k-mer genome-wide association study for anthracnose and BCMV resistance in a Phaseolus vulgaris Andean Diversity Panel.

Access to broad genomic resources and closely linked marker-trait associations for common beans (Phaseolus vulgaris L.) can facilitate development of improved varieties with increased yield, improved market quality traits, and enhanced disease resistance. The emergence of virulent races of anthracnose (caused by Colletotrichum lindemuthianum) and bean common mosaic virus (BCMV) highlight the need for improved methods to identify and incorporate pan-genomic variation in breeding for disease resistance. We sequenced the P. vulgaris Andean Diversity Panel (ADP) and performed a genome-wide association study (GWAS) to identify associations for resistance to BCMV and eight races of anthracnose. Historical single nucleotide polymorphism (SNP)-chip and phenotypic data enabled a three-way comparison between SNP-chip, reference-based whole genome shotgun sequence (WGS)-SNP, and reference-free k-mer (short nucleotide subsequence) GWAS. Across all traits, there was excellent concordance between SNP-chip, WGS-SNP, and k-mer GWAS results-albeit at a much higher marker resolution for the WGS data sets. Significant k-mer haplotype variation revealed selection of the linked I-gene and Co-u traits in North American breeding lines and cultivars. Due to structural variation, only 9.1 to 47.3% of the significantly associated k-mers could be mapped to the reference genome. Thus, to determine the genetic context of cis-associated k-mers, we generated draft whole genome assemblies of four ADP accessions and identified an expanded local repertoire of disease resistance genes associated with resistance to anthracnose and BCMV. With access to variant data in the context of a pan-genome, high resolution mapping of agronomic traits for common bean is now feasible.

Cell-type-aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway of Catharanthus roseus (Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, the C. roseus MIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell-type specificity of MIA biosynthesis is achieved is poorly understood. We generated single-cell multi-omics data from C. roseus leaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)-binding site profiles, we constructed a cell-type-aware gene regulatory network for MIA biosynthesis. We showcased cell-type-specific TFs as well as cell-type-specific cis-regulatory elements. Using motif enrichment analysis, co-expression across cell types, and functional validation approaches, we discovered a novel idioblast-specific TF (Idioblast MYB1, CrIDM1) that activates expression of late-stage MIA biosynthetic genes in the idioblast. These analyses not only led to the discovery of the first documented cell-type-specific TF that regulates the expression of two idioblast-specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell-type-specific metabolic regulation.

Chromosome-scale Salvia hispanica L. (Chia) genome assembly reveals rampant Salvia interspecies introgression.

Salvia hispanica L. (Chia), a member of the Lamiaceae, is an economically important crop in Mesoamerica, with health benefits associated with its seed fatty acid composition. Chia varieties are distinguished based on seed color including mixed white and black (Chia pinta) and black (Chia negra). To facilitate research on Chia and expand on comparative analyses within the Lamiaceae, we generated a chromosome-scale assembly of a Chia pinta accession and performed comparative genome analyses with a previously published Chia negra genome assembly. The Chia pinta and Chia negra genome sequences were highly similar as shown by a limited number of single nucleotide polymorphisms and extensive shared orthologous gene membership. However, there is an enrichment of terpene synthases in the Chia pinta genome relative to the Chia negra genome. We sequenced and analyzed the genomes of 20 Chia accessions with differing seed color and geographic origin revealing population structure within S. hispanica and interspecific introgressions of Salvia species. As the genus Salvia is polyphyletic, its evolutionary history remains unclear. Using large-scale synteny analysis within the Lamiaceae and orthologous group membership, we resolved the phylogeny of Salvia species. This study and its collective resources further our understanding of genomic diversity in this food crop and the extent of interspecies hybridizations in Salvia.

Targeted genotyping-by-sequencing of potato and data analysis with R/polyBreedR.

Mid-density targeted genotyping-by-sequencing (GBS) combines trait-specific markers with thousands of genomic markers at an attractive price for linkage mapping and genomic selection. A 2.5K targeted GBS assay for potato (Solanum tuberosum L.) was developed using the DArTag technology and later expanded to 4K targets. Genomic markers were selected from the potato Infinium single nucleotide polymorphism (SNP) array to maximize genome coverage and polymorphism rates. The DArTag and SNP array platforms produced equivalent dendrograms in a test set of 298 tetraploid samples, and 83% of the common markers showed good quantitative agreement, with RMSE (root mean squared error) <0.5. DArTag is suited for genomic selection candidates in the clonal evaluation trial, coupled with imputation to a higher density platform for the training population. Using the software polyBreedR, an R package for the manipulation and analysis of polyploid marker data, the RMSE for imputation by linkage analysis was 0.15 in a small half-diallel population (N = 85), which was significantly lower than the RMSE of 0.42 with the random forest method. Regarding high-value traits, the DArTag markers for resistance to potato virus Y, golden cyst nematode, and potato wart appeared to track their targets successfully, as did multi-allelic markers for maturity and tuber shape. In summary, the potato DArTag assay is a transformative and publicly available technology for potato breeding and genetics.

