Kyujung Van

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

BackgroundR 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.ResultsA 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.ConclusionsThe 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.

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.

Acclimation of Chlamydomonas to changing carbon availability

Aquatic organisms, including Chlamydomonas reinhardtii, are faced with a variable supply of dissolved inorganic carbon (Ci). Accordingly, C. reinhardtii has the ability to acclimate to the changing Ci supply through a variety of responses, including induction of a CO2 concentrating mechanism (CCM) when Ci is limiting. The CCM uses active Ci uptake to accumulate a high internal concentration of bicarbonate, which is dehydrated by a specific thylakoid carbonic anhydrase to supply CO2, the substrate used in photosynthesis. In addition to the changes demonstrably related to the function of the CCM, C. reinhardtii exhibits several other acclimation responses to limiting Ci, such as changes in cellular organization and induction or upregulation of several genes. A key area currently under investigation is how C. reinhardtii cells recognize the change in Ci or CO2 concentration, and transduce that signal into needed gene expression changes. Mutational analyses are proving very useful for learning more about the CCM and about the acclimation response to changes in Ci availability. Cloning of the gene disrupted in cia5, a mutant apparently unable to acclimate to limiting Ci, has opened opportunities for more rapid progress in understanding the signal transduction pathway. The Cia5 gene appears to encode a transcription factor that may control, either directly or indirectly, much of the gene expression responses to limiting Ci in C. reinhardtii. Several additional new mutants with potential defects in the signal transduction pathway have been isolated, including three new alleles of cia5.

The lipoxygenase gene family: a genomic fossil of shared polyploidy between Glycine max and Medicago truncatula

BackgroundSoybean lipoxygenases (Lxs) play important roles in plant resistance and in conferring the distinct bean flavor. Lxs comprise a multi-gene family that includes GmLx1, GmLx2 and GmLx3, and many of these genes have been characterized. We were interested in investigating the relationship between the soybean lipoxygenase isozymes from an evolutionary perspective, since soybean has undergone two rounds of polyploidy. Here we report the tetrad genome structure of soybean Lx regions produced by ancient and recent polyploidy. Also, comparative genomics with Medicago truncatula was performed to estimate Lxs in the common ancestor of soybean and Medicago.ResultsTwo Lx regions in Medicago truncatula showing synteny with soybean were analyzed. Differential evolutionary rates between soybean and Medicago were observed and the median Ks values of Mt-Mt, Gm-Mt, and Gm-Gm paralogs were determined to be 0.75, 0.62, and 0.46, respectively. Thus the comparison of Gm-Mt paralogs (Ks = 0.62) and Gm-Mt orthologs (Ks = 0.45) supports the ancient duplication of Lx regions in the common ancestor prior to the Medicago-Glycine split. After speciation, no Lx regions generated by another polyploidy were identified in Medicago. Instead tandem duplication of Lx genes was observed. On the other hand, a lineage-specific duplication occurred in soybean resulting in two pairs of Lx regions. Each pair of soybean regions was co-orthologous to one Lx region in Medicago. A total of 34 Lx genes (15 MtLxs and 19 GmLxs) were divided into two groups by phylogenetic analysis. Our study shows that the Lx gene family evolved from two distinct Lx genes in the most recent common ancestor.ConclusionThis study analyzed two pairs of Lx regions generated by two rounds of polyploidy in soybean. Each pair of soybean homeologous regions is co-orthologous to one region of Medicago, demonstrating the quartet structure of the soybean genome. Differential evolutionary rates between soybean and Medicago were observed; thus optimized rates of Ks per year should be applied for accurate estimation of coalescence times to each case of comparison: soybean-soybean, soybean-Medicago, or Medicago-Medicago. In conclusion, the soybean Lx gene family expanded by ancient polyploidy prior to taxon divergence, followed by a soybean- specific duplication and tandem duplications, respectively.

Dynamic Rearrangements Determine Genome Organization and Useful Traits in Soybean

Soybean (Glycine max) is a paleopolyploid whose genome has gone through at least two rounds of polyploidy and subsequent diploidization events. Several studies have investigated the changes in genome structure produced by the relatively recent polyploidy event, but little is known about the ancient polyploidy due to the high frequency of gene loss after duplication. Our previous study, regarding a region responsible for bacterial leaf pustule, reported two homeologous Rxp regions produced by the recent whole-genome duplication event. In this study, we identified the full set of four homeologous Rxp regions (ranging from 1.96 to 4.60 Mb) derived from both the recent and ancient polyploidy events, and this supports the quadruplicated structure of the soybean genome. Among the predicted genes on chromosome 17 (linkage group D2), 71% of them were conserved in a recently duplicated region, while 21% and 24% of duplicated genes were retained in two homeologous regions formed by the ancient polyploidy. Furthermore, comparative analysis showed a 2:1 relationship between soybean and Medicago truncatula, since M. truncatula did not undergo the recent polyploidy event that soybean did. Unlike soybean, M. truncatula homeologous regions were highly fractionated and their synteny did not exist, revealing different rates of diploidization process between the two species. Our data show that extensive synteny remained in the four homeologous regions in soybean, even though the soybean genome experienced dynamic genome rearrangements following paleopolyploidy events. Moreover, multiple Rxp quantitative trait loci on different soybean chromosomes actually comprise homeologous regions produced by two rounds of polyploidy events.

