Results
The HindⅢfragment patterns of ct-DNA from distantly related plants are presented in Figure 1 to illustrate that there was little resemblance between different classes（monocot and dicot），subclasses（spinach and pumpkin）or orders（tobacco and pumpkin）.Within families（tobacco and tomato；not shown）；however，some similarities began to appear.This variability among ct-DNA restriction patterns is apparent with all restriction enzymes used even when relatively few fragments are generated.
Figure 1 Restriction fragment patterns of ct-DNA from various plants generated by digestion with HindⅢ.
1—corn；2—spinach；3—tobacco；4—pumpkin.Fragments were separated by electrophoresis for 18 h in 1%agarose gels
In contrast to the heterogenetiy of ct-DNA restriction fragments observed among the more distantly related plants of Figure 1，the ct-DNA prepared from over 20 species of Nicotiona exhibit remarkable similartry wth all restriction enzymes used thus far.When relatively few fragments of ct-DNA are generated，many Nicotiana species display very similar patterns（see Figure 2）.The molecular weights summed from fragments generated by Smal or Xhol are shown in Table 1 and are in close agreement with the reported value of 9.6×107daltons for N.tabacum ct-DNA（Jurgenson and Bourque，1980）.Considerably greater variation among ct-DNA restriction patterns is obtained with EcoRI which generates more than 40 distinct fragments（see Figure 3）.Even though the identifiable EcoRI fragments account for only 63%to 72%of the chloroplast genome（see Table 1），the greater frequency of its restriction site increases significantly the fraction of base pairs sampled and the likelihood of observing ct-DNA differences.Thus，EcoRI was chosen for routine analyses of ct-DNA.
Figure 2 Restriction fragment pattern of ct-DNA from differen tNicotian aspecies generated by digestion with Xhol（1—3）and Smal（4—6）.1 and 4—N.otophora；2 and 5—N.glauca；3 and 6—N.excelsior；7—SmaI fragments of lambda DNA and 8—EcoRI fragments of lambda DNA.Fragments were separated by electrophoresis for 18 h in 0.8%agarose gels
Figure 3 Restriction fragment patterns of ct-DNA from six species of Nicotiana generated by digestion with EcoRI.1—N.excelsior；2—N.glauca；3—N.gossei；4—N.otophora；5—N.tomentosiformis；6—N.paniculata and 7—EcoRI and HindⅢdouble digest of lambda DNA.Fragments were separated by electrophoresis for 42 h in 1.5%agarose gels
Table 1 Molecular weight of chloroplast DNA from several Nicotiana species determined from single and double enzyme digests of ct-DNA.Lambda DNA fragments produced by Smal，Xhol，BamHI，and EcoRI were used as molecular weight markers.In order to separate the larger fragments for accurate molecular weight determination，photographs were taken of the 0.8%agarose gels arter 20，40，and 60 h of electrophoresis
Figure 4 Microdensitometer tracing of restriction fragment pattern of ct-DNA from N.tomentosiformis generated by EcoRI.The fragments were separated in a 1.5%agarose gel slab at 1.25 V/cm for 42 h
Many or the well-separated EcoRI bands are common to all of these Nicotiana specics；however，certain fragments，most obviously those larger than 2 megadaltons are quite variable（see Figure 3）.Figure 4 illustrates a densitometer tracing of the EcoRI fragment pattern of N.tomentosiformis ct-DNA.Most or these restriction fragments（1，2，3，5，10，13，and 14 for instance）are found in ct-DNA from every Nicotiana species.Amone those conserved fragments.EcoRI fragments 5 and 10 have recently been reported to contain the ribosomal RNA genes（Sugiura and Kusuda，1979）which have undergone little divergence during evolution（Thomas and Tewari，1974）.Those fragments which are not common to all Nicotiana species are listed in Table 2 along with variable EcoRI fragments from several other species.A major portion of these fragments may be considered moderately conserved since they occur in species from several ct-DNA groups（fragments 7 or 9，for example）.Others（such as fragments 3a，6 or 8）occur in only a few of the species examined.Based upon these data，ct-DNA fragments totaling 15 to 25 mcegadaltons can vary among Nicotiana species and，or these，at least 13.5 megadalions are frequently present in species from several major ct-DNA groups.Thus much or the Nicotiana chloroplast genome is apparently highly conserved.(https://www.daowen.com)
The distribution of variable EcoRI fragments（see Figure 3）was used as a basis for organizing these Nicotiana species into groups.Similar groupings were also obtained from analyses of BamHI fragments（see Figure 5（a））which，like the EcoRI ct-DNA groups，could in turn be associated with the isoelectric focusing patterns of the large subunit of RuBPCasc（Chen et al.，1976）.As can be seen from Figure 3 and Table 2.EcoRI fragments 6，8，and 12 are unique to RuBPCase group A（see Table 3）.This group includes all Nicotiana species indigenous to Australia.Although some diversity in fragment pattern among these species exists in this groups Ⅱ were designated as ct-DNA group I.The ct-DNA groups Ⅱ and Ⅲ，which are analogous to RuBPCase groups B and C（see Table 3），have nearly all of the moderately conserved fragments which occur among the various ct-DNA groups.Even though groups Ⅱ and Ⅲ are very similar，separate group assignments were maintained because of the remarkable uniformity of restriction patterns within group Ⅲ.For example，all species in group Ⅲ have identical isoelectric focusing of the large subunit of RuBPCase and appear to have identical ct-DNA（see Figure 5（b））except for N.langsdorffii.This species exhibited minor differences in intensity and band position of EcoRI fragments ranging in molecular weight from 0.7 to 1.25×106 daltons.
Table 2 Size classes of EcoRI fragments which vary among Nicotiana species（expressed in megadaltons）.Different molecular weights for the same fragment number represent real variations in fragment size for which separate identification numbers have not yet been assigned.Lambda DNA fragments produced by single and double digestion with EcoRI and HindⅢ were used for molecular weight determinaiions of fragments separated by electrophoresis for 42 h in 1.5%agarose slab gels
Figure 5 An association of the isoelectric focusing patterns of RuBPCase large subunits（top）with BamHI（a）or EcoRI（b）restriction fragment patterns of ct-DNA from different species of Nicotiana（bottom）.
（a）1—EcoRI and HindⅢ double digest of lambda DNA；2—N.gossei；3—N.glauca；4—N.tabacum；5—N otophora.（b）1—N.tabacum；2—N.sylvestris；3—N.Iangsdorffii；4—N.longtflora.The variable EcoRI fragment region in（b）is marked with brace.The restriction fragments were separated by electrophoresis for 42 h in a 1.5%agarose gel slab
Unlike members of the other ct-DNA groups，the six species examined in RuBPCase group D present a more complex pattern of ct-DNA variation.According to EcoRI fragment patterns，these species form three subgroups：（IVa）N.setchellii，N tomentosa and N.tomentosiformis；（IVb）N.otophora；（IVc）N.knightiana and N.paniculata.Based upon the data in Table 2 N.otophora，which shares fragments 4，7，15，and 16 with group IVa and fragments 9，11，lla，15，16，and 18 with group IVc，appears to be intermediate between these two groups.
