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Germination involves highly dynamic transcriptional programs as the cells of seeds reactivate and express the functions necessary to establish in the environment. Individual cell types have distinct roles within the embryo, so must therefore have cell-type specific gene expression and gene regulatory networks. We can better understand how the functions of different cell types are established and contribute to the embryo by determining how cell-type specific transcription begins and changes through germination. Here we describe a temporal analysis of the germinating Arabidopsis embryo at single-cell resolution. We define the highly dynamic cell-type specific patterns of gene expression and how these relate to changing cellular function as germination progresses. Underlying these are unique gene regulatory networks and transcription factor activity. We unexpectedly discover that most embryo cells transition through the same initial transcriptional state early in germination, after which cell-type specific gene expression is established. Furthermore, our analyses support previous findings that the earliest events leading to the induction of embryo growth take place in the vasculature. Overall, our study constitutes a general framework to characterise Arabidopsis cell states through embryo growth, allowing investigation of different genotypes and other plant species whose seed strategies may differ.

Indigenous crops also known as orphan crops are key contributors to food security, which is becoming increasingly vulnerable with the current trend of population growth and climate change. They have the major advantage that they fit well into the general socio-economic and ecological context of developing world agriculture. However, most indigenous crops did not benefit from the Green Revolution, which dramatically increased the yield of major crops such as wheat and rice. Here, we describe the Tef Improvement Project, which employs both conventional- and molecular-breeding techniques to improve tef—an orphan crop important to the food security in the Horn of Africa, a region of the world with recurring devastating famines. We have established an efficient pipeline to bring improved tef lines from the laboratory to the farmers of Ethiopia. Of critical importance to the long-term success of this project is the cooperation among participants in Ethiopia and Switzerland, including donors, policy makers, research institutions, and farmers. Together, European and African scientists have developed a pipeline using breeding and genomic tools to improve the orphan crop tef and bring new cultivars to the farmers in Ethiopia. We highlight a new variety, Tesfa, developed in this pipeline and possessing a novel and desirable combination of traits. Tesfa’s recent approval for release illustrates the success of the project and marks a milestone as it is the first variety (of many in the pipeline) to be released., Euphytica, 214 (2), ISSN:0014-2336, ISSN:1573-5060

Summary Cannabis (Cannabis sativa L.) is one of the oldest cultivated plants purported to have unique medicinal properties. However, scientific research of cannabis has been restricted by the Single Convention on Narcotic Drugs of 1961, an international treaty that prohibits the production and supply of narcotic drugs except under license. Legislation governing cannabis cultivation for research, medicinal and even recreational purposes has been relaxed recently in certain jurisdictions. As a result, there is now potential to accelerate cultivar development of this multi‐use and potentially medically useful plant species by application of modern genomics technologies. Whilst genomics has been pivotal to our understanding of the basic biology and molecular mechanisms controlling key traits in several crop species, much work is needed for cannabis. In this review we provide a comprehensive summary of key cannabis genomics resources and their applications. We also discuss prospective applications of existing and emerging genomics technologies for accelerating the genetic improvement of cannabis.

Advances in next generation sequencing (NGS)-based methodologies have accelerated the identifications of simple genetic variants such as point mutations and small insertions/deletions (InDels). Structural variants (SVs) including large InDels and rearrangements provide vital sources of genetic diversity for plant breeding. However, their analysis remains a challenge due to their complex nature. Consequently, novel NGS-based approaches are needed to rapidly and accurately identify SVs. Here, we present an NGS-based bulked-segregant analysis (BSA) technique called Sat-BSA (SVs associated with traits) for identifying SVs controlling traits of interest in crops. Sat-BSA targets allele frequencies at all SNP positions to first identify candidate genomic regions associated with a trait, which is then reconstructed by long reads-based local de novo assembly. Finally, the association between SVs, RNA-seq-based gene expression patterns and trait is evaluated for multiple cultivars to narrow down the candidate genes. We applied Sat-BSA to segregating F2 progeny obtained from crosses between turnip cultivars with different tuber colors and successfully isolated two genes harboring SVs that are responsible for tuber phenotypes. The current study demonstrates the utility of Sat-BSA for the identification of SVs associated with traits of interest in species with large and heterozygous genomes.

