Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I–V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses.     

谷子MYB类转录因子SiMYB42提高转基因拟南芥低氮胁迫耐性

Myeloblastosis (MYB)类转录因子是高等植物中最大的转录因子家族之一,在植物发育及防御反应过程中发挥重要作用,还参与植物对干旱等非生物胁迫的响应。谷子(Setaria italica L.)起源于中国,具有抗旱、耐瘠薄的特性,是研究单子叶作物非生物胁迫抗性的理想材料。本研究对耐低氮胁迫谷子品种郑204经低氮处理后进行转录组分析,鉴定出一个在低氮胁迫条件下明显上调的MYB类转录因子SiMYB42。系统发育树结果表明,SiMYB42属于R2R3-MYB亚族,具有2个MYB保守域;表达模式分析显示,SiMYB42在低氮、高盐、干旱和ABA胁迫条件下表达量显著上调;亚细胞定位、quantitative real-time PCR及转录激活活性分析结果表明,SiMYB42蛋白定位于植物的细胞核和细胞膜中,主要在谷子的叶部或根部表达,具有转录激活活性;基因功能分析结果表明,在正常条件下,转SiMYB42基因拟南芥与野生型Columbia-0拟南芥(WT)无明显差异,但在低氮条件下,转SiMYB42基因拟南芥的主根长、根系表面积及鲜重均显著高于WT,结果证明SiMYB42基因可以提高转基因植物对低氮胁迫的耐性;下游基因表达分析结果显示,在转SiMYB42基因拟南芥中,参与植物氮素转运的硝酸盐转运基因NRT2.1、NRT2.4和NRT2.5的表达水平均高于WT,启动子分析结果显示NRT2.1、NRT2.4和NRT2.5基因启动子序列中均具有MYB结合位点。以上结果证明,SiMYB42可以通过调控下游硝酸盐转运体基因的表达提高植物在低氮条件下的耐性。本研究揭示了SiMYB42基因在低氮胁迫反应途径中的作用,为进一步了解谷子低氮胁迫响应的调控网络奠定了基础。     

Unraveling 14-3-3 Proteins in C4 Panicoids with Emphasis on Model Plant Setaria italica Reveals Phosphorylation-Dependent Subcellular Localization of RS Splicing Factor

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.     

Reference genes for quantitative real-time PCR analysis in the model plant foxtail millet (Setaria italica L.) subjected to abiotic stress conditions

Reference genes are standards for quantifying gene expression through quantitative real-time PCR (qRT-PCR); however, the variation observed in their expression levels is the major hindrance towards realising their effective use. Hence, a systematic validation of reference genes is required to ensure proper normalization. However, no such study has been conducted in foxtail millet [Setaria italica (L.)], which has recently emerged as a model crop for genetic and genomic studies. In the present study, 8 commonly used reference genes were evaluated, including 18S ribosomal RNA, elongation factor-1α, Actin2, alpha tubulin, beta tubulin, translation factor, RNA polymerase II and adenine phosphoribosyl transferase. Expression stability of candidate internal control genes was investigated under salinity and dehydration treatments. The results obtained suggested a wide range of Ct values and variable expression of all reference genes. geNorm and NormFinder analysis had revealed that Act2 and RNA POL II are suitable reference genes for salinity stress-related studies and EF-1α and RNA POL II are appropriate internal controls for dehydration stress-related expression analyses. These qualified reference genes has also been validated for relative quantification of 14-3-3 expression analysis which demonstrated their applicability. Thus, this is the first report on selection and validation of superior reference genes for qRT-PCR in foxtail millet under different abiotic stress conditions.     

Association of an allele-specific marker with dehydration stress tolerance in foxtail millet suggests SiDREB2 to be an important QTL

Dehydration-responsive element binding (DREB) genes assist in improving stress tolerance of plants by activating the expression of several stress-responsive genes. Therefore, development of functional markers for useful alleles utilizing DREB genes is crucial for crop improvement strategies. Earlier we reported a synonymous single nucleotide polymorphism (SNP) associated with dehydration tolerance at 558^th bp (an A/G transition) in the SiDREB2 gene of foxtail millet (Setaria italica L.) and developed an allele-specific marker (ASM) for SiDREB2. In the present study, we validated this ASM using a set of 122 foxtail accessions, of which 45 were investigated in an earlier study. The QTL associated with SiDREB2 contributed to ~20 % of the total phenotypic variation (PV) for relative water content (RWC) and this signified the importance of this QTL for dehydration tolerance in foxtail millet.     

