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Background: Soybean downy mildew (SDM), caused by Peronospora manshurica (Pm), is a major fungal disease in soybean. To date, little is known regarding the defense mechanism at molecular level and how soybean plants response to Pm infection. In this study, differential gene expression in SDM-resistant (HR) and SDM-susceptible (HS) genotype was analyzed by RNA-seq to identify differentially expressed genes (DEGs) following Pm infection. Results: Of a total of 55,017 genes mapped to the soybean reference genome sequences, 2581 DEGs were identified. Clustering analysis of DEGs revealed that these genes could be grouped into 8 clusters with distinct expression patterns. Functional annotation based on gene ontology (GO) and KEGG analysis indicated they involved in diverse metabolism pathways. Of particular interest were the detected DEGs participating in SA/ROS and JA signalling transduction and plant/pathogen interaction. Conclusion: Totally, 52 DEGs with P value < 0.001 and log2 fold change > 2 or < − 2 upon fungal inoculation were identified, suggesting they were SDM defense responsive genes. These findings have paved way in further functional characterization of candidate genes and subsequently can be used in breeding of elite soybean varieties with better SDM-resistance. [ABSTRACT FROM AUTHOR]

Background: The MADS-box transcription factors are an ancient family of genes that regulate numerous physiological and biochemical processes in plants and facilitate the development of floral organs. However, the functions of most of these transcription factors in soybean remain unknown. Results: In this work, a MADS-box gene, GmAGL1, was overexpressed in soybean. Phenotypic analysis showed that GmAGL1 overexpression not only resulted in early maturation but also promoted flowering and affected petal development. Furthermore, the GmAGL1 was much more effective at promoting flowering under long-day conditions than under short-day conditions. Transcriptome sequencing analysis showed that before flowering, the photoperiod pathway photoreceptor CRY2 and several circadian rhythm genes, such as SPA1, were significantly down-regulated, while some other flowering-promoting circadian genes, such as GI and LHY, and downstream genes related to flower development, such as FT, LEAFY, SEP1, SEP3, FUL, and AP1, were up-regulated compared with the control. Other genes related to the flowering pathway were not noticeably affected. Conclusions: The findings reported herein indicate that GmAGL1 may promote flowering mainly through the photoperiod pathway. Interestingly, while overexpression of GmAGL1 promoted plant maturity, no reduction in seed production or oil and protein contents was observed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Aldehyde dehydrogenases (ALDHs) represent a group of enzymes that detoxify aldehydes by facilitating their oxidation to carboxylic acids, and have been shown to play roles in plant response to abiotic stresses. However, the comprehensive analysis of ALDH superfamily in soybean (Glycine max) has been limited. Results: In present study, a total of 53 GmALDHs were identified in soybean, and grouped into 10 ALDH families according to the ALDH Gene Nomenclature Committee and phylogenetic analysis. These groupings were supported by their gene structures and conserved motifs. Soybean ALDH superfamily expanded mainly by whole genome duplication/segmental duplications. Gene network analysis identified 1146 putative co-functional genes of 51 GmALDHs. Gene Ontology (GO) enrichment analysis suggested the co-functional genes of these 51 GmALDHs were enriched (FDR < 1e-3) in the process of lipid metabolism, photosynthesis, proline catabolism, and small molecule catabolism. In addition, 22 co-functional genes of GmALDHs are related to plant response to water deprivation/water transport. GmALDHs exhibited various expression patterns in different soybean tissues. The expression levels of 13 GmALDHs were significantly up-regulated and 14 down-regulated in response to water deficit. The occurrence frequencies of three drought-responsive cis-elements (ABRE, CRT/DRE, and GTGCnTGC/G) were compared in GmALDH genes that were up-, down-, or non-regulated by water deficit. Higher frequency of these three cis-elements was observed for the group of up-regulated GmALDH genes as compared to the group of down- or non- regulated GmALDHs by drought stress, implying their potential roles in the regulation of soybean response to drought stress. Conclusions: A total of 53 ALDH genes were identified in soybean genome and their phylogenetic relationships and duplication patterns were analyzed. The potential functions of GmALDHs were predicted by analyses of their co-functional gene networks, gene expression profiles, and cis-regulatory elements. Three GmALDH genes, including GmALDH3H2, GmALDH12A2 and GmALDH18B3, were highly induced by drought stress in soybean leaves. Our study provides a foundation for future investigations of GmALDH gene function in soybean. [ABSTRACT FROM AUTHOR]

Background: Genetic resistance of soybean [Glycine max (L.) Merr] against Aphis glycines provides effective management of this invasive pest, though the underlying molecular mechanisms are largely unknown. This study aimed to investigate genome-wide changes in gene expressions of soybean near-isogenic lines (NILs) either with the Rag5 allele for resistance or the rag5 allele for susceptibility to the aphid following infestation with soybean aphid biotype 2. Results: The resistant (R)-NIL responded more rapidly to aphid infestation than the susceptible (S)-NIL, with differential expressions of 2496 genes during first 12 h of infestation (hai), compared to the aphid-free control. Although the majority of the differentially expressed genes (DEGs) in the R-NIL also responded to aphid infestation in S-NIL, overall the response time was longer and/or the magnitude of change was smaller in the S-NIL. In addition, 915 DEGs in R-NIL continued to be regulated at all time points (0, 6, 12, and 48 hai), while only 20 DEGs did so in S-NIL. Enriched gene ontology of the 2496 DEGs involved in plant defense responses including primary metabolite catalysis, oxidative stress reduction, and phytohormone-related signaling. By comparing R- vs. S-NIL, a total of 556 DEGs were identified. Of the 13 genes annotated in a 120-kb window of the Rag5 locus, two genes (Glyma.13 g190200 and Glyma.13 g190600) were differentially expressed (upregulated in S- or R-NIL), and another gene (Glyma.13 g190500) was induced up to 4-fold in the R-NIL at 6 and 12 h following aphid infestation. Conclusions: This study strengthens our understanding of the defense dynamics in compatible and incompatible interactions of soybean and soybean aphid biotype 2. Several DEGs (e.g., Glyma.13 g190200, Glyma.13 g190500, and Glyma.13 g190600) near the Rag5 locus are strong candidate genes for further investigations. [ABSTRACT FROM AUTHOR]

