New Guinea Impatiens Moala, Pascua
Objective

• Develop DNA fingerprinting protocols for New Guinea Impatiens.

• Test numerical systems for validating polymorphisms.

• Develop a resource for New Guinea Impatiens cultivar identification.

New Guinea Impatiens Apollon, Prepona

New Guinea Impatiens Prepona, Apollon

DNA Fingerprinting of Floral Crops by Amplified Fragment Length Polymorphism: New Guinea Impatiens

J.H. Lyerly, E.R. Parks, and J. W. Moyer

Introduction
New Guinea impatiens (Impatiens hawkeri) have risen to popularity as ornamental plants for beds, borders, and baskets over the last 30 years. The number of commercially available cultivars increased steadily after their initial release in 1972, and by the mid-1980s New Guinea impatiens became a popular bedding plant. Series currently available provide gardeners with a variety of foliage and flower colors.

Figure 1. NGI cultivars Moala (red) and Pascua (pink).
New Guinea Impatiens Moala, Pascua
A concern for breeders in the industry is cultivar protection. Many cultivars have similar flower and foliage colors and can be difficult to distinguish based on morphology alone. Morphological characteristics may be expressed later in development, such as flower color, or be influenced by environmental factors including sun exposure, nutrition, and contact with diseases or pests. These factors make cultivar identification based on morphology difficult and costly, as plants must be maintained until all the needed characteristics develop. Molecular markers provide a reliable and cost-effective alternative. Molecular markers have been used successfully for assessing parentage, examining genetic relationships among and within species, and for marker-assisted breeding.
New Guinea Impatiens cultivar Apollon
Figure 2. NGI cultivar Apollon.

Amplified fragment length polymorphism (AFLP) is a highly informative assay for evaluating plant genomes, as it can be used to generate fingerprints for any DNA regardless of complexity. AFLP relies on PCR amplification of an arbitrary set of restriction fragments distributed randomly throughout the genome, producing a large number of high quality markers without the need for prior sequence knowledge. AFLP has been used to examine genetic relationships among other crop species, including lettuce (Hill et al., 1996) and eggplant (Mace et al., 1999).

The objective of this research is to further the understanding of NGI genetics using current genomic science methods. This project proposes to develop a molecular method for identifying New Guinea impatiens cultivars, and to use the method to examine the genetic relationships between these cultivars.
AFLP polymorphism between New Guinea Impatiens cultivars
Figure 3. An example of AFLP polymorphism between cultivars listed in the table below. Arrows indicate selected fragments.

Data Analysis

  • A spreadsheet was created containing the scores of present (1) or absent (0) for each fragment of interest generated from each primer pair. Plants were processed in duplicate, creating a pair of scores for each fragment.
  • The pairs of scores were compared individually to determine if the fragment should be kept in the analysis.
  • Fragments with a high level of consistency were kept.

Data from selected fragments was entered into a statistical program for genetic analysis using Jaccard’s coefficient of similarity.

Results
Fragment Selection
The focus for the project has been to identify polymorphisms that are potentially useful for fingerprinting NGI and select those most closely linked to cultivar identity. Initial AFLP analysis with seven primer combinations and 10 cultivars yielded a total of 121 polymorphic bands, ranging from 12 to 23 bands per primer combination.

A larger cultivar set (20) was evaluated with the same primer combinations to eliminate any polymorphisms not consistently present in replicate samples. Any bands that were not consistently scored as present or absent in duplicate plants from the same cultivar would be eliminated from the fingerprinting list. This additional testing would ensure that the final set of chosen bands would yield consistent and reliable results when the fingerprinting technique was used on new cultivars.

Following the initial validation, 76 fragments were selected as candidates for fingerprinting.

Forty-three cultivars were selected for further validation of the 76 polymorphic bands. The AFLP process was carried out with replicate samples. In this analysis, fragments that scored consistently in 90% of the samples were kept, and all other fragments were discarded. Forty polymorphic bands were identified for DNA fingerprinting. Due to missing data, 38 of the 40 identified bands were used in the analysis. Jaccard’s Coefficient of Similarity ranged from 0.70 to 1.0 for 35 duplicates.

Table 1. Table of scoring for numbered fragments 1 – 3 with the listed cultivars. Cultivars are listed in order of their position on the gel image from left to right.

New Guinea Impatiens Moala
Figure 4. NGI cultivar Moala from table 1.
Conclusions
  • A reliable protocol for fingerprinting NGI was developed that would differentiate between cultivars and generate reproducible results.
  • Examination of the matching percentages for both fragments and cultivars identified the most reliable fragments and pinpointed variable cultivars quickly.
  • Data analysis focusing on genetic relationships revealed that most cultivar duplicates paired together as expected in the phylogram generated from the similarity coefficient data.
  • Results from this study indicate that NGI cultivars can have high levels of genetic variation.
These results raise important questions regarding the genetic variability within a cultivar as well as the relationships between NGI cultivars.