an integrated transcriptome atlas of the crop model...

An integrated transcriptome atlas of the crop modelGlycine max, and its use in comparative analyses in plants

Marc Libault1,*, Andrew Farmer2, Trupti Joshi3, Kaori Takahashi1, Raymond J. Langley2, Levi D. Franklin3, Ji He4, Dong Xu3,

Gregory May2 and Gary Stacey1

1Division of Plant Sciences, National Center for Soybean Biotechnology, C.S. Bond Life Sciences Center,

University of Missouri, Columbia, MO 65211, USA,2National Center for Genome Resources, 2935 Rodeo Park Drive East, Santa Fe, NM 87505, USA,3Computer Science Department, C.S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA, and4Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA

Received 18 January 2010; revised 25 March 2010; accepted 31 March 2010; published online 14 May 2010.*For correspondence (fax +573 884 9676; e-mail [email protected]).

SUMMARY

Soybean (Glycine max L.) is a major crop providing an important source of protein and oil, which can also be

converted into biodiesel. A major milestone in soybean research was the recent sequencing of its genome. The

sequence predicts 69 145 putative soybean genes, with 46 430 predicted with high confidence. In order to

examine the expression of these genes, we utilized the Illumina Solexa platform to sequence cDNA derived

from 14 conditions (tissues). The result is a searchable soybean gene expression atlas accessible through a

browser (http://digbio.missouri.edu/soybean_atlas). The data provide experimental support for the transcrip-

tion of 55 616 annotated genes and also demonstrate that 13 529 annotated soybean genes are putative

pseudogenes, and 1736 currently unannotated sequences are transcribed. An analysis of this atlas reveals

strong differences in gene expression patterns between different tissues, especially between root and aerial

organs, but also reveals similarities between gene expression in other tissues, such as flower and leaf organs.

In order to demonstrate the full utility of the atlas, we investigated the expression patterns of genes implicated

in nodulation, and also transcription factors, using both the Solexa sequence data and large-scale qRT-PCR.

The availability of the soybean gene expression atlas allowed a comparison with gene expression documented

in the two model legume species, Medicago truncatula and Lotus japonicus, as well as data available for

Arabidopsis thaliana, facilitating both basic and applied aspects of soybean research.

Keywords: soybean, gene expression atlas, comparative genomic, transcription factors, nodulation.

INTRODUCTION

After grasses, legumes are the most economically impor-

tant plant family based on their consumption in human and

animal nutrition. In addition, the use of legumes in biofuel

production will further increase the economic impact of this

plant family. These characteristics justify a substantial effort

by the research community to better understand legume

biology. An attribute of most legumes is the development of

a symbiotic interaction with soil bacteria (rhizobia) that fix

and assimilate atmospheric dinitrogen (atmN2). This symbi-

osis is based on the chemical recognition of diffusible sig-

nals by both partners, which determines the specificity of

the interaction (Oldroyd and Downie, 2008). For example,

the recognition of the lipo-chitin Nod factor, produced by

rhizobia, by the root hair cells of the compatible host leads

to plant morphological and biochemical changes (e.g. root

hair cell curling, cortical cell division, induction of Nod

factor-responsive plant genes and calcium spiking in root

hair cells). These changes are the first signs of the devel-

opment of a new plant organ, the nodule, where the bac-

teria differentiate into bacteroids and reduce atmN2. In

exchange, the plant provides a steady supply of carbon to

the bacteroids.

As part of the effort to better understand legume biology,

the genome sequences of three legume species are now

complete, or nearly complete: that is, Lotus japonicus

(Lotus; http://www.kazusa.or.jp/lotus), Glycine max (soy-

bean; http://www.phytozome.net/soybean) and Medicago

truncatula (Medicago; http://www.medicago.org/genome).

Schmutz et al. (2010) recently described the complete soy-

bean genome sequence. In each case, a large number of

86 ª 2010 The AuthorsJournal compilation ª 2010 Blackwell Publishing Ltd

The Plant Journal (2010) 63, 86–99 doi: 10.1111/j.1365-313X.2010.04222.x

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genes were predicted. The availability of these genome

sequences now enables a variety of functional genomic

methods to characterize these genes and their related

functions. For example, large-scale cDNA sequencing tech-

nologies [e.g. 454 Life Sciences (Margulies et al., 2005) and

Illumina Solexa platforms (Bennett et al., 2005)] provide a

means to accurately profile gene expression (e.g. Libault

et al., 2010). In the past, gene expression atlases were

established in Arabidopsis thaliana (Schmid et al., 2005),

Oryza sativa (Nobuta et al., 2007; Jiao et al., 2009), M. trun-

catula (Benedito et al., 2008) and L. japonicus (Hogslund

et al., 2009) by using massive, parallel-signature sequencing

and array-hybridization technologies.

In this study, the high-throughput Illumina Solexa

sequencing platform was used to develop a gene expression

atlas of the soybean genome. cDNAs derived from a total of

nine different soybean tissues were sequenced. Included in

the soybean gene atlas are five additional data sets,

described by Libault et al. (2010), for a combined total of

14 different conditions (tissues). This provides an unprece-

dented coverage of the transcriptome, including documen-

tation of expression from annotated pseudogenes and

unannotated genes, and also provides accurate quantifica-

tion of low abundant transcripts (Cheung et al., 2006; Weber

et al., 2007; Libault et al., 2010). To demonstrate the utility of

the soybean gene expression atlas, we focused specifically

on expression in root hair cells, as well as on meristem-

specific genes and expression of transcription factor (TF)

genes. The results from the soybean gene expression atlas

were also compared with previously published expression

data from A. thaliana, M. truncatula and L. japonicus. For

example, the comparison to the well-annotated A. thaliana

genome identified putative soybean genes involved in the

determination of floral organs and the maintenance of the

shoot apical meristem (SAM). The availability of the soybean

gene expression atlas should facilitate additional studies on

the basic biology of soybean, while also supporting applied

research to improve soybean agronomic performance.

