# Genomic Resources and Tools for Gene Function Analysis in Potato

Authors: Glenn J. Bryan; Ingo Hein
Journal: International Journal of Plant Genomics
 (2008)

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## Abstract

Potato, a highly heterozygous tetraploid, is undergoing an exciting phase of genomics resource development.  The potato research community has established extensive genomic resources, such as large expressed sequence tag (EST) data collections, microarrays and other expression profiling platforms, and large-insert genomic libraries.  Moreover, potato will now benefit from a global potato physical mapping effort, which is serving as the underlying resource for a full potato genome sequencing project, now well underway.  These tools and resources are having a major impact on potato breeding and genetics.  The genome sequence will provide an invaluable comparative genomics resource for cross-referencing to the other Solanaceae, notably tomato, whose sequence is also being determined.   Most importantly perhaps, a potato genome sequence will pave the way for the functional analysis of the large numbers of potato genes that await discovery.  Potato, being easily transformable, is highly amenable to the investigation of gene function by biotechnological approaches.  Recent advances in the development of Virus Induced Gene Silencing (VIGS) and related methods will facilitate rapid progress in the analysis of gene function in this important crop.


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## Body

## 1. Introduction

Cultivated potato, the  world’s third
most important human food crop, is a tetraploid outbreeder and suffers acutely from
inbreeding depression. Genetic mapping is generally performed at the diploid
level, using highly heterozygous clones as parents, and several diploid maps of
potato have been generated [1], including one of the densest plant genetic maps
[2]. Considerable progress has also been made in working at the tetraploid
level [3, 4]. These efforts have led to
the development of large numbers of molecular markers of all of the main types,
which in some cases allow comparison of different potato maps or between potato
and the closely related tomato. Genetic
mapping has also led to knowledge of locations of many potato genes, notably
those conferring resistance to many of the pests and pathogens that present a
threat to potato [5] and genes influencing tuber traits [6]. Despite these
advances, the lack of described mutational variation for potato is a
disadvantage of its outbreeding mating habit, and renders genetic
complementation problematic for the majority of genes. However, potato is relatively
easy to transform, and so technologies such as overexpression and antisense technology
are options for investigating gene function. Results of such experiments are
not always so easy to interpret, and improved methods for functional analysis
are critical to the future of potato breeding and genetics.This article provides an overview of
genomics resources currently available for potato, and the likely future
developments in this area, paying particular emphasis to tools being developed
for investigating gene function.


## 2. Basic Facts about The Potato Genome

Cultivated potato has a chromosome
number of 2n = 4x = 48, and a haploid genome size of ~850 Mb, roughly six times
that of Arabidopsis thaliana and twice
the size of the rice genome [7]. Although
small chromosome size has been a limitation for cytogenetic analysis in potato,
notable advances have been made using pachytene chromosomes and extended DNA
“fibres” for fluorescence in situ
hybridization (FISH) [8]. The potato genome is very similar in size to its
close relative tomato, and genetic maps of the two species show high levels of macrocolinearity
[9]. Information on how well the two genomes
are conserved at the microsyntenic level should start to become available as
outputs from the respective genome projects accumulate. The tomato genome mainly
comprises low-copy-number sequences, which diverged rapidly in evolutionary
time [10]. Schweizer et al. [11], who characterised
the potato genome in terms of the amounts of different classes of repetitive
DNA, suggest that the more highly repeated sequences comprise only 4–7% of the
potato genome, suggesting that it was relatively devoid of highly repetitive
DNA sequences, thus supporting the earlier tomato study. It is also known that
the majority of tomato heterochromatin is found in centromeric regions with
almost all of the euchromatic DNA located distally in long uninterrupted
tracts, a structural feature likely to be true of potato [12]. Gene isolation and recent BAC-end sequencing
efforts are providing the first detailed glimpses of the genome structure in
potato. Using BAC-end sequence and full BAC sequence data, it has also been
shown that potato (34%) contains considerably less repetitive DNA than tomato
(46%), this difference being consistent with relative genome sizes of the two
crops (850 versus 1000 Mb, resp.) [13].


## 3. Structural Genomics Resources for Potato

### 3.1. Est Resources

The generation of large expressed
sequence tag (EST) collections is a primary route for large-scale gene discovery.
There have been several efforts to generate EST resources for potato [14–16].
The potato gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=potato)
contains almost 220000 ESTs, assembled into more than 30 000 “contigs” with
over 26 000 singletons. These efforts, while not exhaustive, comprise a major
genomics resource for potato researchers, perhaps comprising between 50–70% of the total potato
gene “repertoire.” These ESTs will form an important source not only for the
discovery of candidate genes and genetic markers, but also for the development
of microarrays, until the whole genome sequence becomes available in potato. For
instance, EST data from a number of different genotypes are also a rich source for
the discovery of single nucleotide polymorphism (SNP) and simple sequence
repeat (SSR) markers. For example, Tang et al. [17] demonstrate how large
numbers of “eSNPs” can be mined from EST data using an SNP discovery pipeline
(QualitySNP).


### 3.2. Large-Insert Genomic Libraries and Physical Maps

Bacterial
artificial chromosome (BAC) libraries have become the main vehicle for performing
map-based gene cloning and physical mapping in potato. Several BAC libraries have
been constructed from cultivated potato [18] and some of its wild relatives,
for example, the diploids Solanum bulbocastanum [19], Solanum pinnatisectum [20],
and the Mexican hexaploid Solanum demissum [21]. These libraries represent a potentially useful resource for the study of
comparative genome organisation and evolution in potato and the wider
Solanaceae. A BAC library has been constructed from the male parent (RH89-039-16)
of the cross used to make the ultra-high-density (UHD) genetic map of potato
with 10 000 loci [2], and is being used for construction of a genome-wide potato
physical map. Other significant developments arising from the use of these BAC
libraries include the use of BAC clones and fluorescence in situ hybridization (FISH) to
develop chromosome-specific cytogenetic DNA markers for chromosome
identification in potato [22].


