Coleoptile is a sheath-like structure unique to monocots and is easily observed in sorghum. Colored coleoptiles have been shown to protect plants against abiotic and biotic stresses. The purpose of this study was to identify factors controlling coleoptile color in sorghum. We phenotyped the sorghum mini core accessions for coleoptile color in two environments, determined the anthocyanin content of each color of selected accessions, carried out a genome-wide association analysis and identified a candidate gene. The phenotypic analysis showed that 95 (40% of 235) accessions were green, 28 (12%) were purple and 42 (18%) were red in both 2022 and 2023. About 12% of the accessions changed from green to red due to environmental conditions. The anthocyanin content analysis showed a positive correlation between intensity of coleoptile color and anthocyanin levels. A genome-wide association analysis identified two candidate genes, Sobic.006G175700 and Sobic.006G175500, mapped to this trait in a single locus on chromosome 6. An orthologous comparison, together with mapping, sequence analysis and qPCR, identified Sobic.006G175700 as Rs1, the gene determining the sorghum coleoptile color. The haplotype analysis with SNPs from both coding and upstream regions of Sobic.006G175700 indicates that the predominant haplotypes can differentiate between green and colored coleoptile colors. This information can be used for marker-assisted selection of desired coleoptile colors in sorghum.

Grain size is a crucial agronomic trait that significantly impacts yield potential in sorghum (Sorghum bicolor), making it a key focus for genetic improvement. In this study, we investigated the genetic basis of grain size variation using two contrasting sorghum accessions, PI302232 (small grain, Sg) and PI563512 (large grain, Lg). The 1000-grain weight, grain length, and grain width of Lg were 3.63-fold, 1.22-fold, and 1.65-fold higher than Sg, respectively. The 1000-grain weight in the F₂ segregating population derived from the cross Sg and Lg parents exhibited the highest phenotypic variation and followed a normal distribution in the three traits. Using bulked segregant analysis sequencing (BSA-seq) with small- and large-grain bulks from the F₂ population, two major quantitative trait loci (QTLs) for grain size were identified on chromosomes 1 and 3. Fine mapping with SSR markers narrowed the qGS1 locus to a 1.03 Mb region on chromosome 1 (Chr01: 22,001,448–23,035,593), containing 49 candidate genes. To narrow down potential candidate genes, transcriptome analysis of spike tissues from Sg and Lg at 0 and 14 days after heading revealed 3719 differentially expressed genes (DEGs), primarily enriched in “starch and sucrose metabolism” and “phenylpropanoid biosynthesis” pathways. Integrating fine mapping intervals and RNA-seq data, 6 DEGs were identified within the qGS1 region. Quantitative real-time PCR confirmed that 6 genes exhibited different expression at two stages. The expression and bioinformatics analysis showed Sobic.001G230700 was the most likely candidate gene for the qGS1 locus. This study provides new insights into the genetic regulation of grain size and a new target to improve grain size in sorghum.

Brown midrib (bmr) mutants are frequently associated with unfavorable agronomic traits. In this study, we identified a novel brown midrib mutant, bmr34, which exhibited distinct brown coloration in roots, stems, and leaf midribs. Although most classic bmr mutants show undesirable agronomic performance, this mutant displays altered lignin accumulation and has important potential for forage quality and biomass utilization, providing a key genetic resource for lignin regulatory research in sorghum. Compared to the wild-type, bmr34 showed no significant differences in five major agronomic traits; however, lignin content was significantly reduced. Bulked segregant analysis (BSA) using an F₂ population derived from a cross between bmr34 and the wild type Tx623 mapped the candidate region to chromosome 4. Further sequencing analysis identified a single nucleotide substitution (C → T; reverse strand G → A) at position 5,731,348 within the 5′ splice site of the third intron of Sobic.004G071000 in the mapping interval. KASP marker analysis demonstrated complete co-segregation between the mutation site and the bmr phenotype. Sequence analysis also revealed that this G → A substitution resulted in aberrant splicing and a 33-bp insertion in the third exon, which introduced a premature stop codon. Notably, the normally spliced transcript still accounted for approximately 36.2% of total transcripts in bmr34, indicating partial retention of wild-type transcript processing. These results demonstrate that bmr34 represents a novel weak allele of Bmr6, providing new insights into splice-site mutations and their contribution to lignin biosynthesis regulation in sorghum.