Verification of Parental Lines

A widely used example of Quality Control is to verify the identity of parental lines before making crosses.

• Compare marker results from samples taken from crossing nursery to standard
• Use 20 to 30 SNP markers (depends on level of genetic diversity among the parents)
• Recommended criterion for acceptance – not more than 2 markers different
• Requires having standards in place, e.g. having genotyped the parental lines enough to feel confidence in the genotype
• Works best with inbred lines

To help you better understand how this works, let's follow an example of verifying parental lines in a bean breeding program.  In this case two samples of each bean line were tested with 20 SNP markers.

Table 2 shows the results of using the 20 SNP markers to check the identity of two bean lines, AFR298 and AFR731.. From the results we can see that both samples of AFR298 give the exact same marker pattern results, but for the two AFR731 samples four SNPs give different results between the two samples (highlighted in red). This makes us question the identity of line AFR731 as all samples from that line should be the same genotype. The coefficient of similarity is a calculation of the similarities and differences between two sets of data and normally calculated using different software programs.

There are several possible explanations for what would cause a mismatch in marker pattern seen in samples of the same germplasm being tested, such as seen in this bean breeding example for line AFR731 and sometimes seen in real life cases. The possibilities would include:

• Packaging error
• Planting error in previous generation
• Labeling error
• Harvesting error
• SNP calling error (very small, and can be confirmed or ruled out by running duplicate samples)

The important thing then is knowing when to be concerned with this type of unexpected marker data.  Here are some general guidelines you can follow to help you in making the decision of whether or not to question the identity of a parent line being tested:

• Theoretically inbred lines should be 100% identical at every locus for every SNP
• But what if 2 lines are 99% identical? 98%
• You must know your material and decide what level of mismatch (if any) you think is acceptable. For example, if you are simply looking to create a new variety for marketing, it may not be that important that the parents are exactly what you think they are, but if you are creating a new variety specifically from a well-known parent, and may need to prove the new line is a derivation of that parent, then you may want a very low, or non-existent, mismatch rate.

In addition to improving QA/QC in parental lines, markers can be used in other plant generations throughout a breeding program. The use of markers and morphological traits allows breeder to discard selfs by clearly discriminating homozygotes (accidental selfs) from plants that we know should be heterozygous. Two key advantages of utilizing markers in this way include:

• More efficient use of resources in succeeding generations by not crossing the wrong plants
• Important in BC projects, which are dependent upon plants being a backcross to the recurrent parent

A couple of general methodology guidelines for using markers to verify F1 and BCF1 lines are:

• Apply 20 to 30 SNP markers to all F1s across populations as in parental screening
• Select 2 to 4 SNP markers for each cross based on polymorphism observed in parents

To better understand these ideas we will now use several case study examples where SNP markers are used to verify various plant materials resulting from controlled crosses.

In this first example, we have two bean parents which are crossed and we want to verify that the F1 offspring truly did result from this cross. Table 3 shows the genotyping results of testing 20 SNP markers on five F1 lines derived from a cross between the two parents BAT93 and SER16. F1 lines should all be heterozygotes containing one allele from each parent. Here we can see that line F1-5 has unexpected genotypes at four out of the 20 SNP markers and therefore is most likely an error. The errant results are highlighted in red [data provided by Hamer Paschal].