Discovery of Genome-Wide DNA Polymorphisms in a Landrace Cultivar of Japonica Rice by Whole-Genome Sequencing

Molecular breeding approaches are of growing importance to crop improvement. However, closely related cultivars generally used for crossing material lack sufficient known DNA polymorphisms due to their genetic relatedness. Next-generation sequencing allows the identification of a massive number of DNA polymorphisms such as single nucleotide polymorphisms (SNPs) and insertions–deletions (InDels) between highly homologous genomes. Using this technology, we performed whole-genome sequencing of a landrace of japonica rice, Omachi, which is used for sake brewing and is an important source for modern cultivars. A total of 229 million reads, each comprising 75 nucleotides of the Omachi genome, was generated with 45-fold coverage and uniquely mapped to 89.7% of the Nipponbare genome, a closely related cultivar. We identified 132,462 SNPs, 16,448 insertions and 19,318 deletions between the Omachi and Nipponbare genomes. An SNP array was designed to validate 731 selected SNPs, resulting in validation rates of 95 and 88% for the Omachi and Nipponbare genomes, respectively. Among the 577 SNPs validated in both genomes, 532 are entirely new SNP markers not previously reported between related rice cultivars. We also validated InDels on a part of chromosome 2 as DNA markers and successfully genotyped five japonica rice cultivars. Our results present the methodology and extensive data on SNPs and InDels available for whole-genome genotyping and marker-assisted breeding. The polymorphism information between Omachi and Nipponbare is available at NGRC_Rice_Omachi (http://www.nodai-genome.org/oryza_sativa_en.html)

The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay

<p>Abstract</p> <p>Background</p> <p>SPO11 is a key protein for promoting meiotic recombination, by generating chromatin locus- and timing-specific DNA double-strand breaks (DSBs). The DSB activity of SPO11 was shown by genetic analyses, but whether SPO11 exerts DSB-forming activity by itself is still an unanswered question. DSB formation by SPO11 has not been detected by biochemical means, probably because of a lack of proper protein-folding, posttranslational modifications, and/or specific SPO11-interacting proteins required for this activity. In addition, plants have multiple SPO11-homologues.</p> <p>Results</p> <p>To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a <it>Drosophila </it>bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of <it>Arabidopsis </it>AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in <it>Arabidopsis</it>, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells.</p> <p>Conclusions</p> <p>The <it>Drosophila </it>bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function.</p

Guanosine tetra- and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline

Chloroplasts possess bacterial-type systems for transcription and translation. On the basis of the identification of a Chlamydomonas reinhardtii gene encoding a RelA-SpoT homolog (RSH) that catalyzes the synthesis of guanosine tetra- or pentaphosphate [(p)ppGpp], we have previously suggested the operation of stringent control in the chloroplast genetic system. Although RSH genes have also been identified in several higher plants, the activities of the encoded enzymes and their mode of action in chloroplasts have remained uncharacterized. We have now characterized the intrinsic (p)ppGpp synthase activity of chloroplast extracts prepared from pea (Pisum sativum). Fractionation by ultracentrifugation suggested that the (p)ppGpp synthase activity of a translationally active chloroplast stromal extract was associated with 70S ribosomes. Furthermore, this enzymatic activity was inhibited by tetracycline, as was the peptide elongation activity of the extract. Structural comparisons between rRNA molecules of Escherichia coli and pea chloroplasts revealed the conservation of putative tetracycline-binding sites. These observations demonstrate the presence of a ribosome-associated (p)ppGpp synthase activity in the chloroplasts of a higher plant, further implicating (p)ppGpp in a genetic system of chloroplasts similar to that operative in bacteria

Genome-wide DNA polymorphisms in seven rice cultivars of temperate and tropical japonica groups.

Elucidation of the rice genome is expected to broaden our understanding of genes related to the agronomic characteristics and the genetic relationship among cultivars. In this study, we conducted whole-genome sequencings of 6 cultivars, including 5 temperate japonica cultivars and 1 tropical japonica cultivar (Moroberekan), by using next-generation sequencing (NGS) with Nipponbare genome as a reference. The temperate japonica cultivars contained 2 sake brewing (Yamadanishiki and Gohyakumangoku), 1 landrace (Kameji), and 2 modern cultivars (Koshihikari and Norin 8). Almost >83% of the whole genome sequences of the Nipponbare genome could be covered by sequenced short-reads of each cultivar, including Omachi, which has previously been reported to be a temperate japonica cultivar. Numerous single nucleotide polymorphisms (SNPs), insertions, and deletions were detected among the various cultivars and the Nipponbare genomes. Comparison of SNPs detected in each cultivar suggested that Moroberekan had 5-fold more SNPs than the temperate japonica cultivars. Success of the 2 approaches to improve the efficacy of sequence data by using NGS revealed that sequencing depth was directly related to sequencing coverage of coding DNA sequences: in excess of 30× genome sequencing was required to cover approximately 80% of the genes in the rice genome. Further, the contigs prepared using the assembly of unmapped reads could increase the value of NGS short-reads and, consequently, cover previously unavailable sequences. These approaches facilitated the identification of new genes in coding DNA sequences and the increase of mapping efficiency in different regions. The DNA polymorphism information between the 7 cultivars and Nipponbare are available at NGRC_Rices_Build1.0 (http://www.nodai-genome.org/oryza_sativa_en.html)

Structure-Based in Vitro Engineering of the Anthranilate Synthase, a Metabolic Key Enzyme in the Plant Tryptophan Pathway

Rice (Oryza sativa) anthranilate synthase α-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions