Rapidly growing genetics and bioinformatics studies provide us with an opportunity to obtain a global view of the genetic basis of traits, but also give a challenge to the function validation of candidate genes. CRISPR/Cas9 is an emerging and efficient tool for genome editing. To construct expression clones for the CRISPR/Cas9, most current methods depend on traditional cloning using Gateway reaction or specific type IIS restriction enzymes and DNA ligation, based on multiple steps of PCR. We developed a system for introducing sgRNA expression cassette(s) directly into plant binary vectors in one step. In this system, one sgRNA expression cassette(s) is generated by an optimized multiplex PCR, in which an overlapping PCR took place. Whilst, two sgRNA expression cassettes were amplified in a single round of PCR. Subsequently, an LR or Golden gate reaction was set up with unpurified PCR product and befitting destination vector. We are able to construct expression clones within 36 h, which greatly improves efficiency and saves cost. Furthermore, the efficiency of this system was verified by an agrobacterium-mediated genetic transformation in rice. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing.

Zingiberaceae plants are widely used in the food and pharmaceutical industries; however, research on the chemical composition and interspecific differences in the metabolome and volatilome of Zingiberaceae plants is still limited. In this study, seven species of Zingiberaceae plants were selected, including Curcuma longa L., Zingiber officinale Rosc., Alpinia officinarum Hance, Alpinia tonkinensis Gagnep, Amomum tsaoko Crevost et Lemarie, Alpinia hainanensis K. Schum. and Amomum villosum Lour. Myristica fragrans Houtt. was also selected due to its flavor being similar to that of the Zingiberaceae plant. The metabolome and volatilome of selected plants were profiled by widely targeted approaches; 542 volatiles and 738 non-volatile metabolites were detected, and β-myrcene, α-phellandrene and α-cadinene were detected in all the selected plants, while chamigren, thymol, perilla, acetocinnamone and cis-α-bisabolene were exclusively detected in certain Zingiberaceae plants. Differential analysis showed that some terpenoids, such as cadalene, cadalene-1,3,5-triene, cadalene-1,3,8-triene and (E)-β-farnesene, and some lipids, including palmitic acid, linoleic acid and oleic acid were amongst the most varied compounds in Zingiberaceae plants. In conclusion, this study provided comprehensive metabolome and volatilome profiles for Zingiberaceae plants and revealed the metabolic differences between these plants. The results of this study could be used as a guide for the nutrition and flavor improvement of Zingiberaceae plants.

The plant metabolome is characterized by extensive diversity and is often regarded as a bridge between genome and phenome. Here we report metabolic and phenotypic genome-wide studies (mGWAS and pGWAS) in rice grain that, in addition to previous metabolic GWAS in rice leaf and maize kernel, show both distinct and overlapping aspects of genetic control of metabolism within and between species. We identify new candidate genes potentially influencing important metabolic and/or morphological traits. We show that the differential genetic architecture of rice metabolism between different tissues is in part determined by tissue specific expression. Using parallel mGWAS and pGWAS we identify new candidate genes potentially responsible for variation in traits such as grain colour and size, and provide evidence of metabotype-phenotype linkage. Our study demonstrates a powerful strategy for interactive functional genomics and metabolomics in plants, especially the cloning of minor QTLs for complex phenotypic traits.

As one of the most important crops in the world, rice (Oryza sativa) is a model plant for metabolome research. Although many studies have focused on the analysis of specific tissues, the changes in metabolite abundance across the entire life cycle have not yet been determined. In this study, combining both targeted and nontargeted metabolite profiling methods, a total of 825 annotated metabolites were quantified in rice samples from different tissues covering the entire life cycle. The contents of metabolites in different tissues of rice were significantly different, with various metabolites accumulating in the plumule and radicle during seed germination. Combining these data with transcriptome data obtained from the same time period, we constructed the Rice Metabolic Regulation Network. The metabolites and co-expressed genes were further divided into 12 clusters according to their accumulation patterns, with members within each cluster displaying a uniform and clear pattern of abundance across development. Using this dataset, we established a comprehensive metabolic profile of the rice life cycle and used two independent strategies to identify novel transcription factors-namely the use of known regulatory genes as bait to screen for new networks underlying lignin metabolism and the unbiased identification of new glycerophospholipid metabolism regulators on the basis of tissue specificity. This study thus demonstrates how guilt-by-association analysis of metabolome and transcriptome data spanning the entire life cycle in cereal crops provides novel resources and tools to aid in understanding the mechanisms underlying important agronomic traits.

