Development, validation and genetic analysis of a large soybean SNP genotyping array.

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under-use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high-density genetic mapping and genome-wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome-wide association analyses. More than four million high-quality SNPs identified from high-depth genome re-sequencing of 16 soybean accessions and low-depth genome re-sequencing of 31 soybean accessions were used to select 180,961 SNPs for creation of the Axiom(®) SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170,223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene-rich chromosomal regions suggest that this array may be suitable for genome-wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.

Complete genome sequence of a novel endornavirus isolated from hot pepper.

The complete genome of a putative new endornavirus infecting hot peppers (Capsicum annuum) was determined to be 14,729 nt in size, including 12 cytosines at the 3' end. The hot pepper-infecting virus has the highest nucleotide sequence similarity (94% query cover and 72% identity) to bell pepper endornavirus (BPEV) isolated from the cultivar Yolo Wonder in the USA (GenBank accession no. JN019858). The putative single, large open reading frame encodes a 4,884-amino-acid-long polyprotein that contains four putative functional domains: a viral methyltransferase, a viral RNA helicase, a glycosyltransferase, and an RNA-dependent RNA polymerase. A phylogenetic tree based on whole polyprotein sequences confirmed the close evolutionary relationship of the studied endornavirus to BPEV. The hot pepper-infecting virus also has a nick at nt position 975. Taken together, these results suggest that this virus belongs to a new species in the genus Endornavirus (family Endornaviridae), for which the name hot pepper endornavirus (HPEV) is proposed.

Genome sequence of a recombinant brassica yellows virus infecting Chinese cabbage.

RNA from a Chinese cabbage plant (Brassica campestris ssp. pekinensis) showing leaf malformation and mottling was labeled and hybridized to a DNA chip capable of detecting plant viruses and viroids. Probes specific for beet mild yellowing virus (BMYV) and beet western yellows virus (BWYV) yielded positive results, suggesting that the plant was infected by a polerovirus. Primers designed from the sequences of the positive probes were used to amplify and sequence one portion of the viral genome. This sequence showed a 90 % or greater identity to several poleroviruses, including BMYV, BWYV, beet chlorosis virus (BChV) and turnip yellows virus (TuYV). The complete genome sequence of the Chinese cabbage-infecting polerovirus consisted of 5,666 nt and was most closely related to brassica yellows virus (BrYV; 94 % identity). The virus was named BrYV-Cheongsong (BrYV-CS). However, ORF3, ORF4 and the 5' half of ORF5 of BrYV-CS were more closely related to those of TuYV, BWYV, BChV and BMYV than to those of BrYV. Interestingly, a recombination event (positions 3531-4819 in BrYV-CS) was detected when this sequence was aligned with those of BrYV and TuYV. This region showed the highest sequence identity to that of TuYV (94 % identity) and had greater than 93 % identity to those of BWYV, BChV and BMYV, but it shared only 81 % identity with that of BrYV. Taken together, the genomes of BrYV-CS and BrYV are closely related. However, the structural genes in the 3' half of the genome of BrYV-CS are more closely related to those of other poleroviruses.

Whole-genome sequencing and intensive analysis of the undomesticated soybean (Glycine soja Sieb. and Zucc.) genome.

The genome of soybean (Glycine max), a commercially important crop, has recently been sequenced and is one of six crop species to have been sequenced. Here we report the genome sequence of G. soja, the undomesticated ancestor of G. max (in particular, G. soja var. IT182932). The 48.8-Gb Illumina Genome Analyzer (Illumina-GA) short DNA reads were aligned to the G. max reference genome and a consensus was determined for G. soja. This consensus sequence spanned 915.4 Mb, representing a coverage of 97.65% of the G. max published genome sequence and an average mapping depth of 43-fold. The nucleotide sequence of the G. soja genome, which contains 2.5 Mb of substituted bases and 406 kb of small insertions/deletions relative to G. max, is ∼0.31% different from that of G. max. In addition to the mapped 915.4-Mb consensus sequence, 32.4 Mb of large deletions and 8.3 Mb of novel sequence contigs in the G. soja genome were also detected. Nucleotide variants of G. soja versus G. max confirmed by Roche Genome Sequencer FLX sequencing showed a 99.99% concordance in single-nucleotide polymorphism and a 98.82% agreement in insertion/deletion calls on Illumina-GA reads. Data presented in this study suggest that the G. soja/G. max complex may be at least 0.27 million y old, appearing before the relatively recent event of domestication (6,000∼9,000 y ago). This suggests that soybean domestication is complicated and that more in-depth study of population genetics is needed. In any case, genome comparison of domesticated and undomesticated forms of soybean can facilitate its improvement.

