Dissecting the molecular basis of spike traits by integrating gene regulatory networks and genetic variation in wheat.

Spike architecture influences both grain weight and grain number per spike, which are the two major components of grain yield in bread wheat (Triticum aestivum L.). However, the complex wheat genome and the influence of various environmental factors pose challenges in mapping the causal genes that affect spike traits. Here, we systematically identified genes involved in spike trait formation by integrating information on genomic variation and gene regulatory networks controlling young spike development in wheat. We identified 170 loci that are responsible for variations in spike length, spikelet number per spike, and grain number per spike through genome-wide association study and meta-QTL analyses. We constructed gene regulatory networks for young inflorescences at the double ridge stage and the floret primordium stage, in which the spikelet meristem and the floret meristem are predominant, respectively, by integrating transcriptome, histone modification, chromatin accessibility, eQTL, and protein-protein interactome data. From these networks, we identified 169 hub genes located in 76 of the 170 QTL regions whose polymorphisms are significantly associated with variation in spike traits. The functions of TaZF-B1, VRT-B2, and TaSPL15-A/D in establishment of wheat spike architecture were verified. This study provides valuable molecular resources for understanding spike traits and demonstrates that combining genetic analysis and developmental regulatory networks is a robust approach for dissection of complex traits.

Nutrient Metabolism Pathways Analysis and Key Candidate Genes Identification Corresponding to Cadmium Stress in Buckwheat through Multiomics Analysis.

Fagopylum tatarium (L.) Gaertn (buckwheat) can be used both as medicine and food and is also an important food crop in barren areas and has great economic value. Exploring the molecular mechanisms of the response to cadmium (Cd) stress can provide the theoretical reference for improving the buckwheat yield and quality. In this study, perennial tartary buckwheat DK19 was used as the experimental material, its key metabolic pathways in the response to Cd stress were identified and verified through transcriptomic and metabolomic data analysis. In this investigation, 1798 metabolites were identified through non-targeted metabolomic analysis containing 1091 up-regulated and 984down-regulated metabolites after treatment. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differential metabolites was significantly enriched in galactose metabolism, glycerol metabolism, phenylpropane biosynthesis, glutathione metabolism, starch and sucrose metabolism. Linkage analysis detected 11 differentially expressed genes (DEGs) in the galactose metabolism pathway, 8 candidate DEGs in the lipid metabolism pathway, and 20 candidate DEGs in the glutathione metabolism pathway. The results of our study provided useful clues for genetically improving the resistance to cadmium by analyzing the molecular mechanism of cadmium tolerance in buckwheat.

Analysis of Hormone Regulation on Seed Germination of Coix Based on Muli-Omics Analysis.

Seed germination is an important stage of growth and reproduction and plays an important role in the life cycle of spermatophyte. It is co-determined by both genetic and environmental factors, and plant hormone regulation may be a highly conservative mechanism. Coix lachryrma-jobi (coix) is a grain with balanced nutrition for medicine and food and has substantial production value. It is an important part of agricultural production, and the efficiency of seed germination after sowing is a key link. In this study, coix species "small white shell Xingren" was used as the experimental material, and changes in gene expression levels and metabolite enrichment in seeds were identified by transcriptome and metabonomic analysis before and after seed germination. A total of 599 metabolites, including those from amino acid metabolism, sugar metabolism, and fatty acid metabolism, were significantly increased in germinating coix. Simultaneously, 10,929 differentially expressed genes (DEGs) were identified, and functional clusters of genes were also significantly clustered in hormone-signaling and glucose and fatty acid metabolism. In addition, this study found that a considerable number of hormone-signaling genes were significantly up-regulated during seed germination, activating multiple metabolic processes. The results of our conjoint analysis of multi omics showed that glucose and fatty acid metabolism played an important role in seed germination under hormone regulation.

Integrated transcriptome and metabonomic analysis of key metabolic pathways in response to cadmium stress in novel buckwheat and cultivated species.

Introduction: Buckwheat (Fagopyrum tataricum), an important food crop, also has medicinal uses. It is widely planted in Southwest China, overlapping with planting areas remarkably polluted by cadmium (Cd). Therefore, it is of great significance to study the response mechanism of buckwheat under Cd stress and further develop varieties with excellent Cd tolerance. Methods: In this study, two critical periods of Cd stress treatment (days 7 and 14 after Cd treatment) of cultivated buckwheat (Pinku-1, named K33) and perennial species (F. tatari-cymosum Q.F. Chen) (duoku, named DK19) were analyzed using transcriptome and metabolomics. Results: The results showed that Cd stress led to changes in reactive oxygen species (ROS) and the chlorophyll system. Moreover, Cd-response genes related to stress response, amino acid metabolism, and ROS scavenging were enriched or activated in DK19. Transcriptome and metabolomic analyses highlighted the important role of galactose, lipid (glycerophosphatide metabolism and glycerophosphatide metabolism), and glutathione metabolism in response to Cd stress in buckwheat, which are significantly enriched at the gene and metabolic levels in DK19. Discussion: The results of the present study provide valuable information for a better understanding of the molecular mechanisms underlying Cd tolerance in buckwheat and useful clues for the genetic improvement of drought tolerance in buckwheat.

