Assessment of Genetic Diversity of Pawpaw Cultivars with Inter-Simple Sequence Repeat Markers Kirk W. Pomper*, Sheri B. Crabtree, and Snake C. Jones Kentucky State University
What is Pawpaw? Asimina triloba (L.) Dunal Diploid (x = 9, 2n = 18) Native tree fruit in Kentucky Fruit has tropical-like flavor High nutritional value high in essential vitamins, minerals, amino acids Alternative Cash Crop for tobacco farmers
Native Range of Pawpaw
The Pawpaw The only temperate members of the tropical Custard Apple or Annonaceae family are in the genus Asimina. Other members of the family: custard apple (Annona reticulata L.) cherimoya (A. cherimola Mill.) sweetsop or sugar apple (A. squamosa L.) atemoya (A. squamosa x A. cherimola) soursop (A. muricata L.)
The Pawpaw Members of the Asimina genus that are native to the United States: A. triloba (L.) Dunal (pawpaw) A. incana (Bartr.) Exell. A. longifolia Kral, A. obovata (Willd.) Nash A. pygmaea (Bartr.) Dunal A. reticulata Shuttlw. ex Chapman A. tetramera Small A. parviflora (Michx.) Dunal.
USDA National Clonal Germplasm Repository for Pawpaw 1700 trees from 66 different geographic regions and 16 different states
Why is the level of genetic diversity in pawpaw being investigated? Genetic diversity is a critical component of biodiversity and adjustment to the environment Genetic resources themselves are a rich potential source of useful genetic traits Repository Research Efforts: Evaluate genetic diversity in native range Examine genetic relationships in cultivars Efficiently improving diversity of the repository collection Selection of germplasm in breeding programs
KSU Molecular Genetics Laboratory Established in 1999 Goals: 1) Germplasm collection and assessment of genetic diversity 2) Construction of genetic maps 3) Breeding for improved cultivars 4) Fingerprinting of pawpaw cultivars
Previous genetic research on pawpaw Huang et al. (1997 and 1998) found moderate level of diversity in wild accessions and cultivars using isozymes in the repository collection. Huang et al. (2000) determined genetic diversity was moderate to high in six populations using RAPD than when using isozymes.
Inter Simple Sequence Repeat PCR (ISSR-PCR) Relies on the ubiquity of simple sequence repeats (SSRs) in eukaryotic genomes [e.g. (GT) n or (GATA) n ] A terminally anchored primer specific to a particular SSR is used to amplify between two opposed SSRs of the same type Polymorphism's occur when one genome is missing one of the SSRs or has deletion-insertion that modifies the distance between repeats Have longer primers, higher annealing temperatures, and a higher degree of polymorphisms than RAPDs (Zietkiewicz et al. 1994; Gupta et al. 1994) UBC 857: 5 ACACACACACACACACYG
Objectives To identify inter-simple sequence repeat (ISSR) markers that segregate in a simple Mendelian fashion Use these markers to assess genetic diversity in 19 pawpaw cultivars
Methods and Materials Parent Cross of A. triloba: Controlled cross: 1-7-1 x 2-54 20 resulting progeny 10 progeny of the reciprocal cross 2-54 x 1-7-1 Young leaves were collected in July from trees at the KSU farm DNA was extracted from young leaf tissue via the method of Pomper et al. (1999)
Random Amplified Polymorphic DNA (RAPD) OPB-07: 5 GGTGACGCAG5 94 o C 5 min 5:00 hold 94 o C 1:00 45 cycles 36 o C 1:30 54 o C 0:30 72 o C 2:00 72 o C 15:00 hold 4 o C 94 o C 5 min 5:00 94 o C 0:45 50 o C 1:00 72 o C 2:00 72 o C 10:00 Inter Simple Sequence Repeat (ISSR) UBC 857: 5 ACACACACACACACACYG 4 o C
