BACKGROUND
r genes are a key component of gene interactions between plants and biotrophic bacteria and often function to regulate resistance to bacterial invasion  <cit> .
recent studies have proposed the ‘zigzag model’ to describe the resistance of plants in the context of the co-evolution between invader and host  <cit> .
the first phase of plant defense is pathogen molecular pattern-triggered immunity  by which the immune system of the plant recognizes a broad range of pathogens with conserved molecular patterns, thereby conferring non-host resistance.
in the second phase, effector-triggered immunity  detects effectors injected into the plant cell with the type iii secretion system  of bacteria.
usually, eti results in an amplified pti response, also known as the hypersensitive reaction .
among the known types of r proteins, the most common are those that contain a nucleotide-binding site and leucine-rich repeat  domain  <cit> .
in the arabidopsis col- <dig> genome,  <dig> proteins were predicted to have nbs-lrr domains and about two-thirds of those were included in a subgroup with at least one known r protein or col- <dig> ortholog of an r protein  <cit> .
in soybean, although research on r proteins conferring resistance to diverse diseases is somewhat lacking, nbs-lrr genes with a coiled-coil motif  were reported to co-segregate with the rpg1-b locus which confers resistance to strains of pseudomonas syringae pv.
glycinea <cit> .
notably, an nbs-lrr gene with toll/interleukin- <dig> receptor homology  was reported to restrict nodulation in soybean  <cit> .
although nodulation is a symbiotic interaction rather than an interaction between pathogen and host, this observation may indicate that r genes control microbe entry into soybean plants.
therefore, nbs-lrr genes, which are located throughout the soybean genome, may be involved in recognizing the presence of pathogens and ultimately conferring resistance.
plant perception of bacterial invasion is thought to be accomplished by the plant surveillance system, which recognizes specific pathogen-generated effectors and triggers the plant cell to undergo hr  <cit> .
transcriptome analysis is a powerful tool for studying the role of r genes in disease resistance.
recently, the availability of next-generation sequencers, such as the gs-flx  <dig> titanium and illumina-ga, has enabled the evaluation of expression levels of all predicted genes in a given plant provided that the sequence of the reference genome is available.
after the whole genome draft sequence of soybean was made publicly available at phytozome , soybean transcriptome data could be retrieved for all predicted genes  <cit> .
a previous study presented transcriptome profiling of soybean near-isogenic lines  between bacterial leaf pustule ) –susceptible and –resistant lines and suggested that differential defense-related gene expression in response to xag may contribute to blp resistance in soybean  <cit> .
in this study, we examined the correlation and co-localization between the number of nbs-lrr genes and the number of disease resistance qtl on each chromosome and surveyed the functional redundancy of disease resistance genes on the recently duplicated regions known to harbor nbs-lrr genes.
moreover, we investigated the effects of xag inoculation on gene expression in time-course experiments with susceptible and resistant nils, which revealed a significant increase in the expression of nbs-lrr genes located on qtl previously associated with blp resistance.
RESULTS
distribution of genome-wide nbs-lrr genes and disease resistance qtl
to define the nbs-lrr genes of the soybean genome, we used whole genome information available at phytozome   <cit> .
based on the protein family databases, such as pfam, panther, kog, and kegg, a total of  <dig>  mapped genes have been annotated according to the gmax_109_annotation_info.txt at phytozome.
to retrieve the putative nbs-lrr genes within the soybean genome, the genes with lrr pfam ids pf <dig>  pf <dig> and pf <dig> were selected and further refined to include genes containing nbs domains .
among  <dig>  lrr genes identified in soybean,  <dig> genes were defined as putative nbs-lrr genes.
a total of  <dig> nbs-lrr genes were located on soybean chromosomes , whereas an additional  <dig> nbs-lrr genes were positioned on scaffolds.
nbs-lrr genes were further sorted by their n-terminal domains using homology to already classified arabidopsis nbs-lrr genes and included  <dig> tir-nbs-lrrs,  <dig> cc-nbs-lrrs, and  <dig> other nbs-lrrs .
* more than half of the disease resistance qtl co-localized with nbs-lrr genes within the 2-mb flanking region.
