Suk-Ha Lee

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.

Genome-wide dissection of AP2/ERF and HSP90 gene families in five legumes and expression profiles in chickpea and pigeonpea

Summary APETALA2/ethylene response factor (AP2/ERF) and heat‐shock protein 90 (HSP90) are two significant classes of transcription factor and molecular chaperone proteins which are known to be implicated under abiotic and biotic stresses. Comprehensive survey identified a total of 147 AP2/ERF genes in chickpea, 176 in pigeonpea, 131 in Medicago, 179 in common bean and 140 in Lotus, whereas the number of HSP90 genes ranged from 5 to 7 in five legumes. Sequence alignment and phylogenetic analyses distinguished AP2, ERF, DREB, RAV and soloist proteins, while HSP90 proteins segregated on the basis of their cellular localization. Deeper insights into the gene structure allowed ERF proteins to be classified into AP2s based on DNA‐binding domains, intron arrangements and phylogenetic grouping. RNA‐seq and quantitative real‐time PCR (qRT‐PCR) analyses in heat‐stressed chickpea as well as Fusarium wilt (FW)‐ and sterility mosaic disease (SMD)‐stressed pigeonpea provided insights into the modus operandi of AP2/ERF and HSP90 genes. This study identified potential candidate genes in response to heat stress in chickpea while for FW and SMD stresses in pigeonpea. For instance, two DREB genes (Ca_02170 and Ca_16631) and three HSP90 genes (Ca_23016, Ca_09743 and Ca_25602) in chickpea can be targeted as potential candidate genes. Similarly, in pigeonpea, a HSP90 gene, C.cajan_27949, was highly responsive to SMD in the resistant genotype ICPL 20096, can be recommended for further functional validation. Also, two DREB genes, C.cajan_41905 and C.cajan_41951, were identified as leads for further investigation in response to FW stress in pigeonpea.

Genomic resources in mungbean for future breeding programs

Among the legume family, mungbean (Vigna radiata) has become one of the important crops in Asia, showing a steady increase in global production. It provides a good source of protein and contains most notably folate and iron. Beyond the nutritional value of mungbean, certain features make it a well-suited model organism among legume plants because of its small genome size, short life-cycle, self-pollinating, and close genetic relationship to other legumes. In the past, there have been several efforts to develop molecular markers and linkage maps associated with agronomic traits for the genetic improvement of mungbean and, ultimately, breeding for cultivar development to increase the average yields of mungbean. The recent release of a reference genome of the cultivated mungbean (V. radiata var. radiata VC1973A) and an additional de novo sequencing of a wild relative mungbean (V. radiata var. sublobata) has provided a framework for mungbean genetic and genome research, that can further be used for genome-wide association and functional studies to identify genes related to specific agronomic traits. Moreover, the diverse gene pool of wild mungbean comprises valuable genetic resources of beneficial genes that may be helpful in widening the genetic diversity of cultivated mungbean. This review paper covers the research progress on molecular and genomics approaches and the current status of breeding programs that have developed to move toward the ultimate goal of mungbean improvement.

Genome-wide analysis of mutations in a dwarf soybean mutant induced by fast neutron bombardment

Fast neutron (FN) bombardment is a powerful mutagen that can be effectively employed for functional genomics studies in the post-genome era. In soybean, dwarfism is a desirable agricultural characteristic that improves lodging resistance. In the present study, we selected a dwarf mutant soybean among approximately 10,000 M4 progeny lines derived from FN-irradiated seeds of cultivar Williams 82. This dwarf mutant exhibited reduced plant height, only approximately 20xa0% of wild type. Using mutant plants homozygous for this dwarf phenotype, we performed whole genome sequencing by Illumina HiSeq to identify the deletion site responsible for the dwarfism. Comparative sequence analysis by mapping the mutant reads to the soybean reference genome sequence (wild type) predicted 13 large deletion regions. Among these, three loci (designated del1–3, del2–3, and del3–15) were validated by two complementary PCRs using two allele-specific reverse primers, respectively. We found that the del1–3 and del2–3 loci are positioned in non-coding regions of chromosome 3. In del3–15, the mutated allele has an 803-bp deletion including the first partial exon of Glyma15g05831 (peroxidase superfamily protein) on chromosome 15, resulting in the loss of a start codon. Reverse transcription-PCR analysis revealed that the expression of the gene Glyma15g05831 was completely abolished in the dwarf mutant. A lack of peroxidase (which catalyzes the generation of reactive oxygen species) coupled with indole-3-acetic acid oxidation may be responsible for the dwarfing of this mutant.

