The genome of Chenopodium quinoa : Nature : Nature Research. No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Quinoa sequencing and assembly. We sequenced Chenopodium quinoa Willd. DNA was extracted from leaf and flower tissue of a single plant, as described in the “Preparing Arabidopsis Genomic DNA for Size- Selected ~2. SMRTbell Libraries” protocol (http: //www. Shared- Protocol- Preparing- Arabidopsis- DNA- for- 2. SMRTbell- Libraries. DNA was purified twice with Beckman Coulter Genomics AMPure XP magnetic beads and assessed by standard agarose gel electrophoresis and Thermo Fisher Scientific Qubit Fluorometry. Single- Molecule Real- Time (SMRT) cells were run on the Pac. Bio RS II system with the P6- C4 chemistry by DNALink (Seoul). De novo assembly was conducted using the smrtmake assembly pipeline (https: //github. Pacific. Biosciences/smrtmake) and the Celera Assembler, and the draft assembly was polished using the quiver algorithm. DNA was also sequenced using an Illumina Hi. Seq 2. 00. 0 machine. For this, DNA was extracted from leaf tissue of a single soil- grown plant using the Qiagen DNeasy Plant Mini Kit. PE) libraries were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina. Sequencing reads were processed with Trimmomatic (v. The remaining high- quality reads were assembled with Velvet (v. Integrating Bio. Nano optical maps with the Pac. Bio assembly. High- molecular- weight DNA was isolated and labelled from leaf tissue of three- week old quinoa plants according to standard Bio. Nano protocols, using the single- stranded nicking endonuclease Nt. The metabolic response of plants to oxygen deficiency. Carlos Antônio Ferreira de Sousa 1,* and Ladaslav Sodek 2. 1 Empresa Brasileira de Pesquisa Agropecuária. Bsp. QI. Labelled DNA was imaged automatically using the Bio. Nano Irys system and de novo assembled into consensus physical maps using the Bio. Nano Irys. View analysis software. The final de novo assembly used only single molecules with a minimum length of 1. Pac. Bio- Bio. Nano hybrid scaffolds were identified using Irys. View’s hybrid scaffold alignment subprogram. Chicago library preparation and sequencing. Using the same DNA prepared for Pac. Bio sequencing, a Chicago library was prepared as described previously. The library was sequenced on an Illumina Hi. Seq 2. 50. 0. Scaffolding the Pac. Bio and Bio. Nano assemblies with Hi. Rise. Chicago sequence data (in FASTQ format) was used to scaffold the Pac. Bio- Bio. Nano hybrid assembly using Hi. Rise, a software pipeline designed specifically for using Chicago data to assemble genomes. Chicago library sequences were aligned to the draft input assembly using a modified SNAP read mapper (http: //snap. The separations of Chicago read pairs mapped within draft scaffolds were analysed by Hi. Rise to produce a likelihood model, and the resulting likelihood model was used to identify putative mis- joins and score prospective joins. Kurmi . Homozygous high- and low- saponin F2 lines were identified by planting 1. F3 seeds derived from each F2 line, harvesting F4 seed from these F3 plants, and then performing foam tests on the F4 seed. Phenotyping was validated using gas chromatography/mass spectrometry (GC/MS). RNA was extracted from inflorescences containing a mixture of flowers and seeds at various stages of development from the parents and 4. F3 progeny. RNA extraction and Illumina sequencing were performed as described above. Sequencing reads from all lines were trimmed using Trimmomatic and mapped to the reference assembly using Top. Hat. 44, and SNPs were called using SAMtools mpileup (v. For linkage mapping, markers were assigned to linkage groups on the basis of the grouping by Join. Map v. 4. 1. Using the maximum likelihood algorithm of Join. Map, the order of the markers was determined; using this as start order and fixed order, regression mapping in Join. Map was used to determine the c. M distances. Genes differentially expressed between bitter and sweet lines and between green and red lines were identified using default parameters of the Cuffdiff function of the Cufflinks program. Atlas . Bitter and sweet F2 lines were identified by performing foam and taste tests on the F3 seed. Port Manteaux churns out silly new words when you feed it an idea or two. Enter a word (or two) above and you'll get back a bunch of portmanteaux created by jamming.