## 3.1. Est Resources

The generation of large expressed
sequence tag (EST) collections is a primary route for large-scale gene discovery.
There have been several efforts to generate EST resources for potato [14–16].
The potato gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=potato)
contains almost 220000 ESTs, assembled into more than 30 000 “contigs” with
over 26 000 singletons. These efforts, while not exhaustive, comprise a major
genomics resource for potato researchers, perhaps comprising between 50–70% of the total potato
gene “repertoire.” These ESTs will form an important source not only for the
discovery of candidate genes and genetic markers, but also for the development
of microarrays, until the whole genome sequence becomes available in potato. For
instance, EST data from a number of different genotypes are also a rich source for
the discovery of single nucleotide polymorphism (SNP) and simple sequence
repeat (SSR) markers. For example, Tang et al. [17] demonstrate how large
numbers of “eSNPs” can be mined from EST data using an SNP discovery pipeline
(QualitySNP).


## 3.2. Large-Insert Genomic Libraries and Physical Maps

Bacterial
artificial chromosome (BAC) libraries have become the main vehicle for performing
map-based gene cloning and physical mapping in potato. Several BAC libraries have
been constructed from cultivated potato [18] and some of its wild relatives,
for example, the diploids Solanum bulbocastanum [19], Solanum pinnatisectum [20],
and the Mexican hexaploid Solanum demissum [21]. These libraries represent a potentially useful resource for the study of
comparative genome organisation and evolution in potato and the wider
Solanaceae. A BAC library has been constructed from the male parent (RH89-039-16)
of the cross used to make the ultra-high-density (UHD) genetic map of potato
with 10 000 loci [2], and is being used for construction of a genome-wide potato
physical map. Other significant developments arising from the use of these BAC
libraries include the use of BAC clones and fluorescence in situ hybridization (FISH) to
develop chromosome-specific cytogenetic DNA markers for chromosome
identification in potato [22].


## 4. Gene Isolation in Potato

### 4.1. Map-Based Approach

Mapping efforts in potato have also led
to the generation of knowledge concerning the genetic architecture of a number
of characters, including pest and disease resistance, tuber quality traits,
dormancy, tuber shape, and colour. Also, several potato genes have been
isolated using a map-based approach [18, 23, 24], with most of these aimed at
isolation of major genes for resistance to the more serious pests and pathogens
of potato, the late blight pathogen Phytophthora infestans (Mont. de Bary), potato cyst nematodes (PCN), and potato virus X (PVX). These activities
have necessitated the development of dense genetic maps around the target
resistance loci, as well as concomitant generation of genomic resources, such
as BAC libraries. These gene cloning efforts have afforded early glimpses into
the structure of the potato genome, through the sequencing of a considerable
number of large-insert clones. For instance, a study of Gpa2/Rx1 resistance gene “cluster” provided important information
concerning the evolution and structure of R gene loci and has shown beyond any doubt that resistances to different
pests/pathogens can be coded by structurally similar genes from the same gene cluster.TheR3 locus, which maps to a
cluster of genes for resistance against P.
infestans and other resistance genes on the short arm of chromosome XI, has
shown to comprise two very tightly linked resistance genes (R3a and R3b) with distinct specificities against P. infestans [25]. The R3 locus was found to be syntenic with
the I2 locus of tomato, and a
comparative approach was used to isolate R3a,
which is constitutively expressed along with some of its paralogous genes [26].
It is highly likely that the same approach will allow the future isolation of other P. infestans resistance genes on the
same chromosome. Similarly, there are now determined efforts to isolate genes
from late blight resistance “hotspots” on other potato chromosomes. A notable
example is the recent work on potato chromosome IV, whereby several resistance
genes against P. infestans map to the
same locus [27–29].These are but a few of several successful map-based gene isolation
efforts, but these illustrate how comparative genomics, either between
different potato genotypes or between different Solanaceous plant species, can be
used as a tool for accelerating the normally laborious task of gene isolation,
and they bode well for the future of Solanaceae genomic research. As knowledge of
the genome structure of potato and tomato increases, the isolation of such genes
should become more facile.


### 4.2. Candidate Gene Approach

A candidate gene approach has also
been used for isolating plant genes that underlie specific traits [30]. In
potato, cloning of the gene Gro1-4,
which confers resistance to pathotype Ro1 of the cyst nematode Globodera rostochiensis, has been achieved
using a joint candidate gene/mapping approach [31]. The gene was found to
colocalise in a large segregating population with a marker derived from a
“resistance-gene-like” sequence. The marker was used to isolate 15 members of a
closely related gene family from genomic libraries. By taking into account all
available information (inheritance patterns in resistant and susceptible
germplasm, mapping data, DNA sequence information), it was possible to reduce
the number of candidates to three genes, which were subsequently tested for
complementation of a susceptible phenotype by stable transformation. The identified functional gene, a member of
the TIR-NBS-LRR class, differs from susceptible members of the same family by
29 amino acid changes. This approach may
be used in future for isolation of other resistance genes/QTLs conferring
partial and durable resistance to the major potato pests and pathogens.Another
example of the use of candidate gene approach in potato is the isolation ofP gene that encodes anthocyanin
biosynthetic enzyme flavonoid 3′,5′-hydroxylase (f3′5′h), and is
responsiblefor the production of blue/purple anthocyanin
pigments in tissues like tubers, flowers, or stems [32]. In this study, a Petunia
f3′5′h gene was used to screen a potato cDNA library prepared from
purple-coloured flowers and stems. Six positively hybridizing cDNA clones were
sequenced and all appeared to be derived from a single gene that shared 85%
sequence identity at the amino acid level with Petunia f3′5′h. The potato gene
cosegregated with purple tuber colour in a diploid population and was found to
be expressed in tuber skin only in the presence of the anthocyanin regulatory
locus I. One of the f3′5′h cDNA clone that was placed under the control of a
doubled CaMV 35S promoter was also used for transformation of the red-skinned
cultivar “Desiree.” Tuber and stem
tissues that were coloured red in Desiree were purple in nine of 17
independently transformed lines, confirming the hypothesis that the transformed
gene corresponded to the P locus.In another study, DNA sequence
variation was analysed at the invGE/GF locus (duplicate invertase genesInvGE and InvGF) on potato chromosome IX which colocalizes with a
cold-sweetening QTL [33]. The study focused on 188 tetraploid potato cultivars,
which were assessed for chip quality and tuber starch content. Two closely
correlated invertase alleles, invGE-f and invGF-d, were associated with
better chip quality in three breeding populations, and one allele (invGF-b) was associated with lower tuber
starch content. The potato invGE gene
was also found to be orthologous to the tomato invertase gene Lin5, causal for a fruit-sugar-yield
QTL. These results suggested that natural variation for sugar yield in tomato
fruits and that for sugar content in potato tubers are controlled by functional
variants of orthologous invertase genes.These few examples clearly
demonstrate the potential of using the candidate gene approach in potato. It is
also clear that the extensive knowledge of tuber biochemistry and the large
number of potato gene sequences should enable its further application for tuber
quality traits.