Gene expression profiling for seed protein and oil synthesis during early seed development in soybean

Soybean (Glycine max L.) is one of the most important crops because of its high seed contents of protein and oil. However, little is known about the molecular regulation of seed filling during accumulation of seed protein and oil storage products. We identified soybean homologs of Arabidopsis genes involved in metabolic pathways of carbon precursors, protein, and oil, and analyzed gene expression patterns in immature seeds at 1 and 2xa0weeks after flowering (WAF). G. max undergoes two rounds of whole-genome duplication; the number of genes involved in the three synthesis pathways is more than two times higher than that in Arabidopsis. Among these genes, five were conserved as single-copy genes and 44 were high copy gene families consisting of more than seven homolog members. We identified five differentially expressed genes in immature seeds aged between 1 and 2 WAF, including CELL WALL INVERTASE, BRANCHED-CHAIN AMINO ACID TRANSAMINASE, AMINO ACID PERMEASE, ALDEHYDE REDUCTASE, and BIOTIN CARBOXYL CARRIER PROTEIN. Expression analysis of the duplicated genes on the synteny block revealed that the duplicated genes involved in protein synthesis had stronger positive correlations between their expression patterns than those of oil synthesis genes. This study provides novel insights into the molecular details of genes associated with soybean seed protein and oil synthesis pathways. These genes can be used as tools to improve seed nutrient composition.

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.

Underlying genetic variation in the response of cultivated and wild soybean to enhanced ultraviolet-B radiation

Reductions in stratospheric ozone have resulted in increased levels of ultraviolet-B (UV-B, 280–315xa0nm) radiation reaching the earth’s surface. UV-B radiation can damage plant DNA and photosynthetic machinery. Therefore, enhanced UV-B levels would seriously affect crop production in several regions of the world. Here, we provide a comprehensive report of intraspecific variability in the response of soybean to UV-B radiation and the genetic variation underlying UV-B resistance. We evaluated 140 genotypes, including 94 Glycine max and 46 G. soja accessions, for their sensitivity to supplemental UV-B radiation. Differences in responses to elevated UV-B levels, such as changes in leaf area and aerial biomass, were observed among soybean genotypes, supporting the notion that there is intraspecific variability in this response in G. soja as well as G. max. To explore how genetic variation contributes to differences in UV-B resistance, we sequenced the most UV-B resistant (IT162669) and sensitive (Cheongjakong 3) genotypes at the whole-genome level. Based on their homology and functional annotation, 137 genes were determined to be “UV-B-related genes” in soybean, harboring almost 100 high-confidence single nucleotide polymorphisms between the two genotypes. Interestingly, we identified four genes with non-synonymous mutations that are related to plant protection mechanisms, such as the UV-protection, DNA damage repair and DNA damage tolerance pathways. Our results provide valuable information about UV-B-resistant soybean genotypes, UV-B-related soybean genes, and sequence variations between resistant and sensitive genotypes.

Comprehensive transcriptome analysis of Lactuca indica, a traditional medicinal wild plant

Lactuca indica Linn. (Indian lettuce) is an undomesticated, traditional medicinal plant belonging to the Compositae family. Here, we performed transcriptome assembly and functional annotation of this medicinal plant, along with secondary metabolite analysis. Sesquiterpene lactones, the most abundant secondary metabolites in Lactuca species, are mainly responsible for the medicinal properties of Lactuca. We therefore measured the levels of lactucin, a primary sesquiterpene lactone, in 61 accessions of L. indica, which varied from 1.9xa0μg/g to 98.7xa0μg/g. De novo transcriptome assembly yielded 73,300 unigenes from 127 million reads based on 12.9xa0Gb of data. In total, 28,970 and 34,519 unigenes were annotated using the Swiss-Prot and TAIR10 databases, respectively. The most highly enriched Gene Ontology term for these unigenes was metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 97 significant pathways; the top two pathways were metabolic pathways and biosynthesis of secondary metabolites. Among the 9743 unigenes mapped to 97 significant KEGG pathways, 52 complete unigenes encoding 23 key enzymes in the terpenoid backbone pathway were identified. We identified 1685 transcription factor genes belonging to 53 families and 8830 simple sequence repeat (SSR) loci based on the transcripts. The high-quality transcriptomic and metabolic data on this indigenous herb obtained in this study represent a valuable genetic resource for breeding an L. indica cultivar with excellent pharmaceutical benefits.

Identification of QTLs for branching in soybean (Glycine max (L.) Merrill)

In soybean, the number of branches directly affects total pod number per plant. In this study, we sought to identify QTLs and candidate genes associated with branching in 200 F6 recombinant inbred lines derived from a cross between Jiyu69 and SS0404-T5-76, which exhibit significant differences in branch number. Using a high-resolution genetic map constructed using the BARCSoySNP6K chip, we detected a novel QTL and confirmed three known QTLs related to branching, as well as two known QTLs for total pod number. Two of the QTLs conferring branching, including a major QTL on chromosome six with an R2 value of 14.5%, were co-localized with QTLs associated with total pod number. Although several of the QTLs we identified for the two traits were located near identified QTLs, the high-resolution map enabled us to significantly narrow down the genomic regions for these QTLs (from 26xa0Mb to 460xa0kb at most), facilitating identification of promising candidate genes. From the QTL regions we identified, we selected six candidate genes, mostly encoding transcription factors regulating expression of gene networks involved in axillary branching via interactions with the auxin hormone network, including a TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factor (BRANCHED1: BRC1) and a homeobox-leucine zipper protein (REVOLUTA: REV). The results of this study will help breeders improve soybean yield by increasing the branch number using marker-assisted selection, and will facilitate identification of the causative genes for branching.