A de novo evolved gene contributes to rice grain shape difference between indica and japonica.

The role of de novo evolved genes from non-coding sequences in regulating morphological differentiation between species/subspecies remains largely unknown. Here, we show that a rice de novo gene GSE9 contributes to grain shape difference between indica/xian and japonica/geng varieties. GSE9 evolves from a previous non-coding region of wild rice Oryza rufipogon through the acquisition of start codon. This gene is inherited by most japonica varieties, while the original sequence (absence of start codon, gse9) is present in majority of indica varieties. Knockout of GSE9 in japonica varieties leads to slender grains, whereas introgression to indica background results in round grains. Population evolutionary analyses reveal that gse9 and GSE9 are derived from wild rice Or-I and Or-III groups, respectively. Our findings uncover that the de novo GSE9 gene contributes to the genetic and morphological divergence between indica and japonica subspecies, and provide a target for precise manipulation of rice grain shape.

Genetic Diversification of Starch Branching Enzymes during Maize Domestication and Improvement.

Elucidating the genetic basis of starch pasting and gelatinization properties is crucial for enhancing the quality of maize and its utility as feed and industrial raw material. In maize, ZmSBE genes encode important starch branching enzymes in the starch biosynthesis pathway. In this study, we re-sequenced the genomic sequences of ZmSBEI, ZmSBEIIa, ZmSBEIIb, and ZmSBEIII in three lines called 335 inbred lines, 68 landrace lines, and 32 teosinte lines. Analyses of nucleotide polymorphisms and haplotype diversity revealed differences in the selection patterns of ZmSBEI, ZmSBEIIa, ZmSBEIIb, and ZmSBEIII during maize domestication and improvement. A marker-trait association analysis of inbred lines detected 22 significant loci, including 18 SNPs and 4 indels significantly associated with three maize starch physicochemical properties. The allele frequencies of two variants (SNP17249C and SNP5055G) were examined in three lines. The frequency of SNP17249C in ZmSBEIIb was highest in teosinte lines, followed by landrace lines, and inbred lines, whereas there were no significant differences in the frequency of SNP5055G in ZmSBEIII among the three lines. These results suggest that ZmSBE genes play an important role in the phenotypic variations in the starch physicochemical properties in maize. The genetic variants detected in this study may be used to develop functional markers for improving maize starch quality.

GWAS and Transcriptome Analysis Reveal Key Genes Affecting Root Growth under Low Nitrogen Supply in Maize.

Nitrogen (N) is one of the most important factors affecting crop production. Root morphology exhibits a high degree of plasticity to nitrogen deficiency. However, the mechanisms underlying the root foraging response under low-N conditions remain poorly understood. In this study, we analyzed 213 maize inbred lines using hydroponic systems and regarding their natural variations in 22 root traits and 6 shoot traits under normal (2 mM nitrate) and low-N (0 mM nitrate) conditions. Substantial phenotypic variations were detected for all traits. N deficiency increased the root length and decreased the root diameter and shoot related traits. A total of 297 significant marker-trait associations were identified by a genome-wide association study involving different N levels and the N response value. A total of 51 candidate genes with amino acid variations in coding regions or differentially expressed under low nitrogen conditions were identified. Furthermore, a candidate gene ZmNAC36 was resequenced in all tested lines. A total of 38 single nucleotide polymorphisms and 12 insertions and deletions were significantly associated with lateral root length of primary root, primary root length between 0 and 0.5 mm in diameter, primary root surface area, and total length of primary root under a low-N condition. These findings help us to improve our understanding of the genetic mechanism of root plasticity to N deficiency, and the identified loci and candidate genes will be useful for the genetic improvement of maize tolerance cultivars to N deficiency.

Primary root response to combined drought and heat stress is regulated via salicylic acid metabolism in maize.