Single Locus Segregation and Chi-Square Tests for 11 ISSR Markers in a Pawpaw Cross Parents Progeny ISSR banding ISSR Phenotype (genotype) Expected Locus phenotype Genotype 1 (+/+ or +/-) 0 (-/-) ratio X 2 UBC812-0640 1 0 (+/+ or +/- -/-) 15 19 1:1 0.53 UBC825-0760 1 1 (+/+ or +/- +/+ or +/-) 22 12 3:1 2.17 UBC825-0696 1 0 (+/+ or +/- -/-) 13 21 1:1 2.13 UBC841-1075 1 0 (+/+ or +/- -/-) 19 15 1:1 0.53 UBC841-0480 1 0 (+/+ or +/- -/-) 17 17 1:1 0.00 UBC853-1930 1 0 (+/+ or +/- -/-) 18 16 1:1 0.13 UBC860-0875 1 0 (+/+ or +/- -/-) 13 21 1:1 2.13 UBC860-0940 1 0 (+/+ or +/- -/-) 17 17 1:1 0.00 UBC873-0480 1 0 (+/+ or +/- -/-) 18 16 1:1 0.13 UBC873-0800 1 1 (+/+ or +/- +/+ or +/-) 21 13 3:1 3.60 UBC880-1150 1 1 (+/+ or +/- +/+ or +/-) 23 11 3:1 1.11
Genetic Background of Cultivars Cultivar Genetic background 1. Cales Creek Wild seedling from Summers Co., West Virginia. 2. Davis Wild seedling from Eaton Rapids, Mich. 3. Greenriver Belle Wild seedling from Hart County, Kentucky. 4. IXL Seedling of Overleese female Davis male selected in Eaton Rapids, Mich. 5. Middletown Wild seedling from Middletown, Ohio 6. Mitchell Wild seedling from Iuka, Ill. 7. NC-1 Davis female Overleese male selected in Ontario, Canada. 8. Overleese Seedling from Rushville, Ind. 9. PA-Golden(#1) Seedling from G.A. Zimmerman collection selected in Amherst, NY 10. Prolific Seedling from Eaton Rapids, Mich. 11. Rebecca s Gold Seedling from Eaton Rapids, Mich. 12. SAA-Zimmerman Seedling from G.A. Zimmerman collection selected in Amherst, NY. 13. Sue Wild seedling from Indiana 14. Sunflower Wild seedling from Chanute, Kans. 15. Sweet Alice Wild seedling from West Virginia. 16. Taylor Wild seedling from Eaton Rapids, Mich. 17. Taytwo Wild seedling from Eaton Rapids, Mich. 18. Wells Cultivated seedlings (open pollinated) from Salem, Ind. 19. Wilson Wild seedling from Cumberland, Ky.
ISSR markers scored in 19 pawpaw cultivars Cultivar Marker 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 UBC812-0640 1 1 0 0 1 0 1 1 0 0 1 0 0 0 0 0 1 1 0 UBC825-0760 1 0 0 0 1 0 0 0 1 0 0 0 1 1 0 0 0 0 0 UBC825-0696 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 UBC841-480 0 1 0 1 0 0 0 1 0 1 0 1 0 0 1 1 1 0 1 UBC841-1075 0 0 1 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 UBC853-1930 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 UBC860-0875 0 0 1 0 0 0 1 0 0 1 1 0 0 1 0 1 0 0 0 UBC860-0940 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 UBC873-0480 0 1 0 1 0 0 0 1 1 1 0 0 0 0 0 0 1 0 0 UBC880-1150 1 1 0 0 1 0 1 1 0 0 1 0 0 0 0 0 1 1 1 1 1 1 0 A B 1 0 1 1
Comparison of genetic variation of pawpaw with plant species having the same characteristics Species characteristics Polymorphic locus (%) (P) y Expected heterzygosity (H e ) Life form: long-lived woody perennial z 64.7 ± 2.7 0.177 ± 0.010 Regional distribution: widespread z 58.9 ± 3.1 0.202 ± 0.015 Geographic range: temperate z 48.5 ± 1.5 0.146 ± 0.000 Breeding system: outcrossing-animal z 50.1 ± 2.0 0.167 ± 0.010 Seed dispersal: animal ingested z 45.7 ± 3.9 0.176 ± 0.019 Mode of reproduction: sexual and asexual z 43.8 ± 3.7 0.138 ± 0.016 Average of all characteristics z 51.2 ± 8.2 0.168 ± 0.023 California cherimoya x 73.3 0.330 ± 0.064 California and Spanish cherimoya w 44.8 0.183 ± 0.044 Cultivated pawpaw (Isozymes) v 44.4 0.166 ± 0.048 Pawpaw wild accessions (Isozymes) u 43.5 0.172±0.013 Pawpaw wild accessions (RAPDs) t 64 0.249±0.022 Culitvated pawpaw (RAPDs) r -- 0.285±0.160 Cultivated pawpaw (ISSRs) 80 0.358±0.205
Dice, 1945
ISSR Summary Utilizing 10 of the ISSR markers with19 pawpaw cultivars revealed a moderate to high level of genetic diversity (P = 80; H e = 0.358) Diversity values were higher than those reported for cultivated pawpaw using isozyme and RAPD markers Additional and more reproducible ISSR markers will be needed to accurately fingerprint all cultivars and separate remaining genetic identities