexpensive de novo assembly of next generation sequence data into high-quality assemblies that can be directly compared using whole genome alignment to provide an unbiased assessment. Using this approach, we are able to accurately assess the ‘pan-genome’ of three divergent rice varieties and document several megabases of each genome absent in the other two. Many of the genome-specific loci are annotated to contain genes, reflecting the potential for new biological properties that would be missed by standard reference-mapping approaches. We further provide a detailed analysis of several loci associated with agriculturally important traits, including the S5 hybrid sterility locus, the Sub1 submergence tolerance locus, the LRK gene cluster associated with improved yield, and the Pup1 cluster associated with phosphorus deficiency, illustrating the utility of our approach for biological discovery. All of the data and software are openly available to support further breeding and functional studies of rice and other species.     

The simple repeat poly(dT-dG).poly(dC-dA) common to eukaryotes is absent from eubacteria and archaebacteria and rare in protozoans

Genomic DNA from a wide variety of prokaryotic and eukaryotic organisms has been assayed for the simple repeat sequence poly(dT-dG).poly(dC-dA) by Southern blotting and DNA slot blot hybridizations. Consistent with findings of others, we have found the simple alternating sequence to be present in multiple copies in all organisms in the animal kingdom (e.g., mammals, reptiles, amphibians, fish, crustaceans, insects, jellyfish, nematodes). The TG element was also found in lower eukaryotes (Saccharomyces cerevisiae, Neurospora crassa, and Dictyostelium discoideum) and at a much lower frequency in protozoans (Oxytricha fallux and Tetrahymena thermophila). The sequence was also repeated in high copy number in a higher plant (Zea mays) as well as at very high levels in a unicellular green alga (Chlamydomonas reinhardi). Although the copy number of the repeat per haploid genome was generally proportional to genome size, there was a greater-than-1,000-fold variation in the number of (TG)25/100-kb genomic DNA. By contrast, no eu-or archaebacterium--including Myxococcus xanthus, whose life cycle is very similar to that of the slime mold Dictyostelium discoideum, and Halobacter volcanii, whose genome contains other repeated sequences-- was found whose genomic DNA contained this sequence in detectable amounts. A computer search also failed to find the TG element in human mitochondrial DNA.      

Inactivation of the CTD phosphatase-like gene OsCPL1 enhances the development of the abscission layer and seed shattering in rice

Although susceptibility to seed shattering causes severe yield loss during cereal crop harvest, it is an adaptive trait for seed dispersal in wild plants. We previously identified a recessive shattering locus, sh-h , from the rice shattering mutant line Hsh that carries an enhanced abscission layer. Here, we further mapped sh-h to a 34-kb region on chromosome 7 by analyzing 240 F₂ plants and five F₃ lines from the cross between Hsh and Blue&Gundil. Hsh had a point mutation at the 3' splice site of the seventh intron within LOC_Os07g10690, causing a 15-bp deletion of its mRNA as a result of altered splicing. Two transferred DNA (T-DNA) insertion mutants and one point mutant exhibited the enhanced shattering phenotype, confirming that LOC_Os07g10690 is indeed the sh-h gene. RNA interference (RNAi) transgenic lines with suppressed expression of this gene exhibited greater shattering. This gene, which encodes a protein containing a conserved carboxy-terminal domain (CTD) phosphatase domain, was named Oryza sativa CTD phosphatase-like 1 ( OsCPL1 ). Subcellular localization and biochemical analysis revealed that the OsCPL1 protein is a nuclear phosphatase, a common characteristic of metazoan CTD phosphatases involved in cell differentiation. These results demonstrate that OsCPL1 represses differentiation of the abscission layer during panicle development.     

Identification of quantitative trait loci for physical and chemical properties of rice grain

Quantitative trait loci (QTL) associated with six physical traits of cooked rice and seven chemical properties of rice grain were identified using a recombinant inbred (RI) population of rice evaluated over 3 years at the National Honam Agricultural Research Institute in Korea. The RI population consisted of 164 lines derived from a cross between Milyang23 and Gihobyeo, and the genetic map consisted of 414 molecular markers. A total of 49 QTL were identified for the 13 physico-chemical properties using composite interval mapping. Of these, 13 QTL were identified for 2 or more years, while 36 were detected in only 1 year. Five QTL were identified over all 3 years and will be useful for marker-assisted improvement of rice grain quality in Korea. The two QTL with the highest LOD scores, adhesiveness1.2 and potassium content7.1, provide a valuable starting point for positional cloning of genes underlying these QTL.     

The extent of linkage disequilibrium in rice (Oryza sativa L.)