RESULTS AND DISCUSSION

Sequence-based transcriptome atlas of soybean:

an overview

We used the Illumina Solexa sequencing platform to quan-

tify the expression of soybean genes (i.e. the number of

sequence reads/million reads aligned) in nine different

conditions: root hair cells isolated 84 and 120 h after sowing

(HAS), root tip, root, mature nodules, leaves, SAM, flower

and green pods. Our choice to include root hair cells isolated

at two different time points in this analysis was motivated by

the changes in their transcriptome during development

(Libault et al., 2010). Between 4.18 and 6.84 million reads of

around 36 bp were generated for each of the nine condi-

tions. Among them, 45.8–82.6% of the reads aligned with

less than five loci on the soybean genome (Table 1). Such

variation resulted from the high and low numbers of

unaligned and repetitive reads (i.e. from matches with more

than five loci) in pod (54.2% of the total reads) and flower

samples (17.4% of the total reads), respectively. We classi-

fied the sequence reads aligned with less than five loci on

the soybean genome into two different groups based on the

number of matches identified against the soybean genome

[i.e. non-unique reads (from two to five loci) and unique

reads (only one soybean locus); Table 1]. To insure accuracy

in the quantification of expression in the different tissues

tested, only the sequence reads matching uniquely against

the soybean genome were used. A total of 51 529 annotated

soybean genes (74.5% of the 69 145 putative, annotated

soybean genes) were found to be expressed in at least one

condition (Table S1). Included in the present analysis are

five additional data sets described by Libault et al. (2010) –

i.e. root hairs harvested 12, 24 and 48 h after Bradyrhizobi-

um japonicum inoculation (HAI); 24-HAI mock-inoculated

root hairs; and 48-HAI inoculated stripped roots (Table S2) –

resulting in the documentation of expression for a total of

52 947 annotated genes. No gene expression in any of the 14

conditions was detected for 16 198 annotated genes,

Table 1 Distribution of Illumina-Solexa36-bp reads according to their alignmentagainst the Glycine max (soybean)genome Sample

G. maxunique

G. maxnon-unique(2–5 matches)

Unaligned andhighly repetitivereads (>5 matches) Total reads

Root tip 3 235 689 850 750 1 068 142 5 154 581Root 3 790 433 884 257 1 432 754 6 107 44484-HAS root hairs 2 828 246 719 626 2 063 637 5 611 509120-HAS root hairs 4 086 965 1 052 457 1 698 787 6 838 209Nodule 3 401 083 936 037 1 999 389 6 336 509Leaves 2 813 916 1 202 914 1 279 012 5 295 842Shoot apicalmeristem

3 947 566 1 041 894 1 488 700 6 478 160

Flower 3 372 444 902 730 901 116 5 176 290Green pods 1 462 809 453 340 2 268 639 4 184 788

HAS, h after sowing.

Soybean transcriptome atlas 87

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suggesting that these genes were not expressed, were

expressed at a level below our detection limit or were

expressed only under highly restricted conditions (Table

S2). The data also shows expression from 7314 different

soybean loci currently lacking any gene annotation (Table

S2). Considering only the nine conditions sequenced as part

of the current study, the data demonstrate expression from

7174 currently unannotated regions (Table S1). A number of

root hair genes were found to be specifically expressed upon

inoculation with B. japonicum, as documented by Libault

et al. (2010).

The soybean genome annotation, as described by

Schmutz et al. (2010), refers to 46 430 soybean genes

predicted with high confidence, with the remaining genes

predicted with low confidence. We compared our gene list

for which no detectable expression was found across 14

conditions with the list of low-confidence genes. From the

list of 16 198 putative genes lacking expression, 12 673

(78.2%) were predicted with low confidence in the current

soybean genome annotation (Table S3). The presence of an

expressed sequence tag (EST) or full-length cDNA sequence

led to the annotation of the remaining 3525 genes with high

confidence (Table S3). Having reviewed the conditions in

which these 3525 transcripts were detected, we conclude

these genes were expressed under highly restricted condi-

tions, such as at very specific stages of organ development

or in specific response to abiotic stress, such as drought

stress. Therefore, it is likely that most of the 12 673 low-

confidence genes, which lack expression, are pseudogenes.

Soybean is an allotetraploid that has undergone at least

two rounds of whole genome duplication, with the most

recent having occurred approximately 13 Mya (Schlueter

et al., 2004, 2007; Gill et al., 2009). In a previous study, we

demonstrated cases in which the homeologous gene pairs

showed significant divergence in their expression (Libault

et al., 2010). In order to examine this on a whole genome

basis, we established syntenic relationships between 19 533

annotated genes (28.2% of the annotated soybean genes) to

establish their homeology (Table S4). Among the 12 673

predicted pseudogenes, we identified homeologs expressed

at some level in all conditions tested for only 61 (<1%;

Table S5). Such results are consistent with current theories

of gene evolution, where, after whole genome duplication,

gene fates include silencing or neofunctionalization of one

of the two copies (Adams, 2007).

A number of sequence reads matched against the 7314

loci currently lacking gene annotation (Table S2). The

majority of these loci (7127) were found in regions assem-

bled as part of the chromosome pseudomolecules, whereas

the remainder (187) were located on currently unanchored

scaffolds. In a previous study, we demonstrated the use of

high-throughput cDNA sequencing to improve the current

soybean genome annotation (Libault et al., 2010). Therefore,

we mined 20 kbp of the genomic DNA sequence around

each of the 7127 regions found to have gene expression.

Using FGENESH, we predicted putative protein-coding

genes for 6059 of the 7127 loci (85%). Among them, 4323

of the gene predictions overlapped existing annotated

genes, resulting in the 5¢ or 3¢ expansion of the currently

annotated cDNA sequences (Table S6). The remaining 1736

genes predicted by FGENESH did not overlap currently

annotated genes, suggesting the existence of new protein-

coding genes. We used Interproscan (Zdobnov and Apwe-

iler, 2001) software to identify the signature domains of the

encoded proteins: 542 and 1194 genes encode protein with

and without conserved domains, respectively (Table S7).

Altogether, our analysis suggested that 57 352 soybean

genes are transcribed (i.e. 55 616 out of the 69 145 putative

genes in the current, published soybean genome annota-

tion; the remaining 13 529 are putative pseudogenes, plus

1736 newly annotated genes).

Tissue-specific gene expression

Benedito et al. (2008) noticed large differences in the tran-

scriptome between one M. truncatula organ compared with

another, based on a number of DNA microarray hybridiza-

tions. Similarly, Schmid et al. (2005) and Aceituno et al.