A multi-omics integrative network map of maize.

Networks are powerful tools to uncover functional roles of genes in phenotypic variation at a system-wide scale. Here, we constructed a maize network map that contains the genomic, transcriptomic, translatomic and proteomic networks across maize development. This map comprises over 2.8 million edges in more than 1,400 functional subnetworks, demonstrating an extensive network divergence of duplicated genes. We applied this map to identify factors regulating flowering time and identified 2,651 genes enriched in eight subnetworks. We validated the functions of 20 genes, including 18 with previously unknown connections to flowering time in maize. Furthermore, we uncovered a flowering pathway involving histone modification. The multi-omics integrative network map illustrates the principles of how molecular networks connect different types of genes and potential pathways to map a genome-wide functional landscape in maize, which should be applicable in a wide range of species.

Ectopic Expression of the Rice Grain-Size-Affecting Gene GS5 in Maize Affects Kernel Size by Regulating Endosperm Starch Synthesis.

Maize is one of the most important food crops, and maize kernel is one of the important components of maize yield. Studies have shown that the rice grain-size affecting gene GS5 increases the thousand-kernel weight by positively regulating the rice grain width and grain grouting rate. In this study, based on the GS5 transgenic maize obtained through transgenic technology with specific expression in the endosperm, molecular assays were performed on the transformed plants. Southern blotting results showed that the GS5 gene was integrated into the maize genome in a low copy number, and RT-PCR analysis showed that the exogenous GS5 gene was normally and highly expressed in maize. The agronomic traits of two successive generations showed that certain lines were significantly improved in yield-related traits, and the most significant changes were observed in the OE-34 line, where the kernel width increased significantly by 8.99% and 10.96%, the 100-kernel weight increased by 14.10% and 10.82%, and the ear weight increased by 13.96% and 15.71%, respectively; however, no significant differences were observed in the plant height, ear height, kernel length, kernel row number, or kernel number. In addition, the overexpression of the GS5 gene increased the grain grouting rate and affected starch synthesis in the rice grains. The kernels' starch content in OE-25, OE-34, and OE-57 increased by 10.30%, 7.39%, and 6.39%, respectively. Scanning electron microscopy was performed to observe changes in the starch granule size, and the starch granule diameter of the transgenic line(s) was significantly reduced. RT-PCR was performed to detect the expression levels of related genes in starch synthesis, and the expression of these genes was generally upregulated. It was speculated that the exogenous GS5 gene changed the size of the starch granules by regulating the expression of related genes in the starch synthesis pathway, thus increasing the starch content. The trans-GS5 gene was able to be stably expressed in the hybrids with the genetic backgrounds of the four materials, with significant increases in the kernel width, 100-kernel weight, and ear weight. In this study, the maize kernel size was significantly increased through the endosperm-specific expression of the rice GS5 gene, and good material for the functional analysis of the GS5 gene was created, which was of great importance in theory and application.

Dissecting the Genetic Basis of Flowering Time and Height Related-Traits Using Two Doubled Haploid Populations in Maize.

In the field, maize flowering time and height traits are closely linked with yield, planting density, lodging resistance, and grain fill. To explore the genetic basis of flowering time and height traits in maize, we investigated six related traits, namely, days to anthesis (AD), days to silking (SD), the anthesis-silking interval (ASI), plant height (PH), ear height (EH), and the EH/PH ratio (ER) in two locations for two years based on two doubled haploid (DH) populations. Based on the two high-density genetic linkage maps, 12 and 22 quantitative trait loci (QTL) were identified, respectively, for flowering time and height-related traits. Of these, ten QTLs had overlapping confidence intervals between the two populations and were integrated into three consensus QTLs (qFT_YZ1a, qHT_YZ5a, and qHT_YZ7a). Of these, qFT_YZ1a, conferring flowering time, is located at 221.1-277.0 Mb on chromosome 1 and explained 10.0-12.5% of the AD and SD variation, and qHT_YZ5a, conferring height traits, is located at 147.4-217.3 Mb on chromosome 5 and explained 11.6-15.3% of the PH and EH variation. These consensus QTLs, in addition to the other repeatedly detected QTLs, provide useful information for further genetic studies and variety improvements in flowering time and height-related traits.

Genomic Prediction across Structured Hybrid Populations and Environments in Maize.

