Zhengli Liu

Introduction: Spruce spider mite is a primary insect pest of Chinese chestnut in China and seriously influences its yield and quality. However, the current management against this mite is costly and poorly effective. In previous research, we bred several foxtail millet materials for interplanting with chestnut tree, and found that they had high levels of crude protein (CP) in leaves and attracted spruce spider mite to feed on the leaves, thereby reducing chestnut damage.Methods: In this study, four foxtail millet varieties with significant differences in leaf crude protein content were used for high-throughput sequencing and identification of genes associated with leaf crude protein content. Gene enrichment analyses were carried out to comprehend the functions of these genes and the biological processes in which they are involved. In addition, transcription factors (TFs) were evaluated.Results: 435 differentially expressed genes (DEGs) were identified, suggesting their potential role in crude protein accumulation. Some differentially expressed genes were found to be associated with nitrogen metabolism and ubiquitin-mediated proteolysis pathways. Moreover, we identified 40 TF genes categorized into 11 transcription factor families.Discussion: Our findings represent an important resource that clarifies the mechanisms of accumulation and control of leaf crude protein in foxtail millet, and provide an opportunity for suppression of spruce spider mite attack on Chinese chestnut by interplanting with foxtail millet varieties with high concentrations of leaf crude protein.

A stereo interplanting system with foxtail millet beneath chestnut trees is an effective planting method to raise the utilization of land in chestnut orchards, increase yields and improve quality of chestnut nuts. Consequently, exploration of genes involved in shade tolerance response in foxtail millet and breeding shade-tolerant varieties have become urgent issues. In this study, RNA-seq of leaf samples from two shade-tolerant varieties and three shade-intolerant varieties of foxtail millet at the booting stage was performed. Comparisons between the varieties revealed that 70 genes were commonly differentially expressed. Moreover, the ratio of net photosynthetic rate under shaded environment to that under light environment could be used as an indicator of shade tolerance. Subsequently, weighted gene co-expression network analysis was employed to construct a co-expression network and modules were correlated with this ratio. A total of 375 genes were identified as potentially relevant to shade tolerance, among which nine genes were also present in the 70 differentially expressed genes, which implied that they were good candidates for genes involved in shade tolerance. Our results provide valuable resources for elucidation of the molecular mechanisms underlying shade tolerance and will contribute to breeding of shade-tolerant foxtail millet that are adapted to the shaded environment under chestnut trees.

Background: Chinese chestnut is an economically important tree species whose yield and quality are seriously affected by red spider attack. Tannins is one of the most important class secondary metabolites in plants, and is closely associated with plant defense mechanisms against insect and herbivory. In our previous studies, it was revealed that several low-tannin foxtail millet varieties growing under the Chinese chestnut trees could attract red spiders to feed on their leaves and protect the chestnut trees from the infestation of red spiders, meanwhile, the growth and yield of foxtail millet plants themselves were not greatly affected. Results: To identify genes related to leaf tannin content and selection of foxtail millet germplasm resources with low tannin content for interplanting with Chinese chestnut and preventing the red spider attack, the leaves of 4 varieties with different levels of tannin content were harvested for comparative transcriptome analysis. In total, 335 differentially expressed genes (DEGs) were identified. For acquisition of gene functions and biological pathways they involved in, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed, and several DEGs were found to possibly participate in the tannins biosynthesis pathway and transport processes of precursors. In addition, according to the PlantTFDB database, some transcription factors were predicted among the DEGs, suggesting their role in regulation of tannins biosynthesis pathway. Conclusion: Our results provide valuable gene resources for understanding the biosynthesis and regulation mechanisms of tannins in foxtail millet, and pave the way for speeding up the breeding of low-tannin varieties through marker-assisted selection, which could be utilized for interplanting with Chinese chestnut trees to confer protection against red spider attack.

