Molecular markers for determining tobacco Nicotiana tabacum L. varieties

Abstract

In Thailand, imported and local tobacco varieties have different regulations in tariff collection. However, there are major legal and technical problems on how to distinguish between the two groups of tobacco varieties. In this study, Inter-Simple Sequence Repeat (ISSR) method was introduced to study genetic relationships among totally 66 imported and local tobacco varieties grown in Thailand. Of 20 screened primers, UBC-810, UBC-813, UBC-817, and UBC-818 primers were found giving polymorphic PCR patterns and were selected to determine genetic relationships among 24 imported varieties. Although the relationships among them were not so clear from the ISSR results, most of Turkish varieties were found more closely related to each other than to Burley or Virginia varieties. Moreover, the two best ISSR primers (UBC-807 and UBC-836) generated specific PCR bands which could separate two local tobacco varieties (Chorlare 1 and Chorlare 2) from 53 imported varieties. PCR optimisations were also performed to increase PCR specificity. Genetic relationship trees based on NJ and UPGMA methods revealed only four pairs of tobacco varieties which had Bootstrap supporting-values higher than 50%, i.e. Chorlare 1 and Chorlare 2, Nison and Petkhangsink, Ya and Local Nakhon Si Thammarat, and NC37NF (Virginia) and Ky-10 (Burley). Among all 13 local tobacco varieties collected and examined, only the pair of Chorlare 1 and 2 was distantly separated from other imported varieties, suggesting their long-history of being grown in Thailand. The other 11 local varieties were found morphologically and genetically similar to some imported varieties and could have been originated from such varieties. In conclusion, although ISSR technique could not give clear relationships between tobacco varieties in this study, it would be developed to be a simple, effective molecular marker to distinguish some long-time cultivated local varieties from the imported ones.