Detection of Tobacco mosaic tobamovirus resistance gene (N-gene) in tobacco genotypes using gene specific markers.

Abstract

Tobacco mosaic tobamovirus (TMV) is one of the most destructive viral diseases that threaten worldwide tobacco production. Use of host resistance appears to be the best way to control the disease. The use of specific primers is important and useful in the selection of the TMV resistant gene. The objective of the study was to screen specific primers involved in detecting resistance gene against TMV in both flue cured and dark fire cured tobacco. For this screening, four specific primers namely N1/N2, AS1/AS2, E1/E2 and SD1/SD2 primers were used to detect the resistance gene in flue cured and dark fire flue cured tobacco. Di-oxy ribonucleic acid (DNA) was extracted from young leaves of tobacco plants and quantified by spectrophotometer. A CHL test was carried out to determine DNA quality. Polymerase chain reaction (PCR) reaction mix and amplifying conditions for the four specific primer pairs were optimised to detect TMV resistant gene in tobacco samples. Results showed that out of four sets of primers, two primers N1/N2 and E1/E2 detected the TMV resistant gene in flue cured and dark fire cured tobacco. The two selected specific PCR based primers for TMV resistance were validated in dark fire cured tobacco in backcross one (BC1) and worked. Primers AS1/AS2 and SD1/SD2 did not produce expected band products, therefore, they failed to detect the N gene. The PCR assay is a cost effective, quick diagnostic technique, which is helpful in screening varieties which are resistant to TMV in tobacco breeding. Identification of specific primers linked to the N gene will be a useful tool in plant breeding for TMV resistance gene screening, evaluation and confirmation of resistance in large host plant populations in tobacco.