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Kirk W. Pomper and Sheri B. Crabtree

The propagation of clonal rootstocks for the pawpaw [Asimina triloba (L.) Dunal] has been unsuccessful; therefore, nurseries currently graft cultivars onto rootstock derived from locally available seed of diverse genetic origin. Great variation in pawpaw scion growth and suckering is observed with this seedling rootstock and grafted trees are also slow to come into production, often producing fruit 5 to 6 years after planting. In an effort to develop superior seedling rootstocks for pawpaw cultivars, seedstock was evaluated from the cultivars PA-Golden (#1), Sunflower, Susquehanna, NC-1, K8-2, and commercially available seed (RVT). The objectives of the experiment were to determine if: 1) genotype and seed size influenced vigor of greenhouse-grown container seedlings; 2) graft compatibility and growth rate of scions would vary by rootstock; and 3) scion precocity was influenced by various seedling rootstocks in the field. Greenhouse experiments were conducted in 2002, 2003, and 2004, and each year all seedstocks had a high germination percentage (95%) and uniform size within each seedstock. Seed size was an important factor in determining vigor in container production. Seed of `Sunflower' was large (1.65 g/seed) and consistently produced vigorous seedlings that were able to be chipbudded within 6 months. Seed of the selection K8-2 was smallest (0.94 g/seed) and showed the least vigor. Bud take was similar for the scions `Sunflower' and `Susquehanna' on all rootstocks. Twenty-one weeks after budding, scions of `Sunflower' were about 30% larger than scions of `Susquehanna'; however, source of seedling rootstock did not affect growth of either cultivar. Field evaluations have begun with the two cultivars budded onto the six rootstocks.

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

The North American pawpaw [Asiminatriloba (L.) Dunal] is a tree fruit native to the eastern and midwestern areas of the United States. The fruit has a rich, unique flavor and pawpaw has great potential as a new fruit crop. Kentucky State University (KSU) in Frankfort is the site for the USDA National Clonal Germplasm Repository (NCGR) for Asimina species, containing over 1700 accessions from 17 different states. The objective of this study was to evaluate the diversity in reproductive characteristics for pawpaw accessions in the KSU-USDA repository orchard from six geographic regions (Ind.-site 1, Ind.-site 2, Ky., Md., N.Y., and W.Va.). Data were collected in 2002, 2003, and 2004 for trunk cross-sectional area, total number of flowers, length of flowering, flowering peak, fruit set, total number of clusters, total number of fruit, number of fruit per cluster, average fruit weight, yield, yield efficiency, length of harvest, harvest peak, and growing degree days required for ripening. Significant differences in characteristics were found within and among populations for the various regions. Accessions from Kentucky and West Virginia had the latest flowering peaks. Trees from Maryland had the highest fruit weight in 2002, whereas the West Virginia population produced the largest fruit in 2004. The New York accessions consistently had the latest harvest peak and required the fewest growing degree days for ripening. Correlations were also found between several vegetative and reproductive characteristics. This study suggests that a significant level of reproductive diversity exists within KSU's repository collection that could be used in future breeding strategies for cultivar improvement.

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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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Danielle Rascoe, Kirk W. Pomper*, Jeremiah Lowe, Sheri B. Crabtree, Har Mahdeem and Tejender S. Kochhar

The genus Asimina has the only temperate representatives of the tropical Annonaceae, or Custard Apple family, and includes eight species that are indigenous to North America. The North American pawpaw Asimina triloba (L.) Dunal has the largest edible fruit native to the United States and is the best-known of these species. The USDA National Clonal Germplasm Repository for Asimina species is located at Kentucky State Univ. (KSU); therefore, assessment of genetic diversity is an important research priority for KSU. The inter-simple sequence repeat PCR (ISSR-PCR) methodology has been used successfully to characterize genetic diversity within and among populations of many plant species. The objective of this study was to assess the utility of ISSR markers in evaluating genetic relationships in members of the Asimina genus, as well as closely related tropical relatives in the Annona genus. Leaf samples were collected from three plants each of Asimina longifolia, A. obovata, A. parviflora, A. reticulata, A. tetramera and A. triloba. Leaf samples were also collected from three plants each of Annona cherimola, A. squamosa, A. reticulata, A. muricata, A. glabra, A. diversifolia, and A. montana. DNA was extracted from leaf samples and subjected to ISSR-PCR using the REDExtract-N-Amp™ Plant PCR Kit. DNA samples were screened with ISSR primers using the Univ. of British Columbia microsatellite primer set #9. Three primers, UBC812, UBC841, and UBC873 were found to produce 84 scorable ISSR markers and allowed the determination of genetic relationships among Asimina and Annona members examined.