Genomic prediction (GP) across different populations and environments should be enhanced to increase the efficiency of crop breeding. In this study, four populations were constructed and genotyped with DNA chips containing 55,000 SNPs. These populations were testcrossed to a common tester, generating four hybrid populations. Yields of the four hybrid populations were evaluated in three environments. We demonstrated by using real data that the prediction accuracies of GP across structured hybrid populations were lower than those of within-population GP. Including relatives of the validation population in the training population could increase the prediction accuracies of GP across structured hybrid populations drastically. G × E models (including main and genotype-by-environment effect) had better performance than single environment (within environment) and across environment (including only main effect) GP models in the structured hybrid population, especially in the environment where yields had higher heritability. GP by implementing G × E models in two cross-validation schemes indicated that, to increase the prediction accuracy of a new hybrid line, it would be better to field-test the hybrid line in at least one environment. Our results would be helpful for designing training population and planning field testing in hybrid breeding.

Genome-Wide Analysis of the PIN Auxin Efflux Carrier Gene Family in Coffee.

Coffee is one of the most popular beverages around the world, which is mainly produced from the allopolyploid Coffea arabica. The genomes of C. arabica and its two ancestors C. canephora and C. eugenioides have been released due to the development of next generation sequencing. However, few studies on C. arabica are related to the PIN-FORMED (PIN) auxin efflux transporter despite its importance in auxin-mediated plant growth and development. In the present study, we conducted a genome-wide analysis of the PIN gene family in the three coffee species. Totals of 17, 9 and 10 of the PIN members were characterized in C. Arabica, C. canephora and C. eugenioides, respectively. Phylogenetic analysis revealed gene loss of PIN1 and PIN2 homologs in C. arabica, as well as gene duplication of PIN5 homologs during the fractionation process after tetraploidy. Furthermore, we conducted expression analysis of PIN genes in C. arabica by in silico and qRT-PCR. The results revealed the existence of gene expression dominance in allopolyploid coffee and illustrated several PIN candidates in regulating auxin transport and homeostasis under leaf rust fungus inoculation and the tissue-specific expression pattern of C. arabica. Together, this study provides the basis and guideline for future functional characterization of the PIN gene family.

Effects of Nonstarch Genetic Modifications on Starch Structure and Properties.

This paper examines if, in maize, starch structure and starch-dependent properties might be altered by pleiotropic effects arising from genetic modifications that are not directly related to starch synthesis. The molecular structure, specifically the starch chain-length distributions (CLDs), of two maize lines transformed with Bar (bialaphos resistance) and Cry1c genes (an artificial gene, encoding proteinaceous insecticidal δ-endotoxins) were compared to those of their control lines. The two transgenes are responsible for herbicidal resistance and insect tolerance, respectively. The starch CLDs were measured by enzymatic debranching and measuring the molecular weight distributions of the resulting linear chains. It was found that although all the lines had similar amylose contents, the CLDs of both amylopectin and amylose for Cry1c were noticeably different from the others, having more short amylopectin and long amylose chains. These CLDs are known to affect functional properties, and indeed it was found that the Cry1c transgenic lines showed a lower gelatinization temperature and faster digestion rate than the control or Bar lines. However, a slower digestion rate is nutritionally desirable. Thus, pleiotropic effects from genetic modifications can indirectly but significantly affect the starch synthesis pathway and thus change functional properties of significance for human health.

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector.

Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.

Infection of Embryonic Callus with Agrobacterium Enables High-Speed Transformation of Maize.

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium-mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T0 generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.

Dissecting the genetic architecture of waterlogging stress-related traits uncovers a key waterlogging tolerance gene in maize.

KEY MESSAGE: A key candidate gene, GRMZM2G110141, which could be used in marker-assisted selection in maize breeding programs, was detected among the 16 genetic loci associated with waterlogging tolerance identified through genome-wide association study. Waterlogging stress seriously affects the growth and development of upland crops such as maize (Zea mays L.). However, the genetic basis of waterlogging tolerance in crop plants is largely unknown. Here, we identified genetic loci for waterlogging tolerance-related traits by conducting a genome-wide association study using maize phenotypes evaluated in the greenhouse under waterlogging stress and normal conditions. A total of 110 trait-single nucleotide polymorphism associations spanning 16 genomic regions were identified; single associations explained 2.88-10.67% of the phenotypic variance. Among the genomic regions identified, 14 co-localized with previously detected waterlogging tolerance-related quantitative trail loci. Furthermore, 33 candidate genes involved in a wide range of stress-response pathways were predicted. We resequenced a key candidate gene (GRMZM2G110141) in 138 randomly selected inbred lines and found that variations in the 5'-UTR and in the mRNA abundance of this gene under waterlogging conditions were significantly associated with leaf injury. Furthermore, we detected favorable alleles of this gene and validated the favorable alleles in two different recombinant inbred line populations. These alleles enhanced waterlogging tolerance in segregating populations, strongly suggesting that GRMZM2G110141 is a key waterlogging tolerance gene. The set of waterlogging tolerance-related genomic regions and associated markers identified here could be valuable for isolating waterlogging tolerance genes and improving this trait in maize.