DNA Sequencing in Full Bloom

By Tom Parisi

Petunias

On a bitter cold day this past January, Tom Sims, associate professor of biological sciences, is surrounded by signs of spring—hundreds of delicate blossoms in shades of purple, pink, and white sprouting in an old greenhouse on campus at NIU’s Montgomery Hall.

This is petunia central. And the spadework done here could help usher in a new era in floriculture, one producing varieties of flowers with color schemes, fragrances, and robustness never before imagined. Less apparent but just as important, a new generation of 21st century molecular biologists is being raised here as well.

Sims, who thinks of the petunias as “purple and green rats,” heads NIU’s Plant Molecular Biology Center. He also leads a large international research effort that one would more likely associate with the world flower capital of Aalsmeer, Netherlands, than “corn-is-king” DeKalb, Illinois.

For the past two years, Sims has been coordinating the Petunia Genome Project, which has nearly completed sequencing the DNA of two wild varieties of the petunia. About 20 universities and institutes worldwide are collaborating on the project, including Cornell, Michigan State, and Purdue, along with the Free University of Amsterdam, University of Verona in Italy, and University of Bern in Switzerland.

Why pick petunias?

Professor Tom Sims

Professor Tom Sims

Petunia hybrida, the garden variety of the flower, is the top floriculture bedding plant in the United States, with total wholesale sales of $132 million in 2011, according to U.S. Department of Agriculture statistics.

“Unlike agricultural and vegetable crops, the application of genomics-related technologies to floricultural crops has lagged significantly,” says Alan Blowers, biotechnology project manager for Ball Horticultural Company in West Chicago. “The availability of the petunia genome sequences will play a major role in ushering in the genomics era for floriculture.”

Experts say the sequencing information will lead to identification and isolation of genes that confer traits, such as fragrance, petal color, time to flower, and flower longevity. But what makes the petunia particularly interesting to scientists is  the fact that it is a model species for basic research in plant biology.

“It’s easier to figure out genetic processes in model systems than in others,” Sims explains, adding that understanding the plant’s genetics will help scientists better understand other plants. Information gained from the Petunia Genome Project, for example, will be applicable to closely related crops, such as tomato, potato, pepper, and eggplant. So data from the project could help scientists eventually produce better-tasting and hardier fruits and vegetables.

Researchers also believe the sequencing will lead to an illumination of the phenomenon of self-incompatibility, which prevents many species of plants from fertilizing themselves.

“Self-incompatibility is an important trait that we’d like to better understand in a lot of fruit-bearing trees as well,” Sims says. “But it’s much easier to conduct genetic research on annual flowers than on trees, which can’t be easily manipulated in a laboratory and have a much longer growth cycle.”

Conventional vs. genetic breeding

Of course, humans have been manipulating the breeding of plants for more than 10,000 years. Just about all fruits and vegetables found in grocery stores are the products of conventional breeding. Modern-day corn, for example, does not even grow in the wild. It is believed to be derived from a Mexican grass called teosinte, which produces small ears with only about a dozen kernels encased in a hard outer shell. You wouldn’t touch it at a picnic.

“Conventional breeding recombines genomes, but you don’t know how the changes occur at the genetic level,” Sims says. “With current technology, we could put in or rearrange just a small number of genes in a specific region of the genome and know exactly what we’re getting.”

Most garden-variety petunias are the result of crossbreeding and are not found in the wild. The first hybrids were produced in the 1800s by crossing two wild species native to Brazil: Petunia inflata, a bee-pollinated plant with purple flowers,  and Petunia axillaris, a moth-pollinated plant with white flowers that open only at night. The Petunia Genome Project targets those two parent species.

Sims has long been part of an international community of scientists and commercial breeders specializing in the plant, and his peers asked him to coordinate the sequencing project when it became more financially feasible two years ago. The sequencing project is being accomplished at a cost of about $75,000, using the shared might of high-powered computers and sequencing machines at research institutions worldwide. Roughly five years ago, the same sequencing project would have cost at least $500,000, Sims says.

