Submitted to: Sugar Tech
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/28/2007
Publication Date: 8/9/2007
Citation: Pan, Y.-B., Scheffler, B.S., Richard, Jr., E.P. 2007. High-throughput genotyping of commercial sugarcane clones with microsatellite (SSR) DNA markers. Sugar Tech. 9:176-181. Interpretive Summary: Traditionally, sugarcane breeders insure the identity of the varieties being used in their breeding programs by visually inspecting them for basic anatomical and morphological characteristics. The climatic conditions where these varieties are being grown may often influence some of these characteristics and different varieties, in particular those from the same geographical region, may appear to be identical. Hence, variety mis-identification can be a problem when these varieties are exchanged among the different breeding stations. By adopting a high throughput leaf tissue DNA extraction procedure, a liquid handling robot, and 384-well sample plates, we have demonstrated that DNA fingerprints of sugarcane varieties can be quickly produced that are genetically stable and not influenced by the environments from which the DNA was obtained. We were able to produce molecular fingerprints from 21 different DNA markers for 116 Louisiana sugarcane varieties; some with multiple samples collected from two, three, or four different locations. A total of 184 DNA fingerprints were established and stored in our molecular genotyping database. An alignment of these DNA fingerprints has revealed that almost all of the Louisiana varieties currently being used in the US’s sugarcane breeding programs share a greater than 70% genetic similarity. We also identified ten varieties in these breeding programs that were mis-identified and eliminated them before they were used in the crossing program. By using this high throughput DNA fingerprinting procedure, the U.S. sugarcane breeders are now able to insure the true genetic identity of their parental varieties before utilizing them in their crossing programs. This will ultimately insure that progeny produced are from crosses designed with the highest probability of producing superior individuals.
Technical Abstract: To ensure the identity of Louisiana sugarcane parental clones, we genotyped 116 clones with 21 microsatellite (SSR) DNA markers. A total of 184 leaf samples were collected from four locations (C, H, L, and Q), including 20 from five quadric clones at four locations, 30 from ten triplicate clones at three locations, 66 from 33 duplicate clones at two locations, and 68 stand-alone clones at one location. Leaf DNA was extracted using a high throughput (HT) procedure with a daily output of 576 leaf DNA samples per person. Using 384-well reaction plates and a liquid-handling station, the HT-DNA samples were subjected to PCR, and fluorescence-labeled PCR products were fractionated through capillary electrophoresis (CE) yielding raw data of nine SSR markers for the 184 sugarcane samples per day for genotyping analysis. A total of 144 distinctive SSR alleles were scored. The presence or absence of these SSR alleles in a sugarcane clone was recorded into a DNA sequence of A’s (presence) and C’s (absence) to represent its SSR genotype. Alignment of the 184 SSR genotypes produced homology and phylogenetic trees that revealed the following information: 1) Except for three clones, all Louisiana commercial clones shared a 70% or more genetic similarity; 2) One quadric clone of Ho 95-988 had a SSR genotype not matching with any of the other clones; 3) One triplicate clone of L 03-374 had a different SSR genotype; 4) The SSR genotypes of the duplicate clones of Ho 02-653, HoCP 02-652, HoCP 03-703, L 03-364, and TucCP 77-42 did not match; 5) At Location C, HoCP 85-845 was mis-labeled as TucCP 77-42; 6) At Location H, HoCP 02-639 had the same SSR genotype as that of LCP 85-384, and 7) At Location L, L 92-312 had the same SSR genotype as that of L 99-321. The LA clones that showed different genetic identities from the original locations were considered mis-labeled and removed before the crossing season. The SSR genotypes are stored in the laboratory’s local database and are being used to verify clone identity, assess the fidelity of crosses, and determine the paternity of polycrosses. The HT-SSR genotyping procedure should have general utility in other sugarcane breeding programs.