Genetic Variability in <i>Nicotianatabacum</i> and <i>Nicotiana</i> Species as Revealed by RAPD Markers: 1. Development of the RAPD Procedure


762 (*) Perustitza IST P 1E Bright IST G 19 (*) Havanna IST T 720 (*) Samsun Katerini 2/5 (*) 

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Figure 1 .

 1 Figure 1. Amplification patterns obtained with primer OPA01 of N. tabacum T 1116 line DNA extracted with four different methods; A = CTAB method, B = Edwards method, C = Nucleon phytopure system, D = Goring method modified. Three different DNA preparations were amplified for each method. Molecular weight (Kbp) markers are as indicated. 




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Figure 2 .

 2 Figure 2. Effect of different amount of DNA template on amplification pattern. The amplifications were performed with primer OPA01 (a) and primer OPA13 (b) in a reaction volume of 25 µL. Molecular weight (Kbp) markers are as indicated. 


Figure 4 .

 4 Figure 4. Effect of dNTP concentration on amplification pattern. The amplifications were performed with primer OPA01 (a) and primer OPA13 (b) in a reaction volume of 25 µL. Molecular weight (Kbp) markers are as indicated. 


Figure 5 .

 5 Figure 5. Effect of primer concentration on amplification pattern. The amplifications were performed with primer OPA01 (a) and primer OPA13 (b) in a reaction volume of 25 µL. Molecular weight (Kbp) markers are as indicated. 


Figure 6 .

 6 Figure 6. Amplification profiles obtained with six primers in different PCR thermal programs. Thermal program fixed conditions: Initial denaturation at 94 °C for 12 min, in the cycle denaturation at 94 °C for 0.5 min and annealing at 36 °C for 1 min. Thermal program variated conditions: 1.) extension time in cycle: 72 °C for 1 min (lanes ABCD), 72 °C for 2 min (lanes EFGH); 2.) number of cycles: 45 cycles (lanes AB, EF), 55 cycles (lanes CD, GH); 3.) final extension: 72 °C for 0 min (lanes AC; EG), 72 °C for 10 min (lanes BD, FH). 


Figure 9 .

 9 Figure 9. RAPD profiles generated by primer OPL17 with DNAs extracted from 12 tobacco lines. The following lines are included from lane 1 to 12: Burley IST G 14, Burley IST 23, Bright IST G 19, Bright IST MS 82, Maryland Md 609, Kentucky IST T 762, Havanna IST T 720, Xanthi IST X 2H, Erzegovina IST E 8A, Perustitza IST Pr 61, Samsun Katerini 2/5, Samsun T 1116. The arrow indicates a polymorphic product of about 0.8 Kbp. 


Figure 7 .

 7 Figure 7. RAPD profiles generated by primer OPE20 with DNAs extracted from 12 tobacco lines. The following lines are included from lane 1 to 12: Burley IST G 14, Burley IST 23, Bright IST G 19, Bright IST MS 82, Maryland Md 609, Kentucky IST T 762, Havanna IST T 720, Xanthi IST X 2H, Erzegovina IST E 8A, Perustitza IST Pr 61, Samsun Katerini 2/5, Samsun T 1116. The arrow indicates a polymorphic product of about 0.75 Kbp. 


Figure 8 .

 8 Figure 8. RAPD profiles generated by primer OPL07 with DNAs extracted from 12 tobacco lines. The following lines are included from lane 1 to 12: Burley IST G 14, Burley IST 23, Bright IST G 19, Bright IST MS 82, Maryland Md 609, Kentucky IST T 762, Havanna IST T 720, Xanthi IST X 2H, Erzegovina IST E 8A, Perustitza IST Pr 61, Samsun Katerini 2/5, Samsun T 1116. The arrow indicates a polymorphic product of about 0.6 Kbp. 


Figure 10 .

 10 Figure 10. RAPD profiles generated by primer OPA01 with DNAs extracted from 13 Nicotiana species. The following species are included from lane 1 to 13: N. benevidesii, N. rustica, N. glutinosa, N. tabacum (Samsun T 1116), N. alata, N. debney,N. amplexicaulis, N. velutina, N. occidentalis, N. simulans, N. megalosiphon, N. rotundifolia, N. suaveolens. 


Figure 11 .

 11 Figure 11. RAPD profiles generated by primer OPA13 with DNAs extracted from 13 Nicotiana species. The following species are included from lane 1 to 13: N. benevidesii, N. rustica, N. glutinosa, N. tabacum (Samsun T 1116), N. alata, N. debney, N. amplexicaulis, N. velutina, N. occidentalis, N. simulans, N. megalosiphon, N. rotundifolia, N. suaveolens. 


Table 2 . Nicotiana species examined in this study

 2 
	GKP\GNPGP

* Samsun line T1116 
Table 3 . Yields of DNA from different extraction procedures.

 3 
	Method	Yield (µg/mg of fresh tissue)
	CTAB method	278 ± 40
	Edwards method	219 ± 40
	Nucleon phytopure system	196 ± 20
	Goring method	197 ± 35
	Data represent average ± standard deviation calculated
	from three experiments with 10 replications each

Table 4 . Evaluated and optimum concentrations for RAPD assay.

 4 
		Concentration	
	Variable		
		Evaluated	Optimum
	DNA template (ng/25 µL)	20406080	20
	MgCl 2 (mM)	1.52.03.04.05.0	2
	dNTP (µM) (each)	50100150200250300	100
	Primer (µM)	0.20.40.60.81.01.2	0.4

Table 5 . PCR thermal profiles assayed.

 5 
	Number of cycles	Initial denaturation 94 °C	Denaturation 94 °C	Annealing 36 °C	Extension 72 °C	Final extension 72 °C
						0 min
					1 min	
	45	12 min	0.5 min	1 min		10 min
						0 min
					2 min	
						10 min
						0 min
					1 min	
	55	12 min	0.5 min	1 min		10 min
						0 min
					2 min	
						10 min

Table 6 . Adopted conditions of PCR thermal profile.

 6 
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	UK\GTCPIKPIHTQOVQ-DR2TKOGTUVJCVRTQ	PQVTGXGCNVJG RTGUGPEGQHRQN[OQTRJKEDCP.U(WT
	.WEG. HCKPV DCP.KPI RCVVGTPU QT .K. PQV TGRTQ.WEG	VJGT CPCN[UKU YCU ECTTKG. QWV YKVJ QPN[ QH VJG
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			Cycle (no 45)			
	Initial denaturation	Denaturation	Annealing	Extension	Final extension
	94 °C	94 °C	36 °C	72 °C	72	°C
	12 min	0.5 min	1 min	1 min	10 min
L Del Piano, M Abet, C Sorrentino, F Acanfora, E Cozzolino, A Di Muro
Genetic Variability in <i>Nicotianatabacum</i> and <i>Nicotiana</i> Species as Revealed by RAPD Markers: 1. Development of the RAPD Procedure