
2Genetic Engineering and Biotechnology Research Institute, Minufiya University, Egypt
- Minimal amounts of gDNA are required,
- No prior genome sequence data are necessary for primer construction,
- Markers are randomly distributed throughout the genome, and
- Many bands are generated that potentially provide a large number of polymorphisms [10,11].
Ten smut resistant sugarcane clones were obtained from Sugar Crops Research Institute, Giza, Egypt (GT 54-9, C9/0.5Kr-11, C9/0.5Kr-32, C9/0.5Kr-42, C9/0.5Kr-46, C9/1Kr-3, C9/2Kr-1, C9/3Kr-63, C9/3Kr-69 and C9/3Kr-8) and 10 susceptible clones (C9/2Kr-19, C9/2Kr-4, C9/3Kr-38, C9/3Kr-45, C9/3Kr-47, C9/3Kr-52, C9/3Kr-56, C9/3Kr-70, C9/1Kr-3 and C9/3Kr-79). All clones were selected from a former mutation induced clones by gamma radiation to the moderately resistant sugarcane cultivar GT 54-9.
Encore Adaptor | Tru 9I Adaptor |
(EcoRI-1) 5′-CTCGTAGACTGCGTACC-′3 | (Tru9I-1) 5′-GACGATGAGTCCTGAG-′3 |
(EcoRI-2) 5′-AATTGGTACGCAGTCTAC-′3 | (Tru9I -2)5′-TACTCAGGACTCAT-′3 |
Eco RI adaptors (5 μΜ (conc)) | Tru 9I adaptors (50 μM (conc)) |
10 μl 100 μM RI.sequence 1 | 100 μl 100 μM Tru 9I . sequence 1 |
10 μl 100 μM RI. sequence2 | 100 μl 100 μM Tru 9I . sequence 2 |
2 μl 1 M Tris-HCl (pH 8.0) | 2 μl 1 M Tris-HCl (pH 8.0) |
2 μl 5M NaCl | 2 μl 5 M NaCl |
0.4 μl 0.5M EDTA | 0.4 μl 0.5 M EDTA |
175.6 μl ddH2O |
Two methods were used for restriction/ligation reaction. In the first method digestion and ligation were done in three separate steps while, in the snd method, the restriction/ligation reaction was done in one step.
Pre-selective PCR was performed using Preselective primers carrying a single 30-selective nucleotide. Core sequences of the Eco RI and Tru 9I primers (Table 3) are described in [8].
Primer Name | Sequence |
Eco RI+A | 5′-gactgcgtaccaattca-3′ |
Tru 9I +C | 5′-gatgagtcctgagtaac-3′ |
Selective PCR was carried out using selective primers (Table 4) carrying three 3′-selective nucleotides. Primer combinations used (Table 5) were previously identified as being suitable combinations for sugarcane [12].
Eco RI primers | Sequence | Tru 9I primer | Sequence |
Eco-ACA: | 5′-gactgcgtaccaattcaca-3′ | Tru 9I -CTG | 5′-gatgagtcctgagtaactg-3′ |
Eco-ACC | 5′-gactgcgtaccaattcacc-3′ | Tru 9I -CTA | 5′-gatgagtcctgagtaacta-3′ |
Eco-ACG: | 5′-gactgcgtaccaattcacg-3′ | Tru 9I -CTC | 5′-gatgagtcctgagtaactc-3′ |
Tru 9I -CTT | 5′-gatgagtcctgagtaactt-3′ |
Eco RI-ACG | Eco RI-ACC | Eco RI-ACA | |
Tru 9I -CTG | x | ||
Tru 9I -CTA | x | x | |
Tru 9I -CTC | x | x | x |
Tru 9I -CTT | x | x |
Reagents | µl/one rxn | Final conc. |
Water | 8.1 µl | - |
10X PCR buffer (w/15 mM MgCl2) | 2.0 µl | 1X (1.5 mM MgCl2) |
5 mM dNTPs | 0.4 µl | 200 µM each |
Eco RI I -A## primer (1 µM) | 1.0 µl | 0.046 µM |
Tru 9I -C## primer (5 µM) | 1.0 µl | 0.275 µM |
DNA polymerase (5 U/µl) | 0.4 µl | 2 U/rxn |
Total | 15.0 µl |
The reaction was stopped by adding an equal volume of loading dye (98% formamide, 10 mM EDTA pH 8.0, bromophenol blue 0.25%, xylene cyanol 0.25%). The mixture was denatured for 3 min at 90°C and stored on ice. Subsequently, 8 µl per sample was loaded on a 5% acrylamide/bis acrylamide (19:1) and 7.5 M urea gel, in 100 mM Tris, 100 mM boric acid, 2 mM EDTA, pre-run for 10 min at 140 W. The gel runs for 2 h at 140 W. The 46 samples were run on the same gel, with one gel run per primer combination.
