Sampling and sequencing <\/p>\n
The cultivated and wild rice accessions were all from large collections of rice accessions preserved at the China National Rice Research Institute in Hangzhou, China, and the National Institute of Genetics in Mishima, Japan. The accessions were selected on the basis of the germplasm database records of phenotypic data and sampling localities to maximize genetic and geographic diversity. The collection was maintained by selfing in the laboratories. For each accession, genomic DNA from a single plant was used for sequencing, and seeds derived from the same plant were used for following field trials. In total, the genomes of 1,083 O. sativa accessions, 446 O. rufipogon accessions and 15 accessions of outgroup species were sequenced on the Illumina Genome Analyzer IIx generating 73-bp (or 117-bp) paired-end reads, each to approximately onefold (for O. sativa accessions), twofold (for O. rufipogon accessions) or threefold (for outgroup species) coverage. The detailed information, including geographic origin and sequencing coverage of the rice accessions, was listed in Supplementary Tables 2, 7 and 8. Library construction and sequencing of these accessions were performed as described21. One representative accession of O. rufipogon, W1943, was sequenced on the Illumina HiSeq2000, generating 100-bp paired-end reads with 100-fold genome coverage. An amplification-free method of library preparation49 was used in deep sequencing of the rice accession, which reduced the incidence of duplicate sequences, thus facilitating genome assembly and variation analysis. <\/p>\n
The paired-end reads of all the rice accessions were aligned against the rice reference genome (IRGSP 4.0) using the software Smalt (version 0.4) with the parameters of -pair 50, 700 and -mthresh 50. SNPs were called using the Ssaha Pileup package (version 0.5) with detailed procedure described previously21. Genotypes of the rice accessions, including 1,083 O. sativa accessions, 446 O. rufipogon accessions and 15 accessions of outgroup species, were further called at the SNP sites from the Ssaha Pileup outputs. The genotype calls in 15 accessions of outgroup species were used to determine the ancestral states of SNPs in O. sativa and O. rufipogon. SNPs in coding regions, which were defined based on the gene models in the RAP-DB (release 2), were then annotated to be synonymous or non-synonymous for calculating the non-synonymous\/synonymous ratio and dN\/dS ratio. The genotype data set of the 1,529 rice accessions (1,083 O. sativa accessions and 446 O. rufipogon accessions) was generated on the basis of the calls in each rice accession. Seven sets of genome sequences, which included bacterial-artificial-chromosome-based Sanger sequences and high-coverage resequencing data, were used to assess the accuracy of the genotype data sets (Supplementary Table 6). The wild rice accessions with sequencing coverage >9 were selected to investigate the heterozygosity based on the overlapped reads that were aligned onto the reference sequence. For each accession, the proportion of heterozygosity genotypes was calculated at the polymorphic sites (Supplementary Table 3). <\/p>\n
The software Haploview was used to calculate linkage disequilibrium with default settings, using SNPs with information in 446 O. rufipogon accessions50. Pairwise r2 was calculated for all the SNPs and then averaged across the whole genome. The matrix of pairwise genetic distance derived from simple SNP-matching coefficients was used to construct phylogenetic trees using the software PHYLIP51 (version 3.66). The software TreeView and MEGA5 were used for visualizing the phylogenetic trees. Principal component analysis of the SNPs was performed using the software EIGENSOFT52. The sequence diversity statistics () and the population-differentiation statistics (FST) were computed using a 100-kb window. The value of was calculated for each group in O. rufipogon and O. sativa, respectively, and the ratio of in the full population (or each clade) of O. rufipogon to that in the full population (or corresponding subspecies) of O. sativa was used to detect selective sweeps. The genomic regions where both O. rufipogon and O. sativa show a low level of genetic diversity were excluded for further analysis. To adopt appropriate thresholds to reduce the false-positive rate but also retain true selection signals, thresholds were chosen on the basis of both whole-genome permutation tests and signals at known loci. Permutation tests were performed to estimate the genome-wide type I error rate and determine the threshold to call selective sweeps (see Supplementary Information section 2 for details)53. The method cross-population extended haplotype homozygosity (XP-EHH) was also tested for detecting selective sweeps using the software xpehh54 (http:\/\/hgdp.uchicago.edu\/Software\/<\/a>) (Supplementary Fig. 20). The genetic distance between two clades was computed based on the matrix of pairwise genetic distance, where the distance of all pairs of accessions from the two clades were retrieved and averaged. A custom Perl script was developed to plot all O. rufipogon accessions, using the public geographic information of world borders from the Thematic Mapping data set (version 0.3). The computational simulations under different demographic scenarios were performed using the program SFS_CODE40. <\/p>\n For the O. rufipogon population, approximately five seeds for each accession from the collection of wild rice were germinated and planted in the experimental field (in Sanya, China at N 18.65, E 109.80) from March 2011. The leaf sheath colour was observed and scored directly and the tiller angle was measured for each plant. The mapping population of 210 backcross inbred lines (BILs) and 61 chromosome segment substitution lines (CSSLs) was derived from a cross between O. sativa ssp. indica cv. Guangluai-4 and O. rufipogon accession W1943. The BILs were developed by one generation of backcross to Guangluai-4 followed with six generations of self-fertilization. The CSSLs were developed by five generations of backcross to Guangluai-4 followed with three generations of self-fertilization. Phenotyping was conducted in the experimental field (in Shanghai, China at N 