Institute of Crop Sciences

QTL IciMapping is freely available public software capable of building high-density linkage maps and mapping quantitative trait loci (QTL) in biparental populations. Eight functionalities are integrated in this software package: (1) BIN: binning of redundant markers; (2) MAP: construction of linkage maps in biparental populations; (3) CMP: consensus map construction from multiple linkage maps sharing common markers; (4) SDL: mapping of segregation distortion loci; (5) BIP: mapping of additive, dominant, and digenic epistasis genes; (6) MET: QTL-by-environment interaction analysis; (7) CSL: mapping of additive and digenic epistasis genes with chromosome segment substitution lines; and (8) NAM: QTL mapping in NAM populations. Input files can be arranged in plain text, MS Excel 2003, or MS Excel 2007 formats. Output files have the same prefix name as the input but with different extensions. As examples, there are two output files in BIN, one for summarizing the identified bin groups and deleted markers in each bin, and the other for using the MAP functionality. Eight output files are generated by MAP, including summary of the completed linkage maps, Mendelian ratio test of individual markers, estimates of recombination frequencies, LOD scores, and genetic distances, and the input files for using the BIP, SDL, and MET functionalities. More than 30 output files are generated by BIP, including results at all scanning positions, identified QTL, permutation tests, and detection powers for up to six mapping methods. Three supplementary tools have also been developed to display completed genetic linkage maps, to estimate recombination frequency between two loci, and to perform analysis of variance for multi-environmental trials.

Here we analyse genetic variation, population structure and diversity among 3,010 diverse Asian cultivated rice (Oryza sativa L.) genomes from the 3,000 Rice Genomes Project. Our results are consistent with the five major groups previously recognized, but also suggest several unreported subpopulations that correlate with geographic location. We identified 29 million single nucleotide polymorphisms, 2.4 million small indels and over 90,000 structural variations that contribute to within- and between-population variation. Using pan-genome analyses, we identified more than 10,000 novel full-length protein-coding genes and a high number of presence–absence variations. The complex patterns of introgression observed in domestication genes are consistent with multiple independent rice domestication events. The public availability of data from the 3,000 Rice Genomes Project provides a resource for rice genomics research and breeding. Analyses of genetic variation and population structure based on over 3,000 cultivated rice (Oryza sativa) genomes reveal subpopulations that correlate with geographic location and patterns of introgression consistent with multiple rice domestication events.

Drought and salinity are major abiotic stresses to crop production. Here, we show that overexpression of stress responsive gene SNAC1 (STRESS-RESPONSIVE NAC 1) significantly enhances drought resistance in transgenic rice (22-34% higher seed setting than control) in the field under severe drought stress conditions at the reproductive stage while showing no phenotypic changes or yield penalty. The transgenic rice also shows significantly improved drought resistance and salt tolerance at the vegetative stage. Compared with WT, the transgenic rice are more sensitive to abscisic acid and lose water more slowly by closing more stomatal pores, yet display no significant difference in the rate of photosynthesis. SNAC1 is induced predominantly in guard cells by drought and encodes a NAM, ATAF, and CUC (NAC) transcription factor with transactivation activity. DNA chip analysis revealed that a large number of stress-related genes were up-regulated in the SNAC1-overexpressing rice plants. Our data suggest that SNAC1 holds promising utility in improving drought and salinity tolerance in rice.

Flowering time is a complex trait that controls adaptation of plants to their local environment in the outcrossing species Zea mays (maize). We dissected variation for flowering time with a set of 5000 recombinant inbred lines (maize Nested Association Mapping population, NAM). Nearly a million plants were assayed in eight environments but showed no evidence for any single large-effect quantitative trait loci (QTLs). Instead, we identified evidence for numerous small-effect QTLs shared among families; however, allelic effects differ across founder lines. We identified no individual QTLs at which allelic effects are determined by geographic origin or large effects for epistasis or environmental interactions. Thus, a simple additive model accurately predicts flowering time for maize, in contrast to the genetic architecture observed in the selfing plant species rice and Arabidopsis.

