Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (6): 955-967.doi: 10.3864/j.issn.0578-1752.2019.06.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Development and Application of the Functional Marker for the Broad-Spectrum Blast Resistance Gene PigmR in Rice

WANG FangQuan1,2,CHEN ZhiHui1,2,XU Yang1,2,WANG Jun1,2,LI WenQi1,2,FAN FangJun1,2,CHEN LiQin1,TAO YaJun1,2,ZHONG WeiGong1,YANG Jie1,2()   

  1. 1 Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement/Jiangsu High Quality Rice R & D Center, Nanjing 210014;
    2 Yangzhou University/Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou 225009, Jiangsu
  • Received:2018-11-18 Accepted:2019-01-18 Online:2019-03-16 Published:2019-03-22
  • Contact: Jie YANG E-mail:yangjie168@aliyun.com

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Abstract:

【Objective】Rice blast is one of the most serious rice diseases in the world. The objective of this study was to develop the functional marker for broad-spectrum blast resistance gene PigmR in rice. The marker improved the application of PigmR in blast resistance rice breeding. 【Method】The characteristics of PigmR were analyzed by Snapgene 2.3.2, and the specific function markers were designed with Oligo 7. To avoid mistake results of the PCR amplification failure, the functional marker was optimized by the specific primers of Actin1 as a reference. The parent materials, the monogenic lines of Lijiangxintuanheigu (LTH), the bridge materials, and the BC1F3 population lines of Nangeng 53045/Gumei 4 were identified by the functional markers. The blast isolates in this study were the mixed representative strains of rice blast in Jiangsu Province (2018-4, 2018-65, 2018-102, 2018-222, and 2018-241). The tested isolates were transplanted into RCA medium, cultured at 25 ℃ for 7 d, and irradiated for 72 h. After spores were produced, they were washed with sterile water and then formulated 30-40 spores per field in 10×10 microscope. The mixed spores were injected into each panicle with 1 mL of blast isolates solution at 3-4 days before heading. The resistance was investigated after the rice grains were matured.【Result】Eight pairs of molecular markers were designed, according to the sequence difference of PigmR and PigmS, Pigm-R4. By molecular detected, the function marker of PigmR, GMR-3, could specifically amplify PigmR from Gumei 4 with 98 bp fragment, and no fragment was amplified in the samples without PigmR. The functional marker was optimized by different concentrations of GMR-3 and Actin1-1 (a marker for the internal reference gene Actin1), results shown that the marker consist of 0.4 μmol·L -1 GMR-3 and 0.1 μmol·L -1 Actin1-1 had the best effect. The functional marker was named GMRA. Samples carrying PigmR were amplified the expected size of 146 and 98 bp by GMRA. By contrast, samples without PigmR were only amplified a 146 bp fragment. The 229 rice materials were detected with GMRA, only Gumei 4 amplified the size of 146 and 98 bp, others only amplified a 146 bp fragment. Furthermore, the results detected the monogenic lines of LTH by GMRA suggested that the marker had a strong specificity and could effectively distinguish PigmR from homology genes, such as Pi9, Piz, and Piz-t. Moreover, three donor materials carrying PigmR were obtained from 240 bridge materials by GMRA. By molecular marker-assisted selection (MAS) of GMRA, PigmR was transferred to the good eating quality rice cultivar Nangeng53045 by backcrossing. The BC1F3 plants with PigmR showed resistant/middle resistant to the panicle blast, while others showed high susceptible. It was suggested that PigmR observably improved the resistance of Nangeng53045-Pigm lines in the panicle blast.【Conclusion】In conclusion, the functional marker of PigmR can be effectively used for genetic improvement in breeding and germplasm screening.

