Farhad Ghavami Chief Science Officer, Eurofins BioDiagnostics

The development of next-generation sequencing technologies and the ongoing reduction of sequencing costs have made genotyping by sequencing (GBS) a valuable approach to find the variants in DNA to identify genetic improvements within plant breeding programs.

Variations in DNA molecules have been used as molecular markers for years. Today, the use of molecular markers is not limited to plant breeding projects and is a key component of an effective quality-control process. Having an accurate and cost-effective way of genotyping seed and plant materials is of great value to the seed industry.

Most polymerase chain reaction, commonly known as PCR, based platforms are good for genotyping a few markers (1-100). Array-based platforms are suitable when thousands of markers are required for high-density genotyping, but when hundreds of markers are needed for genotyping, there was no affordable, high-throughput method available. This was one of the main reasons that genomic selection has not been practiced extensively in the seed industry.

Targeted GBS technology fills this void by providing high quality genotyping capabilities at an economical cost per sample to unlock the potential of yield and quality traits hidden in breeding populations.

Three GBS Methods

* GBS via restriction enzyme. This is the first GBS method developed. Using different restriction enzymes, the genome is digested, and only the fragments with certain sizes will be amplified and sequenced. Application: For research and development projects and when there is no information available on the genome. Thousands to millions of markers can be genotyped and discovered. It is slower and more costly than other methods of GBS.

* GBS via sequence capture. The genome is either physically or enzymatically fragmented into small fragments. The targets are then captured by probes attached to beads or arrays and sequenced. Application: It can be used for hundreds to thousands of markers but is not as cost-effective as amplicon-based GBS.

* GBS via targeted amplification, or amplicon-based sequencing. Hundreds of regions in the genome are amplified using multiplex PCR approach. The PCR fragments are then sequenced. Application: A very cost-effective method for genotyping using 100 to 3,000 markers. This would cover most of the marker-assisted breeding applications including genomic selection, marker-assisted backcrossing and marker-assisted selection for qualitative and quantitative traits all together.

The objective of all three methods is to reduce the complexity of the genome to a few hundred or thousand targets. Only those targets are sequenced. Amplicon-based sequencing is the most cost-effective method of all. By using different sequence barcodes, the amplicons from many samples can be barcoded, pooled and sequenced together to reduce the per-sample cost of sequencing. Eurofins has the capability and capacity to perform all three GBS methods.

Amplicon-based sequencing is the best method to address most of the breeding program needs in crops. Testing by researchers at Cornell University and elsewhere has demonstrated that genomic selection (prediction) in corn, wheat, soybean and many other crops only need genotyping by 500-3,000 markers. Therefore, Amplicon-based GBS is considered the best genotyping method to address this need. This technology can change the dynamic of plant breeding, quickly advancing a company’s breeding objectives. In the future, this technology is likely to replace most of the current genetic quality testing.