Maize genetic diversity has been used to understand the molecular basis of phenotypic variation and to improve agricultural efficiency and sustainability. We crossed 25 diverse inbred maize lines to the B73 reference line, capturing a total of 136,000 recombination events. Variation for recombination frequencies was observed among families, influenced by local (cis) genetic variation. We identified evidence for numerous minor single-locus effects but little two-locus linkage disequilibrium or segregation distortion, which indicated a limited role for genes with large effects and epistatic interactions on fitness. We observed excess residual heterozygosity in pericentromeric regions, which suggested that selection in inbred lines has been less efficient in these regions because of reduced recombination frequency. This implies that pericentromeric regions may contribute disproportionally to heterosis.

Genome editing of model organisms is essential for gene function analysis and is thus critical for human health and agricultural production. The current technologies used for genome editing include ZFN (zinc-finger nuclease), meganucleases, TALEN (Transcription activator-like effector nucleases), etc . 1 . These technologies can generate double stranded breaks (DSBs) to either disrupt gene function through generation of premature stop codons by non-homologous end joining (NHEJ) pathway, or to facilitate gene targeting through homologous recombination (HR) with an incoming template. Recently, a new technology for genome editing, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) systems, has been developed 2 . CRISPR/Cas systems are adaptive defense systems in prokaryotic organisms to fight against alien nucleic acids 3 . The spacer sequences acquired from foreign DNA are positioned between host repeats, and transcribed together as CRISPR RNA (crRNA). In the type II CRISPR system, a single nuclease Cas9, guided by a dual-crRNA:tracrRNA, is sufficient to cleave cognate DNA homologous to the spacer 2 . Efficient cleavage also requires the presence of protospacer adjacent motif (PAM) 5′-NGG-3′ following the spacer sequence. The dual-crRNA:tracrRNA has been further streamlined to a single RNA chimera, called sgRNA (single guide RNA) 2 . Compared with protein-guided technologies, CRISPR/Cas system is much easier to implement, as only short guide RNAs need to be customized to target the genes of interest. Up to now, the CRISPR/Cas system has been successfully applied to efficient genome editing in many eukaryotic organisms including human 1 , mice 4 , zebra fish 5 , fly 6 , worm 7 , and yeast 8 . However, the application of CRISPR/Cas system in plants has not been reported. Rice ( Oryza sativa L.) is a major staple crop in the grass family (Poaceae), feeding half of the world's population. Rice is also used as a model monocot plant for biological studies because it has a relatively small genome compared to other cereal crops and is easy to be manipulated genetically. We demonstrate in this study that the CRISPR/Cas technology can achieve efficient targeted mutagenesis in transgenic rice. Our work paves the way for large-scale genome editing in rice, which is important for quality improvement and yield increase of rice. To accommodate the CRISPR/Cas system to Agrobacterium -mediated plant transformation, we designed Gateway ™ binary T-DNA vectors for co-expression of CAS9 and guide RNA (either sgRNA or dual-crRNA:tracrRNA, see Figure 1A ). Gene-specific spacer sequence was cloned into entry vectors for expression of guide RNA ( Supplementary information, Figure S1 and Data S1 ), which was then cloned into destination vectors containing the CAS9 expression cassette. To reconstitute Cas9 ribonucleoprotein complex in the nucleus, Cas9 was attached with a nuclear localization signal, and guide RNAs were driven by pol III type promoter of U3 snRNA. CAS9 coding sequence was codon-optimized for expression in rice ( Supplementary information, Figure S2 ), and was driven by the maize Ubiquitin ( Ubi ) promoter. To facilitate Cas9 binding and R-loop formation, we chose the sgRNA with the secondary structure containing a dangling spacer, an extended hairpin region and a long 3′ end ( Figure 1B ). Figure 1 Targeted mutagenesis in rice using CRISPR-Cas system. (A) Schematic diagram of the vectors used in this study. CAS9 is cloned into the destination vector. Guide RNAs are cloned into entry vectors. Modular design of vectors permits multiplex genome editing. (B) Schematic showing the folding of sgRNA and co-folding of crRNA and tracrRNA. Spacer sequences for targeting DGU.US or CAO1 are also shown. The long hairpin and long tail are used to maintain structural stability. (C) Schematic representation of the DGU.US system to detect the generation of DSB. DSB generated by Cas9 between the direct repeats (U) can be repaired through SSA, thus restoring the GUS function. PAM is underlined. (D) GUS staining after co-transformation of DGU.US with (left) or without the CRISPR construct (right). Scale bar, 0.5 mm. (E) Leaf blade of wild type (WT) and transformants with cao1 phenotype. (F) Targeted mutagenesis of CAO1 gene. The position of target site is shown on the gene structure. The spacer sequence is shaded in gray, and the PAM is underlined. Sequencing results of mutant alleles are aligned to the reference genome sequence. The indels are shown in red letters or dashes. The sequence of the 92-bp insertion in CAO1 gene is 5′-TCG TTC CCT TTC ATC AAG TGG GTA AGG TAG GCG AAC CAC TTA AGC TTT GCC CCC CAC CTC CTT TGG GGA TCC CTA CTT ACC CGA GCT TCC GG-3′. (G) Chlorophyll analysis in WT and transgenic plants. (H) Targeted mutagenesis of LAZY1 gene. The position of target site is shown on the gene structure. The spacer sequence is shaded in gray, and the PAM