An improved photochemical derived methods for protein silver staining proposed by [17] with slight modification. All procedures were performed at room temperature with a constant agitation of the bath while all the staining steps were performed. After the completion of staining procedure, the gel was rinsed twice in water over 10 min and left to dry vertically overnight. The dried gel was preserved at room temperature; the gel scanned using the Adobe Photoshop™ program (Adobe Systems, Mountain View, Ca, USA). The Adobe Photoshop program was used as needed to increase the contrast between bands and background staining.


Primer combination (Eco RI/Tru 9I) | Number of amplified bands |
ACC/CTT | 60 |
ACC/CTG | 51 |
ACC/CTC | 42 |
ACA/CTA | 43 |
ACA/CTC | 43 |
ACA/CTG | 56 |
ACG/CTA | 50 |
ACG/CTC | 46 |
Total | 390 |

- N Singh N., et al. “Smut disease assessment by PCR and microscopy in inoculated tissue cultured sugarcane cultivars”. Plant Science 167.5 (2004): 987-994.
- M Martinez., et al. “Changes of some chemical parameters, involved in sucrose recovery from sugarcane juices, related to the susceptibility or resistance of sugarcane plants to smut (Ustilago scitaminea)”. International Sugar Journal 102.1221 (2000): 445-448.
- P Kumar., et al. “Potential of Molecular Markers in Plant Biotechnology”. Plant Omics Journal 2.4 (2009): 141-162.
- D Botstein., et al. “Construction of a genetic linkage map in man using restriction fragment length polymorphisms”. The American Journal of Human Genetics 32.3 (1980): 314-331.
- Williams JG., et al. “DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18.22 (1990): 6531-6535.
- M Litt and Luty JA. “A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene”. The American Journal of Human Genetics 44.3 (1989): 397-401.
- Weber JL, and May PE. “Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction”. The American Journal of Human Genetics 44.3 (1989): 388-396.
- Pieter Vos., et al. “AFLP: a new technique for DNA fingerprinting”. Nucleic Acids Research 23.21 (1995): 4407-4414.
- C Maiden M., et al. “Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms”. Proceedings of the National Academy of Sciences of the United States of America 95.6 (1998): 3140-3145.
- M Maheswaran., et al. “Polymorphism, distribution, and segregation of AFLP markers in a doubled haploid rice population”. Theoretical and Applied Genetics 94.1 (1997): 39-45.
- Ulrich MG and L Wolfenbarger. “AFLP genotyping and fingerprinting”. Trends in Ecology & Evolution 14.10 (1999): 389-394.
- P Besse., et al. “Assessing genetic diversity in a sugarcane germplasm collection using an automated AFLP analysis”. Genetica 104.2 (1998): 143-153.
- Xu M L., et al. “High-resolution mapping of loci conferring resistance to sugarcane mosaic virus in maize using RFLP, SSR, and AFLP markers”. Molecular and General Genetics 261.3 (1999): 574-581.
- Thokoane LN and Rutherford RS. “cDNA-AFLP differential display of sugarcane (Saccharum spp, hybrids) genes induced by challenge with the fungal pathogen Ustilago scitaminea (sugarcane smut)”. Proceedings of the South African Sugar Technologists' Association 75 (2001): 104-107.
- Que You-Xiong., et al .“Isolation and identification of differentially expressed genes in sugarcane infected by Ustilago scitaminea”. Acta Agronomica Sinica 35.3 (2009): 452-458.
- A Khaled K and H Esh AM. “High-quality genomic DNA impurities-free from sugar crops and other plant tissue”. Guilin, P.R. China: Proc. Internl. Symp. on Technologies to Improve Sugar Productivity in Developing Countries, 2008. 330-332.
- J Bassam B., et al. “Fast and sensitive silver staining of DNA in polyacrylamide gels”. Analytical Biochemistry 196.1 (1991): 80-83.
- B Brugmans., et al. “A new and versatile method for the successful conversion of AFLP™ markers into simple single locus markers”. Nucleic Acids Research 31.10 (2003): e55.
- Raboin LM., et al. “Undertaking genetic mapping of sugarcane smut resistance”. Proceedings of the South African Sugar Technologists 75 (2001): 94-98.
- K Butterfield M., et al. “Application of gene discovery to varietal improvement in sugarcane”. South African Journal of Botany 70.1 (2004): 167-172.
- Xianming Wei., et al. “Associations between DNA markers and resistance to diseases in sugarcane and effects of population structure”. Theoretical and Applied Genetics 114.1 (2006): 155-164.
- Yu-hui Huang., et al. “AFLP analysis of sugarcane mutants”. Southwest China Journal of Agricultural Sciences 20.4 (2007): 727-731.
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