Background: BES/BZR family genes have vital roles in plant growth, development, and adaptation to environmental stimuli. However, they have not yet been characterized and systematically analyzed in wheat and foxtail millet.Results: In the current study, five common and two uniqueBES/BZRgenes were identified by genome-wide analysis in wheat and foxtail millet, respectively. These genes were unevenly distributed on 14 and five chromosomes of wheat and foxtail millet, respectively, and clustered in two subgroups in a phylogenetic analysis. The BES/BZR gene family members in each subgroup contained similar conserved motifs. Investigation ofcis-acting elements and expression profile analysis revealed that theBES/BZRgenes were predominantly expressed in leaf tissues of wheat and foxtail millet seedlings and responded to brassinosteroid, abscisic acid, and NaCl treatments.Conclusions: Our results provide a basis for future studies on the function and molecular mechanisms of the BES/BZR gene family in wheat, foxtail millet, and other plants.

Background: Long noncoding RNAs (lncRNAs) have been reported to play critical roles in diverse growth and development processes in plants. However, the systematic identification and characterization of lncRNAs in foxtail millet is nearly blank.Results: In this study, we performed high-throughput sequencing of young spikelets from four foxtail millet varieties in different yield levels at booting stage. As a result, a total of 12,378 novel lncRNAs were identified, and 70 were commonly significantly differentially expressed in comparisons between high-yield varieties and conventional varieties, suggesting that they involved in yield formation and regulation in foxtail millet. Functional analysis revealed that among the 70 significantly differentially expressed lncRNAs, 67 could transcriptionally modulate target genes in cis and in trans. Moreover, 18 lncRNAs related to grain yield in foxtail millet were predicted to function as miRNA target mimics and regulate gene expression by competing for the interaction between miRNAs and their target mRNAs.Conclusion: Our results will provide materials for elucidation of the molecular mechanisms of lncRNAs participate in yield regulation, and will contribute to high yield foxtail millet breeding.

Due to the maternal inheritance of cytoplasm, using foxtail millet [Setaria italica (L.) P. Beauv.] male sterile lines with a single cytoplasmic source as the female parent will inevitably lead to a narrow source of cytoplasm in hybrids, which may make them vulnerable to infection by cytoplasm-specific pathogens, ultimately leading to destructive yield losses. To assess cytoplasmic genetic diversity in plants, molecular markers derived from chloroplast DNA (cpDNA) have been used. However, such markers have not yet been applied to foxtail millet. In this study, we designed and screened nine pairs of polymorphic foxtail millet-specific primers based on its completely sequenced cpDNA. Using these primers, we analyzed the genetic diversity and cytoplasmic types of 130 elite foxtail millet parental lines collected in China. Our results revealed that the cytoplasmic genetic diversity of these accessions was low and needs to be increased. The parental lines were divided into four cytoplasmic types according to population structure analysis and a female parent-derivative evolutionary graph, indicating that the cytoplasmic types of elite foxtail millet lines were rather limited. A principal component analysis (PCA) plot was linked with the geographic and ecological distribution of accessions for each cytoplasmic type, as well as their basal maternal parents. Collectively, our results suggest that enriching cytoplasmic sources through the use of accessions from diverse ecological regions and other countries as the female parent may improve foxtail millet breeding programs, and prevent infection by cytoplasm-specific pathogens.

The complexity of the leaf constitution of foxtail millet (Setaria italica (L.) P. Beauv.) makes it difficult to obtain high-purity cpDNA. Here, we developed a protocol to isolate high-quality cpDNA from foxtail millet and other crops. The new protocol replaces previous tissue grinding and homogenization by enzyme digestion of tiny leaf strips to separate protoplasts from leaf tissue and protects chloroplasts from damage by undue grinding and homogenization and from contamination of cell debris and nuclear DNA. Using the new protocol, we successfully isolated high-quality cpDNAs for whole-genome sequencing from four foxtail millet cultivars, and comparative analysis revealed that they were approximately 27‰ longer than their reference genome. In addition, six cpDNAs of four other species with narrow and thin leaf blades, including wheat (Triticum aestivum L.), maize (Zea may L.), rice (Oryza sativa L.) and sorghum (Sorghum bicolor (L.) Moench), were also isolated by our new protocol, and they all exhibited high sequence identities to their corresponding reference genomes. A maximum-likelihood tree based on the chloroplast genomes we sequenced here was constructed, and the result was in agreement with previous reports, confirming that these cpDNA sequences were available for well-supported phylogenetic analysis and could provide valuable resources for future research.