DNA Markers Provide Road Signs

One of the advanced techniques for conventional wheat breeding used by Colorado State University (CSU) is DNA marker-assisted selection (MAS). This technique uses DNA markers to identify traits by looking at the wheat plant’s DNA.

“DNA markers are proxies that allow us to select for traits of interest,” said CSU Wheat Breeder Dr. Scott Haley. “By virtue of having a known DNA sequence in the wheat genome, we can establish linkage with traits of interest. As a complement to selection for the trait itself, we can select by absence or presence of the DNA marker.”

The markers are not the DNA that causes the trait, but are like road signs on the DNA that are always associated with that trait. If researchers scan the DNA and find the marker, they know they have the trait.

Haley said that CSU has been using MAS in the wheat breeding program since 2003, though advances in DNA marker technologies and increased funding from the Colorado Wheat Administrative Committee (CWAC) have allowed considerable expansion of this effort since 2007.

DNA markers are like road signs on the DNA that indicate whether a plant has a particular trait.

DNA markers are like road signs on the DNA that indicate whether a plant has a particular trait.

“It helps us select traits, but more importantly it helps us select for trait combinations,” Haley said. “In the greenhouse, we can collect a very small amount of tissue, determine if the plant has the markers, and do back-crossing with the plants that have the markers and throw out the ones that don’t.”

Once the DNA marker for a specific trait has been determined, it is easier to scan thousands of plants for that particular trait.

This is especially helpful in regard to traits that aren’t readily evident in the plant. Wheat breeders can see if a plant is tall or short, or if it is resistant to rust. Other traits, such as baking quality, or resistance to pre-harvest sprouting are not noticeable to the naked eye.

“MAS allows us to select for traits for which we don’t have reliable, convenient or available screening methods. One example is Ug99 stem rust resistance. We don’t work with actual rust pathogens here on campus, so we have to do our selection for that trait with markers,” Haley said.

“We also use markers to select for quality related traits, like the dough strength trait in Snowmass (hard white winter wheat). DNA markers allow us to test for that trait in a young plant rather than testing the quality after the grain is harvested. The quality lab requires about 100 grams of wheat, so we have to plant a bunch of plants to collect enough grain. It takes time to grow the plants, harvest the grain, and mill it. With MAS, we can determine if the seedling of one plant has the trait, and it costs us less than a dollar to do that test.”

Haley said that no varieties developed using MAS have been released yet by CSU, but MAS has been used in the breeding process to confirm presence of favorable traits.

“By using DNA markers, we were able to confirm Snowmass had a specific dough strength gene.  We also used DNA markers in the selection process for Brawl CL Plus (hard red winter wheat), to ensure that the plants had markers for both herbicide tolerance (Clearfield®) genes. We did this during the seed purification process, to make sure the variety was 100 percent pure for both Clearfield® genes.

“We developed a stripe rust resistant version of Ripper using DNA markers, and this was in the variety trial in 2011. In addition to being highly resistant to stripe rust it yielded well, but for some reason its test weight was poor, so we discarded it from release consideration. We sure are crossing the heck out of it though,” Haley said.

Haley said MAS helps refine the breeding process.

“The process of developing a wheat variety involves developing an inbred line through multiple generations of inbreeding through self-pollination. Through this process we apply selection to discard the plants we don’t want and keep those that we do. Where markers really have their power is in identifying trait combinations among those that we select,” Haley said.

Using MAS, the CSU wheat breeding team can test plants at the seedling stage to see if they have successfully combined two desirable traits, such as the dough strength trait and the wheat streak mosaic virus resistance trait. They don’t have to infect the plants with the virus and do a quality test to see if they have both traits.

“And you can put a stripe rust resistance gene on top of that. MAS doesn’t really help with speeding up development time of new experimental lines, but we can develop lines with optimum combinations of genes more reliably and precisely than we can with conventional techniques,” Haley said.

CSU is currently using MAS on thousands of lines in the breeding program. Traditional breeding, doubled haploid production, and single-seed descent are used to produce lines, and then they are screened for desirable markers using MAS. Each season, 3,000 lines (prospective varieties) are screened with MAS. Haley said that the breeding team is screening those 3,000 lines for several different markers.

