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A new research approach that allowed scientists to rapidly identify the gene
responsible for high sodium levels in certain naturally occurring plant
populations could have applications for the study of a wide variety of other
important plant properties, December 2006.

The approach, a combination of new and existing technologies, may offer
researchers a tool to pinpoint genetic differences many times faster than
currently possible and help shed light on the likely origin of such
differences.

"We've combined a variety of techniques to get at the gene behind a specific
trait," said David Salt, a horticulture professor at Purdue University. "If
picked up broadly, the approach could have an important impact on the
activities of other laboratories."

The method allowed Salt's research team to determine differences within a
single gene that drives a specific trait among naturally occurring plant
populations ? a finding that can take years with current methods, he said.

Salt's method combines the new technology of DNA microarrays with
information from a large genetic database in order to sidestep the lengthy
processes previously used to identify similar genetic variations. Salt
employed his methodology to identify a sodium-regulating gene in the
extensively studied Arabidopsis thaliana, a wild mustard plant.

Salt said this approach could allow scientists to better understand the
genetic basis of naturally occurring variations. These variations occur in
the manifestation of tangible traits, or phenotypes, within a single
species. Phenotypic differences can include anything from flower color to
cold sensitivity or sodium concentration. This ubiquitous tendency of
individuals and populations to vary is termed natural variation.
Evolutionary theory proposes that differences among populations can arise
for evolutionarily favorable, or adaptive, reasons. If the differences
between populations become great enough, they might lead to the development
of a new species, called speciation.

The mechanism of speciation, however, remains poorly understood. Salt said
this approach could hopefully shed light on the process.

"By looking at natural variation, which we assume to be adaptive, we might
be able to better understand why the organism evolved to be that way," Salt
said. "This could be of value in many areas of biology."

Salt's findings were published earlier this month in the online journal PLoS
Genetics.

Salt studies the composition of elements and ions, tiny charged particles,
in plants. Called ionomics, the study of a plant's elemental composition is
important for understanding their physiology, Salt said.

Since plants are immobile, they must make the most of their environment ?
the water, sunlight and soil where they are ? to survive. Plants' ability to
survive and thrive is tied to their ability to take up the right chemicals,
usually in ionic form, from the soil.

Salt uses the database, known as the Purdue Ionomics Information Management
System (PiiMS), to find "candidate genes," or genes that warrant further
study. He combines this knowledge with results from DNA microarrays, small
chips that can identify miniscule genetic differences between populations of
a single species.

In the Arabidopsis study, Salt identified the gene, called AtHKT1,
responsible for elevated sodium levels in two wild populations of the plant.
The study began with a simple observation: Two populations of Arabidopsis
from coastal regions of Spain and Japan had inexplicably high levels of
sodium.

"So, the question became, 'Why?' But to get there, we had to first answer a
series of simpler questions," Salt said.

The first question was how those plants differ from the "garden-variety"
Arabidopsis. This is not a simple question, he said, which is why so few
studies have been published concerning the precise genetic basis of natural
variation.

The initial difficulty is that to date only one variety of Arabidopsis has
had its genetic material sequenced. But this particular variety, called
Col-0 (so named because it is indigenous to Colombia), is not genetically
identical to all other populations of Arabidopsis, Salt said.

For an answer, Salt used DNA microarrays to detect genes that varied in the
two coastal populations. He cross-referenced this information with the
database to seek out genetic differences that may play a role in regulating
sodium levels.

Salt found that the costal populations had a different version of the gene
called AtHKT1, which previous studies have shown helps govern the process in
which sodium is prevented from rising out of the plant's roots.

Further experiments showed that AtHKT1 is genetically associated with sodium
tolerance, which could help explain why the gene is found in coastal
populations where there may be elevated salt levels.

"It could just be a coincidence that these coastal populations, where soils
naturally have higher sodium concentrations, have a defective version of a
gene involved in sodium regulation," Salt said, "But it also may not be. We
are currently in the process of answering the original question of why. This
methodology has gotten us very close to an explanation."

Sodium chloride, or table salt, is generally toxic to plants at
significantly high concentrations. Salt said this study will help his team
better understand the way in which plants process sodium.

Postdoctoral researchers Ana Rus and Ivan Baxter co-authored the paper. Salt
is currently investigating the potential origin of the defective AtHKT1
gene.

He continues to add to his database, compiling thousands of samples a week.
His database records what Arabidopsis genes have been "knocked out," or
mutated, and lists the corresponding levels of 17 different elements in each
plant. A paper describing this PiiMS database has been accepted for
publication in the journal Plant Physiology. The database can be accessed
online at [www.purdue.edu].

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