How does parent verification work?

The November issue of the Register contained a Down to the Genes article focused on parentage testing.  Here we republished a blog post with more in depth information on how the parentage analysis works and a hands-on example where readers can work through who is the correct sire.

By Jackie Atkins, Ph.D., Director of Science and Education

Originally published on the ASA Science blog, Mar 7, 2016.

Whether you use parent verification to confirm your breeding records, distinguish between AI and pasture bred calves, or to sort through progeny from a multi-sire pasture, parental validation is an important test for breeders.  Roughly 10% of pedigrees reported across all breed associations are inaccurate.  These are frequently honest mistakes but never-the-less, vital to fix and make informed breeding decisions.  The theory behind parental validation is to determine if an animal could have inherited specific DNA markers from their reported parents.  If the animal could not inherit a specific DNA Marker from the sire or dam, then the parent is excluded.  With enough qualified markers, an animal is confirmed as the sire or dam of the offspring.

In the past, 15 microsatellite markers (also called Short Tandem Repeats or STRs) were used to confirm parentage but these were costly and not always reliable tests.  Most new parental validation is completed with single nucleotide polymorphisms or SNPs (pronounced “snips”).  The SNPs are locations in the DNA that vary from one animal to another.  The SNP parentage test uses over 100 SNP markers to either qualify or exclude an individual as a parent.  Each animal has two markers at each SNP location (one on each pair of chromosomes representing one from each parent).

Let’s work through a hypothetical example using SNPs for parental validation.  We have test results for SNPs on a calf, two possible sires, and the likely dam.  We look at each SNP and see if the calf could have inherited that SNP from the possible parents and also if the mating (the parents analyzed together) could have passed along those SNPs.  The following diagram and table show four SNP markers for the individuals and the conclusions drawn.

Slide1

Example of 4 SNP markers for a calf and his potential parents.  See the table and text for explanation of the analysis.

Slide2

Hypothetical SNP markers at 4 locations for a calf and his potential parents.  See text for further explanation for the conclusions drawn.

Working through each SNP marker, we look if the calf could have inherited his DNA markers (genotype; options are A, T, C, or G) from the potential parents.  At SNP 1, the calf has a genotype of A/T.  This means the calf inherited an “A” from one parent and a “T” from the other parent.  Both the Dam and Sire 1 are homozygous T and SNP1.  This means they could only have passed along a T at this location.  The potential dam and Sire 1 could not have conceived the calf together as neither could have passed along an “A” at this location.  However, we can’t exclude either the dam or Sire 1 based on this information, we just know that they can’t be confirmed as the parents together.

Continuing through our analysis, we cannot exclude any of the parents with SNPs 2 and 3 because the pairing of both sires with the dam could pass on the calf’s SNP markers.  Looking at SNP 4 is helpful.  The calf has two “T”s at SNP 4, one from his dam and one from his sire.  Sire 1 has two “A”s at SNP 4 so he could not have passed along a T at this location.  Sire 1 is excluded based on this comparison.  If Sire 1 is excluded, then the potential Dam and Sire 2 work for the remaining SNP locations.  The parental validation at the lab uses the same logical progression as this simple example except it uses approximately 100 SNP markers for the analysis.

 

DNA Parentage Results – What if animal doesn’t qualify to the Parent(s)?

For some breeders the DNA process is similar to waiting for a present to arrive.  The DNA sample is submitted to the lab and from that moment forward, the anticipation starts to build.  Will the animal submitted qualify to the parents? Will it test homo polled? Homo black? Free of genetic defects?  The not knowing, it’s absolutely heart wrenching, stressful and can just about drive you crazy during the 3-4 week wait.  However, whether the results leave you feeling excited or let down, it’s important to understand the process so lets take a moment and walk through how DNA parentage results are reported from the lab to ASA, uploaded and then emailed/mailed to the member.

Lab Reports Results to ASA

Every business day the lab uploads results that are ready to their online information system named LIMS.  Each day ASA checks LIMS and downloads any results that are available.  Results are uploaded to Herdbook Services and the membership account is invoiced for the testing.  If the sire/dam of the animal being tested has DNA on file with ASA, the lab will run a comparison to the parent(s) and determine whether the animal verifies to sire/dam. There are three critical pieces on the parentage report: sire qualify, dam qualify and sire/dam qualify as a mated pair.

There are 4 possible outcomes if both the sire and the dam have DNA on file with ASA

  • the animal qualifies to both sire/dam
  • the animal does not qualify to sire/dam (result is an exclusion)
  • the animal qualifies to dam but does not qualify to sire – (result is an exclusion)
  • the animal qualifies to sire but does not qualify to dam – (result is an exclusion)

Once the records are uploaded/submitted, an animal that is registered/on file and marked as an exclusion, is automatically suspended pending DNA verification or a pedigree update.

If only one or the other(sire/dam) has DNA on file, the animal is only compared to that parent. If the parent animal does not qualify, the exclusion procedure is followed as described above.

If neither parent has DNA on file, the animal’s DNA parentage is simply placed on file and uploaded.

Member notification

If the member has an email address on the account, an automated email is sent out displaying the invoicing information. These automated notifications do not contain any parentage result information, they simply display the amount invoiced to the account.  If your animal is registered/on file in the database you can go in to Herdbook Services, view the animal pedigree and look for the results designation on the animal record.  For an explanation on what to look for, please view the blog post on DNA results.

DNA parentage reports are then generated and emailed to members as attachments or mailed out the following business day.

What happens if the parentage report says the animal did not qualify to the parent(s)?

  1. If it’s an ET animal, we’re going to ask for other mating possibilities.
  2. If it’s a single birth animal we’re going to ask if there are any other sire/dam possibilities.

If you’re the breeder on this animal, go back and double check your records and if your response is, “no, there are no other possibilities” then you do have the option to submit a *second sample on the animal.  If you submitted a blood sample the first time, we recommend sending in a hair collector card for the second submission or vice-versa.

If there are other sire/dam possibilities, those will be forwarded to the lab for comparison, this process is called a re-check.  Re-checks typically take 24-48 hours to complete, depending on lab workload. If animal qualifies to the sire/dam on the re-check, the pedigree is updated and unsuspended.

If the animal/ET was purchased, you will need to check with the seller/breeder/sales management team and ask them if there are other possible matings.  If they respond as mentioned above, you have the option to submit a *second sample on the animal.

*Please note – If you submit a second sample and the animal tests the same way it did the first time, i.e. does not qualify to parent(s), you will be invoiced for both DNA parentage tests.  If the second sample qualifies to parent(s) as originally stated we will refund the amount invoiced for the second set of results.

Keep in mind, each exclusion case is unique and in certain cases, forces us to leave our comfort zones and consider even the most remote of possibilities.  For some people it’s helpful to think of DNA as a giant puzzle and with that in mind, it often takes more than one person to sort out how all the pieces fit together. Take a moment and consider all the people involved in the process of flushing a donor dam or AI breeding a dam.  The wrong semen straw gets pulled for the flush, the petri dishes on the flushed donors get switched, the ET straws get mis-labeled, the dam gets bred with two straws and it doesn’t get written down, the wrong dam loads in the chute first and it doesn’t get written down or caught.  The list goes on and on. None of us ever like to admit that we are all human and mistakes do happen(me least of all) but I can make you a promise that as issues arise,  ASA will work diligently with you each step of the way  to resolve and try to make real world sense of the situation.

If you have any questions/concerns on a particular DNA case please do not hesitate to email dna@simmgene.com or call 406.587.4531.