** regression analyses were done between the number of nbs-lrr genes and the number of qtl within the 2-mb flanking region of nbs-lrr genes.
to investigate the distribution of nbs-lrr genes in the soybean genome, the chromosomal location of the genes was plotted on a circular genome map at the inner-most layer .
the nbs-lrr gene distribution was biased and clustered on chromosomes   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  more than half of the  <dig> nbs-lrr genes  were located on these six chromosomes, suggesting that the nbs-lrr gene clusters are a result of duplication events .
moreover, the disease resistance qtl locations identified using corresponding marker information, were also highly biased and were similar to the locations of the nbs-lrr genes.
among the  <dig> reported disease resistance qtl on soybase ,  <dig> qtl were selected by the corresponding marker information.
figure  <dig> depicts the location of these disease resistance qtl on the third layer from the innermost layer.
disease resistance qtl tended to be located in the 2-mb regions flanking the physical locations of the nbs-lrr genes, especially the highly clustered regions .
for example, the highly clustered region of nbs-lrr genes on chr  <dig> harbored  <dig> qtl conferring fungal resistance and  <dig> qtl conferring resistance to nematodes.
on chr  <dig>   <dig> nbs-lrr genes were clustered, and  <dig> fungal resistance qtl and  <dig> nematode resistance qtl were found near the clustered genes.
in the case of chr  <dig>   <dig> fungal resistance and  <dig> nematode resistance qtl were located within the 2-mb flanking region of a gene cluster containing  <dig> nbs-lrr genes .
although not all disease resistance qtl were located near nbs-lrr genes, most qtl and nbs-lrr genes tended to co-localize.
about 63% of disease related qtl were positioned within the 2-mb flanking region of an nbs-lrr gene .
linear regression analysis revealed that there were significant correlations between the number of nbs-lrr genes and the number of disease resistance qtl within the 2-mb region flanking an nbs-lrr gene with observed r <dig> values of  <dig>  and a p-value lower than  <dig>  .
additionally, qtl for soybean “leaflet shape” traits were selected to understand the correlation between nbs-lrr genes and different types of qtl rather than disease-related  .
moreover, the total of  <dig> qtl, which were assigned to soybean trait terms, soy: <dig>   was chosen to investigate whether there were other putative effects of nbs-lrr genes .
from those of selected, a total of  <dig> qtl were positioned in the 2-mb flanking regions of nbs-lrr genes and identified that the majority of these traits was soybean morphology and anatomy traits  and soybean stress resistance traits .
the indication showed some nbs-lrr genes could affect many different quantitative traits or in the interaction between disease resistance and other agronomically important traits.
recent duplication of nbs-lrr containing genomic regions
to further support the functional correlation between the nbs-lrr genes and disease resistance qtl, we investigated recently duplicated regions in the soybean genome, which could contain genes that perform redundant functions depending on their nature.
figure  <dig> depicts the recently duplicated region of soybean as a transparent colored ribbon on a circular genome map.
table  <dig> provides a list of disease resistance qtl within  <dig> mb flanking of the recently duplicated regions and nbs-lrr genes within recently duplicated regions were also retrieved.
there were a total of  <dig> nbs-lrr genes within  <dig> recently duplicated genomic regions and these regions contained the duplicated disease resistance qtl.
among those recently duplicated regions, four regions  had the same number of nbs-lrr genes.
an additional four duplicated regions  showed a difference of only one gene.
in these regions, the copy number of nbs-lrr genes on each side was similar.
* chromosome a and a’ represent the recently duplicated region.
** fungal resistance qtl: sclerotinia stem rot , sudden death syndrome , brown stem rot , phytophthora sojae infection ; nematode resistance qtl: soybean cyst nematode , peanut root-knot nematode , southern root-knot nematode , javanese root-knot nematode ; bacterial leaf pustule resistance qtl : ocs-g, sdl <dig>  8ra, ocs-f, lmg <dig>
among these eight duplicated regions,  <dig>   <dig>   <dig> and  <dig> had disease resistance qtl with distinct specificities for divergent diseases .
for example, the recently duplicated region,  <dig>  had duplicated the nbs-lrr genes, glyma01g <dig> and glyma01g <dig> for chr  <dig> and glyma09g <dig>  glyma09g <dig> and glyma09g <dig> for chr  <dig>  the qtl for nematode resistance resides on chr  <dig>  while the qtl for fungal resistance is on chr  <dig>  these examples indicate that the nbs-lrr genes probably retained their biotic resistance functions after duplication, although they would probably have acquired novel specificities as well.
the other duplicated regions  had disease resistance qtl that targeted the same disease agents and other diseases as well.
however, two recently duplicated regions  had a distinct number of nbs-lrr genes, which may have resulted from a tandem duplication occurring independently on one side of the duplicated region.
one side of these duplicated regions contained a higher number of nbs-lrr genes and additional or different source of disease resistance qtl compared to the other side.
for example, the recently duplicated region,  <dig>  has only one duplicated nbs-lrr gene  on chr  <dig> and  <dig> tandemly duplicated genes  on chr  <dig>  several disease resistances qtl also segregate as follows:  <dig> qtl for resistance to sclerotinia  are located on chr  <dig>  while  <dig> qtl for resistance to brown stem rot  of fungi and  <dig> qtl for resistance to soybean cyst nematode  are found on chr  <dig>
the blp resistance qtl that provides bacterial resistance against xag also overlapped with the recently duplicated nbs-lrr regions.
a recently duplicated region on chr  <dig>  has two nbs-lrr genes, glyma08g <dig> and glyma05g <dig>  on each side .
