Carotenoid biosynthesis and accumulation are important in determining nutritional and commercial value of crop products. Yellow pigmentation of mature kernels caused by carotenoids is considered a vital quality trait in foxtail millet, an ancient and widely cultivated cereal crop across the world. Genomic regions associated with yellow pigment content (YPC), lutein and zeaxanthin in foxtail millet grains were identified by genome-wide association analysis (GWAS), and SiPSY1 (Phytoene synthase 1 which regulates formation of the 40-carbon backbone of carotenoids) was confirmed as the main contributor to all three components by knockout and overexpression analysis. SiPSY1 was expressed in seedlings, leaves, panicles, and mature seeds, and was subcellularly localized to chloroplasts. Transcription of SiPSY1 in 15 DAP immature grains was responsible for YPC in mature seeds. Selection of SiPSY1 combined with increased YPC in mature grains during domestication of foxtail millet was confirmed. Haplotype analysis suggested that expression level of SiPSY1 could be a selection target for future breeding programs, and a KASP marker was developed for selection of favorable SiPSY1 alleles in breeding. The results of this work will benefit nutritional and commercial improvement of foxtail millet varieties, as well as other cereal crops.

Foxtail millet (Setaria italica) is an important C₄ model crop; however, due to its high-density planting and high stature, lodging at the filling stage resulted in a serious reduction in yield and quality. Therefore, it is imperative to identify and deploy the genes controlling foxtail millet plant height. In this study, we used a semi-dwarf line 263A and an elite high-stalk breeding variety, Chuang 29 to construct an F₂ population to identify dwarf genes. We performed transcriptome analysis (RNA-seq) using internode tissues sampled at three jointing stages of 263A and Chuang 29, as well as bulk segregant analysis (BSA) on their F₂ population. A total of 8918 differentially expressed genes (DEGs) were obtained from RNA-seq analysis, and GO analysis showed that DEGs were enriched in functions such as "gibberellin metabolic process" and "oxidoreductase activity", which have previously been shown to be associated with plant height. A total 593 mutated genes were screened by BSA-seq method. One hundred and seventy-six out of the 593 mutated genes showed differential expression levels between the two parental lines, and seven genes not only showed differential expression in two or three internode tissues but also showed high genomic variation in coding regions, which indicated they play a crucial role in plant height determination. Among them, we found a gibberellin biosynthesis related GA20 oxidase gene (Seita.5G404900), which had a single-base deletion at the third exon, leading to the frameshift mutation at 263A. Cleaved amplified polymorphic sequence assay and association analysis proved the single-base deletion in Seita.5G404900 co-segregated with dwarf phenotype in two independent F₂ populations planted in entirely different environments. Taken together, the candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height, and the molecular marker will be useful for marker-assisted dwarf breeding.

Boron (B) is an essential micronutrient for vascular plant growth. Both B deficiency and toxicity can impair tissue development in diverse plant species, but little is known about the effect of B on reproductive panicle development and grain yield. In this study, a mutant of Setaria italica exhibiting necrotic panicle apices was identified and designated as sibor1. Sequencing revealed a candidate gene, SiBOR1, with a G-to-A alteration at the seventh exon. Knockout transgenic lines generated by clustered regularly interspaced short palindromic repeats and their associated protein-9 also had necrotic panicles, verifying the function of SiBOR1. SiBOR1 encoded a membrane-localized B efflux transporter, co-orthologous to the rice BOR1 protein. SiBOR1 was dominantly expressed in panicles and displayed a distinct expression pattern from those of its orthologs in other species. The induced mutation in SiBOR1 caused a reduction in the B content of panicle primary branches, and B deficiency-associated phenotypes such as thicker cell walls and higher cell porosity compared with Yugu 1. Transcriptome analysis indicated that differentially expressed genes involved in cell wall biogenesis, jasmonic acid synthesis, and programmed cell death response pathways were enriched in sibor1. qPCR analysis identified several key genes, including phenylalanine ammonia-lyase (SiPAL) and jasmonate-ZIM-domain (SiJAZ) genes, responsive to B-deficient conditions. These results indicate that SiBOR1 helps to regulate panicle primary branch development to maintain grain yield in S. italica. Our findings shed light on molecular mechanisms underlying the relationship between B transport and plant development in S. italica.

