What's in a Name? Great Diversity

Is every wheat called “Bobwhite” the same as others that share the name? Not really, as scientists in CIMMYT’s Applied Biotechnology Center (ABC) determined this past year. In fact, the differences between the Bobwhite sister lines can be considerable.

Does it matter? It does to Bent Skovmand, head of CIMMYT’s Wheat Germplasm Bank. “I got tired of going to conferences and hearing people say, ‘Bobwhite is a Swiss wheat,’ ‘Bobwhite is an Israeli wheat,’ ‘Bobwhite came from here or there,’” he says. “They simply didn’t know where it came from.”

This was too much for Skovmand, who participated in groundbreaking work by Sanjaya Rajaram, now director of CIMMYT’s Wheat Program, on the Bobwhite wheat cross in the early 1970s. Skovmand’s interest went beyond getting the credits right. It engendered collaboration with ABC molecular geneticist Marilyn Warburton and cell biologist Alessandro Pellegrineschi that identified several “supertransformable” wheat lines and new possibilities for molecular fingerprinting in maintaining genetic diversity.

“Bobwhite is the generic name for a wheat cross,” explains Skovmand. “We use such names because it’s difficult to recite long pedigrees, and the names serve as a type of shorthand.” CIMMYT produced 129 Bobwhite sister lines that merited preservation in its genebank and were used by breeding programs worldwide. Lines that were not named by a national breeding program reverted to being called Bobwhite. It is easy to see how this could lead to confusion.

Molecular fingerprinting, Skovmand surmised, could clearly document the identity of the 129 Bobwhite sister lines and address a key question for his genebank: Have fingerprinting techniques advanced to the point where they can be efficiently applied to genetic resource conservation?

CIMMYT’s wheat genebank stores more than 155,000 accessions. Skovmand’s dilemma is to determine how many and which related accessions must be conserved to retain genetic diversity. When a number of lines appear to be representative of a landrace or cross, they can be put together, or “bulked,” in the bank. While this strategy is economical and efficient, Skovmand has resisted it because valuable genetic traits can be lost when bulking is too broad. If fingerprinting could accurately distinguish the 129 Bobwhite sister lines and their genetic relationships, it would help determine when and how to bulk selections.

Bobwhite’s identity problem also surfaced among researchers using the lines for transformation (genetic modification) experiments. “We started receiving reports from some labs about low rates of transformation with Bobwhite and encouraging reports from other labs also using a Bobwhite,” observes Pellegrineschi. “Did these different results come from the genetic diversity of the lines, or from the techniques and protocols used by the labs?”

Skovmand, Warburton, and Pellegrineschi went to work. Skovmand provided a full range of Bobwhite lines to the two scientists. Pellegrineschi set about transforming the lines with a simple selectable marker (which signals whether a transformation has been successful), while Warburton assessed the potential for a large-scale fingerprinting service at the ABC.

Research Collaboration to Benefit
Wheat Farmers Worldwide

CIMMYT is a core member of the Australian Cooperative Research Centre for Molecular Plant Breeding (CRC-MPB), launched in 1997. Other members include the University of Adelaide, Southern Cross University, the South Australian Research and Development Institute, and the Victorian Department of Natural Resources and the Environment.

This partnering, says David Hoisington, director of CIMMYT’s Applied Biotechnology Center (ABC), “brings the knowledge of leading experts in molecular genetic analysis for wheat right to CIMMYT’s door. Clearly this knowledge and the resources they provide will help us develop products that directly benefit our clients in the developing world.” At the same time, CIMMYT provides the CRC-MPB with access to its extensive germplasm collection as well as its own widely respected expertise in wheat research, while serving as a conduit to a range of international organizations and resources.

Alessandro Pellegrineschi, ABC cell biologist, appreciates the relationship. “The genes they’ve provided for experiments on enhanced quality traits, male sterility in wheat (which would promote the development of highly productive hybrid wheats), and disease resistance may prove extremely valuable as research progresses,” he declares. “And the CRC-MPB support for our Bobwhite work was critical to identifying the supertransformable lines.”