the disease resistance qtl that provides resistance against the ocs-g isolate of blp and against sclero, was located on one side of the duplicated regions, while the qtl that provides resistance against the lmg <dig> isolate of blp and sclero was located on the other side of the duplicated region  <cit> .
another example of a recently duplicated region , which harbored nbs-lrr genes on both sides, contained a blp resistance qtl against ocs-g, sdl <dig> and sds 7- <dig> isolates on one side of the duplicated region and a disease resistance qtl against the 8ra isolate of blp and sclero on the other side of the duplicated region.
the nbs-lrr genes in recently duplicated regions showed variable synonymous substitution rates  ranging from  <dig>  to  <dig> .
these values may reflect the order of each duplication event, and herein we propose models to explain the order of duplications.
two models are suggested to explain the duplication orders .
in the first model, the nbs-lrr genes were first tandemly duplicated, then subjected to selective pressures over a significant evolutionary time period, and finally were copied to another chromosome.
genes adhering to this model would show high ks values between tandemly duplicated genes, but low ks values between paralogs of recently duplicated regions.
in the second model, a single nbs-lrr gene was duplicated to another chromosome first and then tandemly duplicated independently.
this would yield a pattern that would be the reverse of the first model.
to determine which of these two models is more likely, we estimated the order of the recent duplication and tandem duplication events by estimating the ks values between paralogs and between the tandemly duplicated regions .
the paralogs of recently duplicated regions of id  <dig> and  <dig> showed average ks values of  <dig>  and  <dig> , respectively, which are similar to the values reported for recently duplicated paralogs   <cit> .
tandemly duplicated regions showed a higher ks value; hence, we infer that these two regions underwent the duplication according to the first model.
notably, on a recently duplicated region , we found an anciently duplicated region that showed a ks value of  <dig> , which is consistent with that the value in a previous report   <cit> .
the ks values of the paralogous region and tandemly duplicated region were  <dig>  and  <dig> , respectively.
this region might have undergone a duplication process in accordance with the second model after an ancient duplication event.
the paralog of id  <dig> had a ks value of  <dig>  and the tandem duplicated regions had ks values of  <dig>  for one region and  <dig>  for the other two regions, suggesting a mixture of the two models.
the duplication region id  <dig> had ks values of  <dig>  and  <dig>  for each of the paralogous regions;  <dig>  for the tandemly duplicated region on chr  <dig> ; an average of  <dig>  on chr  <dig> ; and an average of  <dig>  on chr  <dig>  .
the greater ks value of group  <dig> than that of groups  <dig> and  <dig> support a mixed model in which the tandem duplication of group  <dig> occurred first, followed by the recent duplication between chr  <dig> and chr  <dig>  and then finally the tandem duplication of groups  <dig> and  <dig>
transcriptome analysis of nbs-lrr genes
to explore the disease resistance functions of the nbs-lrrs further, we took advantage of previously generated transcriptome data for blp-treated nils  <cit> .
the locations of nbs-lrr genes, which were differentially expressed between the resistant nil and the susceptible nil, are shown on the circle map .
in total,  <dig> nbs-lrr genes scattered over  <dig> chromosomes  showed significant expression changes between the resistance and susceptible nils.
among  <dig> nbs-lrr genes, five nbs-lrr genes were clustered together at the end of chr  <dig> with sudden death syndrome  and scn resistance qtl.
additionally, the soybean rust resistance locus, rpp <dig>  were reported to be located around this region   <cit>  .
notably, two nbs-lrrs were clustered on chr  <dig>  and the blp resistance qtl was also located in the 2-mb flanking region.
to validate the transcriptome data from illumina-ga, we performed qrt-pcr with four nbs-lrr genes and one rin4-like gene .
the expression patterns were consistent with the rna-seq rpkm values and qrt-pcr values for each gene.
the differences in gene expression between nils were also confirmed to be significant by qrt-pcr , which shows that the transcriptome analysis of the nbs-lrrs was reliable.
discussion
nbs-lrr chromosomal distribution
nbs-lrr genes were distributed on several chromosomes on the soybean genome.
especially, chr  <dig> had the highest number of nbs-lrr genes as well as the highest number of disease resistance qtl.
the other chromosomes containing a biased number of nbs-lrrs also had a tendency to harbor disease resistance qtl.
the number of nbs-lrr genes and the number of disease resistance qtl within the 2-mb flanking regions of the genes were significantly correlated.
a similar correlation has been reported in other plants.
nbs profiling to identify and map resistance analogues  in apple revealed that  <dig> out of  <dig> nbs profiling-derived markers mapped close to a major qtl  <cit> .
barley rgas co-segregated with known disease resistance loci  <cit> .
in potato, nbs-lrr genes from a bac library and  <dig> resistance loci co-localized with tir-nbs-lrrs, most of which underlie qtl  <cit> .