Background: One of the overarching goals of soybean breeding is to develop lines that combine increased yield with improved quality characteristics. High-density-marker QTL mapping can serve as an effective strategy to identify novel genomic information to facilitate crop improvement. In this study, we genotyped a recombinant inbred line (RIL) population (Zhonghuang 24 × Huaxia 3) using a restriction-site associated DNA sequencing (RAD-seq) approach. A high-density soybean genetic map was constructed and used to identify several QTLs that were shown to influence six yield-related and two quality traits. Results: A total of 47,472 single-nucleotide polymorphisms (SNPs) were detected for the RILs that were integrated into 2639 recombination bin units, with an average distance of 1.00 cM between adjacent markers. Forty seven QTLs for yield-related traits and 13 QTLs for grain quality traits were found to be distributed on 16 chromosomes in the 2 year studies. Among them, 18 QTLs were stable, and were identified in both analyses. Twenty six QTLs were identified for the first time, with a single QTL (qNN19a) in a 56 kb region explaining 32.56% of phenotypic variation, and an additional 10 of these were novel, stable QTLs. Moreover, 8 QTL hotpots on four different chromosomes were identified for the correlated traits. Conclusions: With RAD-sequencing, some novel QTLs and important QTL clusters for both yield-related and quality traits were identified based on a new, high-density bin linkage map. Three predicted genes were selected as candidates that likely have a direct or indirect influence on both yield and quality in soybean. Our findings will be helpful for understanding common genetic control mechanisms of co-localized traits and to select cultivars for further analysis to predictably modulate soybean yield and quality simultaneously. [ABSTRACT FROM AUTHOR]

Background: Soybean (Glycine max L. Merr.) cyst nematode (SCN, Heterodera glycines I,) is a major pest of soybean worldwide. The most effective strategy to control this pest involves the use of resistant cultivars. The aim of the present study was to investigate the genome-wide genetic architecture of resistance to SCN HG Type 2.5.7 (race 1) in landrace and elite cultivated soybeans. Results: A total of 200 diverse soybean accessions were screened for resistance to SCN HG Type 2.5.7 and genotyped through sequencing using the Specific Locus Amplified Fragment Sequencing (SLAF-seq) approach with a 6.14-fold average sequencing depth. A total of 33,194 SNPs were identified with minor allele frequencies (MAF) over 4%, covering 97% of all the genotypes. Genome-wide association mapping (GWAS) revealed thirteen SNPs associated with resistance to SCN HG Type 2.5.7. These SNPs were distributed on five chromosomes (Chr), including Chr7, 8, 14, 15 and 18. Four SNPs were novel resistance loci and nine SNPs were located near known QTL. A total of 30 genes were identified as candidate genes underlying SCN resistance. Conclusions: A total of sixteen novel soybean accessions were identified with significant resistance to HG Type 2.5. 7. The beneficial alleles and candidate genes identified by GWAS might be valuable for improving marker-assisted breeding efficiency and exploring the molecular mechanisms underlying SCN resistance. [ABSTRACT FROM AUTHOR]

Background: In recent years, the damage caused by bacterial pathogens to major crops has been increasing worldwide. Pseudomonas syringae is a widespread bacterial species that infects almost all major crops. Different P. syringae strains use a wide range of biochemical mechanisms, including phytotoxins and effectors of the type III and type IV secretion systems, which determine the specific nature of the pathogen virulence. Results: Strains 1845 (isolated from dicots) and 2507 (isolated from monocots) were selected for sequencing because they specialize on different groups of plants. We compared virulence factors in these and other available genomes of phylogroup 2 to find genes responsible for the specialization of bacteria. We showed that strain 1845 belongs to the clonal group that has been infecting monocots in Russia and USA for a long time (at least 50 years). Strain 1845 has relatively recently changed its host plant to dicots. Conclusions: The results obtained by comparing the strain 1845 genome with the genomes of bacteria infecting monocots can help to identify the genes that define specific nature of the virulence of P. syringae strains. [ABSTRACT FROM AUTHOR]

Background: Of the approximately two hundred sequenced plant genomes, how many and which ones were sequenced motivated by strictly or largely scientific considerations, and how many by chiefly economic, in a wide sense, incentives? And how large a role does publication opportunity play? Results: In an integration of multiple disparate databases and other sources of information, we collect and analyze data on the size (number of species) in the plant orders and families containing sequenced genomes, on the trade value of these species, and of all the same-family or same-order species, and on the publication priority within the family and order. These data are subjected to multiple regression and other statistical analyses. We find that despite the initial importance of model organisms, it is clearly economic considerations that outweigh others in the choice of genome to be sequenced. Conclusions: This has important implications for generalizations about plant genomes, since human choices of plants to harvest (and cultivate) will have incurred many biases with respect to phenotypic characteristics and hence of genomic properties, and recent genomic evolution will also have been affected by human agricultural practices. [ABSTRACT FROM AUTHOR]

Background: Soybean [Glycine max (L.) Merrill] is one of the most important legumes cultivated worldwide, and Brazil is one of the main producers of this crop. Since the sequencing of its reference genome, interest in structural and allelic variations of cultivated and wild soybean germplasm has grown. To investigate the genetics of the Brazilian soybean germplasm, we selected soybean cultivars based on the year of commercialization, geographical region and maturity group and resequenced their genomes. Results: We resequenced the genomes of 28 Brazilian soybean cultivars with an average genome coverage of 14.8X. A total of 5,835,185 single nucleotide polymorphisms (SNPs) and 1,329,844 InDels were identified across the 20 soybean chromosomes, with 541,762 SNPs, 98,922 InDels and 1,093 CNVs that were exclusive to the 28 Brazilian cultivars. In addition, 668 allelic variations of 327 genes were shared among all of the Brazilian cultivars, including genes related to DNA-dependent transcription-elongation, photosynthesis, ATP synthesis-coupled electron transport, cellular respiration, and precursors of metabolite generation and energy. A very homogeneous structure was also observed for the Brazilian soybean germplasm, and we observed 41 regions putatively influenced by positive selection. Finally, we detected 3,880 regions with copy-number variations (CNVs) that could help to explain the divergence among the accessions evaluated. Conclusions: The large number of allelic and structural variations identified in this study can be used in marker-assisted selection programs to detect unique SNPs for cultivar fingerprinting. The results presented here suggest that despite the diversification of modern Brazilian cultivars, the soybean germplasm remains very narrow because of the large number of genome regions that exhibit low diversity. These results emphasize the need to introduce new alleles to increase the genetic diversity of the Brazilian germplasm. [ABSTRACT FROM AUTHOR]

