Foxtail Millet Improvement Research Articles

Identification, Molecular Characteristics, and Evolution of GRF Gene Family in Foxtail Millet (Setaria italica L.).

Growth-regulating factor (GRF) is a multigene family that plays a vital role in the growth and development of plants. In the past, the GRF family of many plants has been studied. However, there is not a report about identification and evolution of GRF in foxtail millet (Setaria italia). Here, we identified 10 GRF genes in foxtail millet. Seven (70.00%) were regulated by Sit-miR396, and there were 19 optimal codons in GRFs of foxtail millet. Additionally, we found that WGD or segmental duplication have affected GRFs in foxtail millet between 15.07 and 45.97 million years ago. Regarding the GRF gene family of land plants, we identified a total of 157 GRF genes in 15 representative land plants. We found that GRF gene family originated from Group E, and the GRF gene family in monocots was gradually shrinking. Also, more loss resulted from the small number of GRF genes in lower plants. Exploring the evolution of GRF and functional analysis in the foxtail millet help us to understand GRF better and make a further study about the mechanism of GRF. These results provide a basis for the genetic improvement of foxtail millet and indicate an improvement of the yield.

Efficient Genome Editing in Setaria italica Using CRISPR/Cas9 and Base Editors.

The genome editing toolbox based on CRISPR/Cas9 has brought revolutionary changes to agricultural and plant scientific research. With the development of stable genetic transformation protocols, a highly efficient genome editing system for foxtail millet (Setaria italica) is required. In the present study, we use the CRISPR/Cas9 single- and multi-gene knockout system to target the SiFMBP, SiDof4, SiBADH2, SiGBSS1, and SiIPK1 genes in the foxtail millet protoplasts to screen out highly efficient targeted sgRNAs. Then, we recovered homozygous mutant plants with most of the targeted genes through an Agrobacterium-mediated genetic transformation of foxtail millet. The mutagenesis frequency in the T0 generation was as high as 100%, and it was passed stably on to the next generation. After screening these targeted edited events, we did not detect off-target mutations at potential sites. Based on this system, we have achieved base editing successfully using two base editors (CBE and ABE) to target the SiALS and SiACC genes of foxtail millet. By utilizing CBE to target the SiALS gene, we created a homozygous herbicide-tolerant mutant plant. The current system could enhance the analysis of functional genomics and genetic improvement of foxtail millet.

Under Rainfed Effect of Foxtail Millet [Setaria italica (L.) beauv] Germplasms Evaluation on Genotypic Variance, Correlation and Path Analysis

Background: Setaria italica is an essential crop in the developing world and yield levels are low. To improve the yield, the utilization of diverse germplasm in a breeding program is vital. Methods: The present study aimed to characterize 50 genotypes in the first year and out of 50, 10 genotypes in the second year using multivariant traits. Wide variation was observed in both the qualitative and quantitative characteristics. Analysis of variance revealed the presence of significant differences for most symptoms. Higher estimates of GCV were followed for grain yields, followed by panicle lengths and organic outcomes. Higher estimates of PCV were observed for plant height and leaf length, followed by leaf width. Low GCV and PCV were recorded as leaf length, 50% flowering in days. PCV study indicated direct selection based on characters, panicle weight, test weight and strawweight showed a high and positive effect on grain yield per plant in both rainy and summer season indicating the proper relationship between these characters with grain yield per plant, which helps indirect selection for these traits thus in improving the grain yield per plant. Variability in foxtail millet germplasm allows plant breeders to effectively select specific donor lines for genetic improvement of foxtail millet. Result: Best 5 genotypes were found to be: Kangni-7 greater than Kangni-1 greater than Kangni-6 greater than Kangni-5 greater than Kangni-4.

Phenotypic responses of foxtail millet (Setaria italica) genotypes to phosphate supply under greenhouse and natural field conditions.

Food insecurity is a looming threat for the burgeoning world population. Phosphorus (P), which is absorbed from soil as inorganic phosphate (Pi), is an essential macronutrient for the growth of all agricultural crops. This study reports phenotype analysis for P responses in natural field and greenhouse conditions, using 54 genotypes of foxtail millet (Setaria italica) representing wide geographic origins. The genotype responses were assessed in natural field conditions in two different seasons (monsoon and summer) under Pi-fertilized (P+) and unfertilized (P-) soil for eight above-ground traits. Enormous variations were seen among the genotypes in phenotypic responses for all the measured parameters under low P stress conditions. Variations were significant for plant height, leaf number and length, tillering ability and seed yield traits. Genotypes ISe 1234 and ISe 1541 were P+ responders, and the genotypes ISe 1181, ISe 1655, ISe 783 and ISe 1892 showed tolerance to low P for total seed yield. Genotypes that performed well under P- conditions were almost as productive as genotypes that performed well under P+ conditions suggesting some genotypes are well adapted to nutrient-poor soils. In the greenhouse, most of the genotypes produced changes in root architecture that are characteristic of P- stress, but to differing degrees. Significant variation was seen in root hair density and root hair number and in fresh and dry weight of shoot and root under P- stress. However, there was not much difference in the shoot and root total P and Pi levels of five selected high and low responding genotypes. We noticed contrasting responses in the greenhouse and natural field experiments for most of these genotypes. The leads from the study form the basis for breeding and improvement of foxtail millet for better Pi-use efficiency.

