Author Correction: Increasing CO2 threatens human nutrition.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Soybean BARCSoySNP6K: An assay for soybean genetics and breeding research.

The limited number of recombinant events in recombinant inbred lines suggests that for a biparental population with a limited number of recombinant inbred lines, it is unnecessary to genotype the lines with many markers. For genomic prediction and selection, previous studies have demonstrated that only 1000-2000 genome-wide common markers across all lines/accessions are needed to reach maximum efficiency of genomic prediction in populations. Evaluation of too many markers will not only increase the cost but also generate redundant information. We developed a soybean (Glycine max) assay, BARCSoySNP6K, containing 6000 markers, which were carefully chosen from the SoySNP50K assay based on their position in the soybean genome and haplotype block, polymorphism among accessions and genotyping quality. The assay includes 5000 single nucleotide polymorphisms (SNPs) from euchromatic and 1000 from heterochromatic regions. The percentage of SNPs with minor allele frequency >0.10 was 95% and 91% in the euchromatic and heterochromatic regions, respectively. Analysis of progeny from two large families genotyped with SoySNP50K versus BARCSoySNP6K showed that the position of the common markers and number of unique bins along linkage maps were consistent based on the SNPs genotyped with the two assays; however, the rate of redundant markers was dramatically reduced with the BARCSoySNP6K. The BARCSoySNP6K assay is proven as an excellent tool for detecting quantitative trait loci, genomic selection and assessing genetic relationships. The assay is commercialized by Illumina Inc. and being used by soybean breeders and geneticists and the list of SNPs in the assay is an ideal resource for SNP genotyping by targeted amplicon sequencing.

Increasing CO2 threatens human nutrition.

Dietary deficiencies of zinc and iron are a substantial global public health problem. An estimated two billion people suffer these deficiencies, causing a loss of 63 million life-years annually. Most of these people depend on C3 grains and legumes as their primary dietary source of zinc and iron. Here we report that C3 grains and legumes have lower concentrations of zinc and iron when grown under field conditions at the elevated atmospheric CO2 concentration predicted for the middle of this century. C3 crops other than legumes also have lower concentrations of protein, whereas C4 crops seem to be less affected. Differences between cultivars of a single crop suggest that breeding for decreased sensitivity to atmospheric CO2 concentration could partly address these new challenges to global health.

Genetic dissection of domestication-related traits in soybean through genotyping-by-sequencing of two interspecific mapping populations.

KEY MESSAGE: A total of 132 domestication-related QTLs, of which 41 were novel, were identified through genotyping-by-sequencing of two Glycine max × Glycine soja populations. Soybean [Glycine max (L.) Merr.] was domesticated in East Asia from the wild progenitor Glycine soja. The domestication process led to many distinct morphological changes that adapt it to cultivation. These include larger seeds, erect growth, larger stem diameter, reduced pod shattering, and altered growth habit. The objective of this study was to identify QTLs controlling key domestication-related traits (DRTs) using interspecific mapping populations. A total of 151 RILs from Williams 82 × PI 468916 and 510 RILs from Williams 82 × PI 479752 were utilized for QTL mapping. These lines were genotyped using a genotyping-by-sequencing protocol which resulted in approximately 5000 polymorphic SNP markers. The number of QTLs detected for each of the eleven DRTs ranged between 0-4 QTLs in the smaller Williams 82 × PI 468916 population and 3-16 QTLs in the larger Williams 82 × PI 479752 population. A total of 132 QTLs were detected, of which 51 are associated with selective sweeps previously related to soybean domestication. These QTLs were detected across all 20 chromosomes within 42 genomic regions. This study identifies 41 novel QTLs not detected in previous studies using smaller populations while also confirming the quantitative nature for several of the important DRTs in soybeans. These results would enable more effective use of the wild germplasm for soybean improvement.

Dt2 is a gain-of-function MADS-domain factor gene that specifies semideterminacy in soybean.

