Popcorn varieties are agronomically sub-optimal and genetically limited compared to other maize subspecies. To increase genetic diversity and improve popcorn agronomics, dent germplasm has been introduced to popcorn with limited success and generally, major loss of popping. Between 2013 and 2018, 12 Quality Protein Popcorn (QPP) inbreds containing Quality Protein Maize (QPM) and popcorn germplasm were produced that maintained popping while carrying the opaque-2 allele conferring elevated kernel lysine. This is an opportune trait in the growing market for healthier snacks and a model for mining QPM traits into popcorn. We crossed QPP inbreds to explore the effects of heterosis on popcorn protein, popping quality, and plant agronomics and selected hybrids for further production. To rank and intermediately prescreen hybrids, we utilized a novel hybrid-ranking model adapted from a rank summation index while examining the inbred general combining ability and hybrid specific combining ability estimates for all traits. We observed a biological manifestation of heterosis by categorizing hybrids by pedigree that resulted in a stepwise progression of trait improvement. These results corroborated our hybrid selection and offered insight in basic heterosis research. Estimates for popcorn quality and agronomic trait covariances also suggest the synergistic introgression of highly vitreous dent maize (QPM) into popcorn, providing a likely explanation for the successfully maintained vitreous endosperm, protein quality and popping traits in line with a remodeled proteome. QPP hybrids maintained improved amino acid profiles although different popping methods variably affected popcorn's protein bound and free amino acid levels. This preliminary screening of QPP hybrids is enabling further quantitative selection for large-scale, complex trait comparison to currently marketed elite popcorn varieties.

A one way reproductive barrier exists between most popcorn varieties and dent corn varieties grown in the United States. This barrier is predominantly controlled by the ga1 locus. Using data from a diverse population of popcorn accessions pollinated by a dent corn tester, we found that the non-reciprocal pollination barrier conferred by ga1 is more complex than previously described. Individual accessions ranged from 0% to 100% compatible with dent corn pollen. Using conventional genotyping-by-sequencing data from 371 popcorn accessions carrying Ga1-s, seven significant modifiers of dent pollen compatibility were identified on five chromosomes. One locus may either be a nonfunctional ga1 allele present within popcorn, or second necessary gene for the reproductive barrier in genetic linkage with ga1, while the other modifiers are clearly genetically unlinked. The existence of ga1 modifiers segregating in a popcorn genetic background may indicate selective pressure to allow gene flow between populations, which should be incorporated into future models of the impact of genetic incompatibility loci on gene flow in natural and agricultural plant populations.

The effects of fiber, complex carbohydrates, lipids, and small molecules from food matrices on the human gut microbiome have been increasingly studied. Much less is known about how dietary protein can influence the composition and function of the gut microbial community. Here, we used near-isogenic maize lines of conventional popcorn and quality-protein popcorn (QPP) to study the effects of the opaque-2 mutation and associated quality-protein modifiers on the human gut microbiome. Opaque-2 blocks the synthesis of major maize seed proteins (α-zeins), resulting in a compensatory synthesis of new seed proteins that are nutritionally beneficial with substantially higher levels of the essential amino acids lysine and tryptophan. We show that QPP lines stimulate greater amounts of butyrate production by human gut microbiomes in in vitro fermentation of popped and digested corn from parental and QPP hybrids. In human gut microbiomes derived from diverse individuals, bacterial taxa belonging to the butyrate-producing family Lachnospiraceae, including the genera Coprococcus and Roseburia were consistently increased when fermenting QPP vs. parental popcorn lines. We conducted molecular complementation to further demonstrate that lysine-enriched seed protein can stimulate growth and butyrate production by microbes through distinct pathways. Our data show that organisms such as Coprococcus can utilize lysine and that other gut microbes, such as Roseburia spp., instead, utilize fructoselysine produced during thermal processing (popping) of popcorn. Thus, the combination of seed composition in QPP and interaction of protein adducts with carbohydrates during thermal processing can stimulate the growth of health-promoting, butyrate-producing organisms in the human gut microbiome through multiple pathways.

Tandem duplication gives rise to copy number variation and subsequent functional novelty among genes as well as diversity between individuals in a species. Functional novelty can result from either divergence in coding sequence or divergence in patterns of gene transcriptional regulation. Here, we investigate conservation and divergence of both gene sequence and gene regulation between the copies of the α-zein gene family in maize inbreds B73 and W22. We used RNA-seq data generated from developing, self-pollinated kernels at three developmental stages timed to coincide with early and peak zein expression. The reference genome annotations for B73 and W22 were modified to ensure accurate inclusion of their respective α-zein gene models to accurately assess copy-specific expression. Expression analysis indicated that although the total expression of α-zeins is higher in W22, the pattern of expression in both lines is conserved. Additional analysis of publicly available RNA-seq data from a diverse population of maize inbreds also demonstrates variation in absolute expression, but conservation of expression patterns across a wide range of maize genotypes and α-zein haplotypes.