This paper describes a simple, low cost and reliable DNA template
preparation protocol for polymerase chain reaction (PCR) using immature
leaves from peanut seeds or leaves from field-grown plants. The
technique may find wide utility in studies involving PCR-based
molecular markers, rapid screening for transformants and gene cloning

Bao, Z.M.

Tang, Y.Y.

Wang, C.T.

Wang, X.Z.

Xu, Y.Z.

Yu, S.L.

Zhang, J.C.

Bioline International

Electronic Journal of Biotechnology ISSN: 0717-3458                                                                                 Vol.12 No.2,  Issue of April 15, 2009 © 2009 by Pontificia Universidad Católica de Valparaíso -- Chile                               Received October 23, 2008 / Accepted January 23, 2009 This paper is available on line at http://www.ejbiotechnology.info/content/vol12/issue2/full/11/ DOI: 10.2225/vol12-issue2-fulltext-11                                                                                                                            TECHNICAL NOTE    A rapid and cheap protocol for preparation of PCR templates in peanut Chuan Tang Wang Ocean University of China Qingdao 266003, P R China Xiu Zhen Wang Shandong Peanut Research Institute Qingdao 266100, PR China Yue Yi Tang Shandong Peanut Research Institute Qingdao 266100, PR China Jian Cheng Zhang Shandong Peanut Research Institute Qingdao 266100, PR China Shan Lin Yu Shandong Peanut Research Institute Qingdao 266100, PR China Jian Zhi Xu Shandong Peanut Research Institute Qingdao 266100, PR China Zhen Min Bao* Ocean University of China Qingdao 266003, PR China Tel: 86 532 8203196 E-mail: zmbao@ouc.edu.cn Financial support: Financial support from the earmarked fund for Modern Agro-industry Technology Research System (MATRS) Peanut Program, MOA, China, China Natural Science Foundation (Grant No. 30300224), 863 New and High Technology Project (Grant No. 2002CCC03200, 2006AA10A114), New and High Technology Innovation Foundation of Shandong Academy of Agricultural Sciences (Grant No. 2006 YCX013), and Young Scientists Foundation of Shandong Academy of Agricultural Sciences (Grant No.2007YQN007) is gratefully acknowledged. We are in debt to Prof Rong Hua Tang for his generosity in providing wild peanuts. Keywords: DNA extraction, groundnut, PCR, peanut. Abbreviations: GXAS: Gaungxi academy of agricultural sciencesITS: internal transcribed spacer MAS: marker-assisted selection PCR: polymerase chain reaction PVP: polyvinylpyrrolidone SCAR: sequence-characterized region SSR: simple sequence repeat  This paper describes a simple, low cost and reliable DNA template preparation protocol for polymerase chain reaction (PCR) using immature leaves from peanut seeds or leaves from field-grown plants. The technique may find wide utility in studies involving PCR-based molecular markers, rapid screening for transformants and gene cloning. *Corresponding author Conventional DNA extraction protocols usually involve many steps; for plants species with abundant secondary metabolites, a lengthy procedure of over 10 steps is generally needed (Weising et al. 2005; Sarwat et al. 2006). The cultivated peanut and its wild relatives in the genus Arachis are species with carbohydrates and poly-phenols. Several DNA extraction and purification protocols for   Wang, C.T. et al.  2peanut have been published and proved competent in restriction digestion and Southern hybridization (Kochert et al. 1991; Choi et al. 1999; Burow et al. 2001; Wang et al. 2002; Chen et al. 2008). However, it is still difficult, within a short period of time, to deal with large number of samples even with sophisticated stainless steel beads and a grinding machine. This inevitably impedes the wide application of marker-based selection and fast screening for transformants in peanut.  Here we described a rapid, simple and cost-effective method for preparation of polymerase chain reaction (PCR) templates for peanut, which may facilitate marker-assisted selection (MAS) and gene mapping using simple sequence repeat (SSR) or sequence-characterized region (SCAR), screening of transgenic peanut plants and homology-based cloning as well.  