Confirming clonal identity: A case study in blueberries

Authors: Hummer, Kim; Bassil, Nahla

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/27/2022
Publication Date: 1/31/2023
Citation: Hummer, K.E., Bassil, N.V. 2023. Confirming clonal identity: A case study in blueberries. Acta Horticulturae. 1357:129-136. https://doi.org/10.17660/ActaHortic.2023.1357.19.
DOI: https://doi.org/10.17660/ActaHortic.2023.1357.19

Interpretive Summary: Horticultural research and industries, including blueberry production, are contingent upon correct cultivar identity. This identity must be maintained by clonal propagation through skilled nursery techniques such as cuttings, grafting, layering, and tissue culture. Breeding, the development of improved cultivars, requires confidence in the identity of parental lines within the genepool so that continued advancement can achieve planned program goals. Yet, considering human and machine fallibility, errors are inherent in these practices. Hasty production, such as forcing high re-propagation numbers in short timeframes, can increase the number of accidental mis-propagation, mis-labeling, or mis-interpretation events. These errors can reverberate through many generation cycles. The staff at the United States Department of Agriculture (USDA), Agricultural Research Service, National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, are highly concerned about preserving the correct clonal identity of the diverse blueberry cultivars under their management. Genotypic identity is critical to genebank management and operations. Previously, identity confirmation was determined through the morphology of key taxonomic traits as compared with written descriptions, and visually as determined by expert botanical and horticultural taxonomists. Now, simple sequence repeat (SSR)-based molecular tools have been developed for DNA-based “fingerprinting”. The objectives of this study were to develop a procedural system of cultivar identity confirmation and to suggest consistent ontology. As a discussion example, we present a recent blueberry cultivar identity study of 140 popular blueberry cultivars sampled from three sources. First, fingerprinting-sets of five or 10 tri-nucleotide-containing SSRs were used to genotype the samples. Unique reference fingerprints for each cultivar were established. Parentage analysis, looking at progenitors and progeny, was performed as were comparisons of samples between sources. Genotyping followed by parentage analysis detected correct cultivars, homonyms, synonyms, and incorrect samples. The samples separated into four categories: true to type (TTT) where morphology, SSR markers, and parentage analysis agreed; identity ok (IDOK) where markers agreed but parentage analysis was incomplete; identity question (IDQ) where allele composition did not match parentage and more testing is needed to confirm identity; identity wrong (IDX) where incorrect identity is confirmed by parentage analysis and replacement with TTT is required. In this study, 85% of the 140 NCGR cultivars were TTT or IDOK. The 15% IDQ or IDX will be replaced from original TTT sources. Ideally, with sufficient financial resources, each time a plant is re-propagated, a molecular test that confirms TTT should be performed. We have observed breeder collections with equivalent or greater identity error percentages to that reported here. In summary, molecular markers used in conjunction with parentage analysis, is a strong tool for identity confirmation and should be applied as frequently as re-propagation.

Technical Abstract: Horticultural research and industries, including blueberry production, are contingent upon correct cultivar identity. This identity must be maintained by clonal propagation through skilled nursery techniques such as cuttings, grafting, layering, and tissue culture. Breeding, the development of improved cultivars, requires confidence in the identity of parental lines within the genepool so that continued advancement can achieve planned program goals. Yet, considering human and machine fallibility, errors are inherent in these practices. Hasty production, such as forcing high re-propagation numbers in short timeframes, can increase the number of accidental mis-propagation, mis-labeling, or mis-interpretation events. These errors can reverberate through many generation cycles. The staff at the United States Department of Agriculture (USDA), Agricultural Research Service, National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, are highly concerned about preserving the correct clonal identity of the diverse blueberry cultivars under their management. Genotypic identity is critical to genebank management and operations. Previously, identity confirmation was determined through the morphology of key taxonomic traits as compared with written descriptions, and visually as determined by expert botanical and horticultural taxonomists. Now, simple sequence repeat (SSR)-based molecular tools have been developed for DNA-based “fingerprinting”. The objectives of this study were to develop a procedural system of cultivar identity confirmation and to suggest consistent ontology. As a discussion example, we present a recent blueberry cultivar identity study of 140 popular blueberry cultivars sampled from three sources. First, fingerprinting-sets of five or 10 tri-nucleotide-containing SSRs were used to genotype the samples. Unique reference fingerprints for each cultivar were established. Parentage analysis, looking at progenitors and progeny, was performed as were comparisons of samples between sources. Genotyping followed by parentage analysis detected correct cultivars, homonyms, synonyms, and incorrect samples. The samples separated into four categories: true to type (TTT) where morphology, SSR markers, and parentage analysis agreed; identity ok (IDOK) where markers agreed but parentage analysis was incomplete; identity question (IDQ) where allele composition did not match parentage and more testing is needed to confirm identity; identity wrong (IDX) where incorrect identity is confirmed by parentage analysis and replacement with TTT is required. In this study, 85% of the 140 NCGR cultivars were TTT or IDOK. The 15% IDQ or IDX will be replaced from original TTT sources. Ideally, with sufficient financial resources, each time a plant is re-propagated, a molecular test that confirms TTT should be performed. We have observed breeder collections with equivalent or greater identity error percentages to that reported here. In summary, molecular markers used in conjunction with parentage analysis, is a strong tool for identity confirmation and should be applied as frequently as re-propagation.