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components from various commercial AFLP kits. AFLP Primers; EcoRI and MseI were adopted for the study
(Table 3).
Table 3 AFLP primer combinations and nucleotide sequences
Primer combination
Nucleotide sequences
AAC+CAG
EcoRI -5´- GACTGCGTACCAATTCA - 3´
MseI -5´- GATGAGTCCTGAGTAAC - 3´
AAC+CAA
EcoRI -5´- GACTGCGTACCAATTCA- 3´
MseI -5´-GATGAGTCCTGAGTAAC - 3´
AAC+CAC
Eco
RI -5´-GACTGCGTACCAATTCA- 3´
Mse
I -5´- GATGAGTCCTGAGTAAC - 3´
3.5 Digestion of genomic DNA and ligation of oligonucleotide adapters
Digestion of genomic DNA by the restriction enzymes EcoRI and MseI and ligation of oligonucleotide adapters
were accomplished in a single reaction mixture of 11 μL. Prior to each use, the adaptor pairs were pre-heated to
95ºC for 5 min, and then allowed to cool slowly for ten minute at room temperature of 25ºC. The mixture was
incubated overnight at room temperature in order to digest template DNA completely.
3.6 Pre-selective PCR amplification of DNA
Pre-selective PCR amplification was performed using the Applied Biosystems AFLP kit. The 20 μL reaction
contained 4 μL of the diluted restricted/ligated DNA and 16 μL of a mixture with 1 μL of EcoRI+A and MseI+C
AFLP pre-selective primers with 15 μL of AFLP core mix. The PCR program for the pre-selective amplification
was: 72ºC for 3 min, followed by 20 repetitive cycles of 94ºC for 20 s, 56ºC for 30 s, and 72ºC for 2 mins, with a
final hold at 60ºC for 30 mins.
3.7 Selective PCR amplification of DNA
For selective PCR amplification of restriction fragments, primers were prepared for recognition of EcoRI and
MseI adapters. Fragments are visualized by attaching a D4, D3 or D2 WellRED™ dye to the 5´ end of each EcoRI
selective amplification primer without modification ofMseI primer.
3.8 Preparation of DNA amplification fragments for separation of PCR product by polyacrylamide gel
electrophoresis (PAGE)
Loading solution was prepared with a 100-base-pair (bp) DNA size standard labeled with WellRED™ dye D1
(approximately 100:1; Beckman Coulter 608082 and 608098). This solution was thoroughly mixed by vortexing
for a minimum of two minutes. A 30-μL aliquot of this cocktail was added to 1.5 μL of the selective amplification
product and loaded into the prepared well.
3.9 Data scoring and analysis of AFLP product
Total bands were scored visually from three primer combinations and polymorphic bands were observed as
presence (1) or absence (0). Also, cluster analysis and dendrogram was constructed using Powermarker software
V3.25, to reveal the phylogenetic relationship following UPGMA method of Jaccard’s similarity coefficient.
References
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Amaranthus palmeri
) Populations from North Carolina and Georgia, Weed Sci., 61(1): 136-145
Ayodele J.T., and Olajide O.S., 2011, Proximate and Amino Acid Composition of
Celosia argentea
Leaves, J. Basic Appl. Sci., 19(1): 162-165
Denton O.A., 2004,
Celosia argentea
L. In: Grubben, G.J.H. & Denton, O.A. (Editors). PROTA 2, Vegetables/Légumes. PROTA, Wageningen Netherlands
Esayas A., and Bryngelsson T., 2006, Inverse sequence-tagged repeat (ISTR) analysis of genetic variability in forest coffee (
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