therefore, the association between nbs-lrrs and disease resistance loci or qtl might be a common feature of these crop species.
the qtl located near the nbs-lrr cluster were not specific for one particular disease, but rather for several pathogens and pests , and were often closely associated.
interestingly, bacterial genomes have been reported to possess pathogenicity islands on which functionally similar genes are clustered together  <cit> .
these clusters are thought to result from evolution of the mechanisms involved in invasion strategy.
the major driving force behind this evolution would be tandem duplication and mobile dna-like transposases and integrases  <cit> .
notably, mutator-like element  transposase and the mudr  family transposase domain was located within the nbs-lrr gene, glyma16g <dig>  on chr  <dig>  moreover, this region of glyma16g <dig> had the highest number of clustered nbs-lrr genes.
this finding is consistent with that of a previous study on peanut, which found a high density of transposable elements in bac clones that contained nbs-lrr genes.
these transposable elements might be responsible for the intra-chromosomal duplication and broadening of host specificity  <cit> .
recent duplication of the soybean genome and the redundancy of the disease resistance function
sequencing of the whole soybean genome  revealed the actual locations of recently duplicated regions  <cit> .
so far the functional redundancy between duplicated regions has been reported for several traits in soybean including seed protein, oil and blp resistance  <cit> .
according to our analysis, disease resistance was also redundant between recently duplicated regions.
interestingly, some duplicated genome regions contained the same number of nbs-lrr genes, while other duplicated regions had completely different numbers of nbs-lrr genes for both duplicated sides .
sequence divergence of gene families may occur after the duplication process.
specifically, r genes frequently contain numerous copies throughout genome, most likely as a result of unequal crossing over  <cit> .
from the proposed models, the possibility of a combination of tandem duplication and inter-chromosomal duplication suggests that the duplication histories of nbs-lrr genes could be one of the factors involved in the diversification of disease-resistant qtl near these genes .
however, not all regions with nbs-lrr genes harbored disease-resistant qtl, which might be due to a lack of sufficient disease resistance qtl or to nbs-lrr genes with functions unrelated to disease resistance.
transcriptome analysis of the nbs-lrr genes on the blp-resistant nil set
the transcriptome analysis of a xag-treated soybean nil set showed that two nbs-lrr genes with significant fold-changes in expression between the blp-resistant and the blp-susceptible nil were in close proximity to the previously reported blp-resistant qtl.
however,  <dig> nbs-lrr genes were not near disease resistance qtl.
a major gene conferring resistance to blp on chr  <dig> may affect the expression in different types of nbs-lrr genes.
it is also possible that rna-seq analysis might be sensitive to detect the expression of unrelated genes.
among several nbs-lrr genes showing significantly different expression levels between resistant and susceptible nils, six genes  were annotated as rpm <dig> , which was reported to guard the rin <dig> protein after its modification by effectors secreted from invading bacteria.
among the one rin <dig> gene and four rin4-like genes identified by the rna-seq data, only one  showed the log <dig> fold-change of  <dig> , between the susceptible and resistant nils.
this gene had a portion of arabidopsis rin <dig> and a conserved domain for effector recognition .
although the transcriptional profile does not mirror the actual function of the protein, this result suggests that the partial rin <dig> gene might be involved in the resistance reaction acting together with rpm <dig>  in tomato, the existence of partial proteins with only an effector-binding domain was proposed as part of a ‘decoy model,’ such as for pto and rcr <dig>  <cit> .
it is also shown are the fold changes occurring between resistant nil and susceptible nil in hourly increments after xag inoculation .
the transcriptomes from several ncbi deposited data were also analyzed to figure out differentially expressed nbs-lrr genes by various disease attacks .
rna-seq data of soybean cultivar williams  <dig> inoculated with phakopsora pachyrhizi   and cdna array data of williams  <dig> and pi  <dig> inoculated with sclerotinia sclerotiorum  were used  <cit> .
since the soybean symbiotic relationship with bradyrhizobium japonicum is very important for formulating nodules in nitrogen fixation, we also used cdna array data of ss2- <dig>  supernodulating soybean mutant, response to this bacterium   <cit> .
among the differentially expressed nbs-lrr genes in each transcriptome set, glyma01g <dig> was differentially expressed at all four transcriptomes and glyma05g <dig> and glyma06g <dig> were differentially expressed at three transcriptomes, suggesting that those nbs-lrr genes may work against broad range of pathogen attack.
some of nbs-lrr genes had specific responses to different sources of inoculum.
