High throughput screening of mutagenized populations

Abstract

Efficient methods are disclosed for the high throughput identification of mutations in genes in members of mutagenized populations. The methods comprise DNA isolation, pooling, amplification, creation of libraries, high throughput sequencing of libraries, preferably by sequencing-by-synthesis technologies, identification of mutations and identification of the member of the population carrying the mutation and identification of the mutation.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention, in the fields of molecular biology and genetics relates to improved strategies for identifying mutations in populations, based on the use of high throughput sequencing technologies. The invention further provides for kits that can be applied in the methods.[0003]2. Description of the Background Art[0004]Populations carrying mutations, either induced or naturally occurring are used in modern genomics research to identify genes affecting traits of importance by reverse genetics approaches. This is in particular applicable for plants and crops of agronomic importance, but such populations are also useful, for other organisms such as yeast, bacteria etc. Other organisms, such as animals, birds, mammals etc can also be used, but these populations are typically more cumbersome to obtain or to control. Nevertheless, it is observed that the invention described herein is of a very general nature, and can b...

AI Technical Summary

Benefits of technology

[0024]The present inventors found that using high throughput sequencing strategies, the above-mentioned goals were achieved and mutagenized populations, such as TILLING populations, populations wherein mutations have been introduced using (synthetic) mutagenic or DNA damaging oligonucleotides or, i.e. by Targeted Nu...

Problems solved by technology

Other organisms, such as animals, birds, mammals etc can also be used, but these populations are typically more cumbersome to obtain or to control.

The rate-limiting step is the screening work associated with identification of, respectively, organisms carrying a mutation in the gene of interest.

The challenge therefore is to identify one (or several) plants with loss-of-function mutations in this gene.

However, a limitation of CEL I screening is that not all identified mutations affect gene function (e.g., silent substitutions) and this is not known until the PCR products of individual plants in a positive pool are sequenced.

Nevertheless, the CEL I mediated screening method is cost-saving compared to sequencing PCR products of all plants separately.

Another limitation is that CEL I screening involves running gels and scoring, a relatively cumbersome process that requires confirmation of mutations from the second strand as gel-patte...

Images