Aiko Uemura

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.

Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops. DOI: http://dx.doi.org/10.7554/eLife.08709.001

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

The Draft Genome of Hop (Humulus lupulus), an Essence for Brewing

The female flower of hop (Humulus lupulus var. lupulus) is an essential ingredient that gives characteristic aroma, bitterness and durability/stability to beer. However, the molecular genetic basis for identifying DNA markers in hop for breeding and to study its domestication has been poorly established. Here, we provide draft genomes for two hop cultivars [cv. Saazer (SZ) and cv. Shinshu Wase (SW)] and a Japanese wild hop [H. lupulus var. cordifolius; also known as Karahanasou (KR)]. Sequencing and de novo assembly of genomic DNA from heterozygous SW plants generated scaffolds with a total size of 2.05 Gb, corresponding to approximately 80% of the estimated genome size of hop (2.57 Gb). The scaffolds contained 41,228 putative protein-encoding genes. The genome sequences for SZ and KR were constructed by aligning their short sequence reads to the SW reference genome and then replacing the nucleotides at single nucleotide polymorphism (SNP) sites. De novo RNA sequencing (RNA-Seq) analysis of SW revealed the developmental regulation of genes involved in specialized metabolic processes that impact taste and flavor in beer. Application of a novel bioinformatics tool, phylogenetic comparative RNA-Seq (PCP-Seq), which is based on read depth of genomic DNAs and RNAs, enabled the identification of genes related to the biosynthesis of aromas and flavors that are enriched in SW compared to KR. Our results not only suggest the significance of historical human selection process for enhancing aroma and bitterness biosyntheses in hop cultivars, but also serve as crucial information for breeding varieties with high quality and yield.

Colletotrichum orbiculare Secretes Virulence Effectors to a Biotrophic Interface at the Primary Hyphal Neck via Exocytosis Coupled with SEC22-Mediated Traffic

This work shows that the cucumber anthracnose fungus Colletotrichum orbiculare accumulates its virulence effectors in the plant–pathogen interfacial region around the neck of primary biotrophic hyphae developed in host cells. It also describes that the pathogen secretes the effectors toward the interfacial region via SEC22-dependent traffic and SEC4-mediated exocytosis. The hemibiotrophic pathogen Colletotrichum orbiculare develops biotrophic hyphae inside cucumber (Cucumis sativus) cells via appressorial penetration; later, the pathogen switches to necrotrophy. C. orbiculare also expresses specific effectors at different stages. Here, we found that virulence-related effectors of C. orbiculare accumulate in a pathogen–host biotrophic interface. Fluorescence-tagged effectors accumulated in a ring-like region around the neck of the biotrophic primary hyphae. Fluorescence imaging of cellular components and transmission electron microscopy showed that the ring-like signals of the effectors localized at the pathogen–plant interface. Effector accumulation at the interface required induction of its expression during the early biotrophic phase, suggesting that transcriptional regulation may link to effector localization. We also investigated the route of effector secretion to the interface. An exocytosis-related component, the Rab GTPase SEC4, localized to the necks of biotrophic primary hyphae adjacent to the interface, thereby suggesting focal effector secretion. Disruption of SEC4 in C. orbiculare reduced virulence and impaired effector delivery to the ring signal interface. Disruption of the v-SNARE SEC22 also reduced effector delivery. These findings suggest that biotrophy-expressed effectors are secreted, via the endoplasmic reticulum-to-Golgi route and subsequent exocytosis, toward the interface generated between C. orbiculare and the host cell.

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.

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.

Genome sequencing of the staple food crop white Guinea yam enables the development of a molecular marker for sex determination

BackgroundRoot and tuber crops are a major food source in tropical Africa. Among these crops are several species in the monocotyledonous genus Dioscorea collectively known as yam, a staple tuber crop that contributes enormously to the subsistence and socio-cultural lives of millions of people, principally in West and Central Africa. Yam cultivation is constrained by several factors, and yam can be considered a neglected “orphan” crop that would benefit from crop improvement efforts. However, the lack of genetic and genomic tools has impeded the improvement of this staple crop.ResultsTo accelerate marker-assisted breeding of yam, we performed genome analysis of white Guinea yam (Dioscorea rotundata) and assembled a 594-Mb genome, 76.4% of which was distributed among 21 linkage groups. In total, we predicted 26,198 genes. Phylogenetic analyses with 2381 conserved genes revealed that Dioscorea is a unique lineage of monocotyledons distinct from the Poales (rice), Arecales (palm), and Zingiberales (banana). The entire Dioscorea genus is characterized by the occurrence of separate male and female plants (dioecy), a feature that has limited efficient yam breeding. To infer the genetics of sex determination, we performed whole-genome resequencing of bulked segregants (quantitative trait locus sequencing [QTL-seq]) in F1 progeny segregating for male and female plants and identified a genomic region associated with female heterogametic (male = ZZ, female = ZW) sex determination. We further delineated the W locus and used it to develop a molecular marker for sex identification of Guinea yam plants at the seedling stage.ConclusionsGuinea yam belongs to a unique and highly differentiated clade of monocotyledons. The genome analyses and sex-linked marker development performed in this study should greatly accelerate marker-assisted breeding of Guinea yam. In addition, our QTL-seq approach can be utilized in genetic studies of other outcrossing crops and organisms with highly heterozygous genomes. Genomic analysis of orphan crops such as yam promotes efforts to improve food security and the sustainability of tropical agriculture.

A chloroplast-localized protein LESION AND LAMINA BENDING affects defence and growth responses in rice

Understanding how plants allocate their resources to growth or defence is of long-term importance to the development of new and improved varieties of different crops. Using molecular genetics, plant physiology, hormone analysis and Next-Generation Sequencing (NGS)-based transcript profiling, we have isolated and characterized the rice (Oryza sativa) LESION AND LAMINA BENDING (LLB) gene that encodes a chloroplast-targeted putative leucine carboxyl methyltransferase. Loss of LLB function results in reduced growth and yield, hypersensitive response (HR)-like lesions, accumulation of the antimicrobial compounds momilactones and phytocassanes, and constitutive expression of pathogenesis-related genes. Consistent with these defence-associated responses, llb shows enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae). The lesion and resistance phenotypes are likely to be caused by the over-accumulation of jasmonates (JAs) in the llb mutant including the JA precursor 12-oxo-phytodienoic acid. Additionally, llb shows an increased lamina inclination and enhanced early seedling growth due to elevated brassinosteroid (BR) synthesis and/or signalling. These findings show that LLB functions in the chloroplast to either directly or indirectly repress both JA- and BR-mediated responses, revealing a possible mechanism for controlling how plants allocate resources for defence and growth.