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Background Seeds are the economic basis of oilseed crops, especially soybeans, the most widely cultivated oilseed crop worldwide. Seed development is accompanied by a multitude of diverse cellular processes, and revealing the underlying regulatory activities is critical for seed improvement. Results In this study, we profiled the transcriptomes of developing seeds at 20, 25, 30, and 40 days after flowering (DAF), as these stages represent critical time points of seed development from early to full development. We identified a set of highly abundant genes and highlighted the importance of these genes in supporting nutrient accumulation and transcriptional regulation for seed development. We identified 8925 differentially expressed genes (DEGs) that exhibited temporal expression patterns over the course and expression specificities in distinct tissues, including seeds and nonseed tissues (roots, stems, and leaves). Genes specific to nonseed tissues might have tissue-associated roles, with relatively low transcript abundance in developing seeds, suggesting their spatially supportive roles in seed development. Coexpression network analysis identified several underexplored genes in soybeans that bridge tissue-specific gene modules. Conclusions Our study provides a global view of gene activities and biological processes critical for seed formation in soybeans and prioritizes a set of genes for further study. The results of this study help to elucidate the mechanism controlling seed development and storage reserves.

Soybean cyst nematode (SCN, Heterodera glycine Ichinohe) is the most damaging soybean pest worldwide and management of SCN remains challenging. The current SCN resistant soybean cultivars, mainly developed from the cultivated soybean gene pool, are losing resistance due to SCN race shifts. The domestication process and modern breeding practices of soybean cultivars often involve strong selection for desired agronomic traits, and thus, decreased genetic variation in modern cultivars, which consequently resulted in limited sources of SCN resistance. Wild soybean (Glycine soja) is the wild ancestor of cultivated soybean (Glycine max) and it’s gene pool is indisputably more diverse than G. max. Our aim is to identify novel resistant genetic resources from wild soybean for the development of new SCN resistant cultivars. In this study, resistance response to HG type 2.5.7 (race 5) of SCN was investigated in a newly identified SCN resistant ecotype, NRS100. To understand the resistance mechanism in this ecotype, we compared RNA seq-based transcriptomes of NRS100 with two SCN-susceptible accessions of G. soja and G. max, as well as an extensively studied SCN resistant cultivar, Peking, under both control and nematode J2-treated conditions. The proposed mechanisms of resistance in NRS100 includes the suppression of the jasmonic acid (JA) signaling pathway in order to allow for salicylic acid (SA) signaling-activated resistance response and polyamine synthesis to promote structural integrity of root cell walls. Our study identifies a set of novel candidate genes and associated pathways involved in SCN resistance and the finding provides insight into the mechanism of SCN resistance in wild soybean, advancing the understanding of resistance and the use of wild soybean-sourced resistance for soybean improvement.

Soybean [ Glycine max (L.) Merr.] is an important crop rich in vegetable protein and oil, and is a staple food for human and animals worldwide. However, soybean plants have been challenged by soybean cyst nematode (SCN, Heterodera glycines ), one of the most damaging pests found in soybean fields. Applying SCN-resistant cultivars is the most efficient and environmentally friendly strategy to manage SCN. Currently, soybean breeding and further improvement in soybean agriculture are hindered by severely limited genetic diversity in cultivated soybeans. G . soja is a soybean wild progenitor with much higher levels of genetic diversity compared to cultivated soybeans. In this study, transcriptomes of the resistant and susceptible genotypes of the wild soybean, Glycine soja Sieb & Zucc, were sequenced to examine the genetic basis of SCN resistance. Seedling roots were treated with infective second-stage juveniles (J2s) of the soybean cyst nematode (HG type 2.5.7) for 3, 5, 8 days and pooled for library construction and RNA sequencing. The transcriptome sequencing generated approximately 245 million (M) high quality (Q > 30) raw sequence reads (125 bp in length) for twelve libraries. The raw sequence reads were deposited in NCBI sequence read archive (SRA) database, with the accession numbers SRR5227314-25. Further analysis of this data would be helpful to improve our understanding of the molecular mechanisms of soybean-SCN interaction and facilitate the development of diverse SCN resistance cultivars.