Significance Guinea yam is an important staple tuber crop in West Africa, where it contributes to the sustenance and sociocultural lives of millions of people. Understanding the genetic diversity of Guinea yam and its relationships with wild relatives is important for improving this important crop using genomic information. A recent genomics study proposed that Guinea yam originated from a wild relative, the rainforest species Dioscorea praehensilis . Our results based on sequencing of 336 Guinea yam accessions do not support this notion; rather, our results indicate a hybrid origin of Dioscorea rotundata from crosses between the savannah species Dioscorea abyssinica and D. praehensilis.

Opium poppy (Papaver somniferum) is one of the world’s oldest medicinal plants and a versatile model system to study secondary metabolism. However, our knowledge of its genetic diversity is limited, restricting utilization of the available germplasm for research and crop improvement. We used genotyping-by-sequencing to investigate the extent of genetic diversity and population structure in a collection of poppy germplasm consisting of 91 accessions originating in 30 countries of Europe, North Africa, America, and Asia. We identified five genetically distinct subpopulations using discriminate analysis of principal components and STRUCTURE analysis. Most accessions obtained from the same country were grouped together within subpopulations, likely a consequence of the restriction on movement of poppy germplasm. Alkaloid profiles of accessions were highly diverse, with morphine being dominant. Phylogenetic analysis identified genetic groups that were largely consistent with the subpopulations detected and that could be differentiated broadly based on traits such as number of branches and seed weight. These accessions and the associated genotypic data are valuable resources for further genetic diversity analysis, which could include definition of poppy core sets to facilitate genebank management and use of the diversity for genetic improvement of this valuable crop.

Yam is a collective name of tuber crops belonging to the genus Dioscorea. Yam is important not only as a staple food crop but also as an integral component of society and culture of the millions of people who depend on it. However, due to its regional importance, yam has long been regarded as an “orphan crop” lacking a due global attention. Although this perception is changing with recent advances in genomics technologies, domestication processes of most yam species are still ambiguous. This is mainly due to the complicated evolutionary history of Dioscorea species caused by frequent hybridization and polyploidization, which is possibly caused by dioecy that imposed obligate outcrossing to the species of Dioscorea. In this chapter, we provide an overview of the evolution of Dioscorea and address the domestication of yam from population genomics perspectives by focusing on the processes of hybridization and polyploidization. A review is given to the recent population genomics studies on the hybrid origin of D. rotundata in West and Central Africa, the global dispersion of D. alata through human migrations, and the whole-genome duplication of the South America species of D. trifida. In the end, we give a summary of current understanding of sex-determination system in Dioscorea.

Significance Guinea yam is an important staple tuber crop in West Africa, where it contributes to the sustenance and sociocultural lives of millions of people. Understanding the genetic diversity of Guinea yam and its relationships with wild relatives is important for improving this important crop using genomic information. A recent genomics study proposed that Guinea yam originated from a wild relative, the rainforest species Dioscorea praehensilis. Our results based on sequencing of 336 Guinea yam accessions do not support this notion; rather, our results indicate a hybrid origin of Dioscorea rotundata from crosses between the savannah species Dioscorea abyssinica and D. praehensilis., White Guinea yam (Dioscorea rotundata) is an important staple tuber crop in West Africa. However, its origin remains unclear. In this study, we resequenced 336 accessions of white Guinea yam and compared them with the sequences of wild Dioscorea species using an improved reference genome sequence of D. rotundata. In contrast to a previous study suggesting that D. rotundata originated from a subgroup of Dioscorea praehensilis, our results suggest a hybrid origin of white Guinea yam from crosses between the wild rainforest species D. praehensilis and the savannah-adapted species Dioscorea abyssinica. We identified a greater genomic contribution from D. abyssinica in the sex chromosome of Guinea yam and extensive introgression around the SWEETIE gene. Our findings point to a complex domestication scenario for Guinea yam and highlight the importance of wild species as gene donors for improving this crop through molecular breeding.

White Guinea yam (Dioscorea rotundata) is an important staple tuber crop of West Africa. However, its origin remains unclear. In this study, we re-sequenced 336 accessions of white Guinea yam and compared them with the sequences of the wildDioscoreaspecies using an improved reference genome sequence ofD. rotundata. Our results suggest a hybrid origin of white Guinea yam from crosses between the rainforest wild speciesD. praehensilisand the savannah-adaptedD. abyssinica. We identified a higher genomic contribution fromD. abyssinicain the sex chromosome of Guinea yam and an extensive introgression around theSWEETIEgene. Our findings point to a complex domestication scenario for Guinea yam and highlight the importance of wild species as gene donors for improvement of this crop through molecular breeding.