31.13, E 121.28) from May to October, 2011. The fifteen traits that we phenotyped for this study include germination rate, tiller angle, heading date, stigma colour, the degree of stigma exsertion, plant height, panicle length, the degree of shattering, awn length, grain number per panicle, grain length, grain width, grain weight per 1,000 grains, hull colour and pericarp colour. The degree of stigma exsertion was scored based on the observation of ~20 randomly sampled spikelets of each line, on a scale of 13 (no, incomplete or complete exertion). Seed germination rate was measured by using mature seeds which were placed in a plastic Petri dishes kept at 30C in the dark for 48h5. Other traits were phenotyped and scored as described previously21, 22, 29. <\/p>\n For the genotype data set in O. rufipogon, genotypes of 446 O. rufipogon accessions were called specifically at the ~5 million SNP sites that were polymorphic in the O. rufipogon population. In the panel for GWAS, only the SNPs that have a minor allele frequency (MAF) of more than 5% and contain genotype calls of more than 100 accessions were left for subsequent imputation. The k-nearest neighbour algorithm-based imputation method was used for inferring missing calls21. The specificity of the genotype data set before and after imputation was assessed using three sets of genome sequences (Supplementary Table 6). Association analysis was conducted using the compressed mixed linear model55. The top five principle components were used as fixed effects and the matrix of genetic distance was used to model the variancecovariance matrix of the random effect. Permutation tests were used to define the threshold of association signals of the GWAS in the wild rice population. A total of 20 permutation analyses were performed (10 independent permutation tests for each of the two traits, sheath colour and tiller angle), which resulted in two association signals with the thresholds we set53. Hence, there were an average of 0.1 false positives (that is, totally two false positives in 20 permutation tests) in a single whole-genome scanning analysis. Simulation tests were used to compare the performance of GWAS between the populations of cultivated and wild rice. <\/p>\n Genomic DNA of each line in the mapping population was sequenced on the Illumina Genome Analyzer IIx, each to approximately 0.5 coverage. Both parents of the population, Guangluai-4 and W1943, were sequenced with at least 20 genome coverage, in a previous work21 and in this study, respectively. SNP identification between parents was conducted as described previously41. Genotype calling, recombination breakpoint determination and bin map construction was performed using the software SEG-Map (http:\/\/www.ncgr.ac.cn\/software\/SEG\/<\/a>). QTL analysis of the fifteen traits was conducted with the composite interval mapping (CIM) method implemented in the software Windows QTL Cartographer56 (version 2.5) with a window size of 10cM and a step size of 2cM. QTL with LOD value higher than 3.5 were called, of which the location was described according to its LOD peak location. The phenotypic effect (r2) of each QTL was computed using Windows QTL Cartographer. QTLs located within selective sweep regions were further used to associate the selected regions with their biological functions. It needs to be noted that we adopted a stringent threshold in the QTL calling (LOD>3.5), and the genomic regions with LOD ranging from 2.5 to 3.5 may include many minor QTLs (the threshold was set to 2.5 in most studies). <\/p>\n The genome of W1943 was assembled by using a custom pipeline integrating Phusion2 (clustering the raw reads into different groups)57 and Phrap (then assembling all the reads in each group to generate contigs)58. The N50 length of the entire assembly was calculated for the initial contigs with small contigs of <200bp excluded. All the full-length complementary DNA sequences46 of W1943 were aligned with the final assembly of W1943 genome sequence using the software GMAP59 (version 6) with the parameters -K 15000 and -k 0.97. The resulting contigs from whole-genome de novo assembly were anchored to the rice reference genome sequence (IRGSP4.0) using the software MUMmer60 (version 3). <\/p>\n Gene models of the genome of the wild rice W1943 were predicted using the software Fgenesh that was set for a monocot model61 (version 2.0). The resulting proteome of W1943 was compared with protein sequences in Rice Genome Annotation Project (version 7.0) using BLASTP with a cutoff of a minimum of 95% identity. Sequence variants, including SNPs, indels and imbalanced substitutions, were called using the diffseq program in the EMBOSS package62. Indels of large size were called from the alignment results of MUMmer. Effects of the sequence variants were predicted according to the gene models of Nipponbare in the RAP-DB (release 2) across the rice genome. For indels in genic regions and SNPs with large effect around the domestication loci, the effects were mainly based on the reference gene models. <\/p>\n The sequence reads of 1,083 O. sativa accessions, 446 O. rufipogon accessions and 15 outgroup accessions were then aligned against assembled genome sequences of W1943 using the same parameters with those against the reference Nipponbare genome sequences. Genotypes of each accession were called at all sequence variant sites (including SNPs, indels and imbalanced substitutions that were detected from assembled sequences), based on the alignment outputs against the two genome sequences. The allele frequencies at the sequence variant sites were calculated for each clade of O. sativa and O. rufipogon. In each clade, variant sites with information of less than 10 accessions (less than 2 for the outgroups) were then excluded for computing allele frequencies, namely no data available. <\/p>\n<\/p>\n See more here: Sampling and sequencing The cultivated and wild rice accessions were all from large collections of rice accessions preserved at the China National Rice Research Institute in Hangzhou, China, and the National Institute of Genetics in Mishima, Japan. Continue reading
\nA map of rice genome variation reveals the origin of cultivated rice<\/a><\/p>\n","protected":false},"excerpt":{"rendered":"