Deleterious effects of climate change and human activities, as well as diverse environmental stresses, present critical challenges to food production and the maintenance of natural diversity. These challenges may be met by the development of novel crop varieties with increased biotic or abiotic resistance that enables them to thrive in marginal lands. However, considering the diverse interactions between crops and environmental factors, it is surprising that evolutionary principles have been underexploited in addressing these food and environmental challenges. Compared with domesticated cultivars, crop wild relatives (CWRs) have been challenged in natural environments for thousands of years and maintain a much higher level of genetic diversity. In this review, we highlight the significance of CWRs for crop improvement by providing examples of CWRs that have been used to increase biotic and abiotic stress resistance/tolerance and overall yield in various crop species. We also discuss the surge of advanced biotechnologies, such as next‐generation sequencing technologies and omics, with particular emphasis on how they have facilitated gene discovery in CWRs. We end the review by discussing the available resources and conservation of CWRs, including the urgent need for CWR prioritization and collection to ensure continuous crop improvement for food sustainability.

Soybean cyst nematode (SCN) is the most damaging pest of soybean worldwide. The molecular mechanism of SCN resistance remains largely unknown. We conducted a global RNA-seq comparison between a resistant genotype (S54) and a susceptible genotype (S67) of Glycine soja, the wild progenitor of soybean, to understand its regulatory network in SCN defense. The number of differentially expressed genes (DEGs) in S54 (2,290) was much larger than that in S67 (555). A number of defense-related genes/pathways were significantly induced only in S54, while photosynthesis and several metabolic pathways were affected in both genotypes with SCN infection. These defense-associated DEGs were involved in pathogen recognition, calcium/calmodulin-mediated defense signaling, jasmonic acid (JA)/ethylene (ET) and sialic acid (SA)-involved signaling, the MAPK signaling cascade, and WRKY-involved transcriptional regulation. Our results revealed a comprehensive regulatory network involved in SCN resistance and provided insights into the complex molecular mechanisms of SCN resistance in wild soybean.

A set of nested association mapping (NAM) families was developed by crossing 40 diverse soybean [ (L.) Merr.] genotypes to the common cultivar. The 41 parents were deeply sequenced for SNP discovery. Based on the polymorphism of the single-nucleotide polymorphisms (SNPs) and other selection criteria, a set of SNPs was selected to be included in the SoyNAM6K BeadChip for genotyping the parents and 5600 RILs from the 40 families. Analysis of the SNP profiles of the RILs showed a low average recombination rate. We constructed genetic linkage maps for each family and a composite linkage map based on recombinant inbred lines (RILs) across the families and identified and annotated 525,772 high confidence SNPs that were used to impute the SNP alleles in the RILs. The segregation distortion in most families significantly favored the alleles from the female parent, and there was no significant difference of residual heterozygosity in the euchromatic vs. heterochromatic regions. The genotypic datasets for the RILs and parents are publicly available and are anticipated to be useful to map quantitative trait loci (QTL) controlling important traits in soybean.

Background Soybean seed weight is not only a yield component, but also a critical trait for various soybean food products such as sprouts, edamame, soy nuts, natto and miso. Linkage analysis and genome-wide association study (GWAS) are two complementary and powerful tools to connect phenotypic differences to the underlying contributing loci. Linkage analysis is based on progeny derived from two parents, given sufficient sample size and biological replication, it usually has high statistical power to map alleles with relatively small effect on phenotype, however, linkage analysis of the bi-parental population can’t detect quantitative trait loci (QTL) that are fixed in the two parents. Because of the small seed weight difference between the two parents in most families of previous studies, these populations are not suitable to detect QTL that have considerable effects on seed weight. GWAS is based on unrelated individuals to detect alleles associated with the trait under investigation. The ability of GWAS to capture major seed weight QTL depends on the frequency of the accessions with small and large seed weight in the population being investigated. Our objective was to identify QTL that had a pronounced effect on seed weight using a selective population of soybean germplasm accessions and the approach of GWAS and fixation index analysis. Results We selected 166 accessions from the USDA Soybean Germplasm Collection with either large or small seed weight and could typically grow in the same location. The accessions were evaluated for seed weight in the field for two years and genotyped with the SoySNP50K BeadChip containing >42,000 SNPs. Of the 17 SNPs on six chromosomes that were significantly associated with seed weight in two years based on a GWAS of the selective population, eight on chromosome 4 or chromosome 17 had significant Fst values between the large and small seed weight sub-populations. The seed weight difference of the two alleles of these eight significant SNPs varied from 8.1 g to 11.7 g/100 seeds in two years. We also identified haplotypes in three haplotype blocks with significant effects on seed weight. These findings were validated in a panel with 3753 accessions from the USDA Soybean Germplasm Collection. Conclusion This study highlighted the usefulness of selective genotyping populations coupled with GWAS and fixation index analysis for the identification of QTL with substantial effects on seed weight in soybean. This approach may help geneticists and breeders to more efficiently identify major QTL controlling other traits. The major regions and haplotypes we have identified that control seed weight differences in soybean will facilitate the identification of genes regulating this important trait. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3922-0) contains supplementary material, which is available to authorized users.

