AFLP-RGA Markers in Comparison with RGA and AFLP in Cultivated Tetraploid Cotton Jinfa Zhang,* Youlu Yuan, Chen Niu, Doug J. Hinchliffe, Yingzhi Lu, Shuxun Yu, Richard G. Percy, Mauricio Ulloa, and Roy G. Cantrell ABSTRACT Disease resistance (R) genes have been isolated from many plant species and R genes with domains of nucleotide binding sites (NBS) and leucine-rich repeats (LRR) represent the largest R gene family. The objective of this investigation was to test a resistance gene analog (RGA) anchored marker system, called amplified fragment length polymorphism (AFLP)-RGA in cotton (Gossypium spp.). The AFLP- RGA analysis uses one degenerate RGA primer designed from various NBS and LRR domains of R genes in combination with one selective AFLP primer in a PCR reaction. Out of a total of 446 AFLP- RGA bands amplified by 22 AFLP-RGA primer combinations, 76 (17.0%) and 37 (8.3%) were polymorphic within four G. hirsutum L. genotypes and four G. barbadense L. cotton genotypes, respec- tively. The number of polymorphic AFLP-RGA bands (256) between G. hirsutum and G. barbadense was much higher (57.4%). This level of polymorphism mirrors that of AFLP. The genetic similarity among the eight genotypes based on AFLP-RGA or AFLP lead to similar results in genotype grouping at the species and intraspecies level. However, RGA markers amplified by only degenerate RGA primers could not discriminate several genotypes. AFLP-RGA offers a great flexibility for numerous primer combinations in a genome-wide search for RGAs. Due to the distribution of RGAs or RGA clusters in the plant genome, genome-wide AFLP-RGA analysis provides a useful resource for candidate gene mapping of R genes for disease resistance in cotton. W ITH A BETTER understanding of the general genome structures of higher organisms, primers derived from simple sequence repeats (SSRs), conserved re- gions of transposons, or retrotransposons were used in combination with random or AFLP primers to de- velop a number of modified marker systems such as retrotransposon–microsatellite amplified polymorphism, inter-retrotransposon amplified polymorphism, sequence specific amplification polymorphism, random amplified microsatellite polymorphism (RAMP)/digested RAMP, selective amplification of microsatellite polymorphic loci, and microsatellite-AFLP (Weising et al., 2005). Most of these markers represent random samples of the genome and have been used in various areas including genetic di- versity, germplasm fingerprinting, linkage and quantitative trait locus (QTL) mapping, gene isolation, and marker- assisted selection in breeding. However, in the quest for genes responsible for evolutionary traits and plant phe- notypes, functional markers from transcribed regions of the genome have recently gained more attention. Sequence-related amplified polymorphism (SRAP) (Li and Quiros, 2001) and targeted region amplified polymorphism (TRAP) (Hu and Vick, 2003) were two recent attempts to target gene regions in a high-through- put fashion. Many sequence-tagged site (STS), cleaved amplified polymorphism, single nucleotide polymor- phism, and SSR markers have also been developed from genes or expressed sequence tags in many species. In a technique recently designated single feature poly- morphism (Borevitz et al., 2003), portions of gene sequences have been used as oligonucleotides for mi- croarray hybridizations with labeled genomic DNA to simultaneously reveal genomic variations in thousands of genes. However, one of the prerequisites for single feature polymorphism is the availability of gene chips for the species of interest. Of the many disease resistance (R) genes isolated in numerous plant species, R genes with domains of NBS and LRR represent the largest R gene family (Martin et al., 2003). Recent genome analyses identified approxi- mately 150 and 500 NBS-LRR genes in Arabidopsis (Meyers et al., 2003) and rice (Oryza sativa L.) (Monosi et al., 2004), respectively. The conserved NBS domain comprising the P loop, the kinase-2 motif, and the GLPL motif has enabled the isolation of disease resistance analogs (RGAs) from numerous plant species (reviewed in Martin et al., 2003). Genetic diversity of the RGAs in relation to their origin, evolution, and germplasm diversity have been extensively investigated (Leister et al., 1996; Kanazin et al., 1996; Yu et al., 1996; Chen et al., 1998; Collins et al., 1998; Mago et al., 1999; Grube et al., 2000; Pan and Wendel, 2001; Graham et al., 2002; Rossi et al., 2003; Trognitz and Trognitz, 2005). The genetic and physical mapping of RGAs and their expression and relationships with R genes and QTL have been reported in a number of plant species (Fourmann et al., 2001; Huettel et al., 2002; Penuela et al., 2002; Quint et al., 2003; Di Gaspero and Cipriani, 2003; Hunger et al., 2003; Liu and Ekramoddoullah, 2003; Radwan et al., 2004; J.F. Zhang, C. Niu, and Y.Z. Lu, Dep. of Plant and Environ. Sci., Box 30003, New Mexico State Univ., Las Cruces, NM 88003; Y.L. Yuan and S.X. Yu, Key Laboratory of Cotton Genetic Improvement, Ministry of Agriculture, China Cotton Research Institute, Anyang, Henan, China; D.J. Hinchliffe, USDA-ARS, Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, LA 70124; R.G. Percy, U.S. Arid- Land Agricultural Research Center, USDA-ARS, 21881 N. Cardon Lane, Maricopa, AZ 85239; M. Ulloa, WCIS Res. Unit, Cotton En- hancement Program, USDA, Shafter, CA 93262; R.G. Cantrell, Cotton Inc., Cary, NC 27513. Received 15 Apr. 2006. *Corresponding author ( jinzhang@nmsu.edu). Published in Crop Sci. 47:180–187 (2007). Crop Breeding & Genetics doi:10.2135/cropsci2006.04.0249 ª Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: AFLP, amplified fragment length polymorphism; GS, genetic similarities; LRR, leucine-rich repeats; NBS, nucleotide binding sites; RGA, resistance gene analog; QTL, quantitative trait locus; SRAP, sequence-related amplified polymorphism; SSRs, simple sequence repeats; STS, sequence tagged site; TRAP, targeted region amplified polymorphism. Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved. 180 Published online January 22, 2007