© 2018 The Author(s). Background: The genome-wide expression profile of genes in different tissues/cell types and developmental stages is a vital component of many functional genomic studies. Transcriptome data obtained by RNA-sequencing (RNA-Seq) is often deposited in public databases that are made available via data portals. Data visualization is one of the first steps in assessment and hypothesis generation. However, these databases do not typically include visualization tools and establishing one is not trivial for users who are not computational experts. This, as well as the various formats in which data is commonly deposited, makes the processes of data access, sharing and utility more difficult. Our goal was to provide a simple and user-friendly repository that meets these needs for data-sets from major agricultural crops. Description: AgriSeqDB ( https://expression.latrobe.edu.au/agriseqdb ) is a database for viewing, analysing and interpreting developmental and tissue/cell-specific transcriptome data from several species, including major agricultural crops such as wheat, rice, maize, barley and tomato. The disparate manner in which public transcriptome data is often warehoused and the challenge of visualizing raw data are both major hurdles to data reuse. The popular eFP browser does an excellent job of presenting transcriptome data in an easily interpretable view, but previous implementation has been mostly on a case-by-case basis. Here we present an integrated visualisation database of transcriptome data-sets from six species that did not previously have public-facing visualisations. We combine the eFP browser, for gene-by-gene investigation, with the Degust browser, which enables visualisation of all transcripts across multiple samples. The two visualisation interfaces launch from the same point, enabling users to easily switch between analysis modes. The tools allow users, even those without bioinformatics expertise, to mine into data-sets and understand the behaviour of transcripts of interest across samples and time. We have also incorporated an additional graphic download option to simplify incorporation into presentations or publications. Conclusion: Powered by eFP and Degust browsers, AgriSeqDB is a quick and easy-to-use platform for data analysis and visualization in five crops and Arabidopsis. Furthermore, it provides a tool that makes it easy for researchers to share their data-sets, promoting research collaborations and data-set reuse.

Contents Summary 540 I. Introduction 540 II. There are different types of sRNA mobility 541 III. Mechanisms of sRNA movement 541 IV. Long-distance, shoot-root, mobile siRNAs influence DNA methylation in recipient tissues 541 V. Classes of interactions between shoot-root mobile siRNAs and DNA methylation 542 VI. Loci targeted directly and indirectly by shoot-root mobile siRNAs are associated with different histone modifications 543 VII. Is mobile siRNA-regulated DNA methylation important in specific tissues or under specific conditions? 543 VIII. Mobile sRNAs can be used to modify plant traits 544 IX. Conclusions 544 Acknowledgements 544 References 544 SUMMARY: RNA-directed DNA methylation (RdDM) at cytosine residues regulates gene expression, silences transposable elements and influences genome stability. The mechanisms responsible for RdDM are guided to target loci by small RNAs (sRNAs) that can move within plants cell to cell and long distance. Here we discuss recent advances in the understanding of interactions between mobile sRNAs and DNA methylation. We describe the mechanisms of sRNA movement, the differences between known classes of mobile sRNA-DNA methylation interactions and the limits of current knowledge. Finally, we discuss potential applications of mobile sRNAs in modifying plant traits.

Dioecy (distinct male and female individuals) combined with scarce to non-flowering are common features of cultivated yam (Dioscorea spp.). However, the molecular mechanisms underlying flowering and sex determination in Dioscorea are unknown. We conducted SuperSAGE transcriptome profiling of male, female and monoecious individuals to identify flowering and sex-related genes in white Guinea yam (D. rotundata). SuperSAGE analysis generated a total of 20,236 unique tags, of which 13,901 were represented by a minimum of 10 tags. Of these, 88 tags were significantly differentially expressed in male, female and monoecious plants. Of the 88 differentially expressed SuperSAGE tags, 18 corresponded to genes previously implicated in flower development and sex determination in multiple plant species. We validated the SuperSAGE data with quantitative real-time PCR (qRT-PCR)-based analysis of the expression of four candidate genes. Our findings suggest that mechanisms of flowering and sex determination are likely conserved in Dioscorea. We further investigated the flowering patterns of 1938 D. rotundata accessions representing diverse geographical origins over two years, revealing that over 85% of the accessions are either male or non-flowering, and that less than 15% are female, while monoecious plants are rare. Intensity of flowering appeared to be a function of sex, with male plants flowering more abundantly than female ones. Candidate genes identified in this study can be targeted with the aim to induce regular flowering in poor to non-flowering cultivars. Findings of the study provide important inputs for further studies aiming to overcome the challenge of flowering in yams and to improve the efficiency of yam breeding.

