Conclusions

Several remarkable features are evident from this study.First，a single restriction enzyme（SmaI）can uncover sufficient variation of ct-DNA among Nicotiana species to construct a sequence of its evolution.It is a simple procedure and does not need to radioactively label the fragments or a second enzyme digestion.Second，a sequential gain，as well as elimination，of the SmaI site is a unique feature of ct-DNA evolution in Nicotiana.Third，the great majority of mutations are clustered in one region to form a“hot spot”.Fourth，most of the variability is due to point mutation but deletion and inversion are also implicated.However，recombination has been suggested but only as a rare event（Kung et al.，1981）.Finally，there is a close correlation between the ct-DNA evolution and speciation in Nicotiana indicating a high degree of cooperation between organelle and nuclear genomes in evolution as well as in expression（Kung，1977）.

Nicotiana species studied here cover a wide range with respect to their biochemistry，cytogenetics，morphology and distribution.They include many key species having 12 pairs and 24 pairs of chromosomes indigenous to both America and Australia.Particular attention was focused on the ancestral linkage between those two continents as well as the parentage and descendant relationship during the course of ct-DNA evolution.Results obtained from the restriction fragment analysis of ct-DNA clearly demonstrate that the ancestor of N.noctiflora is the bridging species between the Western Hemisphere and Australian populations.(https://www.daowen.com)

Results presented here also demonstrate that restriction fragment analysis of ct-DNA is a powerful tool and can provide important information in the study of evolution that exceeds many other means available today（Brown et al.，1979）.This is demonstrated by the formulation of a sequence of ct-DNA evolution involving several key Nicotiana species（see Figure 4）.Moreover，by selecting proper restriction enzymes such as SmaI to treat several Nicotiana species a restriction map of ct-DNA can be constructed in a manner similar to using several enzymes to digest a single type of organelle DNA.