Agriculture Reference
In-Depth Information
on and takes advantage of the knowledge available
in the literature and databases on the physiology,
biochemistry, and molecular genetics of a trait of
interest. It is biased, in that it makes assumptions
about the identity and function of the genes under-
lying the trait, but does not require a sequenced
genome and is feasible with limited human and fi-
nancial resources (Huitema
et al
., 2004).
In the postgenome era, the candidate gene
approach is facilitated by the possibility of iden-
tifying DNA variation within specific domains of
interest in a specific gene. Scientific publications,
gene banks, and genome browsers are available
to find information on a trait of interest in other
plant species and to provide lists of possible can-
didate genes and their DNA or protein sequences,
as well as an identification code to find the gene
model in a genome browser or gene bank. The
availability of the potato genome browser en-
ables us to use an amino acid sequence to find
orthologous sequences and determine if these
genes have similar functions in potato.
The gene function can be further analyzed
with programs such as “Smart Domain” (Schultz
et al
., 1998, 2012). With Smart Domain, it is
possible to identify important protein domains
within the query sequence and determine a
smaller stretch within the gene, in which a SNP
causing an amino acid replacement could affect
the trait of interest. By designing primers to
amplify such specific domains of a gene in po-
tato, it is possible to identify meaningful SNPs.
The candidate gene approach can now be
even more specific with the availability of infor-
mation from RNASeq technology (Wang
et al
.,
2009); RNASeq provides expression profiles for
genes from samples extracted from different tis-
sues or organs of the plant, or from the same tis-
sues under different environmental conditions.
These data allow a viewer to determine if a can-
didate gene is suitably expressed in the organ of
interest; for example, meiosis-specific gene ex-
pression in pollen mother cells.
The success of the candidate gene approach
to identify genes of interest is therefore based on
the reliable phenotypic characterization of a popu-
lation, to determine phenotypic extremes for the
trait of interest. The selected extremes can serve as
the base for sequencing the specific domain in
order to identify allelic differences in SNPs. If a par-
ticular SNP produces a change in the amino acid
sequence (i.e. a non-synonymous SNP), and this
change affects the protein structure and possibly
its function, this SNP could be used for further ap-
plications, such as SNP genotyping for large popu-
lation characterization or genetic engineering.
To apply the candidate gene approach in po-
tato, due to its heterozygosity it is necessary to
clone multiple amplicons of the domain for each
selection for reliable sequencing of all possible
allelic versions of the SNP. Once a true SNP is de-
termined, this SNP is used to design allele-specific
primers and screen the whole population by allelic
discrimination and determine if the SNP is linked
significantly to the trait of interest. In an optimal
case, the gene of interest might bear SNPs that
were included in the Illumina SNP chip for potato
(Hamilton
et al
., 2011); if that is the case, a pheno-
typically characterized population can be screened
directly using the potato SNP chip to determine if
the SNP is meaningful for the trait of interest. If so,
the SNP could also be used to design allele-specific
primers for allelic discrimination in larger popula-
tions. A third possibility is that a candidate gene
can also be physically near a SNP that is in the
Infinium 8303 Potato Array. If the gene of interest
is close to a SNP within the SNP chip that is linked
significantly to the trait of interest, then linkage
analysis of the candidate gene would determine if
the SNP is informative for screening a population
for the trait of interest by using the SNP chip.
17.6 Transgenics
While molecular markers and other molecular
applications have been successful in characteriz-
ing existing genetic variation within species,
plant biotechnology generates new genetic di-
versity that often extends beyond species bound-
aries (Shewry
et al
., 2008). The introduction of
single genes with the hope of delivering im-
proved traits to potato through transgenic re-
search has been attempted with a range of genes
from various organisms (Veilleux and De Jong,
2007). The first wave of commercially available
transgenic potatoes with Colorado potato beetle
resistance, PVY, and
Potato leaf roll virus
resist-
ance was released in the mid-1990s, but was
subsequently removed from the market due to
international discrimination against genetically
modified potato (Kaniewski and Thomas, 2004).
In the interim, scientific research has continued