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Li Lu, Kirk W. Pomper, Jeremiah D. Lowe and Sheri B. Crabtree

Pawpaw (Asimina triloba) is a tree fruit native to eastern North America, which is in the early stages of domestication. Most early 20th century pawpaw cultivars have been lost; however, recent cultivar releases and potential new releases may have enhanced genetic diversity. The objective of this study was to compare the genetic variation exhibited among older and new pawpaw cultivars and Kentucky State University (KSU) advanced selections using simple sequence repeat (SSR) markers. Polymorphic microsatellite marker analysis was conducted with nine older pawpaw cultivars, six recently released PawPaw Foundation (PPF) cultivars, and nine KSU advanced selections. Using 18 microsatellite loci, a total of 179 alleles were amplified in the set of 24 genotypes. The major allele frequency (0.13 to 0.96), number of genotypes (two to 23), and allele size (96 to 341 bp) varied greatly by locus. Eighteen loci were highly polymorphic, as indicated by high expected heterozygosity (He = 0.71) and observed heterozygosity (Ho = 0.65) values as well as high polymorphism information content (polymorphism information content = 0.69). The dinucleotide SSR (GA and CA motifs) loci were more polymorphic than trinucleotide (ATG and AAT motifs) SSRs. The PPF cultivars and KSU advanced selections were more closely grouped genetically than with older cultivars. Older cultivars displayed the greatest genetic diversity (Ho = 0.69). The pawpaw cultivar base of older and PPF cultivars does appear to be genetically diverse. However, KSU advanced selections contain unique pawpaw germplasm that should enhance the genetic base of cultivars if these selections are released to the public.

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Sheri B. Crabtree, Kirk W. Pomper, Desmond R. Layne and R. Neal Peterson

The pawpaw [Asimina triloba (L.) Dunal] is a tree fruit native to the eastern United States with potential as an alternative crop for small farmers. The Pawpaw Regional Variety Trial (PRVT) was established in 1993 by Kentucky State University (KSU) and the PawPaw Foundation (PPF) and includes 28 cultivars and advanced selections planted at 12 sites. The PRVT was established at the KSU Research Farm in Frankfort, Ky., in 1998. Data has been collected on the KSU-PRVT annually since its inception. The first fruit were produced in 1999, with Middletown, Mitchell, Overleese, and Sunflower being the most precocious varieties. A frost occurred in early April 2000, decimating the crop, with only eight fruit being produced across the orchard. In 2001, 12% of the trees produced fruit, with PA-Golden having the best early production. In 2002, 68% of trees in the PRVT fruited, producing a total of 3,500 fruit. Selections with the largest fruit (over 200 g) were Susquehanna, 5-5, 4-2, and 1-7-2. In 2003, a spring frost destroyed most of the flowers and developing fruit. Only 32 out of the 224 trees in the PRVT retained fruit, a total of only 131 fruit in the entire orchard. In 2004, the PRVT produced about 25,000 fruit across the entire orchard. Selections 4-2 and 7-90 produced the largest fruit, over 200 g. Shenandoah, 10-35, and 8-20 were the highest-yielding clones, all producing over 15 kg of fruit per tree. In 2005, spring frosts and a severe summer drought diminished fruit set and retention in the PRVT, with the orchard producing 8,900 fruit. Selections 4-2, 5-5, and Susquehanna produced the largest fruit, all weighing over 200 g. The highest yielding selections were 10-35, PA-Golden, and 1-7-2, all producing over 8 kg of fruit per tree.

