Sensitivity of PCR method for detection of ogura male-sterile cytoplasm in Brassica napus L.

Tom XXI Rośliny Oleiste 2000 Marcin Matuszczak, Beata Gazecka, Jan Krzymański Instytut Hodowli i Aklimatyzacji Roślin, Zakład Roślin Oleistych w Poznaniu Sensitivity of PCR method for detection of ogura male-sterile cytoplasm in Brassica napus L. Czułość metody PCR stosowanej dla wykrywania męskosterylnej cytoplazmy typu ogura w rzepaku Key words: Słowa kluczowe: winter oilseed rape, Brassica napus L., PCR, reaction sensitivity, SCAR marker, CMS ogura rzepak ozimy, Brassica napus L., reakcja PCR, czułość reakcji, marker SCAR, CMS ogura Obecnie możliwe jest stosowanie metody wykorzystującej reakcję PCR do wykrywania męskosterylnej cytoplazmy typu ogura (CMS ogura) w rzepaku. Dla odpowiedniej interpretacji wyników uzyskiwanych w praktyce ważne jest określenie czułości używanej metody. W tym celu na podstawie wcześniejszych analiz wybrano dwie próbki nasion posiadające męskosterylną cytoplazmę typu ogura oraz pozbawione takiej cytoplazmy. Izolowano DNA z prób mieszanych złożonych z siewek obu rodzajów w różnych, ściśle określonych proporcjach. Próby te stanowią model sytuacji, w której badany materiał jest w różnym stopniu zanieczyszczony obcymi nasionami. Izolowano także oddzielne próby DNA z siewek każdego rodzaju. Próby te łączono ze sobą w różnych proporcjach dla uzyskania prób mieszanych o ściśle określonej zawartości każdego ze składników, a także wykonano liczne rozcieńczenia obu prób, aby zbadać wpływ zawartości obu typów DNA w próbie oraz stężenia próby na intensywność otrzymanego prążka. Dla wszystkich prób wykonano reakcje PCR przy użyciu starterów specyficznych dla męskosterylnej cytoplazmy typu ogura przy zachowaniu stałych warunków reakcji oraz tych samych odczynników. Po zebraniu wszystkich wyników sporządzono schemat, na którym obecność lub brak prążka charak- The detection of ogura male-sterile cytoplasm (CMS ogura) in Brassica napus L. is now possible using PCR method. For further studies of this method the sensitivity test was done. Two individual plants with and without ogura malesterile cytoplasm were chosen and the seed samples were collected. Then combined DNA samples were prepared using different ratios of plant material of each type (seedlings were used for DNA extractions). This was the simulation of DNA contamination caused by the presence of one or more foreign seeds mixed with the proper seed sample. Another set of DNA samples was prepared by mixing of CMS ogura (-) and CMS ogura (+) DNA samples or by diluting them. This was done to simulate the effects of DNA sample contamination that could happen after extraction and also to find the lowest detection level for ogura male-sterile cytoplasm. For all prepared DNA samples PCR reactions were performed using constant reaction parameters and reagents. The results were presented as the map where the presence or absence of the CMS ogura specific band can be correlated with the concentration of template and the CMS ogura (-) : CMS ogura (+) ratio in the sample. On the basis of this map it was proved that the best results allowing to distinguish between CMS ogura (-) and CMS ogura (+) seedlings are

