Genome-wide association study reveals marker-trait associations for major agronomic traits in proso millet (Panicum miliaceum L.).

MAIN CONCLUSION: The pilot-scale genome-wide association study in the US proso millet identified twenty marker-trait associations for five morpho-agronomic traits identifying genomic regions for future studies (e.g. molecular breeding and map-based cloning). Proso millet (Panicum miliaceum L.) is an ancient grain recognized for its excellent water-use efficiency and short growing season. It is an indispensable part of the winter wheat-based dryland cropping system in the High Plains of the USA. Its grains are endowed with high nutritional and health-promoting properties, making it increasingly popular in the global market for healthy grains. There is a dearth of genomic resources in proso millet for developing molecular tools to complement conventional breeding for developing high-yielding varieties. Genome-wide association study (GWAS) is a widely used method to dissect the genetics of complex traits. In this pilot study of the first-ever GWAS in the US proso millet, 71 globally diverse genotypes of 109 the US proso millet core collection were evaluated for five major morpho-agronomic traits at two locations in western Nebraska, and GWAS was conducted to identify single nucleotide polymorphisms (SNPs) associated with these traits. Analysis of variance showed that there was a significant difference among the genotypes, and all five traits were also found to be highly correlated with each other. Sequence reads from genotyping-by-sequencing (GBS) were used to identify 11,147 high-quality bi-allelic SNPs. Population structure analysis with those SNPs showed stratification within the core collection. The GWAS identified twenty marker-trait associations (MTAs) for the five traits. Twenty-nine putative candidate genes associated with the five traits were also identified. These genomic regions can be used to develop genetic markers for marker-assisted selection in proso millet breeding.

Hyperspectral Imaging and Machine Learning as a Nondestructive Method for Proso Millet Seed Detection and Classification.

Millet is a small-seeded cereal crop with big potential. There are many different cultivars of proso millet (Panicum miliaceum L.) with different characteristics, bringing forth the issue of sorting which are important for growers, processors, and consumers. Current methods of grain cultivar detection and classification are subjective, destructive, and time-consuming. Therefore, there is a need to develop nondestructive methods for sorting the cultivars of proso millet. In this study, the feasibility of using near-infrared (NIR) hyperspectral imaging (900-1700 nm) to discriminate between different cultivars of proso millet seeds was evaluated. A total of 5000 proso millet seeds were randomly obtained and investigated from the ten most popular cultivars in the United States, namely Cerise, Cope, Earlybird, Huntsman, Minco, Plateau, Rise, Snowbird, Sunrise, and Sunup. To reduce the large dimensionality of the hyperspectral imaging, principal component analysis (PCA) was applied, and the first two principal components were used as spectral features for building the classification models because they had the largest variance. The classification performance showed prediction accuracy rates as high as 99% for classifying the different cultivars of proso millet using a Gradient tree boosting ensemble machine learning algorithm. Moreover, the classification was successfully performed using only 15 and 5 selected spectral features (wavelengths), with an accuracy of 98.14% and 97.6%, respectively. The overall results indicate that NIR hyperspectral imaging could be used as a rapid and nondestructive method for the classification of proso millet seeds.

Variation in Leaf Chlorophyll Concentration in Response to Nitrogen Application Across Maize Hybrids in Contrasting Environments.

Leaf chlorophyll concentration was measured for 84 publicly available maize hybrids grown under three nitrogen fertilizer treatments in two contrasting environments in Nebraska. The effect of nitrogen treatment on chlorophyll response was found to be significant (p < 0.05) for both locations. In Scottsbluff, chlorophyll concentrations increased significantly with increasing nitrogen rate, while no significant difference was found between medium and high nitrogen in Lincoln. Within equivalent nitrogen treatments, chlorophyll was more abundant in Lincoln than Scottsbluff for nearly every hybrid. Hybrid response was not consistent between environments, with approximately 11% of variance explained by genotype by environment interaction.

SNP discovery in proso millet (Panicum miliaceum L.) using low-pass genome sequencing.