Male sterility is a common biological phenomenon in plant kingdom and has been used to generate male-sterile lines, which are important genetic resources for commercial hybrid seed production. Although increasing numbers of male-sterility genes have been identified in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), few male-sterility-related genes have been characterized in foxtail millet (Setaria italica). In this study, we isolated a male-sterile ethyl methanesulfonate-generated mutant in foxtail millet, no pollen 1 (sinp1), which displayed abnormal Ubisch bodies, defective pollen exine and complete male sterility. Using bulk segregation analysis, we cloned SiNP1 and confirmed its function with CRISPR/Cas9 genome editing. SiNP1 encoded a putative glucose-methanol-choline oxidoreductase. Subcellular localization showed that the SiNP1 protein was preferentially localized to the endoplasmic reticulum and was predominantly expressed in panicle. Transcriptome analysis revealed that many genes were differentially expressed in the sinp1 mutant, some of which encoded proteins putatively involved in carbohydrate metabolism, fatty acid biosynthesis, and lipid transport and metabolism, which were closely associated with pollen wall development. Metabolome analysis revealed the disturbance of flavonoids metabolism and fatty acid biosynthesis in the mutant. In conclusion, identification of SiNP1 provides a candidate male-sterility gene for heterosis utilization in foxtail millet and gives further insight into the mechanism of pollen reproduction in plants.

Two potential BRASSINAZOLE RESISTANT 1 (BZR1) homologs were downregulated by brassinosteroids (BRs) in Setaria italica roots. Functional analysis showed that BR regulates the dephosphorylation and nuclear localization of SiBZR1 and that SiBZR1 binds conserved BZR1-recognizing cis elements. In comparison with the wild type, SiBZR1-overexpressing S. italica seedlings were more sensitive to BR-inhibited primary root growth and drought stress, indicating that SiBZR1 is a positive regulator of BR signaling and a negative regulator of drought tolerance in S. italica. PLETHORA-LIKE 1 (SiPLT-L1) was found to be a direct target gene of SiBZR1 in S. italica roots. The expression of SiPLT-L1 was downregulated by SiBZR1. SiPLT-L1-overexpressing S. italica was less sensitive to BR-inhibited root growth and more tolerant to drought stress, possibly owing to the upregulation of drought-inducible Dehydrin-family genes.

Retrotransposons account for a large proportion of the genome and genomic variation, and play key roles in creating novel genes and diversifying the genome in many eukaryotic species. Although retrotransposons are abundant in plants, their roles had been underestimated because of a lack of research. Here, we characterized a gibberellin Acid (GA)-insensitive dwarf mutant, 84133, in foxtail millet. Map-based cloning revealed a 5.5-kb Copia-like retrotransposon insertion in DWARF1 (D1), which encodes a DELLA protein. Transcriptional analysis showed that the Copia retrotransposon mediated the transcriptional reprogramming of D1 leading to a novel N-terminal-deleted truncated DELLA transcript that was putatively driven by Copia's LTR, namely D1-TT, and another chimeric transcript. The presence of D1-TT was confirmed by protein immunodetection analysis. Furthermore, D1-TT protein was resistant to GA₃ treatment compared with the intact DELLA protein due to its inability to interact with the GA receptor, SiGID1. Overexpression of D1-TT in foxtail millet resulted in dwarf plants, confirming that it determines the dwarfism of 84133. Thus, our study documents a rare instance of long terminal repeat (LTR) retrotransposon-mediated transcriptional reprograming in the plant kingdom. These results shed light on the function of LTR retrotransposons in generating new gene functions and genetic diversity.

China is a leading country in the production of several minor cereals such as foxtail millet, Job's tears, naked oat, and naked barley. Sorghum and proso millet have also contributed greatly to Chinese agriculture. Foxtail millet, sorghum, barley, and proso millet were widely grown as major crops 60 years ago, and the reduction in their cultivation area reflects historical changes in Chinese agriculture over the past decades. Systematic germplasm collections from the 1950s to the 1990s gathered more than 66,690 accessions of these minor cereals, and for some of them, the Chinese germplasm collections are the largest in the world; for example, the 27,700 accessions of foxtail millet. Germplasm evaluations of each cereal species have focused mainly on drought tolerance, nutritional quality, and resistance to their main diseases. Comparisons among lines and selection of those with desirable traits were the main breeding methods for minor cereals in the 1950s and 1960s, but these methods were replaced by crossbreeding in the 1970s. Newly developed cultivars have greatly changed the production situation, and many super cultivars have become milestones in crop breeding history. In this review, we describe the distribution and ecoregions, origin and domestication, and landmark varieties of several minor cereals in China. Nearly all of the minor cereals are drought-tolerant and fertilizer-efficient. The requirements for environmentally friendly crops and a more diverse food supply for humans and animals provide new opportunities to cultivate minor cereals in the drier and warmer environmental conditions that are predicted in the future.