In diploid organisms, each gene locus is composed of two parental alleles, which would interact with each other for determining the phenotypic variation. Better understanding of the allele-specific expression (ASE) in farm animals is much important to explore the genetic basis underlying economically important traits, which have been poorly understood yet. In this study, genome-wide analysis was applied to explore the spatiotemporal pattern of ASE in the F1 hybrids of chicken. First, meat- and egg-type chickens were selected for producing a full-sib F1 hybrid population (n = 57). Then, genome resequencing of two parents and 38 offspring were performed and liver and breast muscle samples (n = 38) were subjected to strand-specific RNA sequencing (ssRNA-seq) for ASE detection at 1, 28, and 56 days of age, respectively. The results accurately identified a total of 465 informative genes that could be distinguished with respect to their parental origins. There were 0.4% - 4.1% of informative genes showing ASE, and 57 of them were found across different tissues and time points. Besides, most ASE genes in chickens were tissue-specific, and no matter what the time-point pattern of one ASE gene, the same parental allele of this gene almost showed consistently higher or lower expression across all time points in the same type tissue. In conclusion, this study indicated that most of ASE genes were tissue-specific and time-dependent.

Plants have evolved many metabolites to meet the demands of growth and adaptation. Although strigolactones (SLs) play vital roles in controlling plant architecture, their function in regulating plant metabolism remains elusive. Here we report the integrative metabolomic and transcriptomic analyses of two rice SL mutants, d10 (a biosynthesis mutant) and d14 (a perception mutant). Both mutants displayed a series of metabolic and transcriptional alterations, especially in the lipid, flavonoid, and terpenoid pathways. Levels of several diterpenoid phytoalexins were substantially increased in d10 and d14, together with the induction of terpenoid gene cluster and the corresponding upstream transcription factor WRKY45, an established determinant of plant immunity. The fact that WRKY45 is a target of IPA1, which acted as a downstream transcription factor of SL signaling, suggests that SLs contribute to plant defense through WRKY45 and phytoalexins. Moreover, our data indicated that SLs may modulate rice metabolism through a vast number of clustered or tandemly duplicated genes. Our work revealed a central role of SLs in rice metabolism. Meanwhile, integrative analysis of the metabolome and transcriptome also suggested that SLs may contribute to metabolite-associated growth and defense.

Flavonoids are widely distributed in plants and play important roles in many biological processes. Citrus fruits are rich dietary sources of flavonoids. However, there have been very few reports about the comprehensive metabolic profile and natural diversity of flavonoids in different tissues of various Citrus cultivars. In this study, based on the 7416 metabolic signals detected with non-targeted metabolomics approach, Principal Component Analysis revealed the flavedo has the largest differences from other tissues in metabolite levels; as many as 198 flavonoid signals were then detected in 62 Citrus germplasms from 5 species mainly cultivated worldwide, while 117 flavonoids were identified, including 39 polymethoxylated flavonoids (PMFs), 7 flavones, 10 C-O-glycosylflavonoids, 44 O-glycosylflavonoids, 10 C-glycosylflavonoids and 7 newly annotated O-glycosylpolymethoxylated flavonoids. Tissue-specific accumulations were observed: O-glycosylated flavonoids were abundant in all fruit tissues, while PMFs were accumulated preferentially in the flavedo. Among different species, mandarins had the highest levels of PMFs and O-glycosylpolymethoxylated flavonoids, followed by sweet oranges. Based on the flavonoid profiles, 62 germplasms could be clearly grouped into five distinct clusters via hierarchical clustering analysis, which were perfectly matched with their species, with sweet oranges and mandarins clustering closely and being further away from other three species.

