The APETALA2/ethylene-responsive element binding factor (AP2/ERF) family is one of the largest transcription factor (TF) families in plants that includes four major sub-families, namely AP2, DREB (dehydration responsive element binding), ERF (ethylene responsive factors) and RAV (Related to ABI3/VP). AP2/ERFs are known to play significant roles in various plant processes including growth and development and biotic and abiotic stress responses. Considering this, a comprehensive genome-wide study was conducted in foxtail millet (Setaria italica L.). A total of 171 AP2/ERF genes were identified by systematic sequence analysis and were physically mapped onto nine chromosomes. Phylogenetic analysis grouped AP2/ERF genes into six classes (I to VI). Duplication analysis revealed that 12 (∼7%) SiAP2/ERF genes were tandem repeated and 22 (∼13%) were segmentally duplicated. Comparative physical mapping between foxtail millet AP2/ERF genes and its orthologs of sorghum (18 genes), maize (14 genes), rice (9 genes) and Brachypodium (6 genes) showed the evolutionary insights of AP2/ERF gene family and also the decrease in orthology with increase in phylogenetic distance. The evolutionary significance in terms of gene-duplication and divergence was analyzed by estimating synonymous and non-synonymous substitution rates. Expression profiling of candidate AP2/ERF genes against drought, salt and phytohormones revealed insights into their precise and/or overlapping expression patterns which could be responsible for their functional divergence in foxtail millet. The study showed that the genes SiAP2/ERF-069, SiAP2/ERF-103 and SiAP2/ERF-120 may be considered as potential candidate genes for further functional validation as well for utilization in crop improvement programs for stress resistance since these genes were up-regulated under drought and salinity stresses in ABA dependent manner. Altogether the present study provides new insights into evolution, divergence and systematic functional analysis of AP2/ERF gene family at genome level in foxtail millet which may be utilized for improving stress adaptation and tolerance in millets, cereals and bioenergy grasses.

C2H2 type of zinc finger transcription factors (TFs) play crucial roles in plant stress response and hormone signal transduction. Hence considering its importance, genome-wide investigation and characterization of C2H2 zinc finger proteins were performed in Arabidopsis, rice and poplar but no such study was conducted in foxtail millet which is a C4 Panicoid model crop well known for its abiotic stress tolerance. The present study identified 124 C2H2-type zinc finger TFs in foxtail millet (SiC2H2) and physically mapped them onto the genome. The gene duplication analysis revealed that SiC2H2s primarily expanded in the genome through tandem duplication. The phylogenetic tree classified these TFs into five groups (I-V). Further, miRNAs targeting SiC2H2 transcripts in foxtail millet were identified. Heat map demonstrated differential and tissue-specific expression patterns of these SiC2H2 genes. Comparative physical mapping between foxtail millet SiC2H2 genes and its orthologs of sorghum, maize and rice revealed the evolutionary relationships of C2H2 type of zinc finger TFs. The duplication and divergence data provided novel insight into the evolutionary aspects of these TFs in foxtail millet and related grass species. Expression profiling of candidate SiC2H2 genes in response to salinity, dehydration and cold stress showed differential expression pattern of these genes at different time points of stresses.

MYB proteins represent one of the largest transcription factor families in plants, playing important roles in diverse developmental and stress-responsive processes. Considering its significance, several genome-wide analyses have been conducted in almost all land plants except foxtail millet. Foxtail millet (Setaria italica L.) is a model crop for investigating systems biology of millets and bioenergy grasses. Further, the crop is also known for its potential abiotic stress-tolerance. In this context, a comprehensive genome-wide survey was conducted and 209 MYB protein-encoding genes were identified in foxtail millet. All 209 S. italica MYB (SiMYB) genes were physically mapped onto nine chromosomes of foxtail millet. Gene duplication study showed that segmental- and tandem-duplication have occurred in genome resulting in expansion of this gene family. The protein domain investigation classified SiMYB proteins into three classes according to number of MYB repeats present. The phylogenetic analysis categorized SiMYBs into ten groups (I - X). SiMYB-based comparative mapping revealed a maximum orthology between foxtail millet and sorghum, followed by maize, rice and Brachypodium. Heat map analysis showed tissue-specific expression pattern of predominant SiMYB genes. Expression profiling of candidate MYB genes against abiotic stresses and hormone treatments using qRT-PCR revealed specific and/or overlapping expression patterns of SiMYBs. Taken together, the present study provides a foundation for evolutionary and functional characterization of MYB TFs in foxtail millet to dissect their functions in response to environmental stimuli.

