The Stiff Stalk heterotic pool is a foundation of US maize seed parent germplasm and has been heavily utilized by both public and private maize breeders since its inception in the 1930s. Flowering time and plant height are critical characteristics for both inbred parents and their test crossed hybrid progeny. To study these traits, a 6-parent multiparent advanced generation intercross population was developed including maize inbred lines B73, B84, PHB47 (B37 type), LH145 (B14 type), PHJ40 (novel early Stiff Stalk), and NKH8431 (B73/B14 type). A set of 779 doubled haploid lines were evaluated for flowering time and plant height in 2 field replicates in 2016 and 2017, and a subset of 689 and 561 doubled haploid lines were crossed to 2 testers, respectively, and evaluated as hybrids in 2 locations in 2018 and 2019 using an incomplete block design. Markers were derived from a practical haplotype graph built from the founder whole genome assemblies and genotype-by-sequencing and exome capture-based sequencing of the population. Genetic mapping utilizing an update to R/qtl2 revealed differing profiles of significant loci for both traits between 635 of the DH lines and 2 sets of 570 and 471 derived hybrids. Genomic prediction was used to test the feasibility of predicting hybrid phenotypes based on the per se data. Predictive abilities were highest on direct models trained using the data they would predict (0.55-0.63), and indirect models trained using per se data to predict hybrid traits had slightly lower predictive abilities (0.49-0.55). Overall, this finding is consistent with the overlapping and nonoverlapping significant quantitative trait loci found within the per se and hybrid populations and suggests that selections for phenology traits can be made effectively on doubled haploid lines before hybrid data is available.

Panicum virgatum L. (switchgrass) is a polyploid, perennial grass species that is native to North America, and is being developed as a future biofuel feedstock crop. Switchgrass is present primarily in two ecotypes: a northern upland ecotype, composed of tetraploid and octoploid accessions, and a southern lowland ecotype, composed of primarily tetraploid accessions. We employed high-coverage exome capture sequencing (~2.4 Tb) to genotype 537 individuals from 45 upland and 21 lowland populations. From these data, we identified ~27 million single-nucleotide polymorphisms (SNPs), of which 1 590 653 high-confidence SNPs were used in downstream analyses of diversity within and between the populations. From the 66 populations, we identified five primary population groups within the upland and lowland ecotypes, a result that was further supported through genetic distance analysis. We identified conserved, ecotype-restricted, non-synonymous SNPs that are predicted to affect the protein function of CONSTANS (CO) and EARLY HEADING DATE 1 (EHD1), key genes involved in flowering, which may contribute to the phenotypic differences between the two ecotypes. We also identified, relative to the near-reference Kanlow population, 17 228 genes present in more copies than in the reference genome (up-CNVs), 112 630 genes present in fewer copies than in the reference genome (down-CNVs) and 14 430 presence/absence variants (PAVs), affecting a total of 9979 genes, including two upland-specific CNV clusters. In total, 45 719 genes were affected by an SNP, CNV, or PAV across the panel, providing a firm foundation to identify functional variation associated with phenotypic traits of interest for biofuel feedstock production.

The stiff-stalk heterotic group in Maize (Zea mays L.) is an important source of inbreds used in U.S. commercial hybrid production. Founder inbreds B14, B37, B73, and, to a lesser extent, B84, are found in the pedigrees of a majority of commercial seed parent inbred lines. We created high-quality genome assemblies of B84 and four expired Plant Variety Protection (ex-PVP) lines LH145 representing B14, NKH8431 of mixed descent, PHB47 representing B37, and PHJ40, which is a Pioneer Hi-Bred International (PHI) early stiff-stalk type. Sequence was generated using long-read sequencing achieving highly contiguous assemblies of 2.13-2.18 Gbp with N50 scaffold lengths >200 Mbp. Inbred-specific gene annotations were generated using a core five-tissue gene expression atlas, whereas transposable element (TE) annotation was conducted using de novo and homology-directed methodologies. Compared with the reference inbred B73, synteny analyses revealed extensive collinearity across the five stiff-stalk genomes, although unique components of the maize pangenome were detected. Comparison of this set of stiff-stalk inbreds with the original Iowa Stiff Stalk Synthetic breeding population revealed that these inbreds represent only a proportion of variation in the original stiff-stalk pool and there are highly conserved haplotypes in released public and ex-Plant Variety Protection inbreds. Despite the reduction in variation from the original stiff-stalk population, substantial genetic and genomic variation was identified supporting the potential for continued breeding success in this pool. The assemblies described here represent stiff-stalk inbreds that have historical and commercial relevance and provide further insight into the emerging maize pangenome.

Background

Maize stover is an important source of crop residues and a promising sustainable energy source in the United States. Stalk is the main component of stover, representing about half of stover dry weight. Characterization of genetic determinants of stalk traits provide a foundation to optimize maize stover as a biofuel feedstock. We investigated maize natural genetic variation in genome-wide association studies (GWAS) to detect candidate genes associated with traits related to stalk biomass (stalk diameter and plant height) and stalk anatomy (rind thickness, vascular bundle density and area).

Results

Using a panel of 942 diverse inbred lines, 899,784 RNA-Seq derived single nucleotide polymorphism (SNP) markers were identified. Stalk traits were measured on 800 members of the panel in replicated field trials across years. GWAS revealed 16 candidate genes associated with four stalk traits. Most of the detected candidate genes were involved in fundamental cellular functions, such as regulation of gene expression and cell cycle progression. Two of the regulatory genes (Zmm22 and an ortholog of Fpa) that were associated with plant height were previously shown to be involved in regulating the vegetative to floral transition. The association of Zmm22 with plant height was confirmed using a transgenic approach. Transgenic lines with increased expression of Zmm22 showed a significant decrease in plant height as well as tassel branch number, indicating a pleiotropic effect of Zmm22.

Conclusion

Substantial heritable variation was observed in the association panel for stalk traits, indicating a large potential for improving useful stalk traits in breeding programs. Genome-wide association analyses detected several candidate genes associated with multiple traits, suggesting common regulatory elements underlie various stalk traits. Results of this study provide insights into the genetic control of maize stalk anatomy and biomass.

Switchgrass ( is a perennial native North American grass present in two ecotypes: upland, found primarily in the northern range of switchgrass habitats, and lowland, found largely in the southern reaches of switchgrass habitats. Previous studies focused on a diversity panel of primarily northern switchgrass, so to expand our knowledge of genetic diversity in a broader set of North American switchgrass, exome capture sequence data were generated for 632 additional, primarily lowland individuals. In total, over 37 million single nucleotide polymorphisms (SNPs) were identified and a set of 1.9 million high-confidence SNPs were obtained from 1169 individuals from 140 populations (67 upland, 65 lowland, 8 admixed) were used in downstream analyses of genetic diversity and population structure. Seven separate population groups were identified with moderate genetic differentiation [mean fixation index (Fst) estimate of 0.06] between the lowland and the upland populations. Ecotype-specific and population-specific SNPs were identified for use in germplasm evaluations. Relative to rice ( L.), maize ( L.), soybean [ (L.) Merr.], and Gaertn., analyses of nucleotide diversity revealed a high degree of genetic diversity (0.0135) across all individuals, consistent with the outcrossing mode of reproduction and the polyploidy of switchgrass. This study supports the hypothesis that repeated glaciation events, ploidy barriers, and restricted gene flow caused by flowering time differences have resulted in distinct gene pools across ecotypes and geographic regions. These data provide a resource to associate alleles with traits of interest for forage, restoration, and biofuel feedstock efforts in switchgrass.