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At PAG, a Stampede of New SNP Chips Highlighted for Studying Buffalo, Wheat, Ornamentals

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The deluge continues. Year after year, new SNP genotyping arrays have been introduced at the Plant and Animal Genome conference, held annually in San Diego, and this year's meeting was no exception, as new tools were introduced for studying buffalo, wheat, ornamental plants, and many other species of interest to ag researchers.

While the use of SNP genotyping chips was highlighted in numerous scientific sessions and posters, they were also at the center of a number of company workshops, where vendors Illumina and Affymetrix pledged to make new catalog SNP chips available in the coming year.

Both Affy and Illumina already have sizable menus of genotyping chips available. Illumina currently offers arrays to study 25 different species, including bovine, goat, ovine, porcine, canine, chicken, apple, maize, tomato, grapevine, potato, and wheat. Affy has also expanded its number of ag-focused arrays in recent years, as researchers can now use its catalog products to study bovine, chicken, rice, lettuce, pepper, and buffalo.

Affy's Axiom Buffalo Genotyping Array is among its latest agrigenomics offerings. Launched a few weeks ago, the product contains 90,000 common and rare markers found in multiple water buffalo species and breeds, including Mediterranean, Murrah, Jaffarabadi, and Nili-Ravi.

According to the company, it developed the array in collaboration with the International Buffalo Genome Consortium, which includes Parco Tecnologico Padano in Italy, Iowa State University, and the United States Department of Agriculture's Agricultural Research Service. Affy claimed in a recent statement to be the only firm offering a chip for biomarker discovery and marker-assisted selection in buffalo, "one of the most important beef and dairy animals in Italy, Brazil, and South Asia."

John Williams, PTP's science director, described the need for such an array during an Affy workshop at PAG. According to Williams, there are about 158 million buffalo worldwide, in comparison to 1.3 billion cattle, a billion sheep, and about 600 million goats. Buffalo provide 5 percent of the world's milk supply, and are a source of meat and transport, especially in developing countries. The content for the Axiom Buffalo Genotyping Array evolved out of the Buffalo Genome Project, which included the de novo sequencing and assembly of a Mediterranean buffalo genome, and then the SNP discovery and selection for a genotyping panel.

Williams said during his talk that the 90,000 SNPs on the chip were selected from a total of 16 million SNPs, and that the markers were weighted to reflect each of the four featured breeds. He also said that the group intends to publish the animal's genome when it is finished annotating it.

Williams told BioArray News following the workshop that Affy launched the chip over the holidays after a "quick turnaround" – once the consortium selected the content for the array, it took about a month and a half for the chip to become commercially available. In terms of demand, Williams said that the consortium "has had interest from countries that rely on buffalo, such as India and Brazil" and that demand in these countries could be similar to bovine, as the array covers breeds specific to each country where buffalo are raised.

Still, the array is not yet applicable to all countries that rely on buffalo, as the content on the chip covers only river buffalo breeds, not swamp buffalo breeds, which are more commonly used in Southeast Asian countries like the Philippines, Indonesia, and Malaysia. According to Williams, river buffalo have 50 chromosomes while swamp buffalo have 46. He said he does not know how the Axiom Buffalo Genotyping Array would work with swamp buffalo samples, but said it should be possible to adapt the array in the future to contain content that would be "useful for third-world economies."

Now that the array is available, Williams said that PTP and the Italian Buffalo Breeders Association plan to use the chip in an association study for lactation traits. Buffalo is an important animal to Italian breeders because its milk is used in the production of mozzarella cheese.

BreedWheat and GenomeStudio

As most researchers tend to stress the importance of their organism of interest, it's hard to know which staple is most important to breeders, but with catalog SNP chips available for maize and rice, it seems logical that chips for wheat should become available too. For wheat researchers, though, there will be several arrays available, as wheat studies were featured in both Affy and Illumina workshops.

