Using Molecular Markers in Plant Genetics Research

Using Molecular Markers in Plant Genetics Research Unlocking genetic potential for increased productivity Molecular Markers Researchers at Pioneer blaze a new genetic trail.

dentifying molecular markers is ences. Just as smoke rising into the sky the marker and the gene can be inher- like blazing a trail through a plant’s makes it easier to locate a forest fire, ited together,” says Dr. Jim Register, Igenetic makeup and putting up a gene of interest is easier to locate research coordinator of analytical road signs along the way. when a researcher starts with a nearby nucleic acid technologies at Pioneer. Molecular markers, sometimes marker. “We study these differences and use called DNA markers, should be thought “To successfully use a specific marker them to identify the genes we want. of as signs along the DNA trail that pin- to follow a specific trait, the marker must Then, we track those identifiable differ- point the location of desirable genetic be found close enough to the gene of ences to verify we’re making progress traits or indicate specific genetic differ- interest that variations (alleles) of both in developing a particular trait.”

Resistant Plant

DNA G A T C A C G G T A C T C A A T G G A T T T G T T C T A A C T C A A A T A G

GENE "XYZ" (Allele 1)

Polymorphism – using variation for improvement Codes for...... Polymorphism involves the existence of different forms (alleles) of the same gene in plants or a population of plants. These differences are tracked as molecular markers to identify desired genes and the resulting trait. Most organisms are diploid, meaning they have two copies of each gene — one from each parent. Like brown eyes in humans, one gene usually dominates the other thus determining the inherited trait. This protein helps make plant resistant

Following the Road Look for genetic Identify markers for Select plants with Signs to Improve clues to find specific specific genes and markers/genes for Product Performance genes of interest. determine if they evaluation. can be inherited. 11 2 3 What’s the difference? or difference, is the clue researchers need localize genes and follow markers associ- Molecular markers are first identified as to find the gene of interest. ated with other traits such as maturity, short fragments or “strings” of DNA For example, markers associated with plant height, insect resistance, grain oil located in a specific position on a chro- genes involved in disease resistance have content, etc. mosome. “We are able to use a particular been identified in corn and soybeans. fragment of DNA as a marker when we Differences between the DNA sequences Getting the desired results can detect differences in that fragment’s of these genes can be responsible for “Using the latest marker technologies, DNA sequence between multiple plants making a plant sensitive or resistant to a scientists are able to determine right in or plant lines,” Register says. particular disease. And differences in DNA the lab which plants have economically According to Register, these variations sequences near the gene can be used as beneficial traits faster,” Register says. “This in DNA sequence, called polymorphisms, markers to locate the gene and track the allows us to select plants based on the traits can be associated or linked with different desired results in breeding programs. they possess even before going to field forms (alleles) of nearby genes involved Besides disease resistance research, trials. The time saved allows us to move with particular traits. The polymorphism, this same technology can be used to improved products to the market faster.”

Susceptible Plant Polymorphism

T A T C A C G G A C T C A C T G G A T T T G T T T A A C T C A A A A G T G

GENE "XYZ" (Allele 2)

Codes for......

The order of individual nucleotides identifies the domi- nant gene and the differences between the genes. Also, the nucleotides supply the code for protein production resulting in the plant expressing a particular trait. It’s the differences in these nucleotide strings that modify or alter the plant’s trait expression. Altered protein has modified function

Develop hybrids and Test hybrids and Test plant product Release improved varieties from these varieties to determine performance that products to market. parents. if markers/genes were includes desired gene. inherited. 4 5 2 6 7 Repeat that Again Repetition in DNA sequences leads researchers to meaningful genes.

