Agriculture Reference
In-Depth Information
An alternative instrument for transcrip-
tome analysis is the cDNA-AFLP method, where
cDNA rather than genomic DNA is used in con-
junction with AFLP to generate transcrip-
tome-derived fragments (TDFs), which allow the
monitoring of differentially expressed genes
among tissues, genotypes, or treatments applied
(Ritter
et al
., 2008). Among
in vitro
plantlets,
mature leaves, and dormant tubers of potato,
most primer combinations yielded one or two
segregating TDFs (Ritter
et al
., 2008). Henriquez
and Daayf (2010) combined this technique with
subtractive hybridization to remove constitu-
tively expressed genes and common transcripts
between treatments, allowing the identification
of
41
differentially expressed TDFs from the
interaction between potato and
P. infestans
.
The publication of the potato genome (Po-
tato Genome Sequencing Consortium, 2011)
along with the potato genome browser (
http://
searches.shtml
)
has expanded the possibility of
potato transcriptomics by providing a reference
genome along with gene-specific transcriptome
tracks derived from RNASeq data generated
from multiple tissues and treatments (
Fig. 17.1
)
(Massa
et al
., 2011). The amount of data gener-
ated by RNASeq overshadows what can be ob-
tained from microarrays. Novel candidate genes
for plant-pathogen interactions have been iden-
tified by sequencing the transcriptome of com-
patible and incompatible potato-
P. infestans
interactions (Gao
et al
., 2011). Of course, cDNA
prepared from RNA isolation does not discrimin-
ate between translated and non-translated RNA.
Use of RNA derived from isolated polysomes will
provide a more accurate picture of the transla-
tome (Mustroph
et al
., 2009). Another method-
ology for differentiating classes of RNA has been
to separate total RNA using polyacrylamide gel
electrophoresis in order to select small RNA (
20-
30
nt), ligate to adapters, then use reverse tran-
scription and amplification to provide a “DNA
Colony Template Library” for deep sequencing
(Hwang
et al
., 2011c). Application of this meth-
odology to potato plants exposed to drought
stress allowed the identification of several fam-
ilies of microRNAs associated with the stress re-
sponse (Hwang
et al
., 2011a,b,c). SAGE (serial
analysis of gene expression) or DeepSAGE repre-
sents another transcriptomic approach using
next-generation sequencing (Velculescu
et al
.,
1995). Gyetvai
et al
. (2012) applied SAGE tech-
nology to capture the transcriptomes of compat-
ible and incompatible interactions between
potato and
P. infestans
to identify transcriptomic
changes that revealed candidate genes for plant
host-pathogen interactions.
17.
4
Proteomics
Two-dimensional protein gel electrophoresis has
been used to compare the proteomes of potato
under various environmental conditions, in re-
action to disease agents, and to examine possible
unintended changes resulting from transgene
introduction. Initially, tuber proteins were classi-
fied and characterized to determine cultivar differ-
ences (Bauw
et al
., 2006), those localized to the
amyloplast (Stensballe
et al
., 2008), changes over
the life cycle of potato tubers during growth and
development (Lehesranta
et al
., 2006; Agrawal
et al
., 2008) and during the postharvest aging pro-
cess (Delaplace
et al
., 2009). Proteins involved in
plant defense have been demonstrated to be lo-
calized primarily in the skin of potato (Barel and
Ginzberg, 2008). Only fertility management
among a three-factorial experiment comparing
two cover crops in organic and conventional pro-
duction schemes significantly affected the prote-
ome of potato cultivar Santé (Lehesranta
et al
.,
2007). A catalog of differentially expressed pro-
teins in the petioles of
Solanum andigena
exposed to
long or short day conditions has been compiled
and categorized (Shah
et al
., 2011). Proteins
extracted from vacuoles of field-grown tubers of
potato cultivar Kuras have been catalogued and
classified by Jørgensen
et al
. (2011).
Proteomic analyses have also been con-
ducted on potato in conjunction with pathogen
interactions and abiotic stress. Liu and Halter-
man (2009) identified
12
protein spots that dif-
ferentiated susceptible from transgenic cultivar
Katahdin after both were inoculated with the
late blight pathogen,
P. infestans
. Katahdin had
been transformed with a single copy of the
RB
gene (a late blight resistance gene cloned from
Solanum bulbocastanum
) to confer resistance.
Similarly, Barzic and Com (2012) observed that
13
proteins were upregulated in a resistant culti-
var after exposure to bacterial soft rot (
Pectobac-
terium atrosepticum
). Thirty proteins were found