## 4.1. Map-Based Approach

Mapping efforts in potato have also led
to the generation of knowledge concerning the genetic architecture of a number
of characters, including pest and disease resistance, tuber quality traits,
dormancy, tuber shape, and colour. Also, several potato genes have been
isolated using a map-based approach [18, 23, 24], with most of these aimed at
isolation of major genes for resistance to the more serious pests and pathogens
of potato, the late blight pathogen Phytophthora infestans (Mont. de Bary), potato cyst nematodes (PCN), and potato virus X (PVX). These activities
have necessitated the development of dense genetic maps around the target
resistance loci, as well as concomitant generation of genomic resources, such
as BAC libraries. These gene cloning efforts have afforded early glimpses into
the structure of the potato genome, through the sequencing of a considerable
number of large-insert clones. For instance, a study of Gpa2/Rx1 resistance gene “cluster” provided important information
concerning the evolution and structure of R gene loci and has shown beyond any doubt that resistances to different
pests/pathogens can be coded by structurally similar genes from the same gene cluster.TheR3 locus, which maps to a
cluster of genes for resistance against P.
infestans and other resistance genes on the short arm of chromosome XI, has
shown to comprise two very tightly linked resistance genes (R3a and R3b) with distinct specificities against P. infestans [25]. The R3 locus was found to be syntenic with
the I2 locus of tomato, and a
comparative approach was used to isolate R3a,
which is constitutively expressed along with some of its paralogous genes [26].
It is highly likely that the same approach will allow the future isolation of other P. infestans resistance genes on the
same chromosome. Similarly, there are now determined efforts to isolate genes
from late blight resistance “hotspots” on other potato chromosomes. A notable
example is the recent work on potato chromosome IV, whereby several resistance
genes against P. infestans map to the
same locus [27–29].These are but a few of several successful map-based gene isolation
efforts, but these illustrate how comparative genomics, either between
different potato genotypes or between different Solanaceous plant species, can be
used as a tool for accelerating the normally laborious task of gene isolation,
and they bode well for the future of Solanaceae genomic research. As knowledge of
the genome structure of potato and tomato increases, the isolation of such genes
should become more facile.


## 4.2. Candidate Gene Approach

A candidate gene approach has also
been used for isolating plant genes that underlie specific traits [30]. In
potato, cloning of the gene Gro1-4,
which confers resistance to pathotype Ro1 of the cyst nematode Globodera rostochiensis, has been achieved
using a joint candidate gene/mapping approach [31]. The gene was found to
colocalise in a large segregating population with a marker derived from a
“resistance-gene-like” sequence. The marker was used to isolate 15 members of a
closely related gene family from genomic libraries. By taking into account all
available information (inheritance patterns in resistant and susceptible
germplasm, mapping data, DNA sequence information), it was possible to reduce
the number of candidates to three genes, which were subsequently tested for
complementation of a susceptible phenotype by stable transformation. The identified functional gene, a member of
the TIR-NBS-LRR class, differs from susceptible members of the same family by
29 amino acid changes. This approach may
be used in future for isolation of other resistance genes/QTLs conferring
partial and durable resistance to the major potato pests and pathogens.Another
example of the use of candidate gene approach in potato is the isolation ofP gene that encodes anthocyanin
biosynthetic enzyme flavonoid 3′,5′-hydroxylase (f3′5′h), and is
responsiblefor the production of blue/purple anthocyanin
pigments in tissues like tubers, flowers, or stems [32]. In this study, a Petunia
f3′5′h gene was used to screen a potato cDNA library prepared from
purple-coloured flowers and stems. Six positively hybridizing cDNA clones were
sequenced and all appeared to be derived from a single gene that shared 85%
sequence identity at the amino acid level with Petunia f3′5′h. The potato gene
cosegregated with purple tuber colour in a diploid population and was found to
be expressed in tuber skin only in the presence of the anthocyanin regulatory
locus I. One of the f3′5′h cDNA clone that was placed under the control of a
doubled CaMV 35S promoter was also used for transformation of the red-skinned
cultivar “Desiree.” Tuber and stem
tissues that were coloured red in Desiree were purple in nine of 17
independently transformed lines, confirming the hypothesis that the transformed
gene corresponded to the P locus.In another study, DNA sequence
variation was analysed at the invGE/GF locus (duplicate invertase genesInvGE and InvGF) on potato chromosome IX which colocalizes with a
cold-sweetening QTL [33]. The study focused on 188 tetraploid potato cultivars,
which were assessed for chip quality and tuber starch content. Two closely
correlated invertase alleles, invGE-f and invGF-d, were associated with
better chip quality in three breeding populations, and one allele (invGF-b) was associated with lower tuber
starch content. The potato invGE gene
was also found to be orthologous to the tomato invertase gene Lin5, causal for a fruit-sugar-yield
QTL. These results suggested that natural variation for sugar yield in tomato
fruits and that for sugar content in potato tubers are controlled by functional
variants of orthologous invertase genes.These few examples clearly
demonstrate the potential of using the candidate gene approach in potato. It is
also clear that the extensive knowledge of tuber biochemistry and the large
number of potato gene sequences should enable its further application for tuber
quality traits.