(2008) concluded that the A. thaliana transcriptome strongly

varied from one organ to another. These studies suggest

that the identity of specific plant organs is derived from the

respective transcriptome. In soybean, across the nine tis-

sues tested in the current study, the number of annotated/

unannotated sequences transcribed was similar from one

tissue to another (min. 52.4% in pod; max. 61.2% in the

SAM; Table 2). Altogether, these percentages were slightly

lower than those reported in M. truncatula (55–63%;

Benedito et al., 2008) and A. thaliana tissues (55–67%;

Schmid et al., 2005). Such differences might be a direct

consequence of the non-negligible number of putative

pseudogenes mentioned above, and might also reflect the

residual background or cross-hybridization existing when

using array hybridization technology. A similar number of

soybean genes were expressed in a single cell type (root

hair) and in multicellular organs (e.g. 45 717, 40 034, 43 377

and 46 173 soybean genes were expressed in flower, pod,

84- and 120-HAS root hair cells, respectively; Table 2). Jiao

et al. (2009) previously reported that transcripts undetect-

able in cDNA derived from shoot, root or germinated seeds

could be detected if mRNA was sampled from a single cell

type from this organ. Therefore, we hypothesize that the

heterogeneous population of differentiated cells composing

a soybean organ results in a larger diversity of expressed

sequences, but also in the poor detection of low-abundance

transcripts. In contrast, cDNA derived from the single cell

root hairs allows for the detection of low-abundance tran-

scripts, because of a lack of dilution from other tissues, and

the homogeneity of the tissue sampled. Apparently, these

opposing factors result in approximately the same number

88 Marc Libault et al.


of transcripts sequenced from a single cell type and multi-

cellular organ samples.

To better establish the identity of the different soybean

tissues, we generated a heat map based on the correlation

between their transcriptomes (Figure 1a). Based on this

map, the nine organs can be divided into three different

groups: (i) root tip, root and root hairs; (ii) SAM, pod, flower

and leaf; and (iii) nodule. The lack of correlation between

root-related tissues and aerial organs was previously

reported by Benedito et al. (2008) in M. truncatula. These

results are likely to reflect the divergence in function

between the root and aerial portions of the plant. Consistent

with this notion, other tissues show significant overlap in

their transcriptomes. For example, gene expression in the

soybean pod and SAM was strongly correlated (Figure 1a).

The transcription profile can also reflect development. For

example, the flower and leaf transcriptomes were closely

correlated. In 1790, Goethe hypothesized that floral organs

were modified leaves (Coen, 2001). Indeed, four MADS-box

TF genes named SEPALLATA1–4 (SEP1, SEP2, SEP3 and

SEP4, previously named AGL2, AGL4, AGL9 and AGL3) were

characterized for their role in the acquisition of floral organ

identity, as sep mutants develop leaf-like organs instead of

flowers (Honma and Goto, 2001; Pelaz et al., 2001; Ditta

et al., 2004). These results suggest that organ-specific gene

expression could be the result of the action of relatively few

regulatory genes.

The soybean nodule transcriptome showed little correla-

tion with other organs, with the exception of mature roots. It

is interesting to note that the soybean root hair transcrip-

tome was not strongly correlated with that of the whole root,

nor with any of the other soybean tissues analyzed (Fig-

ure 1a). This is likely to reflect the specialization of this single

cell type, but also the tissue dilution that occurred by

sampling the other organs, especially the roots.

In a previous study, Aceituno et al. (2008) showed that the

Arabidopsis organ transcriptomes were not strongly

affected in response to environmental changes. Therefore,

the unique transcriptomic patterns exhibited by the various

soybean organs are likely to reflect their unique identity, and

are not the result of specific environmental conditions.

Therefore, in order to better understand soybean organ

development, we analyzed the soybean gene expression

atlas to identify those genes that were ubiquitously

expressed across the nine tissues, and those showing a

very high level of tissue-specific expression. The results of

this analysis showed that 58 703 soybean genome loci,

including both annotated and unannotated regions, were

expressed in at least one of the nine soybean tissues.

Roughly half of these genes (28 374) were transcribed

ubiquitously (Table S8). In theory, organ identity could

depend on both the level of expression of ubiquitously

expressed genes and the organ-specific expression of

selected genes. To address this issue, we first compared

the overall expression levels of the 28 374 ubiquitous genes

between the nine conditions (Figure 1b). As shown in

Figure 1, this analysis revealed significant differences in

the absolute expression levels of the 28 374 ubiquitously

expressed genes. These data also leave the impression that

few, if any, soybean genes are stably expressed in the

various soybean tissues. In order to examine this directly,

we included the additional five conditions from the publi-

cation by Libault et al. (2010) to define genes constitutively

expressed by the following criteria: (i) the gene was

expressed in all 14 conditions tested; (ii) the fold change in

the relative expression levels was not higher than three

between conditions where genes were the most and the

least expressed. These criteria identified 2532 putative

constitutive genes (Figure S1; Table S9). Among these,

PFAM, KOG or PANTHER conserved domains were identi-

fied for 2187 genes, leading to the identification of 140 TF

genes [2.5% of the 5671 predicted TF genes in the soybean

genome; Schmutz et al., 2010; Libault et al., 2009a; PFAM,

KOG and PANTHER domain predictions are available from

ftp://ftp.jgi-psf.org/pub/JGI_data/Glycine_max/Glyma1/Gly-

ma1_domains). Such a relatively low number is a direct

reflection of the specific role of TF genes in the determina-

tion of plant organ identity.

Table 2 Distribution of expressed and notexpressed annotated and unannotatedsequences across nine Glycine max (soy-bean) tissues

Number of expressed sequencesNumber of silenced sequences(i.e. no transcript detected)

Annotatedsequences (%)

Unannotatedsequences (%)

Annotatedsequences (%)

Unannotatedsequences (%)

Root hair 84 HAS 38 645 (50.54) 4732 (6.19) 30 500 (39.89) 2582 (3.38)Root hair 120 HAS 40 849 (53.43) 5324 (6.97) 28 296 (37.01) 1990 (2.60)Root tip 36 882 (48.24) 4624 (6.05) 32 263 (42.20) 2690 (3.52)Root 40 576 (53.07) 5126 (6.71) 28 569 (37.37) 2188 (2.86)Nodule 36 369 (47.57) 4438 (5.81) 32 776 (42.87) 2876 (3.76)Leaf 37 600 (49.18) 4518 (5.91) 31 545 (41.26) 2796 (3.66)Shoot apical meristem 41 415 (54.17) 5341 (6.99) 27 730 (36.27) 1973 (2.58)Flower 40 863 (53.44) 4854 (6.35) 28 282 (36.99) 2460 (3.22)Pod 36 325 (47.51) 3709 (4.85) 32 820 (42.92) 3605 (4.72)



We also sought to identify soybean transcripts expressed

solely in one soybean organ. These genes were classified

into four groups depending on their tissue specificity:

preferentially (‡3- and <10-fold changes between the expres-

sion levels of the most highly expressed and second most

highly expressed genes), specifically (‡10- and <100-fold

change), very specifically (‡100- and <1000-fold change) and

exclusively identified in one tissue (‡1000-fold change).

