Research: Genetic Snapshots

U.S. Researchers are examining the genetics of switchgrass in order to identify traits that will improve the plants’ conversion to ethanol

Corn has captured public attention as the crop with potential to quench North America’s thirst for ethanol and other such biofuels.  Another fuel-friendly crop is switchgrass. USDA Agricultural Research Service (ARS) research shows that an acre of biomass (stems and leaves) from this perennial grass has the potential to yield 300 to 800 gallons of ethanol.
That’s a promising estimate, but more research is needed to improve the conversion technology used and to make the plant biomass easier and less costly to convert into ethanol.  Conversion is done by breaking down the plant’s cell walls into sugars and then fermenting them.

One approach is to develop new switchgrass varieties with traits geared to producing ethanol rather than traditional uses, like feeding cattle, anchoring soil, or restoring grasslands.

“The ideal switchgrass for bioenergy production would have low input requirements, good stand establishment – especially the first year – high yield, and excellent conversion-to-ethanol properties,” says Gautam Sarath, a molecular biologist in ARS’s Grain, Forage, and Bioenergy Research Unit (GFBRU) in Lindcoln, Neb.

Building Living Libraries
To expedite breeding efforts, Sarath and collaborators generate tens of thousands of genetic “snapshots” of switchgrass in action – from the moment it sprouts from seed to the time it prepares for overwintering.

The snapshots are actually fragments of genetic material called messenger RNA (mRNA).  In plant cells, mRNA delivers instructions for making proteins and carrying out other tasks assigned by DNA – the so-called blueprint for life.

Extracting mRNA from switchgrass offers a glimpse of how this molecular workhorse does the bidding of DNA at particular growth stages or physiological moments in development. A later step, a technique called “microarray analysis” allows scientists to visually identify which genes were active when they plucked the mRNA from the grass’s tissues.
The mRNA is difficult to work with outside of cells. So, the researchers create a more stable version — complementary DNA (cDNA). Using standard biotech methods, they insert the cDNA into specially engineered plasmids, which can be propagated in E. coli bacteria. Plasmids are circular molecules of DNA found outside chromosomes.

Thus engineered, the bacteria are cultured on plates, where they form thousands of colonies. At this stage, they become known as “libraries,” because each bacterial colony contains a plasmid with a unique cDNA from switchgrass.

Since 2003, Sarath and collaborator Paul Twigg of the University of Nebraska-Kearney have produced several cDNA libraries from switchgrass. From these, Christian Tobias, a molecular  biologist at ARS’s Genomics and Gene Discovery Research Unit in Albany, Calif. has determined the structure or sequence of some 12,000 previously unknown switchgrass gene fragments.

In a preliminary analysis of the sequences, Tobias and co-investigators grouped about 65 per cent of the new sequences into clusters based on commonalities in their structures. Each of these groups may prove to be a unique gene. The sequence fragments were then compared with databases containing well-characterized genes to provide insight into the possible function of each new switchgrass sequence.

“A closer examination of fragments within clusters revealed that some seemed to have some slight variations. These variations are of interest because they might lead us to a trait that we want to investigate further,” Tobias points out. “These sequence variations reflect and reveal a portion of the genetic variability within the world’s switchgrass gene pool and can be both associated with desirable traits and used in breeding and switchgrass-improvement programs.”

Tobias and Sarath posted the gene sequences to publicly accessible databases on the Internet in 2005. This treasure trove of new discoveries is the most extensive catalogue of switchgrass  genes yet available for scientists everywhere to use. Researchers can, with the aid of computers, quickly compare and contrast the structure of switchgrass genes to those of other grasses or  other forms of life.

Sarath and Twigg have pinpointed a cluster of 12 to 14 genes regulating production and deposition of lignin, a molecular “glue” that binds components of plant cell walls. Sarath notes that bioenergy researchers are keen on weakening lignin’s grip — either through conventional breeding or genetic engineering — to free up more sugars from cell walls for fermenting into ethanol.

The team’s original cDNA libraries came from a single switchgrass variety. Others will be added,  including lowland bioenergy-switchgrass types from a breeding and economic evaluation program run by GFBRU research leader Kenneth P. Vogel and rangeland scientist Robert Mitchell.

Helping Hands
The scientists are sending these libraries and RNA to the U.S. Department of Energy’s Joint Genome Institute in Walnut Creek, California. There, fast, state-of-the-art gene-sequencing instruments will identify up to a half-million switchgrass sequence fragments, called “expressed sequence tags” (ESTs), within the next three years. These sequences will be compared with those from other plants — particularly other grasses, such as corn and rice — providing invaluable data.
 
“These ESTs will give us the tools to really understand, or look for, genes important for breeding purposes,” adds Sarath. For this ambitious venture, he and Tobias have already supplied several of the requisite cDNA libraries for the institute’s ultrafast analyses. n