## 5. Potato Genome Sequencing

The ultra-high-density (UHD) genetic map of potato [2] forms the underlying
framework for construction of a genome-wide physical map of the potato genome.
Physical map construction is being carried out in two phases. First,
approximately 73 000 clones from a BAC-library have been fingerprinted using a
nonselective AFLP-based method. The fingerprint data has been used to assemble
the RH BACs into roughly 7000 BAC contigs, with a similar number of
“singletons” (i.e., single BAC clones). The second phase entails anchoring of
the contigs and single BACs to the UHD map using a BAC pooling method, which
should also reduce the number of contigs and increase the average contig size.
Subsequent contiging will use a reduced stringency alignment approach which
will reduce the number of contigs still further. The integrated genetic and
physical map will be the main platform, which will be used for obtaining the
DNA sequence of the potato genome. It is expected that approximately 1800
contigs will be anchored to the genetical map, and these scaffolds will be the
starting point for genome sequencing. A
BAC-end sequence resource, comprising more than 140 000 reads, has also been
generated for the project [13]. The ongoing tomato and potato sequencing
projects will have huge implications for those working in the Solanaceae, and will
further sharpen the requirement for functional genomics tools.


## 6. Analysis of Potato Gene Expression

A
wide range of gene expression technologies have been used by potato researchers.
Expression analysis is a discipline that is still very much in transition and
it is likely to undergo significant development in the future, notably with
recent developments in “next generation” sequencing (NGS) technologies, which
have the potential to radically change the way gene discovery is performed.


### 6.1. Cdna-Aflp

The cDNA-AFLP
technique has been used to study gene expression from stolon formation to
sprouting in a range of different tissues during the potato tuber life cycle [34, 35]. Approximately 18 000 transcript-derived
fragments (TDFs) were observed, and over 200 “process specific” TDFs belonging
to different stages of potato tuber life cycle were isolated and sequenced. The
sequence similarities of these TDFs to known genes give insights into the kinds
of processes occurring during tuberisation, dormancy, and sprouting. This
technique is extremely sensitive and can detect differences among gene family
members indistinguishable by Northern blotting. A useful advance has been the
realization that a large proportion of cDNA-AFLP fragments show genetic polymorphism
in segregating populations and can be mapped as transcriptome-derived genetic markers
[36]. Importantly, these markers show less centromeric clustering than AFLP
markers derived directly from genomic DNA and appear to be targeted
specifically to transcriptionally active regions of the genome. This method has
been used to perform a large scale survey of genes differentially expressed
during the tuber life cycle, and the isolation of some of their promoter
regions [37]. Many genes expressed in the tuber life cycle are involved in
defence, stress, storage, and signal transduction pathways. Twelve cis-acting
elements were identified, and are known to be responsive to environmental
stimuli known to play an important role during the tuber life cycle (light,
sugars, hormones, etc.). More recently, a potato transcription map, based on
cDNA-AFLP and containing approximately 700 TDFs, has been generated [38]. One
of the disadvantages of cDNA-AFLP is that it does not provide gene sequence
information and requires laborious isolation of gene fragments from
polyacrylamide gels for sequence characterization.


### 6.2. Sage

Serial
analysis of gene expression (SAGE), which generates short cDNA sequence tags [39, 40]
using a concatemerization-based method, has been used to examine global gene
expression in potato tubers, generating 58 322 sequence tags (of length 19 nucleotides)
of which 22 233 were unique [41]. Putative functions were assigned to almost
700 of those tags occurring at least ten times and roughly 70% matched each known
potato EST sequence. This technology has the advantage over microarray
technology in being an “open” technology, with the possibility of discovering
“new” transcripts. Rapid amplification of complementary DNA ends (RACE) cloning was used
to verify the reliability of SAGE tag annotation using EST sequences from more
than one cultivar. Seventy two per cent of tags represented genes that
participated in a known biological process, with the largest group (43%)
consisting of transcripts active in physiological processes, about half of
which were involved in metabolism. There were no transcripts found which were
involved in photosynthesis. Of the 50 most abundant transcripts from the mature
tuber, protease inhibitors were the dominant class, which is in good agreement
with previous EST projects [14, 15].The
methodologies described briefly in this section are alternatives to the microarrays,
which may ultimately be replaced by NGS methods. For example, Emrich et al. [42]
recently demonstrated how such technologies can be used to extend significantly
the EST resources for maize. The authors used a laser capture microdissection
method to isolate rare transcripts from shoot apical meristems and then
sequenced the corresponding cDNAs using 454 technology. This type of approach
could be used in potato to identify transcripts not present in current EST
databases or to extend the range of potato germplasm represented, currently
limited to a few cultivars. All expression studies share the “problem” that
they are only indicative of the function of particular genes or sets of genes
in biological processes, and require functional analyses whereby the function
of the candidate genes are compromised or exaggerated in some way (e.g.,
overexpression, silencing). This issue will be addressed in a subsequent
section of this article.


## 6.1. Cdna-Aflp

The cDNA-AFLP
technique has been used to study gene expression from stolon formation to
sprouting in a range of different tissues during the potato tuber life cycle [34, 35]. Approximately 18 000 transcript-derived
fragments (TDFs) were observed, and over 200 “process specific” TDFs belonging
to different stages of potato tuber life cycle were isolated and sequenced. The
sequence similarities of these TDFs to known genes give insights into the kinds
of processes occurring during tuberisation, dormancy, and sprouting. This
technique is extremely sensitive and can detect differences among gene family
members indistinguishable by Northern blotting. A useful advance has been the
realization that a large proportion of cDNA-AFLP fragments show genetic polymorphism
in segregating populations and can be mapped as transcriptome-derived genetic markers
[36]. Importantly, these markers show less centromeric clustering than AFLP
markers derived directly from genomic DNA and appear to be targeted
specifically to transcriptionally active regions of the genome. This method has
been used to perform a large scale survey of genes differentially expressed
during the tuber life cycle, and the isolation of some of their promoter
regions [37]. Many genes expressed in the tuber life cycle are involved in
defence, stress, storage, and signal transduction pathways. Twelve cis-acting
elements were identified, and are known to be responsive to environmental
stimuli known to play an important role during the tuber life cycle (light,
sugars, hormones, etc.). More recently, a potato transcription map, based on
cDNA-AFLP and containing approximately 700 TDFs, has been generated [38]. One
of the disadvantages of cDNA-AFLP is that it does not provide gene sequence
information and requires laborious isolation of gene fragments from
polyacrylamide gels for sequence characterization.