is underlined. Sequencing results of mutant alleles are aligned to the reference genome sequence. The indels are shown in red letters or dashes. (I) Tiller angle comparison between WT and transgenic plant. Full size image To test whether the CRISPR/Cas system can be applied in plant cells, we first investigated whether the system can generate DSB in rice callus using the DGU.US as a reporter ( Supplementary information, Figure S3 ). GUS activity can be restored through single strand annealing (SSA) upon DNA cleavage between the repeat regions in the DGU.US reporter 9 ( Figure 1C ). We designed both sgRNA and dual-crRNA:tracrRNA to target the reporter. The result showed that strong GUS staining spots were detected in rice calli after the CRISPR/Cas system and the reporter were co-transformed through particle bombardment, whereas no GUS signal was observed in those transformed with the reporter alone ( Figure 1D ). Both sgRNA and dual-crRNA:tracrRNA were effective in this assay, indicating that pre-crRNA can be properly processed in rice (plant) cells. To further test whether the CRISPR/Cas technology can be used to specifically disrupt an endogenous gene in rice, we designed sgRNAs and dual-crRNA:tracrRNAs targeting either the second exon of the CHLOROPHYLL A OXYGENASE 1 ( CAO1 ) gene or the third exon of the LAZY1 gene, and transformed them into Kitaake , a japonica rice variety with short life cycle 10 . The seedlings of the loss-of-function mutant cao1 show a pale green leaf phenotype due to defective synthesis of Chlorophyll b (Chl b), which is easily observed at an early developmental stage, whereas the la1 mutant, loss-of-function mutant of LAZY1 gene, exhibited a pronounced tiller-spreading phenotype that can be observed after tillering stage 11 . We obtained 30 independent transgenic lines for sgRNA construct and 45 lines for dual-crRNA:tracrRNA construct for the CAO1 gene. We found that some transgenic plants displayed pale green leaf blades ( Figure 1E ), and we conducted genotyping analysis on these lines using gene-specific primers. The result showed that either loss of peak/gain of peak or overlapping peak around the target site was observed in the sequencing chromatograms ( Figure 1F and Supplementary information, Figure S4 ), which confirmed mutations in the CAO1 gene. The occurrence of indels at 3-4-bp upstream of PAM is consistent with the location of Cas9 cleavage site. We randomly selected transgenic lines with biallelic mutation, and measured the contents of Chl a and Chl b 12 . The result showed that Chl b content was reduced to a marginal level, consistent with the cao1 phenotype ( Figure 1G ). We obtained only 12 independent transgenic lines for sgRNA construct for LAZY1 gene. Sequencing analysis showed that 11 out of 12 lines bear mutations in the specific region of LAZY1 gene, confirming the disruption of LAZY1 gene ( Figure 1H and Supplementary information, Figure S5 ). Appearance of tiller-spreading phenotypes in 6 homogenous mutation lines further supported the conclusion ( Figure 1I ). Notably, in the case of sgRNA, about 83.3% and 91.6% of the independent lines of the T1 transgenic rice beared mutations in CAO1 and LAZY1 , respectively, among which 4 lines (13.3%) for CAO1 and 6 lines (50%) for LAZY1 beared biallelic mutations ( Supplementary information, Table S1 ). These results indicate that transgenic rice with mutated gene of interest can be easily generated in the T1 generation by using CRISPR/Cas technology, and that the high efficiency of targeted mutagenesis can be achieved in different genes. This high efficiency is possibly due to the unique feature of the CRISPR/Cas system, i.e., different from ZFN or TALEN, Cas9-mediated cleavage tolerates DNA methylation 13 . This gives the CRISPR/Cas technology leverage for genome editing in plants with high GC content in the genome such as rice. It seems that sgRNA constructs generate targeted mutagenesis more efficiently than dual-crRNA:tracrRNA constructs ( Supplementary information, Table S1 ), the mechanism of which needs to be investigated in the future. To facilitate selection of spacers to target other rice genes, we also used criteria reported recently, which exclude potential mismatches of the first 8 nucleotides of the 20-nt spacer region and leaky activity on 5′-NAG-3′ PAM 8 , and designed more than 3.6 million spacers ( Supplementary information, Data S2 ). We showed that transgenic rice with mutations in specific genes could be generated through the CRISPR/Cas technology in a straightforward manner. Our strategy for vector construction is modular, efficient, and expandable for multiplex gene editing. Because dual expression of pre-crRNA:tracrRNA can be correctly processed in the plant cells although the mutagenesis efficiency is relatively low in the two genes that we tested ( Figure 1 and Supplementary information, Table S1 ), the entry vectors that we designed has the potential to host two sgRNAs or multiple spacers, in an array of crRNA. The facile genome editing at specific sites in rice will speed up functional characterization of rice genes, especially for those genes with family members, and will greatly promote the effort to improve rice quality and yield through agricultural biotechnology. The compiled list of the specific spacers for each gene in rice genome will also help to accelerate the application of the CRISPR/Cas system in rice. Further work will be focused on whether the CRISPR/Cas system can be applied successfully to other cereals with bigger and more complicated genomes such as maize and wheat. Meanwhile, further modification of the CRISPR/Cas system, e.g., removal of the components of the CRISPR/Cas system after the target genes are mutated, will promote the application of this new technology in agriculture.

