Hiroki Takagi

Genome sequencing reveals agronomically important loci in rice using MutMap

The majority of agronomic traits are controlled by multiple genes that cause minor phenotypic effects, making the identification of these genes difficult. Here we introduce MutMap, a method based on whole-genome resequencing of pooled DNA from a segregating population of plants that show a useful phenotype. In MutMap, a mutant is crossed directly to the original wild-type line and then selfed, allowing unequivocal segregation in second filial generation (F2) progeny of subtle phenotypic differences. This approach is particularly amenable to crop species because it minimizes the number of genetic crosses (n = 1 or 0) and mutant F2 progeny that are required. We applied MutMap to seven mutants of a Japanese elite rice cultivar and identified the unique genomic positions most probable to harbor mutations causing pale green leaves and semidwarfism, an agronomically relevant trait. These results show that MutMap can accelerate the genetic improvement of rice and other crop plants.

QTL‐seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations

The majority of agronomically important crop traits are quantitative, meaning that they are controlled by multiple genes each with a small effect (quantitative trait loci, QTLs). Mapping and isolation of QTLs is important for efficient crop breeding by marker-assisted selection (MAS) and for a better understanding of the molecular mechanisms underlying the traits. However, since it requires the development and selection of DNA markers for linkage analysis, QTL analysis has been time-consuming and labor-intensive. Here we report the rapid identification of plant QTLs by whole-genome resequencing of DNAs from two populations each composed of 20-50 individuals showing extreme opposite trait values for a given phenotype in a segregating progeny. We propose to name this approach QTL-seq as applied to plant species. We applied QTL-seq to rice recombinant inbred lines and F2 populations and successfully identified QTLs for important agronomic traits, such as partial resistance to the fungal rice blast disease and seedling vigor. Simulation study showed that QTL-seq is able to detect QTLs over wide ranges of experimental variables, and the method can be generally applied in population genomics studies to rapidly identify genomic regions that underwent artificial or natural selective sweeps.

MutMap+: Genetic Mapping and Mutant Identification without Crossing in Rice

Advances in genome sequencing technologies have enabled researchers and breeders to rapidly associate phenotypic variation to genome sequence differences. We recently took advantage of next-generation sequencing technology to develop MutMap, a method that allows rapid identification of causal nucleotide changes of rice mutants by whole genome resequencing of pooled DNA of mutant F2 progeny derived from crosses made between candidate mutants and the parental line. Here we describe MutMap+, a versatile extension of MutMap, that identifies causal mutations by comparing SNP frequencies of bulked DNA of mutant and wild-type progeny of M3 generation derived from selfing of an M2 heterozygous individual. Notably, MutMap+ does not necessitate artificial crossing between mutants and the wild-type parental line. This method is therefore suitable for identifying mutations that cause early development lethality, sterility, or generally hamper crossing. Furthermore, MutMap+ is potentially useful for gene isolation in crops that are recalcitrant to artificial crosses.

Light-induced increase in free Mg2+ concentration in spinach chloroplasts: measurement of free Mg2+ by using a fluorescent probe and necessity of stromal alkalinization.

Free Mg(2+) in chloroplasts may contribute to the regulation of photosynthetic enzymes, but adequate methodology for the determination of free Mg(2+) concentration ([Mg(2+)]) in chloroplasts has been lacking. We measured internal chloroplast [Mg(2+)] by using a Mg-sensitive fluorescent indicator, mag-fura-2. In intact, dark-kept spinach chloroplasts, internal [Mg(2+)] was estimated to be 0.50 mM, and illumination caused an increase in [Mg(2+)] to 2.0mM in the stroma. The light-induced increase in [Mg(2+)] was inhibited by a blocker of driven electron transport and uncouplers. The K(+)-specific ionophore valinomycin inhibited the [Mg(2+)] increase in the absence of external K(+), and addition of KCl restored the [Mg(2+)] increase. NH(4)Cl, which induces stromal alkalinization, enhanced the [Mg(2+)] increase. A Ca(2+)-channel blocker, ruthenium red, inhibited the [Mg(2+)] increase, but LaCl(3) had no effect. These results indicate that stromal alkalinization is essential for light-induced increase in [Mg(2+)]. This system for measuring internal chloroplast [Mg(2+)] might provide a suitable system for assay of Mg(2+) transport activity of chloroplast membranes.