Similar to Arabidopsis thaliana, the wild soybeans (Glycine soja) and many cultivars exhibit indeterminate stem growth specified by the shoot identity gene Dt1, the functional counterpart of Arabidopsis TERMINAL FLOWER1 (TFL1). Mutations in TFL1 and Dt1 both result in the shoot apical meristem (SAM) switching from vegetative to reproductive state to initiate terminal flowering and thus produce determinate stems. A second soybean gene (Dt2) regulating stem growth was identified, which, in the presence of Dt1, produces semideterminate plants with terminal racemes similar to those observed in determinate plants. Here, we report positional cloning and characterization of Dt2, a dominant MADS domain factor gene classified into the APETALA1/SQUAMOSA (AP1/SQUA) subfamily that includes floral meristem (FM) identity genes AP1, FUL, and CAL in Arabidopsis. Unlike AP1, whose expression is limited to FMs in which the expression of TFL1 is repressed, Dt2 appears to repress the expression of Dt1 in the SAMs to promote early conversion of the SAMs into reproductive inflorescences. Given that Dt2 is not the gene most closely related to AP1 and that semideterminacy is rarely seen in wild soybeans, Dt2 appears to be a recent gain-of-function mutation, which has modified the genetic pathways determining the stem growth habit in soybean.

Genome-wide association study for flowering time, maturity dates and plant height in early maturing soybean (Glycine max) germplasm.

BACKGROUND: Soybean (Glycine max) is a photoperiod-sensitive and self-pollinated species. Days to flowering (DTF) and maturity (DTM), duration of flowering-to-maturity (DFTM) and plant height (PH) are crucial for soybean adaptability and yield. To dissect the genetic architecture of these agronomically important traits, a population consisting of 309 early maturity soybean germplasm accessions was genotyped with the Illumina Infinium SoySNP50K BeadChip and phenotyped in multiple environments. A genome-wide association study (GWAS) was conducted using a mixed linear model that involves both relative kinship and population structure. RESULTS: The linkage disequilibrium (LD) decayed slowly in soybean, and a substantial difference in LD pattern was observed between euchromatic and heterochromatic regions. A total of 27, 6, 18 and 27 loci for DTF, DTM, DFTM and PH were detected via GWAS, respectively. The Dt1 gene was identified in the locus strongly associated with both DTM and PH. Ten candidate genes homologous to Arabidopsis flowering genes were identified near the peak single nucleotide polymorphisms (SNPs) associated with DTF. Four of them encode MADS-domain containing proteins. Additionally, a pectin lyase-like gene was also identified in a major-effect locus for PH where LD decayed rapidly. CONCLUSIONS: This study identified multiple new loci and refined chromosomal regions of known loci associated with DTF, DTM, DFTM and/or PH in soybean. It demonstrates that GWAS is powerful in dissecting complex traits and identifying candidate genes although LD decayed slowly in soybean. The loci and trait-associated SNPs identified in this study can be used for soybean genetic improvement, especially the major-effect loci associated with PH could be used to improve soybean yield potential. The candidate genes may serve as promising targets for studies of molecular mechanisms underlying the related traits in soybean.

Artificial selection of mutations in two nearby genes gave rise to shattering resistance in soybean.

Resistance to pod shattering is a key domestication-related trait selected for seed production in many crops. Here, we show that the transition from shattering in wild soybeans to shattering resistance in cultivated soybeans resulted from selection of mutations within the coding sequences of two nearby genes - Sh1 and Pdh1. Sh1 encodes a C2H2-like zinc finger transcription factor that promotes shattering by repressing SHAT1-5 expression, thereby reducing the secondary wall thickness of fiber cap cells in the abscission layers of pod sutures, while Pdh1 encodes a dirigent protein that orchestrates asymmetric lignin distribution in inner sclerenchyma, creating torsion in pod walls that facilitates shattering. Integration analyses of quantitative trait locus mapping, genome-wide association studies, and allele distribution in representative soybean germplasm suggest that these two genes are primary modulators underlying this domestication trait. Our study thus provides comprehensive understanding regarding the genetic, molecular, and cellular bases of shattering resistance in soybeans.