Three new pieces of equipment recently purchased by the wheat breeding team using royalty funds from Colorado Wheat Research Foundation (CWRF) seed sales are contributing to this effort.

“The liquid handling system is essentially a robot,” Haley said. The device is used to quickly and efficiently dispense reagents into 96- or 384-well plates. A plate is a flat square plastic plate with “wells” which essentially are small test tubes. The new liquid handling system can work with different sizes of plates, from 96 wells to 384 wells (see picture below).

Two of the plates used in the liquid handling system, one with 96 wells (front) and one with 384 wells.

Two of the plates used in the liquid handling system, one with 96 wells (front) and one with 384 wells.

The liquid handling system aids in DNA extractions and polymerase chain reactions (PCRs), tests which are the backbone for MAS. PCRs amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence so that it can be detected.

“The new liquid handling system allows us to do 384 PCRs at a time. Different reagents have to be put in there, plus the DNA, enzymes, and other things, and this can be done automatically with this system. This greatly increases our efficiency,” Haley said.  Previously, the reactions had to be set up manually with pipettes, measuring the different components into the small wells in a plate. The liquid handling system is much more efficient and reduces the potential for errors.

For DNA extractions, “it would take eight days to do 50 plates with 96 samples in each plate,” said Emily Hudson, CSU research associate, “The liquid handling system will do that in half the time or less.”

The second piece of equipment is a DNA fragment analyzer, which is used to separate DNA proteins by size. By looking at the results, the breeding team can tell if that plant has the DNA markers for desirable traits.

The DNA analyzer can test up to 96 samples from different varieties at a time to see if they have the marker for the gene that gives the Snowmass hard white winter wheat variety its dough strength, for example. The researcher sets up a plate with DNA fragments from each variety they want to test for that trait. The plate, with its 96 wells, attaches to 96 capillaries in the fragment analyzer, and the capillaries pull out a very tiny amount of each sample. The machine then sorts the DNA in each sample based on size. The results are sent to a computer, and are then printed out in a graphical form.

CSU Research Associate Emily Hudson demonstrates the new liquid handling system.

CSU Research Associate Emily Hudson demonstrates the new liquid handling system.

The fragment analyzer takes the place of the gel electrophoresis apparatus, which separates DNA by size by applying an electrical field to move the DNA through a gel matrix. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. An image like a photograph is then made of the resulting sorted proteins (see picture below).

The third new piece of equipment is a plate reader. The plate reader is about the size of a desktop printer and looks like a printer. It is used to read the contents of the wells in the rectangular plastic plates.

If CSU develops a mutant line with a beneficial trait, that mutation is present on only one base pair out of the millions of base pairs of DNA. That one base pair can’t be detected on the fragment analyzer, because the difference between mutants and non-mutants is a change on only one base in the DNA sequence.

A gel electrophoresis image, with the DNA molecules sorted by size.

A gel electrophoresis image, with the DNA molecules sorted by size.

Previously, DNA would be extracted, PCR performed and the PCR products would be sent out for sequencing to determine which plants had the mutation of interest and which plants did not. Those plants with the mutation would be used in crosses; those without the mutation would be discarded.

Sample output from the new DNA fragment analyzer. DNA size is represented by graphs.

Sample output from the new DNA fragment analyzer. DNA size is represented by graphs.

Now, CSU wheat researchers are working on detection systems involving fluorescence that can be analyzed using the plate reader. Mutants will show up one color and wild types (or non-mutants) will show up a different color. This process saves money as well as time.

The plate reader also gives accurate readings of DNA concentrations after extraction. This allows for more precise downstream reactions using the DNA.

Haley said some of the traits his team is looking for include herbicide tolerance, resistance to a variety of diseases and insects (stripe, leaf, and stem rust resistance including Ug99; wheat streak mosaic virus; Russian wheat aphid; wheat stem sawfly; wheat curl mite), and several quality-related traits (gluten strength, pre-harvest sprouting tolerance, and polyphenol oxidase (PPO) content (PPO causes darkening in food products).

The goal is to incorporate more than one desirable traits into a variety; to have a “stack” of desirable traits for the farmer.

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