These criteria identified 5313, 1374, 147 and nine genes that

were preferentially, specifically, highly specifically and

exclusively expressed in one tissue, respectively (Figure 2a;

Table S10). Benedito et al. (2008) reported that M. truncatula

seeds and nodules possessed the largest number of tissue-

specific genes. Hogslund et al. (2009) found that L. japoni-

cus flowers exhibited the highest degree of tissue-specific

gene expression. In soybean, the largest numbers of tissue-

specific genes were identified in nodules and flowers (1465

and 1145 genes, respectively; ‡3-fold change; Figure 2b).

Using more stringent parameters, soybean nodule, flower

and pod were the organs that were strongly enriched in

highly tissue-specific genes (61, 54 and 29 genes, respec-

tively; ‡100-fold change; Figure 2b). Given the lack of

correlation in overall gene expression between the nodule

transcriptome and the other tissues sampled (Figure 1), it

was not surprising to identify this tissue among those

showing the highest level of organ-specific gene expression.

In contrast, it would appear that the correlation in the overall

level of gene expression between flowers and leaves

(Figure 1) hides a significant level of flower-specific gene

expression (1145 flower-specific genes; ‡3-fold change).

These genes are clearly strong candidates for determining

the specific functional components of the flower. The overall

soybean transcriptome was also mapped relative to the

position of the respective genes in the assembled soybean

genome. As an aid to visualization of these data, we

established a color-code map for each chromosome, and

for each tissue, to reflect the overall gene expression level

(Figure 3). These data, as well as the data from the earlier

Libault et al. (2010) study, can best be viewed as part of the

soybean genome browser available at http://digbio.

missouri.edu/soybean_atlas. Visualizing the data in this

way rapidly demonstrates that most of the protein-coding

genesandalso themoststronglyexpressedgenesare located

on the chromosome arms, whereas expression from the

less gene-dense pericentromeric regions is much reduced.

Root hair and meristem-specific soybean genes

Root hairs are single cell extensions of the root epidermis,

and play a key role in water and nutrient uptake. However,

in legumes, they play a secondary role as the primary site

for rhizobial infection, leading to the development of

nitrogen-fixing nodules. Root hairs also exhibit polar cell

expansion. In a previous study, we identified around 2000

soybean genes regulated in root hair cells in response to

B. japonicum infection (Libault et al., 2010). In order to

extend our understanding of the soybean root hair cell, we

also sought to identify genes that were specifically

expressed in root hairs. Using the same criteria outlined

above, we identified 451 soybean sequences that were

preferentially expressed in root hairs, including 69 and

three root hair-specific and highly specific genes, respec-

tively (Table S11). Using PFAM, KOG and PANTHER

domain predictions, we predicted the functions of 304 of

the 451 annotated genes. Some gene families are clearly

over-represented in this list of root hair-specific genes. For

example, cellulase (three genes, 1%), pectinesterase (four

genes, 1.3%), peroxidase (eight genes, 2.6%) and extensin

genes (four genes, 1.3%) were gene families preferentially

84HAS RH

120HAS RH

Root

Root tip

Nodule

SAM

Leaf

Flower

Pod

84HAS RH

120HAS RH

Root

Root tip

Root t

ip

Root t

ip

Nodule

SAM

Leaf

Flower

Pod

–0.75–1

–0.5–0.250

0.751

0.50.25

–0.75–1

–0.5–0.250

0.751

0.50.25

(a)

(b)

Figure 1. Comparison of the transcriptomes of various Glycine max (soy-

bean) tissues.

Ward hierarchical clustering of log2 transformed gene distribution in nine

diverse soybean organs [root hair cells isolated 84 and 120 h after sowing,

root tip, root, mature nodules, leaves, shoot apical meristem (SAM), flower

and green pods], based on Pearson correlation coefficients. The entire

soybean tissue transcriptome (a) or the 28 374 annotated soybean genes

identified to be expressed in all nine tissues (b) were used to generate two

distinct maps. The color scale indicates the degree of correlation (green, low

correlation; red, strong correlation). The heat map was generated using

JMP GENOMICS 4.0.



expressed in root hairs (v2 < 1 · e)50). These families rep-

resented only 0.06% (28 genes), 0.3% (144 genes), 0.4%

(205 genes) and 0.03% (16 genes) of the 47 724 soybean

annotated genes for which predicted functions were

established. It is likely that the expression of these gene

families reflects the polar growth of the root hair cells,

where continuous cell wall expansion is required, and

where reactive oxygen species are essential (Baumberger

et al., 2001, 2003; Bucher et al., 2002; Carol and Dolan,

2006).

Shoot apical and root meristems are the locations of the

intense cell division required for plant growth. We combined

the transcriptomes of these two meristematic tissues to

identify 28 soybean genes that were preferentially expressed

in the soybean meristematic zones (Table S11). Among

these, 18 genes encode proteins with conserved domains,

Annotated and unannotated sequences

Unannotated sequences

TF genes

0

1000

2000

3000

4000

5000

6000

7000

8000(a)

(b)

Tissue specificity

Gen

e n

um

ber

Fold-change

3 10 100 1000

6843

1

9

68

156213

120

1530899

6240

Gen

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ber

3 fold-change cut-off 10 fold-change cut-off

0

200

400

600

800

0

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100 fold-change cut-off 1000 fold-change cut-off

0

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ber

Figure 2. Gene expression specificity across

nine Glycine max (soybean) tissues.

(a) All soybean transcripts (dashed grey line),

unannotated transcripts (black line) and tran-

scription factor transcripts (grey line) were clas-

sified into four groups according to their tissue

specificity: preferentially (‡3- and <10-fold

changes between the expression levels of the

most highly expressed and second most highly

expressed genes), specifically (‡10- and <100-

fold changes), very specifically (‡100- and

<1000-fold changes) and exclusively identified

in one tissue (‡1000-fold change).

(b) Distribution of the number of overall soy-

bean transcripts in the nine different soybean

tissues tested according to their level of speci-

ficity (3-, 10-, 100- and 1000-fold change cut-off).



including three encoding a predicted kinesin, a regulator of

cytokinesis (Muller et al., 2006). In addition, eight transcrip-

tional and translational regulators (e.g. bHLH, SBP, Zf-HD

TFs; RNA polymerase subunit, PIWI and ribosomal protein)

were also preferentially expressed in soybean meristematic

zones, suggesting strong transcriptional and translational

activities, which are probably also involved in maintaining

the high cell division rate and in controlling cell determina-

tion, differentiation and elongation.

