Development of AFLP molecular markers for cultivar identification of cured tobacco leaves

Abstract

Tobacco cultivars grown in Thailand are separated to 2 major groups: local and imported cultivars. However, current approaches to distinguish tobacco cultivars are not accurate enough, particularly for dry-cured tobacco. In this study AFLP molecular marker was introduced to investigate genetic differences between cured leaf-materials of 23 tobacco cultivars. The AFLP result showed that three primer-pairs (E[subscript AAG] / M[subscript CAA], E[subscript AAG] / M[subscript CGC] and E[subscript ACT] / M[subscript CAG]) gave a total of 139 AFLP fragments, of which 103 (74.1%) were polymorphic bands. Among these primers, E[subscript ACT] / M[subscript CAG] primer gave both highest number of AFLP-PCR fragments (59 bands) and polymorphic bands (83.1%). Furthermore, this primer-combination produced one cultivar-specific band of Virginia cultivars. This cultivar-specific marker would be useful in identification and confirmation of Virginia genotype in tobacco trade. Genetic relationship trees based on NJ and UPGMA techniques revealed clusterings similar to each other. Almost all local cultivars were closely related with each other and also with Burley and Turkish imported cultivars. The supgrouping of local cultivars tended to form following their cultivating regions. Among the imported cultivars, their clusterings were not only based on cultivating area but also on the imported cultivar groups (Virginia, Burley and Turkish). Virginia cultivars were clustered separately from other local and imported cultivars. In conclusion, this AFLP marker technique could separate Virginia cultivars from other cultivars grown in Thailand. And these results could be a fundamental information for other tobacco genetic researches in the future