An improved protocol of QTL-seq, an NGS-based method for bulked segregant analysis we previously developed in rice, allowed successful mapping of QTLs of interest in the highly heterozygous genome of B. rapa, demonstrating the power of this elegant method for genetic analyses in heterozygous species of economic importance. Recent advances in next-generation sequencing (NGS) and the various NGS-based methods developed for rapidly identifying candidate genes of interest have accelerated genetic analysis mainly in the model plants rice and Arabidopsis. Brassica rapa includes several economically important crops such as Chinese cabbage, turnip and various leafy vegetables. The application of NGS-based approaches for the analysis of B. rapa has been limited mainly due to its highly heterozygous genome and poor quality of the reference genome sequence currently available for this species. In this study, we have improved QTL-seq, a method for NGS-based bulked segregant analysis we previously developed in rice, extending its applicability for accelerating the genetic analysis and molecular breeding of B. rapa. Addition of new filters to the original QTL-seq pipeline allowed removal of spurious single-nucleotide polymorphisms caused by alignment/sequencing errors and variability between parents, significantly improving accuracy of the analysis. As proof of principle, we successfully applied the new approach to identify candidate genomic regions controlling flowering and trichome formation using segregating F2 progeny obtained from crosses made between cultivars of B. rapa showing contrasting phenotypes for these traits. We strongly believe that the improved QTL-seq method reported here will extend the applicability of NGS-based genetic analysis not only to B. rapa but also to other plant species of economic importance with heterozygous genomes.

Due to the recent advances in DNA sequencing technologies, whole genome sequencing (WGS)-based approaches are now accelerating the pace of our research toward a better understanding of host-pathogen interactions. Using WGS-based methods, we have isolated three avirulence ( AVR ) genes: AVR-Pia , AVR-Pii and AVR-Pik from Magnaporthe oryzae and two cognate rice resistance ( R -) genes: Pia and Pii . We briefly review our current understanding of the interactions between AVR-Pia and Pia , AVR-Pik and Pik , and AVR-Pii and Pii .

BackgroundThe genome-wide expression profile of genes in different tissues/cell types and developmental stages is a vital component of many functional genomic studies. Transcriptome data obtained by RNA-sequencing (RNA-Seq) is often deposited in public databases that are made available via data portals. Data visualization is one of the first steps in assessment and hypothesis generation. However, these databases do not typically include visualization tools and establishing one is not trivial for users who are not computational experts. This, as well as the various formats in which data is commonly deposited, makes the processes of data access, sharing and utility more difficult. Our goal was to provide a simple and user-friendly repository that meets these needs for datasets from major agricultural crops.DescriptionAgriSeqDB (https://expression.latrobe.edu.au/agriseqdb), is a database for viewing, analysing and interpreting developmental and tissue/cell-specific transcriptome data from several species, including major agricultural crops such as wheat, rice, maize, barley and tomato. The disparate manner in which public transcriptome data is often warehoused and the challenge of visualizing raw data are both major hurdles to data reuse. The popular eFP browser does an excellent job of presenting transcriptome data in an easily interpretable view, but previous implementation has been mostly on a case-by-case basis. Here we present an integrated visualisation database of transcriptome datasets from six species that did not previously have public-facing visualisations. We combine the eFP browser, for gene-by-gene investigation, with the Degust browser, which enables visualisation of all transcripts across multiple samples. The two visualisation interfaces launch from the same point, enabling users to easily switch between analysis modes. The tools allow users, even those without bioinformatics expertise, to mine into datasets and understand the behaviour of transcripts of interest across samples and time. We have also incorporated an additional graphic download option to simplify incorporation into presentations or publications.ConclusionPowered by eFP and Degust browsers, AgriSeqDB is a quick and easy-to-use platform for data analysis and visualization in five crops and Arabidopsis. Furthermore, it provides a tool that makes it easy for researchers to share their datasets, promoting research collaborations and dataset reuse.