The primary root is the first organ to perceive the stress signals for abiotic stress. In this study, maize plants subjected to drought, heat and combined stresses displayed a significantly reduced primary root length. Metabolic and transcriptional analyses detected 72 and 5,469 differentially expressed metabolites and genes in response to stress conditions, respectively. The functional annotation of differentially expressed metabolites and genes indicated that primary root development was mediated by pathways involving phenylalanine metabolism, hormone metabolism and signaling under stress conditions. Furthermore, we found that the concentration of salicylic acid and two precursors, shikimic acid and phenylalanine, showed rapid negative accumulation after all three stresses. The expression levels of some key genes involved in salicylic acid metabolism and signal transduction were differentially expressed under stress conditions. This study extends our understanding of the mechanism of primary root responses to abiotic stress tolerance in maize.

Genetic and molecular factors in determining grain number per panicle of rice.

It was suggested that the most effective way to improve rice grain yield is to increase the grain number per panicle (GN) through the breeding practice in recent decades. GN is a representative quantitative trait affected by multiple genetic and environmental factors. Understanding the mechanisms controlling GN has become an important research field in rice biotechnology and breeding. The regulation of rice GN is coordinately controlled by panicle architecture and branch differentiation, and many GN-associated genes showed pleiotropic effect in regulating tillering, grain size, flowering time, and other domestication-related traits. It is also revealed that GN determination is closely related to vascular development and the metabolism of some phytohormones. In this review, we summarize the recent findings in rice GN determination and discuss the genetic and molecular mechanisms of GN regulators.

Nucleotide polymorphisms of the maize ZmFWL7 gene and their association with ear-related traits.

Plant fw2.2-like (FWL) genes, encoding proteins harboring a placenta-specific eight domain, have been suggested to control fruit and grain size through regulating cell division, differentiation, and expansion. Here, we re-sequenced the nucleotide sequences of the maize ZmFWL7 gene, a member of the FWL family, in 256 elite maize inbred lines, and the associations of nucleotide polymorphisms in this locus with 11 ear-related traits were further detected. A total of 175 variants, including 159 SNPs and 16 InDels, were identified in the ZmFWL7 locus. Although the promoter and downstream regions showed higher nucleotide polymorphism, the coding region also possessed 61 SNPs and 6 InDels. Eleven polymorphic sites in the ZmFWL7 locus were found to be significantly associated with eight ear-related traits. Among them, two nonsynonymous SNPs (SNP2370 and SNP2898) showed significant association with hundred kernel weight (HKW), and contributed to 7.11% and 8.62% of the phenotypic variations, respectively. In addition, the SNP2898 was associated with kernel width (KW), and contributed to 7.57% of the phenotypic variations. Notably, the elite allele T of SNP2370 was absent in teosintes and landraces, while its frequency in inbred lines was increased to 12.89%. By contrast, the frequency of the elite allele A of SNP2898 was 3.12% in teosintes, and it was raised to 12.68% and 19.92% in landraces and inbred lines, respectively. Neutral tests show that this locus wasn't artificially chosen during the process of domestication and genetic improvement. Our results revealed that the elite allelic variants in ZmFWL7 might possess potential for the genetic improvement of maize ear-related traits.

The plant streptolysin S (SLS)-associated gene B confers nitroaromatic tolerance and detoxification.

Nitroaromatic compounds, as the important chemical feedstock, have caused widespread environmental contaminations, and exhibited high toxicity and mutagenic activity to nearly all living organisms. The clean-up of nitroaromatic-contaminated soil and water has long been a major international concern. Here, we uncovered the role of a novel nitroreductase family gene, streptolysin S (SLS)-associated gene B (SagB), in enhancing nitroaromatic tolerance and detoxification of plants, and its potential application in phytoremediation of nitroaromatic contaminations. The expression of both the Arabidopsis and rice SagB genes is significantly induced by multiple hazardous nitroaromatic substances, including explosive pollutant 2,4,6-trinitrotoluene (TNT), natural compound 1-nitropyrene (1-NP) and herbicide pendimethalin (Pen). In vitro and in vivo evidences revealed that plant SagBs possess activities in degradation of these nitroaromatic substances. Arabidopsis and rice transgenic assays suggested that plant SagB genes increase tolerance and detoxification of nitroaromatic through facilitating its transformation to the amino derivative. More importantly, overexpression of plant SagBs increase their ability in TNT uptake, and remove more TNT from the growth culture. Our findings shed novel insights into a plant endogenous nitroreductase-mediated nitroaromatic tolerance and detoxification, and provide a new gene target for phytoremediation of nitroaromatic-contaminated environments.