Background: A landmark in soybean research, Glyma1.01, the first whole genome sequence of variety Williams 82 (Glycine max L. Merr.) was completed in 2010 and is widely used. However, because the assembly was primarily built based on the linkage maps constructed with a limited number of markers and recombinant inbred lines (RILs), the assembled sequence, especially in some genomic regions with sparse numbers of anchoring markers, needs to be improved. Molecular markers are being used by researchers in the soybean community, however, with the updating of the Glyma1.01 build based on the high-resolution linkage maps resulting from this research, the genome positions of these markers need to be mapped. Results: Two high density genetic linkage maps were constructed based on 21,478 single nucleotide polymorphism loci mapped in the Williams 82 x G. soja (Sieb. & Zucc.) PI479752 population with 1083 RILs and 11,922 loci mapped in the Essex x Williams 82 population with 922 RILs. There were 37 regions or single markers where marker order in the two populations was in agreement but was not consistent with the physical position in the Glyma1.01 build. In addition, 28 previously unanchored scaffolds were positioned. Map data were used to identify false joins in the Glyma1.01 assembly and the corresponding scaffolds were broken and reassembled to the new assembly, Wm82.a2.v1. Based upon the plots of the genetic on physical distance of the loci, the euchromatic and heterochromatic regions along each chromosome in the new assembly were delimited. Genomic positions of the commonly used markers contained in BARCSOYSSR_1.0 database and the SoySNP50K BeadChip were updated based upon the Wm82.a2.v1 assembly. Conclusions: The information will facilitate the study of recombination hot spots in the soybean genome, identification of genes or quantitative trait loci controlling yield, seed quality and resistance to biotic or abiotic stresses as well as other genetic or genomic research. [ABSTRACT FROM AUTHOR]

Background: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. Results: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. Conclusions: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding. [ABSTRACT FROM AUTHOR]

Background: Transcriptomics reveals the existence of transcripts of different coding potential and strand orientation. Alternative splicing (AS) can yield proteins with altered number and types of functional domains, suggesting the global occurrence of transcriptional and post-transcriptional events. Many biological processes, including seed maturation and desiccation, are regulated post-transcriptionally (e.g., by AS), leading to the production of more than one coding or noncoding sense transcript from a single locus. Results: We present an integrated computational framework to predict isoform-specific functions of plant transcripts. This framework includes a novel plant-specific weighted support vector machine classifier called CodeWise, which predicts the coding potential of transcripts with over 96 % accuracy, and several other tools enabling global sequence similarity, functional domain, and co-expression network analyses. First, this framework was applied to all detected transcripts (103,106), out of which 13 % was predicted by CodeWise to be noncoding RNAs in developing soybean embryos. Second, to investigate the role of AS during soybean embryo development, a population of 2,938 alternatively spliced and differentially expressed splice variants was analyzed and mined with respect to timing of expression. Conserved domain analyses revealed that AS resulted in global changes in the number, types, and extent of truncation of functional domains in protein variants. Isoform-specific co-expression network analysis using ArrayMining and clustering analyses revealed specific sub-networks and potential interactions among the components of selected signaling pathways related to seed maturation and the acquisition of desiccation tolerance. These signaling pathways involved abscisic acid- and FUSCA3-related transcripts, several of which were classified as noncoding and/or antisense transcripts and were co-expressed with corresponding coding transcripts. Noncoding and antisense transcripts likely play important regulatory roles in seed maturation- and desiccation-related signaling in soybean. Conclusions: This work demonstrates how our integrated framework can be implemented to make experimentally testable predictions regarding the coding potential, co-expression, co-regulation, and function of transcripts and proteins related to a biological process of interest. [ABSTRACT FROM AUTHOR]

Background: Despite the relevance of the eukaryotic endoplasmic reticulum (ER)-stress response as an integrator of multiple stress signals into an adaptive response, knowledge about these ER-mediated cytoprotective pathways in soybean (Glycine max) is lacking. Here, we searched for genes involved in the highly conserved unfolded protein response (UPR) and ER stress-induced plant-specific cell death signaling pathways in the soybean genome. Methods: Previously characterized Arabidopsis UPR genes were used as prototypes for the identification of the soybean orthologs and the in silico assembly of the UPR in soybean, using eggNOG v4.0 software. Functional studies were also conducted by analyzing the transcriptional activity of soybean UPR transducers. Results: As a result of this search, we have provided a complete profile of soybean UPR genes with significant predicted protein similarities to A. thaliana UPR-associated proteins. Both arms of the plant UPR were further examined functionally, and evidence is presented that the soybean counterparts are true orthologs of previously characterized UPR transducers in Arabidopsis. The bZIP17/bZI28 orthologs (GmbZIP37 and GmbZIP38) and ZIP60 ortholog (GmbZIP68) from soybean have similar structural organizations as their Arabidopsis counterparts, were induced by ER stress and activated an ERSE- and UPRE-containing BiP promoter. Furthermore, the transcript of the putative substrate of GmIREs, GmbZIP68, harbors a canonical site for IRE1 endonuclease activity and was efficiently spliced under ER stress conditions. In a reverse approach, we also examined the Arabidopsis genome for components of a previously characterized ER stress-induced cell death signaling response in soybean. With the exception of GmERD15, which apparently does not possess an Arabidopsis ortholog, the Arabidopsis genome harbors conserved GmNRP, GmNAC81, GmNAC30 and GmVPE sequences that share significant structural and sequence similarities with their soybean counterparts. These results suggest that the NRP/GmNAC81 + GmNAC30/VPE regulatory circuit may transduce cell death signals in plant species other than soybean. Conclusions: Our in silico analyses, along with current and previous functional data, permitted generation of a comprehensive overview of the ER stress response in soybean as a framework for functional prediction of ER stress signaling components and their possible connections with multiple stress responses. [ABSTRACT FROM AUTHOR]

Background: Protein functional diversity at the post-transcriptional level is regulated through spliceosome mediated pre-mRNA alternative splicing (AS) events and that has been widely demonstrated to be a key player in regulating the functional diversity in plants. Identification and analysis of AS genes in cereal crop plants are critical for crop improvement and understanding regulatory mechanisms. Results: We carried out the comparative analyses of the functional landscapes of the AS using the consensus assembly of expressed sequence tags and available mRNA sequences in four cereal plants. We identified a total of 8,734 in Oryza sativa subspecies (ssp) japonica, 2,657 in O. sativa ssp indica, 3,971 in Sorghum bicolor, and 10,687 in Zea mays AS genes. Among the identified AS events, intron retention remains to be the dominant type accounting for 23.5 % in S. bicolor, and up to 55.8 % in O. sativa ssp indica. We identified a total of 887 AS genes that were conserved among Z. mays, S. bicolor, and O. sativa ssp japonica; and 248 AS genes were found to be conserved among all four studied species or ssp. Furthermore, we identified 53 AS genes conserved with Brachypodium distachyon. Gene Ontology classification of AS genes revealed functional assignment of these genes in many biological processes with diverse molecular functions. Conclusions: AS is common in cereal plants. The AS genes identified in four cereal crops in this work provide the foundation for further studying the roles of AS in regulation of cereal plant growth and development. The data can be accessed at Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/). [ABSTRACT FROM AUTHOR]

