Kirk W. Pomper,1 Sheri B. Crabtree,2 Shawn P. Brown,3 Snake C. Jones,3 and Tera M. Bonney4 Kentucky State University, Land Grant Program, 129 Atwood Research Facility, Frankfort, KY 40601-2355
Desmond R. Layne5
Clemson University, Department of Horticulture, Clemson, SC 29634-0375
ADDITIONAL INDEX WORDS. kentucky banana, ISSR, molecular markers, DNA finger printing, cultivar identification
ABSTRACT. The pawpaw [Asimina triloba (L.) Dunal.] is a tree fruit native to many areas of the southeastern and midwestern 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 methodology has a higher level of discrimination in evaluating genetic diversity in pawpaw and/or pawpaw has greater levels of genetic diversity than previously found.
The genus Asimina is the only temperate representative of the tropical Annonaceae, or Custard Apple family, and includes eight species, Asimina triloba (L.) Dunal., A. parviflora (Michx.) Dunal., A. incana (Bartr.) Exell., A. obovata (Willd.) Nash, A. reticulata Shuttlw. ex Chapman, A. tetramera Small, A. pygmaea (Bartr.) Dunal, and A. longifolia Kral, that are indigenous to North America (Kral, 1960; Peterson, 1991). The best-known species is Asimina triloba, the North American pawpaw, which has the largest edible fruit native to the United States (Darrow, 1975). This species is diploid [n = 2x = 18, (Bowden, 1948; Kral, 1960)], outcrosses (Willson and Schemske, 1980), and is pollinated by flies and beetles (Faegri and van der Piji, 1971). The pawpaw fruit is very nutritious (Peterson et al., 1982) and has an almost tropical aroma and smooth, custard-like texture, with flavors reminiscent of a combination of mango, banana, and pineapple (Layne, 1996; Shiota, 1991). The high fruit quality and attractive ornamental appearance give the pawpaw great potential as a commercial tree fruit or as a component in landscapes (Layne, 1996; Pomper et al., 1999).
From about 1900 to 1960, at least 56 pawpaw cultivars were selected and named (Peterson, 1991). However, fewer than 20 of these selections remain, with many being lost from cultivation through neglect, abandonment of collections, and loss of records necessary for identification (Peterson, 1991). Since 1960, additional pawpaw cultivars have been selected from the wild or developed as a result of breeding efforts of hobbyists. More than 40 cultivars are currently commercially available (Jones et al., 1998). The loss of cultivars over the last century may have led to erosion in the genetic base of current pawpaw cultivars (Huang et al., 1997).
Received 1 Nov. 2002. Accepted 12 Mar. 2003. This research was supported by U.S. Dept. of Agriculture, Cooperative State Research, Education, and Extension Service Agreement no. KYX-9702815.
1Principal investigator of horticulture and curator, USDA National Clonal Germ-plasm Repository for Asimina spp., Kentucky State University, and President of The PawPaw Foundation. To whom reprint requests should be addressed.2Research assistant, Horticulture Program, Kentucky State University.3Former research assistant, Horticulture Program, Kentucky State University.4Former graduate student, Horticulture Program, Kentucky State University and Dept. of Horticulture at the Univ. of Kentucky.5Assistant professor of pomology and extension tree fruit specialist.
records necessary for identification (Peterson, 1991). Since 1960, additional pawpaw cultivars have been selected from the wild or developed as a result of breeding efforts of hobbyists. More than 40 cultivars are currently commercially available (Jones et al., 1998). The loss of cultivars over the last century may have led to erosion in the genetic base of current pawpaw cultivars (Huang et al., 1997).
In 1994, Kentucky State University (KSU) was designated as a satellite repository for Asimina preservation in the U.S. Department of Agriculture (USDA), National Plant Germplasm System (NPGS). Germplasm evaluation, preservation, and dissemination have been a high priority at KSU since that time. The repository orchards currently contain over 1700 accessions collected from the wild in 17 states and more than 40 cultivars. One of the goals of the repository is to assess levels of genetic diversity in native populations and commercially available cultivars. Another goal is to acquire unique germplasm to include in our repository collection. Such material could be useful in future pawpaw breeding efforts.
A range of molecular markers has been used in attempts to evaluate genetic diversity in pawpaw. Rogstad et al. (1991) used a minisatellite probe, M13, to determine the genetic variation in pawpaw collected in five states. Using data from one to 22 samples per population, these authors examined genetic variation at sixteen sites both within and among populations. They determined that genetic variation is very low within populations, but moderate genetic variation occurred between populations, attributed to the geographical range of the species. They concluded that the low level of genetic variation within populations might be due to clonality or inbreeding. However, inbreeding is considered to be rare in pawpaw's reproductive biology, because it is most likely self-incompatible and therefore may require out-crossing (Norman et al., 1992; Peterson, 1991). Huang et al. (1997) used