CONCLUSIONS
we analyzed and compared the locations of nbs-lrr genes and disease resistance qtl in the soybean genome to determine the relationship between nbs-lrr genes and resistance functions that have been reported in model plants.
the correlation between nbs-lrr genes and disease resistance qtl in the 2-mb regions flanking nbs-lrr genes was high enough to support the notion that these genes participate in disease resistance.
since the recently duplicated nbs-lrr genes also retained disease resistance qtl, their functional relevance to disease resistance is even more convincing.
we also proposed a model for the order of nbs-lrr gene duplications that includes recent duplication and tandem duplication events.
moreover, in the rna-seq data of a soybean blp-resistant and -susceptible nil set treated with xag, we observed a significant fold-change in nbs-lrr gene expression between the nils.
two of the differentially expressed nbs-lrr genes were located in the previously reported blp-resistant qtl region, indicating that nbs-lrr genes might be located downstream of the rxp locus that confers resistance to blp  <cit> .
by developing molecular markers for nbs-lrr domains, it should be possible to integrate disease resistance against a diverse range of pathogens to generate an elite soybean cultivar.

 r genes are a key component of gene interactions between plants and biotrophic bacteria and often function to regulate resistance to bacterial invasion. recent studies have proposed the ‘zigzag model’ to describe the resistance of plants in the context of the co-evolution between invader and host. the first phase of plant defense is pathogen molecular pattern-triggered immunity by which the immune system of the plant recognizes a broad range of pathogens with conserved molecular patterns, thereby conferring non-host resistance. in the second phase, effector-triggered immunity detects effectors injected into the plant cell with the type iii secretion system of bacteria. usually, eti results in an amplified pti response, also known as the hypersensitive reaction. among the known types of r proteins, the most common are those that contain a nucleotide-binding site and leucine-rich repeat domain. in the arabidopsis col- genome, proteins were predicted to have nbs-lrr domains and about two-thirds of those were included in a subgroup with at least one known r protein or col- ortholog of an r protein. in soybean, although research on r proteins conferring resistance to diverse diseases is somewhat lacking, nbs-lrr genes with a coiled-coil motif were reported to co-segregate with the rpg1-b locus which confers resistance to strains of pseudomonas syringae pv. glycinea. notably, an nbs-lrr gene with toll/interleukin- receptor homology was reported to restrict nodulation in soybean. although nodulation is a symbiotic interaction rather than an interaction between pathogen and host, this observation may indicate that r genes control microbe entry into soybean plants. therefore, nbs-lrr genes, which are located throughout the soybean genome, may be involved in recognizing the presence of pathogens and ultimately conferring resistance. plant perception of bacterial invasion is thought to be accomplished by the plant surveillance system, which recognizes specific pathogen-generated effectors and triggers the plant cell to undergo hr. transcriptome analysis is a powerful tool for studying the role of r genes in disease resistance. recently, the availability of next-generation sequencers, such as the gs-flx titanium and illumina-ga, has enabled the evaluation of expression levels of all predicted genes in a given plant provided that the sequence of the reference genome is available. after the whole genome draft sequence of soybean was made publicly available at phytozome, soybean transcriptome data could be retrieved for all predicted genes. a previous study presented transcriptome profiling of soybean near-isogenic lines between bacterial leaf pustule ) –susceptible and –resistant lines and suggested that differential defense-related gene expression in response to xag may contribute to blp resistance in soybean. in this study, we examined the correlation and co-localization between the number of nbs-lrr genes and the number of disease resistance qtl on each chromosome and surveyed the functional redundancy of disease resistance genes on the recently duplicated regions known to harbor nbs-lrr genes. moreover, we investigated the effects of xag inoculation on gene expression in time-course experiments with susceptible and resistant nils, which revealed a significant increase in the expression of nbs-lrr genes located on qtl previously associated with blp resistance.  distribution of genome-wide nbs-lrr genes and disease resistance qtl to define the nbs-lrr genes of the soybean genome, we used whole genome information available at phytozome. based on the protein family databases, such as pfam, panther, kog, and kegg, a total of mapped genes have been annotated according to the gmax_109_annotation_info.txt at phytozome. to retrieve the putative nbs-lrr genes within the soybean genome, the genes with lrr pfam ids pf pf and pf were selected and further refined to include genes containing nbs domains. among lrr genes identified in soybean, genes were defined as putative nbs-lrr genes. a total of nbs-lrr genes were located on soybean chromosomes, whereas an additional nbs-lrr genes were positioned on scaffolds. nbs-lrr genes were further sorted by their n-terminal domains using homology to already classified arabidopsis nbs-lrr genes and included tir-nbs-lrrs, cc-nbs-lrrs, and other nbs-lrrs. * more than half of the disease resistance qtl co-localized with nbs-lrr genes within the 2-mb flanking region. ** regression analyses were done between the number of nbs-lrr genes and the number of qtl within the 2-mb flanking region of nbs-lrr genes. to investigate the distribution of nbs-lrr genes in the soybean genome, the chromosomal location of the genes was plotted on a circular genome map at the inner-most layer. the nbs-lrr gene distribution was biased and clustered