Utilizing machine learning and bioinformatics analysis to identify drought-responsive genes affecting yield in foxtail millet.

Drought stress is a major constraint on crop development, potentially causing huge yield losses and threatening global food security. Improving Crop's stress tolerance is usually associated with a yield penalty. One way to balance yield and stress tolerance is modification specific gene by emerging precision genome editing technology. However, our knowledge of yield-related drought-tolerant genes is still limited. Foxtail millet (Setaria italica) has a remarkable tolerance to drought and is considered to be a model C4 crop that is easy to engineer. Here, we have identified 46 drought-responsive candidate genes by performing a machine learning-based transcriptome study on two drought-tolerant and two drought-sensitive foxtail millet cultivars. A total of 12 important drought-responsive genes were screened out by principal component analysis and confirmed experimentally by qPCR. Significantly, by investigating the haplotype of these genes based on 1844 germplasm resources, we found two genes (Seita.5G251300 and Seita.8G036300) exhibiting drought-tolerant haplotypes that possess an apparent advantage in 1000 grain weight and main panicle grain weight without penalty in grain weight per plant. These results demonstrate the potential of Seita.5G251300 and Seita.8G036300 for breeding drought-tolerant high-yielding foxtail millet. It provides important insights for the breeding of drought-tolerant high-yielding crop cultivars through genetic manipulation technology.

Proteomic analysis of leaves and roots during drought stress and recovery in Setaria italica L.

Drought is a major environmental factor that limits agricultural crop productivity and threatens food security. Foxtail millet is a model crop with excellent abiotic stress tolerance and is consequently an important subject for obtaining a better understanding of the molecular mechanisms underlying plant responses to drought and recovery. Here the physiological and proteomic responses of foxtail millet (cultivar Yugu1) leaves and roots to drought treatments and recovery were evaluated. Drought-treated foxtail millet exhibited increased relative electrolyte leakage and decreased relative water content and chlorophyll content compared to control and rewatering plants. A global analysis of protein profiles was evaluated for drought-treated and recovery treatment leaves and roots. We also identified differentially abundant proteins in drought and recovery groups, enabling comparisons between leaf and root tissue responses to the conditions. The principal component analysis suggested a clear distinction between leaf and root proteomes for the drought-treated and recovery treatment plants. Gene Ontology enrichment and co-expression analyses indicated that the biological responses of leaves differed from those in roots after drought and drought recovery. These results provide new insights and data resources to investigate the molecular basis of tissue-specific functional responses of foxtail millet during drought and recovery, thereby significantly informing crop breeding.

Molecular evolution, diversification, and expression assessment of MADS gene family in Setaria italica, Setaria viridis, and Panicum virgatum.

KEY MESSAGE: This paper sheds light on the evolution and expression patterns of MADS genes in Setaria and Panicum virgatum. SiMADS51 and SiMADS64 maybe involved in the ABA-dependent pathway of drought response. The MADS gene family is a key regulatory factor family that controls growth, reproduction, and response to abiotic stress in plants. However, the molecular evolution of this family is rarely reported. Here, a total of 265 MADS genes were identified in Setaria italica (foxtail millet), Setaria viridis (green millet), and Panicum virgatum (switchgrass) and analyzed by bioinformatics, including physicochemical characteristics, subcellular localization, chromosomal position and duplicate, motif distribution, genetic structure, genetic evolvement, and expression patterns. Phylogenetic analysis was used to categorize these genes into M and MIKC types. The distribution of motifs and gene structure were similar for the corresponding types. According to a collinearity study, the MADS genes have been mostly conserved during evolution. The principal cause of their expansion is segmental duplication. However, the MADS gene family tends to shrink in foxtail millet, green millet, and switchgrass. The MADS genes were subjected to purifying selection, but several positive selection sites were also identified in three species. And most of the promoters of MADS genes contain cis-elements related to stress and hormonal response. RNA-seq and quantitative Real-time PCR (qRT-PCR) analysis also were examined. SiMADS genes expression levels are considerably changed in reaction to various treatments, following qRT-PCR analysis. This sheds fresh light on the evolution and expansion of the MADS family in foxtail millet, green millet, and switchgrass, and lays the foundation for further research on their functions.

Effects of temperature and microwave on the stability of the blast effector complex APikL2A/sHMA25 as determined by molecular dynamics analyses.

Magnaporthe oryzae is the causal agent of rice blast, and understanding how abiotic stress affects the resistance of plants to this disease is useful for designing disease control strategies. In this paper, the effects of temperature and microwave irradiation on the effector complex comprising APikL2A from M. oryzae and sHMA25 from foxtail millet were investigated by molecular dynamics simulations using the GROMACS software package. While the structure of APikL2A/sHMA25 remained relatively stable in a temperature range of 290 K (16.85 °C) to 320 K (46.85 °C), the concave shape of the temperature-dependent binding free energy curve indicated that there was maximum binding affinity between APikL2A and sHMA25 at 300 K-310 K. This coincided with the optimum infectivity temperature, thus suggesting that coupling of the two polypeptides may play a role in the infection process. A strong oscillating electric field destroyed the structure of APikL2A/sHMA25, although it was stable and not susceptible to weak electric fields.

Exploring the SiCCT Gene Family and Its Role in Heading Date in Foxtail Millet.