382 Marcin Matuszczak... terystycznego dla CMS ogura odpowiada określonym wartościom stężenia matrycy oraz proporcji CMS ogura (-) : CMS ogura (+) w próbie. Analiza tak przedstawionych wyników pozwala stwierdzić, że do rozróżniania siewek CMS ogura (-) i CMS ogura (+) najlepsze są próby DNA rozcieńczone od 10 do 10 2 razy. Użycie prób stężonych może prowadzić do uzyskania fałszywie negatywnych wyników ze względu na wysoką zawartość inhibitorów, które blokują reakcję. Z drugiej strony, poniżej poziomu rozcieńczenia wynoszącego 10 2 razy stężenie matrycowego DNA jest zbyt niskie, toteż otrzymane wyniki są niepewne. Uzyskane dane pozwalają na lepszą interpretację wyników prowadzonych rutynowo analiz. obtained for the dilutions of templates ranged from 10 to 10 2 times. The result obtained with the use of concentrated template can be false negative because of the presence of inhibitors in the sample. On the other hand, below the level of 10 2 times dilution obtained results become unreliable. All data presented here can be useful for calibration of the method for detection of ogura male-sterile cytoplasm. Introduction It was found that PCR reaction can be a convenient tool for early detection of ogura male-sterile cytoplasm in Brassica napus L. In our laboratory this method is now used as the routine test for elimination of self-sown plants and for checking the identity of the lines or plants possessing CMS ogura cytoplasm. Two primers complementary to the sequences of mitochondrial DNA at the region linked with CMS ogura were designed (Krishnasamy and Makaroff 1993; Sigareva and Earle 1997). The single product of PCR reaction (~500bp) is easily detected for plants with ogura male-sterile cytoplasm (CMS(+) plants). On the contrary, no products can be detected for the plants without ogura male-sterile cytoplasm (CMS(-) plants). The result of the test is not influenced by the presence of any other nuclear or cytoplasmic genes in the studied plant. When using the described test, obtained results were sometimes difficult to interpret. The band corresponding to the product of PCR reaction was faint, and it was not sure if the studied plant contains ogura male-sterile cytoplasm or not. As the reliable results are extremely important for breeders, investigation of this problem was necessary. Our main objective was to check the effect of sample dilution on the final result of the analysis. Moreover, the effects of DNA sample contamination on the obtained results were also studied. Such contamination is possible during DNA extraction or seed preparation and we tried to determine if such case can generate ambiguous results.

Sensitivity of PCR method for detection of ogura... 383 Plant material Materials and methods On the basis of some earlier experiments, two individual plants with and without CMS ogura cytoplasm - were chosen and the seed samples were collected. The seeds were put on Petri dishes for germination. Then combined DNA samples were prepared using different ratios of plant material of each type (five days old seedlings were used for DNA extractions). This was done to simulate the effect of DNA contamination caused by the presence of one or more foreign seeds mixed with the proper seed sample. As a control another set of DNA samples was prepared by mixing of CMS ogura ( ) and CMS ogura (+) DNA samples. DNA extraction Plant material was ground to powder in a mortar with liquid nitrogen. DNA was extracted as previously described (Doyle and Doyle 1990) with small modifications. The powder was poured with hot (65 C) extraction buffer 2 CTAB [2%(w/v) CTAB, 100mM Tris-HCl (ph 8.0), 20 mm EDTA, 1.4M NaCl, 1%(w/v) PVP, 1%(v/v) β-mercaptoethanol]. Probes were incubated at 65 C for 30min. and then, after mixing with one volume of chloroform-octanol 24:1 (v/v), were centrifuged for 10min. at 11500 g. DNA from the collected aqueous phase was precipitated by adding of two thirds volume of isopropanol. After 10min. of centrifugation the supernatant was discarded and the pellet was resuspended in TE buffer containing 40µg/ml of RNAse A. After digestion of RNA contaminations (at 37 C for 1 hour), DNA was again precipitated with isopropanol and rinsed with 70% ethanol. Then the pellet of DNA was resuspended in TE buffer. Dilution of DNA samples and the quality test For all extracted DNA samples 10 times dilutions were prepared. These diluted samples served as templates parallel with original ones. Moreover, seven series of templates were prepared, each of them originated from the sample with different CMS ogura (-) : CMS ogura (+) DNA ratio: A pure CMS(-), B (-)9:1(+), C (-)8:2(+), D (-)6:4(+), E (-)5:5(+), F (-)1:9(+), G pure CMS(+). All series were prepared by subsequent dilutions of initial, concentrated sample and consisted of the following samples: not diluted, 2 times diluted, 3 times diluted, 5 times diluted, 10 times diluted, 50 times diluted, 10 2 times diluted, 10 3 times diluted, 10 4 times diluted and 10 5 times diluted. The quality and concentration of all templates were tested using separation on 0.8% agarose gel in TBE buffer followed by ethidium bromide staining. The gels were photographed under the UV light using Kodak Digital Science EDAS system.