on chromosomes and more than half of the nbs-lrr genes were located on these six chromosomes, suggesting that the nbs-lrr gene clusters are a result of duplication events. moreover, the disease resistance qtl locations identified using corresponding marker information, were also highly biased and were similar to the locations of the nbs-lrr genes. among the reported disease resistance qtl on soybase, qtl were selected by the corresponding marker information. figure depicts the location of these disease resistance qtl on the third layer from the innermost layer. disease resistance qtl tended to be located in the 2-mb regions flanking the physical locations of the nbs-lrr genes, especially the highly clustered regions. for example, the highly clustered region of nbs-lrr genes on chr harbored qtl conferring fungal resistance and qtl conferring resistance to nematodes. on chr nbs-lrr genes were clustered, and fungal resistance qtl and nematode resistance qtl were found near the clustered genes. in the case of chr fungal resistance and nematode resistance qtl were located within the 2-mb flanking region of a gene cluster containing nbs-lrr genes. although not all disease resistance qtl were located near nbs-lrr genes, most qtl and nbs-lrr genes tended to co-localize. about 63% of disease related qtl were positioned within the 2-mb flanking region of an nbs-lrr gene. linear regression analysis revealed that there were significant correlations between the number of nbs-lrr genes and the number of disease resistance qtl within the 2-mb region flanking an nbs-lrr gene with observed r values of and a p-value lower than. additionally, qtl for soybean “leaflet shape” traits were selected to understand the correlation between nbs-lrr genes and different types of qtl rather than disease-related. moreover, the total of qtl, which were assigned to soybean trait terms, soy: was chosen to investigate whether there were other putative effects of nbs-lrr genes. from those of selected, a total of qtl were positioned in the 2-mb flanking regions of nbs-lrr genes and identified that the majority of these traits was soybean morphology and anatomy traits and soybean stress resistance traits. the indication showed some nbs-lrr genes could affect many different quantitative traits or in the interaction between disease resistance and other agronomically important traits. recent duplication of nbs-lrr containing genomic regions to further support the functional correlation between the nbs-lrr genes and disease resistance qtl, we investigated recently duplicated regions in the soybean genome, which could contain genes that perform redundant functions depending on their nature. figure depicts the recently duplicated region of soybean as a transparent colored ribbon on a circular genome map. table provides a list of disease resistance qtl within mb flanking of the recently duplicated regions and nbs-lrr genes within recently duplicated regions were also retrieved. there were a total of nbs-lrr genes within recently duplicated genomic regions and these regions contained the duplicated disease resistance qtl. among those recently duplicated regions, four regions had the same number of nbs-lrr genes. an additional four duplicated regions showed a difference of only one gene. in these regions, the copy number of nbs-lrr genes on each side was similar. * chromosome a and a’ represent the recently duplicated region. ** fungal resistance qtl: sclerotinia stem rot, sudden death syndrome, brown stem rot, phytophthora sojae infection; nematode resistance qtl: soybean cyst nematode, peanut root-knot nematode, southern root-knot nematode, javanese root-knot nematode; bacterial leaf pustule resistance qtl: ocs-g, sdl 8ra, ocs-f, lmg among these eight duplicated regions, and had disease resistance qtl with distinct specificities for divergent diseases. for example, the recently duplicated region, had duplicated the nbs-lrr genes, glyma01g and glyma01g for chr and glyma09g glyma09g and glyma09g for chr the qtl for nematode resistance resides on chr while the qtl for fungal resistance is on chr these examples indicate that the nbs-lrr genes probably retained their biotic resistance functions after duplication, although they would probably have acquired novel specificities as well. the other duplicated regions had disease resistance qtl that targeted the same disease agents and other diseases as well. however, two recently duplicated regions had a distinct number of nbs-lrr genes, which may have resulted from a tandem duplication occurring independently on one side of the duplicated region. one side of these duplicated regions contained a higher number of nbs-lrr genes and additional or different source of disease resistance qtl compared to the other side. for example, the recently duplicated region, has only one duplicated nbs-lrr gene on chr and tandemly duplicated genes on chr several disease resistances qtl also segregate as follows: qtl for resistance to sclerotinia are located on chr while qtl for resistance to brown stem rot of fungi and qtl for resistance to soybean cyst nematode are found on chr the blp resistance qtl that provides bacterial resistance against xag also overlapped with the recently duplicated nbs-lrr regions. a recently duplicated region on chr has two nbs-lrr genes, glyma08g and glyma05g on each side. the disease resistance qtl that provides resistance against the ocs-g isolate of blp and against sclero, was located on one side of the duplicated regions, while the qtl that provides resistance against the lmg isolate of blp and sclero was located on the other side of the duplicated region. another example of a recently duplicated region, which harbored nbs-lrr genes on both sides, contained a blp resistance qtl against ocs-g, sdl and sds 7- isolates on one side of the duplicated region and a disease resistance qtl against the 8ra isolate of blp and sclero on the other side of the duplicated region. the nbs-lrr genes in recently duplicated regions showed variable synonymous substitution rates ranging from to. these values may reflect the order of each