The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum).

Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold-forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via an inferred acylated intermediate. This work provides a genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into iridoid biosynthesis in insects.

Chromosome-scale genome assembly of the 'Munstead' cultivar of Lavandula angustifolia.

OBJECTIVES: Lavandula angustifolia (English lavender) is commercially important not only as an ornamental species but also as a major source of fragrances. To better understand the genomic basis of chemical diversity in lavender, we sequenced, assembled, and annotated the 'Munstead' cultivar of L. angustifolia. DATA DESCRIPTION: A total of 80 Gb of Oxford Nanopore Technologies reads was used to assemble the 'Munstead' genome using the Canu genome assembler software. Following multiple rounds of error correction and scaffolding using Hi-C data, the final chromosome-scale assembly represents 795,075,733 bp across 25 chromosomes with an N50 scaffold length of 31,371,815 bp. Benchmarking Universal Single Copy Orthologs analysis revealed 98.0% complete orthologs, indicative of a high-quality assembly representative of genic space. Annotation of protein-coding sequences revealed 58,702 high-confidence genes encoding 88,528 gene models. Access to the 'Munstead' genome will permit comparative analyses within and among lavender accessions and provides a pivotal species for comparative analyses within Lamiaceae.

Cold stress induces differential gene expression of retained homeologs in Camelina sativa cv Suneson.

Camelina sativa (L.) Crantz, a member of the Brassicaceae, has potential as a biofuel feedstock which is attributable to the production of fatty acids in its seeds, its fast growth cycle, and low input requirements. While a genome assembly is available for camelina, it was generated from short sequence reads and is thus highly fragmented in nature. Using long read sequences, we generated a chromosome-scale, highly contiguous genome assembly (644,491,969 bp) for the spring biotype cultivar 'Suneson' with an N50 contig length of 12,031,512 bp and a scaffold N50 length of 32,184,682 bp. Annotation of protein-coding genes revealed 91,877 genes that encode 133,355 gene models. We identified a total of 4,467 genes that were significantly up-regulated under cold stress which were enriched in gene ontology terms associated with "response to cold" and "response to abiotic stress". Coexpression analyses revealed multiple coexpression modules that were enriched in genes differentially expressed following cold stress that had putative functions involved in stress adaptation, specifically within the plastid. With access to a highly contiguous genome assembly, comparative analyses with Arabidopsis thaliana revealed 23,625 A. thaliana genes syntenic with 45,453 Suneson genes. Of these, 24,960 Suneson genes were syntenic to 8,320 A. thaliana genes reflecting a 3 camelina homeolog to 1 Arabidopsis gene relationship and retention of all three homeologs. Some of the retained triplicated homeologs showed conserved gene expression patterns under control and cold-stressed conditions whereas other triplicated homeologs displayed diverged expression patterns revealing sub- and neo-functionalization of the homeologs at the transcription level. Access to the chromosome-scale assembly of Suneson will enable both basic and applied research efforts in the improvement of camelina as a sustainable biofuel feedstock.

Identification of genes associated with abiotic stress tolerance in sweetpotato using weighted gene co-expression network analysis.

Sweetpotato, Ipomoea batatas (L.), a key food security crop, is negatively impacted by heat, drought, and salinity stress. The orange-fleshed sweetpotato cultivar "Beauregard" was exposed to heat, salt, and drought treatments for 24 and 48 h to identify genes responding to each stress condition in leaves. Analysis revealed both common (35 up regulated, 259 down regulated genes in the three stress conditions) and unique sets of up regulated (1337 genes by drought, 516 genes by heat, and 97 genes by salt stress) and down regulated (2445 genes by drought, 678 genes by heat, and 204 genes by salt stress) differentially expressed genes (DEGs) suggesting common, yet stress-specific transcriptional responses to these three abiotic stressors. Gene Ontology analysis of down regulated DEGs common to both heat and salt stress revealed enrichment of terms associated with "cell population proliferation" suggestive of an impact on the cell cycle by the two stress conditions. To identify shared and unique gene co-expression networks under multiple abiotic stress conditions, weighted gene co-expression network analysis was performed using gene expression profiles from heat, salt, and drought stress treated 'Beauregard' leaves yielding 18 co-expression modules. One module was enriched for "response to water deprivation," "response to abscisic acid," and "nitrate transport" indicating synergetic crosstalk between nitrogen, water, and phytohormones with genes encoding osmotin, cell expansion, and cell wall modification proteins present as key hub genes in this drought-associated module. This research lays the groundwork for exploring to a further degree, mechanisms for abiotic stress tolerance in sweetpotato.