OsCOMT, encoding a caffeic acid O-methyltransferase in melatonin biosynthesis, increases rice grain yield through dual regulation of leaf senescence and vascular development.

Melatonin, a natural phytohormone in plants, plays multiple critical roles in plant growth and stress responses. Although melatonin biosynthesis-related genes have been suggested to possess diverse biological functions, their roles and functional mechanisms in regulating rice grain yield remain largely unexplored. Here, we uncovered the roles of a caffeic acid O-methyltransferase (OsCOMT) gene in mediating rice grain yield through dual regulation of leaf senescence and vascular development. In vitro and in vivo evidence revealed that OsCOMT is involved in melatonin biosynthesis. Transgenic assays suggested that OsCOMT significantly delays leaf senescence at the grain filling stage by inhibiting degradation of chlorophyll and chloroplast, which, in turn, improves photosynthesis efficiency. In addition, the number and size of vascular bundles in the culms and leaves were significantly increased in the OsCOMT-overexpressing plants, while decreased in the knockout plants, suggesting that OsCOMT plays a positive role in vascular development of rice. Further evidence indicated that OsCOMT-mediated vascular development might owe to the crosstalk between melatonin and cytokinin. More importantly, we found that OsCOMT is a positive regulator of grain yield, and overexpression of OsCOMT increase grain yield per plant even in a high-yield variety background, suggesting that OsCOMT can be used as an important target for enhancing rice yield. Our findings shed novel insights into melatonin-mediated leaf senescence and vascular development and provide a possible strategy for genetic improvement of rice grain yield.

Nucleotide Diversity of the Maize ZmCNR13 Gene and Association With Ear Traits.

The maize (Zea mays L.) ZmCNR13 gene, encoding a protein of fw2.2-like (FWL) family, has been demonstrated to be involved in cell division, expansion, and differentiation. In the present study, the genomic sequences of the ZmCNR13 locus were re-sequenced in 224 inbred lines, 56 landraces and 30 teosintes, and the nucleotide polymorphism and selection signature were estimated. A total of 501 variants, including 415 SNPs and 86 Indels, were detected. Among them, 51 SNPs and 4 Indels were located in the coding regions. Although neutrality tests revealed that this locus had escaped from artificial selection during the process of maize domestication, the population of inbred lines possesses lower nucleotide diversity and decay of linkage disequilibrium. To estimate the association between sequence variants of ZmCNR13 and maize ear characteristics, a total of ten ear-related traits were obtained from the selected inbred lines. Four variants were found to be significantly associated with six ear-related traits. Among them, SNP2305, a non-synonymous mutation in exon 2, was found to be associated with ear weight, ear grain weight, ear diameter and ear row number, and explained 4.59, 4.61, 4.31, and 8.42% of the phenotypic variations, respectively. These results revealed that natural variations of ZmCNR13 might be involved in ear development and can be used in genetic improvement of maize ear-related traits.

Exploiting natural variation in crown root traits via genome-wide association studies in maize.

BACKGROUND: Root system architecture (RSA), which is determined by the crown root angle (CRA), crown root diameter (CRD), and crown root number (CRN), is an important factor affecting the ability of plants to obtain nutrients and water from the soil. However, the genetic mechanisms regulating crown root traits in the field remain unclear. METHODS: In this study, the CRA, CRD, and CRN of 316 diverse maize inbred lines were analysed in three field trials. Substantial phenotypic variations were observed for the three crown root traits in all environments. A genome-wide association study was conducted using two single-locus methods (GLM and MLM) and three multi-locus methods (FarmCPU, FASTmrMLM, and FASTmrEMMA) with 140,421 SNP. RESULTS: A total of 38 QTL including 126 SNPs were detected for CRA, CRD, and CRN. Additionally, 113 candidate genes within 50 kb of the significant SNPs were identified. Combining the gene annotation information and the expression profiles, 3 genes including GRMZM2G141205 (IAA), GRMZM2G138511 (HSP) and GRMZM2G175910 (cytokinin-O-glucosyltransferase) were selected as potentially candidate genes related to crown root development. Moreover, GRMZM2G141205, encoding an AUX/IAA transcriptional regulator, was resequenced in all tested lines. Five variants were identified as significantly associated with CRN in different environments. Four haplotypes were detected based on these significant variants, and Hap1 has more CRN. CONCLUSIONS: These findings may be useful for clarifying the genetic basis of maize root system architecture. Furthermore, the identified candidate genes and variants may be relevant for breeding new maize varieties with root traits suitable for diverse environmental conditions.