Long noncoding RNAs underlie multiple domestication traits and leafhopper resistance in soybean.

The origin and functionality of long noncoding RNA (lncRNA) remain poorly understood. Here, we show that multiple quantitative trait loci modulating distinct domestication traits in soybeans are pleiotropic effects of a locus composed of two tandem lncRNA genes. These lncRNA genes, each containing two inverted repeats, originating from coding sequences of the MYB genes, function in wild soybeans by generating clusters of small RNA (sRNA) species that inhibit the expression of their MYB gene relatives through post-transcriptional regulation. By contrast, the expression of lncRNA genes in cultivated soybeans is severely repressed, and, consequently, the corresponding MYB genes are highly expressed, shaping multiple distinct domestication traits as well as leafhopper resistance. The inverted repeats were formed before the divergence of the Glycine genus from the Phaseolus-Vigna lineage and exhibit strong structure-function constraints. This study exemplifies a type of target for selection during plant domestication and identifies mechanisms of lncRNA formation and action.

Ozone exposure response for U.S. soybean cultivars: linear reductions in photosynthetic potential, biomass, and yield.

Current background ozone (O(3)) concentrations over the northern hemisphere's midlatitudes are high enough to damage crops and are projected to increase. Soybean (Glycine max) is particularly sensitive to O(3); therefore, establishing an O(3) exposure threshold for damage is critical to understanding the current and future impact of this pollutant. This study aims to determine the exposure response of soybean to elevated tropospheric O(3) by measuring the agronomic, biochemical, and physiological responses of seven soybean genotypes to nine O(3) concentrations (38-120 nL L(-1)) within a fully open-air agricultural field location across 2 years. All genotypes responded similarly, with season-long exposure to O(3) causing a linear increase in antioxidant capacity while reducing leaf area, light absorption, specific leaf mass, primary metabolites, seed yield, and harvest index. Across two seasons with different temperature and rainfall patterns, there was a robust linear yield decrease of 37 to 39 kg ha(-1) per nL L(-1) cumulative O(3) exposure over 40 nL L(-1). The existence of immediate effects of O(3) on photosynthesis, stomatal conductance, and photosynthetic transcript abundance before and after the initiation and termination of O(3) fumigation were concurrently assessed, and there was no evidence to support an instantaneous photosynthetic response. The ability of the soybean canopy to intercept radiation, the efficiency of photosynthesis, and the harvest index were all negatively impacted by O(3), suggesting that there are multiple targets for improving soybean responses to this damaging air pollutant.

Artificial selection for determinate growth habit in soybean.

Determinacy is an agronomically important trait associated with the domestication in soybean (Glycine max). Most soybean cultivars are classifiable into indeterminate and determinate growth habit, whereas Glycine soja, the wild progenitor of soybean, is indeterminate. Indeterminate (Dt1/Dt1) and determinate (dt1/dt1) genotypes, when mated, produce progeny that segregate in a monogenic pattern. Here, we show evidence that Dt1 is a homolog (designated as GmTfl1) of Arabidopsis terminal flower 1 (TFL1), a regulatory gene encoding a signaling protein of shoot meristems. The transition from indeterminate to determinate phenotypes in soybean is associated with independent human selections of four distinct single-nucleotide substitutions in the GmTfl1 gene, each of which led to a single amino acid change. Genetic diversity of a minicore collection of Chinese soybean landraces assessed by simple sequence repeat (SSR) markers and allelic variation at the GmTfl1 locus suggest that human selection for determinacy took place at early stages of landrace radiation. The GmTfl1 allele introduced into a determinate-type (tfl1/tfl1) Arabidopsis mutants fully restored the wild-type (TFL1/TFL1) phenotype, but the Gmtfl1 allele in tfl1/tfl1 mutants did not result in apparent phenotypic change. These observations indicate that GmTfl1 complements the functions of TFL1 in Arabidopsis. However, the GmTfl1 homeolog, despite its more recent divergence from GmTfl1 than from Arabidopsis TFL1, appears to be sub- or neo-functionalized, as revealed by the differential expression of the two genes at multiple plant developmental stages and by allelic analysis at both loci.

