The pawpaw [Asimina triloba (L.) Dunal] is a native plant found mainly in the southeastern and eastern United States, and its fruit has great potential as a new high-value crop in these regions. Although there are ≈45 named pawpaw cultivars, breeding for improvement of specific traits, such as fruit size and quality, is desirable. Our long-term goal is to utilize molecular marker systems to identify markers that can be used for germplasm diversity analyses and for the construction of a molecular genetic map, where markers are correlated with desirable pawpaw traits. The objective of this study was to identify random amplified polymorphic DNA (RAPD) markers that segregate in a simple Mendelian fashion in a controlled A. triloba cross. DNA was extracted from young leaves collected from field-planted parents and 20 progeny of the cross 1-7 × 2-54. The DNA extraction method used gave acceptable yields of ≈7 μg·g-1 of leaf tissue. Additionally, sample 260/280 ratios were ≈1.4, which indicated that the DNA was of high enough purity to be subjected to the RAPD methodology. Screening of 10-base oligonucleotide RAPD primers with template DNA from the parents and progeny of the cross has begun. We have identified two markers using Operon primer B-07 at 1.1 and 0.9 kb that segregate in a simple Mendelian fashion in progeny of the 1-7 × 2-54 cross. Other primers and controlled crosses will also be screened.
Tera M. Bonney, Shawn P. Brown, Snake C. Jones, Kirk W. Pomper and Robert L. Geneve
Kirk W. Pomper, Sheri B. Crabtree, Shawn P. Brown, Snake C. Jones, Tera M. Bonney and Desmond R. Layne
The pawpaw [Asimina triloba (L.) Dunal.] is a tree fruit native to many areas of the southeastern and mid-western United States. Kentucky State University (KSU) is designated as a satellite repository for Asimina for the U.S. Department of Agriculture (USDA), National Plant Germplasm System (NPGS). An assessment of the level of genetic diversity in cultivated pawpaw would assist in development of the future germplasm repository collection strategies for cultivar improvement. The objectives of this study were to identify intersimple sequence repeat (ISSR) markers that segregate in a simple Mendelian fashion and to use these markers to assess genetic diversity in 19 pawpaw cultivars. Leaf samples from the 34 progeny of controlled crosses (1-7-1 × 2-54 and reciprocal) and the parents were collected, DNA was extracted, and subjected to the ISSR methodology using the University of British Columbia microsatellite primer set #9. Seven primers yielded 11 Mendelian markers with either a 3:1 or 1:1 ratio that was confirmed by chi-square analysis. Analysis of genetic diversity using 10 of the ISSR markers from 19 pawpaw cultivars revealed a moderate to high level of genetic diversity, with a percent polymorphic loci P = 80 and an expected heterozygosity He = 0.358. These diversity values are higher than those reported for cultivated pawpaw using isozyme or randomly amplified polymorphic DNA (RAPD) markers, indicating that the ISSR marker methodolgy has a higher level of discrimination in evaluating genetic diversity in pawpaw and/or pawpaw has greater levels of genetic diversity than previously found.