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Proc. Int. Soc. Sugar Cane Technol., 24: 551-553
2001
DNA SEQUENCE INFORMATION INDEPENDENT TECHNOLOGIES FOR
PLANT GENOMICS
ANDRZEJ KILIAN, SRI KOERNIATI, XIQIN FU, SELVAMEENA RAJAGOPAL,
DAMIEN JACCOUD and KAIMAN PENG
Centre for the Application of Molecular Biology to International Agriculture (CAMBIA),
Canberra, ACT 2601, Australia
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Abstract
CAMBIA'S genomics program has developed two new technologies. The first, the Transgenomics
Initiative (TGI), offers a novel paradigm for biotechnological intervention and is a complement to
the dominant sequence-based paradigm. This program concentrates on developing novel traits and
agriculturally relevant characteristics. TGI offers a foray into the field of 'regulomics', where novel
phenotypes generated through changes in gene regulation will provide researchers with a basis for
mapping and understanding gene networks in plants. The controlled manipulation of expression of
practically any gene in rice offers an opportunity to develop aad test specific hypotheses about
linkages between gene expression and the resulting phenotype. The rice TGI program has focused on
the development of a comprehensive population of Transcriptional Activator Facilitated Enhancer
Trap (TAFET) lines. The second technology, complementary to TGI is the development of new
approaches to high throughput genetic analysis using DNA microarrays. The main technology,
Diversity Arrays (or DArT) offers significantly higher sample throughput and lower cost, as well as
minimal DNA sample requirement compared to other technologies. DArT offers simultaneous
genotyping and pathogen diagnosticsldetection on a single slide. Development of Diversity Arrays
and the application of DArTs to germplasm characterisation will be discussed.
Introduction
Structural genomics, including genetic maps and
complete genome sequences, has made very rapid
progress in recent years. However, the ultimate goal
is functional genomics, which seeks the understanding of the functional role of genome components. Gene disruption mutagenesis (Kumar and
Hirochika, 2001) and, more recently, whole genome
transcriptional analysis (Zhu and Wang, 2000) are
becoming the dominant technologies of this new
field. Both approaches offer advances in our understanding of plant biochemistry and development, yet
seem to have limited capability to deliver outcomes
relevant to plant breeding.
Our ability to effectively capture the outcomes of
current functional genomics programs is constrained
by the reductionism of these approaches, genomic
and functional redundancies (Martienssen and Irish,
1999), and the complexity of genetic interactions in
plants and animals. Even if the gene involved in a
specific process is identified by gene disruption, its
agricultural use is still highly problematic+This is due
to the complexity of the interaction of the gene with
the thousands of other genes functioning in any cell
at any given time.
CAMBIA'S genomics research program is based
on very recent understandings that changes in gene
regulation rather than changes in protein sequence are
a driving force in plant evolution. Using this new
knowledge, we have developed a process in which
novel phenotypes and traits are created by the
changed expression pattern of the plant's own genes.
Instead of searching for novel genes in the genome of
a crop plant's distant relatives (or even outside the
plant kingdom) we are exploiting each genome's
natural ability to evolve solutions to environmental
demands. Our approach will offer breeders an important new tool in the crop improvement process.
Transgenomics-novel approach to trait
generation
The Transgenomics approach is to manipulate
gene expression patterns and networks by using
developmentally defined "foreign" activators andlor
repressors. Transgenomics involves a three-step
process:
Transcriptional Activator Facilitated
Enhancer Trapping (TAFET)
In the first step, a large number of genomic
regions are captured using a specially adapted
'enhancer trap' that employs a transcriptional
activator to generate transactivator 'pattern' lines
(Ferveur et al., 1995). The Transactivator is a protein
capable of gene activation upon binding to its defined
DNA target sequence. Using a range of reporters
developed at CAMBIA, that are capable of monitoring the activity of a transactivator, we are characterising a population of transactivator lines in the
laboratory. We expect to characterise the lines under
field conditions to evaluate the transactivator's
performance in the 'real life' situation.
KEYWORDS: Genomics, Gene Expression, Phenotype, DNA, Germplasm, Diversity Arrays.
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Kilian et a/., Prsc. Int. Soc. Sugar Cane Technsl., 24: 551-553
Genome saturation with activator responsive
short sequence tags
In the second step, short DNA elements called
Upstream Activating Sequences (UAS) are inserted
randomly into a plant genome. The Transactivator
protein recognises these UAS elements and upon
binding to them stimulates the expression of nearby
genes. A large population of 'target' plants is developed, each line containing a number of UAS
elements in its genome.