Genome-Wide Association Study of Major Agronomic Traits in Foxtail Millet (Setaria italica L.) Using ddRAD Sequencing

Foxtail millet (Setaria italica), the second largest cultivated millet crop after pearl millet, is utilized for food and forage globally. Further, it is also considered as a model crop for studying agronomic, nutritional and biofuel traits. In the present study, a genome-wide association study (GWAS) was performed for ten important agronomic traits in 142 foxtail millet core eco-geographically diverse genotypes using 10 K SNPs developed through GBS-ddRAD approach. Number of SNPs on individual chromosome ranged from 844 (chromosome 5) to 2153 (chromosome 8) with an average SNP frequency of 25.9 per Mb. The pairwise linkage disequilibrium (LD) estimated using the squared-allele frequency correlations was found to decay rapidly with the genetic distance of 177 Kb. However, for individual chromosome, LD decay distance ranged from 76 Kb (chromosome 6) to 357 Kb (chromosome 4). GWAS identified 81 MTAs (marker-trait associations) for ten traits across the genome. High confidence MTAs for three important agronomic traits including FLW (flag leaf width), GY (grain yield) and TGW (thousand-grain weight) were identified. Significant pyramiding effect of identified MTAs further supplemented its importance in breeding programs. Desirable alleles and superior genotypes identified in the present study may prove valuable for foxtail millet improvement through marker-assisted selection.

Comprehensive phenotypic and statistical based analysis of foxtail millet (Setaria italic L.) lines for parental line selection

Minor millets are the small-grained cereals that belong to family Poaceae, with local importance as a food source but are often called as underutilized plant species. Foxtail millet is one among the oldest cultivated small diploid, C4 Panicoid, with short life cycle, and inbreeding nature. The genetic diversity of the foxtail millet is important for breeding systems, geographical distribution studies, assist in conservation of genetic resources for high yielding varietal development in breeding programs, expansion of the genetic base and for identification of genes for various phenotypic traits. Keeping in view of this an attempt has made to develop pure lines from local farmers preserved populations and phenotypically characterize them along with four released cultivars. A total of eleven farmers varieties and four released cultivars were evaluated for seven quantitative traits in complete random block desing at net house conditions. The maximum coefficient of variation was observed for panicle exertion, followed by number of tillers/plant and panicle weight. High positive significant correlations was observed for Plant height, panicle length, panicle weight and total seed weight with other traits under study. Panicle exertion exhibit negative correlations with panicle length, panicle weight and no of tillers. The results of principal component analysis (PCA) explains variability of 95.43% in the fifteen genotypes for the traits under study that corresponds to first 4 eigen values with greater than 0.60. The proportions of the total variance explained by the first 4 principal components (PCs) were 42.98%, 71.02%, 85.43% and 95.43% respectively. Wards method based genetic similarity or dissimilarity clustering based on seven morphological traits among the fifteen foxtail millet genotypes was able to separate and grouped them into 2 major clusters I and II. The genetic and phenotypic variability present in the foxtail millet genotypes give opportunity for plant breeders for effective selection of specific donor lines for foxtail millet improvement.

Identification, characterization and validation of core collection of foxtail millet (Setaria italica)

Foxtail millet [Setaria italica (L.) P. Beauv.] is an important minor millet, which is drought tolerant, rich in micronutrients and grown in the marginal and rainfed regions of India. Power core software was used to develop the core collection of foxtail millet using total of 1515 germplasm accessions, emanating from 21 different countries. Eleven quantitative and 13 qualitative traits were utilized to characterize the core collection. Each qualitative and quantitative trait was divided into discrete classes and 223 core entries were selected from them. Selected core entries comprises accessions predominantly from India (75.78%). Average Shannon-Weaver diversity (0.73) and Nei’s diversity index (0.43) of core collection were higher for the entire collection indicating true representation of diversity available in the entire collection. Average polymorphism information content (PIC) value was 0.99. Core accessions were evaluated and validated using analysis of variance (ANOVA), descriptive statistics, clustering based on neighbour joining tree and principal component analysis. Accessions from Andhra Pradesh, Karnataka, Madhya Pradesh, Tamil Nadu, Uttar Pradesh and North-East Region were distributed in almost all the clusters and sub-clusters suggesting lack of discrete relationship between geographic locations and clustering pattern. Thus, a highly diverse core collection representing the diversity of entire germplasm holding from different parts of the world was identified and this can be used for foxtail millet improvement programs as well as to identify trait-specific reference collections.

