A robust set of black walnut microsatellites for parentage and clonal identification

We describe the development of a robust and powerful suite of 12 microsatellite marker loci for use in genetic investigations of black walnut and related species. These 12 loci were chosen from a set of 17 candidate loci used to genotype 222 trees sampled from a 38-year-old black walnut progeny test. The 222 genotypes represent a sampling from the broad geographic distribution of the species. Analysis of the samples using the 12 loci revealed an average expected heterozygosity of 0.83, a combined probability of identity of 3×10⁻¹⁹, and a combined probability of exclusion for paternity analysis of >0.999. The 222 genotyped trees from the progeny test comprised 39 open-pollinated families, 29 of which (having at least five sampled progeny) were used to estimate the outcrossing rate for the progeny trial. The same 29 families were used to construct a Neighbor-Joining dendrogram based upon allele sharing between individuals. The multilocus estimate of the outcrossing rate was 100% (standard error of zero), higher than the 90% level found in previous studies at the embryo stage, suggesting that both artificial and natural selection against selfs may have occurred over the 38-year lifespan of the progeny trial. In the Neighbor-Joining dendrogram, the majority of the putative siblings grouped together in 21 out of the 29 families, showing that the microsatellites were able to discern most of the family structure in the dataset. Our results indicate that errors were sometimes committed during the establishment of the progeny test. This set of microsatellite loci clearly provides a powerful tool for future applications in black walnut.