Domesticated ~10,000 years ago in northern China, Proso millet (Panicum miliaceum L.) is a climate-resilient and human health-promoting cereal crop. The genome size of this self-pollinated allotetraploid is 923 Mb. Proso millet seeds are an important part of the human diet in many countries. In the USA, its use is restricted to the birdseed and pet food market. Proso millet is witnessing gradual demand in the global human health and wellness food market owing to its health-promoting properties such as low glycemic index and gluten-free. The breeding efforts for developing improved proso millet cultivars are hindered by the dearth of genomic resources available to researchers. The publication of the reference genome and availability of cost-effective NGS methodologies could lead to the identification of high-quality genetic variants, which can be incorporated into breeding pipelines. Here, we report the identification of single-nucleotide polymorphisms (SNPs) by low-pass (1×) genome sequencing of 85 diverse proso millet accessions from 23 different countries. The 2 × 150 bp Illumina paired-end reads generated after sequencing were aligned to the proso millet reference genome. The resulting sequence alignment information was used to call SNPs. We obtained 972,863 bi-allelic SNPs after quality filtering of the raw SNPs. These SNPs were used to assess the population structure and phylogenetic relationships among the accessions. Most of the accessions were found to be highly inbred with heterozygosity ranging between .05 and .20. Principal component analysis (PCA) showed that PC1 (principal component) and PC2 explained 19% of the variability in the population. PCA also clustered all the genotypes into three groups. A neighbor-joining tree clustered the genotypes into four distinct groups exhibiting diverse representation within the population. The SNPs identified in our study could be used for molecular breeding and genetics research (e.g., genetic and association mapping, and population genetics) in proso millet after proper validation.

A longitudinal study on morpho-genetic diversity of pathogenic Rhizoctonia solani from sugar beet and dry beans of western Nebraska.

BACKGROUND: Root and stem rot caused by Rhizoctonia solani is a serious fungal disease of sugar beet and dry bean production in Nebraska. Rhizoctonia root rot and crown rot in sugar beet and dry bean have reduced the yield significantly and has also created problems in storage. The objective of this study was to analyze morpho-genetic diversity of 38 Rhizoctonia solani isolates from sugar beet and dry bean fields in western Nebraska collected over 10 years. Morphological features and ISSR-based DNA markers were used to study the morphogenetic diversity. RESULTS: Fungal colonies were morphologically diverse in shapes, aerial hyphae formation, colony, and sclerotia color. Marker analysis using 19 polymorphic ISSR markers showed polymorphic bands ranged from 15 to 28 with molecular weight of 100 bp to 3 kb. Polymorphic loci ranged from 43.26-92.88%. Nei genetic distance within the population ranged from 0.03-0.09 and Shannon diversity index varied from 0.24-0.28. AMOVA analysis based on ΦPT values showed 87% variation within and 13% among the population with statistical significance (p < 0.05). Majority of the isolates from sugar beet showed nearby association within the population. A significant number of isolates showed similarity with isolates of both the crops suggesting their broad pathogenicity. Isolates were grouped into three different clusters in UPGMA based cluster analysis using marker information. Interestingly, there was no geographical correlation among the isolates. Principal component analysis showed randomized distribution of isolates from the same geographical origin. Identities of the isolates were confirmed by both ITS-rDNA sequences and pathogenicity tests. CONCLUSION: Identification and categorization of the pathogen will be helpful in designing integrated disease management guidelines for sugar beet and dry beans of mid western America.

In Vitro Pepsin Digestibility of Cooked Proso Millet ( Panicum miliaceum L.) and Related Species from Around the World.

Thirty-three samples of proso millet ( Panicum miliaceum) with different countries of origin were screened for their pepsin digestibility after cooking to identify samples with high digestibility. The pepsin digestibility of all samples ranged from 26% to 57% (average 32%). There were no apparent differences in protein profiles (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) of samples with the lowest, intermediate, and highest digestibilities. However, liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis revealed a negative correlation between pepsin digestibility and peptides that matched to high molecular weight proteins (24 kDa) from Panicum hallii with regions of contiguous hydrophobic amino acids. Low digestibility upon cooking was also observed for other species from the Panicum genus, such as little millet, switchgrass, and panicgrass, which suggests a unique inherent property of the genus. The results obtained from this study may form a basis for in-depth analysis of proso proteins that may help in developing new cultivars with higher digestibility upon cooking.