CCT transcription factors are involved in the regulation of photoperiod and abiotic stress in Arabidopsis and rice. It is not clear that how CCT gene family expand and regulate heading date in foxtail millet. In this study, we conducted a systematic analysis of the CCT gene family in foxtail millet. Thirty-nine CCT genes were identified and divided into four subfamilies based on functional motifs. Analysis showed that dispersed duplication played a predominant role in the expansion of CCT genes during evolution. Nucleotide diversity analysis suggested that genes in CONSTANS (COL)-like, CCT MOTIF FAMILY (CMF)-like, and pseudoresponse response regulator (PRR)-like subfamilies were subjected to selection. Fifteen CCT genes were colocalized with previous heading date quantitative trait loci (QTL) and genome-wide association analysis (GWAS) signals. Transgenic plants were then employed to confirm that overexpression of the CCT gene SiPRR37 delayed the heading date and increased plant height. Our study first investigated the characterization and expansion of the CCT family in foxtail millet and demonstrated the role of SiPRR37. These results lay a significant foundation for further research on the function of CCT genes and provide a cue for the regulation of heading date.

High-depth resequencing of 312 accessions reveals the local adaptation of foxtail millet.

KEY MESSAGE: Based on the high-density variation map, we identified genome-level evidence for local adaptation and demonstrated that Siprr37 with transposon insertion contributes to the fitness of foxtail millet in the northeastern ecoregion. Adaptation is a robust way through which plants are able to overcome environmental constraints. The mechanisms of adaptation in heterogeneous natural environments are largely unknown. Deciphering the genomic basis of local adaptation will contribute to further improvement in domesticated plants. To this end, we describe a high-depth (19.4 ×) haplotype map of 3.02 million single nucleotide polymorphisms in foxtail millet (Setaria italica) from whole-genome resequencing of 312 accessions. In the genome-wide scan, we identified a set of improvement signals (including the homologous gene of OsIPA1, a key gene controlling ideal plant architecture) related to the geographical adaptation to four ecoregions in China. In particular, based on the genome-wide association analysis results, we identified the contribution of a pseudo-response regulator gene, SiPRR37, to heading date adaptation in foxtail millet. We observed the expression changes of SiPRR37 resulted from a key Tc1-Mariner transposon insertion in the first intron. Positive selection analyses revealed that SiPRR37 mainly contributed to the adaptation of northeastern ecoregions. Taken together, foxtail millet adapted to the northeastern region by regulating the function of SiPRR37, which sheds lights on genome-level evidence for adaptive geographical divergence. Besides, our data provide a nearly complete catalog of genomic variation aiding the identification of functionally important variants.

Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

Expressing an (E)-ß-farnesene synthase in the chloroplast of tobacco affects the preference of green peach aphid and its parasitoid.

(E)-ß-Farnesene (EßF) synthase catalyses the production of EßF, which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids' natural enemies. Many plants possess EßF synthase genes and can release EßF to repel aphids. In order to effectively recruit the plant-derived EßF synthase genes for aphid control, by using chloroplast transit peptide (CTP) of the small subunit of Rubisco (rbcS) from wheat (Triticum aestivum L.), we targeted AaßFS1, an EßF synthase gene from sweet wormwood (Artemisia annua L.), to the chloroplast of tobacco to generate CTP + AaßFS1 transgenic lines. The CTP + AaßFS1 transgenic tobacco plants could emit EßF at a level up to 19.25 ng/day per g fresh tissues, 4-12 fold higher than the AaßFS1 transgenic lines without chloroplast targeting. Furthermore, aphid/parasitoid behavioral bioassays demonstrated that the CTP + AaßFS1 transgenic tobacco showed enhanced repellence to green peach aphid (Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae, thus affecting aphid infestation at two trophic levels. These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EßF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.

Engineering plants for aphid resistance: current status and future perspectives.

KEY MESSAGE: The current status of development of transgenic plants for improved aphid resistance, and the pros and cons of different strategies are reviewed and future perspectives are proposed. Aphids are major agricultural pests that cause significant yield losses of crop plants each year. Excessive dependence on insecticides for aphid control is undesirable because of the development of insecticide resistance, the potential negative effects on non-target organisms and environmental pollution. Transgenic plants engineered for resistance to aphids via a non-toxic mode of action could be an efficient alternative strategy. In this review, the distribution of major aphid species and their damages on crop plants, the so far isolated aphid-resistance genes and their applications in developments of transgenic plants for improved aphid resistance, and the pros and cons of these strategies are reviewed and future perspectives are proposed. Although the transgenic plants developed through expressing aphid-resistant genes, manipulating plant secondary metabolism and plant-mediated RNAi strategy have been demonstrated to confer improved aphid resistance to some degree. So far, no aphid-resistant transgenic crop plants have ever been commercialized. This commentary is intended to be a helpful insight into the generation and future commercialization of aphid-resistant transgenic crops in a global context.

(E)-ß-farnesene synthase genes affect aphid (Myzus persicae) infestation in tobacco (Nicotiana tabacum).

Aphids are major agricultural pests which cause significant yield losses of the crop plants each year. (E)-ß-farnesene (EßF) is the alarm pheromone involved in the chemical communication between aphids and particularly in the avoidance of predation. In the present study, two EßF synthase genes were isolated from sweet wormwood and designated as AaßFS1 and AaßFS2, respectively. Overexpression of AaßFS1 or AaßFS2 in tobacco plants resulted in the emission of EßF ranging from 1.55 to 4.65 ng/day/g fresh tissues. Tritrophic interactions involving the peach aphids (Myzus persicae), predatory lacewings (Chrysopa septempunctata) demonstrated that the transgenic tobacco expressing AaßFS1 and AaßFS2 could repel peach aphids, but not as strongly as expected. However, AaßFS1 and AaßFS2 lines exhibited strong and statistically significant attraction to lacewings. Further experiments combining aphids and lacewing larvae in an octagon arrangement showed transgenic tobacco plants could repel aphids and attract lacewing larvae, thus minimizing aphid infestation. Therefore, we demonstrated a potentially valuable strategy of using EßF synthase genes from sweet wormwood for aphid control in tobacco or other economic important crops in an environmentally benign way.