Breeding crops has always been an art and a science. With genetic markers now playing a larger role in breeding, the science is beginning to outweigh the art. According to the US Department of Agriculture, in the US alone, public spending for agricultural research in 2008 reached $4.0 billion. Genomic analysis tools have changed crop breeding, making it faster, more accurate and more cost effective. Crop breeding has also become a market opportunity for providers of microarray and sequencing instruments.

Marker-assisted selection (MAS) is used to overcome problems of traditional crop breeding. In traditional breeding, an individual plant of the chosen crop that expresses a desired trait, such as better taste or larger size, is selected. Simple traits such as taste or size are easily identified, but more complex traits, known as quantitative traits, such as disease or drought resistance, are more difficult to determine in an individual plant.

Quantitative traits are controlled by multiple genes and environmental factors. The challenge of traditional breeding is singling out the best parental lines and identifying superior offspring for quantitative traits. MAS addresses this problem by using genetic markers that are closely linked to the desired quantitative trait. For example, once a genetic sequence linked to disease resistance is identified, researchers can avoid testing every single offspring plant for disease resistance.

DNA markers have been commonly used since the 1980s as genetic markers for the identification of quantitative traits in plants. DNA markers for quantitative traits offer numerous advantages over conventional phenotype-based alternatives and traditional crop-breeding methods. They are stable and can be detected in all tissues, regardless of age, differentiation and environmental conditions. DNA can be extracted from very young plants and analyzed before the plant expresses the actual trait. Determining a plant’s trait early in its life reduces breeding time.

Single-nucleotide polymorphisms (SNPs) constitute the most abundant molecular markers in plant genomes and are widely distributed throughout each genome, although their number and distribution varies depending on the plant. As a result, of the DNA-marker systems available, SNPs provide the most comprehensive MAS information regarding quantitative traits in crops. SNPs also optimize breeding by minimizing the introduction of deleterious genes around the target gene (known as linkage drag) by identifying undesirable genes in a plant.

SNP microarrays are the preferred technique for large-scale genotyping for MAS. They are available for several traits and crops, including disease resistance in maize and rice. Unlike other genotyping techniques for MAS, such as PCR, gel-based systems and MALDI-TOF MS, microarrays allow for highly multiplexed reactions, resulting in cost and time savings.

Crop microarrays for genotyping are provided by a number of microarray companies, including Illumina and Affymetrix. Illumina provides two configurations for its crop microarrays: the GoldenGate and the Infinium assays. The GoldenGate arrays are available with 384–1,536 SNPs. Infinium assays are higher density, with as many as 4,000 SNPs per array. According to Wilson Grabill, manager of Public Relations at Illumina, Illumina’s BeadChip customers for SNP genotyping for MAS include large and small seed companies, private research institutes and testing labs, and individual and core laboratories in academia.

Demand for crop genotyping microarrays is robust, according to Mr. Grabill. “The demand for BeadChips [for MAS] has been strong across geographies and will continue to be strong for the foreseeable future,” he said. “This is driven primarily by the ever-present, agnostic-to-geography need to improve yield and characteristics of economically important plants and animals.”

Affymetrix provides custom SNP microarrays and tiling microarrays (which cover a whole genome) for MAS applications. Affymetrix’s GeneChip tiling microarrays for crops are used for single feature polymorphism (SFP) genotyping. SFPs are a type of SNP. In addition they are markers for traits as well as SNPs, and are used to analyze loci of crops for which SNPs are less useful, such as recombinant inbred or double haploid crops. Affymetrix GeneChip tiling arrays for crops can hold nearly 23,000 probe sets each.

Affymetrix also provides custom tiling SNP microarrays through its MyGeneChip Program, which was launched early last year. Through the program, the company offers custom arrays for whole genome SNP genotyping for plants. For example, during the program’s launch, the company created a custom Arabidopsis tiling array.

Pricing and collaborations are key features of the SNP microarray market for MAS applications. Mr. Grabill pointed out that the market differs from other microarray markets due to a more price-sensitive customer base. “Some customers, namely those working with smaller numbers of SNPs, tend to be more price sensitive in agricultural markets, largely due to the volume of samples they need to run,” he said.

Partnerships with research institutes and seed companies are important for providers of SNP microarrays for the agriculture market, because sequencing information used for microarray content, such as SNPs, is still being gathered. “Our partnerships in the agricultural space differ in that development of species-specific genotyping products often requires DNA sequencing to develop genotyping content,” said Mr. Grabill. “Our consortia manager works intimately with scientists in the community to incorporate existing content and discover novel variants,” he explained. “Additionally, we bring together members of the community to help drive down price through increasing overall sample volumes.”

Mr. Grabill believes that as the SNP microarray market for MAS matures there will be an increasing need for more species-specific content, an growing number of genome-wide association studies for crops, and an increasing use of service providers, especially as microarrays become more cost effective.

DNA sequencing is also an enabling technology for MAS. Sequencers are used for the discovery of SNPs. The discovery of SNPs requires the sequencing of individual reference crops, followed by the resequencing of other varieties of the crop in order to find variable base pairs. According to Mr. Grabill, sequencers are used for a wide range of MAS applications, including gene expression measurement, methylation detection and de novo sequencing. “Customers tend to be reliant upon discovering SNPs for their species of interest. Many turn to Illumina for our next-generation sequencing instruments—either through purchasing their own instruments or sending samples to service providers,” he said.

The demand for sequencers and sequencing services for MAS applications comes largely from the private sector. “The agricultural market is probably comprised of a larger proportion of ‘for profit’ entities, such as seed companies, fee-for-service organizations and livestock companies, than human sequencing markets,” said Mr. Grabill.

Sequencing service providers are also finding MAS applications lucrative. According to Marco Van Schriek, Crop Team Leader at Keygene NV, a company providing molecular genetic services for agriculture applications, early success with MAS offerings led the company to expand its sequencing capabilities. “Keygene was founded some 20 years ago by a number of plant-breeding companies, knowing that upcoming biotechnology was of interest and something needed to be done.“ After the company found success with its PCR-based SNP genotyping technology, the Amplified Fragment Length Polymorphism platform, it decided to invest in sequencing technology for SNP identification. “When the company was founded, the plant-breeding companies did not know if either MAS or genetic modification would be the method of choice, but MAS was the preferential selection. However, over the last five years or so, we have expanded our [MAS instrument] portfolio quite heavily with high-throughput sequencing machinery.” Keygene currently provides sequencing services for MAS using Illumina and Roche 454 sequencers.

SNP discovery for MAS is also pushing public institutes to invest in sequencers. In January, Illumina sold 128 HiSeq 2000 sequencers (see IBO 4/15/10), its largest sale of sequencers to date, to the Beijing Genomics Institute. According to Mr. Grabill, many of these sequencers were purchased in order to sequence plants.

The use of sequencers and microarrays for genotyping for MAS is also affecting instrument development in other areas. Mr. Van Schriek highlighted the need for improved phenotyping technology to provide data to pair with genotyping data, which will enable more comprehensive trait identification. “As the genotyping costs over the years are coming down quite heavily, the phenotyping costs, to which you link up to your genotypes, are becoming more and more of an issue. That’s why we’ve invested in phenotyping machinery, but this still needs further work,” he told IBO.

Mr. Van Schriek also expressed the need for third-generation sequencing technology. “As whole genome sequences for various crops are becoming available, which is a huge leap forward, the question arises quickly about the variation between various individuals of that crop. Third-generation sequencing makes whole genome resequencing possible at an affordable price, so that you’re able to perform MAS by direct sequencing, which is also called breeding by sequencing.”