Nanoparticles prepared by proso millet protein as novel curcumin delivery system.

Encapsulation of lipophilic bioactive compounds using plant-derived proteins as delivery systems has received increasing interest. In this study, proso millet protein was extracted by either wet milling or 60% (v/v) aqueous ethanol and then used as the wall material to encapsulate curcumin. Millet protein was composed of prolamines that showed a significant difference in surface hydrophobicity depending on extraction procedure. The millet protein-curcumin nanoparticles were spherical with diameter around 250-350nm. The entrapment efficiency ranged from 11.2% to 78.9%, with millet protein extracted by ethanol showing better performance than that by wet milling process. The encapsulated curcumin exhibited a lower degradation rate than free curcumin at 60°C. The encapsulation showed no negative effect on the antioxidant activity of curcumin as assessed by the DPPH and ABTS assays. These results suggested that MP could be a promising encapsulant for the delivery of lipophilic compounds.

Phytoceuticals in Fenugreek Ameliorate VLDL Overproduction and Insulin Resistance via the Insig Signaling Pathway.

SCOPE: This study aims to characterize the effect of fenugreek (Trigonella foenum-graecum) seed and its phytoceutical trigonelline in antimetabolic inflammation and ameliorating overproduction of very low density lipoprotein (VLDL) in insulin resistance. METHODS AND RESULTS: Two groups of genetic hyperlipidemic mice generated by depletion of cAMP responsive element binding protein H (CREBH) are fed either a chow containing 2% fenugreek seed or vehicle for 7 weeks. Q-RT-PCR and immunoblotting analysis demonstrated that fenugreek seed containing diet inhibits hepatic SREBP-1c activation and the subsequent de novo lipogenesis by enhancing expression of insulin-inducible gene-1 (Insig-1) and gene-2 (Insig-2). mRNA expression of PPARα and its target genes that are involved in fatty acid ß-oxidation are also upregulated in the fenugreek seed fed-mice which is accompanied by significantly reduced hepatic lipid accumulation and VLDL secretion, improved endoplasmic reticulum (ER) stress, and ameliorated metabolic inflammation. These actions enhance insulin sensitivity and improve hyperlipidemia. In vitro, treating a rat hepatoma cell line, McA-RH7777 (McA), with trigonelline is able to recapitulate the results observed in vivo. CONCLUSIONS: This study unveils a novel mechanism of fenugreek seed and trigonelline in countering hepatic VLDL overproduction and insulin resistance by enhancing the Insig signaling pathways and ameliorating metabolic inflammatory stress in the liver.

Genotype, environment, seeding rate, and top-dressed nitrogen effects on end-use quality of modern Nebraska winter wheat.

BACKGROUND: Fine-tuning production inputs such as seeding rate, nitrogen (N), and genotype may improve end-use quality of hard red winter wheat (Triticum aestivium L.) when growing conditions are unpredictable. Studies were conducted at the Agronomy Research Farm (ARF; Lincoln, NE, USA) and the High Plains Agricultural Laboratory (HPAL; Sidney, NE, USA) in 2014 and 2015 in Nebraska, USA, to determine the effects of genotype (6), environment (4), seeding rate (3), and flag leaf top-dressed N (0 and 34 kg N ha-1 ) on the end-use quality of winter wheat. RESULTS: End-use quality traits were influenced by environment, genotype, seeding rate, top-dressed N, and their interactions. Mixograph parameters had a strong correlation with grain volume weight and flour yield. Doubling the recommended seeding rate and N at the flag leaf stage increased grain protein content by 8.1% in 2014 and 1.5% in 2015 at ARF and 4.2% in 2014 and 8.4% in 2015 at HPAL. CONCLUSION: The key finding of this research is that increasing seeding rates up to double the current recommendations with N at the flag leaf stage improved most of the end-use quality traits. This will have a significant effect on the premium for protein a farmer could receive when marketing wheat. © 2017 Society of Chemical Industry.