Overexpression of millet ZIP-like gene (SiPf40) affects lateral bud outgrowth in tobacco and millet

A SiPf40 gene was identified from an immature seed cDNA library of foxtail millet (Setaria italica). This gene encodes for a 29.4 KDa protein containing eight potential transmembrane domains and a highly conserved ZIP signature motif typical of ZIPs (zinc or iron transporter proteins) family. Other SiPf40 potential homologous genes have also been identified in rice, maize, wheat and Arabidopsis by Southern analysis. Expression data showed that this gene is preferentially expressed in millet hypocotyl and bud; however, a minimal level of constitutive expression could be detected in other foxtail millet tissues.Overexpression of SiPf40 gene causes extra branches in tobacco and extra tillering in millet associated with vessel enlarging and xylary fibers increasing, whereas the tiller number decreases in SiPf40 gene silenced plants. Moreover, IAA content decreased significantly in shoot apex of the transgenic tobacco overexpressing SiPf40 gene. All together, these morphological alterations indicate that SiPf40 gene is essential for lateral shoots growth.     

Characteristics and Expression Patterns of the Aldehyde Dehydrogenase (ALDH) Gene Superfamily of Foxtail Millet (Setaria italica L.)

Recent genomic sequencing of the foxtail millet, an abiotic, stress-tolerant crop, has provided a great opportunity for novel gene discovery and functional analysis of this popularly-grown grass. However, few stress-mediated gene families have been studied. Aldehyde dehydrogenases (ALDHs) comprise a gene superfamily encoding NAD (P) ⁺-dependent enzymes that play the role of “aldehyde scavengers”, which indirectly detoxify cellular ROS and reduce the effect of lipid peroxidation meditated cellular toxicity under various environmental stresses. In the current paper, we identified a total of 20 ALDH genes in the foxtail millet genome using a homology search and a phylogenetic analysis and grouped them into ten distinct families based on their amino acid sequence identity. Furthermore, evolutionary analysis of foxtail millet reveals that both tandem and segmental duplication contributed significantly to the expansion of its ALDH genes. The exon-intron structures of members of the same family in foxtail millet or the orthologous genes in rice display highly diverse distributions of their exonic and intronic regions. Also, synteny analysis shows that the majority of foxtail millet and rice ALDH gene homologs exist in the syntenic blocks between the two, implying that these ALDH genes arose before the divergence of cereals. Semi-quantitative and real-time quantitative PCR data reveals that a few SiALDH genes are expressed in an organ-specific manner and that the expression of a number of foxtail millet ALDH genes, such as, SiALDH7B1, SiALDH12A1 and SiALDH18B2 are up-regulated by osmotic stress, cold, H₂O₂, and phytohormone abscisic acid (ABA). Furthermore, the transformation of SiALDH2B2, SiALDH10A2, SiALDH5F1, SiALDH22A1, and SiALDH3E2 into Escherichia coli (E.coli) was able to improve their salt tolerance. Taken together, our results show that genome-wide identification characteristics and expression analyses provide unique opportunities for assessing the functional roles of foxtail millet ALDH genes in stress responses.     

Construction of a foxtail millet linkage map and mapping of spikelet-tipped bristles 1(stb1) by using transposon display markers and simple sequence repeat markers with genome sequence information

We attempted genetic analysis and mapping of a gene responsible for the trait “spikelet-tipped bristles” (stb) in foxtail millet, Setaria italica (L.) P.Beauv., as the first step in positional cloning of the gene. This trait is important not only in grain yield such as grain number per panicle of this millet but also in the evolutionary development of the “bristle grass” clade including genera Setaria, Pennisetum and Cenchrus in subfamily Panicoideae. First of all, we confirmed that this trait is controlled by a single recessive gene, using two populations of F2 plants; one was a cross combination between two Taiwanese landraces and the other was a combination between a Taiwanese landrace and a Japanese landrace. Using the latter of the two F2 populations, with transposon display (TD) markers and simple sequence repeat (SSR) markers developed previously, we constructed a genetic map with 13 linkage groups and mapped the responsible gene (stb1) on chromosome 2. We also developed novel SSR markers by using foxtail millet genome sequence information, and we finally constructed nine linkage groups corresponding to nine chromosomes with a total length of 1287.5 cM, and mapped stb1 more precisely on chromosome 2. This work suggests that the foxtail millet genome sequences recently published are useful for developing genome-wide SSR markers for constructing linkage maps and mapping genes in this millet.     

