5. Considerations when selecting a mutant line

When selecting a mutant line, several criteria should be taken into consideration. There will be many lines with point mutations in the gene of interest; therefore, it may not be immediately obvious which line is the best to take forward. Every case will be slightly different and depend on the aim of the study but here we will describe some common usages. We recently published a review which might serve as a useful reference for general info on wheat, available wheat genomic resources available and issues surrounding polyploidy in wheat [Borrill et al 2015] (http://onlinelibrary.wiley.com/doi/10.1111/nph.13533/full).

###Type of mutation The "Consequence" column in the results table will describe the type of mutation predicted based on gene model. As these consequence are predicted based on the IWGSC gene model it is important to assess the annotation of the gene to ensure that this is consistent with the user’s gene model. If the IWGSC gene model is consistent with the user’s gene model, then the predictions will be fine. If the gene model is incomplete or is missing (for example in the case of UCW_Kronos_U and TGAC_Cadenza_U scaffolds, then a manual annotation will be required (see below).

In many cases, the most desirable consequence will be “stop_gained” (premature termination codon), which will most likely result in a loss of function of the gene of interest. It is important to determine where in the protein the stop codon occurs as this might have implications in the phenotype.

If there are no “stop_gained” mutations available in the database, then a “splice_donor_variant” or “splice_acceptor_variant” would be of interest. These are mutations in the GT or AG splice sites that are at the start and end of introns, respectively. A mutation that results in loss of one of these sites often leads to incorrect splicing resulting in a non-functional protein. As before, it is important to understand the context of the mutation and identify downstream GT or AG sequences that could potentially be used as alternative splice sites.

It is normally advisable to select truncation (“stop_gained” or “splice_acceptor/donor_variants”) mutants where possible. If there are none available then missense mutations can be pursued. These are coding sequence mutations that result in a change of amino acid. The effect of these mutations on the protein was predicted using the Sorting Intolerant From Tolerant (SIFT) algorithm which scores the probability that a particular amino acid substitution will be tolerated in the protein. It can be interpreted in the same way as a p-value where a lower SIFT score implies a low probability that the substitution will be tolerated and hence it is classifies as deleterious (e.g. a SIFT score <0.05 implies that the mutation is very likely to affect protein function). More information on how the SIFT algorithm works can be found at http://sift.jcvi.org. Alternatively, highly conserved residues within specific protein domains could be assessed for missense mutations.

It is important to invest time in defining the best mutations to be used in the crossing scheme. As described in Borrill et al 2015:

“This is especially relevant in situations of functional redundancy between homoeologues, where it is necessary to cross individual mutants to generate double or triple knockouts to observe a phenotype. The use of truncations in all homoeologues will generate a complete null across genomes, thus allowing for correct interpretation of the resulting phenotype. However, the use of missense mutations is risky if one of the mutations does not effectively abolish gene function, thereby limiting the phenotypic effect in the double/triple mutant.”

###Zygosity Mutations may be in either a homozygous or heterozygous state. Homozygous mutations have the advantage that they will be present in all seeds and so fewer seeds will need to be screened to confirm mutations. In addition phenotypes can be evaluated immediately in the plants since they should all be homozygous for the desired mutation (if the phenotype is expressed in a single mutant).

Heterozygous mutations require some additional work but have distinct advantages. The sequence information for a particular mutant comes from a single M2 plant and the seeds which are shipped to users are either M4 or M5 seed (two or three additional self-pollinated generations). Heterozygous mutations will most likely still be segregating in the M4 or M5 seed so a larger number of seeds will need to be screened to identify homozygous mutations. Despite this apparent drawback, the opportunity to identify both homozygous mutant and wild type plants provides a proper experimental control in case the user wants to assess phenotypes in this first generation. It is important to consider that some of the mutations that were heterozygous in the M2 may have been fixed in the M4 or M5 seed, whereas other mutations may have been lost through genetic drift.

In all cases it is advisable that users confirm the mutations with the designed KASP assays or alternative methods (for example Sanger sequencing) to ensure the presence of the mutation in the plants that will be phenotyped or used for crossing.

###Population We have developed two populations which we use for complementary purposes. As outlined in Uauy et al 2009:

“We use the tetraploid TILLING population to generate mutants for basic research projects because it is easier and faster to generate complete null mutants. A single generation of crosses between A and B genome mutations, followed by selection of homozygous double mutants in the F2 populations is sufficient to generate null mutants. However, when a targeted mutant has important breeding applications we screen the hexaploid TILLING population for mutations, because hexaploid wheat represents most of the wheat grown around the world (~95%).”

###Category As stated above, the www.wheat-tilling.com output displays by default HetMC5/HomMC3 mutations. If users cannot find an adequate mutation within this category they can click the “Show lower quality mutations” to display mutations classified as HetMC4/HomMC3, HetMC3/HomMC2, MM and RH. As in any category, users are advised to order primers and confirm the mutation in the seeds received.

###Primer availability If several mutant lines have a suitable mutation, one factor to consider is the availability of chromosome_specific KASP genotyping primers (column 20). However, the absence of genotyping primers should not stop you from selecting a mutation if it is otherwise fit for purpose as primers for KASP and alternative genotyping methods can be designed manually.