Background: Crop improvement always involves selection of specific alleles at genes controlling traits of agronomic importance, likely resulting in detectable signatures of selection within the genome of modern soybean (Glycine max L. Merr.). The identification of these signatures of selection is meaningful from the perspective of evolutionary biology and for uncovering the genetic architecture of agronomic traits. Results: To this end, two populations of soybean, consisting of 342 landraces and 1062 improved lines, were genotyped with the SoySNP50K Illumina BeadChip containing 52,041 single nucleotide polymorphisms (SNPs), and systematically phenotyped for 9 agronomic traits. A cross-population composite likelihood ratio (XP-CLR) method was used to screen the signals of selective sweeps. A total of 125 candidate selection regions were identified, many of which harbored genes potentially involved in crop improvement. To further investigate whether these candidate regions were in fact enriched for genes affected by selection, genome-wide association studies (GWAS) were conducted on 7 selection traits targeted in soybean breeding (grain yield, plant height, lodging, maturity date, seed coat color, seed protein and oil content) and 2 non-selection traits (pubescence and flower color). Major genomic regions associated with selection traits overlapped with candidate selection regions, whereas no overlap of this kind occurred for the non-selection traits, suggesting that the selection sweeps identified are associated with traits of agronomic importance. Multiple novel loci and refined map locations of known loci related to these traits were also identified. Conclusions: These findings illustrate that comparative genomic analyses, especially when combined with GWAS, are a promising approach to dissect the genetic architecture of complex traits. [ABSTRACT FROM AUTHOR]

Background: Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression. Results: In total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains. Conclusion: The study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases. [ABSTRACT FROM AUTHOR]

Background: Since drought can seriously affect plant growth and development and little is known about how the oscillations of gene expression during the drought stress-acclimation response in soybean is affected, we applied Illumina technology to sequence 36 cDNA libraries synthesized from control and drought-stressed soybean plants to verify the dynamic changes in gene expression during a 24-h time course. Cycling variables were measured from the expression data to determine the putative circadian rhythm regulation of gene expression. Results: We identified 4866 genes differentially expressed in soybean plants in response to water deficit. Of these genes, 3715 were differentially expressed during the light period, from which approximately 9.55% were observed in both light and darkness. We found 887 genes that were either up- or down-regulated in different periods of the day. Of 54,175 predicted soybean genes, 35.52% exhibited expression oscillations in a 24 h period. This number increased to 39.23% when plants were submitted to water deficit. Major differences in gene expression were observed in the control plants from late day (ZT16) until predawn (ZT20) periods, indicating that gene expression oscillates during the course of 24 h in normal development. Under water deficit, dissimilarity increased in all time-periods, indicating that the applied stress influenced gene expression. Such differences in plants under stress were primarily observed in ZT0 (early morning) to ZT8 (late day) and also from ZT4 to ZT12. Stress-related pathways were triggered in response to water deficit primarily during midday, when more genes were up-regulated compared to early morning. Additionally, genes known to be involved in secondary metabolism and hormone signaling were also expressed in the dark period. Conclusions: Gene expression networks can be dynamically shaped to acclimate plant metabolism under environmental stressful conditions. We have identified putative cycling genes that are expressed in soybean leaves under normal developmental conditions and genes whose expression oscillates under conditions of water deficit. These results suggest that time of day, as well as light and temperature oscillations that occur considerably affect the regulation of water deficit stress response in soybean plants. [ABSTRACT FROM AUTHOR]

Background: SWEET (MtN3_saliva) domain proteins, a recently identified group of efflux transporters, play an indispensable role in sugar efflux, phloem loading, plant-pathogen interaction and reproductive tissue development. The SWEET gene family is predominantly studied in Arabidopsis and members of the family are being investigated in rice. To date, no transcriptome or genomics analysis of soybean SWEET genes has been reported. Results: In the present investigation, we explored the evolutionary aspect of the SWEET gene family in diverse plant species including primitive single cell algae to angiosperms with a major emphasis on Glycine max. Evolutionary features showed expansion and duplication of the SWEET gene family in land plants. Homology searches with BLAST tools and Hidden Markov Model-directed sequence alignments identified 52 SWEET genes that were mapped to 15 chromosomes in the soybean genome as tandem duplication events. Soybean SWEET (GmSWEET) genes showed a wide range of expression profiles in different tissues and developmental stages. Analysis of public transcriptome data and expression profiling using quantitative real time PCR (qRT-PCR) showed that a majority of the GmSWEET genes were confined to reproductive tissue development. Several natural genetic variants (non-synonymous SNPs, premature stop codons and haplotype) were identified in the GmSWEET genes using whole genome re-sequencing data analysis of 106 soybean genotypes. A significant association was observed between SNP-haplogroup and seed sucrose content in three gene clusters on chromosome 6. Conclusion: Present investigation utilized comparative genomics, transcriptome profiling and whole genome re-sequencing approaches and provided a systematic description of soybean SWEET genes and identified putative candidates with probable roles in the reproductive tissue development. Gene expression profiling at different developmental stages and genomic variation data will aid as an important resource for the soybean research community and can be extremely valuable for understanding sink unloading and enhancing carbohydrate delivery to developing seeds for improving yield. [ABSTRACT FROM AUTHOR]

Background: Single-stranded non-protein coding small RNAs, 18-25 nucleotides in length, are ubiquitous throughout plants genomes and are involved in post-transcriptional gene regulation. Several types of DNA markers have been reported for the detection of genetic diversity or sequence variation in soybean, one of the most important legume crops in worldwide for seed protein and oil content. Recently, with the available of public genomic databases, there has been a shift from the labor-intensive development of PCR-based markers to sequence-based genotyping and the development of functional markers within genes, often coupled with the use of RNA information. But thus far miRNA-based markers have been only developed in rice and tobacco. Here we report the first functional molecular miRNA marker, miR1511-InDel, in soybean for a specific single copy locus used to assess genetic variation in domesticated soybean (Glycine max [L.] Merr) and its wild progenitor (Glycine soja Sieb. & Zucc.). Results: We genotyped a total of 1,669 accessions of domesticated soybean (G. max) and its wild progenitor G. soja which are native throughout the China and parts of Korea, Japan and Russia. The results indicate that the miR1511 locus is distributed in cultivated soybean and has three alleles in annual wild soybean. Based on this result, we proposed that miR-InDel marker technology can be used to assess genetic variation. The inclusion of geo-reference data with miR1511-InDel marker data corroborated that accessions from the Yellow River basin (Huanghuai) exhibited high genetic diversity which provides more molecular evidence for gene diversity in annual wild soybean and domestication of soybean. Conclusions: These results provide evidence for the use of RNA marker, miRNA1511-InDel, as a soybean-specific functional maker for the study of genetic diversity, genotyping of germplasm and evolution studies. This is also the first report of functional marker developed from soybean miRNA located within the functional region of pre-miRNA1511. [ABSTRACT FROM AUTHOR]

