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Title: Highly polymorphic microsatellite DNA markers for sugarcane germplasm evaluation and variety identity testing

item Pan, Yong-Bao

Submitted to: Sugar Tech
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/31/2006
Publication Date: 12/3/2006
Citation: Pan, Y.-B. 2006. Highly polymorphic microsatellite DNA markers for sugarcane germplasm evaluation and variety identity testing. Sugar Tech. 8(4):246-256.

Interpretive Summary: Based on the DNA sequences of an Australian sugarcane variety Q124 and a French sugarcane variety R570, the International Sugarcane Microsatellite Consortium (ICSB) developed 221 sugarcane microsatellite (SSR) DNA markers in the late 1990s. The general utility of these SSR DNA markers, however, on sugarcane cultivars grown in other geographical areas has never been fully tested thereby limiting the usage of these markers in world sugarcane community. This study was set up to test how these 221 SSR DNA markers perform on five popular U.S. sugarcane varieties using a semi-automatic capillary gel electrophoresis system. The system can detect tiny amounts of DNA products that differ by as little as one base pair unit. As was expected, only 67 (30%) of the 221 SSR DNA markers tested were able to produce detectable amounts of DNA products from the U.S. varieties and the size of these DNA products ranged from 80 to 460 base pairs. There were 467 DNA products in total, of which only 117 were commonly produced by all five varieties. The remaining 350 DNA products were not found to similar extents in the five varieties suggesting that patterns created by the presence or absence of these DNA products could be used to uniquely describe each variety. The frequency of each unique DNA banding pattern among the five varieties produced by any particular SSR marker, which is define as the marker’s polymorphism information content (PIC) value, ranged from 56% to 80%. The results from this study should provide useful information for the sugarcane breeders and geneticists all over the world to promote their research in the areas of sugarcane germplasm molecular evaluation and variety genetic identity testing.

Technical Abstract: The objective of this study was to evaluate the entire set of 221 sugarcane microsatellite (SSR) markers from the International Sugarcane Microsatellite Consortium for their utility on molecular characterization of elite U.S. germplasm. Five elite U.S. sugarcane clones were tested, including two cultivars LCP 85-384 and HoCP 96-540 from Louisiana, two cultivars CP 72-1210 and CP 85-1308 from Florida, and Green German, an active parental clone used in both states. The 5’ ends of all forward primers were labeled with the fluorescent phosphoramidite dye, FAM and PCR-amplified DNA fragments detected using a semi-automatic capillary electrophoresis system. The sizes of DNA fragments were computed accurately by running genotyping software calibrated against 16 fluorescence-labeled DNA size standard fragments. Evaluation criteria included PCR robustness, extent of the presence of irregular peaks, and the polymorphism information content (PIC). Sixty-seven SSR markers (30% of the total) were found to be highly robust, with PIC values ranging from 56% to 80%. Of these, 40 (60%) markers contained dinucleotide repeats, 11 (16%) markers contained trinucleotide repeats, and 16 (24%) markers contained composite repeats. A total of 467 alleles were scored, of which 350 were polymorphic, averaging five polymorphic alleles per marker, with their sizes ranging from 80 to 460 bp. Several of these highly polymorphic SSR markers have proven useful in sugarcane germplasm evaluation, variety identity tests, cross fidelity assessment, and polycross paternity analysis.