The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/β-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs., 植物の枝分かれ制御ホルモン「ストリゴラクトン」の受容メカニズムを解明 --受容体タンパクがストリゴラクトンの受容と不活性化を担うことを発見--. 京都大学プレスリリース. 2019-01-18.

Owing to recent developments in DNA sequencing technologies, whole-genome analysis of crop species has become a routine procedure. In order to improve local rice cultivars adapted to northern Japan, we are applying whole-genome analysis to identify and utilize useful alleles for crossbreeding. Here we show that large-scale generation of genetic resources combined with whole-genome analyses including MutMap and QTL-seq provides a powerful platform for the application of “next-generation breeding” in rice.

Yam (Dioscorea spp.) is a major staple crop widely cultivated for its starchy tubers. To date, very few marker resources are publicly avail - able as tools for genetic and genomic studies of this economically important crop. In this study, 90 simple sequence repeat (SSr) markers were developed from an enriched genomic library of yellow Guinea yam (D. cayenensis Lam.). Cross- amplification revealed that 85 (94.4%) and 51 (56.7%) of these SSrs could be successfully transferred to the two major cultivated species of D. rotundata poir. and D. alata L., respec - tively. polymorphisms in 30 markers selected on the basis of reliability and reproducibility of DNA bands were evaluated using a panel of 12 D. cayenensis, 48 D. rotundata, and 48 D. alata accessions. Accordingly, number of alleles per locus ranged from 2 to 8 in D. cayenensis (mean = 3.9), 3 to 30 in D. rotundata (mean = 13.9), and 2 to 22 in D. alata (mean = 12.1). The average observed and expected heterozygosi- ties were 0.156 and 0.634 ( D. cayenensis), 0.326 and 0.853 (D. rotundata), and 0.247 and 0.836 (D. alata), respectively. Clustering based on six SSrs that were polymorphic in at least four of the five cultivated Dioscorea species studied, including D. cayenensis, D. rotundata, D. alata, D. dumetorum (Kunth) pax., and D. bulbifera L., detected groups consistent with the phyloge - netic relationships of the species except for D. dumetorum. These new SSr markers are invalu - able resources for applications such as genetic diversity analysis and marker-assisted breeding.

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.

Background Root 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. Results To 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. Conclusions Guinea 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. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0419-x) contains supplementary material, which is available to authorized users.

Supplementary data including world yam production statistics [94] (Table S1), summary of genome sequence reads (Table S2), summary of genome assembly (Table S3), CEGMA result [95] (Table S4), BUSCO result (Table S5), summary of chloroplast genome assembly (Table S6), data on RAD-based linkage analysis and anchoring of scaffolds (Tables S7â S9), validation of genome assembly (Tables S10, S11), summary of RNA-seq data (Table S12), summary of assembly of transcripts (Table S13), number of lectin genes in the genomes of D. rotundata and three species (Table S14), segregation of sex in F1 derived from a cross between two accessions (Table S15), summary statistics of bulk DNA sequencing and its analysis (Tables S16, S17), BLAST result of female-specific region against TDr96_F1 reference genome (Table S18), summary of simple sequence repeats (Table S19), sequences of primers used for RAD-seq (Table S20). (PPTX 137 kb)

The significance of plastid terminal oxidase (PTOX) in phytoene desaturation and chloroplast function has been demonstrated using PTOX-deficient mutants, particularly in Arabidopsis. However, studies on its role in monocots are lacking. Here, we report cloning and characterization of the rice (Oryza sativa) PTOX1 gene. Using Ecotype Targeting Induced Local Lesions IN Genomes (EcoTILLING) and TILLING as forward genetic tools, we identified the causative mutation of an EMS mutant characterized by excessive tillering, semi-dwarfism and leaf variegation that corresponded to the PTOX1 gene. The tillering and semi-dwarf phenotypes of the ptox1 mutant are similar to phenotypes of known strigolactone (SL)-related rice mutants, and both phenotypic traits could be rescued by application of the synthetic SL GR24. The ptox1 mutant accumulated phytoene in white leaf sectors with a corresponding deficiency in β-carotene, consistent with the expected function of PTOX1 in promoting phytoene desaturase activity. There was also no accumulation of the carotenoid-derived SL ent-2'-epi-5-deoxystrigol in root exudates. Elevated concentrations of auxin were detected in the mutant, supporting previous observations that SL interaction with auxin is important in shoot branching control. Our results demonstrate that PTOX1 is required for both carotenoid and SL synthesis resulting in SL-deficient phenotypes in rice.