Genetic variation in a tepary bean (Phaseolus acutifolius A. Gray) diversity panel reveals loci associated with biotic stress resistance.

Tepary bean (Phaseolus acutifolius A. Gray), indigenous to the arid climates of northern Mexico and the Southwest United States, diverged from common bean (Phaseolus vulgaris L.), approximately 2 million years ago and exhibits a wide range of resistance to biotic stressors. The tepary genome is highly syntenic to the common bean genome providing a foundation for discovery and breeding of agronomic traits between these two crop species. Although a limited number of adaptive traits from tepary bean have been introgressed into common bean, hybridization barriers between these two species required the development of bridging lines to alleviate this barrier. Thus, to fully utilize the extant tepary bean germplasm as both a crop and as a donor of adaptive traits, we developed a diversity panel of 422 cultivated, weedy, and wild tepary bean accessions which were then genotyped and phenotyped to enable population genetic analyses and genome-wide association studies for their response to a range of biotic stressors. Population structure analyses of the panel revealed eight subpopulations and the differentiation of botanical varieties within P. acutifolius. Genome-wide association studies revealed loci and candidate genes underlying biotic stress resistance including quantitative trait loci for resistance to weevils, common bacterial blight, Fusarium wilt, and bean common mosaic necrosis virus that can be harnessed not only for tepary bean but also common bean improvement.

Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Chromosome-scale assembly of the Verbenaceae species Queen's Wreath (Petrea volubilis L.).

OBJECTIVES: Petrea volubilis, a member of the Order Lamiales and the Verbenaceae family, is an important horticultural species that has been used in traditional folk medicine. To provide a genome sequence for comparative studies within the Order Lamiales that includes important families such as Lamiaceae (mints), we generated a long-read, chromosome-scale genome assembly of this species. DATA DESCRIPTION: Using a total of 45.5 Gb of Pacific Biosciences long read sequence, we generated a 480.2 Mb assembly of P. volubilis, of which, 93% is chromosome anchored. Representation of genic regions was robust with 96.6% of the Benchmarking of Universal Single Copy Orthologs present in the genome assembly. A total of 57.8% of the genome was annotated as a repetitive sequence. Using a gene annotation pipeline that included refinement of gene models using transcript evidence, 30,982 high confidence genes were annotated. Access to the P. volubilis genome will facilitate evolutionary studies in the Lamiales, a key order of Asterids that includes significant crop and medicinal plant species.

Genome Report: Genome sequence of 1S1, a transformable and highly regenerable diploid potato for use as a model for gene editing and genetic engineering.

Availability of readily transformable germplasm, as well as efficient pipelines for gene discovery are notable bottlenecks in the application of genome editing in potato. To study and introduce traits such as resistance against biotic and abiotic factors, tuber quality traits and self-fertility, model germplasm that is amenable to gene editing and regeneration is needed. Cultivated potato is a heterozygous autotetraploid and its genetic redundancy and complexity makes studying gene function challenging. Genome editing is simpler at the diploid level, with fewer allelic variants to consider. A readily transformable diploid potato would be further complemented by genomic resources that could aid in high throughput functional analysis. The heterozygous Solanum tuberosum Group Phureja clone 1S1 has a high regeneration rate, self-fertility, desirable tuber traits and is amenable to Agrobacterium-mediated transformation. We leveraged its amenability to Agrobacterium-mediated transformation to create a Cas9 constitutively expressing line for use in viral vector-based gene editing. To create a contiguous genome assembly, a homozygous doubled monoploid of 1S1 (DM1S1) was sequenced using 44 Gbp of long reads generated from Oxford Nanopore Technologies (ONT), yielding a 736 Mb assembly that encoded 31,145 protein-coding genes. The final assembly for DM1S1 represents a nearly complete genic space, shown by the presence of 99.6% of the genes in the Benchmarking Universal Single Copy Orthologs (BUSCO) set. Variant analysis with Illumina reads from 1S1 was used to deduce its alternate haplotype. These genetic and genomic resources provide a toolkit for applications of genome editing in both basic and applied research of potato.

Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a dynamic evolutionary trajectory.

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites.

Leveraging prior biological knowledge improves prediction of tocochromanols in maize grain.

With an essential role in human health, tocochromanols are mostly obtained by consuming seed oils; however, the vitamin E content of the most abundant tocochromanols in maize (Zea mays L.) grain is low. Several large-effect genes with cis-acting variants affecting messenger RNA (mRNA) expression are mostly responsible for tocochromanol variation in maize grain, with other relevant associated quantitative trait loci (QTL) yet to be fully resolved. Leveraging existing genomic and transcriptomic information for maize inbreds could improve prediction when selecting for higher vitamin E content. Here, we first evaluated a multikernel genomic best linear unbiased prediction (MK-GBLUP) approach for modeling known QTL in the prediction of nine tocochromanol grain phenotypes (12-21 QTL per trait) within and between two panels of 1,462 and 242 maize inbred lines. On average, MK-GBLUP models improved predictive abilities by 7.0-13.6% when compared with GBLUP. In a second approach with a subset of 545 lines from the larger panel, the highest average improvement in predictive ability relative to GBLUP was achieved with a multi-trait GBLUP model (15.4%) that had a tocochromanol phenotype and transcript abundances in developing grain for a few large-effect candidate causal genes (1-3 genes per trait) as multiple response variables. Taken together, our study illustrates the enhancement of prediction models when informed by existing biological knowledge pertaining to QTL and candidate causal genes.

Keeping time in the dark: Potato diel and circadian rhythmic gene expression reveals tissue-specific circadian clocks.

The circadian clock is an internal molecular oscillator and coordinates numerous physiological processes through regulation of molecular pathways. Tissue-specific clocks connected by mobile signals have previously been found to run at different speeds in Arabidopsis thaliana tissues. However, tissue variation in circadian clocks in crop species is unknown. In this study, leaf and tuber global gene expression in cultivated potato under cycling and constant environmental conditions was profiled. In addition, we used a circadian-regulated luciferase reporter construct to study tuber gene expression rhythms. Diel and circadian expression patterns were present among 17.9% and 5.6% of the expressed genes in the tuber. Over 500 genes displayed differential tissue specific diel phases. Intriguingly, few core circadian clock genes had circadian expression patterns, while all such genes were circadian rhythmic in cultivated tomato leaves. Furthermore, robust diel and circadian transcriptional rhythms were observed among detached tubers. Our results suggest alternative regulatory mechanisms and/or clock composition is present in potato, as well as the presence of tissue-specific independent circadian clocks. We have provided the first evidence of a functional circadian clock in below-ground storage organs, holding important implications for other storage root and tuberous crops.

Combining GWAS and TWAS to identify candidate causal genes for tocochromanol levels in maize grain.

Tocochromanols (tocopherols and tocotrienols, collectively vitamin E) are lipid-soluble antioxidants important for both plant fitness and human health. The main dietary sources of vitamin E are seed oils that often accumulate high levels of tocopherol isoforms with lower vitamin E activity. The tocochromanol biosynthetic pathway is conserved across plant species but an integrated view of the genes and mechanisms underlying natural variation of tocochromanol levels in seed of most cereal crops remains limited. To address this issue, we utilized the high mapping resolution of the maize Ames panel of ∼1,500 inbred lines scored with 12.2 million single-nucleotide polymorphisms to generate metabolomic (mature grain tocochromanols) and transcriptomic (developing grain) data sets for genetic mapping. By combining results from genome- and transcriptome-wide association studies, we identified a total of 13 candidate causal gene loci, including 5 that had not been previously associated with maize grain tocochromanols: 4 biosynthetic genes (arodeH2 paralog, dxs1, vte5, and vte7) and a plastid S-adenosyl methionine transporter (samt1). Expression quantitative trait locus (eQTL) mapping of these 13 gene loci revealed that they are predominantly regulated by cis-eQTL. Through a joint statistical analysis, we implicated cis-acting variants as responsible for colocalized eQTL and GWAS association signals. Our multiomics approach provided increased statistical power and mapping resolution to enable a detailed characterization of the genetic and regulatory architecture underlying tocochromanol accumulation in maize grain and provided insights for ongoing biofortification efforts to breed and/or engineer vitamin E and antioxidant levels in maize and other cereals.

Transcriptome-wide association and prediction for carotenoids and tocochromanols in fresh sweet corn kernels.