Bioinformatic identification and experimental validation of miRNAs from foxtail millet (Setariaitalica)

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet (Setaria italica), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5′-RLM-RACE) method.     

Infraspecific variation and systematics of cultivatedSetaria italica,foxtail millet (Poaceae)

Foxtail millet (Setaria italica,) is grown as a cereal in southern Europe and in temperate, subtropical, and tropical Asia. Its closest wild relative isS. italica ssp.viridis (green foxtail). Green foxtail is native to temperate Eurasia, but was introduced and became widely established as a weed in temperate and warmer parts of the Americas. Spontaneous and cultivatedS. italica cross to produce fertile hybrids. Derivatives of such crosses, resembling foxtail millet in some inflorescence traits but with efficient natural seed dispersal, accompany the cereal across its range of cultivation. Giant green foxtail of Europe and the American corn belt is a weed of hybrid origin. Foxtail millet was domesticated in the highlands of central China; remains of cultivated foxtail millet are known from the Yang-shao culture period dating back some 5,000 yrs. Comparative morphology suggests that foxtail millet spread to Europe and India as a cereal soon after its domestication. Three cultivated races are recognized. Moharia, from Europe and southwestern Asia, includes cultivars with 5–52 culms, each bearing several, small, more or less erect inflorescences. Cultivars in race maxima are characterized by plants with 1–8 usually unbranched culms that bear large inflorescences; they occur in Transcaucasian Russia and the Far East. Race indica is intermediate in culm number (ave. 6.6) and inflorescence size between races moharia and maxima, and is cultivated in southern Asia.     

Microsatellite Variations of Elite Setaria Varieties Released during Last Six Decades in China

Crop improvement is a multifaceted micro-evolutionary process, involving changes in breeding approaches, planting configurations and consumption preferences of human beings. Recent research has started to identify the specific genes or genomic regions correlate to improved agronomic traits, however, an apparent blank between the genetic structure of crop elite varieties and their improving histories in diverse modern breeding programs is still in existence. Foxtail millet (Setaria italica) was one of the earliest cereal crops to be domesticated and served as a staple crop for early civilizations in China, where it is still widely grown today. In the present trial, a panel of foxtail millet elite varieties, which were released in the last sixty years in different geographical regions of China, was characterized using microsatellite markers (SSRs). A clear separation of two subpopulations corresponding to the two eco-geographical regions of foxtail millet production in China was identified by the dataset, which also indicated that in more recently released elite varieties, large quantities of accessions have been transferred from spring-sowing to summer-sowing ecotypes, likely as a result of breeding response to planting configurations. An association mapping study was conducted to identify loci controlling traits of major agronomic interest. Furthermore, selective sweeps involved in improvement of foxtail millet were identified as multi-diverse minor effect loci controlling different agronomic traits during the long-term improvement of elite varieties. Our results highlight the effect of transition of planting configuration and breeding preference on genetic evolvement of crop species.     

Mapping of Quantitative Trait Locus (QTLs) that Contribute to Germination and Early Seedling Drought Tolerance in the Interspecific Cross Setaria italica×Setaria viridis

Drought tolerance is an important breeding target for enhancing the yields of grain crop species in arid and semi-arid regions of the world. Two species of Setaria, domesticated foxtail millet (S. italica) and its wild ancestor green foxtail (S. viridis) are becoming widely adopted as models for functional genomics studies in the Panicoid grasses. In this study, the genomic regions controlling germination and early seedling drought tolerance in Setaria were identified using 190 F₇ lines derived from a cross between Yugu1, a S. italica cultivar developed in China, and a wild S. viridis genotype collected from Uzbekistan. Quantitative trait loci were identified which contribute to a number of traits including promptness index, radical root length, coleoptile length and lateral root number at germinating stage and seedling survival rate was characterized by the ability of desiccated seedlings to revive after rehydration. A genetic map with 128 SSR markers which spans 1293.9 cM with an average of 14 markers per linkage group of the 9 linkage groups was constructed. A total of eighteen QTLs were detected which included nine that explained over 10% of the phenotypic variance for a given trait. Both the wild green foxtail genotype and the foxtail millet cultivar contributed the favorite alleles for traits detected in this trial, indicating that wild Setaria viridis populations may serve as a reservoir for novel stress tolerance alleles which could be employed in foxtail millet breeding.