Analysis of relationships by gRandma
By utilizing kinship estimates from COLONY 2.0 or Sequoia, including GG, UANN, and FS relationships, we were able to identify families for 36 out of 47 F2 individuals. However, the families for the remaining 11 F2 individuals were still unknown. In contrast, gRandma, a specialized software for estimating GG trio relationships, was able to estimate grandparents for almost all F2 individuals. gRandma failed to infer any GG relationship for only two of the F2 individuals, but Sequoia estimated that they had a UANN relationship with an F1 individual (mA1-4) of the A1 family, which supports the hypothesis that these F2 individuals were produced by breeding between F1 individuals of the A1 family.
The algorithm used by gRandma is unique because it considers GG trios instead of just GG pairs. Originally developed to estimate GG relationships between wild-caught grandchildren and hatchery grandparents, gRandma was designed to detect crossbreeding when adult sampling in the wild is limited (Delomas and Campbell, 2021). As a result, it was initially intended for cases where only one set of grandparents, paternal or maternal, was sampled. However, in this study, where both paternal and maternal grandparents were sampled, gRandma was able to estimate both sets of grandparents (T. A. Delomas, pers. comm.). However, because Delomas and Campbell (2021) initially assumed a low probability of false positives from trios other than GG, it is possible for a false-positive trio to yield a high LLR when, for example, the potential grandparents include siblings (Delomas, pers. comm.). As the COLONY 2.0 analysis revealed that the target population in this study contained many siblings in the grandparents’ generation, we incorporated a Mendelian incompatibilities (MI) threshold in addition to the LLR value. This is because a trio with a high LLR and low MI is more likely to be reliable, as a true GG trio would have both a high LLR and low MI, whereas a false-positive trio would have a high LLR and high MI. Moreover, we have information on the relationships between grandparents, such as if they are FS and HS, and which individuals were possibly interbred, so we were able to identify whether the inferred trios were false positives.