Updated foxtail millet genome assembly and gene mapping of nine key agronomic traits by resequencing a RIL population.

Foxtail millet (Setaria italica) provides food and fodder in semi-arid regions and infertile land. Resequencing of 184 foxtail millet recombinant inbred lines (RILs) was carried out to aid essential research on foxtail millet improvement. A total 483 414 single nucleotide polymorphisms were determined. Bin maps were constructed based on the RILs’ recombination data. Based on the high-density bin map, we updated Zhanggu reference with 416 Mb after adding 16 Mb unanchored scaffolds and Yugu reference with some assembly error correction and 3158 gaps filled. Quantitative trait loci (QTL) mapping of nine agronomic traits was done based on this RIL population, five of which were controlled by a single gene. Meanwhile, two QTLs were found for plant height, and a candidate gene showed 89% identity to the known rice gibberellin-synthesis gene sd1. Three QTLs were found for the trait of heading date. The whole genome resequencing and QTL mapping provided important tools for foxtail millet research and breeding. Resequencing of the RILs could also provide an effective way for high-quality genome assembly and gene identification.

Genomic organization and structural diversity of germin-like protein coding genes in foxtail millet (Setaria italica L.)

Germin-like protein (GLP) genes in plants constitute a multigene family that functions in a variety of biological processes, such as plant growth and development and respond to abiotic and biotic stresses. Available genome sequences of foxtail millet (Setaria italica) have facilitated the identification and delineation of the GLP gene family, which is perhaps the first report in any millet crop. A total of 20 SiGLP genes were mapped in six of the nine foxtail millet chromosomes. The majority of these SiGLP genes, except six, clustered into five known germin (GER) groups and revealed group-specific variations in their gene structure and conserved protein motifs. The SiGLP genes shared homologous relationship within and across plant genomes as per their syntenic chromosomal regions, indicating gene duplication and expansion in course of plant evolution. Gene expression dynamics revealed four out of the 20 SiGLP genes having a putative functional role in different tissues. The presence of diverse cis-acting regulatory elements in the putative SiGLP promoter sequences and homology-based 3-D protein structure predictions revealed their possible functional diversity. The present work encompasses a molecular insight into the organization and functions of SiGLP gene family members. This may serve as a platform for functional analysis and their efficient utilizations in genetic improvement of foxtail millet and other related crops.

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.

Culturing of immature inflorescences and Agrobacterium-mediated transformation of foxtail millet (Setaria italica)

In previous reports, we developed an Agrobacterium -mediated transformation system for foxtail millet. Here, we report optimization of the system through improvement of the regeneration system efficiency and optimization of conditions for gene delivery. Immature inflorescences explants of foxtail millet cv. Jigu 11 varying in length (0.5 to 1.0, 1.1 to 1.5, 1.6 to 2.0 and >2.0 cm) were cultured on modified MS medium for callus induction and regeneration. The highest embryogenic callus-formation efficiency (90.72%) was achieved with 0.5 to 1.0 cm long inflorescences and 25 days old calli induced from 0.5 to 1.0 cm long immature inflorescences gave rise to the highest differentiation frequency (90.93%). In addition, factors affecting T-DNA delivery were examined by transient β-glucuronidase (GUS) expression. Calli induced from younger explants (0.5 to 1.0 cm immature inflorescences) were optimal. Agrobacterium tumefaciens strain LBA4404 performed significantly better than EHA105. Co-cultivation at 22°C with 0.15 g/l dithiothreitol (DTT) in the infection solution and co-cultivation medium led to higher GUS transient expression efficiency than with other treatments. Using this optimized procedure, the lysine-rich protein encoding gene SBgLR from potato was transformed into foxtail millet cv. Jigu 11 with 5.5% transformation efficiency. The procedure described here will be useful for genetic improvement of foxtail millet. Key words : Foxtail millet, regeneration, Agrobacterium-mediated transformation, temperature, dithiothreitol (DTT).

Sequence-based novel genomic microsatellite markers for robust genotyping purposes in foxtail millet [Setaria italica (L.) P. Beauv.