MutMap accelerates breeding of a salt-tolerant rice cultivar.

445 two chromosome substitution lines (CSSLs), SL502 and SL503, harboring chromosomal segments of Nona Bokra in the genetic background of Koshihikari, a cultivar closely related to Hitomebore10,11. We compared the growth of hst1, SL502 and SL503 at 0.75% NaCl (Supplementary Fig. 2). The 18 and 43% improved growth of hst1 as compared to SL503 and SL502, respectively, showed that this was a good starting point for breeding a salt-tolerant rice cultivar. For rapid identification of the mutation conferring salinity tolerance in hst1, we used MutMap, a method based on wholegenome resequencing of bulked DNA of F2 segregants1. The hst1 line was crossed to WT Hitomebore to generate F1 progeny, and F2 progeny were derived from self-pollination of the F1 progeny. Two-week-old F2 seedlings were treated with water containing 0.75% NaCl. The progeny segregated in a 133:54 ratio for salinity-susceptible and salinitytolerant phenotypes, respectively, conforming to a 3:1 segregation ratio (chi-squared test: χ2 = 2.2 × 10-16, nonsignificant) and thereby indicating that the salinity tolerance of hst1 is conferred by a single recessive mutation. We combined DNA from 20 F2 progeny that had the salinity-tolerance phenotype and applied whole-genome resequencing using an Illumina GAIIx DNA sequencer. We obtained a total of 7.34 Gbp of short (75-bp) reads (Supplementary Table 1) that were aligned to the Hitomebore reference sequence (DDBJ Sequence Read Archive DRA000927), resulting in the identification of 1,005 SNP positions. For each SNP position, the value of SNP-index (the ratio of short reads harboring SNPs different from the reference1) was obtained and a graph relating SNP positions and SNP-index was generated for all 12 rice chromosomes (Fig. 2a, Supplementary Fig. 3). The causative SNP should be shared by all the mutant F2 plants and therefore have a SNP-index = 1, whereas SNPs unrelated to the mutant phenotype should segregate in a 1:1 ratio among the F2 progeny, resulting in a SNP-index of ~0.5. MutMap applied to hst1 To the Editor: Following the 2011 earthquake and tsunami that affected Japan, >20,000 ha of rice paddy field was inundated with seawater, resulting in salt contamination of the land. As local rice landraces are not tolerant of high salt concentrations, we set out to develop a salttolerant rice cultivar. We screened 6,000 ethyl methanesulfonate (EMS) mutant lines of a local elite cultivar, ‘Hitomebore’, and identified a salt-tolerant mutant that we name hitomebore salt tolerant 1 (hst1). In this Correspondence, we report how we used our MutMap method1 to rapidly identify a loss-of-function mutation responsible for the salt tolerance of hst1 rice. The salttolerant hst1 mutant was used to breed a salt-tolerant variety named ‘Kaijin’, which differs from Hitomebore by only 201 singlenucleotide polymorphisms (SNPs). Field trials showed that it has the same growth and yield performance as the parental line under normal growth conditions. Notably, production of this salt-tolerant mutant line ready for delivery to farmers took only two years using our approach. Although soluble salts, such as nitrates and potassium salts, are common components of soil and essential plant nutrients, their accumulation above specific threshold concentrations can substantially affect plant growth. There is considerable variation among plants with respect to their tolerance of salinity, and rice is considered the most sensitive of all the cereals2. Yields of paddy rice start to decline at salinity levels >3 dS m–1 (measured by the electrical conductivity of the extract, ECe), beyond which a 12% reduction in yield is expected for every 1 dS m–1 increase in ECe. Soil salinity affects >6% of world’s total land area, causing yield losses as a result of both osmotic and ionic stresses to crop plants2. Soil salinization due to the flooding of agricultural lands by seawater has become an additional concern since the 2004 Indian Ocean tsunami4. In 2011, Japan was hit by the Great Tohoku Earthquake, which triggered a devastating tsunami, altogether claiming the lives of more than 15,000 people. The tsunami extended more than 5 km inland on the Sendai Plain of Miyagi Prefecture, one of the main rice-production regions in Japan5. An environmental impact assessment study conducted in the same area over a period of 2–7 months after the tsunami revealed wide spatial variation in the salinity level of ponded water, with ECe ranging from 0.31 to 68.2 mS cm–1 (ref. 6). Although salt concentration gradually decreased, it was too high for rice production to resume in October, 2011. To restore rice production in tsunamiaffected areas of the Tohoku region of Japan, we set out to develop and deliver a salt-tolerant rice cultivar from a line suited to local agronomic conditions. First, we carried out a genetic screen for salt tolerance using seeds pooled from approximately 6,000 independent EMS-mutagenized lines of Hitomebore7, (Supplementary Fig. 1). We identified a mutant that survived with 1.5% NaCl supplied to the soil with irrigation water for 7 days, which we designated hitomebore salt tolerant 1 (hst1). Seeds from a self-pollinated hst1 plant were used to further test the performance of hst1 at different NaCl concentrations (Fig. 1a,b). The hst1 mutant grew better than wild-type (WT) Hitomebore plants at both 0.375% and 0.75% NaCl concentrations, as measured after 14 days of treatment. The 0.375% NaCl treatment caused reductions of 38.4% and 2.9% in the fresh weight of WT and hst1 plants, respectively. At 0.75% NaCl, WT plants dried out, with a 61.5% reduction in fresh weight, whereas hst1 plants remained green with only a 13.2% reduction in fresh weight compared with hst1 plants that received fresh water (Fig. 1b). Previously, the rice SHOOT K+ CONCENTRATION 1 (SKC1) gene, encoding a Na+ transporter, was identified as the main quantitative trait locus (QTL) conferring salt tolerance in the indica cultivar Nona Bokra8,9. This QTL has been used to develop MutMap accelerates breeding of a salt-tolerant rice cultivar CORRESPONDENCE