## 6.2. Sage

Serial
analysis of gene expression (SAGE), which generates short cDNA sequence tags [39, 40]
using a concatemerization-based method, has been used to examine global gene
expression in potato tubers, generating 58 322 sequence tags (of length 19 nucleotides)
of which 22 233 were unique [41]. Putative functions were assigned to almost
700 of those tags occurring at least ten times and roughly 70% matched each known
potato EST sequence. This technology has the advantage over microarray
technology in being an “open” technology, with the possibility of discovering
“new” transcripts. Rapid amplification of complementary DNA ends (RACE) cloning was used
to verify the reliability of SAGE tag annotation using EST sequences from more
than one cultivar. Seventy two per cent of tags represented genes that
participated in a known biological process, with the largest group (43%)
consisting of transcripts active in physiological processes, about half of
which were involved in metabolism. There were no transcripts found which were
involved in photosynthesis. Of the 50 most abundant transcripts from the mature
tuber, protease inhibitors were the dominant class, which is in good agreement
with previous EST projects [14, 15].The
methodologies described briefly in this section are alternatives to the microarrays,
which may ultimately be replaced by NGS methods. For example, Emrich et al. [42]
recently demonstrated how such technologies can be used to extend significantly
the EST resources for maize. The authors used a laser capture microdissection
method to isolate rare transcripts from shoot apical meristems and then
sequenced the corresponding cDNAs using 454 technology. This type of approach
could be used in potato to identify transcripts not present in current EST
databases or to extend the range of potato germplasm represented, currently
limited to a few cultivars. All expression studies share the “problem” that
they are only indicative of the function of particular genes or sets of genes
in biological processes, and require functional analyses whereby the function
of the candidate genes are compromised or exaggerated in some way (e.g.,
overexpression, silencing). This issue will be addressed in a subsequent
section of this article.


## 7. Microarrays: Tools for High-Throughput Gene Expression Analysis

### 7.1. Cdna Microarrays

The available potato EST resources
comprise an unknown but significant fraction of the gene complement of potato,
and are derived from several genotypes, tissues, and environmental influences. A nonredundant set of 10 000 of these ESTs was
used by the Institute for Genomic Research (TIGR) to develop a cDNA potato
microarray that was made available to the research community at minimal cost. Moreover,
the same organisation offered a transcription profiling service to allow the
evaluation of these arrays by a wide range of users working on different
Solanaceous plant species asking different biological questions. This allowed
generation of massive microarray data that is publicly available (http://www.tigr.org/tdb/potato/profiling_service2.shtml#AProcedure).
However, this platform had the disadvantage of containing a very small
proportion of the potato gene repertoire. Moreover, as the “TIGR array” was based
on spotted cDNAs, it was inherently difficult to achieve a high level of
reproducibility. Rensink et al. [43] have used this platform to identify genes
involved in abiotic stress responses, with more than 3000 genes found to be
significantly up- or downregulated in response to at least one of the stress
conditions used (cold, heat, salt). In another detailed study, expression of
1315 genes during tuber development was examined, where transient changes in
gene expression were found to be relatively uncommon and several new genes were
found to be differentially expressed during tuber development [44]. These
studies, while informative, highlight the dilemma faced by plant molecular
biologists in prioritizing genes for further study from a large number of
candidate genes in the absence of genetic information and mutations in target
trait genes.


### 7.2. Oligonucleotide Microarrays

Long oligonucleotide arrays that have
been manufactured by various technology providers have also been found useful
in potato since the use of short oligonucleotide arrays may lead to
misinterpretations due to high degree of allelic heterozygosity in this crop. For
this purpose, the potato oligo chip initiative (POCI) has selected the Agilent
“44K feature platform” system, which was made available for use in 2006. This
system is very flexible and allows for redesign of the array as more gene
sequence information becomes available. Kloosterman
et al. [45] described the design of this platform and demonstrated its utility
by analyzing different stages of tuber initiation and growth.


## 7.1. Cdna Microarrays

The available potato EST resources
comprise an unknown but significant fraction of the gene complement of potato,
and are derived from several genotypes, tissues, and environmental influences. A nonredundant set of 10 000 of these ESTs was
used by the Institute for Genomic Research (TIGR) to develop a cDNA potato
microarray that was made available to the research community at minimal cost. Moreover,
the same organisation offered a transcription profiling service to allow the
evaluation of these arrays by a wide range of users working on different
Solanaceous plant species asking different biological questions. This allowed
generation of massive microarray data that is publicly available (http://www.tigr.org/tdb/potato/profiling_service2.shtml#AProcedure).
However, this platform had the disadvantage of containing a very small
proportion of the potato gene repertoire. Moreover, as the “TIGR array” was based
on spotted cDNAs, it was inherently difficult to achieve a high level of
reproducibility. Rensink et al. [43] have used this platform to identify genes
involved in abiotic stress responses, with more than 3000 genes found to be
significantly up- or downregulated in response to at least one of the stress
conditions used (cold, heat, salt). In another detailed study, expression of
1315 genes during tuber development was examined, where transient changes in
gene expression were found to be relatively uncommon and several new genes were
found to be differentially expressed during tuber development [44]. These
studies, while informative, highlight the dilemma faced by plant molecular
biologists in prioritizing genes for further study from a large number of
candidate genes in the absence of genetic information and mutations in target
trait genes.


## 7.2. Oligonucleotide Microarrays

Long oligonucleotide arrays that have
been manufactured by various technology providers have also been found useful
in potato since the use of short oligonucleotide arrays may lead to
misinterpretations due to high degree of allelic heterozygosity in this crop. For
this purpose, the potato oligo chip initiative (POCI) has selected the Agilent
“44K feature platform” system, which was made available for use in 2006. This
system is very flexible and allows for redesign of the array as more gene
sequence information becomes available. Kloosterman
et al. [45] described the design of this platform and demonstrated its utility
by analyzing different stages of tuber initiation and growth.


