Rapid breeding results with TILLING

A modified TILLING-technology was developed for the potato that takes its species-specific traits (autotetraploid, heterozygous, vegetative reproduction) into account. The goal of the TILLING-technology is to further develop the potato and assess the pros and cons of different practical potato breeding methods.

Exemplarily, the enzyme genes for potato starch metabolism were selected. The products of these enzymes are involved in the biosynthesis of different starch types in the potato bulb. Clones were identified that carry new target gene alleles. These clones are currently being processed.

The genetic repertoire of sexually compatible wild types may be fallen back on if the existing varieties and breeding clones do not have the resources that new challenges in potato cultivation demand. However, aside from the desired genes/alleles they also carry genes that lessen the yield and important quality traits. These unwanted genes have to be removed in a tedious and perhaps decade long pre-breeding.

Since this is a very time consuming and expensive process alternative approaches were sought to make new alleles for practical potato breeding available. Recently, a new technology has been developed the so-called TILLING (Targeting Induced Local Lesions in Genomes) technology which makes new alleles for breeding available.

The TILLING-method has been modified for the potato. Key elements are the screening of mutant populations of diploid potatoes after mutations have been induced using Sanger-sequencing of the target gene. Different gene alleles can differ in their insertion/deletion
polymorphisms. These are especially found in the intron region.

To be able to definitely demonstrate possible mutations in the target gene of heterozygous organisms, the target gene alleles from both crossing partners have to carry the same InDel-pattern. If this is not the case, the sequences will shift against each other and can no longer be analyzed. In diploid potatoes the EMS-generated alleles can easily be detected by sequencing. This is because the new single nucleotide polymorphisms (SNPs) and the original sequence are found at a ratio of 1:1. Therefore, the seed production for EMS mutagenesis has to be done based on diploid parents. In addition, they have to be sexually compatible. This is not necessarily the case for diploid potatoes because of their system of gametophytic self-incompatibility.

Depending on the breeding goal the crossing partners are chosen from the segments fresh produce, processing, flakes, or starch of a diploid elite-breeding program. Thus, the new alleles are directly inserted into the desired genetic background. This is an important time-saving aspect of practical potato breeding.

The seeds that arise from this crossing will be treated with EMS. They are then used to establish a population of several thousand potato clones. These are obtained by vegetative reproduction. Sequencing the respective target gene of the individual EMS-clones is done by high through-put methods. The identified breeding clones with new alleles are then placed in tissue culture. Here with suitable methods the genomes are doubled. Tetraploid plants are generated and have direct application in practical potato breeding.

In practice the work with TILLING-populations from diploid potatoes has proven successful. In the meantime, various potato starch metabolism target genes have been processed. “Missense” and “nonsense” mutations have been induced and identified that have found application in practical potato breeding. In cooperation with a breeding farm, for example, a “nonsense” mutation was identified that affects a splice-site in the gene for starch granule-bound starch synthetase 1 (GBSS 1).

In the course of a relatively short breeding cycle homozygous amylase-free starch potatoes were bred. This characteristic significantly increases the value, because it enables the optimal use of the resulting starch without the removal or modification of amylase which would be necessary with regular starch for various applications (e.g. paper, textile, and glue manufacturing). Despite the positive experience with the TILLING-method for diploid potatoes, a technology was developed to produce tetraploid TILLING populations. In contrast to diploid populations, they can be stored long-term in seed form.

Thus, the continuous preparation of new TILLING populations is not necessary. Furthermore, identified mutants can be introduced directly into variety breeding without complex tissue culture steps. Screening such populations is currently still difficult, but in the near future the “next generation sequencing” will be used for routine and inexpensive screening of TILLING-populations. The technology that has been used so far will be replaced. Disadvantages are no longer expected in screening tetraploid potato TILLING-populations.

Bioplant, Biotechnological Research Laboratory GmbH

Dr. habil. Eckhard Tacke
Brüggerfeld 44
29574 Ebstorf

www.bioplant.de