MATERIALS AND METHODS Materials For primary evaluation of the protocol, totally 7 peanut genotypes were utilized. These included 3 peanut cultivars (I8B4, Luhua14 and L-1), 2 interspecific derivitives [6-33 (Silihong x Arachis rigonii) and L7-1 (Silihong x A. glabrata)] and 1 chemical mutant of L7-1 (m5) (Table 1). For further evaluation, 8 accessions of peanut wild relatives (field-grown plants), 2 peanut landraces (Yingkousilihong and Xingchengdahuasheng) and an interspecific derivative 1-4 (Silihong x Arachis glabrata) (immature leaflets from seeds) were used (Table 2). PREPARATION OF PCR TEMPLATES When a seed is available, the immature leaflets (embryonic tissue from a non-germinated seed, 2.2 ~3.2 mg) may be used as the starting material. The leaflets were collected and placed into a 1.5 ml Eppendorf tube, and 20, 40 or 60 µl of 0.25 mol/L sodium hydroxide (NaOH) was added. The leaflets were then smashed using a thin-walled PCR tube mounted with a 1 ml pipette tip as a pestle. The mixture was boiled for 30 sec. Then 80, 160 or 240 µl of Tris-HCl (pH 7.6) with5 mg/ml of polyvinylpyrrolidone (PVP) (4 times the volume of NaOH added) was added followed by boiling for 2 min. After centrifugation at 10,000 RPM for 5 min, the supernatant was collected and stored at 4ºC for use within a week, or stored at -20ºC for several months. Leaves from peanut plants grown in glasshouse or field may also be used, but the unexpanded or freshly expanded leaflets on the apex of branches/stems are most preferable. The unused end (without a tip) of a ball point pen refill was utilized as a hole puncher. The leaf disc (6.5 mm2) thus prepared may be handled using the alkali-lysis protocol, just as immature leaflets from a seed, which was described above. PCR To amplify the internal transcribed spacer (ITS) of peanut, 2 µl DNA template was used in a 25 µl reaction using Tiangen 2 x Taq PCR MasterMix (Tiangen Biotech) and the primer a and primer b as recommended by Wang et al. (1999). The PCR profile consisted of a pre-denaturation of 94ºC for 3 min, 35 cycles of 94ºC for 50 sec, 55ºC for 1 min, and 72ºC for 1.5 min, and a final extension of 7 min. To amplify the β-tubulin gene-derived DNA regions, Tiangen 2 x Taq PCR MasterMix and primer pairs TBPfex1/TBPrex1 and TBPfin2/TBPrin2 were used. The primer pairs and PCR profile were the same as Breviario et al. (2007). Agarose gel electrophoresis of PCR products After amplification was complete, 3 µl of liquid from the PCR tube were run on a 1.0% agarose gel (1 x TBE) for 40 min at 100 V, and the gel was subsequently stained using  Figure 1. PCR products resolved on a 1.0% agarose gel. M: Tiangen D2000 DNA marker. The numbers used in the figure were the same as in the first column of Table 1. Genotypes, source of leaflets, primer pairs and treatments were listed in Table 1.  A rapid and cheap protocol for preparation of PCR templates in peanut  3ethidium bromide and visualized under UV light to check for the presence of PCR products.  DNA sequencing The PCR products of primer pairs a/b were recovered using the E.Z.N.A. Cycle Pure Kit, and DNA sequencing was performed on an ABI 3730XL sequencer using the primer a or b.    Figure 2. ITS of A. duranensis PI263133 (A19).    Figure 3. ITS of A. pusilla PI289628 (A10). Wang, C.T. et al.  4RESULTS AND DISCUSSION Primary evaluation of the protocol All of the materials/treatments produced distinct bands of expected size on agarose gel (Figure 1). The PCR products resulting from 20, 40 or 60 μl NaOH treatment were all acceptable; therefore, in subsequent studies only the 60 μl NaOH treatment was used. The concentration of the PCR templates prepared using 60 μl NaOH was around 3 ng/μl. Further evaluation of the protocol All of the 11 peanut accessions (Table 2) gave good results, regardless of the starting materials. Clear bands were visualized on agarose gel, even with some wild accessions whose leaflets coated with thick cuticle. The bands were recovered and directly sequenced. Two of the trace files (partial) were shown in Figure 2 and Figure 3. The sequencing results verified that the PCR template preparation protocol was successful.  CONCLUDING REMARKS To the best of our knowledge, this is the first report on high through-put preparation of PCR template in peanut. The present protocol was developed on the basis of the alkali-lysis method as proposed by Wang et al. (1993) with some modifications. The boiling steps and the PVP component were included to ensure better and repeatable results. Chenault et al. (2007) reported a non-destructive seed sampling method for PCR-based analysis with potential in marker assisted selection and transgene screeningin peanut, where as little as 20 mg of peanut seed sample was enough for PCR template preparation; however, 20 steps are still needed. Using the present protocol, peanut DNA can be extracted in a relatively short period of time whenever a Table 1. Peanut materials used for primary evaluation of the protocol.  