Transposable elements (TEs) are major components of plant genome and are reported to play significant roles in functional genome diversity and phenotypic variations. Several TEs are highly polymorphic for insert location in the genome and this facilitates development of TE-based markers for various genotyping purposes. Considering this, a genome-wide analysis was performed in the model plant foxtail millet. A total of 30,706 TEs were identified and classified as DNA transposons (24,386), full-length Copia type (1,038), partial or solo Copia type (10,118), full-length Gypsy type (1,570), partial or solo Gypsy type (23,293) and Long- and Short-Interspersed Nuclear Elements (3,659 and 53, respectively). Further, 20,278 TE-based markers were developed, namely Retrotransposon-Based Insertion Polymorphisms (4,801, ∼24%), Inter-Retrotransposon Amplified Polymorphisms (3,239, ∼16%), Repeat Junction Markers (4,451, ∼22%), Repeat Junction-Junction Markers (329, ∼2%), Insertion-Site-Based Polymorphisms (7,401, ∼36%) and Retrotransposon-Microsatellite Amplified Polymorphisms (57, 0.2%). A total of 134 Repeat Junction Markers were screened in 96 accessions of Setaria italica and 3 wild Setaria accessions of which 30 showed polymorphism. Moreover, an open access database for these developed resources was constructed (Foxtail millet Transposable Elements-based Marker Database; ). Taken together, this study would serve as a valuable resource for large-scale genotyping applications in foxtail millet and related grass species.

Bambara groundnut (Vigna subterranea (L.) Verdc.) is an African legume and is a promising underutilized crop with good seed nutritional values. Low temperature stress in a number of African countries at night, such as Botswana, can effect the growth and development of bambara groundnut, leading to losses in potential crop yield. Therefore, in this study we developed a computational pipeline to identify and analyze the genes and gene modules associated with low temperature stress responses in bambara groundnut using the cross-species microarray technique (as bambara groundnut has no microarray chip) coupled with network-based analysis. Analyses of the bambara groundnut transcriptome using cross-species gene expression data resulted in the identification of 375 and 659 differentially expressed genes (p<0.01) under the sub-optimal (23°C) and very sub-optimal (18°C) temperatures, respectively, of which 110 genes are commonly shared between the two stress conditions. The construction of a Highest Reciprocal Rank-based gene co-expression network, followed by its partition using a Heuristic Cluster Chiseling Algorithm resulted in 6 and 7 gene modules in sub-optimal and very sub-optimal temperature stresses being identified, respectively. Modules of sub-optimal temperature stress are principally enriched with carbohydrate and lipid metabolic processes, while most of the modules of very sub-optimal temperature stress are significantly enriched with responses to stimuli and various metabolic processes. Several transcription factors (from MYB, NAC, WRKY, WHIRLY & GATA classes) that may regulate the downstream genes involved in response to stimulus in order for the plant to withstand very sub-optimal temperature stress were highlighted. The identified gene modules could be useful in breeding for low-temperature stress tolerant bambara groundnut varieties.

14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides interesting information on their gene structure, protein domains, phylogenetic and evolutionary relationships, and expression patterns during abiotic stresses and hormonal treatments, which could be useful in choosing candidate members for further functional characterization. In addition, demonstration of interaction between Si14-3-3 and SiRSZ21A provides novel clues on the involvement of 14-3-3 proteins in the splicing events.

No abstract

Foxtail millet has recently been regarded as a model crop for studying the systems biology of millets and bioenergy grass species. For expediting the functional genomic studies in this model crop as well as in the related millets and bioenergy grasses, we have developed a comprehensive transcription factor database. Our foxtail millet transcription factors database (FmTFDb: http://59.163.192.91/FmTFDb/index.html ) encompasses 2,297 putative TFs in 55 families along with its sequence features, chromosomal locations, tissue-specific gene expression data, gene ontology (GO) assignment, and phylogeny. FmTFDb is intended to provide the users an unrestricted public access in retrieving and visualizing the individual members of a TF family through a set of query interfaces and analysis tools, including the BLAST search, annotation query interfaces, and tools to identify enriched GO terms and to visualize physical maps. This FmTFDb will serve as a promising central resource for researchers as well as breeders who are dedicated towards crop improvement of millets and bioenergy grasses.