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Several bioactive components of the human diet have major effects on composition and function of the gut microbiome, but no systematic framework exists for understanding variation in microbiome-active components amid the vast amount of genotypic and phenotypic variation within a given species of food crop. Here we present a powerful new approach for complex trait analysis of Microbiome-Active Traits (MATs) in food crops. Capitalizing on a novel automated in vitro microbiome screening (AiMS) methodology to quantify human gut microbiome phenotypes after fermentation of grain from genetically diverse lines, we show how microbiome phenotypes can be used as quantitative traits for genetic analysis. Quantitative Trait Locus (QTL) analysis of AiMS-based phenotypes across grain samples from 294 sorghum (Sorghum bicolor) recombinant inbred lines identified significant QTLs at 10 different genomic regions that collectively control MATs affecting 16 different microbial taxa. Segregation analysis and validation in Near-Isogenic Lines (NILs) confirmed that overlapping QTL peaks for microbiome phenotypes, seed color, and tannin concentration are driven by variation in the Tan2 (chromosome 2) and Tan1 (chromosome 4) regulators of the tannin biosynthetic pathway. Candidate genes at other QTLs suggest that variation in a diverse array of plant molecules can drive MATs.

Background:Waxy starches contain >90% amylopectin and are derived from grain crops carrying naturally-occurring mutations that block amylose biosynthesis. The absence of amylose in waxy starches produces unique physiochemical properties that are desirable for food processing, but the effects of increased amylopectin/amylose ratios in waxy starches on the gut microbiome and physiological characteristics of the host are not well characterized. Here, we used a whole-grain model with isogenic pairs of wild type sorghum lines and their waxy derivatives to test the hypothesis that major differences in amylose/amylopectin ratio produce significant effects on the human gut microbiome. Results:Fermentation of grain from waxy versus wild type derivatives produced substantial differences in overall microbiome composition, abundances of multiple taxa, and production of microbial metabolites (butyrate). Several of the taxonomic and metabolic signatures of fermentations from parental versus waxy lines were shared across fermentations with microbiomes from different human donors, including reduced levels of butyrate production and lower abundances of Roseburia and other amylolytic, butyrate-producing members of Lachnospiraceae in fermentations of waxy lines. Using a human microbiome-associated mouse model, we also detected significant differences in microbiome composition in animals fed low-fiber diets supplemented with 20% grain from isogenic pairs of parental versus waxy derivatives of sorghum. Remarkably, these microbiome changes were accompanied by significant differences in weight gain, with animals consuming waxy sorghum gaining significantly more weight. Conclusions:We conclude that the benefits of waxy starches on food functionality can have trade-off effects on the gut microbiome and host physiology that could be particularly relevant in human populations consuming large amounts of waxy grains.

In sorghum (Sorghum bicolor) and other C4 grasses, brown midrib (bmr) mutants have long been associated with plants impaired in their ability to synthesize lignin. The brown midrib 30 (Bmr30) gene, identified using a bulk segregant analysis and next-generation sequencing, was determined to encode a chalcone isomerase (CHI). Two independent mutations within this gene confirmed that loss of its function was responsible for the brown leaf midrib phenotype and reduced lignin concentration. Loss of the Bmr30 gene function, as shown by histochemical staining of leaf midrib and stalk sections, resulted in altered cell wall composition. In the bmr30 mutants, CHI activity was drastically reduced, and the accumulation of total flavonoids and total anthocyanins was impaired, which is consistent with its function in flavonoid biosynthesis. The level of the flavone lignin monomer tricin was reduced 20-fold in the stem relative to wild type, and to undetectable levels in the leaf tissue of the mutants. The bmr30 mutant, therefore, harbors a mutation in a phenylpropanoid biosynthetic gene that is key to the interconnection between flavonoids and monolignols, both of which are utilized for lignin synthesis in the grasses.

In sorghum (Sorghum bicolor) and other C4 grasses, brown midrib (bmr) mutants have long been associated with plants impaired in their ability to synthesize lignin. The brown midrib 30 (Bmr30) gene, identified using a bulk segregant analysis and next-generation sequencing, was determined to encode a chalcone isomerase (CHI). Two independent mutations within this gene confirmed that loss of its function was responsible for the brown leaf midrib phenotype and reduced lignin concentration. Loss of the Bmr30 gene function, as shown by histochemical staining of leaf midrib and stalk sections, resulted in altered cell wall composition. In the bmr30 mutants, CHI activity was drastically reduced, and the accumulation of total flavonoids and total anthocyanins was impaired, which is consistent with its function in flavonoid biosynthesis. The level of the flavone lignin monomer tricin was reduced 20-fold in the stem relative to wild type, and to undetectable levels in the leaf tissue of the mutants. The bmr30 mutant, therefore, harbors a mutation in a phenylpropanoid biosynthetic gene that is key to the interconnection between flavonoids and monolignols, both of which are utilized for lignin synthesis in the grasses.