384 Marcin Matuszczak... PCR analysis For all prepared DNA samples the PCR reaction was done using two primers specific to the mitochondrial DNA regions correlated with CMS ogura cytoplasm (Krishnasamy and Makaroff 1993, Sigareva and Earle 1997). The sequences of the primers were 5 GTC GTT ATC GAC CTC GCA AGG 3 and 5 GTC AAA GCA ATT GGG TTC AC 3. The reaction mixture (25 µl) contained: PCR buffer, 1.25 mm MgCl 2, 0.2 mm dntps, 1.5 units of Taq polymerase (MBI Fermentas), 5 pmoles of each primer and template DNA (5 µl for each sample). Amplification was conducted in the Biometra UNO II thermocycler with the following thermal profile: initial denaturation for 3 min. at 90 C; 34 cycles of 30 sec. at 92 C, 30 sec. at 65 C and 1 min. 30 sec. at 70 C; final amplification for 2 min. at 70 C (Miko³ajczyk et al. 1998). The reaction products were analyzed using separation on 1.4% agarose gel in TBE buffer followed by ethidium bromide staining. The gels were photographed under the UV light using Kodak Digital Science EDAS system. The size marker used was Lambda DNA digested by HindIII and EcoRI (MBI Fermentas). Results and discussion The seedlings were collected and DNAs were extracted from both CMS(-) and CMS(+) as well as from nine mixed samples. The diluted samples were also prepared. The templates were then checked on agarose gels (fig. 1A-C). It was demonstrated that the DNA samples are of equal quality and the concentrations are consistent with previous assumptions. They can be considered as PCR ready. The same conditions were maintained for all the PCR reactions, including concentration and origin of all reagents as well as thermocycler manufacturer, model and thermal parameters. The concentration of template was the only variable factor. The negative control (with no template) was also made to check the reagents for the presence of any contaminations. The products of PCR reaction were analyzed on agarose gels (fig. 2A-B, 3A-G). The significant differences were observed for results obtained using concentrated and 10 times diluted templates (compare fig. 2A and 2B). As it was shown (fig. 3A-G, table 1), 50 times dilution of samples gives even better efficiency, but below that concentration it goes down again. Below the level of 10 2 times dilution obtained results become unreliable with totally non-informative no band cases for 10 5 diluted template.

Sensitivity of PCR method for detection of ogura... 385 [bp] λ/hindiii/ /EcoRI KD 6:4 (-) 9:1 8:2 7:3 5:5 4:6 3:7 2:8 1:9 (+) (-)* (+)* λ/hindiii/ /EcoRI A B C Figure 1. Samples of template DNA separated on 0.8% agarose gel. (A) 10 times diluted samples of different CMS(-) : CMS(+) ratio. Pure CMS(-) and CMS(+) samples are marked (-) and (+), respectively. Mixed samples are described with the ratio the first number is for CMS(-) and the second is for CMS(+). K D is a control without DNA. (B) pure CMS(-) samples of different concentrations. (C) pure CMS(+) samples of different concentrations. DNA concentrations are expressed as dilutions of initial sample that is marked Conc. Samples marked with asterisk were prepared using 20 seeds instead of 10. The size of DNA fragments is expressed in base pairs (bp). Próby DNA rzepaku obserwowane po rozdziale na 0,8% żelu agarozowym. (A) Próby, które powstały w wyniku mieszania tkanek CMS(-) i CMS(+) w różnych proporcjach w czasie izolacji; na żelu analizowano próby 10 razy rozcieńczone. Czyste próby CMS(-) i CMS(+) oznaczone są jako (-) oraz (+). Próby mieszane oznaczone są jako proporcja CMS(-) : CMS(+). K D kontrola bez DNA. (B) Czyste próby CMS(-) o różnych stężeniach DNA. (C) Czyste próby CMS(+) o różnych stężeniach DNA. Stężenia prób opisano jako rozcieńczenia wyjściowej próby oznaczonej Conc. Próby oznaczone gwiazdką powstały przez izolację DNA z 20 siewek zamiast 10. Wielkości fragmentów wyrażono w parach zasad (bp).

386 Marcin Matuszczak... [bp] λ/hindiii/ /EcoRI KD 6:4 K (-) 9:1 8:2 7:3 5:5 4:6 3:7 2:8 1:9 (+) (-)* (+)* λ/hindiii/ /EcoRI A B Figure 2. PCR reaction products separated on 1.4% agarose gel. Samples of different CMS(-) : CMS(+) ratio. (A) Concentrated samples. (B) 10 times diluted samples. Pure CMS(-) and CMS(+) samples are marked (-) and (+), respectively. Mixed samples are described with the ratio - the first number is for CMS(-) and the second is for CMS(+). K D and K are controls without DNA. The product specific for ogura male-sterility cytoplasm is marked with an arrow. The size of DNA fragments is expressed in base pairs (bp). Produkty reakcji PCR obserwowane po rozdziale na 1,4% żelu agarozowym. Próby o różnych proporcjach matryc CMS(-) i CMS(+). (A) Próby dla matryc stężonych. (B) Próby dla matryc 10 razy rozcieńczonych. Czyste próby CMS(-) i CMS(+) oznaczone są jako (-) oraz (+). Próby mieszane oznaczone są jako proporcja zawartości CMS(-) do zawartości CMS(+). K D oraz K kontrole bez DNA. Produkt specyficzny dla męskosterylnej cytoplazmy typu ogura oznaczono strzałkami. Wielkości fragmentów wyrażono w parach zasad (bp).