Heating Reduces Proso Millet Protein Digestibility via Formation of Hydrophobic Aggregates.

Proso millet protein has reported structural similarities with sorghum. In order to explore the potential of this crop as an alternative protein source for people with gluten sensitivity, in vitro protein digestibility was analyzed. Dehulled proso millet flour was subjected to various processing techniques (dry heating and wet heating). Regardless of the processing technique there was a significant decline in digestibility of protein in proso millet flour when compared with unprocessed flour (from 79.7 ± 0.8% to 42.0 ± 1.2%). Reduced digestibility persisted even when cooking with reducing agents. Heating in the presence of urea (8 M) and guanidine-HCl (4.5 M) prevented the reduction in observed digestibility (urea cooked 77.4 ± 0.8%; guanidine HCl cooked 84.3 ± 0.9%), suggesting formation of hydrophobic aggregates during heating in water. This was supported by an increase in surface hydrophobicity upon cooking. Thus, the proso millet protein, termed panicin, forms hydrophobic aggregates that are resistant to digestion when subjected to heat.

Identification of a candidate gene for the wheat endopeptidase Ep-D1 locus and two other STS markers linked to the eyespot resistance gene Pch1.

Wheat is prone to strawbreaker foot rot (eyespot), a fungal disease caused by Oculimacula yallundae and O. acuformis. The most effective source of genetic resistance is Pch1, a gene derived from Aegilops ventricosa. The endopeptidase isozyme marker allele Ep-D1b, linked to Pch1, has been shown to be more effective for tracking resistance than DNA-based markers developed to date. Therefore, we sought to identify a candidate gene for Ep-D1 as a basis for a DNA-based marker. Comparative mapping suggested that the endopeptidase loci Ep-D1 (wheat), enp1 (maize), and Enp (rice) were orthologous. Since the product of the maize endopeptidase locus enp1 has been shown to exhibit biochemical properties similar to oligopeptidase B purified from E. coli, we reasoned that Ep-D1 may also encode an oligopeptidase B. Consistent with this hypothesis, a sequence-tagged-site (STS) marker, Xorw1, derived from an oligopeptidase B-encoding wheat expressed-sequence-tag (EST) showed complete linkage with Ep-D1 and Pch1 in a population of 254 recombinant inbred lines (RILs) derived from a cross between wheat cultivars Coda and Brundage. Two other STS markers, Xorw5 and Xorw6, and three microsatellite markers (Xwmc14, Xbarc97, and Xcfd175) were also completely linked to Pch1. On the other hand, Xwmc14, Xbarc97, and Xcfd175 showed recombination in the W7984 x Opata85 RIL population suggesting that recombination near Pch1 is reduced in the Coda/Brundage population. In a panel of 44 wheat varieties with known eyespot reactions, Xorw1, Xorw5, and Xorw6 were 100% accurate in predicting the presence or absence of Pch1 whereas Xwmc14, Xbarc97, and Xcfd175 were less effective. Thus, linkage mapping and a germplasm survey suggest that the STS markers identified here should be useful for indirect selection of Pch1.

Construction and characterization of a soybean yeast artificial chromosome library and identification of clones for the Rps6 region.

We report the construction and characterization of the first soybean yeast artificial chromosome (YAC) library using high-molecular weight DNA isolated from leaf nuclei of the cultivar Conrad 94 that carries Phytophthora resistance genes Rps1-k and Rps6. The quality of this library has been evaluated through analysis of 393 randomly selected YAC clones. The library consists of 36864 clones, of which approximately 19956 carry single soybean YACs with an average size of about 285 kb. The library represents approximately five soybean genome equivalents. The probability of finding any soybean sequences from this library is about 0.99. The library was screened for 43 SSR markers representing the whole soybean genome. We were able to identify positive YAC pools for 95% of the SSR markers. Two YAC clones carrying molecular markers linked to the Rps6 gene were identified. The YAC library reported here would be a useful resource for map-based cloning of agronomically important soybean genes and also to complement the effort towards construction of the physical map for the soybean genome.