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 Dti 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. [ABSTRACT FROM AUTHOR]

Background: Soybean cyst nematode (SCN) is the most economically devastating pathogen of soybean. Two resistance loci, Rhg1 and Rhg4 primarily contribute resistance to SCN race 3 in soybean. Peking and PI 88788 are the two major sources of SCN resistance with Peking requiring both Rhg1 and Rhg4 alleles and PI 88788 only the Rhg1 allele. Although simple sequence repeat (SSR) markers have been reported for both loci, they are linked markers and limited to be applied in breeding programs due to accuracy, throughput and cost of detection methods. The objectives of this study were to develop robust functional marker assays for high-throughput selection of SCN resistance and to differentiate the sources of resistance. Results: Based on the genomic DNA sequences of 27 soybean lines with known SCN phenotypes, we have developed Kompetitive Allele Specific PCR (KASP) assays for two Single nucleotide polymorphisms (SNPs) from Glyma08g11490 for the selection of the Rhg4 resistance allele. Moreover, the genomic DNA of Glyma18g02590 at the Rhg1 locus from 11 soybean lines and cDNA of Forrest, Essex, Williams 82 and PI 88788 were fully sequenced. Pairwise sequence alignment revealed seven SNPs/insertion/deletions (InDels), five in the 6th exon and two in the last exon. Using the same 27 soybean lines, we identified one SNP that can be used to select the Rhg1 resistance allele and another SNP that can be employed to differentiate Peking and PI 88788-type resistance. These SNP markers have been validated and a strong correlation was observed between the SNP genotypes and reactions to SCN race 3 using a panel of 153 soybean lines, as well as a bi-parental population, F5--derived recombinant inbred lines (RILs) from G00-3213 x LG04-6000. Conclusions: Three functional SNP markers (two for Rhg1 locus and one for Rhg4 locus) were identified that could provide genotype information for the selection of SCN resistance and differentiate Peking from PI 88788 source for most germplasm lines. The robust KASP SNP marker assays were developed. In most contexts, use of one or two of these markers is sufficient for high-throughput marker-assisted selection of plants that will exhibit SCN resistance. [ABSTRACT FROM AUTHOR]

Background: Root system architecture is important for water acquisition and nutrient acquisition for all crops. In soybean breeding programs, wild soybean alleles have been used successfully to enhance yield and seed composition traits, but have never been investigated to improve root system architecture. Therefore, in this study, high-density single-feature polymorphic markers and simple sequence repeats were used to map quantitative trait loci (QTLs) governing root system architecture in an inter-specific soybean mapping population developed from a cross between Glycine max and Glycine soja. Results: Wild and cultivated soybean both contributed alleles towards significant additive large effect QTLs on chromosome 6 and 7 for a longer total root length and root distribution, respectively. Epistatic effect QTLs were also identified for taproot length, average diameter, and root distribution. These root traits will influence the water and nutrient uptake in soybean. Two cell division-related genes (D type cyclin and auxin efflux carrier protein) with insertion/ deletion variations might contribute to the shorter root phenotypes observed in G. soja compared with cultivated soybean. Based on the location of the QTLs and sequence information from a second G. soja accession, three genes (slow anion channel associated 1 like, Auxin responsive NEDD8-activating complex and peroxidase), each with a nonsynonymous single nucleotide polymorphism mutation were identified, which may also contribute to changes in root architecture in the cultivated soybean. In addition, Apoptosis inhibitor 5-like on chromosome 7 and slow anion channel associated 1-like on chromosome 15 had epistatic interactions for taproot length QTLs in soybean. Conclusion: Rare alleles from a G. soja accession are expected to enhance our understanding of the genetic components involved in root architecture traits, and could be combined to improve root system and drought adaptation in soybean. [ABSTRACT FROM AUTHOR]

Background: Quantitative trait locus (QTL) mapping is an efficient approach to discover the genetic architecture underlying complex quantitative traits. However, the low density of molecular markers in genetic maps has limited the efficiency and accuracy of QTL mapping. In this study, specific length amplified fragment sequencing (SLAF-seq), a new high-throughput strategy for large-scale SNP discovery and genotyping based on next generation sequencing (NGS), was employed to construct a high-density soybean genetic map using recombinant inbred lines (RILs, Luheidou2 × Nanhuizao, F5:8). With this map, the consistent QTLs for isoflavone content across various environments were identified. Results: In total, 23 Gb of data containing 87,604,858 pair-end reads were obtained. The average coverage for each SLAF marker was 11.20-fold for the female parent, 12.51-fold for the male parent, and an average of 3.98-fold for individual RILs. Among the 116,216 high-quality SLAFs obtained, 9,948 were polymorphic. The final map consisted of 5,785 SLAFs on 20 linkage groups (LGs) and spanned 2,255.18 cM in genome size with an average distance of 0.43 cM between adjacent markers. Comparative genomic analysis revealed a relatively high collinearity of 20 LGs with the soybean reference genome. Based on this map, 41 QTLs were identified that contributed to the isoflavone content. The high efficiency and accuracy of this map were evidenced by the discovery of genes encoding isoflavone biosynthetic enzymes within these loci. Moreover, 11 of these 41 QTLs (including six novel loci) were associated with isoflavone content across multiple environments. One of them, qIF20-2, contributed to a majority of isoflavone components across various environments and explained a high amount of phenotypic variance (8.7% - 35.3%). This represents a novel major QTL underlying isoflavone content across various environments in soybean. Conclusions: Herein, we reported a high-density genetic map for soybean. This map exhibited high resolution and accuracy. It will facilitate the identification of genes and QTLs underlying essential agronomic traits in soybean. The novel major QTL for isoflavone content is useful not only for further study on the genetic basis of isoflavone accumulation, but also for marker-assisted selection (MAS) in soybean breeding in the future. [ABSTRACT FROM AUTHOR]