The unavailability of microsatellite markers and saturated genetic linkage map has restricted the genetic improvement of foxtail millet [Setaria italica (L.) P. Beauv.], despite the fact that in recent times it has been documented as a new model species for biofuel grasses. With the objective to generate a good number of microsatellite markers in foxtail millet cultivar 'Prasad', 690 clones were sequenced which generated 112.95kb high quality sequences obtained from three genomic libraries each enriched with different microsatellite repeat motifs. Microsatellites were identified in 512 (74.2%) of the 690 positive clones and 172 primer pairs (pp) were successfully designed from 249 (48.6%) unique SSR-containing clones. The efficacies of the microsatellite containing genomic sequences were established by superior primer designing ability (69%), PCR amplification efficiency (85.5%) and polymorphic potential (52%) in the parents of F(2) mapping population. Out of 172pp, functional 147 markers showed high level of cross-species amplification (~74%) in six grass species. Higher polymorphism rate and broad range of genetic diversity (0.30-0.69 averaging 0.58) obtained in constructed phylogenetic tree using 52 microsatellite markers, demonstrated the utility of markers in germplasm characterizations. In silico comparative mapping of 147 foxtail millet microsatellite containing sequences against the mapping data of sorghum (~18%), maize (~16%) and rice (~5%) indicated the presence of orthologous sequences of the foxtail millet in the respective species. The result thus demonstrates the applicability of microsatellite markers in various genotyping applications, determining phylogenetic relationships and comparative mapping in several important grass species.

Technique for Artificial Hybridization of Foxtail Millet [Setaria italica (L.) Beauv.

The difficulty making crosses and lack of an efficient crossing technique have resulted in a very limited number of genetic studies and, consequently, limited improvement of foxtail millet [Setaria italica (L.) Beauv.]. Objectives of this study were to acquire knowledge of the anthesis habits of foxtail millet and to develop a technique for artificial hybridization of foxtail millet. To understand the process of anthesis, several variables were observed on five plants of each of five cultivars under greenhouse and growth chamber conditions during the summer of 1994. The efficiency of the crossing technique described was evaluated based on seed set from emasculated but non‐pollinated checks, seed set from more than 6400, and percent hybrid plants among 30 F1 plants from each of 21 cross‐combinations. During the summer, anthesis of foxtail occurred between 1800 h and 0700 h, starting the third day, after the emergence of the spike. Few flowers opened and there was practically no anthesis between 0800 h and 1800 h. Anthesis initiated at or about one third down the spike and progressed up to the apex and down to the base. Each spike required from 10 to 13 d to complete anthesis. The number of florets opening on the second day averaged 36, which was an optimum for successful emasculation. Opening and closing of a single flower occurred in about 7 min. The crossing technique described was effective with an average 75% seed set, and more than 90% true hybrid seed or 67.5% hybrid seed set per flower crossed.

Intraspecific and interspecific variation in Setaria revealed by RAPD analysis

Random amplified polymorphic DNA (RAPD) analysis was applied to foxtail millet (Setaria italica) and other Setaria species in order to assess the degree of polymorphism among and within the species and to investigate their genetic relationships. Twenty accessions of foxtail millet, five of green foxtail (S. viridis), five of yellow foxtail (S. glauca), one each of S. germanica, S. verticilliformis, S. pallidifusca, S. macrostachya, S. sphacelata and S. faberi, and one unidentified, were evaluated for variability using a set of 19 random 10-mer primers. A total of 148 markers were scored for all the accessions. Wide variations in banding profiles among species were observed with nearly every primer used. The results showed that foxtail millet and S. glauca had a considerable degree of variation. The geographic differentiation of RAPD patterns in foxtail millet accessions examined seemed to exist. The present study confirmed that foxtail millet is more closely related to green foxtail than to other species in Setaria, providing support for the origin of foxtail millet from S. viridis and the conspecificity of these two species. The results confirmed that S. germanica and S. italica are conspecific and the former can be replaced by the latter. Setaria glauca and S. sphacelata are distinct from foxtail millet, implying that elite traits found in these two species are relatively difficult to be used in foxtail millet improvement. This study demonstrates that RAPD analysis can be used in assessing intraspecific and interspecific variation in foxtail millet and its wild relatives.

Breeding strategy in foxtail millet, Setaria italica (L.P. Beauv.), following interspecific hybridization

An interspecific cross between foxtail millet Setaria italica and its wild relative Setaria viridis was undertaken in order to introduce from the latter triazine resistance in the cultivated plant. Four backcross generations obtained with S. italica as recurrent parent were studied. Results show that only two backcross generations associated with selection are enough to eliminate weedy characters and to return to the cultivated type. S. viridis could thus be an interesting source for improvement of foxtail millet without a complex and time consuming breeding strategy.