Relationship between asparagine metabolism and protein concentration in soybean seed.

The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at maturity. Analysis of a second population of recombinant inbred lines adapted to Ontario associated the elevated free asparagine trait with two of four quantitative trait loci determining population variation for protein concentration, including a major one on chromosome 20 (linkage group I) which has been reported in multiple populations. In the seed coat, levels of asparagine synthetase were high at 50 mg and progressively declined until 150 mg seed weight, suggesting that nitrogenous assimilates are pre-conditioned at early developmental stages to enable a high concentration of asparagine in the embryo. The levels of asparaginase B1 showed an opposite pattern, being low at 50 mg and progressively increased until 150 mg, coinciding with an active phase of storage reserve accumulation. In a pair of genetically related cultivars, ∼2-fold higher levels of asparaginase B1 protein and activity in seed coat, were associated with high protein concentration, reflecting enhanced flux of nitrogen. Transcript expression analyses attributed this difference to a specific asparaginase gene, ASPGB1a. These results contribute to our understanding of the processes determining protein concentration in soybean seed.

Molecular mapping of soybean rust resistance in soybean accession PI 561356 and SNP haplotype analysis of the Rpp1 region in diverse germplasm.

Soybean rust (SBR), caused by Phakopsora pachyrhizi Sydow, is one of the most economically important and destructive diseases of soybean [Glycine max (L.) Merr.] and the discovery of novel SBR resistance genes is needed because of virulence diversity in the pathogen. The objectives of this research were to map SBR resistance in plant introduction (PI) 561356 and to identify single nucleotide polymorphism (SNP) haplotypes within the region on soybean chromosome 18 where the SBR resistance gene Rpp1 maps. One-hundred F(2:3) lines derived from a cross between PI 561356 and the susceptible experimental line LD02-4485 were genotyped with genetic markers and phenotyped for resistance to P. pachyrhizi isolate ZM01-1. The segregation ratio of reddish brown versus tan lesion type in the population supported that resistance was controlled by a single dominant gene. The gene was mapped to a 1-cM region on soybean chromosome 18 corresponding to the same interval as Rpp1. A haplotype analysis of diverse germplasm across a 213-kb interval that included Rpp1 revealed 21 distinct haplotypes of which 4 were present among 5 SBR resistance sources that have a resistance gene in the Rpp1 region. Four major North American soybean ancestors belong to the same SNP haplotype as PI 561356 and seven belong to the same haplotype as PI 594538A, the Rpp1-b source. There were no North American soybean ancestors belonging to the SNP haplotypes found in PI 200492, the source of Rpp1, or PI 587886 and PI 587880A, additional sources with SBR resistance mapping to the Rpp1 region.

Numbers of genes in the NBS and RLK families vary by more than four-fold within a plant species and are regulated by multiple factors.

Many genes exist in the form of families; however, little is known about their size variation, evolution and biology. Here, we present the size variation and evolution of the nucleotide-binding site (NBS)-encoding gene family and receptor-like kinase (RLK) gene family in Oryza, Glycine and Gossypium. The sizes of both families vary by numeral fold, not only among species, surprisingly, also within a species. The size variations of the gene families are shown to correlate with each other, indicating their interactions, and driven by natural selection, artificial selection and genome size variation, but likely not by polyploidization. The numbers of genes in the families in a polyploid species are similar to those of one of its diploid donors, suggesting that polyploidization plays little roles in the expansion of the gene families and that organisms tend not to maintain their 'surplus' genes in the course of evolution. Furthermore, it is found that the size variations of both gene families are associated with organisms' phylogeny, suggesting their roles in speciation and evolution. Since both selection and speciation act on organism's morphological, physiological and biological variation, our results indicate that the variation of gene family size provides a source of genetic variation and evolution.

Effects of chronic elevated ozone concentration on antioxidant capacity, photosynthesis and seed yield of 10 soybean cultivars.