Summary Next-generation sequencing allows the identification of mutations responsible for mutant phenotypes by whole-genome resequencing and alignment to a reference genome. However, when the resequenced cultivar/line displays significant structural variation from the reference genome, mutations in the genome regions missing from the reference (gaps) cannot be identified by simple alignment. Here we report on a method called ‘MutMap-Gap’, which involves delineating a candidate region harboring a mutation of interest using the recently reported MutMap method, followed by de novo assembly, alignment, and identification of the mutation within genome gaps. We applied MutMap-Gap to isolate the blast resistant gene Pii from the rice cv Hitomebore using mutant lines that have lost Pii function. MutMap-Gap should prove useful for cloning genes that exhibit significant structural variations such as disease resistance genes of the nucleotide-binding site-leucine rich repeat (NBSLRR) class.

Contemporary crop improvement relies on the genetic analysis of progeny derived from a cross between different lines with contrasting phenotypes. Such analysis allowed positioning of genes for agronomically important traits, enabling development of DNA makers for marker-assisted selection (MAS). So far the identification of loci for desirable traits have been carried out by linkage analysis using DNA markers. This process required the development of DNA markers that are distributed over the genome as well as the genotyping of each progeny. Due to recent development in next generation sequencing (NGS) technology, whole genome sequencing (WGS) is becoming easier and cheaper. Using NGS, we developed a new method called MutMap that allows rapid isolation of useful alleles from rice mutant lines. An important feature of MutMap is that it does not require marker development. We foresee that the era of genetic markers will be eventually eclipsed by that of WGS applied to all the individuals in the breeding processes.

The rice (Oryza sativa L.) lesion mimic and senescence (lms) EMS-mutant, identified in a japonica cultivar Hitomebore, is characterized by a spontaneous lesion mimic phenotype during its vegetative growth, an accelerated senescence after flowering, and enhanced resistance to rice blast (Magnaporthe oryzae). To isolate the OsLMS gene, we crossed the lms mutant to Kasalath (indica), and used mutant F(2) plants to initially map the candidate region to about 322-kb on the long arm of chromosome 2. Illumina whole-genome re-sequencing of the mutant and aligning the reads to Hitomebore reference sequence within the candidate region delineated by linkage analysis identified a G to A nucleotide substitution. The mutation corresponded to the exon-intron splicing junction of a novel gene that encodes a carboxyl-terminal domain (CTD) phosphatase domain and two double stranded RNA binding motifs (dsRBM) containing protein. By PCR amplification, we confirmed that the mutation causes splicing error that is predicted to introduce a premature stop codon. RNA interference (RNAi) transgenic lines with suppressed expression of LMS gene exhibited the lesion mimic phenotype, confirming that the mutation identified in LMS is responsible for the mutant phenotype. OsLMS shares a moderate amino-acid similarity to the Arabidopsis FIERY2/CPL1 gene, which is known to control many plant processes such as stress response and development. Consistence with this similarity, the lms mutant shows sensitivity to cold stress at the early growth stage, suggesting that LMS is a negative regulator of stress response in rice.

Rice blast caused by an ascomycete fungus Magnaporthe oryzae is the most devastating disease of rice worldwide. Thus understanding of the molecular mechanisms of Magnaporthe-rice interactions is crucial to devise efficient means of disease control. The availability of whole genome sequences of both M. oryzae and rice enables us to analyze their DNA polymorphisms on the genomic scale for the purpose of association genetics as well as for population genomics analysis. Studies on the association between phenotypes-DNA polymorphisms allowed us to isolate three M. oryzae AVR genes, AVR-Pia, AVR-Pii, AVR-Pik/km/kp as well as a rice R-gene, Pia. Population genomics addresses the patterns of DNA polymorphisms to identify the genomic regions under natural selection, a potentially useful tool to isolate the genes involved in plant-pathogen interactions. Recent progress in next-generation sequencing technologies predicts that these approaches would be commonly used to elucidate various host-pathogen interactions.