Genome-wide mapping of NBS-LRR genes and their association with disease resistance in soybean.

BACKGROUND: R genes are a key component of genetic interactions between plants and biotrophic bacteria and are known to regulate resistance against bacterial invasion. The most common R proteins contain a nucleotide-binding site and a leucine-rich repeat (NBS-LRR) domain. Some NBS-LRR genes in the soybean genome have also been reported to function in disease resistance. In this study, the number of NBS-LRR genes was found to correlate with the number of disease resistance quantitative trait loci (QTL) that flank these genes in each chromosome. NBS-LRR genes co-localized with disease resistance QTL. The study also addressed the functional redundancy of disease resistance on recently duplicated regions that harbor NBS-LRR genes and NBS-LRR gene expression in the bacterial leaf pustule (BLP)-induced soybean transcriptome. RESULTS: A total of 319 genes were determined to be putative NBS-LRR genes in the soybean genome. The number of NBS-LRR genes on each chromosome was highly correlated with the number of disease resistance QTL in the 2-Mb flanking regions of NBS-LRR genes. In addition, the recently duplicated regions contained duplicated NBS-LRR genes and duplicated disease resistance QTL, and possessed either an uneven or even number of NBS-LRR genes on each side. The significant difference in NBS-LRR gene expression between a resistant near-isogenic line (NIL) and a susceptible NIL after inoculation of Xanthomonas axonopodis pv. glycines supports the conjecture that NBS-LRR genes have disease resistance functions in the soybean genome. CONCLUSIONS: The number of NBS-LRR genes and disease resistance QTL in the 2-Mb flanking regions of each chromosome was significantly correlated, and several recently duplicated regions that contain NBS-LRR genes harbored disease resistance QTL for both sides. In addition, NBS-LRR gene expression was significantly different between the BLP-resistant NIL and the BLP-susceptible NIL in response to bacterial infection. From these observations, NBS-LRR genes are suggested to contribute to disease resistance in soybean. Moreover, we propose models for how NBS-LRR genes were duplicated, and apply Ks values for each NBS-LRR gene cluster.

Tracing soybean domestication history: From nucleotide to genome.

Since the genome sequences of wild species may provide key information about the genetic elements involved in speciation and domestication, the undomesticated soybean (Glycine soja Sieb. and Zucc.), a wild relative of the current cultivated soybean (G. max), was sequenced. In contrast to the current hypothesis of soybean domestication, which holds that the current cultivated soybean was domesticated from G. soja, our previous work has suggested that soybean was domesticated from the G. soja/G. max complex that diverged from a common ancestor of these two species of Glycine. In this review, many structural genomic differences between the two genomes are described and a total of 705 genes are identified as structural variations (SVs) between G. max and G. soja. After protein families database of alignments and hidden Markov models IDs and gene ontology terms were assigned, many interesting genes are discussed in detail using four domestication related traits, such as flowering time, transcriptional factors, carbon metabolism and disease resistance. Soybean domestication history is explored by studying these SVs in genes. Analysis of SVs in genes at the population-level may clarify the domestication history of soybean.

Fine mapping of a resistance gene to bacterial leaf pustule in soybean.