Despite its status as one of the world's major crops, linkage disequilibrium (LD) patterns have not been systematically characterized across the genome of Asian rice (Oryza sativa). Such information is critical to fully exploit the genome sequence for mapping complex traits using association techniques. Here we characterize LD in five 500-kb regions of the rice genome in three major cultivated rice varieties (indica, tropical japonica, and temperate japonica) and in the wild ancestor of Asian rice, Oryza rufipogon. Using unlinked SNPs to determine the amount of background linkage disequilibrium in each population, we find that the extent of LD is greatest in temperate japonica (probably >500 kb), followed by tropical japonica (approximately 150 kb) and indica (approximately 75 kb). LD extends over a shorter distance in O. rufipogon (<40 kb) than in any of the O. sativa groups assayed here. The differences in the extent of LD among these groups are consistent with differences in outcrossing and recombination rate estimates. As well as heterogeneity between groups, our results suggest variation in LD patterns among genomic regions. We demonstrate the feasibility of genomewide association mapping in cultivated Asian rice using a modest number of SNPs.     

Identification of QTLs associated with physiological nitrogen use efficiency in rice

Demand for low-input sustainable crop cultivation is increasing to meet the need for environment-friendly agriculture. Consequently, developing genotypes with high nutrient use efficiency is one of the major objectives of crop breeding programs. This study was conducted to identify QTLs for traits associated with physiological nitrogen use efficiency (PNUE). A recombinant inbred population (DT-RILs) between Dasanbyeo (a tongil type rice, derived from an indica x japonica cross and similar to indica in its genetic make-up) and TR22183 (a Chinese japonica variety) consisting of 166 F8 lines was developed and used for mapping. A frame map of 1,409 cM containing 113 SSR and 103 STS markers with an average interval of 6.5 cM between adjacent marker loci was constructed using the DT-RILs. The RILs were cultivated in ordinary-N (N-P2O5-K2O = 100-80-80 kg/ha) and low-N (N-P2O5-K2O= 50-80-80 kg/ha) (100 kg/ha) conditions. PNUE was positively correlated with the harvest index and grain yield in both conditions. Twenty single QTLs (S-QTLs) and 58 pairs of epistatic loci (E-QTLs) were identified for the nitrogen concentration of grain, nitrogen concentration of straw, nitrogen content of shoot, harvest index, grain yield, straw yield and PNUE in both conditions. The phenotypic variance explained by these S-QTLs and E-QTLs ranged from 11.1 to 44.3% and from 16.0% to 63.6% , respectively. The total phenotypic variance explained by all the QTLs for each trait ranged from 35.8% to 71.3%, showing that the expression of PNUE and related characters depends significantly upon genetic factors. Both S-QTLs and E-QTLs may be useful for marker-assisted selection (MAS) to develop higher PNUE genotypes.     

Global dissemination of a single mutation conferring white pericarp in rice

Here we report that the change from the red seeds of wild rice to the white seeds of cultivated rice (Oryza sativa) resulted from the strong selective sweep of a single mutation, a frame-shift deletion within the Rc gene that is found in 97.9% of white rice varieties today. A second mutation, also within Rc, is present in less than 3% of white accessions surveyed. Haplotype analysis revealed that the predominant mutation originated in the japonica subspecies and crossed both geographic and sterility barriers to move into the indica subspecies. A little less than one Mb of japonica DNA hitchhiked with the rc allele into most indica varieties, suggesting that other linked domestication alleles may have been transferred from japonica to indica along with white pericarp color. Our finding provides evidence of active cultural exchange among ancient farmers over the course of rice domestication coupled with very strong, positive selection for a single white allele in both subspecies of O. sativa.     