## 8. Functional Studies in Potato

Potato geneticists and breeders have
generated a great deal of information about the location of genes and QTLs coding
for important potato traits, including pest and disease resistance and tuber
traits. The volume of gene sequence information, notably from cDNA sequencing and
the genome project, will increase rapidly in the coming years. Developments in genetics
and structural genomics are beginning to be matched by concomitant development
of functional genomics tools. Potato has
a strong need for a high-density gene map or a genome sequence, to place gene
sequences in their genetic/genomic context. Relatively high-throughput methods
are also needed for testing and assessing gene function. The availability of
mutant populations of potato will also be of tremendous value in this regard
[46]. Potato cultivars are highly heterozygous and contain very high levels of
“genetic load.” It has been estimated that there is one SNP approximately every
25 bp [47]. If individual alleles can be “isolated” in the homozygous condition,
there is no telling what information they would yield about potato biology. The
nonavailability of mutants may largely be overcome by recourse to use of
diploid self-compatible potato clones for the development of mutant populations
or by mining of variant alleles in heterozygous germplasm. Functional studies currently rely on the use
of transformation-based techniques or use of viral vector-mediated gene delivery systems for
the establishment of information regarding gene function. There have been some
recent tantalising developments in functional genetics/genomics tools and
resources for potato. Of course gene expression profiling or microarray studies
have a role to play in the identification of a pool of candidate genes
potentially involved in any given biological process. These methods, in
combination with other functional genomics tools such as RNA interference (RNAi),
virus-induced gene silencing (VIGS), and activation tagged lines, have the
potential to facilitate the identification of the role of thousands of potato
genes over the next several years. Furthermore, combining structural genetics
approaches (such as QTL and candidate gene mapping) with functional genomics
information (such as microarray-derived gene expression data for candidate
genes) has great potential for the dissection of many complex, polygenic potato
traits.


### 8.1. Virus-Induced Gene Silencing (vigs)

Virus-induced gene silencing (VIGS) is
a powerful tool for plant functional genomics. VIGS exploits an RNA-mediated
antiviral defense mechanism in plants. This phenomenon has been exploited for
gene silencing through the use of virus vectors carrying host target genes that
are directed against the corresponding plant mRNAs [48]. VIGS is increasingly
used to generate transient loss-of-function assays, and is a powerful
reverse-genetics tool in functional genomic programs as an alternative to
stable transformation. In potato, two viral vectors, potato virus X (PVX) and tobacco
rattle virus (TRV), have been successfully utilized for VIGS [49, 50]. Faivre-Rampant
et al. [49] have shown that a
binary PVX-based vector, pGR106, [51, 52] is effective in triggering VIGS of
phytoene desaturase (PDS) in both diploid and cultivated tetraploid Solanum species. In this study,
silencing was maintained throughout the foliar tissues and tubers and could
also be triggered and sustained in in
vitro micropropagated tetraploid potato for several cycles and on in
vitro generated microtubers. Similarly, PDS silencing with TRV has been
observed in cultivated potato, as well as the diploid wild species S. bulbocastanum and S. okadae, and the distantly related hexaploid S.
nigrum [50]. In the same study, silencing of known resistance genes (e.g., R1, Rx,
and RB) in normally resistant plants yielded
a compatible interaction in detached leaf tests. A modification of the leaf
inoculation used for both PVX- and TRV-based silencing was demonstrated for TRV
in a so-called “agrodrench” method, in which soil adjacent to the plant root is
drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors
[53]. TRV-based silencing of genes such as PDS, a 20S proteasome subunit (PB7)
or Mg-protoporphyrin chelatase (Chl H) by agrodrench has been shown to be
efficient for different members of the Solanaceae including Nicotiana benthamiana, tomato, pepper,
tobacco, potato, and petunia.N. benthamiana provides a particularly suitable
model system for Solanaceae species, including potato, as it is highly amenable
to manipulations such as VIGS and virus- or Agrobacterium-mediated overexpression
of candidate genes (Figure 1) [54]. Indeed, many silencing studies have been
conducted in N.benthamiana to demonstrate involvement of candidate genes involved
in the plant disease resistance (including the hypersensitive response; HR),
abiotic stress, cellular signaling, and secondary metabolite biosynthesis [55].
Recently, for example, Gilroy et al.
[56], using a combination of VIGS and biochemical approaches, demonstrated that
the cysteine protease cathepsin B is required for the HR. Silencing of
cathepsin B in N. benthamiana prevented programmed cell death (PCD) and compromised disease resistance
induced by Erwinia amylovora and Pseudomonas syringae pv. tomato (Pst)
DC3000, two distinct nonhost bacterial pathogens. It also suppressed the HR
triggered by transient coexpression of potato R3a and Phytophthora
infestansAvr3a genes but did not
compromise the HR triggered by recognition of Cladosporiumfulvum AVR4
by tomato Cf-4. The ease of silencing in N.
benthamiana makes it suitable for large scale VIGS experiments. A study of
192 cDNA-AFLP fragments, expressed during the HR following recognition of Avr4 from C. fulvum by tomato Cf-4,
was conducted in N. benthamiana and
identified 15 Avr4-responsive tomato
(ART) fragments that, when silenced, resulted in a compromised HR induced by
both Avr4 in Cf-4 transgenic plants
and the Inf1 gene from P. infestans [57]. In addition,
silencing of HSP90, a nuclear GTPase, an L19 ribosomal protein, and a
nucleotide binding-leucine rich repeat (NB-LRR)-type protein suppressed the HR [57].
Interestingly, silencing of the NB-LRR-type protein NRC1 not only affected the
Cf-4/Avr4-induced HR and compromised Cf-4-mediated resistance to C. fulvum, but also revealed that this
protein is required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx, and Mi [58].N. benthamiana plants, noninoculated
(a), inoculated with pGR106::PDS to silence endogenous phytoene desaturase
resulting in photo bleaching of leaves (b) and overexpressing GFP via a
pGR106::GFP construct—viewed under UV light to show expression of
GFP (c). Overexpression and coinfiltration of virulent Avr3a KI and avirulent
Avr3a EM with the potato R gene R3a are shown in (d). Naturally occurring PVX
resistance in S. papita (e) and the recognition
of virulent Avr3a KI and avirulent Avr3a EM alleles but not from tdT, used as a
control, in S. chacoense (f).
(a)(b)(c)(d)(e)(f)A
recently developed TRV RNA2 vector, which utilizes ligation-independent cloning
(LIC), has been employed to assess the function of 400 tomato ESTs inN. benthamiana [59]. The function of SlMADS1
and its N. benthamiana homologous
sequences, NbMADS4-1 and -2, was shown during flowering and demonstrated that
NbMADS4-1 and NbMADS4-2 act  nonredundantly in floral development. Silencing of
either gene resulted in loss of organ identity. These studies show the
potential for use of N. benthamiana as a “proxy” species for high-throughput gene function analysis for potato and
other Solanaceae.