A very big jigsaw puzzle

A genome contains a full set of chromosomes (seven for petunia) with all the inheritable traits of an individual organism. Chromosomes are made from double-helix-shaped DNA, which carry all of a cell’s genetic information.

Sequencing requires determining the order of nucleotide bases—the As, Ts, Cs, and Gs (for adenine, thymine, cytosine, and guanine)—that make up an organism’s DNA. The entire petunia genome has 1.3 billion base pairs (A always pairs with T, and C with G), compared to the human genome’s 3 billion.

DNA from each chromosome must be sequenced. Hidden along the seemingly endless strings of genetic letters are sections that make up genes, which provide instructions leading to hereditary characteristics, such as eye or skin color.

First-year master’s student Allison Makulec loads a DNA gel

Master’s student Allison Makulec loads a DNA gel

Using basic techniques, NIU researchers extract DNA from petunia leaves. Because it’s not possible to sequence DNA in a linear fashion from beginning to end, the NIU scientists and their collaborators use a “shotgun method,” sequencing small DNA pieces hundreds of millions of times.

A sequencing machine at the University of Illinois at Urbana-Champaign generates the raw data, and high-end computers and sophisticated programs are used to reassemble the genome.

“It’s like putting together a very, very big jigsaw puzzle with hundreds of millions of pieces,” says Mitrick Johns, associate professor of biological sciences at NIU and a member of the research team who specializes in computer-based analyses of DNA sequence information.

30 trillion calculations per second

Johns is working to discover how the genes in the two different petunia varieties differ. He also is searching for the genes that make one plant become a petunia and another a tomato, potato, or eggplant. Though the plants seem widely different, about 85 percent of their DNA is the same, Johns says.

lthough NIU does not have an expensive sequencing machine, portions of the sequencing are done on campus, along with annotation, which involves finding genes within the sequence and determining their detailed structure.

Johns relies on NIU’s hybrid GPU/CPU supercomputer, or cluster of computers, known as “Gaea” (pronounced GUYuh), for the mythological Greek goddess who was the mother of all. The cluster, which came online in 2012, has a capacity of more than 30 teraflops, meaning it can do more than 30 trillion calculations per second. That kind of computing power comes in handy when comparing two species of petunia with billions of base pairs.

“It’s primarily a matter of speed,” Johns says. “I could do sequencing on a regular personal computer, but the cluster is several hundred times faster.”

Next generation biologists

Two of Johns’ graduate students also are working on the sequencing project, along with three of Professor Sims’ students.

“They are being trained for the genetics of the 21st century, which is based on DNA sequencing,” Johns says. “The general principles in sequencing apply to any organism. Progress in medicine and agriculture will come from sequencing  projects like this.”

Professor Mitrick Johns and doctoral student Jennifer Hintzsche review DNA results

Professor Mitrick Johns and doctoral student Jennifer Hintzsche review DNA results

Jennifer Hintzsche, a doctoral student in biology, plans to make a career in the area of bioinformatics, the blending of biology and computer science. Using a variety of software, the Hinckley, Illinois, native is creating a database of the DNA sequences for assembly and annotation of the petunia genome. “It’s exciting to be part of the entire sequencing project from start to finish with such a large collaboration of great scientists,” Hintzsche says.

“The sequencing of DNA has increased exponentially in the past few years,” she adds. “Consequently, the need for bioinformaticians to analyze this data is also increasing. The knowledge, experience, skill set, and guidance Dr. Johns has given me are invaluable.”

Allison Makulec, a master’s student in biology from Cherry Valley, Illinois, is beginning work on a thesis that will also rely heavily on the Petunia Genome Project. Makulec will map the S-locus, an area in the genome that spans about 6 million base pairs and encodes many of the genes (at least 12) involved in the self-incompatibility response of petunia species. Makulec aims to learn how a self-incompatible plant recognizes and rejects its own pollen.

“We’re still in beginning phases, but hopefully we will be able to determine the structure of that specific area of the genome,” says Makulec, who clearly loves the detective work involved in molecular biology. “It’s the whole idea of being able to find and decode things that are invisible but you know are there.”