Evolutionary History of GS3, a Gene Conferring Grain Length in Rice

Unlike maize and wheat, where artificial selection is associated with an almost uniform increase in seed or grain size, domesticated rice exhibits dramatic phenotypic diversity for grain size and shape. Here we clone and characterize GS3, an evolutionarily important gene controlling grain size in rice. We show that GS3 is highly expressed in young panicles in both short- and long-grained varieties but is not expressed in leaves or panicles after flowering, and we use genetic transformation to demonstrate that the dominant allele for short grain complements the long-grain phenotype. An association study revealed that a C to A mutation in the second exon of GS3 (A allele) was associated with enhanced grain length in Oryza sativa but was absent from other Oryza species. Linkage disequilibrium (LD) was elevated and there was a 95.7% reduction in nucleotide diversity (θ_π) across the gene in accessions carrying the A allele, suggesting positive selection for long grain. Haplotype analysis traced the origin of the long-grain allele to a Japonica-like ancestor and demonstrated introgression into the Indica gene pool. This study indicates a critical role for GS3 in defining the seed morphologies of modern subpopulations of O. sativa and enhances the potential for genetic manipulation of grain size in rice.SEED size and seed number are the major determinants of crop yield in both the cereals and the grain legumes. Seed size was also a target of artificial selection during domestication, where large seeds are generally favored due to ease of harvesting and enhanced seedling vigor (Harlan et al. 1972). In rice, traits related to grain size and appearance have a large impact on market value and play a pivotal role in the adoption of new varieties (Champagne et al. 1999; Juliano 2003). However, different grain quality traits are prized by different local cultures and cuisines and, unlike other cereals such as wheat, barley, and maize that are sold largely in processed forms, the physical properties of rice grains are immediately obvious to consumers (Fitzgerald et al. 2009). Thus, rice offers a unique opportunity to investigate the genetics and evolutionary history of seed size and shape.Cultivated rice (Oryza sativa) was domesticated in Asia from the wild progenitor O. rufipogon Griff. and/or O. nivara Sharma (Ishii et al. 1988; Oka 1988; Dally and Second 1990; Nakano et al. 1992; Chen et al. 1993). Classical studies of the subpopulation structure of O. sativa have identified two primary subspecies or varietal groups, namely Indica and Japonica (Oka 1988; Wang and Tanksley 1989; Sun et al. 2002). Studies that have dated the divergence between the Indica and the Japonica groups indicate that it predates rice domestication by at least 100,000 years (Ma and Bennetzen 2004; Vitte et al. 2004; Zhu and Ge 2005), suggesting that at least two genetically distinct gene pools of O. rufipogon were cultivated and subsequently domesticated.Isozyme and DNA studies revealed that there is additional genetic structure within these two groups, with three subpopulations composing the Japonica varietal group (temperate japonica, tropical japonica, and aromatic, written all in lowercase) and two subpopulations composing the Indica group (indica and aus) (Second 1985; Glaszmann 1987; Garris et al. 2005; Caicedo et al. 2007). While there is great diversity of seed size and shape both within and between the different subpopulations of O. sativa, each subpopulation is popularly associated with a characteristic seed morphology. Temperate japonica varieties are known for their short, round grains, indica and aus for slender grains, and within the aromatic subpopulation [hereafter referred to as Group V varieties, according to the isozyme group designation (Glaszmann 1987)] the group of basmati varieties is highly valued for their very long, slender grains (Juliano and Villareal 1993). Identification of the genes that control the range of seed size variation in rice will offer opportunities to study the evolutionary history and phenotypic diversification of the five subpopulations within O. sativa and also provide valuable targets for genetic manipulation.In rice, four genes contributing to seed or grain size have been identified and characterized. The first, grain size 3 (GS3), was isolated from an indica × indica population and found to encode a novel protein with several conserved domains including a phosphatidylethanolamine-binding protein (PEBP)-like domain, a transmembrane region, a putative tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR) family domain, and a von Willebrand factor type C (VWFC) domain (Fan et al. 2006). A second gene, grain weight 2 (GW2), was found to encode an unknown RING-type protein with E3 ubiquitin ligase activity (Song et al. 2007). The third, grain incomplete filling 1 (GIF1), encodes a cell-wall invertase required for carbon partitioning during early grain filling (Wang et al. 2008). Finally, the recently characterized seed width 5 (SW5) has no apparent homolog in the database but was shown to interact with polyubiquitin in a yeast two-hybrid assay; thus it likely acts in the ubiquitin–proteasome pathway to regulate cell division during seed development (Shomura et al. 2008; Weng et al. 2008).Many genes controlling seed size have also been identified in Arabidopsis and tomato, providing a framework for assembling the genetic pathway that determines this trait in dicotyledonous plants (Chaudhury et al. 2001; Jofuku et al. 2005; Ohto et al. 2005; Sundaresan 2005; Schruff et al. 2006; Yoine et al. 2006; Roxrud et al. 2007; Li et al. 2008; Xiao et al. 2008; Orsi and Tanksley 2009; Zhou et al. 2009). Several of these genes show maternal control by regulating endosperm and/or ovule development (Garcia et al. 2003; Jofuku et al. 2005; Li et al. 2008; Ohto et al. 2005; Xiao et al. 2008).Numerous studies have identified rice QTL associated with grain weight and grain length [www.gramene.org (Ni et al. 2009)]. Ten of these studies identified a seed size QTL located in the pericentromeric region of rice chromosome 3, using both inter- and intraspecific crosses (Li et al. 1997; Yu et al. 1997; Redona and Mackill 1998; Xiao et al. 1998; Kubo et al. 2001; Moncada et al. 2001; Brondani et al. 2002; Xing et al. 2002; Thomson et al. 2003; Li et al. 2004). In interspecific crosses, the wild accessions always contributed the dominant allele for small seed size at this locus. Comparative mapping of QTL controlling seed weight in rice, maize, and sorghum further suggested that orthologous seed size genes at this locus might be associated with domestication in all three crops (Paterson et al. 1995).In the current study, we used positional cloning and transformation to demonstrate that the GS3 gene underlies both the gw3.1 QTL (Thomson et al. 2003; Li et al. 2004) and the lk3 QTL (Kubo et al. 2001). In transformation experiments, we demonstrated for the first time that the dominant allele for small grain size complements the long-grain phenotype and we characterized the spatial expression patterns of the gene at different developmental stages. We undertook an association analysis to examine the relationship between the alleles at GS3 and the observed variation for grain length/size in both wild and cultivated rice. Finally, we examined sequence haplotypes across the GS3 region to look for evidence of selection and to identify the origin of the mutation leading to increased grain length in O. sativa.