QTL-seq for rapid identification of candidate genes for 100-seed weight and root/total plant dry weight ratio under rainfed conditions in chickpea

Summary Terminal drought is a major constraint to chickpea productivity. Two component traits responsible for reduction in yield under drought stress include reduction in seeds size and root length/root density. QTL‐seq approach, therefore, was used to identify candidate genomic regions for 100‐seed weight (100SDW) and total dry root weight to total plant dry weight ratio (RTR) under rainfed conditions. Genomewide SNP profiling of extreme phenotypic bulks from the ICC 4958 × ICC 1882 population identified two significant genomic regions, one on CaLG01 (1.08 Mb) and another on CaLG04 (2.7 Mb) linkage groups for 100SDW. Similarly, one significant genomic region on CaLG04 (1.10 Mb) was identified for RTR. Comprehensive analysis revealed four and five putative candidate genes associated with 100SDW and RTR, respectively. Subsequently, two genes (Ca_04364 and Ca_04607) for 100SDW and one gene (Ca_04586) for RTR were validated using CAPS/dCAPS markers. Identified candidate genomic regions and genes may be useful for molecular breeding for chickpea improvement.

A Stress-Activated Transposon in Arabidopsis Induces Transgenerational Abscisic Acid Insensitivity.

Transposable elements (TEs), or transposons, play an important role in adaptation. TE insertion can affect host gene function and provides a mechanism for rapid increases in genetic diversity, particularly because many TEs respond to environmental stress. In the current study, we show that the transposition of a heat-activated retrotransposon, ONSEN, generated a mutation in an abscisic acid (ABA) responsive gene, resulting in an ABA-insensitive phenotype in Arabidopsis, suggesting stress tolerance. Our results provide direct evidence that a transposon activated by environmental stress could alter the genome in a potentially positive manner. Furthermore, the ABA-insensitive phenotype was inherited when the transcription was disrupted by an ONSEN insertion, whereas ABA sensitivity was recovered when the effects of ONSEN were masked by IBM2. These results suggest that epigenetic mechanisms in host plants typically buffered the effect of a new insertion, but could selectively “turn on” TEs when stressed.

A cytochrome P450, OsDSS1, is involved in growth and drought stress responses in rice (Oryza sativa L.)