BACKGROUND: Studies on composition and functional properties in germplasm collections are important in determining their diversity and suitability for food and non-food applications. The diversity in 65 yam accessions collected from Southern Ethiopia and belonging largely to a yet unknown species and to Dioscorea bulbifera L. (aerial yam) was analyzed based on protein, starch and amylose contents, and pasting properties of tuber flour, applying descriptive and multivariate statistics. RESULT: Starch content varied from 65.2% to 76.6% dry matter, while the protein content range was 6.4–13.4% dry matter. Amylose represented between 7.1% and 30.6% of the starch fraction, and was negatively correlated (P < 0.01) with starch content. UPGMA clustering and principal component analysis clearly distinguished aerial yam from those accessions with underground tubers. The first four principal components accounted for 78% of the total variability, and were highly correlated with pasting parameters. CONCLUSION: The extent of diversity detected among accessions studied showed scope for improving the crop through selection of landraces with desirable characteristics. Similar investigations on tubers grown under different environmental conditions and additional data on physicochemical properties of isolated starch will be useful in evaluating the potential of yam for food and non-food applications. Copyright © 2008 Society of Chemical Industry

Yam (Dioscorea spp.) is widely cultivated in southern Ethiopia, where farmers maintain many named landraces. Nevertheless, little is known about their diversity and species identity. In this study, amplifi ed fragment length polymor- phism (AFLP) markers were used to investi- gate the genetic diversity in 48 yam accessions from Ethiopia, and to assess their relatedness to yam species commonly cultivated in West Africa such as D. alata L., D. bulbifera L., D. cayenensis Lam., and D. rotundata Poir. Ten AFLP primer combinations generated 900 frag- ments, of which 97% were polymorphic. Clus- ter and principal coordinate analyses revealed that the Ethiopian accessions are distinct from the Dioscorea species widely cultivated in West Africa. A separate analysis of the Ethiopian accessions gave six clusters that represented the various maturity groups and the nonfl ow- ering accession in the collection. Analysis of molecular variance (AMOVA) showed that 81% of the variation detected was found within col- lecting areas, while the variation among col- lecting areas contributed only 19%. The groups detected by AFLP markers were highly consis- tent with the local yam classifi cation system and also refl ected the main structure of morphologi- cal diversity.

Yam (Dioscorea spp.) is widely grown in many parts of Ethiopia and plays a vital role in local subsistence. Nevertheless, its diversity has not been studied in detail. A survey covering 339 farm households and eight districts was conducted in the major yam growing regions of Southern Ethiopia to investigate the diversity and distribution of yam landraces using structured and semi-structured questionnaires. A total of 37 named landraces were recorded, with a range from one to six (mean 2.9) on individual farms. Farmers’ decisions regarding the number and type of landraces maintained was influenced by tolerance of the landraces to drought, their maturity time and market demand. Most landraces had limited abundance and distribution, and only a few dominant landraces were widely grown. There was also variation amongst districts with respect to diversity, distribution and abundance of the landraces found. In the majority of the localities surveyed, farmers reported a decreasing trend in the number of landraces maintained on individual farms and in the overall yam production. Besides, in those limited areas where yam production is expanding, farmers are increasingly relying on a few selected landraces that mature early. Findings of this study suggest that local farmers in Wolayita and Gamo-Gofa zones maintain considerable yam diversity that remains to be further explored for sustainable utilization and conservation of the available genetic resources.

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.

The recent accumulation of whole genome sequences (WGS) in a large number of plant species creates new opportunities to use this information for identifying genes/quantitative trait loci (QTL) and to accelerate crop improvement. To this end, we recently developed the MutMap method (Abe et al., Nat Biotechnol 30:174–178, 2012) and its derivatives MutMap+ (Fekih et al., PLoS One 8(7):e68529, 2013) and MutMap-Gap (Takagi et al., New Phytol 200(1):276–283, 2013a), which take full advantage of WGS to efficiently identify mutant genes from EMS mutagenized plant populations. We also reported QTL-seq (Takagi et al., Plant J 74:174–183, 2013b), a WGS-based method for identification of QTL. We applied these methods to rice for rapid identification and discovery of genes of agronomic importance. In this chapter, we introduce these WGS-based methods, MutMap family and QTL-seq, and provide an overview of the genetic analyses that we expect to accelerate crop improvement in rice and other crop species of economic importance.

