TILLING Genes To Improve
Technician Scott Johnson (left) and geneticist Niels Nielsen
identify a mutant after deconvoluting an eightfold pool of mutant DNAs.
A new genetic tool is making it possible to reap many of the
benefits of genetic engineering without its disadvantages, whether real or
perceived. Among these benefits is breeding better crops—that is, plants
that are more nutritious or that can lower risk of developing cancer or food
The technique is called TILLING—for Targeting Induced
Local Lesions In Genomes.
Geneticist Niels Nielsen and biologist Rae Ritchie, both in
the ARS Crop Production and Pest Control
Research Unit on the campus of Purdue University at West Lafayette, Indiana,
are using TILLING to develop soybeans with better seeds. The TILLING strategy
was devised by Steven Henikoff and his colleagues at the Fred Hutchinson Cancer
Research Center in Seattle. Nielsen and Ritchie are focusing first on improving
the oil and protein content of soybeans. Later, they’ll turn to making
soybeans allergen free.
Since 2002, they have been using TILLING to create special
lines of soybeans for breeding. They’re working with Williams 82
soybeans, adopted by other soybean genome researchers as the standard variety
for genome mapping.
First, they soak soybean seeds in a chemical
solution—either ethyl methane sulfonate or nitrosomethylurea—to
Then they plant the seeds, grow the mutant soybean plants,
harvest a seed from each plant, and grow these second-generation seeds the next
summer. Only about 10 percent of the treated seeds survive to this point. From
the second-generation plants they harvest leaves and third-generation seeds.
DNA from the leaf tissue is extracted and placed in 96-well plates. Each well
contains DNA from 8 plants (an “eightfold pool”), so each plate
permits analysis of 768 plants.
Showing Them Where It’s At
DNA prepared from each mutant plant is examined to identify
mutant genes. Here, biologist Rae Ritchie collects leaf punchouts that will be
used for DNA isolation.
Perry Cregan, a geneticist at the ARS Soybean Genomics and
Improvement Laboratory in Beltsville, Maryland, provided Nielsen and Ritchie
with marker genes to use to analyze mutation frequency.
To find mutations in the genes they are interested in, they
make fluorescently tagged primers and use PCR (polymerase chain reaction) to
amplify the gene of interest. Then the PCR products are heated to make the DNA
strands separate. When the solution cools, the denatured DNA strands reunite.
When a mutant strand combines with a normal strand, a mismatch will occur,
forming a bulge, or lesion, at the mutation site.
When a specific enzyme is added to the DNA “soup,”
it causes the strands to be cut at the lesion. The solution is loaded onto a
gel, and the strands are separated by size using a technique called
Shining ultraviolet light on each gel reveals the sizes of the
strands, with the mutant pieces appearing smaller than the others. When a
mutation is found in an eightfold pool, the process is repeated on each of the
eight individuals to determine which plant has the mutation. The seed from this
mutant plant is then sown, and the plants are later tested to see how gene
function may have changed.
Seeds derived from mutant soybeans are loaded on a plot planter
by geneticist Niels Nielsen (middle), technician Scott Johnson, and Miranda
Salla, a research assistant from Dairyland Seed Company.
Finding Out What the Genes Do
The technique of chemically inducing mutations has been around
since the 1930s and has been used by plant breeders to introduce useful genetic
variation into crop plants. For example, it was used to knock out a gene in
rapeseed to make production of edible canola oil possible. But what’s new
about TILLING is that it uses automated, robotic equipment to quickly screen
for mutations—gene by gene, one piece of DNA at a time. These genetic
variations could eliminate or change a gene’s functions.
Nielsen’s TILLING lines of soybeans should offer soybean
breeders and genome researchers a valuable resource, making it easier to
determine a gene’s function, remove undesirable traits, or create
“With TILLING, you can either find out what genes
actually do—so you use the right genes for breeding—or you can find
a mutant gene that has the desired trait,” says Nielsen.
Eliot Herman, an ARS molecular biologist at St. Louis,
Missouri, works on developing hypoallergenic soybeans by eliminating protein
allergens from the seeds. In 2003, he and colleagues nationwide used genetic
engineering to create a soybean line with a gene that knocks out a major seed
allergen. Herman sees TILLING as the next step for creating allergen-free
soybeans, particularly for use in baby food or formula. His genetically
engineered soybeans are presently not acceptable for that purpose.
Soybeans are one of the top eight allergenic foods, along with
peanuts. Food allergies affect 6 to 8 percent of children and 1 to 2 percent of
adults. Many children grow out of some of their food allergies (milk for
example), which accounts for the differences in adult percentages.
Soybean allergens can cause a skin rash, gastric distress, and
other reactions in children and adults and can also affect livestock and,
TILLING can be used to find mutant genes to create a soybean
or other legume line that is free of specific allergens or to find mutant genes
that weaken the allergens so they affect even fewer people. It may be difficult
to eliminate all the allergens; but the goal is to develop strains with reduced
Nielsen and Herman work with Rick Helm, who is with the
Arkansas Children’s Hospital Research Institute/Arkansas Children’s
Nutrition Center in Little Rock. Helm is using piglets to study sensitivity to
food allergens. Like human infants, some weaned piglets are affected by similar
soybean allergens until their immune systems are fully developed.
Helm is also working with Nielsen and Purdue University animal
scientist and geneticist Allan Schinckel to develop lines of swine specifically
bred for allergen-sensitivity studies.
“We’ll breed a line of swine that is highly
sensitive to food allergens in general and a line that is highly tolerant to
them,” Helm says. “Then we can test the effects of specific
allergen-free crops on the piglets.”
Nielsen notes that next year, United States regulations will
mandate labeling of all products with soy as containing a potential
Agricultural Research Service Information Staff.
This research is part of Plant, Microbial, and Insect
Genetic Resources, Genomics, and Genetic Improvement, an ARS National Program
(#301) described on the World Wide Web at www.nps.ars.usda.gov.
Niels Nielsen and
Rae Ritchie are in the USDA-ARS
Production and Pest Control Research Unit, 915 West State St., Room 2-464A,
West Lafayette, IN 47907-2054; phone (765) 494-8057, fax (765) 494-6508.
"TILLING Genes To Improve Soybeans" was
published in the July
2005 issue of Agricultural Research magazine.