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1.2 Genomic DNA Preparation
Tender leaves from donor materials were selected and
genomic DNA was obtained using CTAB method.
(Wang et al., 2003)
1.3 SSR loci selection
SSR markers were from RM series of Cornell
University. PCR amplification was conducted to 55
copies of material. 95 pairs of SSR primer from rice
chromosome were selected for preliminary screening
and 53 pairs of SSR primer were chosen because of
good amplification and clear banding (Table 2).
1.4 PCR amplification and loci detection
Method described by Deng was introduced and
adjusted in PCR for SSR analysis. (Deng et al., 2003)
PCR instrument is MJ PTC-100. 20μL reaction system
was chosen for PCR amplification, which included
25 ng total DNA, 1.5 mmol/L MgCl
2
, 0.2 mmol/L
dNTP, 1.0 U
Taq
DNA polymerase and 0.1 μmol/L
primer. PCR amplification procedure is listed as
follows: 94
℃
5 min, 94
℃
60s, 55
℃
30 s, 72
℃
60s,
35 cycles, 72
℃
10 min, 2/5 proportion of STR
loading buffer, degeneration at 95
℃
for 5 min,
immediate cooling, electrophoresis with 4% PAGE
using 2000V for 1~1.5hrs and then detection with
silver staining method (Panaud et al., 1996).
1.5 Data analysis
Database was set up with number “1” for the allelic
variation detected and “0” for not detected. Formula
proposed by Smith (1997) was used to calculate
polymorphism index content (PIC):
n
1
2
i
p 1 PIC
i
,
n means the number of test materials and P
i
stands for
variation frequency at locus “i”. On the basis of
genetic similarity, calculated as Jaccard (J), dendrogram
was established with UPGMA. Both J and UPGMA
were calculated by NTSYS-PC2.11.
Table 2 Analysis on diversity of SSR loci
Primers
Chromosome No. of
alleles
Size of segment
(bp)
PIC
Primers
Chromosome No. of
alleles
Size of segment
(bp)
PIC
RM9
1
4
124
-
194
0.646
RM214
7
4
114
-
152
0.572
RM259
1
5
156
-
175
0.748
RM125
7
6
107
-
204
0.530
RM23
1
5
134
-
150
0.345
RM248
7
6
80
-
104
0.761
RM208
2
4
166
-
180
0.658
RM210
8
4
141
-
165
0.683
RM250
2
7
154
-
174
0.612
RM331
8
5
149
-
179
0.770
RM324
2
4
135
-
180
0.727
RM38
8
7
246
-
278
0.485
RM53
2
5
168
-
208
0.603
RM72
8
7
152
-
198
0.503
RM475
2
5
188
-
235
0.478
RM201
9
4
144
-
158
0.719
RM227
3
4
99
-
107
0.592
RM105
9
4
131
-
140
0.609
RM282
3
4
128
-
136
0.672
RM215
9
5
147
-
153
0.730
RM85
3
4
85
-
107
0.610
RM219
9
5
192
-
222
0.715
RM545
3
4
212
-
230
0.555
RM257
9
6
121
-
173
0.469
RM251
3
5
114
-
150
0.636
RM278
9
4
131
-
146
0.660
RM55
3
4
217
-
235
0.451
RM271
10
4
92
-
105
0.664
RM7
3
5
168
-
182
0.535
RM228
10
6
108
-
154
0.609
RM261
4
4
122
-
126
0.730
RM222
10
5
199
-
215
0.707
RM280
4
5
148
-
181
0.287
RM258
10
5
133
-
152
0.672
RM470
4
5
83
-
122
0.720
RM332
11
4
162
-
183
0.480
RM252
4
7
196
-
262
0.678
RM202
11
4
158
-
189
0.537
RM335
4
6
104
-
155
0.609
RM21
11
7
132
-
170
0.647
RM340
6
5
119
-
189
0.719
RM206
11
7
128
-
202
0.758
RM541
6
4
156
-
192
0.648
RM224
11
7
124
-
158
0.786
RM50
6
5
180
-
202
0.758
RM101
12
4
258
-
334
0.626
RM204
6
6
106
-
194
0.633
RM19
12
4
219
-
252
0.450
RM528
6
7
232
-
260
0.714
RM270
12
5
104
-
117
0.757
RM276
6
5
85
-
153
0.523
RM235
12
6
96
-
134
0.638
RM234
7
5
133
-
163
0.639