duplication event, and herein we propose models to explain the order of duplications. two models are suggested to explain the duplication orders. in the first model, the nbs-lrr genes were first tandemly duplicated, then subjected to selective pressures over a significant evolutionary time period, and finally were copied to another chromosome. genes adhering to this model would show high ks values between tandemly duplicated genes, but low ks values between paralogs of recently duplicated regions. in the second model, a single nbs-lrr gene was duplicated to another chromosome first and then tandemly duplicated independently. this would yield a pattern that would be the reverse of the first model. to determine which of these two models is more likely, we estimated the order of the recent duplication and tandem duplication events by estimating the ks values between paralogs and between the tandemly duplicated regions. the paralogs of recently duplicated regions of id and showed average ks values of and, respectively, which are similar to the values reported for recently duplicated paralogs. tandemly duplicated regions showed a higher ks value; hence, we infer that these two regions underwent the duplication according to the first model. notably, on a recently duplicated region, we found an anciently duplicated region that showed a ks value of, which is consistent with that the value in a previous report. the ks values of the paralogous region and tandemly duplicated region were and, respectively. this region might have undergone a duplication process in accordance with the second model after an ancient duplication event. the paralog of id had a ks value of and the tandem duplicated regions had ks values of for one region and for the other two regions, suggesting a mixture of the two models. the duplication region id had ks values of and for each of the paralogous regions; for the tandemly duplicated region on chr; an average of on chr; and an average of on chr. the greater ks value of group than that of groups and support a mixed model in which the tandem duplication of group occurred first, followed by the recent duplication between chr and chr and then finally the tandem duplication of groups and transcriptome analysis of nbs-lrr genes to explore the disease resistance functions of the nbs-lrrs further, we took advantage of previously generated transcriptome data for blp-treated nils. the locations of nbs-lrr genes, which were differentially expressed between the resistant nil and the susceptible nil, are shown on the circle map. in total, nbs-lrr genes scattered over chromosomes showed significant expression changes between the resistance and susceptible nils. among nbs-lrr genes, five nbs-lrr genes were clustered together at the end of chr with sudden death syndrome and scn resistance qtl. additionally, the soybean rust resistance locus, rpp were reported to be located around this region. notably, two nbs-lrrs were clustered on chr and the blp resistance qtl was also located in the 2-mb flanking region. to validate the transcriptome data from illumina-ga, we performed qrt-pcr with four nbs-lrr genes and one rin4-like gene. the expression patterns were consistent with the rna-seq rpkm values and qrt-pcr values for each gene. the differences in gene expression between nils were also confirmed to be significant by qrt-pcr, which shows that the transcriptome analysis of the nbs-lrrs was reliable. discussion nbs-lrr chromosomal distribution nbs-lrr genes were distributed on several chromosomes on the soybean genome. especially, chr had the highest number of nbs-lrr genes as well as the highest number of disease resistance qtl. the other chromosomes containing a biased number of nbs-lrrs also had a tendency to harbor disease resistance qtl. the number of nbs-lrr genes and the number of disease resistance qtl within the 2-mb flanking regions of the genes were significantly correlated. a similar correlation has been reported in other plants. nbs profiling to identify and map resistance analogues in apple revealed that out of nbs profiling-derived markers mapped close to a major qtl. barley rgas co-segregated with known disease resistance loci. in potato, nbs-lrr genes from a bac library and resistance loci co-localized with tir-nbs-lrrs, most of which underlie qtl. therefore, the association between nbs-lrrs and disease resistance loci or qtl might be a common feature of these crop species. the qtl located near the nbs-lrr cluster were not specific for one particular disease, but rather for several pathogens and pests, and were often closely associated. interestingly, bacterial genomes have been reported to possess pathogenicity islands on which functionally similar genes are clustered together. these clusters are thought to result from evolution of the mechanisms involved in invasion strategy. the major driving force behind this evolution would be tandem duplication and mobile dna-like transposases and integrases. notably, mutator-like element transposase and the mudr family transposase domain was located within the nbs-lrr gene, glyma16g on chr moreover, this region of glyma16g had the highest number of clustered nbs-lrr genes. this finding is consistent with that of a previous study on peanut, which found a high density of transposable elements in bac clones that contained nbs-lrr genes. these transposable elements might be responsible for the intra-chromosomal duplication and broadening of host specificity. recent duplication of the soybean genome and the redundancy of the disease resistance function sequencing of the whole soybean genome revealed the actual locations of recently duplicated regions. so far the functional redundancy between duplicated regions has been reported for several traits in soybean including seed protein, oil and blp resistance. according to our analysis, disease resistance was also redundant between recently duplicated regions. interestingly, some duplicated genome regions contained the same number of nbs-lrr genes, while other duplicated regions had completely different numbers of nbs-lrr genes for both duplicated