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Central Coffee Research Institute, Coffee Research Station --577117, Dist Chikmagalur, Karnataka, India.
Laboratory of Genetics, Departimento di Biologia Universita di Trieste, P.le Valmaura 9, 34100 Trieste, Italy.
High quality genomic DNA was successfully extracted from coffee seeds using a simple protocol
devoid of liquid nitrogen or freeze-drying and proteinase K. The isolated DNA was quantified using
spectrophotometer and using agarose gel electrophoresis. The DNA was free from polysaccharides,
polyphenols, RNA and other contaminants. The quantity of DNA ranged from 180 to 630 g/g of seed
powder. Quality of DNA was confirmed by digestion using EcoRI, HindIII and PstI restriction
endonucleases and complete digestion was observed. PCR with random decamer primers and
consensus primers of mitochondria and chloroplast DNA and PCR-RFLP revealed the suitability of the
DNA for PCR based marker techniques including diagnostics.
Key words: Coffee seed, DNA extraction, RAPD, PCR-RFLP, molecular diagnostics.
INTRODUCTION
Coffee is one of the most important agricultural comm.odities in international trade. Coffee represents an important source of income for millions of people in coffee
growing countries in Asia, Africa and Latin America.
Many countries depend on the export of this product to
buy goods and equipments. Commercial coffee cultivation relies upon two coffee varieties; Coffea arabica
(Arabica coffee) and Coffea canephora (Robusta coffee).
Arabica produces high quality coffee and contribute about
70% of total world coffee production. Robusta produces
inferior coffee compared to Arabica. Highly priced varieties would benefit from diagnostic tests for authenticity
and provenance in this commodity, as coffee is liable to
be adulterated with the addition of inferior quality of
coffee, husks or chicory (Martellosi et al., 2005). Since
coffee is traded as beans, it is essential to develop a
diagnostic tool using the seed material for identifying the
adulteration that will benefit the consumers worldwide.
Such a diagnostic tool will be useful for the developing
countries that are involved in coffee growing. In addition,
several consuming countries particularly in Europe and
USA are now insisting on GM-free coffee while importing
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Afr. J. Biotechnol.
S/N
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Genotype
Sln.5A
Sln.6
Sln.9
S.795
Hybrid 1
Hybrid 2
Sarchimor
Cauvery
Columbian Catimor
CXR
S.274
Perpurascens robusta
Tree coffee hybrid
Agaro
Cioccie
Tafarikela
Parentage
Devamachi x S.881
S.274 x Kents
HdeT x Tafarikela
S.288 x Kents
S.795 x Sln.9
Sln.9 x S.795
Villarsachi x Hybrido de Timor
Caturra x Hybrido de Timor
Catimor from Columbia
C. congensis x C. canephora
Indian Robusta selection
Natural Arbugogov mutants
C. arabica x C. liberica ( putative hybrid)
Exotic-Ethiopian collection
Exotic-Ethiopian collection
Exotic-Ethiopian collection
Mishra et al.
10 11
10 11
12 13 14 15
16
12 13 14 15 16
M1 1
2 3 4 5
7 8
411
9 10 11 12 13 14 15 16 M2
PCR RFLP
We were able to obtain high quality DNA free from polyphenols and polysaccharides from the coffee seeds.
When spooled out of solution, the DNA is clear or white;
there is no visible coloration. The A260/A280 ratio of the
DNA ranges from 1.7 to 1.85 indicating the isolated DNA
is free from protein and RNA contamination. Yields of the
DNA varied with the cultivars and ranged from 180 to 630
g for 1 g of seed powder (Table 1). The extracted DNA
showed no visible RNA contamination, as determined by
agarose gel electrophoresis and could be digested
completely by EcoRI, Hind III and PstI, which indicated
the suitability of the DNA for RFLP and AFLP analysis
o
(Figure 1). The extracted DNA stored at 20 C for more
than one year showed no deleterious effects and could
be amplified. In order to check the efficiency and
reliability of the method, we first amplified the seed DNA
of coffee using random primers. The PCR amplification
was robust (Figure 2) and on par with leaf DNA. (Data not
shown)
We further tested the suitability coffee seed DNA for
amplification of mitochondrial and chloroplast DNA using
consensus primers (Taberlet, 1991; Dumesure et al.,
1995; Dumolin et al., 1997). In both the cases (Figures 3
and 4), as expected a single robust band was obtained
and this confirmed the earlier observations of various
authors in different crops (Pharmawati et al., 2004;
Vettori et al., 2004; Achere et al, 2004). Further the PCR
412
Afr. J. Biotechnol.
2 3
4 5
6 7
8 9 10 11 12 13 14 15 16 M1
M1 1
9 10 11 12 13 14 15 16
M1 1 2 3
4 5 6
7 8 9 10 11 12 13 14 15 16
ACKNOWLEDGEMENT
The research was carried out by a support grant by
Government of India under Plan Fund Scheme Plant
Improvement and Biotechnology.
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