18,144 cDNA clones derived from different
organs (Kouchi et al. 2004 ), which allowed iden-
tification of 860 and 1,076 genes, respectively,
that were differentially expressed between unin-
fected roots andMesorhizobium loti-infected roots
or nodules. In 2005, a transcriptome analysis
comparing uninfected and nodulating roots was
performed by serial analysis of gene expression
(SAGE) (Asamizu et al. 2005 ). SAGE libraries
from these two tissues contained, respectively,
85,482 and 80,233 SAGE tags, which are short
cDNA fragments unique to each transcript used to
determine transcript level based on their fre-
quency. This analysis revealed more than 800
genes differentially expressed between infected
and uninfected roots. In 2008, a draft genome
sequence of Lotus was published (Sato et al.
2008 ), which enabled production of a new gen-
eration of gene chip containing probes for most
genes.
17.2 The AffymetrixL. japonicus
Genechip
TheLotus japonicusGeneChip is an Affymetrix
custom array, called A-AFFY-90. It contains
52,749 Lotus and 8,710 Mesorhizobium loti
(M. loti)-derived probe sets, each representing a
known or predicted open reading frame (ORF).
Each probe set has a unique identifier consisting
of a gene or clone name, with one of the fol-
lowing suffixes:“_at”meaning antisense target,
“_st”for sense target (i.e., controls),“_s_at”for
probe set matching multiple transcripts,“_a_at”
for genes coding potential alternatively spliced
transcripts and“_x_at”when it was not possible
to design unique probes for the transcript (i.e.,
this probe set is not unique for the gene).
To estimate the coverage of the Affymetrix
GeneChip, we mapped the 47,486 gene sequences
from theL. japonicusGene Index (LjGI version 6)
representing tentative transcripts (TC) and sin-
gleton expressed sequence tags (ESTs) (http://
compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.
pl?gudb=l_japonicus) to probe sets present on the
chip. This revealed that 77 % (36,599/47,486) of
the genes encoding putative proteins in LjGI were
represented on the Affymetrix GeneChip.17.2.1 AnnotationsMapping of Affymetrix probe set sequences to
sequences obtained from the LjGI v6 and from
the annotated genome (Sato et al. 2008 ) via
reciprocal BLAST enabled us to re-annotate
probe set IDs using reciprocal BLAST (Verdier
et al. 2013 ). Each Lotus gene/probe set was
assigned to a different bincode classification of
the Kyoto Encyclopedia of Genes and Genomes
(KEGG) (Kanehisa and Goto 2000 ) and the gene
ontology (GO) (Ashburner et al. 2000 ) consor-
tium (Verdier et al. 2013 ). KEGG and GO
annotations provide information about genes,
gene products, and metabolic pathways that may
be useful to describe putative gene function. For
across-species studies, identification of soybean
and Medicago orthologs for each probe set was
performed using reciprocal BLASTP and the best
hits method combined with soft filtering and
Smith-Waterman alignment options (Moreno-
Hagelsieb and Latimer 2008 ).17.2.2 TFs, Transporters, and LSGsAfter refining probe set annotations, 1,489
probe sets were assigned to putative transcrip-
tion factor genes (from a total of 1,616 TFs
identified in the Lotus genome) and 597 probe
sets were assigned to putative transporter genes
(from a total of 1,087 genes in the Lotus gen-
ome). In the same way that TF and transporter
genes can provide insight into special features
of particular organs, genes that are specifictoa
family of plants can provide insight into unique
biological aspects of that family. Previous
analysis of the Lotus genome identified 1,190
putative legume-specific genes (LSGs) (Sato
et al. 2008 ), which matched 729 probe sets
present on the chip.184 J. Verdier et al.