Jasmonic acid (JA) and its precursors and dervatives, referred as jasmonates (JAs) are important molecules in the regulation of many physiological processes in plant growth and development, and especially the mediation of plant responses to biotic and abiotic stresses. JAs biosynthesis, perception, transport, signal transduction and action have been extensively investigated. In this review, we will discuss the initiation of JA signaling with a focus on environmental signal perception and transduction, JA biosynthesis and metabolism, transport of signaling molecules (local transmission, vascular bundle transmission, and airborne transportation), and biological function (JA signal receptors, regulated transcription factors, and biological processes involved).

Rice blast is one of the most destructive diseases affecting rice worldwide. The adoption of host resistance has proven to be the most economical and effective approach to control rice blast. In recent years, sequence-specific nucleases (SSNs) have been demonstrated to be powerful tools for the improvement of crops via gene-specific genome editing, and CRISPR/Cas9 is thought to be the most effective SSN. Here, we report the improvement of rice blast resistance by engineering a CRISPR/Cas9 SSN (C-ERF922) targeting the OsERF922 gene in rice. Twenty-one C-ERF922-induced mutant plants (42.0%) were identified from 50 T0 transgenic plants. Sanger sequencing revealed that these plants harbored various insertion or deletion (InDel) mutations at the target site. We showed that all of the C-ERF922-induced allele mutations were transmitted to subsequent generations. Mutant plants harboring the desired gene modification but not containing the transferred DNA were obtained by segregation in the T1 and T2 generations. Six T2 homozygous mutant lines were further examined for a blast resistance phenotype and agronomic traits, such as plant height, flag leaf length and width, number of productive panicles, panicle length, number of grains per panicle, seed setting percentage and thousand seed weight. The results revealed that the number of blast lesions formed following pathogen infection was significantly decreased in all 6 mutant lines compared with wild-type plants at both the seedling and tillering stages. Furthermore, there were no significant differences between any of the 6 T2 mutant lines and the wild-type plants with regard to the agronomic traits tested. We also simultaneously targeted multiple sites within OsERF922 by using Cas9/Multi-target-sgRNAs (C-ERF922S1S2 and C-ERF922S1S2S3) to obtain plants harboring mutations at two or three sites. Our results indicate that gene modification via CRISPR/Cas9 is a useful approach for enhancing blast resistance in rice.