### 8.2. Virus andAgrobacterium tumefaciens-Based Overexpression

In
addition to their role in VIGS, virus vectors can be used for overexpressing
genes in plants. The AgrobacteriumPVX-based binary vector pGR106, an
efficient silencing vector for Solanum species, can also be used for
overexpressing genes, as shown for GFP in Figure1 [51, 52]. The search for
novel sources of plant resistance, driven by knowledge of pathogen “effectors”
with avirulent activities, rather than more traditional plant disease
resistance breeding, has been coined “effectoromics” [60]. For example, overexpression of P. infestans effectors in potato
represents an opportunity to seek vital and invariant components of the P. infestans pathogenicity apparatus that
can be targeted for sustainable potato protection. Information emerging from
effectoromic studies will be useful to identify the cognate host R genes as sources of durable disease
resistance and to develop novel control strategies that are intrinsically difficult
for the pathogen to overcome. The discovery of a conserved motif, RxLR, within
many avirulence genes [61, 62] that is required for translocation of the
effectors from pathogen haustoria into the plant cell [63] has had a tremendous
impact on the prediction of pathogen effectors. Overexpression via pGR106 in N. benthamiana of 63 predicted P.infestans extracellular proteins
(Pex) led to the discovery of two novel necrosis-inducing cDNAs,
encoding extracellular proteins belonging to a large and complex
protein family in Phytophthora [64]. Similarly, the recognition of the P. infestans effector Avr3a by the potato R gene R3a [26] could be demonstrated in N. benthamiana [61]. Coinfiltration of N. benthamiana leaves with an A. tumefaciens strain carrying a
construct expressing R3a and a strain
carrying a construct expressing the truncated avirulent Avr3a (Avr3a KI) sequence
via PVX resulted in a confluent cell death response, not observed when
overexpressing the truncated virulent Avr3a (Avr3a EM) sequence (Figure 1). Using
the P. infestans elicitins INF1,
INF2A, and INF2B, the same PVX system has been adapted and optimized to screen
Solanum plants for response to pathogen elicitors [65]. Of 31 potato species
tested, 11 clones of Solanum
huancabambense and Solanum
microdontum responded with HR-like symptoms, which were also observed
following infiltration with purified recombinant INF1, INF2A, and INF2B.Two
similar studies have been reported that utilize the twoAvr3a alleles described above to identify potentially novel
resistance mechanisms within wild potato accessions [66, 67]. One study [66]
utilized PVX to express the different Avr3a alleles in wild Solanum species, whereas  the other [67] utilized Agrobacterium-only-based expression of the Avr3a alleles to circumvent the relative
high level of resistance against PVX within the wild species tested (Figure 1).
These studies identified similar sets of species that recognize both the EM and
KI forms of AVR3a (unpublished data).