imple Sequence Repeats (SSR) markers, “This variation in DNA sequence can be germplasm with other competitors’,” he says. or microsatellite markers, are one of the used just like other types of DNA sequence “We use this technology to protect our intel- S most advanced marker technologies variation to locate nearby genes and follow lectual property and the investment Pioneer available in genetic research today and are specific forms of those genes through product puts into product development research for its a key part of research efforts underway at development,” says Dr. Jim Register, research customers.” Pioneer. SSR markers are stretches of DNA in coordinator of analytical nucleic acid technolo- With SSR markers, Pioneer researchers which the same short nucleotide sequence is gies at Pioneer. “SSR markers are considered are routinely able to analyze the location of repeated over and over. highly polymorphic as the number of repeats genes on chromosomes (positions of genes on Polymorphism, or variation, among SSR can vary greatly among plants. This allows us chromosomes) hundreds of plants at one time. markers is determined by the number of times to detect many different alleles for that marker. The millions of data points generated annually the base sequence repeats (e.g. AGTTAGTT vs. “These highly polymorphic SSR markers help them develop improved products faster AGTTAGTTAGTTAGTT). are also excellent tools for comparing our and with added precision.

Single Sequence Repeats Markers or Microsatellites. Following the Road Signs to Improve Product Performance SSR markers are one to six nucleotide repetitions of the DNA code. These repeats, like an argyle sock pattern, are found most often between genes and don’t alter cell function. These are generally found near a gene of interest and serve as a “marker.” Look for genetic clues to find specific genes of interest. Plant A has four unit SSR near 1 gene of interest Identify markers for specific Plant A genes and determine if they can be inherited. T G A G C A T T T T G T T A 2 G C C G A A T T A T Select plants with markers/ (allele 1) genes for evaluation. Gene of interest 3 Plant B Plant B has seven unit SSR near gene of interest Develop hybrids and varieties from these parents. G G T A A T T T T T T T A 4 A T T A T A T T A T T Test hybrids and varieties to (allele 2) determine if marker/gene was Gene of interest Plant C has three unit SSR inherited. near gene of interest 5 Plant C Test plant product performance

T G that includes desired gene. G A A C T T C T G G A 6 G C C G A A T T A T T Release improved products (allele 3) to market. Gene of interest

7 3 Because DNA fragments have different lengths due to the number of SSR repeats, Pioneer researchers can separate the fragments through Gel Electrophoresis. DNA fragments are placed into the top of a gel that acts as a molecular filter. DNA fragments carry a negative charge and the bottom of the gel has Large a positive charge. Just as positive and negative magnets attract, the DNA population moves through the gel matrix towards the positive charge. of inbred As DNA fragments move through the gel, it sorts according to size with plants, descended the smallest fragments moving farther. This way even fragments that differ from a known by only a single nucleotide base can be separated. male/female cross, is Negatively examined for desirable charged DNA characteristics. fragments tagged with fluorescent dye

Plant A: Susceptible Plant B: Resistant Plant C: Susceptible

Sample taken from each plant in the fragments with test plot. For example, the three 7 SSR repeats plants above. stop here

fragments with 4 SSR repeats stop here Plant B Polymerase Chain Reaction (PCR) Plant A fragments with used to amplify the samples. 3 SSR repeats Plant C stop here

In SSR studies, products in the PCR step T are “tagged” with a fluorescent dye T A which will be used later to identify the T T DNA fragments. A T T T A Gel with multiple “lanes” to analyze T T many plants at once A T T A T T T A T A T T T T A T A T T A T T A T T A T T A A

Actual SSR markers

In the next step, researchers compare agronomic knowledge with DNA knowledge via computer analysis. If plant B with an 7-unit SSR repeat has shown resistance to the disease and this same 7-unit SSR repeat has shown up often in other plants demonstrating disease resistance, then this region of DNA can serve as a marker to track and develop the trait. Plant A Plant B Plant C 4 The One and Only A single base nucleotide substitution can be the key to important crop traits.

llele Specific Hybridization (ASH) the discovery and detection of important discovered, these single-base differences focuses on the most common form genes in crops. can be used as molecular markers for genes A of genetic variation in plants: single Just as the name indicates, SNPs are of interest. nucleotide polymorphism (SNP). Because identified by a single nucleotide base SNPs occur frequently throughout the plant change in the genetic code at a specific Promising possibilities genome, they offer enormous potential in location on the chromosome. Once In recent years, scientists have begun to