Background Soybean is one of the most economically important crops in the world. The cotyledon is the nutrient storage area in seeds, and it is critical for seed quality and yield. Cotyledon mutants are important for the genetic dissection of embryo patterning and seed development. However, the molecular mechanisms underlying soybean cotyledon development are largely unexplored. Results In this study, we characterised a soybean curled-cotyledon (cco) mutant. Compared with wild-type (WT), anatomical analysis revealed that the cco cotyledons at the torpedo stage became more slender and grew outward. The entire embryos of cco mutant resembled the "tail of swallow". In addition, cco seeds displayed reduced germination rate and gibberellic acid (GA3) level, whereas the abscisic acid (ABA) and auxin (IAA) levels were increased. RNA-seq identified 1,093 differentially expressed genes (DEGs) between WT and the cco mutant. The KEGG pathway analysis showed many DEGs were mapped to the hormone biosynthesis and signal transduction pathways. Consistent with assays of hormones in seeds, the results of RNA-seq indicated auxin and ABA biosynthesis and signal transduction in cco were more active than in WT, while an early step in GA biosynthesis was blocked, as well as conversion rate of inactive GAs to bioactive GAs in GA signaling. Furthermore, genes participated in other hormone biosynthesis and signalling pathways such as cytokinin (CK), ethylene (ET), brassinosteroid (BR), and jasmonate acid (JA) were also affected in the cco mutant. Conclusions Our data suggest that multiple phytohormone biosynthesis and signal transduction pathways are reprogrammed in cco, and changes in these pathways may partially contribute to the cco mutant phenotype, suggesting the involvement of multiple hormones in the coordination of soybean cotyledon development. [ABSTRACT FROM AUTHOR]

Background MicroRNAs (miRNAs) regulate various biological processes in plants. Considerable data are available on miRNAs involved in the development of rice, maize and barley. In contrast, little is known about miRNAs and their functions in the development of wheat. In this study, five small RNA (sRNA) libraries from wheat seedlings, flag leaves, and developing seeds were developed and sequenced to identify miRNAs and understand their functions in wheat development. Results Twenty-four known miRNAs belonging to 15 miRNA families were identified from 18 MIRNA loci in wheat in the present study, including 15 miRNAs (9 MIRNA loci) first identified in wheat, 13 miRNA families (16 MIRNA loci) being highly conserved and 2 (2 MIRNA loci) moderately conserved. In addition, fifty-five novel miRNAs were also identified. The potential target genes for 15 known miRNAs and 37 novel miRNAs were predicted using strict criteria, and these target genes are involved in a wide range of biological functions. Four of the 15 known miRNA families and 22 of the 55 novel miRNAs were preferentially expressed in the developing seeds with logarithm (log2) of the fold change of 1.0 ~ 7.6, and half of them were seed-specific, suggesting that they participate in regulating wheat seed development and metabolism. From 5 days post-anthesis to 20 days post-anthesis, miR164 and miR160 increased in abundance in the developing seeds, whereas miR169 decreased, suggesting their coordinating functions in the different developmental stages of wheat seed. Moreover, 8 known miRNA families and 28 novel miRNAs exhibited tissue-biased expression in wheat flag leaves, with the logarithm of the fold changes of 0.1 ~ 5.2. The putative targets of these tissue-preferential miRNAs were involved in various metabolism and biological processes, suggesting complexity of the regulatory networks in different tissues. Our data also suggested that wheat flag leaves have more complicated regulatory networks of miRNAs than developing seeds. Conclusions Our work identified and characterised wheat miRNAs, their targets and expression patterns. This study is the first to elucidate the regulatory networks of miRNAs involved in wheat flag leaves and developing seeds, and provided a foundation for future studies on specific functions of these miRNAs. [ABSTRACT FROM AUTHOR]

Background Brassica juncea is an economically important vegetable crop in China, oil crop in India, condiment crop in Europe and selected for canola quality recently in Canada and Australia. B. juncea (2n = 36, AABB) is an allotetraploid derived from interspecific hybridization between B. rapa (2n = 20, AA) and B. nigra (2n = 16, BB), followed by spontaneous chromosome doubling.Results Comparative genome analysis by genome survey sequence (GSS) of allopolyploid B. juncea with B. rapa was carried out based on high-throughput sequencing approaches. Over 28.35 Gb of GSS data were used for comparative analysis of B. juncea and B. rapa, producing 45.93 % reads mapping to the B. rapa genome with a high ratio of single-end reads. Mapping data suggested more structure variation (SV) in the B. juncea genome than in B. rapa. We detected 2,921,310 single nucleotide polymorphisms (SNPs) with high heterozygosity and 113,368 SVs, including 1-3 bp Indels, between B. juncea and B. rapa. Non-synonymous polymorphisms in glucosinolate biosynthesis genes may account for differences in glucosinolate biosynthesis and glucosinolate components between B. juncea and B. rapa. Furthermore, we identified distinctive vernalization-dependent and photoperiod-dependent flowering pathways coexisting in allopolyploid B. juncea, suggesting contribution of these pathways to adaptation for survival during polyploidization. Conclusions Taken together, we proposed that polyploidization has allowed for accelerated evolution of the glucosinolate biosynthesis and flowering pathways in B. juncea that likely permit the phenotypic variation observed in the crop. [ABSTRACT FROM AUTHOR]

Background Foxtail millet (Setaria italica (L.) Beauv.) is an important gramineous grain-food and forage crop. It is grown worldwide for human and livestock consumption. Its small genome and diploid nature have led to foxtail millet fast becoming a novel model for investigating plant architecture, drought tolerance and C4 photosynthesis of grain and bioenergy crops. Therefore, cost-effective, reliable and highly polymorphic molecular markers covering the entire genome are required for diversity, mapping and functional genomics studies in this model species. Result A total of 5,020 highly repetitive microsatellite motifs were isolated from the released genome of the genotype 'Yugu1' by sequence scanning. Based on sequence comparison between S. italica and S. viridis, a set of 788 SSR primer pairs were designed. Of these primers, 733 produced reproducible amplicons and were polymorphic among 28 Setaria genotypes selected from diverse geographical locations. The number of alleles detected by these SSR markers ranged from 2 to 16, with an average polymorphism information content of 0.67. The result obtained by neighbor-joining cluster analysis of 28 Setaria genotypes, based on Nei's genetic distance of the SSR data, showed that these SSR markers are highly polymorphic and effective. Conclusions A large set of highly polymorphic SSR markers were successfully and efficiently developed based on genomic sequence comparison between different genotypes of the genus Setaria. The large number of new SSR markers and their placement on the physical map represent a valuable resource for studying diversity, constructing genetic maps, functional gene mapping, QTL exploration and molecular breeding in foxtail millet and its closely related species. [ABSTRACT FROM AUTHOR]