No. Genotype Leaflet Primer pair Treatment  1  I8B4  immature  TBPfex1, TBPrex1  40 µl NaOH  2  L-1  immature  TBPfex1, TBPrex1  40 µl NaOH  3  Luhua 14  immature  TBPfex1, TBPrex1  40 µl NaOH  4  6-33  immature  TBPfex1, TBPrex1  40 µl NaOH  5  m5  immature  TBPfex1, TBPrex1  40 µl NaOH  6  I8B4  immature  TBPfin2, TBPrin2  40 µl NaOH  7  L-1  immature  TBPfin2, TBPrin2  40 µl NaOH  8  Luhua14  immature  TBPfin2, TBPrin2  40 µl NaOH  9  6-33D3  immature  TBPfin2, TBPrin2  40 µl NaOH  10  L7-1  immature  a, b  40 µl NaOH  11  m5  immature  a, b  40 µl NaOH  12  L7-1  field-grown  a, b  20 µl NaOH  13  L7-1  field-grown  a, b  40 µl NaOH  14  L7-1  field-grown  a, b  60 µl NaOH  15  m5  field-grown  a, b  20 µl NaOH  16  m5  field-grown  a, b  40 µl NaOH  17  m5  field-grown  a, b  60 µl NaOH Note: The numbers used in the first column were the same as in Figure 1.  A rapid and cheap protocol for preparation of PCR templates in peanut  5seed or a leaflet is available. It should be noted that although the ITS amplified in this report has high a copy number in plant genomes, this does not mean that the protocol is only suitable for DNA segments with high copy number. In fact, we have successfully cloned and sequenced partial fad 2 and conglutin genes using similar protocols (Wang et al. 2001; Wang et al. 2004). In conclusion, we have developed a short protocol, suitable for high through-put preparation of PCR templates in peanut, with no genotypes proved recalcitrant thus far. Neither nitrogen treatment nor organic solvent is needed. It should not only save time and money, but also reduce the possibility of contamination. This protocol may find wide utility in PCR-based applications in peanut. REFERENCES BREVIARIO, Diego; BAIRD, W. Vance; SANGOI, Shail; HILU, Khidir; BLUMETTI, Pietro and GIANI, Silvia. High polymorphism and resolution in targeted fingerprinting with combined β-tubulin introns. Molecular Breeding, October 2007, vol. 20, no. 3, p. 249-259.  BUROW, Mark D.; SIMPSON, Charles E.; STARR, James L. and PATERSON, Andrew H. Transmission genetics of chromatin from a synthetic amphidiploid to cultivated peanut (Arachis hypogaea L.): broadening the gene pool of a monophyletic polyploid species. Genetics, October 2001, vol. 159, no. 2, p. 823-837.  CHEN, J.; HU, X.H.; MIAO, H.R.; CUI, F.G. and YU, S.L. Genomic DNA Extraction with CTAB method and its use for SSR and SRAP. Journal of Peanut Science, 2008, vol. 37, no. 1, p. 29-31.  CHENAULT, K.D.; GALLO, M.; SEIB, J.C. and JAMNE, V.A. A non-destructive seed sampling method for PCR-based analysis in marker assisted selection and transgene screening. Peanut Science, January-June 2007, vol. 34, no. 1, p. 38-43.  CHOI, K.; BUROW, M.D.; CHURCH, G.; BUROW, G.; PATERSON, A.M.; SIMPSON, C.E. and STARR, J.L. Genetics and mechanism of resistance to Meloidogyne arenaria in peanut. 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Identity Plant Introduction No.  Species Section Botanical type  GXAS* No. A 19  263133  A. duranensis  Arachis     GXAS No. A2  338280  A. stenosperma  Arachis     GXAS No. A14   331192  A. correntina  Arachis     GXAS No.A8  210555  A. villosa  Arachis     GXAS No. A7   219824  A. monticola  Arachis     GXAS No. A10  289628  A. pusilla  Heteranthae     GXAS No. A6  289639  A. sp  Uncertain affinity     GXAS No. A13   289637  A. sp  Uncertain affinity     Yingkousilihong (Landrace)  -  -  -  fastigiata  Xingchengdahuasheng (Landrace)  -  -  -  hypogaea  1-4 (Silohong x A. glabrata)  -  -  -  - *GXAS: Gaungxi academy of agricultural sciences.  Wang, C.T. et al.  6for genomic DNA isolation from Terminalia arjuna for genetic diversity analysis. Electronic Journal of Biotechnology, 2006, vol. 9, no. 1, p. 85-90.  WANG, C.T.; YANG, X.D.; JIANG, Y.; ZHANG, J.C. and CHEN, D.X. A simple pretreatment procedure for direct PCR using immature leaves from peanut kernels. 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DNA fingerprinting in plants: principles, methods and applications. 2nd ed. Taylor and Francis Group, 2005. 338 p. ISBN 0-8493-1488-7.  ZIEGELIN, G.; FÜRSTE, J.P. and LANKA, E. TraJ protein of plasmid RP4 binds to a 19-base pair invert sequence repetition within the transfer origin. Journal of Biological Chemistry, July 1989, vol. 264, no. 20, p. 11989-11994.   Note: Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication. Supported by UNESCO / MIRCEN network. 

A rapid and cheap protocol for preparation of PCR templates in peanut

University of Toronto Research Repository

A rapid and cheap protocol for preparation of PCR templates in peanut

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