Moyer | DNA Fingerprinting of Floral Crops by Amplified Fragment Length Polymorphism: Poinsettia


Objective • Develop DNA fingerprinting protocols for Poinsettia genotypes. • Explore methods of validating DNA fragment polymorphisms for cultivar Id. • Provide a resource for the identification of commercial cultivars. • Information will be used as an aid for determining cultivar authenticity.

DNA Fingerprinting of Floral Crops by Amplified Fragment Length Polymorphism:
Poinsettia
E. R. Parks, J. H. Lyerly, and J. W. Moyer


	Figure 1. Subtle differences between cultivars can make them difficult to differentiate using morphological characteristics.

Introduction
Euphorbia pulcherrima, the popular Christmas ornamental poinsettia, is the best selling potted plant in the United States, comprising over 85% of potted plant sales during the holiday season. There are over 175 varieties of cultivated poinsettias, and the thriving $300 million annual business fosters incentive for breeders to develop new varieties. Breeders seek to protect their new cultivars through plant patents in the United States, or Plant Breeder’s Rights in Europe. Both of these require proof of distinctness, uniformity, and stability before protection will be granted. Poinsettia breeders evaluate morphological characteristics such as bract color, growth habit, and time to flower to fulfill the patent requirements.

Figure 2. An example of AFLP polymorphism between cultivars with varying degrees of relatedness. Genotypes: C1, C27, C17, Sel.119, Freedom Red, Freedom Jingle Bells, Freedom Marble, Freedom White, Freedom Pink, Angelika Red, Peterstar, Cortez, Bonita, Sonora, Celebrate 2, Red Glory, Nutcracker Red.

Selection of AFLP polymorphisms
AFLP fragments used for scoring were subject to strict selection criteria . Fragments were selected for analysis from these if they were easily scored on the AFLP image in terms of intensity and separation from other fragments, and were reproducible in two independent amplifications. Figure 2 shows examples of these fragments, indicated by the arrows. A total of 98 AFLP fragments were chosen for analysis from 8 primer combinations.

Data analysis.

Data analysis was performed as described.

Reproducibility of AFLP polymorphisms
To further validate the reproducibility and reliability of the AFLP fingerprints, the intracultivar variation of the AFLP fragments was evaluated. A set of cultivars was chosen for this study, and multiple samples of each were evaluated with the selected primers. The amount of intracultivar variation was different for all the cultivars. This divergence within a cultivar was attributed to inadvertent propagation of variants of the cultivar. Polymorphisms that were variable among sources of a given cultivar were likely to be variable among sources of one or more of the other cultivars; 22 of the fragments varied in one cultivar, and 35 varied in more than one. Of the 98 fragments, eight were consistent in all selections of all cultivars. In total, 57 of the 98 fragments were found to be highly variable between plant from different sources in one or more cultivars. These highly variable fragments were eliminated from AFLP analysis, resulting in 41 validated fragments in the poinsettia AFLP database.

Table 1. Example of validation results for one AFLP primer combination showing variation of polymorphic fragments within chosen cultivars (grey) and validated, non-varibale fragments (yellow). Table 1

Conclusions
A dendrogram was created that included 81 named cultivars in the study, along with the 2 outgroup species. The set of 41 validated AFLP fragments was able to differentiate all but a small number of cultivar comparisons. Those cultivars that could not be distinguished with the set of fragments are easily separated by morphological traits; they are most often color sports of one another. Those cultivars that are the most difficult to distinguish morphologically, such as the many red cultivars in the study, were differentiated with AFLP fingerprinting.

The data in this study shows a high degree of correlation with known relationships. The repeatability of the AFLP banding patterns coupled with the validation of the fragments by testing of multiple samples of various cultivars provides credibility to the AFLP fingerprints and the relationships concluded from them. The AFLP technique has been shown to be an effective and robust tool for determining genetic relationships in poinsettia. AFLP analysis of poinsettia and other floral crops can provide cultivar protection, support in breeding programs, and the potential to develop markers for desirable characteristics.