Background Phytophthora root and stem rot (PRR) of soybean, caused by Phytophthora sojae, is controlled by Rps genes. However, little is known regarding the Rps-induced molecular responses to P. sojae and how they actually overlap. We thus sequenced, analyzed, and compared the transcriptomes of 10 near isogenic lines (NILs), each with a unique Rps gene/allele, and the susceptible parent Williams, pre- and post-inoculation with the pathogen. Results A total of 4,330 differentially expressed genes (DEGs) were identified in Williams versus 2,014 to 5,499 DEGs in individual NILs upon inoculation with the pathogen. Comparisons of the DEGs between the NILs and Williams identified incompatible interaction genes (IIGs) and compatible interaction genes (CIGs). Hierarchical cluster and heatmap analyses consistently grouped the NILs into three clusters: Cluster I (Rps1-a), Cluster II (Rps1-b, 1-c and 1-k) and Cluster III (Rps3-a, 3-b, 3-c, 4, 5, and 6), suggesting an overlap in Rps-induced defense signaling among certain NILs. Gene ontology (GO) analysis revealed associations between members of the WRKY family and incompatible reactions and between a number of phytohormone signaling pathways and incompatible/compatible interactions. These associations appear to be distinguished according to the NIL clusters. Conclusions This study characterized genes and multiple branches of putative regulatory networks associated with resistance to P. sojae in ten soybean NILs, and depicted functional "fingerprints" of individual Rps-mediated resistance responses through comparative transcriptomic analysis. Of particular interest are dramatic variations of detected DEGs, putatively involved in ethylene (ET)-, jasmonic acid (JA)-, (reactive oxygen species) ROS-, and (MAP-kinase) MAPK- signaling, among these soybean NILs, implicating their important roles of these signaling in differentiating molecular defense responses. We hypothesize that different timing and robustness in defense signaling to the same pathogen may be largely responsible for such variations. [ABSTRACT FROM AUTHOR]

Background: The presence of homoeologous sequences and absence of a reference genome sequence make discovery and genotyping of single nucleotide polymorphisms (SNPs) more challenging in polyploid crops. Results: To address this challenge, we constructed reduced representation libraries (RRLs) for two Brassica napus inbred lines and their 91 doubled haploid (DH) progenies using a modified ddRADseq technique. A bioinformatics pipeline termed RFAPtools was developed to discover and genotype SNPs and presence/absence variations (PAVs). Using this pipeline, a pseudo-reference sequence (PRF) containing 180,991 sequence tags was constructed. By aligning sequence reads to the pseudo-reference sequence, allelic SNPs as well as PAVs were identified and genotyped with RFAPtools. Two parallel linkage maps, one SNP bin map containing 8,780 SNP loci and one PAV linkage map containing 12,423 dominant loci, were constructed. By aligning marker sequences to B. rapa sequence scaffolds, whose genome is available, we assigned 44 unassembled sequence scaffolds comprising 8.15 Mb onto the B. rapa chromosomes, and also identified 14 instances of misassembly and eight instances of mis-ordering sequence scaffolds. Conclusions: These results indicate that the modified ddRADseq approach is a cost-effective and simple method to genotype tens of thousands SNPs and PAV markers in a polyploidy plant species. The results also demonstrated that RFAPtools developed in this study are powerful to mine allelic SNPs from homoeologous sequences in polyploids, therefore they are generally applicable in either diploid or polyploid species with or without a reference genome sequence. [ABSTRACT FROM AUTHOR]

Background: Natural antisense transcripts (NATs) are a class of RNAs that contain a sequence complementary to other transcripts. NATs occur widely in eukaryotes and play critical roles in post-transcriptional regulation. Soybean NAT sequences are predicted in the PlantNATsDB, but detailed analyses of these NATs remain to be performed. Results: A total of 26,216 NATs, including 994 cis-NATs and 25,222 trans-NATs, were predicted in soybean. Each sense transcript had 1-177 antisense transcripts. We identified 21 trans-NATs using RT-PCR amplification. Additionally, we identified 179 cis-NATs and 6,629 trans-NATs that gave rise to small RNAs; these were enriched in the NAT overlapping region. The most abundant small RNAs were 21, 22, and 24 nt in length. The generation of small RNAs was biased to one stand of the NATs, and the degradation of NATs was biased. High-throughput sequencing of the degradome allowed for the global identification of NAT small interfering RNAs (nat-siRNAs) targets. 446 target genes for 165 of these nat-siRNAs were identified. The nat-siRNA target could be one transcript of a given NAT, or from other gene transcripts. We identified five NAT transcripts containing a hairpin structure that is characteristic of pre-miRNA. We identified a total of 86 microRNA (miRNA) targets that had antisense transcripts in soybean. Conclusions: We globally identified nat-siRNAs, and the targets of nat-siRNAs in soybean. It is likely that the cis-NATs, trans-NATs, nat-siRNAs, miRNAs, and miRNA targets form complex regulatory networks. [ABSTRACT FROM AUTHOR]

Background: Biological nitrogen fixation in root nodules is a process of great importance to crops of soybean [Glycine max (L.) Merr.], as it may provide the bulk of the plant's needs for nitrogen. Legume nodulation involves several complex steps and, although studied for many decades, much remains to be understood. Results: This research aimed at analyzing the global expression of genes in soybean roots of a Brazilian cultivar (Conquista) inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. To achieve this, we used the suppressive subtractive hybridization (SSH) technique combined with Illumina sequencing. The subtractive library (non-inoculated x inoculated) of soybean roots resulted in 3,210 differentially expressed transcripts at 10 days after inoculation were studied. The data were grouped according to the ontologies of the molecular functions and biological processes. Several classes of genes were confirmed as related to N2 fixation and others were reported for the first time. Conclusions: During nodule formation, a higher percentage of genes were related to primary metabolism, cell-wall modifications and the antioxidant defense system. Putative symbiotic functions were attributed to some of these genes for the first time. [ABSTRACT FROM AUTHOR]