Genome-wide DNA mutations in Arabidopsis plants after multigenerational exposure to high temperatures.

BACKGROUND: Elevated temperatures can cause physiological, biochemical, and molecular responses in plants that can greatly affect their growth and development. Mutations are the most fundamental force driving biological evolution. However, how long-term elevations in temperature influence the accumulation of mutations in plants remains unknown. RESULTS: Multigenerational exposure of Arabidopsis MA (mutation accumulation) lines and MA populations to extreme heat and moderate warming results in significantly increased mutation rates in single-nucleotide variants (SNVs) and small indels. We observe distinctive mutational spectra under extreme and moderately elevated temperatures, with significant increases in transition and transversion frequencies. Mutation occurs more frequently in intergenic regions, coding regions, and transposable elements in plants grown under elevated temperatures. At elevated temperatures, more mutations accumulate in genes associated with defense responses, DNA repair, and signaling. Notably, the distribution patterns of mutations among all progeny differ between MA populations and MA lines, suggesting that stronger selection effects occurred in populations. Methylation is observed more frequently at mutation sites, indicating its contribution to the mutation process at elevated temperatures. Mutations occurring within the same genome under elevated temperatures are significantly biased toward low gene density regions, special trinucleotides, tandem repeats, and adjacent simple repeats. Additionally, mutations found in all progeny overlap significantly with genetic variations reported in 1001 Genomes, suggesting non-uniform distribution of de novo mutations through the genome. CONCLUSION: Collectively, our results suggest that elevated temperatures can accelerate the accumulation, and alter the molecular profiles, of DNA mutations in plants, thus providing significant insight into how environmental temperatures fuel plant evolution.

Integrating transcriptome, co-expression and QTL-seq analysis reveals that primary root growth in maize is regulated via flavonoid biosynthesis and auxin signal transduction.

The primary root is critical for early seedling growth and survival. To understand the molecular mechanisms governing primary root development, we performed a dynamic transcriptome analysis of two maize (Zea mays) inbred lines with contrasting primary root length at nine time points over a 12-day period. A total of 18 702 genes were differentially expressed between two lines or different time points. Gene enrichment, phytohormone content determination, and metabolomics analysis showed that auxin biosynthesis and signal transduction, as well as the phenylpropanoid and flavonoid biosynthesis pathways, were associated with root development. Co-expression network analysis revealed that eight modules were associated with lines/stages, as well as primary or lateral root length. In root-related modules, flavonoid metabolism accompanied by auxin biosynthesis and signal transduction constituted a complex gene regulatory network during primary root development. Two candidate genes (rootless concerning crown and seminal roots, rtcs and Zm00001d012781) involved in auxin signaling and flavonoid biosynthesis were identified by co-expression network analysis, QTL-seq and functional annotation. These results increase our understanding of the regulatory network controlling the development of primary and lateral root length, and provide a valuable genetic resource for improvement of root performance in maize.

Genetic control of root plasticity in response to salt stress in maize.

KEY MESSAGE: GWAS identified 559 significant SNPs associated with the remodelling of the root architecture in response to salt, and 168 candidate genes were prioritized by integrating RNA-seq, DEG and WGCNA data. Salinity is a major environmental factor limiting crop growth and productivity. The root is the first plant organ to encounter salt stress, yet the effects of salinity on maize root development remain unclear. In this study, the natural variations in 14 root and 4 shoot traits were evaluated in 319 maize inbred lines under control and saline conditions. Considerable phenotypic variations were observed for all traits, with high salt concentrations decreasing the root length, but increasing the root diameter. A genome-wide association study was conducted to analyse these traits and their plasticity (relative variation). We detected 559 significant single nucleotide polymorphisms, of which 125, 181 and 253 were associated with the control condition, stress condition and trait plasticity, respectively. A total of 168 of 587 candidate genes identified by genome-wide association study were supported by the differentially expressed genes or co-expression networks. Two candidate genes ZmIAA1 and ZmGRAS43 were validated by resequencing. Among these genes, 130 were detected under stress condition or trait plasticity that involved in diverse biological processes including plant hormone signal transduction, phenylpropanoid biosynthesis and fatty acid biosynthesis. Our findings clarify the root remodelling to salinity, and the identified loci and candidate genes may be important for the genetic improvement of root traits and salt tolerance in maize.