Phenolamides constitute a diverse class of secondary metabolites that are found ubiquitously in plants and have been implicated to play an important role in a wide range of biological processes, such as plant development and defense. However, spatiotemporal accumulation patterns of phenolamides in rice, one of the most important crops, are not available, and no gene responsible for phenolamide biosynthesis has been identified in this species. In this study, we report the comprehensive metabolic profiling and natural variation analysis of phenolamides in a collection of rice germplasm using a liquid chromatography-mass spectrometry-based targeted metabolomics method. Spatiotemporal controlled accumulations were observed for most phenolamides, together with their differential accumulations between the two major subspecies of rice. Further metabolic genome-wide association study (mGWAS) in rice leaf and in vivo metabolic analysis of the transgenic plants identified Os12g27220 and Os12g27254 as two spermidine hydroxycinnamoyl transferases that might underlie the natural variation of levels of spermidine conjugates in rice. Our work demonstrates that gene-to-metabolite analysis by mGWAS provides a useful tool for functional gene identification and omics-based crop genetic improvement.

Phosphorus is an essential macronutrient for plant growth and development. However, the network that affects phosphate (Pi) accumulation in crops is not well established. It is reported here that OsCSLF6, a member of the cellulose synthase-like family (CSLF), which is found only in grasses, is involved in Pi accumulation. The oscslf6 mutants (oscslf6-1 and oscslf6-2) display Pi toxic symptoms and increased Pi accumulation in both roots and shoots under the Pi-sufficient condition, which correlate with the induced expression of Pi transporters in the knockout mutants. Consistent with the over-accumulation of Pi, a significant decrease in primary root length, adventitious root length, and adventitious root number were observed in the oscslf6 mutants when compared with the wild type (WT) under Pi-sufficient conditions. In addition, the sucrose (Suc) level was increased in the oscslf6 mutants and the expression of sucrose synthases (OsSUS4/5) and sucrose transporters (OsSUT1/2/4/OsSweet14) genes were also induced in the shoots of oscslf6 mutants, suggesting that OsCSLF6 may play a role in affecting Pi accumulation by affecting the level of carbon metabolism.

Although considerable progress has been made in identifying the genes regulating accumulation of hormones that are involved in leaf senescence, only a few studies have focused on natural variations in jasmonates content and much less on the underlying genetic basis. Moreover, the epigenetic regulation of jasmonate-mediated leaf senescence remains largely unknown. In this study, we carried out metabolic profiling of a worldwide collection of rice accessions and demonstrated that there are substantial variations in jasmonate levels among these accessions. A subsequent metabolite-based genome-wide association study identified candidates for two major quantitative genes (QTGs), OsPME1 and OsTSD2, affecting the content of jasmonates. Further investigations using a series of relevant mutants and transgenic lines revealed the MeOH-jasmonate cascade plays an important role in regulating leaf senescence. Moreover, we showed that OsSRT1, one of the two Sir2 (silent information regulator 2) homologs in rice, negatively regulates leaf senescence by repressing expression of the biosynthetic genes of this metabolic cascade and at least particially through histone H3K9 deacetylation of OsPME1. Taken together, our results indicate that the MeOH-jasmonates cascade and its epigenetic regulation are crucial for controlling leaf senescence process in rice.

Decoration of phytochemicals contributes to the majority of metabolic diversity in nature, whereas how this process alters the biological functions of their precursor molecules remains to be investigated. Flavones, an important yet overlooked subclass of flavonoids, are most commonly conjugated with sugar moieties by UDP-dependent glycosyltransferases (UGTs). Here, we report that the natural variation of rice flavones is mainly determined by OsUGT706D1 (flavone 7-O-glucosyltransferase) and OsUGT707A2 (flavone 5-O-glucosyltransferase). UV-B exposure and transgenic evaluation demonstrate that their allelic variation contributes to UV-B tolerance in nature. Biochemical characterization of over 40 flavonoid UGTs reveals their differential evolution in angiosperms. These combined data provide biochemical insight and genetic regulation into flavone biosynthesis and additionally suggest that adoption of the positive alleles of these genes into breeding programs will likely represent a potential strategy aimed at producing stress-tolerant plants.

