DNA fingerprinting and genetic diversity of Philippine papaya (Carica papaya L.) using Microsatellite markers

Microsatellites are an imporatnt class of RNA markers which occur frequently and randomly in all eukaryotic DNAs. These are also known as Simple Sequence Repeats (SSRs) because they are characterized as tandem repeats of DNA sequences of 1-6 bp in length. The variation in the number of tandemly repeated core sequence of nucleotides at an SSR locus results in highly polymorphic, multi-allelic markers that are useful in the genetic analyses of a number of organisms such as insects, birds, fish, mouse, cattle and plants. The DNA sequences flanking the SSRs are known to be highly conserved and can provide valuable information in designing SSR primers for the amplification of the SSR using Polymerase Chain Reaction (PCR). Using bioinformatics tools (in silico biology) such as Tandem Repeats Finder (TRF) and Vector NTI, four pairs of primers based on the DNA sequences of papayas were designed. Optimized PCR protocol of these markers yielded a total o f 18 polymorphic entries which were effective in identifying on fingerprinting different papaya genotypes and two wild relatives. The average number of alleles per locus were calculated to be 4.5 while the average polymorphism information content (PIC) of all these alleles is 0.532. Similarity coefficients based on Dice's formula were calculated and the composite of all markers have similarity values ranging from 0.364 to 0.957. Dendrograms were constructed using UPGMA-SAHN to study the genetic diversity of papaya. The composite or average dendrogram of the microsatellite markers showed that there were six major groupings at 85% similarity cut off. There were 26 haplotypes found in the average cluster analysis, and by using Nei's formula for unbiased gene diversity, it was calculated to be 0.5, and the domesticated papaya was clustered at 80% similarity. This value indicates that there is indeed genetic erosion in papaya which may be due to the domestication of the species. The SSR markers derived from this study can distinguish among Carica papaya varieties and other papaya relatives such as C. parviflora and C. quercifolia. In addition, these SSRs markers can be used in marker-assisted breeding program especially in interspecific hybridization.