sides. sequence divergence of gene families may occur after the duplication process. specifically, r genes frequently contain numerous copies throughout genome, most likely as a result of unequal crossing over. from the proposed models, the possibility of a combination of tandem duplication and inter-chromosomal duplication suggests that the duplication histories of nbs-lrr genes could be one of the factors involved in the diversification of disease-resistant qtl near these genes. however, not all regions with nbs-lrr genes harbored disease-resistant qtl, which might be due to a lack of sufficient disease resistance qtl or to nbs-lrr genes with functions unrelated to disease resistance. transcriptome analysis of the nbs-lrr genes on the blp-resistant nil set the transcriptome analysis of a xag-treated soybean nil set showed that two nbs-lrr genes with significant fold-changes in expression between the blp-resistant and the blp-susceptible nil were in close proximity to the previously reported blp-resistant qtl. however, nbs-lrr genes were not near disease resistance qtl. a major gene conferring resistance to blp on chr may affect the expression in different types of nbs-lrr genes. it is also possible that rna-seq analysis might be sensitive to detect the expression of unrelated genes. among several nbs-lrr genes showing significantly different expression levels between resistant and susceptible nils, six genes were annotated as rpm, which was reported to guard the rin protein after its modification by effectors secreted from invading bacteria. among the one rin gene and four rin4-like genes identified by the rna-seq data, only one showed the log fold-change of, between the susceptible and resistant nils. this gene had a portion of arabidopsis rin and a conserved domain for effector recognition. although the transcriptional profile does not mirror the actual function of the protein, this result suggests that the partial rin gene might be involved in the resistance reaction acting together with rpm in tomato, the existence of partial proteins with only an effector-binding domain was proposed as part of a ‘decoy model,’ such as for pto and rcr. it is also shown are the fold changes occurring between resistant nil and susceptible nil in hourly increments after xag inoculation. the transcriptomes from several ncbi deposited data were also analyzed to figure out differentially expressed nbs-lrr genes by various disease attacks. rna-seq data of soybean cultivar williams inoculated with phakopsora pachyrhizi and cdna array data of williams and pi inoculated with sclerotinia sclerotiorum were used. since the soybean symbiotic relationship with bradyrhizobium japonicum is very important for formulating nodules in nitrogen fixation, we also used cdna array data of ss2- supernodulating soybean mutant, response to this bacterium. among the differentially expressed nbs-lrr genes in each transcriptome set, glyma01g was differentially expressed at all four transcriptomes and glyma05g and glyma06g were differentially expressed at three transcriptomes, suggesting that those nbs-lrr genes may work against broad range of pathogen attack. some of nbs-lrr genes had specific responses to different sources of inoculum.  we analyzed and compared the locations of nbs-lrr genes and disease resistance qtl in the soybean genome to determine the relationship between nbs-lrr genes and resistance functions that have been reported in model plants. the correlation between nbs-lrr genes and disease resistance qtl in the 2-mb regions flanking nbs-lrr genes was high enough to support the notion that these genes participate in disease resistance. since the recently duplicated nbs-lrr genes also retained disease resistance qtl, their functional relevance to disease resistance is even more convincing. we also proposed a model for the order of nbs-lrr gene duplications that includes recent duplication and tandem duplication events. moreover, in the rna-seq data of a soybean blp-resistant and -susceptible nil set treated with xag, we observed a significant fold-change in nbs-lrr gene expression between the nils. two of the differentially expressed nbs-lrr genes were located in the previously reported blp-resistant qtl region, indicating that nbs-lrr genes might be located downstream of the rxp locus that confers resistance to blp. by developing molecular markers for nbs-lrr domains, it should be possible to integrate disease resistance against a diverse range of pathogens to generate an elite soybean cultivar. 
moreover, we propose models for how nbs-lrr genes were duplicated, and apply ks values for each nbs-lrr gene cluster. r genes are a key component of genetic interactions between plants and biotrophic bacteria and are known to regulate resistance against bacterial invasion. 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 -induced soybean transcriptome. 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 that flank these genes in each chromosome. nbs-lrr genes co-localized with disease resistance qtl. a total of 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. glycines supports the conjecture that nbs-lrr genes have disease resistance functions in the soybean genome. 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. from these observations, nbs-lrr genes are suggested to contribute to disease resistance in soybean. the significant difference in nbs-lrr gene expression between a resistant near-isogenic line and a susceptible nil after inoculation of xanthomonas axonopodis pv. in addition, nbs-lrr gene expression was significantly different between the blp-resistant nil and the blp-susceptible nil in response to bacterial infection.
 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 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 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 -induced soybean transcriptome.  a total of 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 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.  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. genome duplicationnbs-lrrsoybeantranscriptome analysis 