Karen A. Hudson

Ionomic Screening of Field-Grown Soybean Identifies Mutants with Altered Seed Elemental Composition

Soybean [Glycine max (L.) Merr.] seeds contain high levels of mineral nutrients essential for human and animal nutrition. High throughput elemental profiling (ionomics) has identified mutants in model plant species grown in controlled environments. Here, we describe a method for identifying potential soybean ionomics mutants grown in a field setting and apply it to 975 N‐nitroso‐N‐methylurea (NMU) mutagenized lines. After performing a spatial correction, we identified mutants using either visual scoring of standard score (z‐score) plots or computational ranking of putative mutants followed by visual confirmation. Although there was a large degree of overlap between the methods, each method identified unique lines. The visual scoring approach identified 22 out of 427 (5%) potential mutants, 70% (16 out of 22) of which were confirmed when seeds from the same parent plant were regrown in the field. We also performed simulations to determine an optimal strategy for screening large populations. All data from the screen is available at the Ionomics Hub File Transfer Page (http://www.ionomicshub.org/home/PiiMS/dataexchange).

The Circadian Clock-controlled Transcriptome of Developing Soybean Seeds

A number of metabolic and physiological processes in plants are controlled by the circadian clock, which enables a plant to anticipate daily changes in the environment. Relatively little is known about circadian rhythms in developing seeds, which may be important for determining the extent and timing of nutrient storage in grain. Microarray expression profiling was used to identify genes expressed in developing soybean (Glycine max) seeds that are controlled by the circadian clock. Genes with predicted functions in protein synthesis, fatty acid metabolism, and photosynthesis totaling 1.8% of the mRNAs detected in seed were found to be expressed in a circadian rhythm. Known circadian and light‐controlled promoter elements were identified as over‐represented in the promoters of clock‐controlled seed genes, with the over‐represented elements varying according to the phase of circadian expression. A subset of circadian‐regulated genes were found to be expressed in different phases in developing seeds with respect to leaves from the same plants, many of which have roles in photosynthesis and carbon metabolism. These results help to characterize the genes and processes in seeds that may be regulated by the circadian clock, and provide some insight into organ‐specific phasing of clock controlled gene expression.

A Classification of Basic Helix-Loop-Helix Transcription Factors of Soybean

The complete genome sequence of soybean allows an unprecedented opportunity for the discovery of the genes controlling important traits. In particular, the potential functions of regulatory genes are a priority for analysis. The basic helix-loop-helix (bHLH) family of transcription factors is known to be involved in controlling a wide range of systems critical for crop adaptation and quality, including photosynthesis, light signalling, pigment biosynthesis, and seed pod development. Using a hidden Markov model search algorithm, 319 genes with basic helix-loop-helix transcription factor domains were identified within the soybean genome sequence. These were classified with respect to their predicted DNA binding potential, intron/exon structure, and the phylogeny of the bHLH domain. Evidence is presented that the vast majority (281) of these 319 soybean bHLH genes are expressed at the mRNA level. Of these soybean bHLH genes, 67% were found to exist in two or more homeologous copies. This dataset provides a framework for future studies on bHLH gene function in soybean. The challenge for future research remains to define functions for the bHLH factors encoded in the soybean genome, which may allow greater flexibility for genetic selection of growth and environmental adaptation in this widely grown crop.

Evolutionary divergence of phytochrome protein function in Zea mays PIF3 signaling

Highlight phyB and PIF3 share a common protein-protein signaling mechanism in maize and Arabidopsis. However, phyB1 and phyB2 in maize do not share this mechanism, despite being closely related, indicating recent evolutionary divergence.

Mutations in SACPD-C result in a range of elevated stearic acid concentration in soybean seed.

Soybean oil has a wide variety of uses, and stearic acid, which is a relatively minor component of soybean oil is increasingly desired for both industrial and food applications. New soybean mutants containing high levels of the saturated fatty acid stearate in seeds were recently identified from a chemically mutagenized population. Six mutants ranged in stearate content from 6–14% stearic acid, which is 1.5 to 3 times the levels contained in wild-type seed of the Williams 82 cultivar. Candidate gene sequencing revealed that all of these lines carried amino acid substitutions in the gene encoding the delta-9-stearoyl-acyl-carrier protein desaturase enzyme (SACPD-C) required for the conversion of stearic acid to oleic acid. Five of these missense mutations were in highly conserved residues clustered around the predicted di-iron center of the SACPD-C enzyme. Co-segregation analysis demonstrated a positive association of the elevated stearate trait with the SACPD-C mutation for three populations. These missense mutations may provide additional alleles that may be used in the development of new soybean cultivars with increased levels of stearic acid.