The pattern and rate of genome evolution have profound consequences in organismal evolution. Whole-genome duplication (WGD), or polyploidy, has been recognized as an important evolutionary mechanism of plant diversification. However, in non-model plants the molecular signals of genome duplications have remained largely unexplored. High-throughput transcriptome data from next-generation sequencing have set the stage for novel investigations of genome evolution using new bioinformatic and methodological tools in a phylogenetic framework. Here we compare ten de novo-assembled transcriptomes representing the major lineages of the angiosperm genus Cornus (dogwood) and relevant outgroups using a customized pipeline for analyses. Using three distinct approaches, molecular dating of orthologous genes, analyses of the distribution of synonymous substitutions between paralogous genes, and examination of substitution rates through time, we detected a shared WGD event in the late Cretaceous across all taxa sampled. The inferred doubling event coincides temporally with the paleoclimatic changes associated with the initial divergence of the genus into three major lineages. Analyses also showed an acceleration of rates of molecular evolution after WGD. The highest rates of molecular evolution were observed in the transcriptome of the herbaceous lineage, C. canadensis, a species commonly found at higher latitudes, including the Arctic. Our study demonstrates the value of transcriptome data for understanding genome evolution in closely related species. The results suggest dramatic increase in sea surface temperature in the late Cretaceous may have contributed to the evolution and diversification of flowering plants.

The genetic mechanisms causing seed development by gametophytic apomixis in plants are predominantly unknown. As apomixis is consistently associated with hybridity and polyploidy, these confounding factors may either (a) be the underlying mechanism for the expression of apomixis, or (b) obscure the genetic factors which cause apomixis. To distinguish between these hypotheses, we analyzed the population genetic patterns of diploid and triploid apomictic lineages and their sexual progenitors in the genus Boechera (Brassicaceae). We find that while triploid apomixis is associated with hybridization, the majority of diploid apomictic lineages are likely the product of intra-specific crosses. We then show that these diploid apomicts are more likely to sire triploid apomictic lineages than conspecific sexuals. Combined with flow cytometric seed screen phenotyping for male and female components of apomixis, our analyses demonstrate that hybridization is an indirect correlate of apomixis in Boechera.

Soybean cyst nematode (SCN) is the most destructive soybean pest worldwide. Host plant resistance is the most environmentally friendly and cost-effective way of mitigating SCN damage to soybeans. However, overuse of the resistant soybean (Glycine max (L.) Merr.) cultivars from limited genetic resources has resulted in SCN race shifts in many soybean-growing areas. Thus, exploration of novel sources of SCN resistance and dissection of the genetic basis are urgently needed. In this study, we screened 235 wild soybean (Glycine soja Sieb. & Zucc.) accessions to identify genotypes resistant to SCN HG Type 2.5.7 (race 5), a less investigated type but is prevalent in the southeastern US. We also dissected the genetic basis of SCN resistance using a genome-wide association study with SNPs genotyped by SoySNP50k iSelect BeadChip. In total, 43 resistant accessions (female index < 30) were identified, with ten SNPs being significantly associated with SCN HG 2.5.7 resistance in this wild species. Furthermore, four significant SNPs were localized to linked regions of the known quantitative trait locus (QTL) rhg1 on chromosome 18. The other four SNPs on chromosome 18 and two SNPs on chromosome 19 are novel. Genes encoding disease resistance-related proteins with a leucine-rich region, a mitogen-activated protein kinase (MAPK) on chromosome 18, and a MYB transcription factor on chromosome 19 were identified as promising candidate genes. The identified SNPs and candidate genes will benefit future marker-assisted breeding and dissection of the molecular mechanisms underlying the soybean-SCN interaction.

