Next-generation sequencing-based amplicon sequencing is a novel method for the detection of breeding relevant traits in barley (Hordeum vulgare)

This work presents a novel technique to speed up plant breeding concerning traits of agronomic importance with warranted success and suitable for a large number of samples. An assay, suitable for the high-throughput characterization of nine breeding relevant QTLs (quantitative trait locus – a section in the genome that correlates with variations in a phenotype), could be successfully established and was demonstrated on 16 homozygous barley lines and four heterozygous hybrids. Moreover, the assay was successfully employed for identifying one QTL in maize in two homozygous lines and their corresponding hybrid. The verification of this method provides an initial starting point for the extension of the assay to additional QTLs and other plant species. It could be shown that it is possible to confidently detect relevant polymorphisms and their zygosity in QTLs of interest, which have an impact on the phenotypic characteristic of a plant. This is done by the amplification of PCR products of up to 400 bp, which constitute the relevant QTL sequences, and subsequent next-generation sequencing (NGS). The established assay generates robust and unambiguous data through the alignment to specific reference sequences and an adequate sequencing coverage of each PCR product. An adequate coverage enables the statistically sound analysis of the polymorphisms, especially in heterozygous genotypes. Beginning with the database search, it was confirmed that a sufficient amount of relevant sequence data with annotated polymorphisms and their phenotypic impact are published and could be used for the assay. The effectiveness of the assay was confirmed by a combined genotyping/phenotyping approach. This could successfully connect genotypic data, concerning a pathogen-resistance gene, and a phenotypic resistance in an infection assay. The complete NGS-based assay, starting with the primer design, followed by the DNA extraction from leaf material, a PCR, the preparation of the amplicons for NGS, the sequencing and finally the data evaluation was optimized and thereby simplified to fit the needs for a high-throughput analysis of a large number of plants with minimal effort and cost. The cost for an analysis of a QTL in 96 plants can be kept at 1740 Euro and can be carried out within two days. Within this period of two days, the complete analysis round, starting from the raw plant material and concluding to the selection of plants based on the NGS sequencing data can be performed. Plants can be selected as soon as they have grown enough to extract DNA from leaf tissure, usually 1–2 weeks after planting. Thereby plants can be selected or sorted out quickly with respect to the desired phenotype, which saves expenditure in plant cultivation. The procedure of the analysis has tremendous scale up potential and the respective cost proportionally decreases with increasing sample number. Also a flexible application of the assay with a parallel analysis of different plant species and/or the parallel detection of transgenic events is possible. The scalability as well as the flexible application make the assay interesting for large plant breeding companies that seek large-scale genotyping data for rapid plant selection, as well as medium-size companies and research facilities that are in need for variable types of genotyping data