Sensitivity of PCR method for detection of ogura... 387 [bp] K A B C D E F (A) Pure CMS(-) samples. (B) Samples of the 9 CMS(-) : 1 CMS(+) ratio. (C) Samples of the 8 CMS(-) : 2 CMS(+) ratio. (D) Samples of the 6 CMS(-) : 4 CMS(+) ratio. (E) Samples of the 5 CMS(-) : 5 CMS(+) ratio. (F) Samples of the 1 CMS(-) : 9 CMS(+) ratio. (G) Pure CMS(+) samples. DNA concentrations are expressed as dilutions of initial sample that is marked Conc. K is a control without DNA. The product specific for ogura male-sterility cytoplasm is marked with an arrow. The size of DNA fragments is expressed in base pairs (bp). (A) Czyste próby CMS(-). (B) Próby o proporcji matryc CMS(-) do CMS(+) równej 9 : 1. (C) Próby o proporcji matryc CMS(-) do CMS(+) równej 8 : 2. (D) Próby o proporcji matryc CMS(-) do CMS(+) równej 6 : 4. (E) Próby o proporcji matryc CMS(-) do CMS(+) równej 5 : 5. (F) Próby o proporcji matryc CMS(-) do CMS(+) równej 1 : 9. (G) Czyste próby CMS(+). Stężenia prób opisano jako rozcieńczenia wyjściowej próby oznaczonej Conc. K kontrola bez DNA. Produkt specyficzny dla męskosterylnej cytoplazmy typu ogura oznaczono strzałkami. Wielkości fragmentów wyrażono w parach zasad (bp). G Figure 3. PCR reaction products separated on 1.4% agarose gel. Series of samples with different concentrations Produkty reakcji PCR obserwowane po rozdziale na 1,4% żelu agarozowym. Serie prób o różnych stężeniach matrycowego DNA.

388 Marcin Matuszczak... Table 1 The map of results obtained using PCR reaction detecting CMS ogura cytoplasm, for DNA samples with different concentration and different CMS(-) : CMS(+) ratio Schemat wyników uzyskiwanych w reakcji PCR pozwalającej wykrywać cytoplazmę typu CMS ogura dla prób o różnym stężeniu DNA matrycowego oraz o różnych proporcjach matryc CMS(-) : CMS(+) C C M S (-) : C M S (+) R A T I O O (-) 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 (+) N Conc 0 0 0 0 0? 0 1 1 1 1 1 C 2 0 0 0 1? 0? 1 1 S E 3 0 0 0 1 1? 1 1 T N 5 0 0?? 1 1 1 1 Ę T 10 0 0? 1? 1 1 1 1 1 1 1 1 Ż R 50 0?? 1? 1 1 1 1 E A 10 2 0???? 1 1 1 1 N T 10 3 0 0? 0????? 1? 1? I I 10 4 0 0 0 0 0 0? 0? E O 10 5 0 0 0 0 0 0 0 N P R O P O R C J A C M S (-) : C M S (+) Each row refers to the different DNA concentration in the sample. DNA concentrations are expressed as dilutions of initial sample that is marked as Conc. Each column refers to the different CMS(-) : CMS(+) DNA ratio in the sample. Pure CMS(-) and CMS(+) samples are marked (-) and (+), respectively. Mixed samples are described with the ratio; the first number is for CMS(-) and the second is for CMS(+). Symbols: 0 no band (negative result); 0?,??, 1? faint bands; 1 good band (positive result); no data. Każdy wiersz odpowiada innemu stężeniu DNA w próbie. Stężenia opisano jako rozcieńczenia wyjściowej próby oznaczonej Conc. Każda kolumna odpowiada innej proporcji DNA CMS(-) : CMS(+) w próbie. Czyste próby CMS(-) i CMS(+) oznaczone są jako (-) oraz (+). Próby mieszane oznaczone są jako proporcja CMS(-) : CMS(+). Symbole: 0 brak prążka (wynik negatywny); 0?,??, 1? prążek niewyraźny; 1 prążek wyraźny (wynik pozytywny); brak danych. Patterns Cieniowanie: Positive results for pure CMS(+) samples Wyniki pozytywne dla czystych prób CMS(+) Positive results for mixed samples Wyniki pozytywne dla prób mieszanych Faint bands zone Strefa niewyraźnych prążków Negative results for mixed samples Wyniki negatywne dla prób mieszanych. Negative results for pure CMS(-) samples Wyniki negatywne dla czystych prób CMS(-). Non-informative results (<10 2 dilution) Wyniki niemożliwe do interpretacji (rozc. <10 2 )

