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G.A. Picchioni, C.J. Graham, and A.L. Ulery

Charles Johnson (LSU Agricultural Center) for providing Asimina triloba seedlings used in the study, and Peter Gallagher (Louisiana Tech Univ. Dept. of Agricultural Sciences) for providing use of the study site. We also thank Bobby McCaslin and Denise

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Hideka Kobayashi, Changzheng Wang, and Kirk W. Pomper

( Butkus and Pliszka, 1993 , Haddad et al., 2001 ; McAnulty et al., 2004 ; Rossi et al., 2003 ; Schmidt et al., 2004 ; Weiguang et al., 2006 ). Pawpaw [ Asimina triloba (L.) Dunal] is a small tree found in the temperate woodlands of the Eastern

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Hongwen Huang, Desmond R. Layne, and Thomas L. Kubisiak

Thirty-four extant pawpaw [Asimina triloba (L.) Dunal] cultivars and advanced selections representing a large portion of the gene pool of cultivated pawpaws were investigated using 71 randomly amplified polymorphic DNA (RAPD) markers to establish genetic identities and evaluate genetic relatedness. All 34 cultivated pawpaws were uniquely identified by as few as 14 loci of eight primers. Genetic diversity of the existing gene pool of cultivated pawpaws, as estimated by Nei's gene diversity (He), was similar to that of wild pawpaw populations. The genetic relatedness among the cultivated pawpaws examined by UPGMA cluster analysis separated 34 cultivars and selections into two distinct clusters, a cluster of PPF (The PawPaw Foundation) selections and a cluster including a majority of the extant cultivars selected from the wild and their derived selections. The results are in general agreement with the known selection history and pedigree information available. The consensus fingerprint profile using the genetically defined RAPD markers is a useful and reliable method for establishing the genetic identities of the pawpaw cultivars and advanced selections. This also proved to be an improved discriminating tool over isozyme markers for the assessment of genetic diversity and relatedness. RAPD profiling of data presented in this study provides a useful reference for germplasm curators engaged in making decisions of sampling strategies, germplasm management and for breeders deciding which parents to select for future breeding efforts.

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Hongwen Huang, Desmond R. Layne, and Thomas L. Kubisiak

Twelve, 10-base primers amplified a total of 20 intense and easily scorable polymorphic bands in an interspecific cross of PPF1-5 pawpaw [Asimina triloba (L.) Dunal.] × RET (Asimina reticulata Shuttlew.). In this cross, all bands scored were present in, and inherited from, the A. triloba parent PPF1-5. Nineteen of the 20 bands were found to segregate as expected (1:1 or 3:1) based on chi-square goodness-of-fit tests, and were subsequently used to evaluate genetic diversity in populations of A. triloba collected from six states (Georgia, Illinois, Indiana, Maryland, New York, and West Virginia) within its natural range. Analysis of genetic diversity of the populations revealed that the mean number of alleles per locus was A = 1.64, percent polymorphic loci was P = 64, and expected heterozygosity was He = 0.25. No significant differences were found among populations for any of the polymorphic indices. Partitioning of the population genetic diversity showed that the average genetic diversity within populations was Hs = 0.26, accounting for 72% of the total genetic diversity. Genetic diversity among populations was Dst = 0.10, accounting for 28% of the total genetic diversity. Nei's genetic identity and distance showed a high mean identity of 0.86 between populations. Genetic relationships among the populations examined by unweighted pair-group mean clustering analysis separated the six populations into two primary clusters: one composed of Georgia, Maryland, and New York, and the other composed of Illinois, Indiana, and West Virginia. The Georgia and Indiana populations were further separated from the other populations within each group. This study provides additional evidence that marginal populations within the natural range of A. triloba should be included in future collection efforts to capture most of the rare and local alleles responsible for this differentiation.

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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.

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G.A. Picchioni and C.J. Graham

Asimina triloba (L.) Dunal (pawpaw), a deciduous tree indigenous to the eastern U.S., is being considered as a potential new fruit crop. The difficulty in establishing transplanted pawpaw seedlings has been identified as an important research need for successful cultivation of this species. We have addressed the possible benefits of soil-applied CaSO4 in establishing pawpaw seedlings on acidic, low-Ca orchard soil. Two-year-old seedling rootstocks were planted at a spacing of 1.5 m (within rows) × 5.5 m (between rows), and trickle-irrigated (with N, P, and K) for two growing seasons. Before planting, CaSO4 was applied at rates of 0, 11, and 22 t/ha and incorporated to a depth of 15 cm. Seedling trunk cross-sectional area (TCA) growth increased with increasing CaSO4 application. After the first season, increases in TCA averaged 27% and 44% greater with CaSO4 treatments (11 and 22 t/ha, respectively), as compared to the 0 t/ha treatment. This effect was accentuated by the end of the second season. Average qualitative ratings (based on seedling vigor and appearance) were also improved with CaSO4 treatment. These findings indicate that establishment of pawpaw seedling rootstocks may be improved with Ca fertilization in orchards of low-Ca status. Additional data, including seedling dry matter accumulation, will be presented.