Expression pattern of soybean nodulation-related genes

A unique feature of legumes, including soybean, is their

formation of a novel root organ, the nodule, in response to

rhizobial infection. Previously, Schmutz et al. (2010) anno-

tated approximately 100 soybean genes as those predicted

to play a role in nodulation, based on an extensive review of

the nodulation literature. Among these 100 putative nodu-

lation-related soybean genes, 14 were regulated during root

Figure 3. Color code maps of gene expression across the 20 Glycine max (soybean) chromosomes.

For each chromosome, gene expression (i.e. number of sequence reads per million reads aligned: <0.5, yellow; 0.5–2, orange; 2–5, light green; 5–10, green; 10–25,

greenish brown; 25–50, brown; 50–100, brownish red; 100, red) is indicated for nine different tissues (from top to bottom: root hairs 84 h after sowing, root hairs

120 h after sowing, nodule, root, root tip, shoot apical meristem, leaf, flower and pod). The final color strip at the bottom of each chromosome represents gene

density (i.e. number of genes per 100 kbp; 0–15 or higher fi black-white). These maps were generated by using the comparative map and trait viewer (CMTV)

software.



hair cell infection by B. japonicum (Libault et al., 2010). An

examination of the soybean gene expression atlas showed

that only one, Glyma13g12440 (a putative GmN56 gene;

Schmutz et al., 2010), of the 100 soybean nodulation-related

genes (Table S12) was not expressed in any of the nine tis-

sues sampled. In a previous study of soybean nodulation,

Kouchi and Hata (1995) clearly identified a transcript for

GmN56. Consequently, we looked at the expression of Gly-

ma13g12490 and Glyma13g12500, two homeologous genes

to Glyma13g12440 (Schmutz et al., 2010). Both genes were

expressed and to a significantly higher level in nodules

(Figure S2; Table S12). Therefore, it is likely that the GmN56

EST identified by Kouchi and Hata (1995) arose from either

Glyma13g12490 or Glyma13g12500, and not from Gly-

ma13g12440. Of the remaining 100 putative nodulation-

related genes, 70 genes were not expressed preferentially in

nodules (£3-fold change between nodule and the eight

remaining tissues), including those encoding the putative

Nod factor receptors (NFR1a-b and NFR5a-b), and TFs known

to regulate root hair cell infection (e.g. NSP1 and NSP2)

(Table S12). The induction of the expression of these genes

during root hair infection by B. japonicum (Libault et al.,

2010), but not in mature nodules, is in agreement with their

early role during legume infection (Catoira et al., 2000; Amor

et al., 2003; Madsen et al., 2003; Oldroyd and Long, 2003;

Radutoiu et al., 2003; Kalo et al., 2005; Smit et al., 2005;

Heckmann et al., 2006; Murakami et al., 2006). The remain-

ing 29 genes were preferentially expressed in nodules (‡3-

fold change; Figure S2; Table S12). Among these, 16 and

seven genes were specifically (‡10- and <100-fold changes)

and very specifically (‡100- and <1000-fold changes)

expressed in the nodules (Figure S2; Table S12). Homeolo-

gous pairs of NIN (Glyma04g00210 and Glyma06g00240),

NIN2 (Glyma12g05390 and Glyma11g13390) and CYCLOPS

genes (Glyma01g35260 and Glyma09g34690) were

expressed specifically in soybean nodules (Figure S2; Table

S12). The role of NIN in L. japonicus nodule development

was previously noted by Schauser et al. (1999), whereas

CYCLOPS function during L. japonicus nodule development

was not clearly established (Yano et al., 2008). In addition,

consistent with their initial characterization, 23 encoded

nodulins were also expressed specifically in nodules.

Recently, Haney and Long (2009) identified seven flotillin-

like genes in M. truncatula, which are gene homologs of the

soybean nodulin GmNod53b (Winzer et al., 1999). Two of

the M. truncatula flotillin genes were induced at 24 HAI with

Sinorhizobium meliloti. Utilizing the GmNod53b sequence,

we identified only two, homeologous flotillin genes in soy-

bean (Glyma06g06930 and Glyma04g06830; e-value < e)20).

However, their expression patterns across the nine tissues

were very different. For example, Glyma04g06830 expres-

sion was not detected in any tissues, with the exception of

nodule tissue, where its transcript was barely detected.

Glyma06g06930 was strongly and primarily expressed

in nodules, but also in root hair cells uninoculated by

B. japonicum. In addition, Glyma06g06930 expression was

induced in soybean root hairs at 12 (3.7-fold change), but not

at 24 and 48 HAI, with B. japonicum (Table S2). These data

suggest that the flotillin encoded by Glyma06g06930 is likely

to be orthologous to the genes shown by Haney and Long

(2009) to be crucial to root hair infection by S. meliloti.

Expression patterns of soybean transcription factor genes

The TF genes are of clear interest because they control plant

responses to the environment, as well as developmental

pathways (for a review, see Libault et al., 2009a). For

example, our earlier study (Libault et al., 2010) identified a

number of soybean TF genes in which expression

responded to B. japonicum inoculation. Soybean genes

homologous to MtHAP2.1, MtERN and LjNIN genes, genes

controlling M. truncatula and L. japonicus nodule develop-

ment (Schauser et al., 1999; Combier et al., 2006; Middleton

et al., 2007), were clearly identified based on syntenic rela-

tionships and their nodule-specific expression (Libault et al.,

2009a,b).

The soybean gene expression atlas was mined to identify

TF genes exhibiting tissue-specific expression. This analysis

identified 624 TF genes that were expressed preferentially in

one soybean tissue compared with the eight others, includ-

ing 114, five and one TF genes, specifically, very specifically

and exclusively expressed in one tissue, respectively (Fig-

ure 2a; Table S13).

