Cost Effective Resequencing for Association Genetics in Ryegrass Utilising a Modified Illumina NGS Work-Flow

Advances in DNA sequencing have fundamentally changed the life sciences and dramatic reductions in costs have enabled aspirational goals to be routinely achieved. For applied approaches to crop and livestock improvement maximal benefit of this technology can be achieved by minimising costs. For many situations the implementation of modest numbers of molecular markers or gene sequences will give significant benefits to the improvement program and whole genome profiling is not required. Through the development of a simple cost-effective Illumina library preparation technique for PCR-based amplicons significant cost savings can be achieved for modern sequenced based diagnostics. An experimental candidate gene resequencing approach was undertaken involving 24 known genes in the signalling pathway for flowering of the forage crop perennial ryegrass across 576 plant genotypes to identify associations with using a target enrichment approach. The MyFi^® DNA polymerase was used to generate the amplicons that would become the sequencing library. The MyFi^® DNA polymerase offers high fidelity along with an ability to work on a wide range of templates, reducing the need for repeat work whilst increasing the samples under investigation, whilst still being half the cost of the majority of the current extreme fidelity DNA polymerases. SNPs were identified through BWA reference alignment and called through mpileup and Samtools, after filtering a total of 350 SNPs were identified that gave a range of prediction accuracies, maximal at 0.56.