Cytochrome P450s are among the largest protein coding gene families in plant genomes. However, majority of the genes remain uncharacterized. Here, we report the characterization of dss1, a rice mutant showing dwarfism and reduced grain size. The dss1 phenotype is caused by a non-synonymous point mutation we identified in DSS1, which is member of a P450 gene cluster located on rice chromosome 3 and corresponds to the previously reported CYP96B4/SD37 gene. Phenotypes of several dwarf mutants characterized in rice are associated with defects in the biosynthesis or perception of the phytohormones gibberellins (GAs) and brassinosteroids (BRs). However, both GA and BR failed to rescue the dss1 phenotype. Hormone profiling revealed the accumulation of abscisic acid (ABA) and ABA metabolites, as well as significant reductions in GA19 and GA53 levels, precursors of the bioactive GA1, in the mutant. The dss1 contents of cytokinin and auxins were not significantly different from wild-type plants. Consistent with the accumulation of ABA and metabolites, germination and early growth was delayed in dss1, which also exhibited an enhanced tolerance to drought. Additionally, expressions of members of the DSS1/CYP96B gene cluster were regulated by drought stress and exogenous ABA. RNA-seq-based transcriptome profiling revealed, among others, that cell wall-related genes and genes involved in lipid metabolism were up- and down-regulated in dss1, respectively. Taken together, these findings suggest that DSS1 mediates growth and stress responses in rice by fine-tuning GA-to-ABA balance, and might as well play a role in lipid metabolism.

Transcriptional and Post-transcriptional Mechanisms Limit Heading Date 1 (Hd1) Function to Adapt Rice to High Latitudes

Rice flowering is controlled by changes in the photoperiod that promote the transition to the reproductive phase as days become shorter. Natural genetic variation for flowering time has been largely documented and has been instrumental to define the genetics of the photoperiodic pathway, as well as providing valuable material for artificial selection of varieties better adapted to local environments. We mined genetic variation in a collection of rice varieties highly adapted to European regions and isolated distinct variants of the long day repressor HEADING DATE 1 (Hd1) that perturb its expression or protein function. Specific variants allowed us to define novel features of the photoperiodic flowering pathway. We demonstrate that a histone fold domain scaffold formed by GRAIN YIELD, PLANT HEIGHT AND HEADING DATE 8 (Ghd8) and several NF-YC subunits can accommodate distinct proteins, including Hd1 and PSEUDO RESPONSE REGULATOR 37 (PRR37), and that the resulting OsNF-Y complex containing Hd1 can bind a specific sequence in the promoter of HEADING DATE 3A (Hd3a). Artificial selection has locally favored an Hd1 variant unable to assemble in such heterotrimeric complex. The causal polymorphism was defined as a single conserved lysine in the CCT domain of the Hd1 protein. Our results indicate how genetic variation can be stratified and explored at multiple levels, and how its description can contribute to the molecular understanding of basic developmental processes.

Draft genome sequence of bitter gourd (Momordica charantia), a vegetable and medicinal plant in tropical and subtropical regions

Abstract Bitter gourd (Momordica charantia) is an important vegetable and medicinal plant in tropical and subtropical regions globally. In this study, the draft genome sequence of a monoecious bitter gourd inbred line, OHB3-1, was analyzed. Through Illumina sequencing and de novo assembly, scaffolds of 285.5 Mb in length were generated, corresponding to ∼84% of the estimated genome size of bitter gourd (339 Mb). In this draft genome sequence, 45,859 protein-coding gene loci were identified, and transposable elements accounted for 15.3% of the whole genome. According to synteny mapping and phylogenetic analysis of conserved genes, bitter gourd was more related to watermelon (Citrullus lanatus) than to cucumber (Cucumis sativus) or melon (C. melo). Using RAD-seq analysis, 1507 marker loci were genotyped in an F2 progeny of two bitter gourd lines, resulting in an improved linkage map, comprising 11 linkage groups. By anchoring RAD tag markers, 255 scaffolds were assigned to the linkage map. Comparative analysis of genome sequences and predicted genes determined that putative trypsin-inhibitor and ribosome-inactivating genes were distinctive in the bitter gourd genome. These genes could characterize the bitter gourd as a medicinal plant.