Sample taken from each plant in the test plot The difference in the two DNA sample from each plant placed within gene alleles is very small 1 of 96 wells on 9 plates (9 x 96 = 864). and specific; namely the A PCR reaction is run to magnify the DNA simple substitution of a samples to a working volume. single nucleotide, in this case a "T" (thymine), instead of "C" (cytosine). To discover which version of the gene each plant has, a probe is

CT A G G T A C A T G G T A Allele 1 (resistance)

TCT A G Allele 2 A G A (susceptibility)

Allele 1

Radioactive probe

Nylon membrane with captured DNA from 864 plants is Plates with the hybridized with a 864 DNA samples radioactively are grouped, and labeled probe the DNA samples are which will attached onto a nylon match only with membrane allele 1 of the gene in question.

Segregating DNA samples from populations of plants each of the 864 plants Nylon membrane

Following the Road Look for genetic Identify markers for Select plants with Signs to Improve clues to find specific specific genes and markers/genes for Product Performance genes of interest. determine if they evaluation. can be inherited. 15 2 3 recognize the promise SNPs offer for coordinator of analytical nucleic acid “Using ASH analysis of SNPs, we rou- targeting important genes. Because they technologies at Pioneer. “SNP analysis is tinely differentiate soybean experimental represent the most common type of a ‘yes’ or ‘no’ proposition (Is the sequence lines by a difference of one base nucleotide genetic variation in plants, SNPs can have of interest there or not?). SSR markers in a region of interest associated with SCN significant effects on resistance to disease, measure a varying number of sequence resistance,” says Dr. Daria Schmidt, performance under adverse environmental repeats.” research coordinator in soybean product conditions and other economically development at Pioneer. “With it, we’re important traits. Resistant or not? able to identify soybean plants with SCN “SNPs are particularly valuable SNP has been a key technology used in resistance before we begin field trials. For because they offer the potential of ultra- Pioneer researchers’ work to develop soy- producers that means getting improved high throughput and highly automated bean varieties with resistance to soybean varieties a couple of years faster than field analysis,” says Dr. Jim Register, research cyst nematode (SCN). trials alone would allow.”

Allele 2 designed to match with each Radioactive probe version, according to The membrane is then washed to remove any trace of probe 1. the rules of nucleotide A second probe to match to the second possible allele is then pairing (A to T, C to G). hybridized to the membrane. A final pattern emerges indicating The probes are given a each plant’s inheritance status of the 2 alleles in question. radioactive tag, which has the ability to expose photo- Actual Audorad from ASH study graphic film. ASH Analysis: What does it tell us? Here are examples of results after hybridization C C G T T A with 2 probes.

White: Allele 1 White: Allele 2 G A A C T T is not present not present in T G G T T A in the plant the plant Probe 1 Grey: The plant w Probe 2 Grey: The plant is heterozygous a is heterozygous H for Allele 1 for Allele 2 for Allele 1 s for Allele 2 S (Resistance) h (Susceptibility) A W Black: Black: The plant is The plant is homozygous homozygous for Allele 1 for Allele 2 Membrane Film Membrane Film After the radioactive probe 1 is hybridized to the membrane, The photographic films from it is exposed to photographic film. each hybridization procedure If a plant has one copy of the allele are scanned into a computer, (heterozygous) being tested for, which compares the two films it will make a gray spot on the film. and then tabulates the results. If it has two copies of the allele The final product of a series (homozygous), it will make a of ASH analyses is a compu- black spot on the film. ter spreadsheet of various If it does not contain the allele at all, plant traits, giving the spot will remain white. breeders a shopping list to choose from. Photographic film

Develop hybrids and Test hybrids and Test plant product Release improved varieties from these varieties to determine performance that products to market. parents. if markers/ genes were includes desired gene. inherited. 4 5 6 6 7 For more information: [email protected]

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