Background: The Osa1 Genome Annotation of rice (Oryza sativa L. ssp. japonica cv. Nipponbare) is the product of a semi-automated pipeline that does not explicitly predict pseudogenes. As such, it is likely to mis-annotate pseudogenes as functional genes. A total of 22,033 gene models within the Osa1 Release 5 were investigated as potential pseudogenes as these genes exhibit at least one feature potentially indicative of pseudogenes: lack of transcript support, short coding region, long untranslated region, or, for genes residing within a segmentally duplicated region, lack of a paralog or significantly shorter corresponding paralog. Results: A total of 1,439 pseudogenes, identified among genes with pseudogene features, were characterized by similarity to fully-supported gene models and the presence of frameshifts or premature translational stop codons. Significant difference in the length of duplicated genes within segmentally-duplicated regions was the optimal indicator of pseudogenization. Among the 816 pseudogenes for which a probable origin could be determined, 75% originated from gene duplication events while 25% were the result of retrotransposition events. A total of 12% of the pseudogenes were expressed. Finally, F-box proteins, BTB/POZ proteins, terpene synthases, chalcone synthases and cytochrome P450 protein families were found to harbor large numbers of pseudogenes. Conclusion: These pseudogenes still have a detectable open reading frame and are thus distinct from pseudogenes detected within intergenic regions which typically lack definable open reading frames. Families containing the highest number of pseudogenes are fast-evolving families involved in ubiquitination and secondary metabolism. [ABSTRACT FROM AUTHOR]

Background: Sulforaphane is a natural isothiocyanate available from cruciferous vegetables with multiple characteristics including antioxidant, antitumor and anti-inflammatory effect. Single-molecule real-time (SMRT) sequencing has been used for long-read de novo assembly of plant genome. Here, we investigated the molecular mechanism related to glucosinolates biosynthesis in Chinese kale using combined NGS and SMRT sequencing. Results: SMRT sequencing produced 185,134 unigenes, higher than 129,325 in next-generation sequencing (NGS). NaCl (75 mM), methyl jasmonate (MeJA, 40 μM), selenate (Se, sodium selenite 100 μM), and brassinolide (BR, 1.5 μM) treatment induced 6893, 13,287, 13,659 and 11,041 differentially expressed genes (DEGs) in Chinese kale seedlings comparing with control. These genes were associated with pathways of glucosinolates biosynthesis, including phenylalanine, tyrosine and tryptophan biosynthesis, cysteine and methionine metabolism, and glucosinolate biosynthesis. We found NaCl decreased sulforaphane and glucosinolates (indolic and aliphatic) contents and downregulated expression of cytochrome P45083b1 (CYP83b1), S-alkyl-thiohydroximatelyase or carbon–sulfur lyase (SUR1) and UDP-glycosyltransferase 74B1 (UGT74b1). MeJA increased sulforaphane and glucosinolates contents and upregulated the expression of CYP83b1, SUR1 and UGT74b1; Se increased sulforaphane; BR increased expression of CYP83b1, SUR1 and UGT74b1, and increased glucosinolates contents. The desulfoglucosinolate sulfotransferases ST5abc were decreased by all treatments. Conclusions: We confirmed that NaCl inhibited the biosynthesis of both indolic and aliphatic glucosinolates, while MeJA and BR increased them. MeJA and BR treatments, conferred the biosynthesis of glucosinolates, and Se and MeJA contributed to sulforaphane in Chinese kale via regulating the expression of CYP83b1, SUR1 and UGT74b1. [ABSTRACT FROM AUTHOR]

Background: To continue to meet the increasing demands of soybean worldwide, it is crucial to identify key genes regulating flowering and maturity to expand the cultivated regions into short season areas. Although four soybean genes have been successfully utilized in early maturity breeding programs, new genes governing maturity are continuously being identified suggesting that there remains as yet undiscovered loci governing agronomic traits of interest. The objective of this study was to identify novel loci and genes involved in a diverse set of early soybean maturity using genome-wide association (GWA) analyses to identify loci governing days to maturity (DTM), flowering (DTF) and pod filling (DTPF), as well as yield and 100 seed weight in Canadian environments. To do so, soybean plant introduction lines varying significantly for maturity, but classified as early varieties, were used. Plants were phenotyped for the five agronomic traits for five site-years and GWA approaches used to identify candidate loci and genes affecting each trait. Results: Genotyping using genotyping-by-sequencing and microarray methods identified 67,594 single nucleotide polymorphisms, of which 31,283 had a linkage disequilibrium < 1 and minor allele frequency > 0.05 and were used for GWA analyses. A total of 9, 6, 4, 5 and 2 loci were detected for GWA analyses for DTM, DTF, DTPF, 100 seed weight and yield, respectively. Regions of interest, including a region surrounding the E1 gene for flowering and maturity, and several novel loci, were identified, with several loci having pleiotropic effects. Novel loci affecting maturity were identified on chromosomes five and 13 and reduced maturity by 7.2 and 3.3 days, respectively. Novel loci for maturity and flowering contained genes orthologous to known Arabidopsis flowering genes, while loci affecting yield and 100 seed weight contained genes known to cause dwarfism. Conclusions: This study demonstrated substantial variation in soybean agronomic traits of interest, including maturity and flowering dates as well as yield, and the utility of GWA analyses in identifying novel genetic factors underlying important agronomic traits. The loci and candidate genes identified serve as promising targets for future studies examining the mechanisms underlying the related soybean traits. [ABSTRACT FROM AUTHOR]

Background: Soybean, a major legume crop native to East Asia, presents a wealth of resources for utilization. The basic leucine zipper (bZIP) transcription factors play important roles in various biological processes including developmental regulation and responses to environmental stress stimuli. Currently, little information is available regarding the bZIP family in the legume crop soybean. Results: Using a genome-wide domain analysis, we identified 160 GmbZIP genes in soybean genome, named from GmbZIP1 to GmbZIP160. These 160GmbZIP genes, distributed unevenly across 20 chromosomes, were grouped into 12 subfamilies based on phylogenetic analysis. Gene structure and conserved motif analyses showed that GmbZIP within the same subfamily shared similar intron-exon organizations and motif composition. Syntenic and phylogenetic analyses identified 40 Arabidopsis bZIP genes and 83 soybean bZIP genes as orthologs. By investigating the expression profiling of GmbZIP in different tissues and under drought and flooding stresses, we showed that a majority of GmbZIP (83.44%) exhibited transcript abundance in all examined tissues and 75.6% displayed transcript changes after drought and flooding treatment, suggesting that GmbZIP may play a broad role in soybean development and response to water stress. Conclusions: One hundred sixty GmbZIP genes were identified in soybean genome. Our results provide insights for the evolutionary history of bZIP family in soybean and shed light on future studies on the function of bZIP genes in response to water stress in soybean. [ABSTRACT FROM AUTHOR]