Mutations in the soybean 3-ketoacyl-ACP synthase gene are correlated with high levels of seed palmitic acid

A complete understanding of the biosynthetic pathways involved in the formation of seed oils is an important step in the development of lines with useful oil profiles. Soybean oil has a number of industrial applications and is also a source of edible oils for human consumption. The elongation of palmitic acid to stearic acid by the 3-ketoacyl-ACP synthase II (KASII) enzyme represents a branch point in the pathway of oil biosynthesis in soybean. Three mutant soybean lines were identified which contained elevated levels of palmitic acid ranging from 12.9 to 15.6%. Candidate gene sequencing in these lines revealed that all three contain nucleotide changes in the coding sequence for the KASIIa gene. One mutation caused an early termination in the third exon of KASIIa, and two mutations resulted in amino acid changes in the KASIIa protein. These new mutant alleles of soybean KASIIa may be useful to fine-tune the composition of soybean seed oil using molecular breeding approaches.

The Basic Helix-Loop-Helix Transcription Factor Family in the Sacred Lotus, Nelumbo Nucifera

Nelumbo nucifera (Sacred Lotus) is a basal eudicot with exceptional physiological and metabolic properties including seed longevity, adaptations for an aquatic habit, and floral thermiogenesis. It also occupies a unique position in the phylogeny of land plants and can be a useful species for studies of conserved plant gene families. The basic-helix-loop-helix (bHLH) proteins represent one of the largest transcription factor families in plants and has undergone extensive duplication and expansion during plant evolution. One hundred and seventeen transcript models encoding canonical bHLHs were identified in the sacred lotus genome, as well as several “atypical” bHLH-encoding genes. The canonical bHLH proteins fall into 23 previously characterized subfamilies also present in other sequenced plant genomes, and are expressed as mRNA. Analysis of bHLHs from sacred lotus and other sequenced angiosperms indicates most of these families of bHLHs, along with secondary motifs associated with the bHLH domain, were likely present in the progenitor of flowering plants. The absence of a bHLH subfamily involved in root development in sacred lotus is consistent with the possibility that the development of specialized root structures may be mediated in part by changes in the bHLH families that regulate root development in dicots.

A comparison of genotyping-by-sequencing analysis methods on low-coverage crop datasets shows advantages of a new workflow, GB-eaSy

BackgroundGenotyping-by-sequencing (GBS), a method to identify genetic variants and quickly genotype samples, reduces genome complexity by using restriction enzymes to divide the genome into fragments whose ends are sequenced on short-read sequencing platforms. While cost-effective, this method produces extensive missing data and requires complex bioinformatics analysis. GBS is most commonly used on crop plant genomes, and because crop plants have highly variable ploidy and repeat content, the performance of GBS analysis software can vary by target organism. Here we focus our analysis on soybean, a polyploid crop with a highly duplicated genome, relatively little public GBS data and few dedicated tools.ResultsWe compared the performance of five GBS pipelines using low-coverage Illumina sequence data from three soybean populations. To address issues identified with existing methods, we developed GB-eaSy, a GBS bioinformatics workflow that incorporates widely used genomics tools, parallelization and automation to increase the accuracy and accessibility of GBS data analysis. Compared to other GBS pipelines, GB-eaSy rapidly and accurately identified the greatest number of SNPs, with SNP calls closely concordant with whole-genome sequencing of selected lines. Across all five GBS analysis platforms, SNP calls showed unexpectedly low convergence but generally high accuracy, indicating that the workflows arrived at largely complementary sets of valid SNP calls on the low-coverage data analyzed.ConclusionsWe show that GB-eaSy is approximately as good as, or better than, other leading software solutions in the accuracy, yield and missing data fraction of variant calling, as tested on low-coverage genomic data from soybean. It also performs well relative to other solutions in terms of the run time and disk space required. In addition, GB-eaSy is built from existing open-source, modular software packages that are regularly updated and commonly used, making it straightforward to install and maintain. While GB-eaSy outperformed other individual methods on the datasets analyzed, our findings suggest that a comprehensive approach integrating the results from multiple GBS bioinformatics pipelines may be the optimal strategy to obtain the largest, most highly accurate SNP yield possible from low-coverage polyploid sequence data.

Genome of the long-living sacred lotus (Nelumbo nucifera Gaertn.) - eScholarship

BackgroundSacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan.ResultsThe genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment.ConclusionsThe slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.

Nelumbo nucifera [data set]

BackgroundSacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan.ResultsThe genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment.ConclusionsThe slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.