Discovery of single nucleotide polymorphisms in soybean using primers designed from ESTs

Discovery of single nucleotide polymorphisms (SNPs), including small insertions and deletions (indels), is one of the hot topics in genetic research. SNPs were surveyed using nine soybean genotypes from Korea. Sequence variations in a total of 110 genes from GenBank among the nine genotypes were studied using genomic DNA as a template. Direct fluorescent dideoxynucleotide sequencing data of PCR products from primers designed from soybean ESTs were analyzed by SeqScape software to ensure high accuracy. Approximately 70% of the primer sets produced a single PCR product from which reliable sequence data were obtained, and 23.6% of these had at least one SNP. Overall, a total of 110 ESTs for SNPs were screened in 33,262 bp, consisting of 16,302 bp from coding regions and 16,960 bp from adjacent non-coding regions (5′ UTR, 3′ UTR and introns). SNPs in coding and non-coding regions occurred at a frequency of 1 per 3,260 bp, corresponding to a nucleotide diversity (θ) of 0.00011, and 1 per 278 bp (θ = 0.00128), respectively. This suggested that the higher level of sequence variation in non-coding regions would make them good regions in which to search for SNPs. The SNPs in partial cDNA sequences could be valuable for gene-targeted map construction in soybean.

Sequence Level Analysis of Recently Duplicated Regions in Soybean [Glycine max (L.) Merr.] Genome

A single recessive gene, rxp, on linkage group (LG) D2 controls bacterial leaf-pustule resistance in soybean. We identified two homoeologous contigs (GmA and GmA′) composed of five bacterial artificial chromosomes (BACs) during the selection of BAC clones around Rxp region. With the recombinant inbred line population from the cross of Pureunkong and Jinpumkong 2, single-nucleotide polymorphism and simple sequence repeat marker genotyping were able to locate GmA′ on LG A1. On the basis of information in the Soybean Breeders Toolbox and our results, parts of LG A1 and LG D2 share duplicated regions. Alignment and annotation revealed that many homoeologous regions contained kinases and proteins related to signal transduction pathway. Interestingly, inserted sequences from GmA and GmA′ had homology with transposase and integrase. Estimation of evolutionary events revealed that speciation of soybean from Medicago and the recent divergence of two soybean homoeologous regions occurred at 60 and 12 million years ago, respectively. Distribution of synonymous substitution patterns, Ks, yielded a first secondary peak (mode Ks = 0.10–0.15) followed by two smaller bulges were displayed between soybean homologous regions. Thus, diploidized paleopolyploidy of soybean genome was again supported by our study.

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.

QTL identification of yield-related traits and their association with flowering and maturity in soybean

Two soybean recombinant inbred line populations, Jinpumkong 2 × SS2-2 (J × S) and Iksannamulkong × SS2-2 (I x S) showed population-specific quantitative trait loci (QTLs) for days to flowering (DF) and days to maturity (DM) and these were closely correlated within population. In the present study, we identified QTLs for six yield-related traits with simple sequence repeat markers, and biological correlations between flowering traits and yield-related traits. The yield-related traits included plant height (PH), node numbers of main stem (NNMS), pod numbers per plant (PNPP), seed numbers per pod (SNPP), 100-seed weight (SW), and seed yield per plant (SYPP). Eighteen QTLs for six yield-related traits were detected on nine chromosomes (Chrs), containing four QTLs for PH, two for NNMS, two for PNPP, three for SNPP, five for SW, and two for SYPP. Two highly significant QTLs for PH and NNMS were identified on Chr 6 (LG C2) in both populations where the major flowering gene, E1, and two DF and DM QTLs were located. One other PNPP QTL was also located on this region, explaining 12.9% of phenotypic variation. Other QTLs for yield-related traits showed population-specificity. Two significant SYPP QTLs potentially related with QTLs for SNPP and PNPP were found on the same loci of Chrs 8 (Satt390) and 10 (Sat_108). Also, highly significant positive phenotypic correlations (P < 0.01) were found between DF with PH, NNMS, PNPP, and SYPP in both populations, while flowering was negatively correlated with SNPP and SW in the J × S (P < 0.05) and I × S (P < 0.01) populations. Similar results were also shown between DM and yield-related traits, except for one SW. These QTLs identified may be useful for marker-assisted selection by soybean breeders.