Submitted to: Plant Cell Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/26/2008
Publication Date: 9/18/2008
Citation: Lu, Y.Z., Curtiss, J., Miranda, D., Hughs, S.E., Zhang, J. 2008. ATG-anchored AFLP (ATG-AFLP) analysis in cotton. Plant Cell Reports. 27:1645-1653. Interpretive Summary: This paper describes the modification and testing of an existing gene marker system called amplified fragment length polymorphism (AFLP) by the substitution of a restriction enzyme that recognizes specific gene sites containing ATG. The purpose of the substitution was to increase the AFLP system efficiency as a marker system for genes that are active in coding genetic traits. Two cotton species, upland (Gossypium hirsutum) and Pima (Gossypium barbadense) were used as test species. Results showed that the modified system was able to correctly separate and assign four genotypes into the appropriate two groups while operating as a high throughput marker system for an entire given genome. The modified AFLP system did the appropriate segregation with demonstrated increased efficiency over the original AFLP system.
Technical Abstract: Amplified fragment length polymorphism (AFLP) marker system has had broad applications in biology, genetics, breeding, evolution, and ecology due to its no requirement for prior sequence information, high reproducibility, robustness, and high throughput nature. However, the anonymous AFLP markers are mainly amplified from non-coding regions, limiting their usefulness as a functional marker system for identification of candidate genes underlying quantitative trait variations. To take advantages of the traditional AFLP techniques, we propose substitution of a restriction enzyme that recognizes a restriction site containing ATG, called ATG-anchored AFLP, i.e., ATG-AFLP analysis. In this study, we chose NsiI (recognizing ATGCAT) to replace EcoRI (recognizing GAATTC) in combination with MseI (recognizing TTAA) to completely digest genomic DNA. One specific adaptor, one pre-selective primer and six selective amplification primers for NsiI sites were designed for ligation and PCR. Six NsiI and 8 MseI primers generated a total of 1,780 ATG-AFLP fragments, of which 750 (42%, 15-16 per primer combination) were polymorphic among four genotypes from two cultivated cotton species (Upland cotton, Gossypium hirsutum and Pima cotton, G. barbadense). The number of ATG-AFLP markers was sufficient to separate the four genotypes into two groups, consistent with their evolutionary and breeding history. Our results also showed that ATG-AFLP generated less number of total and polymorphic fragments per primer combination (2-3 vs. 4-5) than conventional AFLP within Upland cotton. Using a recombination inbred line (RIL) population, a total of 62 polymorphic ATG-AFLP markers amplified with 8 primer combinations were mapped to 19 linkage groups with known chromosome anchored simple sequence repeat (SSR) markers. Of the 9 ATG-AFLP fragments cloned and sequenced, 3 were found to be highly homologous to cotton cDNA sequences and 1 to a hypothetical protein, indicating almost half (44.4%) of the ATG-AFLP markers were amplified from gene regions. An in-sillico analysis of cotton EST unigenes confirmed that the ATG-anchored enzyme combination NsiI/MseI did release more cDNA fragments than the EcoRI/MseI combination. Our results demonstrate that a restriction enzyme with recognition sequence containing ATG in combination with a traditional enzyme used in AFLP can be used as a high throughput marker system to search for genome-wide functional markers.