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Kirk W. Pomper*, Joseph G. Masabni, Desmond R. Layne, Sheri B. Crabtree, R. Neal Peterson and Dwight Wolfe

The pawpaw [Asimina triloba (L.) Dunal] has great potential as a new fruit crop. A pawpaw variety trial was established in Fall 1995 in Princeton, Ky. as a joint Kentucky State Univ.-Univ. of Kentucky research effort with the objective to identify superior varieties for Kentucky. A randomized block experimental design was used with 8 replicates of 28 grafted scion selections on seedling rootstock. Cultivars being tested included Middletown, Mitchell, NC-1, Overleese, PA-Golden, Rappahannock, Shenandoah, Sunflower, Susquehanna, Taylor, Tay-two, Wells, and Wilson. The other 15 clones were selections from the PawPaw Foundation. In 2002 and 2003, the following parameters were examined: tree survival, trunk cross-sectional area (TCSA), average fruit weight, total fruit harvested per tree, average fruit per cluster, total yield per tree, and yield efficiency. In 2003, 54% of the trees had survived, with `Susquehanna' (13%) showing the poorest survival. Based on TCSA, most selections displayed excellent vigor, with the exception of the selections: 5-5 and `Overleese'. Average fruit weight was greatest in 1-7-2 (194 g), 1-68 (167g), 4-2 (321 g), 5-5 (225 g), 7-90 (166g), 9-58 (176 g), 10-35 (167 g), NC-1 (180 g), `Sunflower' (204 g), and `Shenandoah' (168g), with the smallest fruit in `Middletown' (70 g), `Wells' (78 g), and `Wilson' (88 g). The selections `Wilson' (81), `Middletown' (75), and `Wells' (70) had the greatest average number of fruit per tree, whereas 4-2 (9), 5-5 (17) and 8-20 (15) the fewest. Yield efficiency and average fruit per cluster also varied greatly among selections. Several pawpaw selections in the trial show promise for production in Kentucky.

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Kirk W. Pomper, Jeremiah D. Lowe, Li Lu, Sheri B. Crabtree, Shandeep Dutta, Kyle Schneider and James Tidwell

Pawpaw [Asimina triloba (L.) Dunal.], a tree fruit native to eastern North America, is in the beginning stages of commercialization. Cultivars available in the early 20th century have been lost, and significant genetic erosion may have occurred. Polymorphic microsatellite marker loci were developed from enriched genomic libraries. Five marker loci were used to fingerprint 28 cultivars and 13 selections. For the 41 genotypes, 102 alleles were amplified and major allele frequency (0.16–0.94), number of genotypes (2–27), and allele size (144–343 bp) varied greatly by locus. Four loci were highly polymorphic, as indicated by values for expected heterozygosity (He), observed heterozygosity (Ho), and polymorphism information content, but only two alleles were detected at locus Pp-C104. A high level of genetic diversity was observed in the studied genotypes. The Ho (0.68) and He (0.70) were similar and indicated few null alleles. In the 41 genotypes, 39 unique fingerprints were observed. These new microsatellite marker loci will be useful for cultivar fingerprinting, management of collections, and investigation of genetic diversity in collections and wild populations. Grouping of genotypes in an unweighted pair group method with arithmetic mean dendrogram was generally consistent with their origins.

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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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Robert L. Geneve, Sharon T. Kester, Kirk W. Pomper, Jonathan N. Egilla, Cynthia L.H. Finneseth, Sheri B. Crabtree and Desmond R. Layne

Pawpaw (Asimina triloba) is an under-exploited small tree with commercial potential as a fruit crop, ornamental tree, and source of secondary products with insecticidal and medicinal properties. It is most often propagated from seeds that are recalcitrant and must be stored moist at a chilling temperature. Seeds display combinational (morphophysiological) dormancy. Endogenous, physiological dormancy is broken by about 100 days of chilling stratification followed by a period of warm moist conditions where the small embryo develops prior to seedling emergence about 45 days after the warm period begins. Pawpaw cultivars with superior fruit characteristics are propagated by grafting onto seedling understocks. The most common practice is chip budding. Other methods of clonal propagation have proven problematic. Pawpaw can be propagated from cuttings, but only in very young seedling stock plants. Micropropagation from mature sources is not yet possible, but shoot proliferation has been accomplished from seedling explants and explants rejuvenated by induction of shoots from root cuttings of mature plants. However, rooting of microcuttings and subsequent acclimatization has not been successful.