Examination of this list of 120 TF genes specifically

expressed in at least one tissue (‡10-fold change) identified

a significant number of C2H2 (Zn) and NIN-like TF genes

expressed preferentially in nodules (Figure 4). As described

above, the role of NIN-like genes in legume nodulation is

well established. However, to date, there is no functional

demonstration of a role for C2H2 (Zn) TF genes during

legume nodulation. Our data suggests that this should be

examined more closely. Members of the Homeodomain TF

family were restricted to the SAM, whereas members of the

LIM, MADS and NAC TF families were preferentially

expressed in flowers, suggesting a specific role for these

TF gene families in the normal development of these tissues

(Figure 4). In A. thaliana, a large number of MADS TF genes,

such as SEP1, SEP2, SEP3, SEP4, APETALA1 (AP1), APET-

ALA3 (AP3), PISTILLATA (PI) and AGAMOUS (AG), are key

regulators of flower development (for a review, see Robles

and Pelaz, 2005). Arabidopsis thaliana Homeodomain TF

genes, such as WUSCHEL (WUS) and SHOOTMERISTEM-

LESS (STM), are important in the formation and mainte-

nance of the SAM (Barton and Poethig, 1993; Endrizzi et al.,

1996; Laux et al., 1996; Mayer et al., 1998). Consequently, we

hypothesized that some of the soybean Homeodomain and

MADS TF genes expressed specifically in the SAM and

flower may be orthologs to WUS and STM, and to SEP1,

SEP2, SEP3, SEP4, AP1, AP3, PI and AG, respectively. In



order to establish this orthology, we looked for syntenic

relationships between these gene families in the A. thaliana

and G. max genomes. With the exception of SEP3 and PI

genes, we identified soybean orthologs of the flower and

SAM-related Arabidopsis genes (Figure S3). In most cases,

the recent duplication of the soybean genome logically led

to the identification of two putative orthologs. More surpris-

ingly, the Glyma18g50900 gene was identified as the

potential ortholog of SEP1 and SEP2, whereas the region

encoding Glyma02g13420 was orthologous to both SEP4

and AP1. Such a surprising result suggested the gene pairs

SEP1/SEP2 and SEP4/AP1 probably diverged from common

gene ancestors before the divergence between soybean and

Arabidopsis. To provide further evidence of the orthology

between the soybean MADS and Homeodomain genes

WUS, STM, SEP1, SEP2, SEP4, AP1, AP3 and AG, we mined

the Arabidopsis gene expression data (Hruz et al., 2008) to

compare the expression profiles of the genes in both

organisms. Similarly to A. thaliana, a significant number of

soybean genes putatively involved in flower development

were strongly but not exclusively expressed in flowers

(Figure 5). Among them, four MADS genes (Gly-

ma01g08150, Glyma02g13420, Glyma04g02980 and Gly-

ma06g02990), orthologs to AtAP1, AtSEP4 and AtAP3,

were identified as specifically expressed in flowers (Fig-

ure S3; Table S13). The function of the remaining four

soybean MADS genes and seven Homeodomain genes

expressed specifically in flower and SAM needs to be

investigated. Altogether, this analysis clearly demonstrates

the usefulness of combining genome and transcriptome

comparisons to identify genes playing critical developmen-

tal roles in soybean.

Taking advantage of this analysis, and to validate the

accurate measurement of soybean gene expression by

Illumina Solexa technology, we compared the Illumina

Solexa data set with transcriptomic analyses performed on

11 soybean tissues using the previously published quanti-

tative RT-PCR primer set library, designed against more than

ABI3/VP1

AP2-EREBP

AS2 AUX-IAA-ARF

bHLH

BZIP

C2C2 (Zn) CO-likeC2C2 (Zn) Dof

C2C2 (Zn) YABBY

C2H2 (Zn)

CAMTACCAAT

DHHC (Zn)

GRAS

HomeodomainHOMEOBOXLIM

MADS

MYB

MYB/HD-like

NAC

NIN-like

SBP SRS

TPR

WRKYZf-HD

SAM(1)Pod

(2)

Nodule(11)

SAM(7)

Flower(1)

Flower(5)

Flower(8)

Pod(2)

Nodule(2)

Flower(5)

Nodule(6)

Figure 4. Distribution of Glycine max (soybean) transcription factor genes expressed specifically in one soybean tissue, based on their family membership.

The sub-pies highlight the distribution of specific transcription factor gene families in the different tissues, based on the specificity of their expression.



1000 soybean regulatory genes, including 652 TF genes

(Libault et al., 2009b). In virtually all cases, the qRT-PCR

results validated the measurements made by Illumina

Solexa sequencing. Full details are provided in Appen-

dix S1.

Comparison of the M. truncatula, L. japonicus and G. max

transcriptome

Glycine max, M. truncatula and L. japonicus probably

diverged around 40 Mya, reflecting the extensive micro-

synteny that exists between their genomes (Choi et al., 2004;

Cannon et al., 2006; Young and Udvardi, 2009). This rela-

tionship provides opportunities to transfer genetic knowl-

edge between these three species. However, such

comparisons also need to allow for divergence in the

expression patterns of orthologous genes during legume

evolution, especially given the more recent whole genome

duplication in soybean (Schlueter et al., 2004, 2007), and the

silencing of homeologous genes (Libault et al., 2009a,b;

present study). Consequently, the use of orthology to

deduce common function among the three legume species

will not only require the establishment of a syntenic

relationship, but also the demonstration of similar gene

expression patterns. This is further evidence for the utility of

gene expression atlases for these three species.

The majority of gene expression data available for

M. truncatula and L. japonicus come from a variety of

Affymetrix microarray experiments. Therefore, as a first

step to compare gene expression from these two species

with that of soybean, we sought to identify the ortholo-

gous genes present on the M. truncatula and L. japonicus

Affymetrix arrays, and their counterparts in soybean. To

simplify this analysis, we focused on the 147 annotated

soybean genes expressed very specifically in only one

Root tip

(b)

(a)

Root hair84HAS

Root hair120HAS

Root

Nodule

SAM

Leaf

Flower

Pod

Arabidopsis thalianaCallusCell culture/primary cell

Sperm cellSeedling

CotyledonsHypocotylRadicleImbibed seed

InflorescenceFlowerSiliqueSeedStemNodeShoot apexCauline leaf

RosetteJuvenile leafAdult leafPetioleSenescent leafHypocotylLeaf primordiaStem

RootLateral rootRoot hair zone

Elongation zoneEndodermisEndodermis + cortexEpid. atrichoblastsLateral root capStele

Root tip

00.91.82.73.64.55.46.37.28.1

9

Figure 5. Gene expression patterns of Arabid-

opsis genes involved in the formation and

maintenance of the shoot apical meristem

(SAM) and the determination of flower organs

(a), and their putative orthologs in Glycine max

(soybean) (b).