Background Cultivated soybean (Glycine max) is a major agricultural crop that provides a crucial source of edible protein and oil. Decreased amounts of saturated palmitic acid and increased amounts of unsaturated oleic acid in soybean oil are considered optimal for human cardiovascular health and therefore there has considerable interest by breeders in discovering genes affecting the relative concentrations of these fatty acids. Using a genome-wide association (GWA) approach with nearly 30,000 single nucleotide polymorphisms (SNPs), we investigated the genetic basis of protein, oil and all five fatty acid levels in seeds from a sample of 570 wild soybeans (Glycine soja), the progenitor of domesticated soybean, to identify quantitative trait loci (QTLs) affecting these seed composition traits. Results We discovered 29 SNPs located on ten different chromosomes that are significantly associated with the seven seed composition traits in our wild soybean sample. Eight SNPs co-localized with QTLs previously uncovered in linkage or association mapping studies conducted with cultivated soybean samples, while the remaining SNPs appeared to be in novel locations. Twenty-four of the SNPs significantly associated with fatty acid variation, with the majority located on chromosomes 14 (6 SNPs) and seven (8 SNPs). Two SNPs were common for two or more fatty acids, suggesting loci with pleiotropic effects. We also identified some candidate genes that are involved in fatty acid metabolism and regulation. For each of the seven traits, most of the SNPs produced differences between the average phenotypic values of the two homozygotes of about one-half standard deviation and contributed over 3% of their total variability. Conclusions This is the first GWA study conducted on seed composition traits solely in wild soybean populations, and a number of QTLs were found that have not been previously discovered. Some of these may be useful to breeders who select for increased protein/oil content or altered fatty acid ratios in the seeds. The results also provide additional insight into the genetic architecture of these traits in a large sample of wild soybean, and suggest some new candidate genes whose molecular effects on these traits need to be further studied. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3397-4) contains supplementary material, which is available to authorized users.

Natural Selection at Work Catching the evolution of a novel function and determining its selective parameters in nature remains an extremely difficult task. Prasad et al. (p. 1081 ) have undertaken this quest documenting the molecular basis of a natural allelic polymorphism and its effects on herbivory and survival in the Arabidopsis relative, Boechera stricta , living in the Rocky Mountains.

The selection and development of a study system for evolutionary and ecological functional genomics (EEFG) depend on a variety of factors. Here, we present the genus Boechera as an exemplary system with which to address ecological and evolutionary questions. Our focus on Boechera is based on several characteristics as follows: (i) native populations in undisturbed habitats where current environments reflect historical conditions over several thousand years; (ii) functional genomics benefitting from its close relationship to Arabidopsis thaliana; (iii) inbreeding tolerance enabling development of recombinant inbred lines, near-isogenic lines and positional cloning; (iv) interspecific crosses permitting mapping for genetic analysis of speciation; (v) apomixis (asexual reproduction by seeds) in a genetically tractable diploid; and (vi) broad geographic distribution in North America, permitting ecological genetics for a large research community. These characteristics, along with the current sequencing of three Boechera species by the Joint Genome Institute, position Boechera as a rapidly advancing system for EEFG studies.

Information about polymorphism, population structure, and linkage disequilibrium (LD) is crucial for association studies of complex trait variation. However, most genomewide studies have focused on model systems, with very few analyses of undisturbed natural populations. Here, we sequenced 86 mapped nuclear loci for a sample of 46 genotypes of Boechera stricta and two individuals of B. holboellii, both wild relatives of Arabidopsis. Isolation by distance was significant across the species range of B. stricta, and three geographic groups were identified by structure analysis, principal coordinates analysis, and distance-based phylogeny analyses. The allele frequency spectrum indicated a genomewide deviation from an equilibrium neutral model, with silent nucleotide diversity averaging 0.004. LD decayed rapidly, declining to background levels in ∼10 kb or less. For tightly linked SNPs separated by, This work was supported by the National Science Foundation (award EF-0723447), the National Institutes of Health (award R01 GM086496-01), Duke University, and the Max-Planck Society.

