Author(s)

Hongshuo Liu, Yasuhiro Ito, Naho Muraida, Yuuka Hayakawa, Kyo Itoyama, Shuichi Ohsato, Wataru Marubashi

Graduate School of Agriculture, Meiji University, Kawasaki, Japan.

Monosomic lines of Nicotiana tabacum are helpful to confirm the location of genes on specific chromosomes. In the cross N. nudicaulis and N. tabacum, hybrid seedlings express lethal symptoms, which are controlled by the S subgenome of N. tabacum. To identify the responsible chromosome, we needed to produce chromosome lacking lines (CLLs) of N. tabacum L. “Red Russian” and use them to cross with N. nudicaulis. From a cross of (N. tabacum × N. tomentosiformis) × N. tabacum, 380 BC1 individuals were obtained. Using a Haplo-Q line (a monosomic line lacking the single linkage group 11) and N. tabacum, we found that qPCR is a simple and reliable screening method for CLLs of N. tabacum. The marker PT30342 is located on linkage group 11, and the -Ct value (Ct Actin - Ct PT30342) was 2.0 for a disomic line and was 1.097 for a Haplo-Q line. By the use of flow cytometry, qPCR and chromosome counting together as a screening method, we identified 6 CLLs lacking 2 to 6 chromosomes. Compared with conventional methods, our method is a rapid technique for making and screening CLLs ofthe S or S/T subgenome of N. tabacum. Further, these CLLs will be useful to identify the location of two or more factors on chromosomes controlling a variety of genetic problems affecting breeding. Here, we only made CLLs of the S or S/T subgenome of N. tabacum. We will use the method established in this study to produce CLLs of the T subgenome of N. tabacum, and gather a full set of CLLs of N. tabacum. qPCR could also be applied to the identification of chromosome aberrations in other plants.

Chromosome Lacking Lines, Quantitative Real-Time Polymerase Chain Reaction, Simple Sequence Repeat Marker, Flow Cytometry, Nicotiana tabacum L.

Liu, H. , Ito, Y. , Muraida, N. , Hayakawa, Y. , Itoyama, K. , Ohsato, S. and Marubashi, W. (2017) Development of a Method to Produce Chromosome Lacking Lines (CLLs) in Nicotiana tabacum L. “Red Russian”. American Journal of Plant Sciences, 8, 2923-2943. doi: 10.4236/ajps.2017.812198.