Gain of function mutagenesis-trait generator
This is the final step in Transgenomics, where
combinatorial matching of transactivator (pattern)
lines from step 1 and UAS tagged (target) lines from
step Occurs. Traditional genetic crossing between
pattern and target lines allows transactivator protein
(from a pattern line) to promote expression of the
genes proximal to UAS elements (from a target line).
This activation of UAS-tagged genes will result in
modified interactions between genes and gene networks, often leading to the emergence of a novel trait
via gain of function mutations. A similar approach
was used to identify gene functions in Drosophila
(Rorth, 1996). For agricultural applications, characters of agricultural importance and need will be identified by screening progenies of genetic crosses
between pattern and target lines. This phase of the
project will be executed through interaction with
plant breeders and physiologists who will be instrumental in developing effective screens and using the
novel genetic materials developed through Transgenomics in their breeding programs.
Currently, our efforts are concentrated on the
molecular characterisation of rice TAFET lines. We
currently have over 3000 TAFET lines that have been
analysed for reporter gene expression (GUS and
GFP) and T-DNA copy number. Of the first 300 lines
analysed, 36 lines have been identified with GUS
reporter gene expression in the root. These lines can
be classified into 12 distinct pattern classes with GUS
expression in the pericycle, vascular bundles, apical
meristem, cap or root hair or a specific combination
of the above. In shoot and leaf tissues, we have
identified 19 patterns with expression in the shoot
base, node, coleoptile, leaf blade, collar, ligule or
auricle, or a combination of the above. Analysis of
floral tissues yielded 55 GUS expressing lines that
could be grouped into 32 'pattern' classes with
expression in the palea, lemma, lodicule, anther
filament, anther sac, pollen style or stigma, or a
combination of the above. In general, a wide range of
interesting expression patterns in practically all
tissueslorgans of TAFET lines has been obtained.
A subset of the TAFET lines was analysed by
Southern blot analysis for transgene copy number.
The Southern blot data indicates that there are 1 to 7
transgene copies per TAFET line, with over 50%
552
lines showing single copy inserts. Nearly 90% of the
TAFET lines had 1-3 copies of the transgene. The
average number of copies in the studied population
was 2.1. Based on these data, we are very confident
of quickly developing a large population of true lines
(homozygotes) with a single copy of the TAFET
transgene.
Diversity array technology
We have adapted the DNA microarray platform to
analyse DNA p~lymorphisms.This genotiping technology is referred to as Diversity Array (DArT) and
combines the highly parallel and automation compatible nature of the microarray platform with a userdefined source, size and complexity of diversity
panels arrayed on the slide (Jaccoud et al., 2001).
~ l i ~the igel ~based
~ separation
~ i ~and~quantitation
steps reduces the cost of analysis per sample and
increases the throughput. DArT is not reliant on DNA
sequence information, which enables applications in
practically any species.
The principle of Diversity &ray technology is
based on assaying for the amount of a specific DNA
fragment in a subgenomic sample which is derived
from the total genomic DNA of an organism or a
population of organisms. A Diversity Panel is created
by cloning and individually arraying a large number
of DNA fragments prepared from subgenomic
samples representing a selected group of genotypes.
Subgenomic samples are prepared by restriction
enzyme digestion of genomic DNA followed by ligation of restriction fragments to adapters and subsequent amplification. Individual DNA fragments are
isolated by cloning. The inserts are then amplified
and arrayed on a solid support. Diversity Panels
created using this method allow genetic fingerprinting of any organism or a group of organisms
belonging to the genepool from which
developed. A fingerprint is deterrmned
a subgenomic sample prepared from the organism(s)
to be assayed to the arrayed nucleic acid fragments.
Two basic analysis formats can be used: in the
first approach (diplex analysis), two subgenomic
samples are compared on a single
~ ~ c o napproach
d
(internal control method), a subgenomic sample is compared to DNA fragment
COmmon to all elements of the array.
To validate Diversity Array Technology we used
rice, an important model for crop plants. The Diversity
Panels were generated using nine rice cultivars. Using
several different restriction enzymes to create Panels
we applied DArT to rice germplasm characterisation
and tracking genome wide methylation changes. Composite Diversity Panels allowed the resolution of complex genomic samples into respective components,
offering genotyping in parallel with pathogen or endosymbiont detection and characterisation.
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Kilian et al., Proc. Int. Soc. Sugar Cane Technol., 24: 551-553
2001
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.laccoud, D., Peng, K., Feinstein, D. and Kilian, A. (2001). Diversity arrays: a solid state technology for sequence information independent
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Kumar, A. and Hirochika, H. (2001). Applications of retrotransposons as genetic tools in plant biology. Trends Plant Sci., 6: 127-134.
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