More accurate, rapid, and easy phenotyping tools are required to match the recent advances in high-throughput genotyping for accelerating breeding and genetic analysis. The conventional data recording in field notebooks and then inputting data to computers for further analysis is inefficient, time-consuming, laborious, and prone to human error. Here, we report WIPPER (for Wireless Plant Phenotyper), a new phenotyping platform that combines field phenotyping and data recording with the aid of Bluetooth communication, thus saving time and labor not only for field data recoding but also for inputting data to computers. Additionally, it eliminates the risk of human error associated with phenotyping and inputting data. We applied WIPPER to 100 individuals of a rice recombinant inbred line (RIL) for measuring leaf width and relative chlorophyll content (SPAD value), and were able to record an accurate data in a significantly reduced time compared with the conventional method of data collection. We are currently using WIPPER for routine management of rice germplasm including recording and documenting information on phenotypic data, seeds, and DNA for their accelerated utilization in crop breeding.

Lesion mimic mutants (LMMs) provide a useful tool to study defense-related programmed cell death (PCD) in plants. Although a number of LMMs have been identified in multiple species, most of the candidate genes are yet to be isolated. Here, we report the identification and characterization of a novel rice (Oryza sativa L.) lesion mimic resembling (lmr) mutant, and cloning of the corresponding LMR gene. The LMR locus was initially delineated to 1.2 Mb region on chromosome 6, which was further narrowed down to 155-kb using insertions/deletions (INDELs) and cleavage amplified polymorphic sequence markers developed in this study. We sequenced the open reading frames predicted within the candidate genomic region, and identified a G–A base substitution causing a premature translation termination in a gene that encodes an ATPase associated with various cellular activities type (AAA-type) protein. RNA interference transgenic lines with reduced LMR transcripts exhibited the lesion mimic phenotype similar to that of lmr plants. Furthermore, expression of the wild-type LMR in the mutant background complemented the lesion phenotype, confirming that the mutation identified in LMR is responsible for the mutant phenotype. The pathogenesis-related (PR) genes PBZ1 and PR1 were induced in lmr, which also showed enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae), suggesting LMR is a negative regulator of cell death in rice. The identification of lmr and cloning of the corresponding LMR gene provide an additional resource for the study of PCD in plants.

We describe a protocol for transient gene expression in rice protoplasts and its application to the study of Magnaporthe oryzae avirulence (AVR) gene function. In this assay the gene encoding the firefly luciferase protein is transfected into rice protoplasts by electroporation together with the candidate AVR genes. The luminescence can then be used to assess the viability of rice protoplasts. The hypersensitive response (HR) caused by the interaction between M. oryzae AVR and rice R genes can subsequently be monitored by recording the decrease in luminescence from the transfected cells.

We describe a protocol for transient gene expression in rice protoplasts and its application to the study of Magnaporthe oryzae avirulence (AVR) gene function. In this assay the gene encoding the firefly luciferase protein is transfected into rice protoplasts by electroporation together with the candidate AVR genes. The luminescence can then be used to assess the viability of rice protoplasts. The hypersensitive response (HR) caused by the interaction between M. oryzae AVR and rice R genes can subsequently be monitored by recording the decrease in luminescence from the transfected cells.

Next-generation sequencing allows the identification of mutations responsible for mutant phenotypes by whole-genome resequencing and alignment to a reference genome. However, when the resequenced cultivar/line displays significant structural variation from the reference genome, mutations in the genome regions missing from the reference (gaps) cannot be identified by simple alignment. Here we report on a method called 'MutMap-Gap', which involves delineating a candidate region harboring a mutation of interest using the recently reported MutMap method, followed by de novo assembly, alignment, and identification of the mutation within genome gaps. We applied MutMap-Gap to isolate the blast resistant gene Pii from the rice cv Hitomebore using mutant lines that have lost Pii function. MutMap-Gap should prove useful for cloning genes that exhibit significant structural variations such as disease resistance genes of the nucleotide-binding site-leucine rich repeat (NBS-LRR) class.