Soybean bacterial leaf pustule (BLP) is a prevalent disease caused by Xanthomonas axonopodis pv. glycines. Fine mapping of the BLP resistant gene, rxp, is needed to select BLP resistant soybean cultivars by marker-assisted selection (MAS). We used a total of 227 recombinant inbred lines (RILs) derived from a cross between 'Taekwangkong' (BLP susceptible) and 'Danbaekkong' (BLP resistant) for rxp fine mapping and two different sets of near isogenic lines (NILs) from Hwangkeumkong x SS2-2 and Taekwangkong x SS2-2 were used for confirmation. Using sequences between Satt372 and Satt486 flanking rxp from soybean genome sequences, eight simple sequence repeats (SSR) and two single nucleotide polymorphism (SNP) markers were newly developed in a 6.2-cM interval. Linkage mapping with the RILs and NILs allowed us to map the rxp region with high resolution. The genetic order of all markers was completely consistent with their physical order. QTL analysis by comparison of the BLP phenotyping data with all markers showed rxp was located between SNUSSR17_9 and SNUSNP17_12. Gene annotation analysis of the 33 kb region between SNUSSR17_9 and SNUSNP17_12 suggested three predicted genes, two of which could be candidate genes of BLP resistance: membrane protein and zinc finger protein. Candidate genes showed high similarity with their paralogous genes, which were located on the duplicated regions obtaining BLP resistance QTLs. High-resolution map in rxp region with eight SSR and two SNP markers will be useful for not only MAS of BLP resistance but also characterization of rxp.

[A case of primary hepatic epithelioid hemangioendothelioma with spontaneous rupture].

Primary hepatic epithelioid hemangioendothelioma is a rare neoplasm of endothelial origin. The clinical manifestations are nonspecific, ranging from complete absence of symptoms to hepatic failure and death. Spontaneous rupture of a hepatic epithelioid hemangioendothelioma is an extremely rare presentation. We present a case of primary hepatic epithelioid hemangioendothelioma in a 65-year-old male patient with alcoholic liver cirrhosis. He was hospitalized due to epigastric pain and multiple liver masses on abdominal ultrasound. Dynamic liver CT imaging revealed multiple peripheral nodular enhanced mass lesions with delayed centripetal enhancement, and the adjacent collection of high-attenuation fluid along the liver capsule. Abdominal tapping revealed blood in the peritoneal cavity. Primary hepatic epithelioid hemangioendothelioma with spontaneous rupture was finally diagnosed based on a histopathologic examination revealing positive immunohistochemical staining for CD34.

Identification of Key Genes for the Precise Classification between Solenopsis invicta and S. geminata Facilitating the Quarantine Process.

One of the 100 worst invasive exotic species, Solenopsis invicta (red imported fire ant), has the possibility to induce an allergic reaction that may eventually cause death from its aggressive stinging. In 2017, S. invicta was found at a container yard in Gamman Port, Busan, South Korea for the first time. It may result in an infestation of fire ants in the Korean environment. After this incident, sensitive quarantine procedures are required to detect possible contamination of fire ants in imported containers. However, currently, fire ant identification relies on phenotypic characteristics. This requires highly trained experts for identification and there are not enough to cover all imported containers. Here, we develop a key molecular marker to distinguish S. invicta from others using the whole genome sequence (WGS) of collected S. invicta from Gamman Port and NCBI-deposited WGS data of S.invicta and S. geminata. The consolidated genotypes of Solenopsis genus successfully indicate the distinguishable gene. The gel-based experimental validation confirmed expected classification and the developed cleaved amplified polymorphic sequences (CAPS) marker also gave a consistent result. Using the CAPS marker derived from our consolidated genotypes, the samples collected from containers in several ports can be easily tested by PCR in a few hours. The quick and easy test would increase not only the labor efficiency but also the environmental safety from fire ants.

The Hypernodulating nts mutation induces jasmonate synthetic pathway in soybean leaves.

