"Frontmatter". In: Plant Genomics and Proteomics

M
OLECULAR
M
APS
As mentioned in previous chapters there are a number of different types of
molecular maps. The genetic map includes the loci ordered with respect to
the frequency with which they recombine. A physical map is a linear order
of the genomic sequence in some form whether it is as a set of ordered BACs
or as a complete genome sequence. The most useful type of molecular map
will be the result of integrating the genetic and the physical maps. When the
genetic markers are used to anchor the physical contigs any locus that is
genetically mapped can then be placed in a specific physical region of the
genome. The ultimate example in plants of an integrated genetic and phy-
sical map is Arabidopsis, for which the complete genome is available.
However, other examples that have large, but as yet incomplete, genomic
resources are maize (http://www.maizemap.org/iMapDB/iMap.html) 
and soybean (http://hbz7.tamu.edu/homelinks/phymap/soybean/
soytool_4.htm).
DNA-based markers have revolutionized the whole process of generat-
ing genetic maps because, for the first time, a large number of loci can be
followed in a single segregating population. The range of genetic markers
that are available includes restriction fragment length polymorphisms
(RFLP), random amplified polymorphic DNAs (RAPD) (Williams et al.,
1990), amplified fragment length polymorphisms (AFLP) (Vos et al., 1995),
single-nucleotide polymorphisms (SNPs), and simple sequence repeats
(SSRs) or microsatellites (Senior and Heun, 1993). The usefulness or neces-
sity of each of the marker systems depends largely on the genetic resources
available for the species under consideration. 
M
ARKER
S
YSTEMS
RFLP
S
These were the first generation of markers and are generally detected by the
hybridization of a probe to restriction-digested genomic DNA. Many of the
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probes are derived from single-copy sequences, with EST sequences being
one rich source of potential probes. One current application of RFLPs is the
generation of a high-density map in wheat. In this example, a series of chro-
mosome deletion lines are subjected to hybridization with labeled cDNAs as
probes (http://wheat.pw.usda.gov/NSF/). Despite there being multiple
copies of the genome present in hexaploid wheat, the different members of
the homologous groups can be identified by the disappearance of a band in
the deletion line even if there were no polymorphisms available for that par-
ticular gene (Figure 8.1). Thus deletion lines are exceptionally useful for
mapping regions that are polymorphism poor. Unfortunately, this type of
deletion resource is not available for many plant species, although a set of
maize-oat addition lines that can be used for mapping maize genes has been
constructed (Okagaki et al., 2001).
1 5 0
8. I
D E N T I F I C AT I O N A N D
M
A N I P U L AT I O N O F
C
O M P L E X
T
R A I T S
1
2
3
4
5
6
a
b
c
d
FIGURE 8.1.
Using deletion lines to map chromosomal locations of ESTs. Lanes a–d
represent restriction-digested DNA from a hexaploid plant (containing 6 chromo-
somes designated 1A, 1A, 2B, 2B, 3C, 3C) and where the DNA was extracted from:
a) Plants that have all the chromosomes; b) plants that are 2B, 2B, 3C, 3C; c) plants
that are 1A, 1A, 3C, 3C; d) plants that are 1A, 1A, 2B, 2B. The Southern blot was
hybridized with an EST sequence that showed no polymorphisms in any of the acces-
sions and so could not be mapped. However, from the use of deletion stocks it is pos-
sible to place the specific EST: bands 1 and 2 are on chromosome 3, bands 4 and 5
are on chromosome 2, and bands 3 and 6 are on chromosome 1. Deletion stocks that
have less than a whole pair of chromosomes missing can be used to place the ESTs
more accurately on the chromosomes. 

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AFLP
S
These markers are RFLPs detected by PCR amplification. The polymor-
phic fragments are observed against the background of all of the possible
sized restriction fragments that can be amplified. Adaptors are added to 
the ends of restriction fragments, and these adaptors are then used as
primers in a PCR reaction. Every possible band should be amplified, and the
complex mixture of bands is separated on gels or through automated
sequencers. The polymorphisms can be cloned and sequenced to generate
sequence-tagged sites (STSs). Potential epigenetic effects resulting from
hyper- or hypomethylated regions of the genome can be investigated by
using methylation-sensitive and -insensitive restriction enzyme isoschizo-
mers. However, as mentioned in Chapter 1 species with large amounts of
DNA (>20 pg per 1C) can be problematic when studying genetic diversity
with AFLP techniques.
RAPD
S
Statistically a sequence of 10 bp should appear once every 10
6
nucleotides.
PCR amplification using genomic DNA as the target and a series of single
random 10-base primers has been very successful in generating large
numbers of polymorphisms (Williams et al., 1990). The methodology can be
used when little other genomic information is known. Unfortunately, the
technique appears to suffer from irreproducibility between laboratories and
sources of thermostable enzyme, although, within a laboratory, reproducible
results can be achieved (Jones et al., 1997). 
M
ICROSATELLITES AND
SSR
S
Microsatellites or SSRs are genetic markers that are derived from short
(usually <6 bp) tandemly repeated sequences such as (GA)
n
, (AAT)
n
, (GT)
n
.
The terms are often used interchangeably, although microsatellites are gen-
erally longer than the 2- to 3-bp unit of the SSRs. This type of sequence is
widely dispersed through most animal and plant genomes and polymor-
phisms are due to the variability in the number of repeats at a given site.
SSRs can be isolated from genomic libraries or enriched genomic libraries
(Panaud et al., 1995) or generated from an analysis of cDNA sequences. They
can be converted into STSs with primers designed in unique regions sur-
rounding the repeat and have become an important source of genetic
markers for many eukaryotic genomes (Panaud et al., 1995), especially when
the primers are designed in a conserved region of a transcribed sequence,
making them applicable over a wide range of taxa.
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S
INGLE
-N
UCLEOTIDE
P
OLYMORPHISMS
Single-nucleotide polymorphisms (SNPs) are DNA sequence variations that
occur when a single nucleotide (A, T, C, or G) in the genome sequence differs
between two individual DNA samples. For example, a SNP might change
the DNA sequence AGGATTCA to AGGATTTA. SNPs can occur in both
coding (gene) and noncoding regions of the genome. Many SNPs have no
effect on cell function because they may not change protein structure (in fact,
any SNP that occurs at the third position in the amino acid codon will have
no effect if it does not change the amino acid sequence of the resulting
protein). Their high frequency (perhaps as high as 2–3% in plant DNAs)
means that they can be particularly useful in linkage mapping (Kristensen
et al., 2001; Lai, 2001). They must be derived from sequence information, and
that information must be obtained from two or more individuals. Informat-
ics tools can be used to compare the sequences and identify variations, but
the raw data in the form of trace files may be important in deciding which
polymorphisms may be real. In the building of unigene sets, these differ-
ences are eliminated in the formation of the consensus sequence and need
to be retrieved. Because there is no a priori way of differentiating between a
true SNP and sequencing errors, each potential SNP must be validated. Even
at a frequency of 1% these polymorphisms would generate an exceptionally
large number of haplotypes if every polymorphism could be inherited inde-
pendently. However, relatively few haplotypes are observed, indicating that
perhaps the rate of SNP production is similar to the rate at which recombi-
nation occurs across the regions of the genome making up the haplotype
blocks. Therefore, SNPs are most likely to be useful for defining haplotypes,
rather than for their information individually, and so the use of SNPs is likely
to involve linkage disequilibrium studies using the haplotype rather than
the use of specific SNPs as individual molecular markers.