Incorporation of parental phenotypic data into multi-omic models improves prediction of yield-related traits in hybrid rice.

Hybrid breeding has been shown to effectively increase rice productivity. However, identifying desirable hybrids out of numerous potential combinations is a daunting challenge. Genomic selection holds great promise for accelerating hybrid breeding by enabling early selection before phenotypes are measured. With the recent advances in multi-omic technologies, hybrid prediction based on transcriptomic and metabolomic data has received increasing attention. However, the current omic-based hybrid prediction has ignored parental phenotypic information, which is of fundamental importance in plant breeding. In this study, we integrated parental phenotypic information into various multi-omic prediction models applied in hybrid breeding of rice and compared the predictabilities of 15 combinations from four sets of predictors from the parents, that is genome, transcriptome, metabolome and phenome. The predictability for each combination was evaluated using the best linear unbiased prediction and a modified fast HAT method. We found significant interactions between predictors and traits in predictability, but joint prediction with various combinations of the predictors significantly improved predictability relative to prediction of any single source omic data for each trait investigated. Incorporation of parental phenotypic data into various omic predictors increased the predictability, averagely by 13.6%, 54.5%, 19.9% and 8.3%, for grain yield, number of tillers per plant, number of grains per panicle and 1000 grain weight, respectively. Among nine models of incorporating parental traits, the AD-All model was the most effective one. This novel strategy of incorporating parental phenotypic data into multi-omic prediction is expected to improve hybrid breeding progress, especially with the development of high-throughput phenotyping technologies.

Adaptive innovation of green plants by horizontal gene transfer.

Horizontal gene transfer (HGT) refers to the movement of genetic material between distinct species by means other than sexual reproduction. HGT has contributed tremendously to the genome plasticity and adaptive evolution of prokaryotes and certain unicellular eukaryotes. The evolution of green plants from chlorophyte algae to angiosperms and from water to land represents a process of adaptation to diverse environments, which has been facilitated by acquisition of genetic material from other organisms. In this article, we review the occurrence of HGT in major lineages of green plants, including chlorophyte and charophyte green algae, bryophytes, lycophytes, ferns, and seed plants. In addition, we discuss the significance of horizontally acquired genes in the adaptive innovations of green plants and their potential applications to crop breeding and improvement.

Natural Variation and Domestication Selection of ZmCKX5 with Root Morphological Traits at the Seedling Stage in Maize.

Root system architecture plays a crucial role in water and nutrient acquisition in maize. Cytokinins, which can be irreversibly degraded by the cytokinin oxidase/dehydrogenase (CKX), are important hormones that regulate root development in plants. In this study, ZmCKX5 was resequenced in 285 inbred lines, 68 landraces, and 32 teosintes to identify the significant variants associated with root traits in maize. Sequence polymorphisms and nucleotide diversity revealed that ZmCKX5 might be selected during domestication and improvement processes. Marker-trait association analysis in inbred lines identified 12 variants of ZmCKX5 that were significantly associated with six root traits, including seed root number (SRN), lateral root length (LRL), total root area (RA), root length in 0 to 0.5 mm diameter class (RL005), total root volume (RV), and total root length (TRL). SNP-1195 explained the most (6.01%) phenotypic variation of SRN, and the frequency of this allele G increased from 6.25% and 1.47% in teosintes and landraces, respectively, to 17.39% in inbred lines. Another significant variant, SNP-1406, with a pleiotropic effect, is strongly associated with five root traits, with the frequency of T allele increased from 25.00% and 23.73% in teosintes and landraces, respectively, to 35.00% in inbred lines. These results indicate that ZmCKX5 may be involved in the development of the maize root system and that the significant variants can be used to develop functional markers to accelerate the improvement in the maize root system.

