ATG-anchored AFLP (ATG-AFLP) analysis in cotton.
Amplified fragment length polymorphism (AFLP) marker system has had broad applications in biology. However, the anonymous AFLP markers are mainly amplified from non-coding regions, limiting their usefulness as a functional marker system. 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 (ATG-AFLP) analysis. In this study, we chose NsiI (recognizing ATGCAT) to replace EcoRI in combination with MseI to completely digest genomic DNA. One specific adaptor, one pre-selective primer and six selective amplification primers for the NsiI site were designed for ligation and PCR. Six NsiI and eight MseI primers generated a total of 1,780 ATG-AFLP fragments, of which 750 (42%) 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, 62 polymorphic ATG-AFLP markers were mapped to 19 linkage groups with known chromosome anchored simple sequence repeat (SSR) markers. Of the nine ATG-AFLP fragments randomly chosen, three were found to be highly homologous to cotton cDNA sequences. An in-silico analysis of cotton and Arabidopsis cDNA confirmed that the ATG-anchored enzyme combination NsiI/MseI did generate more fragments than the EcoRI/MseI combination.
Lu
Yingzhi
Y
Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM, 88003, USA.
Curtiss
Jessica
J
Miranda
Danielle
D
Hughs
Ed
E
Zhang
Jinfa
J
D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D001483:Base Sequence; D002874:Chromosome Mapping; D020090:Chromosome Segregation; D003001:Cloning, Molecular; D016000:Cluster Analysis; D019295:Computational Biology; D004262:DNA Restriction Enzymes / Q000378:metabolism; D020224:Expressed Sequence Tags; D005819:Genetic Markers; D005838:Genotype; D003368:Gossypium / Q000235:genetics*; D007178:Inbreeding; D011110:Polymorphism, Genetic; D011123:Polyploidy; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D005819:Genetic Markers; D004262:DNA Restriction Enzymes