Genetic Variation in Pawpaw Cultivars Using Microsatellite Analysis

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. The North American pawpaw is a tree fruit native to eastern North America, which is in the early stages of domestication (Pomper et al., 2008a, 2008b; Pomper and Layne, 2005). Pawpaws can be grown successfully in U.S. Department of Agriculture plant hardiness zone 5 (minimum of –29 C) through zone 8 (minimum of –7 C) (Kral, 1960). The fruit is very nutritious (Peterson et al., 1982) and has an intense flavor that resembles a combination of banana (Musa acuminate), mango (Mangifera indica), and pineapple (Ananas comosus) (Duffrin and Pomper, 2006; Layne, 1996; Shiota, 1991). This fruit crop has great potential for the processing market (Duffrin and Pomper, 2006; Templeton et al., 2003) as well as fresh market sales at farmers’ markets, on-farm sales, and community-supported agriculture (Pomper and Layne, 2005). This plant also contains annonaceous acetogenins in the twigs, fruit, seeds, roots, and bark tissues (McLaughlin, 2008; Pomper et al., 2009b). These compounds display antitumor, pesticidal, antimalarial, anthelmintic, piscicidal, antiviral, and antimicrobial effects, suggesting many potentially useful applications (McLaughlin, 2008). The pawpaw is diploid [2n = 2x = 18 (Bowden, 1948; Kral, 1960)]. Flowers are strongly protogynous and are likely selfincompatible (Willson and Schemske, 1980). Pawpaws have dark maroon blossoms that occur singly on the previous year’s wood and produce one to nine carpels or oneto 13-fruited clusters (Kral, 1960; Pomper and Layne, 2005). Species whose populations are distributed over a wide geographic region such as Asimina triloba may maintain significant variation among populations (Hamrick and Godt, 1989). In the southern portion of its native range, the distribution of A. triloba overlaps with that of some subtropical Asimina species, and introgression may have occurred. From 1900 to 1950, over 50 pawpaw cultivars were selected from the wild and named; however, only two remain: ‘Sweet Alice’ and ‘Middletown’ (Peterson, 1991, 2003). The rest of the cultivars appear to have been lost through neglect or abandonment of collections or cannot be identified as a result of loss of records. The loss of cultivars over the past century may have resulted in considerable genetic erosion (Huang et al., 1997; Peterson, 1991). Since 1950, additional pawpaw cultivars have been selected from the wild or developed as a result of breeding efforts of hobbyists (Peterson, 1986, 1991). The PawPaw Foundation breeding program released six pawpaw cultivars in 2004 and 2007. The KSU National Clonal Germplasm Repository (NCGR) for Asimina species in Frankfort, KY (a satellite site of the NCGR in Corvallis, OR) has over 2000 accessions from 17 states in the repository orchards that were collected by enthusiasts and KSU personnel. The repository also contains over 45 cultivars that are currently available from nurseries (Pomper et al., 2009a). Maintaining a high level of genetic diversity in pawpaw is important for the long-term genetic improvement of the crop and in minimizing vulnerability to disease. A range of DNA and protein-based genetic marker systems has been used in attempts to evaluate genetic diversity in pawpaw. These marker systems include the minisatellite probe (Rogstad et al., 1991), allozymes (Huang et al., 1997, 1998), random amplified polymorphic DNA [RAPD (Huang et al., 2000, 2003)], amplified fragment length polymorphism [AFLP (Wang et al., 2005)], intersimple sequence repeat [ISSR (Pomper et al., 2003)], and SSR (Pomper et al., 2010). Overall, these studies determined that the genetic variation in both cultivated and wild pawpaws is similar to those of other long-lived, temperate woody perennials characterized by a widespread geographic range, insect-pollinated outcrossing breeding Received for publication 11 July 2011. Accepted for publication 24 Aug. 2011. We thank R. Neal Peterson for reviewing the manuscript and for his helpful comments. Curator, USDA National Clonal Germplasm Repository for Asimina species. Corresponding author. E-mail: [email protected]. J. AMER. SOC. HORT. SCI. 136(6):415–421. 2011. 415 systems, secondary asexual reproduction, and animal-dispersed seed. The objective of this study was to compare the genetic variation exhibited among older and new pawpaw cultivars and KSU advanced selections using SSR markers. Materials and Methods PLANT MATERIAL. Leaf samples were collected from older and new (PPF) pawpaw cultivars and KSU advanced selections (Table 1). Leaf samples were collected in Spring 2009 and 2010 from pawpaw trees located at the KSU-NCGR for Asimina species in Frankfort, KY. DNA EXTRACTION. DNA was extracted from leaves using the DNAMITE Plant Kit (Gel Co., San Francisco, CA). Approximately 2 cm of young leaf tissue was used. DNA concentration and 260/280 nm absorbance ratio were determined with a GeneQuant pro RNA/DNA calculator (GE Healthcare Biosciences, Piscataway, NJ). All samples were stored at –80 C until needed. MICROSATELLITE-ENRICHED LIBRARIES AND PRIMER DESIGN. Genetic Identification Services (GIS, Chatsworth, CA) constructed pawpaw genomic libraries from DNA extracted from the cultivar PA-Golden (#1); libraries were enriched for di-nucleotide repeat GA (Library B) and for trinucleotide repeat ATG (Library C), and AAT (Library G). Inserts were sequenced by GIS using the DYEnamic ET Terminator Cycle Sequencing Kit (GE Healthcare Biosciences) followed by electrophoresis on a Model 377 DNA Sequencer (Applied Biosystems, Foster City, CA). Primers were designed from flanking regions using DesignerPCR, Version 1.03 (Research Genetics, Huntsville, AL) with the parameters of annealing temperature 60 C, GC content 50%, and amplicon size of 100 to 350 bp. Primer pairs were labeled with FAM or HEX and were made by Integrated DNA Technologies (Coralville, IA). SIMPLE SEQUENCE REPEAT–POLYMERASE CHAIN REACTION AMPLIFICATION. The SSR–polymerase chain reaction (PCR) amplification was performed with GoTaq Flexi DNA polymerase (Promega, Madison, WI). The reactions were set up as follows: 4 mL of 5· colorless GoTaq Flexi buffer, 0.4 mL of 10 mM dNTPs solution, 2 mL of 25 mM MgCl2, 0.3 mL of 30 mM forward primer (fluorescence labeled with FAM or HEX) solution and 0.3 mL of 30 mM reverse primer (unlabeled) solution, 0.2 mL of 5 units/mL GoTaq DNA polymerase, 2 mL of diluted 1 ng mL pawpaw Table 1. Genetic background of pawpaw cultivars and selections included in the genetic study. Clone Genetic background