Sensitivity of PCR method for detection of ogura... 389 These results can be well explained using PCR kinetics. As shown by many authors, the real-time PCR techniques can be used for monitoring the progress of the PCR (Higuchi et al. 1992; Higuchi et al. 1993). With the use of these techniques the model of a single amplification plot was established by plotting the increase in fluorescence versus cycle number (fig. 4). The parameter C T (threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. As it was shown, the higher the initial amount of genomic DNA, the sooner accumulated product is detected in the PCR process, and the lower the C T value (Higuchi et al., 1993). The number of cycles used in our experiments is fixed, but it can exceed the C T value, depending on the reaction conditions. The faint bands and the lack of bands are the result of two factors: the presence of inhibitors in the DNA sample that block the activity of Taq polymerase, and the extremely low concentration of DNA with the CMS(+) sequence in the sample. The inhibition of the Taq polymerase activity slows down the whole reaction, so it can reach the exponential phase much later than in normal conditions. Small dilution of the sample decreases the concentration of inhibitor enough to allow the effective amplification. However, further dilution can cause the decrease of starting copy number and the C T value can become higher than the number of cycle used for the reaction. Both factors increase the C T value, making it difficult to produce the required amount of amplified DNA. It can be detected on the gel only as faint bands and in some cases it gives no band at all. 0.90 0.80 (NORMALIZED Rn FLUORESCENCE) 0.70 0.60 0.50 Rn 0.40 0.30 0.20 0.10 0.00 CYCLE NUMBER 0 5 10 15 20 25 30 35 40 CT Figure 4. Model of a single amplification plot - the plot of the detected fluorescence signal versus PCR cycle number Modelowy wykres amplifikacji pojedynczego fragmentu DNA na wykresie pokazano wartość wykrywanego sygnału fluorescencyjnego w zależności od ilości cykli reakcji PCR (Applied Biosystems)

390 Marcin Matuszczak... It should be noted that the highest level of reaction sensitivity can be achieved for the dilutions of templates ranged from 10 to 10 2 times. For that case even one seedling from CMS(+) plant among nine other seedlings used for analysis can be detected (fig. 3B, table 1). As only the faint bands can be obtained in this way, we suggest to analyze both concentrated and diluted templates to get an overall result. On the contrary, the method does not allow to detect any addition of CMS(-) seedlings to the sample where the CMS(+) component predominates. Conclusions The result obtained with the use of concentrated template can be false negative because of the presence of inhibitors in the sample. The best results that allow to distinguish between CMS(-) and CMS(+) seedlings are obtained for the dilutions of templates ranged from 10 to 10 2 times. Only the pure CMS(-) sample gives no band for such dilution. Below the level of 10 2 times dilution obtained results become unreliable - even for pure CMS(-) and CMS(+) samples. The method is useless for detection of CMS(-) seedlings among CMS(+) ones if the latter component predominates. It is possible to detect small addition of CMS(+) seedlings in a sample, but in such case both concentrated and diluted samples should be analyzed to get an overall result. References Doyle J.J., Doyle J.L. 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13-15. Higuchi R., Dollinger G., Walsh P.S., Griffith R. 1992. Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10: 413-417. Higuchi P.M., Fockler C., Dollinger G., Watson R. 1993. Kinetic PCR: Real time monitoring of DNA amplification reactions. Biotechnology 11: 1026-1030. Krishnasamy S., Makaroff C. 1993. Characterization of the radish mitochondrial orf B locus: possible relationship with male sterility in Ogura radish. Curr. Genet. 24: 156-163. Mikołajczyk K., Matuszczak M., Piętka T., Bartkowiak-Broda I., Krzymański J. 1998. Zastosowanie markerów DNA do badań odmian składników mieszańcowych rzepaku. Rośliny Oleiste XIX (2): 463-471. Sigareva M.A., Earle E.D. 1997. Direct transfer of a cold-tolerant Ogura male-sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion. Theor. Appl. Genet. 94: 213-220.