Composite interval mapping (CIM) is the most commonly used method for mapping quantitative trait loci (QTL) with populations derived from biparental crosses. However, the algorithm implemented in the popular QTL Cartographer software may not completely ensure all its advantageous properties. In addition, different background marker selection methods may give very different mapping results, and the nature of the preferred method is not clear. A modified algorithm called inclusive composite interval mapping (ICIM) is proposed in this article. In ICIM, marker selection is conducted only once through stepwise regression by considering all marker information simultaneously, and the phenotypic values are then adjusted by all markers retained in the regression equation except the two markers flanking the current mapping interval. The adjusted phenotypic values are finally used in interval mapping (IM). The modified algorithm has a simpler form than that used in CIM, but a faster convergence speed. ICIM retains all advantages of CIM over IM and avoids the possible increase of sampling variance and the complicated background marker selection process in CIM. Extensive simulations using two genomes and various genetic models indicated that ICIM has increased detection power, a reduced false detection rate, and less biased estimates of QTL effects.

Sequencing and analysing the diploid genome and transcriptome of Aegilops tauschii provide new insights into the role of this genome in enabling the adaptation of bread wheat and are a step towards understanding the very large and complicated hexaploid genomes of wheat species. The hexaploid genome of bread wheat Triticum aestivum, designated AABBDD, evolved as a result of hybridization between three ancestral grasses. Two papers published in the issue of Nature present genome sequences and analysis of two of these wheat progenitors. First, the genome sequence of the diploid wild wheat T. urartu (ancestor of the A genome), which resembles cultivated wheat more strongly than either Aegilops speltoides (the B ancestor) or Ae. tauschii (the D donor). And second, the Ae. tauschii genome, together with an analysis of its transcriptome. These genomes and their analyses will be powerful tools for the study of complex, polyploid wheat genomes and a valuable resource for genetic improvement of wheat. About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD)1,2. Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of baker’s flour2. Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae. tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.

Wild relatives of crops are an important source of genetic diversity for agriculture, but their gene repertoire remains largely unexplored. We report the establishment and analysis of a pan-genome of Glycine soja, the wild relative of cultivated soybean Glycine max, by sequencing and de novo assembly of seven phylogenetically and geographically representative accessions. Intergenomic comparisons identified lineage-specific genes and genes with copy number variation or large-effect mutations, some of which show evidence of positive selection and may contribute to variation of agronomic traits such as biotic resistance, seed composition, flowering and maturity time, organ size and final biomass. Approximately 80% of the pan-genome was present in all seven accessions (core), whereas the rest was dispensable and exhibited greater variation than the core genome, perhaps reflecting a role in adaptation to diverse environments. This work will facilitate the harnessing of untapped genetic diversity from wild soybean for enhancement of elite cultivars.