Sweet corn (Zea mays L.) is consistently one of the most highly consumed vegetables in the United States, providing a valuable opportunity to increase nutrient intake through biofortification. Significant variation for carotenoid (provitamin A, lutein, zeaxanthin) and tocochromanol (vitamin E, antioxidants) levels is present in temperate sweet corn germplasm, yet previous genome-wide association studies (GWAS) of these traits have been limited by low statistical power and mapping resolution. Here, we employed a high-quality transcriptomic dataset collected from fresh sweet corn kernels to conduct transcriptome-wide association studies (TWAS) and transcriptome prediction studies for 39 carotenoid and tocochromanol traits. In agreement with previous GWAS findings, TWAS detected significant associations for four causal genes, ß-carotene hydroxylase (crtRB1), lycopene epsilon cyclase (lcyE), γ-tocopherol methyltransferase (vte4), and homogentisate geranylgeranyltransferase (hggt1) on a transcriptome-wide level. Pathway-level analysis revealed additional associations for deoxy-xylulose synthase2 (dxs2), diphosphocytidyl methyl erythritol synthase2 (dmes2), cytidine methyl kinase1 (cmk1), and geranylgeranyl hydrogenase1 (ggh1), of which, dmes2, cmk1, and ggh1 have not previously been identified through maize association studies. Evaluation of prediction models incorporating genome-wide markers and transcriptome-wide abundances revealed a trait-dependent benefit to the inclusion of both genomic and transcriptomic data over solely genomic data, but both transcriptome- and genome-wide datasets outperformed a priori candidate gene-targeted prediction models for most traits. Altogether, this study represents an important step toward understanding the role of regulatory variation in the accumulation of vitamins in fresh sweet corn kernels.

Phased, chromosome-scale genome assemblies of tetraploid potato reveal a complex genome, transcriptome, and predicted proteome landscape underpinning genetic diversity.

Cultivated potato is a clonally propagated autotetraploid species with a highly heterogeneous genome. Phased assemblies of six cultivars including two chromosome-scale phased genome assemblies revealed extensive allelic diversity, including altered coding and transcript sequences, preferential allele expression, and structural variation that collectively result in a highly complex transcriptome and predicted proteome, which are distributed across the homologous chromosomes. Wild species contribute to the extensive allelic diversity in tetraploid cultivars, demonstrating ancestral introgressions predating modern breeding efforts. As a clonally propagated autotetraploid that undergoes limited meiosis, dysfunctional and deleterious alleles are not purged in tetraploid potato. Nearly a quarter of the loci bore mutations are predicted to have a high negative impact on protein function, complicating breeder's efforts to reduce genetic load. The StCDF1 locus controls maturity, and analysis of six tetraploid genomes revealed that 12 allelic variants of StCDF1 are correlated with maturity in a dosage-dependent manner. Knowledge of the complexity of the tetraploid potato genome with its rampant structural variation and embedded deleterious and dysfunctional alleles will be key not only to implementing precision breeding of tetraploid cultivars but also to the construction of homozygous, diploid potato germplasm containing favorable alleles to capitalize on heterosis in F1 hybrids.

Genome-wide association study suggests an independent genetic basis of zinc and cadmium concentrations in fresh sweet corn kernels.

Despite being one of the most consumed vegetables in the United States, the elemental profile of sweet corn (Zea mays L.) is limited in its dietary contributions. To address this through genetic improvement, a genome-wide association study was conducted for the concentrations of 15 elements in fresh kernels of a sweet corn association panel. In concordance with mapping results from mature maize kernels, we detected a probable pleiotropic association of zinc and iron concentrations with nicotianamine synthase5 (nas5), which purportedly encodes an enzyme involved in synthesis of the metal chelator nicotianamine. In addition, a pervasive association signal was identified for cadmium concentration within a recombination suppressed region on chromosome 2. The likely causal gene underlying this signal was heavy metal ATPase3 (hma3), whose counterpart in rice, OsHMA3, mediates vacuolar sequestration of cadmium and zinc in roots, whereby regulating zinc homeostasis and cadmium accumulation in grains. In our association panel, hma3 associated with cadmium but not zinc accumulation in fresh kernels. This finding implies that selection for low cadmium will not affect zinc levels in fresh kernels. Although less resolved association signals were detected for boron, nickel, and calcium, all 15 elements were shown to have moderate predictive abilities via whole-genome prediction. Collectively, these results help enhance our genomics-assisted breeding efforts centered on improving the elemental profile of fresh sweet corn kernels.

Genomic variation within the maize stiff-stalk heterotic germplasm pool.

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.