Parentage Verification of Blackberry Seedling Populations with a Microsatellite Fingerprinting Set

In an effort to confirm identity by descent, seedling populations representing important blackberry (Rubus sp.) breeding parents from the University of Arkansas (UA) and the USDA-ARS, Oregon were analyzed for trueness-to-type with a fingerprinting set consisting of six trinucleotide-containing simple sequence repeats (SSRs). Six seedling populations from UA and 12 seedling populations from USDA-ARS were initially genotyped with this 6-SSR set. Presence of alleles not found in either parent but in a seedling indicated an off-type individual. This initial analysis with the 6-SSR set revealed 19 off-type seedlings out of 264 from UA and 24 off-types out of 202 seedlings representing 11 USDA-ARS populations. In the 12^th population, ORUS 4647, all 23 seedlings contained alleles found in one of the parents, ‘Obsidian’, but also contained an allele not found in either ‘Obsidian’ or ORUS 2532-1, possibly indicating that the wrong parent plant was sampled for ORUS 2532-1. This hypothesis will be confirmed by genotyping each of the three available ORUS 2532-1 plants. Though the 6-SSR set was able to identify some off-types, it could not differentiate amongst 23 groups of individuals representing 55 seedlings from the UA populations and seven pairs of individuals representing 14 individuals from the USDA-ARS populations. To achieve better resolution, marker RH_MEa0016bC11 was removed and replaced with RH_MEa0008cF01, and two additional markers were added, resulting in a more polymorphic 8-SSR fingerprinting set. With this revised set of primers, undifferentiated samples were reduced to eight in UA material, and there was no reduction for the USDA-ARS material. Genotyping the 14 undifferentiated USDA-ARS samples with 13 additional Rubus SSRs generated similar results for 12 of the samples, indicating good distinguishing ability of this 8-SSR multiplex. This 8-SSR multiplex can be used in future work as a quick screen to confirm parentage in blackberry and ensure that progeny are true to type and that any genetically redundant individuals are excluded.