Inference of Chromosome-Length Haplotypes Using Genomic Data of Three or a Few More Single Gametes.

Compared with genomic data of individual markers, haplotype data provide higher resolution for DNA variants, advancing our knowledge in genetics and evolution. Although many computational and experimental phasing methods have been developed for analyzing diploid genomes, it remains challenging to reconstruct chromosome-scale haplotypes at low cost, which constrains the utility of this valuable genetic resource. Gamete cells, the natural packaging of haploid complements, are ideal materials for phasing entire chromosomes because the majority of the haplotypic allele combinations has been preserved. Therefore, compared with the current diploid-based phasing methods, using haploid genomic data of single gametes may substantially reduce the complexity in inferring the donor's chromosomal haplotypes. In this study, we developed the first easy-to-use R package, Hapi, for inferring chromosome-length haplotypes of individual diploid genomes with only a few gametes. Hapi outperformed other phasing methods when analyzing both simulated and real single gamete cell sequencing data sets. The results also suggested that chromosome-scale haplotypes may be inferred by using as few as three gametes, which has pushed the boundary to its possible limit. The single gamete cell sequencing technology allied with the cost-effective Hapi method will make large-scale haplotype-based genetic studies feasible and affordable, promoting the use of haplotype data in a wide range of research.

Genetic and genomic analysis of the seed-filling process in maize based on a logistic model.

Seed filling is a dynamic process that determines seed size and nutritional quality. This time-dependent trait follows a logistic (S-shaped) growth curve that can be described by a logistic function, with parameters of biological relevance. When compared between genotypes, the filling dynamics variations are explained by the differences of parameter values; as such, the parameter estimates can be considered as "traits" for genetic analysis to identify loci that are associated with the seed-filling process. We carried out genetic and genomic analysis of the seed-filling process in maize, using a recombinant inbred line (RIL) population derived from the two inbred lines with contrasting seed-filling dynamics. We recorded seed dry weight at 14 time points after pollination, spanning the early filling phases to the late maturation stages. Fitting these data to a logistic model allowed for estimating 12 characteristic parameters that can be used to meaningfully describe the seed-filling process. Quantitative trait locus (QTL) mapping of these parameters identified a total of 90 nonredundant loci. Using bulked segregant RNA-sequencing (BSR-seq) analysis, we identified eight genes that showed differential gene expression patterns at multiple time points between the extreme pools, and these genes co-localize with the mapped QTL regions. Two of the eight genes, GRMZM2G391936 and GRMZM2G008263, are implicated in starch and sucrose metabolism, and biosynthesis of secondary metabolites that are well known for playing a vital role in seed filling. This study suggests that the logistic model-based approach can efficiently identify genetic loci that regulate dynamic developing traits.

Integrating GWAS and Gene Expression Analysis Identifies Candidate Genes for Root Morphology Traits in Maize at the Seedling Stage.

Root system plays an essential role in water and nutrient acquisition in plants. Understanding the genetic basis of root development will be beneficial for breeding new cultivars with efficient root system to enhance resource use efficiency in maize. Here, the natural variation of 13 root and 3 shoot traits was evaluated in 297 maize inbred lines and genome-wide association mapping was conducted to identify SNPs associated with target traits. All measured traits exhibited 2.02- to 21.36-fold variations. A total of 34 quantitative trait loci (QTLs) were detected for 13 traits, and each individual QTL explained 5.7% to 15.9% of the phenotypic variance. Three pleiotropic QTLs involving five root traits were identified; SNP_2_104416607 was associated with lateral root length (LRL), root surface area (RA), root length between 0 and 0.5mm in diameter (RL005), and total root length (TRL); SNP_2_184016997 was associated with RV and RA, and SNP_4_168917747 was associated with LRL, RA and TRL. The expression levels of candidate genes in root QTLs were evaluated by RNA-seq among three long-root lines and three short-root lines. A total of five genes that showed differential expression between the long- and short-root lines were identified as promising candidate genes for the target traits. These QTLs and the potential candidate genes are important source data to understand root development and genetic improvement of root traits in maize.