Crops losses to tropospheric ozone (O(3)) in the United States are estimated to cost $1-3 billion annually. This challenge is expected to increase as O(3) concentrations ([O(3)]) rise over the next half century. This study tested the hypothesis that there is cultivar variation in the antioxidant, photosynthetic and yield response of soybean to growth at elevated [O(3)]. Ten cultivars of soybean were grown at elevated [O(3)] from germination through maturity at the Soybean Free Air Concentration Enrichment facility in 2007 and six were grown in 2008. Photosynthetic gas exchange, leaf area index, chlorophyll content, fluorescence and antioxidant capacity were monitored during the growing seasons in order to determine if changes in these parameters could be used to predict the sensitivity of seed yield to elevated [O(3)]. Doubling background [O(3)] decreased soybean yields by 17%, but the variation in response among cultivars and years ranged from 8 to 37%. Chlorophyll content and photosynthetic parameters were positively correlated with seed yield, while antioxidant capacity was negatively correlated with photosynthesis and seed yield, suggesting a trade-off between antioxidant metabolism and carbon gain. Exposure response curves indicate that there has not been a significant improvement in soybean tolerance to [O(3)] in the past 30 years.

Highly variable patterns of linkage disequilibrium in multiple soybean populations.

Prospects for utilizing whole-genome association analysis in autogamous plant populations appear promising due to the reported high levels of linkage disequilibrium (LD). To determine the optimal strategies for implementing association analysis in soybean (Glycine max L. Merr.), we analyzed the structure of LD in three regions of the genome varying in length from 336 to 574 kb. This analysis was conducted in four distinct groups of soybean germplasm: 26 accessions of the wild ancestor of soybean (Glycine soja Seib. et Zucc.); 52 Asian G. max Landraces, the immediate results of domestication from G. soja; 17 Asian Landrace introductions that became the ancestors of North American (N. Am.) cultivars, and 25 Elite Cultivars from N. Am. In G. soja, LD did not extend past 100 kb; however, in the three cultivated G. max groups, LD extended from 90 to 574 kb, likely due to the impacts of domestication and increased self-fertilization. The three genomic regions were highly variable relative to the extent of LD within the three cultivated soybean populations. G. soja appears to be ideal for fine mapping of genes, but due to the highly variable levels of LD in the Landraces and the Elite Cultivars, whole-genome association analysis in soybean may be more difficult than first anticipated.

Analysis of soybean protein-derived peptides and the effect of cultivar, environmental conditions, and processing on lunasin concentration in soybean and soy products.

Soybean, an important source of food proteins, has received increasing interest from the public because of its reported health benefits. These health benefits are attributed to its components, including isoflavones, saponins, proteins, and peptides. Lunasin, Bowman-Birk inhibitor, lectin, and beta-conglycinin are some of the biologically active peptides and proteins found in soybean. This article provides a comprehensive review on the recently used techniques in the analysis and characterization of food bioactive peptides, with emphasis on soybean peptides. The methods used to isolate and purify lunasin from defatted soybean flour were ion-exchange chromatography, ultrafiltration, and gel filtration chromatography. The identity of lunasin was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, matrix-assisted laser desorption ionization-time of flight, and liquid chromatography. The results on the effect of soybean cultivar and environmental factors on lunasin concentration are also reported. The highest lunasin concentration, 11.7 +/- 0.3 mg/g flour, was found in Loda soybean cultivar grown at 23 degrees C; the lowest concentration, 5.4 +/- 0.4 mg/g flour, was found in Imari soybean cultivar grown at 28 degrees C. Lunasin concentration was affected by cultivar-temperature, cultivar-soil moisture, and cultivar-temperature-soil moisture interactions. The variation on lunasin concentration suggests that its content can be improved by breeding, and by optimization of growing conditions. In summary, bioactive peptides can be accurately identified and quantified by using different techniques and conditions. In addition, lunasin concentration in soybean depends mainly on cultivar and to some extent on environmental factors, particularly temperature. Lunasin concentration in soy products was also affected by processing conditions.

Elevation of soybean seed oil content through selection for seed coat shininess.