Abstract Grassland diversity can support sustainable intensification of grassland production through increased yields, reduced inputs and limited weed invasion. We report the effects of diversity on weed suppression from 3 years of a 31‐site continental‐scale field experiment. At each site, 15 grassland communities comprising four monocultures and 11 four‐species mixtures based on a wide range of species' proportions were sown at two densities and managed by cutting. Forage species were selected according to two crossed functional traits, “method of nitrogen acquisition” and “pattern of temporal development”. Across sites, years and sown densities, annual weed biomass in mixtures and monocultures was 0.5 and 2.0 t DM ha −1 (7% and 33% of total biomass respectively). Over 95% of mixtures had weed biomass lower than the average of monocultures, and in two‐thirds of cases, lower than in the most suppressive monoculture (transgressive suppression). Suppression was significantly transgressive for 58% of site‐years. Transgressive suppression by mixtures was maintained across years, independent of site productivity. Based on models, average weed biomass in mixture over the whole experiment was 52% less (95% confidence interval: 30%–75%) than in the most suppressive monoculture. Transgressive suppression of weed biomass was significant at each year across all mixtures and for each mixture. Weed biomass was consistently low across all mixtures and years and was in some cases significantly but not largely different from that in the equiproportional mixture. The average variability (standard deviation) of annual weed biomass within a site was much lower for mixtures (0.42) than for monocultures (1.77). Synthesis and applications . Weed invasion can be diminished through a combination of forage species selected for complementarity and persistence traits in systems designed to reduce reliance on fertiliser nitrogen. In this study, effects of diversity on weed suppression were consistently strong across mixtures varying widely in species' proportions and over time. The level of weed biomass did not vary greatly across mixtures varying widely in proportions of sown species. These diversity benefits in intensively managed grasslands are relevant for the sustainable intensification of agriculture and, importantly, are achievable through practical farm‐scale actions.

A new member of the AP2/ERF transcription factor family, GmERF3, was isolated from soybean. Sequence analysis showed that GmERF3 contained an AP2/ERF domain of 58 amino acids and two putative nuclear localization signal (NLS) domains. It belonged to a group IV protein in the ERF (ethylene response factor) subfamily as typified by a conserved N-terminal motif [MCGGAI(I/L)]. Expression of GmERF3 was induced by treatments with high salinity, drought, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and soybean mosaic virus (SMV), whereas there was no significant GmERF3 mRNA accumulation under cold stress treatment. GmERF3 could bind to the GCC box and DRE/CRT element, and was targeted to the nucleus when transiently expressed in onion epidermal cells. The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast. Ectopic expression of the GmERF3 gene in transgenic tobacco plants induced the expression of some PR genes and enhanced resistance against infection by Ralstonia solanacearum, Alternaria alternata, and tobacco mosaic virus (TMV), and gave tolerance to high salinity and dehydration stresses. Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions. The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.

Abstract Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the ‘pan-genome’ 1 ). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley ( Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions 2 . Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley—comprising landraces, cultivars and a wild barley—that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most devastating diseases in rice worldwide. The dominant resistance gene, Pi-d2 [previously named Pi-d(t)2], present in the rice variety Digu, confers gene-for-gene resistance to the Chinese blast strain, ZB15. Pi-d2 was previously mapped close to the centromere of chromosome 6. In this study, the Pi-d2 gene was isolated by a map-based cloning strategy. Pi-d2 encodes a receptor-like kinase protein with a predicted extracellular domain of a bulb-type mannose specific binding lectin (B-lectin) and an intracellular serine-threonine kinase domain. Pi-d2 is a single-copy gene that is constitutively expressed in the rice variety Digu. Transgenic plants carrying the Pi-d2 transgene confer race-specific resistance to the M. grisea strain, ZB15. The Pi-d2 protein is plasma membrane localized. A single amino acid difference at position 441 of Pi-d2 distinguishes resistant and susceptible alleles of rice blast resistance gene Pi-d2. Because of its novel extracellular domain, Pi-d2 represents a new class of plant resistance genes.

The three most important agronomic traits of rice (Oryza sativa), yield, plant height, and flowering time, are controlled by many quantitative trait loci (QTLs). In this study, a newly identified QTL, DTH8 (QTL for days to heading on chromosome 8), was found to regulate these three traits in rice. Map-based cloning reveals that DTH8 encodes a putative HAP3 subunit of the CCAAT-box-binding transcription factor and the complementary experiment increased significantly days to heading, plant height, and number of grains per panicle in CSSL61 (a chromosome segment substitution line that carries the nonfunctional DTH8 allele) with the Asominori functional DTH8 allele under long-day conditions. DTH8 is expressed in most tissues and its protein is localized to the nucleus exclusively. The quantitative real-time PCR assay revealed that DTH8 could down-regulate the transcriptions of Ehd1 (for Early heading date1) and Hd3a (for Heading date3a; a rice ortholog of FLOWERING LOCUS T) under long-day conditions. Ehd1 and Hd3a can also be down-regulated by the photoperiodic flowering genes Ghd7 and Hd1 (a rice ortholog of CONSTANS). Meanwhile, the transcription of DTH8 has been proved to be independent of Ghd7 and Hd1, and the natural mutation of this gene caused weak photoperiod sensitivity and shorter plant height. Taken together, these data indicate that DTH8 probably plays an important role in the signal network of photoperiodic flowering as a novel suppressor as well as in the regulation of plant height and yield potential.