Etienne Paux of France's National Institute for Agricultural Research, or INRA, described the development of an Axiom genotyping chip for marker-assisted selection in wheat. According to Paux, the new chip was designed in line with the objectives of BreedWheat, a project that brings together 13 public research units from various INRA sites and universities and institutes across France as well as 13 partners from private companies and cooperatives, all to strengthen the country's wheat breeding sector by identifying various traits including yield, quality, and resistance to biotic and abiotic stresses.

Wheat faces some specific challenges, said Paux. In addition to being the "most widely consumed crop in the world," with increasing demand requiring a projected 2 percent increase in production annually by 2050, and yield stagnation due to climate change, the cereal grain is also more complex than maize and rice, being 17 gigabases in size, 40 times the size of the rice genome, and six times the maize genome's size. Because of this, Paux said that wheat researchers will require more SNPs than researchers of other crops to saturate the wheat genome and develop efficient genomics-assisted breeding programs.

While that high number of variants is not compatible with most high-throughput genotyping platforms, Paux and fellow researchers have developed a method based on genotyping of insertion site-based polymorphism markers to identify diagnostic markers for most target loci in the wheat genome. Ultimately, the project was able to design a 420,000-marker array – referred to in his talk as the BreedWheat 420K SNP Chip – on the Axiom platform.

Paux detailed BreedWheat's genotyping options, noting that the project selected Axiom from a candidate group that also included Illumina's Infinium and GoldenGate assays, Sequenom's iPlex platform, Life Technologies' OpenArray, and LGC Genomics/Kbioscience's KASP assay. He said that BreedWheat settled on Affy because of the firm's "flexibility," "efficiency" in scoring SNPs in polyploid species, and the ability to run 96 custom arrays at the same time using its ArrayPlate format. Affy recently announced that it will soon launch a 384-array ArrayPlate option for customers later this quarter, though Paux did not comment on that new product (BAN 1/15/2013).

During an Illumina workshop, Eduard Akhunov, an associate professor at Kansas State University, informed fellow wheat researchers of a different kind of tool that could aid in their research. Akhunov's talk focused on genomic patterns of SNP variation in worldwide wheat populations and provided information on two Illumina-manufactured arrays for wheat studies — an iSelect 9,000-marker array that has been used to genotype 12,000 accessions and an iSelect 90,000-marker chip that has been used to screen 48,000 accessions worldwide.

Akhunov also highlighted new automated polyploidy-calling software in Illumina's GenomeStudio data-analysis tool. According to Akhunov, previous versions of GenomeStudio were designed to analyze diploid genomes, complicating efforts to analyze variation in bread wheat, which has a hexaploid genome. The new software has no inherent assumptions about the genome being analyzed, and relies on researchers to determine the genotype represented by the clusters based on their own knowledge, Akhunov said.

Four on One

Not all agricultural SNP chips in development or use today are focused on increasing global food security. Annemieke Jungerius, head of applied research at Genetwister Technologies, a Dutch biotechnology company, discussed during an Affy workshop the development of an Axiom genotyping array for studying four different ornamental plant species — chrysanthemum, carnation, kalanchoe, and pelargonium.

According to Jungerius, the chip was designed in partnership with the Agribio Group, a multinational plant breeding firm, specifically for plants sold by Fides and Barberet & Blanc, two Agribio subsidiaries. Agribio also acquired a stake in Genetwister last year.

"We wanted to fill the toolbox of Agribio with the best markers and tools for ornamental breeding," said Jungerius of the project. She added that Genetwister selected Affy based on its ability to manufacture arrays containing four different species. The firm selected 200,000 SNPs — 50,000 per species — from an initial dataset of 2 million SNPs. Jungerius noted that the SNPs selected are informative for identifying crosses, linkage maps, and qualitative trait loci.

The company has not yet used them to identify markers useful for future selection, though. Jungerius said that the chip is currently being manufactured and that the company is "looking forward to seeing the genotyping results."

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