Hui Zhi

Grain fat content contributes to nutritional quality in foxtail millet, but its genetic basis remains incompletely understood. We evaluated a 256-line recombinant inbred population derived from Jingu21 and Chuang29 in two environments. Among the individual environments, qFC5 was detected only in Sanya (2022_SY; LOD 5.86; phenotypic variance explained: 9.24%), exceeding the LOD threshold of 3.58. Across-environment mapping using best linear unbiased estimates identified an overlapping chromosome 5 region (LOD: 5.99; phenotypic variance explained: 7.67%), exceeding the threshold of 4.14 from 1000 permutations. Among 248 annotated genes in the 1.80 Mb Sanya interval, Arabidopsis and rice homology, functional annotation and expression information supported prioritization of Seita.5G394800.1 (SiMGT2), a predicted MGT/MRS2 magnesium transporter. Six SiMGT family members were identified. In the parental panicle dataset, SiMGT2 had a higher mean expression in Jingu21 at three sampling stages; only the middle-stage difference remained significant after correction across 18 comparisons. In 534 phenotyped accessions, H1 had a higher grain fat content than H2, but not H3; these comparisons were not corrected for population structure or kinship. The results prioritize SiMGT2 as a candidate associated with grain fat content, with environment-dependent QTL support. Causality and the proposed connection with magnesium homeostasis require functional validation.

Carotenoid content is a key trait that defines the unique characteristics of foxtail millet varieties. Varieties with different levels of carotenoids often show distinct genetic features and nutritional profiles. However, the genetic basis of carotenoid content in foxtail millet remains mostly unknown. In this study, we explored the genetic basis of carotenoid content using a recombinant inbred line (RIL) population of 305 lines derived from two parental accessions, JG21 (high-carotenoid, 16.75 mg·kg⁻¹) and JG25 (low-carotenoid, 0.93 mg·kg⁻¹). The results showed that the RIL population exhibited continuous phenotypic variation and significant transgressive segregation for carotenoid components (lutein, zeaxanthin, β-cryptoxanthin) and kernel color (measured by b* value), with zeaxanthin reaching 8.47 mg·kg⁻¹, significantly surpassing the higher parent (3.44 mg·kg⁻¹) in 24DY. To ensure that enhancing this nutritional trait does not compromise grain yield, we analyzed its relationship with key agronomic traits, testing for pleiotropic trade-offs. Notably, carotenoid content showed no significant correlation with any of the 8 key agronomic traits (r ranged from −0.11 to 0.08, all p > 0.05), suggesting no apparent trade-off, although fine-mapping is needed to separate pleiotropy from tight linkage for concurrent improvement. Genetic modeling analysis revealed that carotenoid content is stably controlled by three major-gene pairs plus polygenes (MX3-AI-A model), with major-gene heritability of 96.65% and polygene heritability of 3.35%. Based on this framework, three elite RILs with >23% higher carotenoid and superior agronomic performance were identified and advanced to marker-assisted backcrossing. These results provide a clear genetic framework and immediate breeding resources for marker-assisted selection, enabling the development of high-yielding, carotenoid-enriched foxtail millet varieties without compromising agronomic value.

Panicle development and grain production in crop species are essential breeding characteristics affected by the synthesis of auxin, which is influenced by flavin monooxygenase-encoding genes such as YUC (YUCCA) family members. In this trial, fourteen YUCs were identified and named uniformly in foxtail millet, an ancient crop species cultivated across the world. The phylogenetic analysis revealed that the SiYUCs were clustered into four subgroups; protein motif and gene structure analyses suggested that the closely clustered SiYUC genes were relatively conserved within each subgroup; while genome mapping analysis indicated that the SiYUC genes were unevenly distributed on foxtail millet chromosomes and colinear with other grass species. Transcription analysis revealed that the SiYUC genes differed greatly in expression pattern in different tissues and contained hormonal/light/stress-responding cis-elements. The haplotype characterization of SiYUC genes indicated many superior haplotypes of SiYUCs correlated with higher panicle and grain weight could be favorably selected by breeding. These results will be useful for the further study of the functional characteristics of SiYUC genes, particularly with regard to the marker-assisted pyramiding of beneficial haplotypes in foxtail millet breeding programs.

The activity of ribonucleotide reductase (RNR), which catalyses the transformation of four ribonucleoside diphosphates (NDPs) to their corresponding deoxyribonucleoside diphosphates (dNDPs), is the main determiner of the cellular concentration of dNTP pools and should be tightly coordinated with DNA synthesis and cell-cycle progression. Constitutively increased or decreased RNR activity interferes with DNA replication and leads to arrested cell cycle progression; however, the mechanisms underlying these disruptive effects in higher plants remain to be uncovered. In this study, we identified a RNR large subunit mutant, sistl1, in Setaria italica (foxtail millet), which exhibited growth retardation as well as striped leaf phenotype, i.e. irregularly reduced leaf vein distances and decreased chloroplast biogenesis. We determined that a Gly₇₃₇ to Glu substitution occurring in the C-terminus of the SiSTL1 protein slightly affected its optimal function, leading in turn to the reduced expression of genes variously involved in the assembly and activation of the DNA pre-replicative complex, elongation of replication forks and S phase entry. Our study provides new insights into how SiSTL1 regulates plant growth, chloroplast biogenesis, and cell cycle progression in Poaceae crops.

