Sorghum (Sorghum bicolor) is a versatile C4 crop and a model for research in family Poaceae. High-quality genome sequence is available for the elite inbred line BTx623, but functional validation of genes remains challenging due to the limited genomic and germplasm resources available for comprehensive analysis of induced mutations. In this study, we generated 6400 pedigreed M4 mutant pools from EMS-mutagenized BTx623 seeds through single-seed descent. Whole-genome sequencing of 256 phenotyped mutant lines revealed >1.8 million canonical EMS-induced mutations, affecting >95% of genes in the sorghum genome. The vast majority (97.5%) of the induced mutations were distinct from natural variations. To demonstrate the utility of the sequenced sorghum mutant resource, we performed reverse genetics to identify eight genes potentially affecting drought tolerance, three of which had allelic mutations and two of which exhibited exact cosegregation with the phenotype of interest. Our results establish that a large-scale resource of sequenced pedigreed mutants provides an efficient platform for functional validation of genes in sorghum, thereby accelerating sorghum breeding. Moreover, findings made in sorghum could be readily translated to other members of the Poaceae via integrated genomics approaches.

As in other cereal crops, the panicles of sorghum (Sorghum bicolor (L.) Moench) comprise two types of floral spikelets (grass flowers). Only sessile spikelets (SSs) are capable of producing viable grains, whereas pedicellate spikelets (PSs) cease development after initiation and eventually abort. Consequently, grain number per panicle (GNP) is lower than the total number of flowers produced per panicle. The mechanism underlying this differential fertility is not well understood. To investigate this issue, we isolated a series of ethyl methane sulfonate (EMS)-induced multiseeded (msd) mutants that result in full spikelet fertility, effectively doubling GNP. Previously, we showed that MSD1 is a TCP (Teosinte branched/Cycloidea/PCF) transcription factor that regulates jasmonic acid (JA) biosynthesis, and ultimately floral sex organ development. Here, we show that MSD2 encodes a lipoxygenase (LOX) that catalyzes the first committed step of JA biosynthesis. Further, we demonstrate that MSD1 binds to the promoters of MSD2 and other JA pathway genes. Together, these results show that a JA-induced module regulates sorghum panicle development and spikelet fertility. The findings advance our understanding of inflorescence development and could lead to new strategies for increasing GNP and grain yield in sorghum and other cereal crops.

Grain number per panicle is an important component of grain yield in sorghum (Sorghum bicolor (L.)) and other cereal crops. Previously, we reported that mutations in multi-seeded 1 (MSD1) and MSD2 genes result in a two-fold increase in grain number per panicle due to the restoration of the fertility of the pedicellate spikelets, which invariably abort in natural sorghum accessions. Here, we report the identification of another gene, MSD3, which is also involved in the regulation of grain numbers in sorghum. Four bulked F₂ populations from crosses between BTx623 and each of the independent msd mutants p6, p14, p21, and p24 were sequenced to 20× coverage of the whole genome on a HiSeq 2000 system. Bioinformatic analyses of the sequence data showed that one gene, Sorbi_3001G407600, harbored homozygous mutations in all four populations. This gene encodes a plastidial ω-3 fatty acid desaturase that catalyzes the conversion of linoleic acid (18:2) to linolenic acid (18:3), a substrate for jasmonic acid (JA) biosynthesis. The msd3 mutants had reduced levels of linolenic acid in both leaves and developing panicles that in turn decreased the levels of JA. Furthermore, the msd3 panicle phenotype was reversed by treatment with methyl-JA (MeJA). Our characterization of MSD1, MSD2, and now MSD3 demonstrates that JA-regulated processes are critical to the msd phenotype. The identification of the MSD3 gene reveals a new target that could be manipulated to increase grain number per panicle in sorghum, and potentially other cereal crops, through the genomic editing of MSD3 functional orthologs.

The maize (Zea mays) mutant Unstable factor for orange1 (Ufo1) has been implicated in the epigenetic modifications of pericarp color1 (p1), which regulates the production of the flavonoid pigments phlobaphenes. Here, we show that the ufo1 gene maps to a genetically recalcitrant region near the centromere of chromosome 10. Transcriptome analysis of Ufo1-1 mutant and wild-type plants identified a candidate gene in the mapping region using a comparative sequence-based approach. The candidate gene, GRMZM2G053177, is overexpressed by >45-fold in multiple tissues of Ufo1-1, explaining the dominance of Ufo1-1 and its phenotypes. In the mutant stock, GRMZM2G053177 has a unique transcript originating within a CACTA transposon inserted in its first intron, and it is missing the first four codons of the wild-type transcript. GRMZM2G053177 expression is regulated by the DNA methylation status of the CACTA transposon, explaining the incomplete penetrance and poor expressivity of Ufo1-1. Transgenic overexpression lines of GRMZM2G053177 (Ufo1-1) phenocopy the p1-induced pigmentation in coleoptiles, tassels, leaf sheaths, husks, pericarps, and cob glumes. Transcriptome analysis of Ufo1 versus wild-type tissues revealed changes in several pathways related to abiotic and biotic stress. Thus, this study addresses the enigma of Ufo1 identity in maize, which had gone unsolved for more than 50 years.

SUMMARY: Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum, a drought and heat‐tolerant C4 species, requires a vast, large‐scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large‐scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)‐induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis‐regulatory elements. A comparison of the distribution of mutations in the large‐scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS‐induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5′ UTR, gene body, and 3′‐UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the K_a/K_s value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB (https://www.depts.ttu.edu/igcast/sorbmutdb.php) and SorghumBase (https://sorghumbase.org/). This current large‐scale sequence‐indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

Sorghum (Sorghum bicolor) is the fifth most important cereal crop worldwide; however, its utilization in food products can be limited due to reduced nutritional quality related to amino acid composition and protein digestibility in cooked products. Low essential amino acid levels and digestibility are influenced by the composition of the sorghum seed storage proteins, kafirins. In this study, we report a core collection of 206 sorghum mutant lines with altered seed storage proteins. Wet lab chemistry analysis was conducted to evaluate the total protein content and 23 amino acids, including 19 protein-bound and 4 non-protein amino acids. We identified mutant lines with diverse compositions of essential and non-essential amino acids. The highest total protein content in these lines was almost double that of the wild-type (BTx623). The mutants identified in this study can be used as a genetic resource to improve the sorghum grain quality and determine the molecular mechanisms underlying the biosynthesis of storage protein and starch in sorghum seeds.