Articles
Confirming clonal identity: a case study in blueberries
Article number
1357_19
Pages
129 – 136
Language
English
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, 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 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.
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 four 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 confirmed TTT sources.
Ideally, with sufficient financial resources, each time a plant is re-propagated, a molecular test that confirms identity 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.
We leave with a caveat that in some cases original germplasm may no longer be extant.
In this case, consensus agreement using pedigrees and parentage analysis are our best tools for identity determination.
This identity must be maintained by clonal propagation through skilled nursery techniques such as cuttings, 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 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.
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 four 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 confirmed TTT sources.
Ideally, with sufficient financial resources, each time a plant is re-propagated, a molecular test that confirms identity 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.
We leave with a caveat that in some cases original germplasm may no longer be extant.
In this case, consensus agreement using pedigrees and parentage analysis are our best tools for identity determination.
Publication
Authors
K.E. Hummer, N.V. Bassil
Keywords
Vaccinium, genetic diversity, fingerprinting set, microsatellite markers, simple sequence repeat (SSR)
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