High throughput analysis for TILLING adapted starch potato breeding - HATZ

For the expression of recessive traits, such as the formation of amylopectin starch (HAP), all 4 copies of the gbssI gene must be inactive in the tetraploid potato. The recessive traits will be lost by crossing in other valuable traits and must be restored in a further crossing step. The combination with additional recessive inactive starch biosynthesis alleles to improve amylopectin quality multiplies the crossing and selection effort. The selection of recessive alleles has so far been done by sequencing. However, with increasing numbers of plants, this is hardly possible and very expensive. For example, if tetraploid clones (4x) that are duplex for one allele AND also duplex for another inactive allele are crossed to combine the two traits, only one in 1,296 plants will be homozygous for the two inactive alleles and express both traits: However, many of these genotypes need to be present because there are approximately about 40 other valuable traits that need to be evaluated and selected.

The goal of this project was to combine the development of shorter breeding cycles with high-throughput analysis for efficient selection of desired genotypes. In addition, the evaluation of the newly developed analytics, i.e. genotyping, was to be carried out using automated biostatistical analysis.

Partners

• Böhm-Nordkartoffel Agrarproduktion GmbH & Co. OHG
• Emslandstärke

The project resulted in significantly shortened breeding cycles. The analysis of the sequence environment of inactive alleles of amylopectin synthesis allowed the development of sensitive SNP detection methods for high-throughput analysis. Single nucleotide polymorphisms (SNPs) are genomic variations at a single nucleotide position in DNA. SNPs are used as molecular markers to associate genomic regions with phenotypic traits. In the case of SNPs induced by TILLING, the SNPs themselves are the cause of the gene KO.

With such molecular markers, genotypes can be determined at the seedling stage in in vitro culture and selected for further crosses or for the production of consumer goods for starch analysis.
For the automated evaluation of the SNP analyses, we used the statistical software fitTetra (Voorrips & Gort link) for automated genotyping of tetraploid organisms with adaptation to different genotyping methods. In addition, we simplified the operation of the software by developing a user-friendly graphical interface, the HATZApp.