DNA sequencing was performed with DNA from the parents and 9. F2 lines, as described above, and sequencing reads were mapped to the reference assembly using BWA. SNPs were called in the parents and in a merged file containing all combined F2 lines. Genotype calls were generated for the 9. F2 genotypes by summing up read counts over a sliding window of 5. Over each 5. 00- variant stretch, all reads with Atlas alleles were summed, and all reads with the Carina Red allele were summed. Markers were assigned to linkage groups using Join. Map, with regression mapping used to obtain the genetic maps per linkage group. Integrated linkage map. The Kurmi . We selected markers from the same scaffold that were in the same 1. The anchor markers on the alternative map received the position of the Kurmi . This process was repeated with anchor markers at the 1. The assumption is that at the 1. On this level, a regression of c. M position on both maps yielded R2 values > 0. Kurmi . Based on these coordinates, custom scripts were used to generate the pseudomolecule assembly and to recoordinate the annotation file. Sequencing and assembly of C. The generated reads were trimmed using the quality- based trimming tool Sickle (https: //github. The trimmed reads were then assembled using the ALLPATHS- LG assembler. Gap. Closer v. 1. N spacers and gap lengths produced by the ALLPATHS- LG assembler. Genome annotation. Repeat families found in the genome assemblies of quinoa, C. Repeat. Masker. 50 was used to discover and identify repeats within the respective genomes. AUGUSTUS5. 1 was used for ab initio gene prediction, using model training based on coding sequences from Amaranthus hypochondriacus, Beta vulgaris, Spinacia oleracea and Arabidopsis thaliana. RNA- seq and isoform sequencing reads generated from RNA of different tissues were mapped onto the reference genome using Bowtie 2 (ref. GMAP5. 3, respectively. Hints with locations of potential intron–exon boundaries were generated from the alignment files with the software package BAM2hints in the MAKER package. MAKER with AUGUSTUS (intron–exon boundary hints provided from RNA- seq and isoform sequencing) was then used to predict genes in the repeat- masked reference genome. To help guide the prediction process, peptide sequences from B. Genes were characterized for their putative function by performing a BLAST search of the peptide sequences against the Uni. Prot database. PFAM domains and Inter. Pro. Scan ID were added to the gene models using the scripts provided in the MAKER package. Re- sequencing. The following quinoa accessions were chosen for DNA re- sequencing: 0. Ollague, Real, Pasankalla (BYU 1. Kurmi, CICA- 1. 7, Regalona (BYU 9. Salcedo INIA, G- 2. DK, Cherry Vanilla (BYU 1. Chucapaca, Ku- 2, PI 6. Ames 2. 21. 57), Atlas and Carina Red. The following accessions of C. Two accessions of C. All sequencing was performed with an Illumina Hi. Seq 2. 00. 0 machine, using either 1. Atlas and Carina Red) or 1. Reads were trimmed using Trimmomatic and mapped to the reference assembly using BWA (v. Read alignments were manipulated with SAMtools, and the mpileup function of SAMtools was used to call SNPs. Identification of orthologous genes. Orthologous and paralogous gene clusters were identified using Ortho. MCL2. 8. Recommended settings were used for all- against- all BLASTP comparisons (Blast+ v. Ortho. MCL analyses. Custom Perl scripts were used to process Ortho. MCL outputs for visualization with Interacti. Venn. 57. Phylogenetic inference. Using Ortho. MCL, orthologous gene sets containing two copies in quinoa and one copy each in C. In total, 7,4. 33 gene sets were chosen, and their amino acid sequences were aligned individually for each set using MAFFT5. The 7,4. 33 alignments were converted into PHYLIP format files by the seqret command in the EMBOSS package. Individual gene trees were then constructed using the maximum likelihood method using proml in PHYLIP6. In addition, the genomic variants of all 2. Supplementary Data 5) relative to the reference sequence were called based on the mapped Illumina reads in 2. BAM files using SAMtools. To call variants in the reference genome (PI 6. Illumina sequencing reads were mapped to the reference assembly. Variants were then filtered using VCFtools. SAMtools, and the qualified SNPs were combined into a single VCF file which was used as an input into SNPhylo. To identify FT homologues, the protein sequence from the A. Filtering for hits with an E value < 1 . One quinoa protein (AUR6. For the construction of the phylogenetic tree, protein sequences from these five quinoa FT homologues were aligned using Clustal Omega. B. Phylogenetic analysis was performed with MEGA6. The JTT model was selected as the best fitting model. The initial phylogenetic tree was estimated using the neighbour joining method (bootstrap value = 5. Gaps/ Missing Data Treatment = Partial Deletion, Cutoff 9. The syntenic relationships between the coding sequences of the chromosomal regions surrounding these FT genes were visualized using the Co. GE6. 5 GEvo tool and the Multi- Genome Synteny Viewer. The alignment of b. HLH domains was performed with Clustal Omega. Mertens et al. 3. The phylogeny was inferred using the maximum likelihood method based on the JTT matrix- based model. Initial trees for the heuristic search were obtained automatically by applying Neighbour- Join and Bio. NJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. All positions containing gaps and missing data were eliminated. Distinguishing and analysing the quinoa sub- genomes. Trimmed PE Illumina sequencing reads that were used for the de novo assembly of C. For every base in the quinoa genome, the depth coverage of properly paired reads from the C.
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