Genevestigator (Hruz et al., 2008) and the soy-

bean gene atlas were mined to establish the

expression pattern of the Arabidopsis and soy-

bean genes, respectively.



tissue (‡100-fold change; Table S10). Subsequently, we

mined the M. truncatula and L. japonicus expression data

for the corresponding orthologs by referencing the respec-

tive gene expression atlases (Benedito et al., 2008; Hogsl-

und et al., 2009). This approach allowed the direct

comparison of 40 soybean genes in five tissues (nodule,

root, leaf, flower and pods) with the corresponding

M. truncatula orthologs in the same five tissues, and the

L. japonicus orthologs in four tissues (nodule, root, leaf

and flower). This comparison showed that 18 soybean

genes share similar tissue specificity with their putative

orthologs in M. truncatula and L. japonicus (Table S14).

This number may simply reflect the difficulty of establish-

ing true orthology, or may reflect subfunctionalization or

neofunctionalization of the remaining 22 soybean putative

orthologs. To better establish orthology, we analyzed

microsynteny between the G. max, M. truncatula and

L. japonicus loci encoding the various putative orthologs.

Significant microsynteny was found between three G. max

and M. truncatula and eight G. max and L. japonicus gene

regions (Figure S4; Table S14). For example, microsynteny

was found between the Glyma01g44660 soybean gene

region and the corresponding regions in both M. trunca-

tula (Medtr5g006680) and L. japonicus (CM0591.50.nd).

These three genes were expressed specifically in flowers.

Interestingly, during our analysis we also highlighted

synteny between legume genes not identified during the

initial screen (Figure S4). For instance, in addition to appar-

ent orthology to Glyma07g16290, LjCM0147.870.nc was also

orthologous to Glyma18g40360, a soybean gene preferen-

tially expressed in the nodules, based on the soybean gene

atlas (Figure S4; Table S2). These three genes are predicted

to encode C2H2 (Zn) TFs, consistent with the previously

mentioned abundant expression of this family of TF genes in

nodule tissue. Microsynteny was found between genes in

G. max and M. truncatula, which have very different expres-

sion patterns. For example, Glyma09g41200, Gly-

ma18g44670 and Glyma18g44680 are soybean genes

expressed specifically in flowers, and lie on a region of the

soybean genome microsyntenic to Medtr7g080300, which

also appears microsyntenic to the soybean loci encoding

Glyma01g32750 and Glyma01g32760, two soybean genes

expressed in a variety of organs (Figure S4; Tables S2).

This example suggests the subfunctionalization of

Glyma01g32750 and Glyma01g32760 after the divergence

of G. max and M. truncatula. As Glyma18g44670–Gly-

ma18g44680 and Glyma01g32750–Glyma01g32760 proba-

bly arose by tandem duplication, we assume that the

subfunctionalization of Glyma01g32750 and Gly-

ma01g32760 occurred after the duplication of the soybean

genome, but before their tandem duplication. The above

example further illustrates the value of genome and tran-

scriptome comparisons that allow interesting conclusions

concerning the orthology of specific genes, and their

evolutionary history. Space prevents us from presenting a

variety of additional examples. At this point, the annotation

of the G. max, M. truncatula and L. japonicus genomes

clearly needs improvement. We predict that the full integra-

tion of the syntenic and transcriptome analysis of these

three genomes will ultimately lead to the systematic iden-

tification of legume orthologs. At that point, it will be

possible to rapidly transfer genetic and functional knowl-

edge derived in one species to the others.

EXPERIMENTAL PROCEDURES

Bacterial cultures

Bradyrhizobium japonicum USDA110 was grown at 30�C for 3 daysin HM medium (Cole and Elkan, 1973), supplemented with yeastextract (0.025%), D-arabinose (0.1%) and chloramphenicol (0.004%).Before plant inoculation, B. japonicum cells were pelleted (2000 g

for 10 min), washed and diluted with sterile water to OD600 = 0.1.

Plant culture

All tissues described below were isolated from soybean G. max (L.)Merr. cultivar ‘Williams 82’ plants. For each tissue, three indepen-dent biological replicates were performed on a different set of plantsto ensure the reproducibility of the plant tissues analyzed (i.e. seedswere sowed three times on different days, and tissues were har-vested as described below).

Soybean seeds were surface sterilized according to the methoddescribed by Wan et al. (2005), and were sowed on nitrogen-freeB&D agar medium (Broughton and Dilworth, 1971). Untreatedroot hair cells and stripped roots used for qRT-PCR were isolatedfrom 3-day-old seedlings, as described by Wan et al. (2005). Asimilar protocol was used to isolate 84- and 120-HAS root hairs(Libault et al., 2010; 84- and 120-HAS root hairs were mock-inoculated root hairs isolated 12 and 48 h after being sprayedwith water).

Other tissues were isolated as described below. The 3-day-oldseedlings were germinated between moist Whatman filter paper.Root tips were harvested on these seedlings. To produce othertissues, germinated seedlings were transferred to the glasshouseunder long-day conditions (16-h day/8-h night) at 27�C on Promix Bxsoil (Premier Horticulture, http://www.premierhort.com). Fourteen-day-old SAM (V2 stage), 18-day-old trifoliate leaves, stem and roots(V2 stage), flowers (R2 stage), and seeds and pods (R6 stage) wereharvested. Nodules were harvested 32 days after the inoculationof 1 ml of B. japonicum suspension (OD600 = 0.1) on transferred3-day-old seedlings.

RNA extraction, DNase treatments, and reverse

transcription

Total RNA was isolated using Trizol Reagent (Invitrogen, http://www.invitrogen.com) according to the manufacturer’s instructions,followed by a chloroform extraction to improve their purity. TotalRNAs were treated and reverse-transcribed differentially regardingthe technology used to quantify cDNA levels.

qRT-PCR. The qRT-PCR reactions including the different controlswere performed as described by Libault et al. (2009b).

Solexa sequencing. For each condition, similar quantities of totalRNA isolated from three independent biological replicates werepooled together. After first- and second-strand cDNA synthesis, the



cDNAs were end repaired prior to ligation of Solexa adaptors. Theproducts were sequenced on a Solexa platform.

Quantitative PCR reaction conditions and data analysis

The qRT-PCR reactions were performed as described by Libaultet al. (2009b). The specificity of primer sets was confirmed byanalyzing the dissociation curve profile of each qRT-PCR amplicon,and the efficiency of primers (Peff) was quantified using LinRegPCR(Ramakers et al., 2003). Cons6, encoding an F-box protein (Libaultet al., 2008), was used to normalize the expression levels of puta-tive soybean regulatory genes. The cycle threshold (Ct) value of thereference gene was subtracted from the Ct values of the test geneanalyzed (DCt). The expression level (E) of each gene was calcu-lated according to the equation: E = Peff

()DCt). The average of

the expression levels between three different replicates wascalculated.

