Interpretive Summary: As sessile organisms, plants have evolved the ability to survive a myriad of stresses such as pests, pathogens, high salinity, drought, and temperature fluctuations. A challenge faced by today’s farmers is the means to deal with these stresses in a way that does not adversely affect production. Crop losses and / or increased production costs can lead to an imbalance in supply and demand, and directly affect the consumer in the form of lower quality products and increased prices. The development of crop plants that have the genetic means to overcome stresses with minimal outside intervention (i.e. – pesticides, herbicides, increased irrigation, etc.) is of great benefit to both the producer and the consumer. Minimizing the use of chemical treatments on a crop field is also less detrimental to neighboring ecosystems and the environment as a whole. Crop plant breeding programs attempt to maintain and increase genetic diversity of a crop plant so that the ability to survive adverse conditions is not lost in the cultivated varieties (cultivars) utilized by farmers. These traits can then be selected for and advanced into cultivars that have other desirable traits such as high yield and high quality. The difficulty in selecting for traits of interest lies in measuring a crop plant’s ability to resist or tolerant a specific stressor.
Molecular markers represent portions of deoxyribonucleic acid (DNA) that may be closely associated with, or linked, to a gene that enables the plant to successfully survive an environmental stress. In some cases, the molecular marker is the gene itself. At present, many types of molecular marker systems are utilized to identify DNA sequences that may prove useful in plant breeding programs, and facilitate selection of desirable traits. The use of molecular markers also minimizes the inadvertent selection of false positives. Here, we report the development of a new marker system that combines the ease and reliability of an existing marker system called amplified fragment length polymorphism (AFLP), with a selective process that identifies possible genes conferring plant disease resistance (R genes) or plant resistance gene analogues (RGAs). The new marker is therefore referred to as RGA-AFLP, and its usability was successfully tested using two species of cultivated cotton, Gossypium hirsutum L. and Gossypium barbadense L. Plants have an immune system composed of many R genes that are constantly replicating and evolving to recognize a diverse number of pathogens, signaling the plant to take the appropriate measures to ensure survival. Resistance genes are widely distributed in the DNA of all plants and RGA-AFLP represents an excellent tool to determine the location of various R genes in the plant genome. Linking R genes or R gene molecular markers to resistance traits will greatly enhance a plant breeder’s ability to combine disease resistance with other traits such as increased yield and higher quality.

Technical 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 families. The objective of this investigation was to test a resistance gene analogue (RGA) -anchored marker system, called RGA-AFLP in cotton. The RGA-AFLP 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 RGA-AFLP bands amplified by 22 RGA-AFLP primer combinations, 76 (17.0%) and 37 (8.3%) were polymorphic within four upland cotton (Gossypium hirsutum L.) genotypes and four Pima cotton (G. barbadense L.) genotypes, respectively. The number of polymorphic RGA-AFLP markers (256) at the interspecific level was much higher (57.4%). The polymorphism of RGA-AFLP was comparable with that of AFLP. The genetic similarity between the eight genotypes based on RGA-AFLP was highly correlated with that measured by AFLP, leading to similar results in genotype grouping at the species and intra-species level. However, resistance gene analogue polymorphism (RGAP) markers amplified by only degenerate RGA primers could not discriminate several genotypes. RGA-AFLP 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 RGA-AFLP analysis provides a useful resource for candidate gene mapping of R genes for disease resistances, and could also serve as chromosomal anchors for mapping of other traits.