Identification of AFLP markers linked to tomato spotted wilt virus resistance in tobacco

Abstract

Tomato spotted wilt virus (TSWV) is a serious disease in several crops such as tobacco (Nicotiana tabacum.), peanut (Arachis hypogaea.), tomato (Lycopersicon esculentum.), and pepper (Capsicum annuum.). One source of resistance in tobacco is the breeding line Polalta, which contains a TSWV resistance gene introgressed from the wild relative Nicotiana alata. However, the resistance is associated with abnormal plant morphology and traditional backcrossing has been ineffective in producing normal plants with TSWV resistance. Molecular marker-assisted backcrossing allows for rapid identification of the plants that are most genetically similar to the recurrent parent and can be used to reduce the size of an introgressed chromosome segment. We applied AFLP (amplified fragment length polymorphism) technology and bulk segregant analysis to identify markers linked to TSWV resistance. One TSWV resistant bulk and two susceptible bulks were constructed by combining DNA from 5 doubled haploid lines from the cross K326 x Polalta for each bulk. A total of 128 primer combinations were used to screen one resistant and two susceptible bulks, and I found 48 potential markers linked to the TSWV resistance. Thirty two markers were selected for further study based on their reproducibility. A population of 88 F2 plants and 23 doubled haploid lines were screened with 32 markers and a 2.5 cM map with 24 markers was constructed. Eleven AFLP fragments between 100 to 400 bp in size linked in coupling phase to TSWV resistance were isolated and sequenced to develop PCR based markers. Four AFLP fragments were successfully converted to sequence characterized amplified region (SCAR) markers.

An F2BC2 population of 160 plants was screened with 5 AFLP coupling phase markers to select resistant plants that have fewer Polalta-derived markers and more K326-derived markers. No resistant line was identified with a reduced introgression from N. alata. In an F2BC3 population of about 200 plants, 8 plants were selected based on an improved phenotype and screened with the 17 AFLP coupling phase markers. Four plants with a reduced introgression were identified and will form the basis for future backcrossing. This approach is expected to facilitate development of a line with TSWV resistance and a normal phenotype.