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Hideka Kobayashi, Changzheng Wang, and Kirk W. Pomper

Pawpaw (Asimina triloba L.), a species of the eastern United States, bears the largest edible fruit of all native trees. Relatively little is known about ripening of pawpaw, and several problems, such as short shelf life and duration of harvesting, hamper pawpaw production. While previous investigations have resulted in identifying physical properties associated with ripening, the effects on phenolic content and antioxidant capacity have not been investigated. The objectives of the study were to investigate changes in phenolic content and antioxidant capacity and to identify physical parameters of pawpaw pulp during ripening. Sample extraction of pawpaw was achieved by adding acetone (2 mL/1 g of sample) to pulp of a pawpaw cultivar, PA Golden, and then vortexing (30 s) and sonicating (15 min) the sample and solvent, prior to centrifugation (15 min) twice at 2987 × g. Folin-Ciocalteu assay and ferric reducing antioxidant power (FRAP) assay were used for the estimation of phenolic content and the antioxidant capacity, respectively. While soluble solid content increased during ripening, the hardness of the fruit decreased, confirming previous reports. The pulp of unripe fruits had the greatest phenolic content (gallic acid eq. 131.2 mg/100 g FW) and antioxidant capacity (Trolox eq. 22.7 μM/g FW), which decreased by about 20% as the fruit ripened. Of three color properties measured, chroma, an estimate of color saturation, increased with ripening, while lightness of pawpaw pulp remained the same. A high correlation was found between chroma and hardness of fruits (r = 0.62), and between phenolic content and antioxidant capacity of pawpaw pulp (r = 0.80), suggesting these parameters can be incorporated into methods to estimate the ripeness of pawpaw fruit.

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Sheri B. Crabtree*, Kirk W. Pomper, and Robert L. Geneve

The pawpaw (Asimina triloba) is the largest fruit native to the U.S. and has potential as a new fruit crop. Few methods are available to clonally propagate pawpaw, with grafting or budding onto a seedling rootstock being the only currently feasible method. Developing new options for clonal propagation of pawpaw could help advance this growing industry. Layering has been used to clonally propagate other difficult to root tree species. The objective of this study was to examine trench layering as a method to clonally propagate pawpaw. A randomized factorial experiment was implemented to examine the roles of plant juvenility and auxin concentration on rooting in a greenhouse trench layering system. Seedlings were defoliated, tipped, and transplanted into trench layering beds at 3, 6, and 12 weeks after emergence. Shoots were etiolated, then girdled and treated with three levels of IBA (0, 5000, and 10,000 ppm). The main effects of age and IBA concentration significantly affected the percentage of shoots producing roots. Juvenility enhanced rooting, with 15% of the shoots of the 3-week-old pawpaw seedlings producing roots, compared to only about 5% of the 12-week-old seedlings rooting. Auxin application to shoots also promoted rooting, with 16% of IBA-treated shoots producing roots, compared to the untreated control, with only 2% of shoots producing roots. There was no significant difference in rooting percentage between the two concentrations of IBA. The treatment combination most successful at promoting root initiation was 10,000 ppm IBA applied to shoots of 3-week-old seedlings, with 31% of shoots rooting.

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Tera M. Bonney, Shawn P. Brown, Snake C. Jones, Kirk W. Pomper, and Robert L. Geneve

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.

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Ying Wang, Gregory L. Reighard, Desmond R. Layne, Albert G. Abbott, and Hongwen Huang

Pawpaw (Asimina triloba) produces the largest fruit native to the United States. Six linkage groups were identified for A. triloba using the interspecific cross [PPF1-5 (A. triloba) × RET (A. reticulata Shuttlw. ex Chapman)], covering 206 centimorgans (cM). A total of 134 dominant amplification fragment length polymorphism (AFLP) markers (37 polymorphic and 97 monomorphic) were employed for estimating the genetic diversity of eight wild populations and 31 cultivars and advanced selections. For the wild populations, the percentage of polymorphic loci over all populations was 28.1% for dominant markers and Nei's genetic diversity (He) were 0.077 estimated by 134 dominant markers. Genetic diversity and the percentage of polymorphic loci estimated using only polymorphic dominant AFLPs were 0.245 and 79%, respectively, which are comparable with other plant species having the same characteristics. Estimated genetic diversity within populations accounted for 81.3% of the total genetic diversity. For cultivars and advanced selections, genetic diversity estimated by 134 dominant markers was similar to that of wild pawpaw populations (He = 0.071). Thirty-one cultivars and advanced selections were delineated by as few as nine polymorphic AFLP dominant loci. Genetic relationships among wild populations, cultivars and advanced selections were further examined by unweighted pair group method with arithmetic mean (UPGMA) of Nei's unbiased genetic distance. The genetic diversity estimated for wild populations using the clustered polymorphic markers was lower than the result estimated using the nonclustered polymorphic markers. Therefore, this study indicates that the number of sampled genomic regions, instead of the number of markers, plays an important role for the genetic diversity estimates.