Conservation of endangered species becomes a critical issue with the increasing rates of extinction. In this study, we use 13 microsatellite loci and 27 single-copy nuclear loci to investigate the population genetics of Boechera fecunda, a rare relative of Arabidopsis thaliana, known from only 21 populations in Montana. We investigated levels of genetic diversity and population structure in comparison to its widespread congener, Boechera stricta, which shares similar life history and mating system. Despite its rarity, B. fecunda had levels of genetic diversity similar to B. stricta for both microsatellites and nucleotide polymorphism. Populations of B. fecunda are highly differentiated, with a majority of genetic diversity existing among populations (F(ST) = 0.57). Differences in molecular diversity and allele frequencies between western and eastern population groups suggest they experienced very different evolutionary histories.

The genus Boechera is a widespread North American group with great potential for studies of ecology and evolution: Boechera is closely related to Arabidopsis and exhibits different ecological and reproductive strategies. Boechera stricta (previously Arabis drummondii) is a morphologically and genetically well-defined, perennial crucifer species. Fifteen natural populations of diploid individuals from the Rocky Mountains were analysed using 21 microsatellite loci. In accordance with our expectation for this predominately inbreeding species, a high F IS value (0.89) was observed. Furthermore, populations of B. stricta were highly differentiated, as indicated by F ST = 0.56. Three clusters were identified using structure- the majority of populations belonged to either the Northern or Southern cluster. Together, the north-south partitioning and evenness of genetic variation across the two clusters suggested multiple refugia for this perennial herb in the Rocky Mountains. Pleistocene glaciation, together with the topographically and climatologically heterogeneous cordillera, has profoundly influenced the genetic architecture of B. stricta. Genetic population structure was also influenced by relatively recent genome admixture at two levels: within species (involving individuals from the Northern and Southern clusters) and between species (with the hybridization of B. stricta and Boechera holboellii). This complexity of population structure at presumably neutral microsatellite loci located throughout the genome in B. stricta provides a baseline against which to test whether functional genetic variation is undergoing local adaptive evolution throughout the natural species range.

Sequence and restriction site analyses of the paternally inherited chloroplast rbcL gene and maternally inherited mitochondrial nad1 fragments from the same set of populations and individuals were used to investigate cytoplasmic composition and population establishment of Pinus densata, a diploid pine that originated through hybridization between P. tabuliformis and P. yunnanensis. Two variable sites and three chlorotypes (TT, TC and GC) were detected on the rbcL gene of the three pines. P. densata harboured the three chlorotypes, two of which (TT, GC) were characteristic of the parental species, respectively. The third chlorotype (TC) was distributed extensively in seven of the 10 P. densata populations analysed, and might represent a mutation type or have been derived from an extinct parent. The distribution of chlorotypes, together with that of mitotypes, indicated that significant founder effect and backcross happened during the population establishment of the hybrid pine. P. tabuliformis and P. yunnanensis had acted as both mother and father donors, i.e. bi-directional gene flow existed between the two parental species in the past. Population differentiation of P. densata is high, as detected from the cytoplasmic genomes: GST = 0.533 for cpDNA and GST = 0.905 for mtDNA. The differences in cytoplasmic composition among the hybrid populations suggest that the local populations have undergone different evolutionary histories.

Previous morphological, allozyme and chloroplast DNA data have suggested that Pinus densata originated through hybridization between P. tabuliformis and P. yunnanensis. In the present study, sequence and restriction site analyses of maternally inherited mitochondrial nad1 intron were used to detect variation patterns in 19 populations of P. tabuliformis, P. yunnanensis and P. densata. A total of three mitotypes (A, B, C) were detected. All but one of the populations of P. yunnanensis possessed mitotype B while all populations of P. tabuliformis had mitotype A. Pinus densata populations, on the other hand, harboured both mitotypes A and B, which are characteristic of P. tabuliformis and P. yunnanensis, respectively. This result gives strong additional evidence supporting the hybrid origin of this diploid pine. The distribution of mitotypes indicated very different mating compositions and evolutionary history among P. densata populations. It seems that local founder populations and backcrosses may have played important roles in the early establishment of P. densata populations. The uplift of the Tibetan Plateau had a significant impact on the distribution of maternal lineages of P. densata populations.

