Location: National Clonal Germplasm RepositoryTitle: An improved 10-SSR pyrus fingerprinting DNA test to confirm parentage [abstract]
Submitted to: HortScience
Publication Type: Abstract Only
Publication Acceptance Date: 9/19/2017
Publication Date: 9/19/2017
Citation: Zurn, J.D., Nyberg, A.M., Bell, R.L., Postman, J.D., Bassil, N.V. 2017. An improved 10-SSR pyrus fingerprinting DNA test to confirm parentage [abstract]. HortScience. September 19-22, 2017, Waikoloa, Hawaii.
Interpretive Summary: The ability to distiguish individuals and confirm pedigrees is an important aspect of maintaining germplasm collections for clonally propigated plants such as pear. In the past, pedigree and identity confirmasion was conducted using morphological observations. This approach is not ideal because fruit is often needed for confirmation, which may not be available, particularly if the variety is being used as a rootstock. To overcome the limitations of using morphology, a DNA fingerprinting set was developed by the European Cooperative Program fro Plant Genetic Resources (ECPGR). This fingerprinting set consists of 12 markers and is conducted in two reactions. The ECPGR DNA fingerprinting set is difficult to use due to the nature of the markers chosen. As such, we created a 10-SSR marker DNA fingerprinting set which can be performed in a single reaction. This new fingerprinting set is easier to score than the ECPGR fingerprinting set and was used to evaluate 712 pear trees. Unfortunately, 12 groups of 26 individuals could not be distinguished from one another. This new 10-SSR DNA fingerprinting set is a useful tool and will be improved to differentiate all indiviuals.
Technical Abstract: Pedigree confirmation is a critical part of breeding, managing genetic resources, and developing genetic mapping populations for out-crossing plants, such as the genus Pyrus. Individuals which are not progeny of a biparental cross can cause mapping errors or result in the costly genotyping of individuals not needed for the study. Moreover, it is important to ensure the identity of individuals within a germplasm repository. The ability to validate pedigree and identity is usually not possible through morphology alone. A DNA test was needed to confirm parentage and identity by descent in three biparental populations developed to identify fire blight resistance loci as part of the United State Department of Agriculture – Specialty Crop Research Initiative (USDA-SCRI) funded RosBREED project and confirm identity within the USDA National Clonal Germplasm Repository (NCGR) Pyrus collection. An existing fingerprinting set developed by the European Cooperative Program for Plant Genetic Resources (ECPGR) consists of 12 di-nucleotide-containing simple sequence repeat (SSR) markers which are amplified in two PCR reactions. Di-nucleotide-containing SSRs often exhibit a number of amplification artifacts such as stutters, split peaks, and binning errors, whereas high-core repeat (3-6 bp motifs) SSRs usually do not. Initially, a single-reaction 9-SSR fingerprinting set consisting of four di-nucleotide-containing SSRs from the ECPGR set and five high-core repeat SSRs was created and used to evaluate the 310 biparental progeny housed at the USDA-Agricultural Research Service (USDA-ARS) Appalachian Fruit Research Station and 412 accession from the Pyrus collection housed at the USDA-ARS NCGR. The 9-SSR fingerprinting set was able to confirm parentage for 255 of the 310 progeny and generated unique profiles for all but 45 individuals. Unique profiles were generated for all but 47 individuals from the Pyrus core collection. The inability to distinguish these 92 individuals prompted the improvement of the 9-SSR fingerprinting set. Therefore, poorly performing markers were removed, and five new high-core repeat SSRs were added to bring the set to 10-SSRs. The number of undifferentiated individuals was reduced from 92 to 12 sets of 26 individuals using the improved SSR set. This 10-SSR fingerprinting set is a useful tool for quick parentage verification in pear and will continue to be improved.