## 8.1. Virus-Induced Gene Silencing (vigs)

Virus-induced gene silencing (VIGS) is
a powerful tool for plant functional genomics. VIGS exploits an RNA-mediated
antiviral defense mechanism in plants. This phenomenon has been exploited for
gene silencing through the use of virus vectors carrying host target genes that
are directed against the corresponding plant mRNAs [48]. VIGS is increasingly
used to generate transient loss-of-function assays, and is a powerful
reverse-genetics tool in functional genomic programs as an alternative to
stable transformation. In potato, two viral vectors, potato virus X (PVX) and tobacco
rattle virus (TRV), have been successfully utilized for VIGS [49, 50]. Faivre-Rampant
et al. [49] have shown that a
binary PVX-based vector, pGR106, [51, 52] is effective in triggering VIGS of
phytoene desaturase (PDS) in both diploid and cultivated tetraploid Solanum species. In this study,
silencing was maintained throughout the foliar tissues and tubers and could
also be triggered and sustained in in
vitro micropropagated tetraploid potato for several cycles and on in
vitro generated microtubers. Similarly, PDS silencing with TRV has been
observed in cultivated potato, as well as the diploid wild species S. bulbocastanum and S. okadae, and the distantly related hexaploid S.
nigrum [50]. In the same study, silencing of known resistance genes (e.g., R1, Rx,
and RB) in normally resistant plants yielded
a compatible interaction in detached leaf tests. A modification of the leaf
inoculation used for both PVX- and TRV-based silencing was demonstrated for TRV
in a so-called “agrodrench” method, in which soil adjacent to the plant root is
drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors
[53]. TRV-based silencing of genes such as PDS, a 20S proteasome subunit (PB7)
or Mg-protoporphyrin chelatase (Chl H) by agrodrench has been shown to be
efficient for different members of the Solanaceae including Nicotiana benthamiana, tomato, pepper,
tobacco, potato, and petunia.N. benthamiana provides a particularly suitable
model system for Solanaceae species, including potato, as it is highly amenable
to manipulations such as VIGS and virus- or Agrobacterium-mediated overexpression
of candidate genes (Figure 1) [54]. Indeed, many silencing studies have been
conducted in N.benthamiana to demonstrate involvement of candidate genes involved
in the plant disease resistance (including the hypersensitive response; HR),
abiotic stress, cellular signaling, and secondary metabolite biosynthesis [55].
Recently, for example, Gilroy et al.
[56], using a combination of VIGS and biochemical approaches, demonstrated that
the cysteine protease cathepsin B is required for the HR. Silencing of
cathepsin B in N. benthamiana prevented programmed cell death (PCD) and compromised disease resistance
induced by Erwinia amylovora and Pseudomonas syringae pv. tomato (Pst)
DC3000, two distinct nonhost bacterial pathogens. It also suppressed the HR
triggered by transient coexpression of potato R3a and Phytophthora
infestansAvr3a genes but did not
compromise the HR triggered by recognition of Cladosporiumfulvum AVR4
by tomato Cf-4. The ease of silencing in N.
benthamiana makes it suitable for large scale VIGS experiments. A study of
192 cDNA-AFLP fragments, expressed during the HR following recognition of Avr4 from C. fulvum by tomato Cf-4,
was conducted in N. benthamiana and
identified 15 Avr4-responsive tomato
(ART) fragments that, when silenced, resulted in a compromised HR induced by
both Avr4 in Cf-4 transgenic plants
and the Inf1 gene from P. infestans [57]. In addition,
silencing of HSP90, a nuclear GTPase, an L19 ribosomal protein, and a
nucleotide binding-leucine rich repeat (NB-LRR)-type protein suppressed the HR [57].
Interestingly, silencing of the NB-LRR-type protein NRC1 not only affected the
Cf-4/Avr4-induced HR and compromised Cf-4-mediated resistance to C. fulvum, but also revealed that this
protein is required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx, and Mi [58].N. benthamiana plants, noninoculated
(a), inoculated with pGR106::PDS to silence endogenous phytoene desaturase
resulting in photo bleaching of leaves (b) and overexpressing GFP via a
pGR106::GFP construct—viewed under UV light to show expression of
GFP (c). Overexpression and coinfiltration of virulent Avr3a KI and avirulent
Avr3a EM with the potato R gene R3a are shown in (d). Naturally occurring PVX
resistance in S. papita (e) and the recognition
of virulent Avr3a KI and avirulent Avr3a EM alleles but not from tdT, used as a
control, in S. chacoense (f).
(a)(b)(c)(d)(e)(f)A
recently developed TRV RNA2 vector, which utilizes ligation-independent cloning
(LIC), has been employed to assess the function of 400 tomato ESTs inN. benthamiana [59]. The function of SlMADS1
and its N. benthamiana homologous
sequences, NbMADS4-1 and -2, was shown during flowering and demonstrated that
NbMADS4-1 and NbMADS4-2 act  nonredundantly in floral development. Silencing of
either gene resulted in loss of organ identity. These studies show the
potential for use of N. benthamiana as a “proxy” species for high-throughput gene function analysis for potato and
other Solanaceae.


## 8.2. Virus andAgrobacterium tumefaciens-Based Overexpression

In
addition to their role in VIGS, virus vectors can be used for overexpressing
genes in plants. The AgrobacteriumPVX-based binary vector pGR106, an
efficient silencing vector for Solanum species, can also be used for
overexpressing genes, as shown for GFP in Figure1 [51, 52]. The search for
novel sources of plant resistance, driven by knowledge of pathogen “effectors”
with avirulent activities, rather than more traditional plant disease
resistance breeding, has been coined “effectoromics” [60]. For example, overexpression of P. infestans effectors in potato
represents an opportunity to seek vital and invariant components of the P. infestans pathogenicity apparatus that
can be targeted for sustainable potato protection. Information emerging from
effectoromic studies will be useful to identify the cognate host R genes as sources of durable disease
resistance and to develop novel control strategies that are intrinsically difficult
for the pathogen to overcome. The discovery of a conserved motif, RxLR, within
many avirulence genes [61, 62] that is required for translocation of the
effectors from pathogen haustoria into the plant cell [63] has had a tremendous
impact on the prediction of pathogen effectors. Overexpression via pGR106 in N. benthamiana of 63 predicted P.infestans extracellular proteins
(Pex) led to the discovery of two novel necrosis-inducing cDNAs,
encoding extracellular proteins belonging to a large and complex
protein family in Phytophthora [64]. Similarly, the recognition of the P. infestans effector Avr3a by the potato R gene R3a [26] could be demonstrated in N. benthamiana [61]. Coinfiltration of N. benthamiana leaves with an A. tumefaciens strain carrying a
construct expressing R3a and a strain
carrying a construct expressing the truncated avirulent Avr3a (Avr3a KI) sequence
via PVX resulted in a confluent cell death response, not observed when
overexpressing the truncated virulent Avr3a (Avr3a EM) sequence (Figure 1). Using
the P. infestans elicitins INF1,
INF2A, and INF2B, the same PVX system has been adapted and optimized to screen
Solanum plants for response to pathogen elicitors [65]. Of 31 potato species
tested, 11 clones of Solanum
huancabambense and Solanum
microdontum responded with HR-like symptoms, which were also observed
following infiltration with purified recombinant INF1, INF2A, and INF2B.Two
similar studies have been reported that utilize the twoAvr3a alleles described above to identify potentially novel
resistance mechanisms within wild potato accessions [66, 67]. One study [66]
utilized PVX to express the different Avr3a alleles in wild Solanum species, whereas  the other [67] utilized Agrobacterium-only-based expression of the Avr3a alleles to circumvent the relative
high level of resistance against PVX within the wild species tested (Figure 1).
These studies identified similar sets of species that recognize both the EM and
KI forms of AVR3a (unpublished data).


## 9. Where Next for Potato?

Potato has entered an exciting new era,
whereby the development of extensive genetic and genomic resources have opened up
many new possibilities for studying important potato traits relevant to potato
agronomy. Concomitant development of similar resources for other Solanaceous
species, notably tomato, and a growing cohesiveness of the Solanaceae research
community, as demonstrated by the “SOL vision” (http://www.sgn.cornell.edu/solanaceae-project/)
bode well for future genomic research of potato and its close relatives. Development
of biotechnological tools for assaying potato gene function is likely to
progress rapidly in the coming years.



---

*Source: 216513-2008-12-18.xml*