Deoxycytidine monophosphate deaminase (DCD) is a key enzyme in the de novo dTTP biosynthesis pathway. Previous studies have indicated that DCD plays key roles in the maintenance of the balance of dNTP pools, cell cycle progression, and plant development. However, few studies have elucidated the functions of the DCD gene in Panicoideae plants. Setaria has been proposed as an ideal model of Panicoideae grasses, especially for C₄ photosynthesis research. Here, a Setaria italica stripe leaf mutant (sistl2) was isolated from EMS-induced lines of “Yugu1,” the wild-type parent. The sistl2 mutant exhibited semi-dwarf, striped leaves, abnormal chloroplast ultrastructure, and delayed cell cycle progression compared with Yugu1. High-throughput sequencing and map-based cloning identified the causal gene SiSTL2, which encodes a DCD protein. The occurrence of a single-base G to A substitution in the fifth intron introduced alternative splicing, which led to the early termination of translation. Further physiological and transcriptomic investigation indicated that SiSTL2 plays an essential role in the regulation of chloroplast biogenesis, cell cycle, and DNA replication, which suggested that the gene has conserved functions in both foxtail millet and rice. Remarkably, in contrast to DCD mutants in C₃ rice, sistl2 showed a significant reduction in leaf cell size and affected C₄ photosynthetic capacity in foxtail millet. qPCR showed that SiSTL2 had a similar expression pattern to typical C₄ genes in response to a low CO₂ environment. Moreover, the loss of function of SiSTL2 resulted in a reduction of leaf ¹³C content and the enrichment of DEGs in photosynthetic carbon fixation. Our research provides in-depth knowledge of the role of DCD in the C₄ photosynthesis model S. italica and proposed new directions for further study of the function of DCD.

Foxtail millet (Setaria italica) is a diploid C4 panicoid species. Because of its prominent drought resistance, small genome size, self-pollination, and short life cycle, foxtail millet has become an ideal model system for studying drought tolerance of crops. MicroRNAs (miRNAs) are endogenous, small RNAs that play important regulatory roles in the development and stress response in plants. In this study, we applied Illumina sequencing to systematically investigate the drought-responsive miRNAs derived from S. italica inbred An04-4783 seedlings grown under control and drought conditions. Degradome sequencing was applied to confirm the targets of these miRNAs at a global level. A total of 81 known miRNAs belonging to 28 families were identified, among which 14 miRNAs were upregulated and four were downregulated in response to drought. In addition, 72 potential novel miRNAs were identified, three of which were differentially expressed under drought conditions. Degradome sequencing analysis showed that 56 and 26 genes were identified as targets of known and novel miRNAs, respectively. Our analysis revealed post-transcriptional remodeling of cell development, transcription factors, ABA signaling, and cellar homeostasis in S.italica in response to drought. This preliminary characterization provided useful information for further studies on the regulatory networks of drought-responsive miRNAs in foxtail millet.

Foxtail millet (Setaria italica (L.) P. Beauv), which belongs to the Panicoideae tribe of the Poaceae, is an important grain crop widely grown in Northern China and India. It is currently developing into a novel model species for functional genomics of the Panicoideae as a result of its fully available reference genome sequence, small diploid genome (2n=18, ~510Mb), short life cycle, small stature and prolific seed production. Argonaute 1 (AGO1), belonging to the argonaute (AGO) protein family, recruits small RNAs and regulates plant growth and development. Here, we characterized an AGO1 mutant (siago1b) in foxtail millet, which was induced by ethyl methanesulfonate treatment. The mutant exhibited pleiotropic developmental defects, including dwarfing stem, narrow and rolled leaves, smaller panicles and lower rates of seed setting. Map-based cloning analysis demonstrated that these phenotypic variations were attributed to a C–A transversion, and a 7-bp deletion in the C-terminus of the SiAGO1b gene in siago1b. Yeast two-hybrid assays and BiFC experiments revealed that the mutated region was an essential functional motif for the interaction between SiAGO1b and SiHYL1. Furthermore, 1598 differentially expressed genes were detected via RNA-seq-based comparison of SiAGO1b and wild-type plants, which revealed that SiAGO1b mutation influenced multiple biological processes, including energy metabolism, cell growth, programmed death and abiotic stress responses in foxtail millet. This study may provide a better understanding of the mechanisms by which SiAGO1b regulates the growth and development of crops.

Setaria italica and its wild ancestor Setaria viridis are emerging as model systems for genetics and functional genomics research. However, few systematic gene mapping or functional analyses have been reported in these promising C4 models. We herein isolated the yellow‐green leaf mutant (siygl1) in S. italica using forward genetics approaches. Map‐based cloning revealed that SiYGL1, which is a recessive nuclear gene encoding a magnesium‐chelatase D subunit (CHLD), is responsible for the mutant phenotype. A single Phe to Leu amino acid change occurring near the ATPase‐conserved domain resulted in decreased chlorophyll (Chl) accumulation and modified chloroplast ultrastructure. However, the mutation enhanced the light‐use efficiency of the siygl1 mutant, suggesting that the mutated CHLD protein does not completely lose its original activity, but instead, gains novel features. A transcriptional analysis of Chl a oxygenase revealed that there is a strong negative feedback control of Chl b biosynthesis in S. italica. The SiYGL1 mRNA was expressed in all examined tissues, with higher expression observed in the leaves. Comparison of gene expression profiles in wild‐type and siygl1 mutant plants indicated that SiYGL1 regulates a subset of genes involved in photosynthesis (rbcL and LHCB1), thylakoid development (DEG2) and chloroplast signaling (SRP54CP). These results provide information regarding the mutant phenotype at the transcriptional level. This study demonstrated that the genetic material of a Setaria species could be ideal for gene discovery investigations using forward genetics approaches and may help to explain the molecular mechanisms associated with leaf color variation.

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.