Symbiotic nitrogen fixation with nitrogen-fixing bacteria in the root nodules is a distinctly beneficial metabolic process in legume plants. Legumes control the nodule number and nodulation zone through a systemic negative regulatory system between shoot and root. Mutation in the soybean NTS gene encoding GmNARK, a CLAVATA1-like serine/threonine receptor-like kinase, causes excessive nodule development called hypernodulation. To examine the effect of nts mutation on the gene expression profile in the leaves, suppression subtractive hybridization was performed with the trifoliate leaves of nts mutant 'SS2-2' and the wild-type (WT) parent Sinpaldalkong2, and 75 EST clones that were highly expressed in the leaves of the SS2-2 mutant were identified. Interestingly, the expression of jasmonate (JA)-responsive genes such as vspA, vspB, and Lox2 were upregulated, whereas that of a salicylate-responsive gene PR1a was suppressed in the SS2-2 mutant. In addition, the level of JA was about two-fold higher in the leaves of the SS2-2 mutant than in those of the WT under natural growth conditions. Moreover, the JA-responsive gene expression persists in the leaves of SS2-2 mutant without rhizobia infection in the roots. Taken together, our results suggest that the nts mutation increases JA synthesis in mature leaves and consequently leads to constitutive expression of JA-responsive genes which is irrelevant to hypernodulation in the root.

Development of a new vector using Soybean yellow common mosaic virus for gene function study or heterologous protein expression in soybeans.

A new vector using Soybean yellow common mosaic virus (SYCMV) was constructed for gene function study or heterologous protein expression in soybeans. The in vitro transcript with a 5' cap analog m7GpppG from an SYCMV full-length infectious vector driven by a T7 promoter infected soybeans (pSYCMVT7-full). The symptoms observed in the soybeans infected with either the sap from SYCMV-infected leaves or pSYCMVT7-full were indistinguishable, suggesting that the vector exhibits equivalent biological activity as the virus itself. To utilize the vector further, a DNA-based vector driven by the Cauliflower mosaic virus (CaMV) 35S promoter was constructed. The complete sequence of the SYCMV genome was inserted into a binary vector flanked by a CaMV 35S promoter at the 5' terminus of the SYCMV genome and a cis-cleaving ribozyme sequence followed by a nopaline synthase terminator at the 3' terminus of the SYCMV genome (pSYCMV-full). The SYCMV-derived vector was tested for use as a virus-induced gene silencing (VIGS) vector for the functional analysis of soybean genes. VIGS constructs containing either a fragment of the Phytoene desaturase (PDS) gene (pSYCMV-PDS1) or a fragment of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) gene (pSYCMV-RbcS2) were constructed. Plants infiltrated with each vector using the Agrobacterium-mediated inoculation method exhibited distinct symptoms, such as photo-bleaching in plants infiltrated with pSYCMV-PDS1 and yellow or pale green coloring in plants infiltrated with pSYCMV-RbcS2. In addition, down-regulation of the transcripts of the two target genes was confirmed via northern blot analysis. Particle bombardment and direct plasmid DNA rubbing were also confirmed as alternative inoculation methods. To determine if the SYCMV vector can be used for the expression of heterologous proteins in soybean plants, the vector encoding amino acids 135-160 of VP1 of Foot-and-mouth disease virus (FMDV) serotype O1 Campos (O1C) was constructed (pSYCMV-FMDV). Plants infiltrated with pSYCMV-FMDV were only detected via western blotting using the O1C antibody. Based on these results, we propose that the SYCMV-derived vector can be used for gene function study or expression of useful heterologous proteins in soybeans.

Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans.

Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of the soybean. Following SYCMV: Glycine max-phytoene desaturase (GmPDS) infiltration, we investigated the effect of photoperiod, inoculation time, concentration of Agrobacterium inoculm, and growth temperature on VIGS efficiency. In addition, the relative expression of GmPDS between non-silenced and silenced plants was measured by qRT-PCR. We found that gene silencing efficiency was highest at a photoperiod of 16/8 h (light/dark) at a growth temperature of approximately 27°C following syringe infiltration to unrolled unifoliolate leaves in cotyledon stage with a final SYCMV:GmPDS optimal density (OD)600 of 2.0. Using this optimized protocol, we achieved high efficiency of GmPDS-silencing in various soybean germplasms including cultivated and wild soybeans. We also confirmed that VIGS occurred in the entire plant, including the root, stem, leaves, and flowers, and could transmit GmPDS to other soybean germplasms via mechanical inoculation. This optimized protocol using a SYCMV-based VIGS system in the soybean should provide a fast and effective method to elucidate gene functions and for use in large-scale screening experiments.

Draft genome sequence of adzuki bean, Vigna angularis.

Adzuki bean (Vigna angularis var. angularis) is a dietary legume crop in East Asia. The presumed progenitor (Vigna angularis var. nipponensis) is widely found in East Asia, suggesting speciation and domestication in these temperate climate regions. Here, we report a draft genome sequence of adzuki bean. The genome assembly covers 75% of the estimated genome and was mapped to 11 pseudo-chromosomes. Gene prediction revealed 26,857 high confidence protein-coding genes evidenced by RNAseq of different tissues. Comparative gene expression analysis with V. radiata showed that the tissue specificity of orthologous genes was highly conserved. Additional re-sequencing of wild adzuki bean, V. angularis var. nipponensis, and V. nepalensis, was performed to analyze the variations between cultivated and wild adzuki bean. The determined divergence time of adzuki bean and the wild species predated archaeology-based domestication time. The present genome assembly will accelerate the genomics-assisted breeding of adzuki bean.

Genome sequence of mungbean and insights into evolution within Vigna species.

Mungbean (Vigna radiata) is a fast-growing, warm-season legume crop that is primarily cultivated in developing countries of Asia. Here we construct a draft genome sequence of mungbean to facilitate genome research into the subgenus Ceratotropis, which includes several important dietary legumes in Asia, and to enable a better understanding of the evolution of leguminous species. Based on the de novo assembly of additional wild mungbean species, the divergence of what was eventually domesticated and the sampled wild mungbean species appears to have predated domestication. Moreover, the de novo assembly of a tetraploid Vigna species (V. reflexo-pilosa var. glabra) provides genomic evidence of a recent allopolyploid event. The species tree is constructed using de novo RNA-seq assemblies of 22 accessions of 18 Vigna species and protein sets of Glycine max. The present assembly of V. radiata var. radiata will facilitate genome research and accelerate molecular breeding of the subgenus Ceratotropis.

RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and -susceptible alleles.

Bacterial leaf pustule (BLP) disease is caused by Xanthomonas axonopodis pv. glycines (Xag). To investigate the plant basal defence mechanisms induced in response to Xag, differential gene expression in near-isogenic lines (NILs) of BLP-susceptible and BLP-resistant soybean was analysed by RNA-Seq. Of a total of 46 367 genes that were mapped to soybean genome reference sequences, 1978 and 783 genes were found to be up- and down-regulated, respectively, in the BLP-resistant NIL relative to the BLP-susceptible NIL at 0, 6, and 12h after inoculation (hai). Clustering analysis revealed that these genes could be grouped into 10 clusters with different expression patterns. Functional annotation based on gene ontology (GO) categories was carried out. Among the putative soybean defence response genes identified (GO:0006952), 134 exhibited significant differences in expression between the BLP-resistant and -susceptible NILs. In particular, pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors and the genes induced by these receptors were highly expressed at 0 hai in the BLP-resistant NIL. Additionally, pathogenesis-related (PR)-1 and -14 were highly expressed at 0 hai, and PR-3, -6, and -12 were highly expressed at 12 hai. There were also significant differences in the expression of the core JA-signalling components MYC2 and JASMONATE ZIM-motif. These results indicate that powerful basal defence mechanisms involved in the recognition of PAMPs or DAMPs and a high level of accumulation of defence-related gene products may contribute to BLP resistance in soybean.