Results and Discussion

Results and Discussion

Nicotiana ct-DNAs exhibit distinct common features，as well as，individual variables in their EcoRI restriction patterns（see Figure 1）.There are five major regions of restriction fragments plus a few minor bands in region 6，as arranged in order of decreasing molecular weight in Figure 1（a）.Each region contains both constant and variable fragments.The frequency of changes and ratio of constant to variable fragments in each region varies from species to species［see Figure 1（a）］.Therefore，the overall ct-DNA restriction pattern in Nicotiana is generally species specific.However，within the species N.tabacum，the normal cultivars possess identical patterns［see Figure 1（b）］.This identity was confirmed by using several restriction enzymes（BamHI，SmaI，HindⅢ，SalI，and EcoRI）in single or double digestion experiments.No variation in restriction patterns of ct-DNA occurs among any of the seven commercial cultivars，Turkish Samsun，Burley 21，NC 95，Ky 14，Md 609，John Williams Broadleaf（JWB），and JWB yellow mutant，analyzed so far.Figure 2 is a densitometer tracing of a typical EcoRI restriction pattern of N.tabacum ct-DNA.There are about 40 identifiable bands，many which are doublets or triplets.Bands 8，9，12，16，17，22，27，29，33，and 39 are bimolar whereas bands 4，15，and 18 are possibly trimolar.The molecular weights of the EcoRI fragments range from 4.96 to 0.48×106 daltons.This pattern is highly reproducible and the corresponding band numbers will be referred to repeatedly throughout this paper.

Chloroplast DNAs from CMS lines of N.tabacum constitute the exceptions to this pattern.They either have an altered ct-DNA pattern differing from both parents，or are identical to the female parent of the original cross as expected from the mode of maternal inheritance in Nicotiana（Kung，1976）.Figure 3 represents an EcoRI SalI and SmaI restriction patterns of ct-DNAs prepared from a CMS line and its parents.This CMS line was derived from the alloplasmic combination between N.plumbaginifolia cytoplasm and N.tabacum chromosomes.The overall patterns of ct-DNA from both parents are similar but there are some noticeable differences.In comparison with N.tabacum the ct-DNA fragment 9 of N.plumbaginifolia is obviously reduced from a doublet to a single band［compare the bracketed regions of lanes 1 and 3 in Figure 3（a）］.The position of fragments 13，14，16，18，19 and 24 have shifted slightly.Also，a new fragment is present between fragments 16 and 17 and the intensities or fragments 28 and 29 are reversed［see Figure 3（a），lane 3］.In comparison to the patterns of its parents the EcoRI restriction pattern of ct-DNA of the CMS line is strikingly different［see Figure 3（a），lane 2］.① It has three unique fragments not present in either parent（marked with white dots）；② fragments 6 and 7 common to both parents have disappeared；③ there are five fragments which are similar to the male parent（marked with black dots）；④ there are five fragments similar to the female parent（marked with arrows）.The presence of new EcoRI fragments and fragments characteristic to both parents in this CMS line indicate recombination or other alterations may have occurred between or within the parental ct-DNAs.

This suggestion is supported by the restriction fragment pattern generated by SmaI［see Figure 3（b）］.When ct-DNA from N.tabacum，N.plumbaginifolia，and the CMS line are treated with SmaI they exhibit distinct differences.N.tabacum has at least 12 SmaI fragments（a—l）similar to that determined by Jurgenson and Bourque（1980）and Jurgenson（1980）.According to Jurgeson's numbering scheme the fragments d，f，h，i，j，and l are doublets［see Figure 3（b），lane 6］.In Figure 3（b）fragment f is resolved as two single bands.N.plumbaginifolia differs from N.tabacum by the presence of bands b'and d'and the absence of bands e，f，g，and a copy of j［see Figure 3（b），lane 4］.As in the case of the EcoRI fragment pattem，the ct-DNA of the CMS line possesses fragments common to both parents.However，the size of fragment e is reduced as compared to that in N.tabacum and there is only a single copy of j as in N.plumbaginifolia［see Figure 3（b），lane 5］.Based on the fragment sizes determined by Rhodes and Kung（1981）the combined molecular weight of bands e，f，g，and j（19.3×10-6）is equivilent to that of bands b'and d'.Therefore，it can be deduced that the ct-DNA of the CMS line may represent a recombined molecule.This assumption is supported by the restriction map of N.tabacum constructed on the basis of SmaI and SalI fragments in which the fragments e，f，g，and j are sequentially arranged adjacent to one of the inverted repeats on the chloroplast genome（Jurgenson and Bourque，1980；Jurgenson，1980）.On the basis of the serial order of fragments e，f，g，and j of Jurgenson（1980），the fragment sizes of Rhodes and Kung（1981），and the evidence provided in this study，the possible site of recombination is proposed to be between SmaI fragments f and g of N.tabacum and b'and d'of N.plumbaginifolia.This proposed site of recombination can account for the observed fragment pattern of the CMS line ct-DNA which contains fragments e and f of the male and fragment d'of the female parents［see Figure 3（b），lane 4］.No CMS lines containing the b'of the female and fragment g of male parent was detected since only one type can survive in a cell（Eberhard，1980）.It should also be noted that no alteration of SalI fragments is observed［see Figure 3（b），lane 1—lane 3］.(https://www.daowen.com)

The second case of ct-DNA alteration was detected from a CMS line originating from a spontaneous，cytoplasmic mutation（Berbec，1974）.As can be noted from Figure 4（a）（compare lane 1 with lane 3）in the altered form the relative proportion of fragments 8 and 9，and 10 and 11（bracket 1）are reversed.Fragments 14 and 21（brackets 2 and 3）are reduced in size and a new fragment，17'（bracket 3），is present.Here，the ct-DNA of N.tabacum［see Figure 4（a），lane 3］was altered to another type resembling N.glauca［see Figure 4（a），lane 2］.

A third case is ct-DNA from a CMS line derived from an interspecific cross between N.glauca and N.tabacum.In this case the EcoRI fragments of ct-DNA of this CMS line follow the expected mode of maternal inheritance.They are identical to those of N.glauca.However，when this was further examined by BamHI enzyme，a single extra fragment（marked with dot in Figure 4（b），lane 2）appeared in the CMS line.

Two other CMS lines showed no alteration of ct-DNA.One is the direct interspecific cross of N.repanda and N.tabacum［see Figure 4（c）］.This was indeed a rare cross since this was the only reported success in the literature（Pittarelli and Stavely，1975）.The analysis of both RuBPcase（data not shown）and ct-DNA confirm the identity of this cross.The other case involves a cross between N.fragrans and N.tabacum（data not shown）.Both cases simply follow the mode of maternal inheritance.