The CRC-MPB also supports research by CIMMYT molecular geneticist Manilal William, who works on identifying molecular markers linked to durable leaf rust resistance genes, and that of Juan José Olivares and Magdalena Salgado, doctoral candidates at the University of Adelaide who are undertaking their dissertation research at CIMMYT. Olivares focuses on the molecular genetics of drought in wheat, and Salgado investigates the engineering of thaumatin-like protein genes into wheat to provide disease resistance.

Equally valuable, Pellegrineschi and William concur, has been their interaction with Australian scientists. “It’s been a fantastic experience,” says Pellegrineschi, “as they come out with stimulating ideas and provide a great deal of valuable information. I believe synergies have been developed that help us all in our research, and I hope we can continue building on this base.”

For more information on CRC-MPB, see
www.molecularplantbreeding.com

Pellegrineschi’s team inserted a marker gene into 200 embryos from each of the 129 sister lines. They screened all embryos to determine the rate of successful transformation. This process was repeated three times for each variety, and an average rate was derived.

“A lot of the lines had a transformation rate of zero,” reports Pellegrineschi, “but we did come up with five lines with transformation rates around 60%, and one line—possibly our premier line for transformation—with a rate of about 70%. ”He confirmed the efficacy of those lines by transforming and screening 2,000 embryos from each line and growing them into plants.

This investigative effort was funded and supported by Australia’s Cooperative Research Centre for Molecular Plant Breeding (see: "Research Collaboration to Benefit Wheat Farmers Worldwide"). Prior to this research, Pellegrineschi relates, in its transformation work the ABC had used a Bobwhite line that could now be categorized as “quite mediocre.” The newly identified line increased the transformation rate about sevenfold. Higher transformation rates translate into more efficient transfer of genes and traits and lower costs. Given that just a year or two ago wheat transformation rates of 5% were considered good and 10% exceptional, these “supertransformable” lines, called MPB-Bobwhite26 and MPB-Bobwhite29, have elicited great interest in CIMMYT and major laboratories worldwide.

Warburton, meanwhile, fingerprinted 101 Bobwhite sister lines using amplified fragment length polymorphism (AFLP) technology. “It was the first opportunity to maximize the efficiency of the procedures for large-scale fingerprinting,” she says, “and the experience gained through the work made it worthwhile.”

The first thing Warburton’s team determined was that although the sister lines came from the same cross and even fourth-and fifth-generation populations, there was enough diversity among them to warrant maintaining them separately in the genebank.

“This result shows how unwise it would have been to bulk these lines in the bank,” Skovmand observes. “If we had done that, we never would have found the highly transformable wheat lines. Obviously the lessons learned here go beyond Bobwhite."

An important serendipitous finding, according to Warburton, was that translocations can greatly skew an analysis of diversity. Translocations are fragments of a chromosome that replace a similar fragment in another species, which is often the result or goal of a wide crossing experiment. “Our AFLP fingerprinting and analysis showed two distinct clusters among the Bobwhite lines, a totally unexpected outcome,” she says. “We discovered that the clustering stemmed from a translocation from rye. Using AFLPs that did not fall in the translocated region, we found that the sister lines did not divide into those clusters and were actually a lot closer to one another.”

By identifying diagnostic markers for these and other translocations, Warburton can look for factors that might distort analyses of genetic relationships between lines, as well as tell breeders whether useful genes from the translocation are present in their latest selections, thereby accelerating breeding. Cytology already allows such diagnostics, but at a higher cost.

Cost, Warburton has confirmed, is a critical factor in DNA fingerprinting. To reduce costs, Warburton is exploring the use of diversity arrays. “A single diversity array reaction provides the same amount of information we’d get from running 38 gels,” she explains. “We’ll be running experiments and cost analyses on this technology to see if diversity arrays are an economical alternative.”

“The Bobwhite research has shown how useful fingerprinting at CIMMYT can be,” concludes Warburton. “If we continue bringing down costs, we hope to help the genebank with its work and extend the benefits of this technology to many more clients, both within and outside of CIMMYT.”

With Bobwhite’s identity crisis resolved and many lessons learned along the way, the three scientists conclude that a hidden benefit of the work was the communication it promoted among them. “Before this study, I don’t think that Marilyn or Alessandro were well versed in selection histories,” says Skovmand, “and I certainly learned a lot about fingerprinting and genetic transformation from them. These interactions can help us recognize other opportunities for collaboration.”