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.

Interactions between breeding system and ploidy affect niche breadth in Solanum.

Understanding the factors driving ecological and evolutionary interactions of economically important plant species is important for agricultural sustainability. The geography of crop wild relatives, including wild potatoes (Solanum section Petota), have received attention; however, such information has not been analysed in combination with phylogenetic histories, genomic composition and reproductive systems to identify potential species for use in breeding for abiotic stress tolerance. We used a combination of ordinary least-squares (OLS) and phylogenetic generalized least-squares (PGLM) analyses to identify the discrete climate classes that make up the climate niche that wild potato species inhabit in the context of breeding system and ploidy. Self-incompatible diploid or self-compatible polyploid species significantly increase the number of discrete climate classes within a climate niche inhabited. This result was sustained when correcting for phylogenetic non-independence in the linear model. Our results support the idea that specific breeding system and ploidy combinations increase niche breadth through the decoupling of geographical range and niche diversity, and therefore, these species may be of particular interest for crop adaptation to a changing climate.

Acylsugars protect Nicotiana benthamiana against insect herbivory and desiccation.

KEY MESSAGE: Nicotiana benthamiana acylsugar acyltransferase (ASAT) is required for protection against desiccation and insect herbivory. Knockout mutations provide a new resource for investigation of plant-aphid and plant-whitefly interactions. Nicotiana benthamiana is used extensively as a transient expression platform for functional analysis of genes from other species. Acylsugars, which are produced in the trichomes, are a hypothesized cause of the relatively high insect resistance that is observed in N. benthamiana. We characterized the N. benthamiana acylsugar profile, bioinformatically identified two acylsugar acyltransferase genes, ASAT1 and ASAT2, and used CRISPR/Cas9 mutagenesis to produce acylsugar-deficient plants for investigation of insect resistance and foliar water loss. Whereas asat1 mutations reduced accumulation, asat2 mutations caused almost complete depletion of foliar acylsucroses. Three hemipteran and three lepidopteran herbivores survived, gained weight, and/or reproduced significantly better on asat2 mutants than on wildtype N. benthamiana. Both asat1 and asat2 mutations reduced the water content and increased leaf temperature. Our results demonstrate the specific function of two ASAT proteins in N. benthamiana acylsugar biosynthesis, insect resistance, and desiccation tolerance. The improved growth of aphids and whiteflies on asat2 mutants will facilitate the use of N. benthamiana as a transient expression platform for the functional analysis of insect effectors and resistance genes from other plant species. Similarly, the absence of acylsugars in asat2 mutants will enable analysis of acylsugar biosynthesis genes from other Solanaceae by transient expression.

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.

The Germ Fraction Inhibits Iron Bioavailability of Maize: Identification of an Approach to Enhance Maize Nutritional Quality via Processing and Breeding.

Improving the nutritional quality of Fe in maize (Zea mays) represents a biofortification strategy to alleviate iron deficiency anemia. Therefore, the present study measured iron content and bioavailability via an established bioassay to characterize Fe quality in parts of the maize kernel. Comparisons of six different varieties of maize demonstrated that the germ fraction is a strong inhibitory component of Fe bioavailability. The germ fraction can contain 27-54% of the total kernel Fe, which is poorly available. In the absence of the germ, Fe in the non-germ components can be highly bioavailable. More specifically, increasing Fe concentration in the non-germ fraction resulted in more bioavailable Fe. Comparison of wet-milled fractions of a commercial maize variety and degerminated corn meal products also demonstrated the inhibitory effect of the germ fraction on Fe bioavailability. When compared to beans (Phaseolus vulgaris) containing approximately five times the concentration of Fe, degerminated maize provided more absorbable Fe, indicating substantially higher fractional bioavailability. Overall, the results indicate that degerminated maize may be a better source of Fe than whole maize and some other crops. Increased non-germ Fe density with a weaker inhibitory effect of the germ fraction are desirable qualities to identify and breed for in maize.

Population Structure and Phylogenetic Relationships in a Diverse Panel of Brassica rapa L.

The crop species Brassica rapa L. has significant economic importance around the world. However, the global distribution and complex evolutionary history of the species has made investigating its genetic population structure difficult. Crop domestication and improvement has resulted in extreme phenotypic diversity and subspecies that are used for oilseed, food for human consumption, and fodder for livestock. These subspecies include the oilseed morphotypes. oleifera (turnip rape), ssp. dichotoma (brown sarson/toria), ssp. trilocularis (yellow sarson); ssp. rapa (turnip); and Asian leafy vegetables ssp. pekinensis (Chinese cabbage), ssp. chinensis (bok choy), ssp. nipposinica (mizuna/mibuna), ssp. rapifera (rapini/broccoli rabe), ssp. narinosa (tatsoi), ssp parachinensis (choy sum), and ssp. perviridis (komatsuna). To date, studies have had insufficient sampling to determine the relationship of all morphotypes, especially oilseed morphotypes, and questions remain over the contribution of morphotype and geographic origin to population structure. We used genotyping-by-sequencing to score 18,272 single nucleotide polymorphism markers in a globally diverse panel of 333 B. rapa National Plant Germplasm System accessions that included 10 recognized subspecies. Our population genetic and phylogenetic analyses were broadly congruent and revealed five subpopulations that were largely reflective of morphotype and geography. These subpopulations were 1. European turnips/oilseed, 2. Asian turnips/oilseed, 3. yellow/brown sarson (ssp. trilocularis and ssp. dichotoma), 4. Chinese cabbage (ssp. pekinensis), and 5. bok choy, choy sum, and tatsoi (ssp. chinensis, ssp. parachinensis, ssp. narinosa). Additionally, we found evidence of polyphyly and/or paraphyly, particularly for oilseed morphotypes (ssp. oleifera and ssp. dichotoma) and turnips. The results of this study have provided improved resolution to the genetic and phylogenetic relationships of subspecies within the species B. rapa. Understanding of these relationships is key to the future genetic study and improvement of this globally important crop species.

Re-evaluating the phylogeny of allopolyploid Gossypium L.

The formation of allopolyploid cotton precipitated a rapid diversification and colonization of dry coastal American tropical and subtropical regions. Previous phylogenetic analyses, combined with molecular divergence analyses, have offered a temporal framework for this radiation, but provide only weak support for some of the resolved branches. Moreover, these earlier analyses did not include the recently recognized sixth polyploid species, G. ekmanianum Wittmack. Here we use targeted sequence capture of multiple loci in conjunction with both concatenated and Bayesian concordance analyses to reevaluate the phylogeny of allopolyploid cotton species. Although phylogenetic resolution afforded by individual genes is often low, sufficient signal was attained both through the concatenated and concordance analyses to provide robust support for the Gossypium polyploid clade, which is reported here.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.