Jung-Kyung Moon

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 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.

Population Structure and Domestication Revealed by High-Depth Resequencing of Korean Cultivated and Wild Soybean Genomes

Despite the importance of soybean as a major crop, genome-wide variation and evolution of cultivated soybeans are largely unknown. Here, we catalogued genome variation in an annual soybean population by high-depth resequencing of 10 cultivated and 6 wild accessions and obtained 3.87 million high-quality single-nucleotide polymorphisms (SNPs) after excluding the sites with missing data in any accession. Nuclear genome phylogeny supported a single origin for the cultivated soybeans. We identified 10-fold longer linkage disequilibrium (LD) in the wild soybean relative to wild maize and rice. Despite the small population size, the long LD and large SNP data allowed us to identify 206 candidate domestication regions with significantly lower diversity in the cultivated, but not in the wild, soybeans. Some of the genes in these candidate regions were associated with soybean homologues of canonical domestication genes. However, several examples, which are likely specific to soybean or eudicot crop plants, were also observed. Consequently, the variation data identified in this study should be valuable for breeding and for identifying agronomically important genes in soybeans. However, the long LD of wild soybeans may hinder pinpointing causal gene(s) in the candidate regions.

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.

Genetic Analysis of Genes Controlling Natural Variation of Seed Coat and Flower Colors in Soybean

Soybean exhibits natural variation in flower and seed coat colors via the deposition of various anthocyanin pigments in the respective tissues. Although pigmentation in seeds or flowers has been well dissected at molecular level in several plant species, the genes controlling natural variation in anthocyanin traits in the soybean are not completely understood. To evaluate the genetic correlation between genetic loci and genes, 8 enzyme-encoding gene families and a transcription factor were localized in a soybean genome-wide genetic map. Among the seed coat color-controlling loci, the genetic location of the gene encoding for W1 was substantiated in the context of the current soybean molecular genetic map and O was postulated to correspond to anthocyanidin reductase. Among the genetic loci that regulate flower pigmentation, the genetic locations of the genes encoding for W1, W4, and Wp were identified, W3 was mapped on soybean linkage group B2 (chromosome 14), and W2 was postulated to correspond to an MYB transcription factor. Correlation studies between the developed markers and 3 color-controlling loci provided important empirical data that should prove useful in the design of marker-assisted breeding schemes as well as future association studies involving soybean.

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.

Sequence Information on Simple Sequence Repeats and Single Nucleotide Polymorphisms through Transcriptome Analysis of Mungbean

Mungbean (Vigna radiata (L.) Wilczek) is a unique species in its ability to fix atmospheric nitrogen, with early maturity, and relatively good drought resistance. We used 454 sequencing technology for transcriptome sequencing. A total of 150 159 and 142 993 reads produced 5 254 and 6 374 large contigs (≥ 500 bp) with an average length of 833 and 853 for Sunhwa and Jangan, respectively. Functional annotation to known sequences yielded 41.34% and 41.74% unigenes for Jangan and Sunhwa. A higher number of simple sequence repeat (SSR) motifs was identified in Jangan (1 630) compared with that of Sunhwa (1 334). A similar SSR distribution pattern was observed in both varieties. A total of 8 249 single nucleotide polymorphisms (SNPs) and indels with 2 098 high-confidence candidates were identified in the two mungbean varieties. The average distance between individual SNPs was approximately 860 bp. Our report demonstrates the utility of transcriptomic data for implementing a functional annotation and development of genetic markers. We also provide large resource sequence data for mungbean improvement programs.

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.

Ln Is a Key Regulator of Leaflet Shape and Number of Seeds per Pod in Soybean

Whether the leaflet shape gene Ln has a pleiotropic effect on the number of seeds per pod (NSPP) trait or not has long been debated in soybean research. This study shows that both the leaflet shape and NSPP traits are regulated by a single gene, which is a homolog of Arabidopsis JAGGED that regulates leaf and flower development, and establishes a novel role for JAGGED in fruit development. Narrow leaflet soybean (Glycine max) varieties tend to have more seeds per pod than broad leaflet varieties. Narrow leaflet in soybean is conferred by a single recessive gene, ln. Here, we show that the transition from broad (Ln) to narrow leaflet (ln) is associated with an amino acid substitution in the EAR motif encoded by a gene (designated Gm-JAGGED1) homologous to Arabidopsis JAGGED (JAG) that regulates lateral organ development and the variant exerts a pleiotropic effect on fruit patterning. The genomic region that regulates both the traits was mapped to a 12.6-kb region containing only one gene, Gm-JAG1. Introducing the Gm-JAG1 allele into a loss-of-function Arabidopsis jagged mutant partially restored the wild-type JAG phenotypes, including leaf shape, flower opening, and fruit shape, but the Gm-jag1 (ln) and EAR-deleted Gm-JAG1 alleles in the jagged mutant did not result in an apparent phenotypic change. These observations indicate that despite some degree of functional change of Gm-JAG1 due to the divergence from Arabidopsis JAG, Gm-JAG1 complemented the functions of JAG in Arabidopsis thaliana. However, the Gm-JAG1 homoeolog, Gm-JAG2, appears to be sub- or neofunctionalized, as revealed by the differential expression of the two genes in multiple plant tissues, a complementation test, and an allelic analysis at both loci.

Novel major quantitative trait loci regulating the content of isoflavone in soybean seeds

Despite their medicinal, pharmaceutical, and nutritional importance of isoflavones, the genetic basis controlling the amounts of isoflavones in soybean seeds is still not well understood. The main obstacle is the great variability in the content of isoflavone in seeds harvested from different environments. In this study, quantitative trait loci (QTL) for the content of different isoflavones including daidzein, genistein, and glycitein were investigated in a population of recombinant inbred lines derived from the cross of “Hwangkeum” (Glycine max) by “IT182932” (Glycine soja). Seeds analyzed were harvested in three different experimental environments. QTL analyses for isoflavone content were conducted by composite interval mapping across a genomewide genetic map. Two major QTL were mapped to soybean chromosomes 5 and 8, which were designated QDZGT1 and QDZGT2, respectively. Both loci have not been previously reported in other isoflavone sources. The results from this study will be useful in cloning genes that can control the contents of isoflavones in soybean and for the development of soybean lines containing a high or low isoflavone content.