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Molecular Plant Breeding 2011, Vol.2, No.12, 83
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sequences of the primers. A 20
-
μL mixture was
prepared for the PCR assay, containing 50 ng of
template DNA, 200 μmol/L dNTPs, 1 U
Taq
DNA
polymerse, 0.3 μmol/L forward SSR primer, 0.3 μmol/L
reverse SSR primer, 50 mmol/L KCl, 10 mmol/L
Tris-HCl (pH 8.3) and 2.5 mmol/L MgCl2. Briefly, the
PCR reaction was performed at 94
℃
for 5 min, then
the amplification was carried out with 32 cycles at a
melting temperature of 94
℃
for 1 min, an annealing
temperature of 55
℃
for 90 sec, and an extension
temperature of 72
℃
for 2 min, followed by an
additional extension at 72
℃
for 5 min. Subsequently,
the PCR products were analyzed on 8% polyacrylam-
ide non-denaturing gels, and the bands were revealed
by silver staining following the electrophoresis (Huo
et al., 2005). The amplifications were done in
duplicate for each analyzed primer.
Table 5 List of coconut-specific microsatellite primer pairs with their sequences
Primer
Repeat type
F
R
Size range Tm
CAC02
(CA)
15
(AG)
7
AGCTTTTTCATTGCTGGAAT
CCCCTCCAATACATTTTTCC
225~240 49
CAC03
(CA)
12
(AG)
14
GGCTCTCCAGCAGAGGCTTAC
GGGACACCAGAAAAAGCC
176~183 55
CAC04
(CA)
19
(AG)
17
CCCCTATGCATCAAAACAAG
CTCAGTGTCCGTCTTTGTCC
185~207 53
CAC06
(AG)
14
(CA)
9
TGTACATGTTTTTTGCCCAA
CGATGTAGCTACCTTCCCC
146~164 49
CAC08
(AG)
10
(CA)
9
ATCACCCCAATACAAGGACA
AATTCTATGGTCCACCCACA
198~290 53
CAC10
(AG)
13
(CA)
9
GATGGAAGGTGGTAATGCTG
GGAACCTCTTTTGGGTCATT
156~163 53
CAC11
(CA)
n
(TA)
n
GATCTTCGGCGTTCCTCA
TCTCCTCAACAATCTGAAGC
144~147 53
CAC13
(CA)
9
(TA)
5
A
(TA)
4
(CA)
6
GGGTTTTTTAGATCTTCGGC
CTCAACAATCTGAAGCATCG
151~153 53
CAC20
(CA)
19
CTCATGAACCAAACGTTATA
CATCATATACATACATGCAACA
124~133 52
CAC21
(CA)
11
AATTGTGTGACACGTAGCC
GCATAACTCTTTCATAAGGGA
149~151 53.7
CAC23
(CA)
8
TGAAAACAAAAGATAGATGTCAG GAAGATGCTTTGATATGGAAC
170~179 53.9
CAC39
(CA)
15
AATTGAGATAAGCAGATCAGT
GTCGGTCTTTATTCAGAAGG
142~166 53.8
CAC52
(CA)
19
TTATTTTCTCCACTTCTGTGG
ATATTACCCATGCACAGTACG
142~160 53.5
CAC56
(CA)
14
ATTCTTTTGGCTTAAAACATG
TGATTTTACAGTTACAAGTTTGG 138~162 53.9
CAC65
(CA)
15
GAAAAGGATGTAATAAGCTGG
TTTGTCCCCAAATATAGGTAG
150~173 53.4
CAC68
(CA)
13
AATTATTTTCTTGTTACATGCATC
AACAGCCTCTAGCAATCATAG3 130~146 54
CAC71
(CA)
17
ATAGCTCAAGTTGTTGCTAGG3
ATATTGTCATGATTGAGCCTC3
172~283 54
CAC72
(CA)
18
TCACATTATCAAATAAGTCTCACA GCTCTCTTTCTCATGCACA
124~132 54
CAC84
(CA)
13
TTGGTTTTTGTATGGAACTCT
AAATGCTAACATCTCAACAGC
150~163 54
CN11A10 (CT)
30
GTTGGAGATTTAATTTTCTTG
CCCAATAATATTTTATAACAG
81~119
46
CN11E10 (GT)
22
(GA)
14
AGAGAGAGTAAATGGGTAAGT
CCCTTTCATTTTTCCTTATTC
99~151
50
CN11E6 (CT)
21
TACTTAGGCAACGTTCCATTC
TAACCAGAAAGCAAAAAGATT
85~128
50
CN1C6
(CT)
1`
TT(GT)
5
AGTATGTGAGTAGGATTATGG
TTCCTTGGACCCTTATCTCTT
175~184 52
CN1G4
(CT)
15
GTCGTCCTATACTCATCATCA
GATGCGTATGAGATGTGAGAG
112~132 54
CN1H2
(GA)
18
TTGATAGGAGAGCTTCATAAC
ATCTTCTTTAATGCTCGGAGT
230~321 52
CN2A4
(CT)
15
TT(CT)
3
CAGGATGGTTCAAGCCCTTAA
GGTGGAAGAGGGAGAGATTGA 87~111
56
CN2A5
(CT)
12
TT(CT)
3
AAGGTGAAATCTATGAACACA
GGCAGTAACACATTACACATG
88~121
50
CNZ01
(CT)
15
(CA)
9
ATGATGATCTCTGGTTAGGCT
AAATGAGGGTTTGGAAGGATT
109~131 52
CNZ02
(GA)
15
CTCTTCCCATCATATACCAGC
ACTGGGGGGATCTTATCTCTG
143~161 56
CNZ03
(GA)
7
CATCTTTCATCATTTAGCTCT
AAACCAAAAGCAAGGAGAAGT 91~97
50
Note: The SSR primers were developed by Perera et al. (1999, 2000), Teulat et al. (2000), and Meerow et al. (2003)