There is a rapidly rising trend in the development and application of molecular marker assays for gene mapping and discovery in field crops and trees. Thus far, more than 50 SNP arrays and 15 different types of genotyping-by-sequencing (GBS) platforms have been developed in over 25 crop species and perennial trees. However, much less effort has been made on developing ultra-high-throughput and cost-effective genotyping platforms for applied breeding programs. In this review, we discuss the scientific bottlenecks in existing SNP arrays and GBS technologies and the strategies to develop targeted platforms for crop molecular breeding. We propose that future practical breeding platforms should adopt automated genotyping technologies, either array or sequencing based, target functional polymorphisms underpinning economic traits, and provide desirable prediction accuracy for quantitative traits, with universal applications under wide genetic backgrounds in crops. The development of such platforms faces serious challenges at both the technological level due to cost ineffectiveness, and the knowledge level due to large genotype-phenotype gaps in crop plants. It is expected that such genotyping platforms will be achieved in the next ten years in major crops in consideration of (a) rapid development in gene discovery of important traits, (b) deepened understanding of quantitative traits through new analytical models and population designs, (c) integration of multi-layer -omics data leading to identification of genes and pathways responsible for important breeding traits, and (d) improvement in cost effectiveness of large-scale genotyping. Crop breeding chips and genotyping platforms will provide unprecedented opportunities to accelerate the development of cultivars with desired yield potential, quality, and enhanced adaptation to mitigate the effects of climate change.

Plants produce a variety of metabolites that have a critical role in growth and development. Here we present a comprehensive study of maize metabolism, combining genetic, metabolite and expression profiling methodologies to dissect the genetic basis of metabolic diversity in maize kernels. We quantify 983 metabolite features in 702 maize genotypes planted at multiple locations. We identify 1,459 significant locus–trait associations (P≤1.8 × 10−6) across three environments through metabolite-based genome-wide association mapping. Most (58.5%) of the identified loci are supported by expression QTLs, and some (14.7%) are validated through linkage mapping. Re-sequencing and candidate gene association analysis identifies potential causal variants for five candidate genes involved in metabolic traits. Two of these genes were further validated by mutant and transgenic analysis. Metabolite features associated with kernel weight could be used as biomarkers to facilitate genetic improvement of maize. Plants produce a variety of metabolites that have a critical role in growth and development. Here, the authors carry out a genome-wide association analysis of metabolites and metabolite features in maize and identify candidate genes involved in secondary metabolism, and a potential biomarker for the genetic improvement of the crop.

Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors.

Cereals high in amylose content (AC) and resistant starch (RS) offer potential health benefits. Previous studies using chemical mutagenesis or RNA interference have demonstrated that starch branching enzyme (SBE) plays a major role in determining the fine structure and physical properties of starch. However, it remains a challenge to control starch branching in commercial lines. Here, we use CRISPR/Cas9 technology to generate targeted mutagenesis in SBEI and SBEIIb in rice. The frequencies of obtained homozygous or bi-allelic mutant lines with indels in SBEI and SBEIIb in T0 generation were from 26.7 to 40%. Mutations in the homozygous T0 lines stably transmitted to the T1 generation and those in the bi-allelic lines segregated in a Mendelian fashion. Transgene-free plants carrying only the frame-shifted mutagenesis were recovered in T1 generation following segregation. Whereas no obvious differences were observed between the sbeI mutants and wild type, sbeII mutants showed higher proportion of long chains presented in debranched amylopectin, significantly increased AC and RS content to as higher as 25.0% and 9.8%, respectively, and thus altered fine structure and nutritional properties of starch. Taken together, our results demonstrated for the first time the feasibility to create high-amylose rice through CRISPR/Cas9-mediated editing of SBEIIb.