Many leguminous species have adapted their seed coat with a layer of powdery bloom that contains hazardous allergens and makes the seeds less visible, offering duel protection against potential predators 1 . Nevertheless, a shiny seed surface without bloom is desirable for human consumption and health, and is targeted for selection under domestication. Here we show that seed coat bloom in wild soybeans is mainly controlled by Bloom1 (B1), which encodes a transmembrane transporter-like protein for biosynthesis of the bloom in pod endocarp. The transition from the 'bloom' to 'no-bloom' phenotypes is associated with artificial selection of a nucleotide mutation that naturally occurred in the coding region of B1 during soybean domestication. Interestingly, this mutation not only 'shined' the seed surface, but also elevated seed oil content in domesticated soybeans. Such an elevation of oil content in seeds appears to be achieved through b1-modulated upregulation of oil biosynthesis in pods. This study shows pleiotropy as a mechanism underlying the domestication syndrome 2 , and may pave new strategies for development of soybean varieties with increased seed oil content and reduced seed dust.

Identification of glycinin and beta-conglycinin subunits that contribute to the increased protein content of high-protein soybean lines.

Seed protein concentration of commercial soybean cultivars calculated on a dry weight basis ranges from approximately 37 to 42% depending on genotype and location. A concerted research effort is ongoing to further increase protein concentration. Several soybean plant introductions (PI) are known to contain greater than 50% protein. These PIs are exploited by breeders to incorporate the high-protein trait into commercial North American cultivars. Currently, limited information is available on the biochemical and genetic mechanisms that regulate high-proteins. In this study, we have carried out proteomic and molecular analysis of seed proteins of LG00-13260 and its parental high-protein lines PI 427138 and BARC-6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that the high-protein lines accumulated increased amounts of beta-conglycinin and glycinins, when compared with Williams 82. High-resolution two-dimensional electrophoresis utilizing pH 4-7 and pH 6-11 ampholytes enabled improved resolution of soybean seed proteins. A total of 38 protein spots, representing the different subunits of beta-conglycinin and glycinin, were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. High-protein was correlated with an increase in the accumulation of most of the subunits representing beta-conglycinin and glycinin. Comparisons of the amino acid profiles of high-protein soybean lines revealed that the concentration of sulfur amino acids, a reflection of protein quality, was not influenced by the protein concentration. Southern blot analysis showed the presence of genotypic variation at the DNA level between PI 427138 and BARC-6 for the genes encoding group1 glycinin, beta-conglycinin, Bowman-Birk inhibitor (BBI), and the Kunitz trypsin inhibitor (KTI). LG00-13260 inherited the allelic variants of the parental line PI 427138 for glycinin, beta-conglycinin, and KTI, while BBI was inherited from the parental line BARC-6. The results of our study indicate that high-seed protein concentration is attributed to greater accumulation of specific components of beta-conglycinin and glycinin subunits presumably mediated by preferential expression of these genes during seed development.

Genome-wide Association Mapping of Qualitatively Inherited Traits in a Germplasm Collection.

Genome-wide association (GWA) has been used as a tool for dissecting the genetic architecture of quantitatively inherited traits. We demonstrate here that GWA can also be highly useful for detecting many major genes governing categorically defined phenotype variants that exist for qualitatively inherited traits in a germplasm collection. Genome-wide association mapping was applied to categorical phenotypic data available for 10 descriptive traits in a collection of ∼13,000 soybean [ (L.) Merr.] accessions that had been genotyped with a 50,000 single nucleotide polymorphism (SNP) chip. A GWA on a panel of accessions of this magnitude can offer substantial statistical power and mapping resolution, and we found that GWA mapping resulted in the identification of strong SNP signals for 24 classical genes as well as several heretofore unknown genes controlling the phenotypic variants in those traits. Because some of these genes had been cloned, we were able to show that the narrow GWA mapping SNP signal regions that we detected for the phenotypic variants had chromosomal bp spans that, with just one exception, overlapped the bp region of the cloned genes, despite local variation in SNP number and nonuniform SNP distribution in the chip set.