Principles of Plant Genetics and Breeding

Structural defect in the gene construct may cause transgenic
breakdown. The phenomenon of progressive
transgenic silencing or failure is not completely
understood. It is suspected that ancillaries of an expression
system (e.g., terminal sequence promoters) may
have a role in the event. Especially, systems using heterologous
sequences from non-related sources (e.g.,
plants having bacterial or viral sequences, instead of
native sequences, or those from closely related species)
are more prone to malfunction, as they become recognized
as foreign DNA and consequently excised or
methylated. It is also suspected that the structural
integrity of the introduced foreign DNA sequence may
be responsible. This relates to the presence or absence
of introns and the nature of the 5′ and 3′ sequences.
Transgenics produced from cDNA clones instead of
genomic sequences lack introns and possibly other 3′
gene sequences. This may reduce the accumulation of
mRNA as some researchers have reported.
Plant genomics
The basic set of chromosomes of an organism is called
its genome. Traditional geneticists generally investigate
single genes, one at a time, as snapshots. Studying
genetics whereby the totality of the genes in an individual
are considered together would be advantageous.
Genes seldom, if ever, work independently in higher
organisms.
What is genomics?
Genomics is the approach of investigating the totality
of genes in an individual as a dynamic system, not as
snapshots, but rather over time, and to determine how
these genes interact and impact biological pathways and
the general physiology of an organism, from a global
perspective (i.e., the “big picture”).
Genomics may be broadly categorized into two –
structural and functional – each with its set of tools
and functions. However, as the field advances, new
terms, categories, and subcategories continue to emerge
in the literature.
Structural (classic) genomics
Structural genomics, at the most basic level, is concerned
with activities at the initial phase of genome analysis –
mapping (the construction of high resolution genetic,
physical, and transcript maps of an organism). The
BIOTECHNOLOGY IN PLANT BREEDING 237
initial phase of genome analysis is the establishment of
a physical map of the genome. The ultimate physical
map of an organism that can be achieved is the complete
sequence of its total DNA (genomic sequence).
Genomic sequencing projects yield linear amino acid
sequences. The most visible genome sequencing project
in recent times is the Human Genome Project. Genome
sequencing projects have been initiated or completed for
some major food crops such as rice, maize, and wheat.
Genome mapping entails determining the order of
genes (or other genetic markers) and the spacing
between them on each chromosome. Two categories of
mapping strategies are employed in genome mapping:
(i) genetic linkage mapping; and (ii) physical mapping.
Genome sequencing
The purpose of DNA sequencing is to determine the
order (sequence) of bases in a DNA molecule. Classic
mapping approaches involve generating and mapping
mutants, thus limiting characterization to those genes
for which mutants have been isolated. rDNA techniques
provide a direct approach to genetic analysis, whereby
a genomic library is created, from which overlapping
clones are then assembled to construct genetic and
physical maps for the entire genome. Ultimately, the
entire genome is sequenced such that all genes are
identified by both their location in the chromosome as
well as their nucleotide sequence.
Allan Maxam and Walter Gilbert invented the chemical
degradation method of DNA sequencing. Another
method, the Sanger enzymatic (dideoxy) method
(chain terminating), named after its inventor, is more
commonly used. However, in these days of more
advanced technology, there are high-throughput methods
of DNA sequencing, making it more convenient for
most researchers to contract sequencing jobs to outside
companies. Capillary array electrophoresis (CAE) systems
coupled with high-sensitivity detection provided
by energy-transfer labeling reagents are now the standard
for high-throughput DNA sequencing facilities.
Genome sequencing is involved and hence often
undertaken on a collaborative basis (i.e., consortium)
over several years. Modern supercomputers and powerful
algorithms are making genome sequencing more
efficient.
Comparative genomics
Biotechnology makes use of certain organisms as models
for comparative research. A fundamental premise of