The relative contribution of advantageous and neutral mutations to the evolutionary process is a central problem in evolutionary biology. Current estimates suggest that whereas Drosophila, mice, and bacteria have undergone extensive adaptive evolution, hominids show little or no evidence of adaptive evolution in protein-coding sequences. This may be a consequence of differences in effective population size. To study the matter further, we have investigated whether plants show evidence of adaptive evolution using an extension of the McDonald-Kreitman test that explicitly models slightly deleterious mutations by estimating the distribution of fitness effects of new mutations. We apply this method to data from nine pairs of species. Altogether more than 2,400 loci with an average length of approximate to 280 nucleotides were analyzed. We observe very similar results in all species; we find little evidence of adaptive amino acid substitution in any comparison except sunflowers. This may be because many plant species have modest effective population sizes.

The angiosperm family Brassicaceae contains both the research model Arabidopsis (Arabidopsis thaliana) and the agricultural genus Brassica. Comparative genomics in the Brassicaceae has largely focused on direct comparisons between Arabidopsis and the species of interest. However, the reduced genome size and chromosome number (n = 5) of Arabidopsis complicates comparisons. Arabidopsis shows extensive genome and chromosome reshuffling compared to its close relatives Arabidopsis lyrata and Capsella rubella, both with n = 8. To facilitate comparative genomics across the Brassicaceae we recently outlined a system of 24 conserved chromosomal blocks based on their positions in an ancestral karyotype of n = 8, rather than by their position in Arabidopsis. In this report we use this system as a tool to understand genome structure and evolution in Boechera stricta (n = 7). B. stricta is a diploid, sexual, and highly self-fertilizing species occurring in mostly montane regions of western North America. We have created an F(2) genetic map of B. stricta based on 192 individuals scored at 196 microsatellite and candidate gene loci. Single-nucleotide polymorphism genotyping of 94 of the loci was done simultaneously using an Illumina bead array. The total map length is 725.8 cM, with an average marker spacing of 3.9 cM. There are no gaps greater than 19.3 cM. The chromosomal reduction from n = 8 to n = 7 and other genomic changes in B. stricta likely involved a pericentric inversion, a chromosomal fusion, and two reciprocal translocations that are easily visualized using the genomic blocks. Our genetic map will facilitate the analysis of ecologically relevant quantitative variation in Boechera. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DU 667459 to DU 708532.

Comparative genomics of Arabidopsis relatives has great potential to improve our understanding of molecular function and evolutionary processes. Recent studies of phylogenetic relationships within Brassicaceae and the publication of a new tribal classification scheme provide an important framework for comparative genomics research. Comparative linkage mapping and chromosome painting in the close relatives of Arabidopsis have inferred an ancestral karyotype of these species. In addition, comparative mapping to Brassica has identified genomic blocks that have been maintained since the divergence of the Arabidopsis and Brassica lineages. Several analyses of conserved non-coding regions have identified putative cis-regulatory sequences, and have highlighted the need for comparative sequencing at greater evolutionary distances. The development of new model species with novel physiological and ecological traits allows analysis of phenotypes that are not available in A. thaliana. Looking towards the future, we suggest a prioritized research agenda for comparative genomics in the Brassicaceae.

Background Pseudostellaria heterophylla produces both closed (cleistogamous, CL) and open (chasmogamous, CH) flowers on the same individual but in different seasons. The production of CH and CL flowers might be in response to environmental changes. To better understand the molecular mechanisms of CH and CL flowering, we compared the transcriptome of the two types of flowers to examine differential gene expression patterns, and to identify gene regulatory networks that control CH and CL flowering. Results Using RNA sequencing, we identified homologues of 428 Arabidopsis genes involved in regulating flowering processes and estimated the differential gene expression patterns between CH and CL flowers. Some of these genes involved in gene regulatory networks of flowering processes showed significantly differential expression patterns between CH and CL flowers. In addition, we identified another 396 differentially expressed transcripts between CH and CL flowers. Some are involved in environmental stress responses and flavonoid biosynthesis. Conclusions We propose how the differential expression of key members of three gene regulatory modules may explain CH and CL flowering. Future research is needed to investigate how the environment impinges on these flowering pathways to regulate CH and CL flowering in P. heterophylla. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2732-0) contains supplementary material, which is available to authorized users.