Solexa read alignment, statistical analysis and data

representation

Illumina Genome Analyzer II image data were base-called andquality filtered using the default filtering parameters of the IlluminaGA Pipeline GERALD stage (Illumina, Inc., http://www.illu-mina.com). Alignments of passing 36-mer reads to all contigs of theGlyma1 8x Soybean Genome assembly (Soybean Genome Project,http://www.jgi.doe.gov) were performed using GSNAP (Wu andNacu, 2010), an alignment program derived from GMAP (Wu andWatanabe, 2005), with optimizations for aligning short transcriptreads from next-generation sequencers to genomic referencesequences. Alignments were processed using the Alpheus pipeline(Miller et al., 2008), keeping only alignments that had at least 34 outof 36 identities, and had no more than five equivalent best hits. Readcounts used in expression analyses were based on the subset ofuniquely aligned reads that also overlapped the genomic spans ofthe Glyma1 gene predictions. Read counts for a given sample werenormalized by using values for a gene’s uniquely aligned readcounts per million reads uniquely aligning within that sample.

The raw and normalized Solexa data are available on http://digbio.missouri.edu/soybean_atlas, whereas the entire set ofSolexa sequences used in our studies can be downloaded fromthe NCBI SRA browser (accession number SRA012188.1; http://www.ncbi.nlm.nih.gov/Traces/sra).

The color code maps of the soybean transcriptome across the 20chromosomes were generated by using the comparative map andtrait viewer (CMTV) software (Sawkins et al., 2004).

Synteny analysis

To establish microsynteny between G. max and A. thaliana, aminoacid sequences of the A. thaliana candidate genes and at least the 20genes surrounding them were blasted against soybean genomesequences. Using a P < e)20 as a cut-off, BLAST results and geneannotation were analyzed manually to established microsynteny.

To compare the gene expression of orthologous genes betweenG. max, M. truncatula and L. japonicus, we first mapped themedicago and lotus Affymetrix probe sets against their respectivegenomes based on NCBI BLASTN searches. Only probe sets with atleast nine matching probes, sited at least 22-bp up- or downstreamof a 4000-bp region, were considered for further analysis. TheBLAST of the predicted soybean transcripts against the MedicagoMt v3.0 (http://www.medicago.org/genome) and Lotus pseudoge-nomes (http://www.kazusa.or.jp/lotus) associated with the mappingof the Medicago and Lotus Affymetrix probe sets led to a directcomparison of the expression of the soybean, Medicago and Lotusgenes. When genes shared a similar tissue specificity, we high-

lighted their orthology by establishing a microsynteny relationshipbetween them using the same methodology described above.

Graphics showing microsynteny relationships were generated byusing CMTV (Sawkins et al., 2004).

ACKNOWLEDGEMENTS

We thank Melanie Mormile, Sandra Thibivilliers and CharlieP. Jones for their critical reading of the manuscript. We also thankChia Rou Yeo for technical assistance and Shaoxing Wang forproviding some total RNA samples. We are also grateful to theMedicago Genome Sequence Consortium (MGSC) for providingM. truncatula genomic sequences. This work was funded by a grantfrom the National Science Foundation (Plant Genome Program,#DBI-0421620). TJ, LDF and DX were supported by United SoybeanBoard grant #8236.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Expression levels of putative soybean (Glycine max)constitutive genes in 14 different conditions (y-axis) compared withthe average of their expression levels across the 14 conditions(x-axis).Figure S2. A total of 29 soybean (Glycine max) nodulation-relatedgenes were expressed preferentially in mature nodules.Figure S3. Syntenic relationship between Glycine max and Arabid-opsis thaliana genes involved in flower organ determination andmaintenance of the shoot apical meristem.Figure S4. Syntenic relationship between Glycine max (soybean),Medicago truncatula and Lotus japonicus genes surrounding soy-bean-nodule- and flower-specific genes.Figure S5. Comparison of the transcriptomes of 1016 soybeanregulatory genes by qRT-PCR tissues.Table S1. Gene expression pattern of predicted and unannotatedGlycine max (soybean) genes in nine different tissues.Table S2. Gene expression pattern of predicted and unannotatedGlycine max (soybean) genes in nine different tissues, and in roothair and stripped roots in response to Bradyrhizobium japonicum.Table S3. Confidence in gene prediction according to Schmutz et al.(2010) of 16 198 Glycine max (soybean) genes not expressed insoybean tissues, and in the early steps of nodulation.Table S4. Gene expression of Glycine max (soybean) homeologousgenes.Table S5. Gene expression of Glycine max (soybean) homeologousgenes relative to putative pseudogenes.Table S6. Unannotated sequence reads that overlap Glycine max(soybean) annotated genes leading to an improvement of thesoybean gene annotation.Table S7. Identification of the signature domains of the 1736proteins encoded by the putative new Glycine max (soybean)genes.Table S8. Expression levels of Glycine max (soybean) sequencesidentified to be ubiquitously expressed across the nine soybeantissues tested.Table S9. Gene expression and function of putative Glycine max(soybean) constitutive genes across 14 different conditions.Table S10. Identification of Glycine max (soybean) transcriptspreferentially (‡3- and <10-fold changes between the expressionlevels of the most highly expressed and second most highlyexpressed genes; yellow), specifically (‡10- and <100-fold changes;orange), very specifically (‡100- and <1000-fold changes; red) andexclusively (‡1000-fold change; purple) identified in one of the ninetissues tested.



Feng

高亮

Feng

高亮

Table S11. Identification of Glycine max (soybean) transcriptspreferentially (‡3- and <10-fold change; yellow), specifically (‡10-and <100-fold change; orange) and very specifically (‡100- and<1000-fold change; red) identified in soybean root hair cells andmeristems.Table S12. Relative gene expression levels of putative Glycinemax (soybean) nodulation-related genes in nine different tissues,including mature nodules.Table S13. Identification of Glycine max (soybean) transcriptionfactor genes preferentially (‡3- and <10-fold changes; yellow),specifically (‡10- and <100-fold changes; orange), very specifically(‡100- and <1000-fold changes; red) and exclusively (>1000-foldchange; purple) expressed in one out of the nine tissues tested.Table S14. Gene expression pattern between Glycine max(soybean), Medicago truncatula and Lotus japonicus orthologousgenes.Table S15. Gene expression of 1016 Glycine max (soybean)regulatory genes in 11 different soybean tissues.Table S16. Identification of tissue-specific Glycine max (soybean)regulatory genes, based on qRT-PCR experiments.Appendix S1. Large-scale qRT-PCR of Glycine max (soybean)transcription factor genes.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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