Plants are considered an important food and nutrition source for humans. Despite advances in plant seed metabolomics, knowledge about the genetic and molecular bases of rice seed metabolomes at different developmental stages is still limited. Here, using Zhenshan 97 (ZS97) and Minghui 63 (MH63), we performed a widely targeted metabolic profiling in seeds during grain filling, mature seeds and germinating seeds. The diversity between MH63 and ZS97 was characterized in terms of the content of metabolites and the metabolic shifting across developmental stages. Taking advantage of the ultra-high-density genetic map of a population of 210 recombinant inbred lines (RILs) derived from a cross between ZS97 and MH63, we identified 4681 putative metabolic quantitative trait loci (mQTLs) in seeds across the three stages. Further analysis of the mQTLs for the codetected metabolites across the three stages revealed that the genetic regulation of metabolite accumulation was closely related to developmental stage. Using in silico analyses, we characterized 35 candidate genes responsible for 30 structurally identified or annotated compounds, among which LOC_Os07g04970 and LOC_Os06g03990 were identified to be responsible for feruloylserotonin and l-asparagine content variation across populations, respectively. Metabolite-agronomic trait association and colocation between mQTLs and phenotypic quantitative trait loci (pQTLs) revealed the complexity of the metabolite-agronomic trait relationship and the corresponding genetic basis.

Volatile organic compounds play essential roles in plant environment interactions as well as determining the fragrance of plants. Although gas chromatography-mass spectrometry-based untargeted metabolomics is commonly used to assess plant volatiles, it suffers from high spectral convolution, low detection sensitivity, a limited number of annotated metabolites, and relatively poor reproducibility. Here, we report a widely targeted volatilomics (WTV) method that involves using a "targeted spectra extraction" algorithm to address spectral convolution, constructing a high-coverage MS2 spectral tag library to expand volatile annotation, adapting a multiple reaction monitoring mode to improve sensitivity, and using regression models to adjust for signal drift. The newly developed method was used to profile the volatilome of rice grains. Compared with the untargeted method, the newly developed WTV method shows higher sensitivity (for example, the signal-to-noise ratio of guaicol increased from 4.1 to 18.8), high annotation coverage (the number of annotated volatiles increased from 43 to 132), and better reproducibility (the number of volatiles in quality control samples with relative standard deviation value below 30.0% increased from 14 to 92 after normalization). Using the WTV method, we studied the metabolic responses of tomato to environmental stimuli and profiled the volatilomes of different rice accessions. The results identified benzothiazole as a potential airborne signal priming tomato plants for enhanced defense and 2-nonanone and 2-heptanone as novel aromatic compounds contributing to rice fragrance. These case studies suggest that the widely targeted volatilomics method is more efficient than those currently used and may considerably promote plant volatilomics studies.

Coconut is an important tropical oil and fruit crop whose evolutionary position renders it a fantastic species for the investigation of the evolution of monocot chromosomes and the subsequent differentiation of ancient plants.

Citrus fruits are widely consumed worldwide in juices or as fresh and provide a broad range of phytonutrients that are important for human health. Here, a citrus multi-omics resource is presented: comprehensive metabolic profiling of various citrus species was performed and metabolic profiles were compared among species, with a focus on the phenylpropanoid metabolic pathway. A metabolite-based genome-wide association analysis (mGWAS) of 154 pummelo accessions was performed using factored spectrally transformed linear mixed models (FaST-LMM) and efficient mixed-model association eXpedited (EMMAX), and the genetic and biochemical basis of metabolomic variation was comprehensively analysed. A metabolite-single nucleotide polymorphism-gene (metabolite-SNP-gene) interaction network was constructed for pummelo, and many candidate loci controlling the synthesis and regulation of bioactive compounds were identified; among these loci, three BAHD malonyltransferases were involved in the malonylation of flavonoid glycosides. Further investigation revealed that an R2R3-MYB transcription factor CgMYB1 positively controls the metabolism of phenylpropanoid molecules, particularly the flavonoid derivatives. This study provides valuable data resources on the metabolic regulatory networks of bioactive components in citrus, in addition to demonstrating an efficient method for metabolic pathway dissection and providing targets for future breeding work with the aim of improving nutritional value.