Key words: rice (Oryza sativa L.), rice blast, Pigm, functional marker, molecular marker-assisted selection

Fig. 1

Sequence alignment of the variation sites between PigmR and parts of its homologous genes"

Table 1

The primers of molecular marker"

标记名称
Marker name		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 理论产物长度
Expected size (bp)
GMR-1 GMR-1F TTCCTTTCTCTTTATCATAATT 180
GMR-1R AGATTCCAACCTGCACTTGCCT
GMS-1 GMS-1F GTCCTTGATCTTTATCAGAAAG 180
GMS-1R TGGTTCCAACCTGCAGAATCTG
GMR-2 GMR-2F TTCCTTTCTCTTTATCATAcTT 180
GMR-2R AGATTCCAACCTGCACTTGCCT
GMS-2 GMS-2F GTCCTTGATCTTTATCAGAcAG 180
GMS-2R TGGTTCCAACCTGCAGAATCTG
GMR-3 GMR-3F AGTTCTACTTACGGAGGAGC 98
GMR-3R AGAATTATGATAAAGAGAAAGGAA
GMS-3 GMS-3F AGATCAACTTATTGAGCAGC 98
GMS-3R AGCTTTCTGATAAAGATCAAGGAC
GMR-4 GMR-4F AGTTCTACTTACGGAGGcGC 98
GMR-4R AGAATTATGATAAAGAGAAAGcAA
GMS-4 GMS-4F AGATCAACTTATTGAGCcGC 98
GMS-4R AGCTTTCTGATAAAGATCAAGcAC
Actin1-1 Actin1-1F ACAAAGTTTTCAACCGGCCTA 146
Actin1-1R CTGGTACCCTCATCAGGCATC
Actin1-2 Actin1-2F AGTCTGGCCCATCCATTGTG 149
Actin1-2R CGGTTGAAAACTTTGTCCACGCTA

Fig. 2

The PCR amplification products of molecular maker for PigmR M: DL2000 marker; 1-8: GMR-1, GMS-1, GMR-2, GMS-2, GMR-3, GMS-3, GMR-4, and GMS-4"

Fig. 3

PCR amplification products of molecular maker for Actin1 M: DL2000 marker; 1-3 represent Gumei 4, Niponbare, and Huanghuazhan"

Fig. 4

Optimization of identification primers for PigmR M: DL2000 marker; 1-12: Represent the combination with the different concentration of primers, including 0.2 μmol·L-1 GMR-3+0 μmol·L-1 Actin1-1, 0 μmol·L-1 GMR-3+0.2 μmol·L-1 Actin1-1, 0.2 μmol·L-1 GMR-3+0.2 μmol·L-1 Actin1-1, 0.1 μmol·L-1 GMR-3+0.1 μmol·L-1 Actin1-1, 0.2 μmol·L-1 GMR-3+0.1 μmol·L-1 Actin1-1, 0.2 μmol·L-1 GMR-3+0.05 μmol·L-1 Actin1-1, 0.4 μmol·L-1 GMR-3+0.1 μmol·L-1 Actin1-1, 0.4 μmol·L-1 GMR-3+0.05 μmol·L-1 Actin1-1, 0.4 μmol·L-1 GMR-3+0.025 μmol·L-1 Actin1-1, 0.8 μmol·L-1 GMR-3+0.1 μmol·L-1 Actin1-1, 0.8 μmol·L-1 GMR-3+0.05 μmol·L-1 Actin1-1, and 0.8 μmol·L-1 GMR-3+0.025 μmol·L-1 Actin1-1"

Fig. 5

Molecular detection for partial varieties by GMRA M: DL2000 marker; 1-20: Represent Gumei 4, Niponbare, Huanghuazhan, IR36, Yuzhenxiang, Nanjing 16, Kasalath, Liangeng 7, Nangeng 9108, Wuyungeng 24, Huageng 5, Yangyugeng 2, Nangeng 44, Nangeng 5055, Jingeng 818, Suxiu 867, Wuyungeng 21, Wuyungeng 27, Huaidao 5, and Nangeng53045"

Table 2

Identification of partial rice materials by GMRA"

编号
No.		 品种
Variety		 粳/籼稻 Geng/Xian 带型
Type		 编号
No.		 品种
Variety		 粳/籼稻 Geng/Xian 带型
Type
1 谷梅4号 Gumei 4 X 2 40 盐粳10号 Yandao 10 G 1
2 日本晴 Niponbare G 1 41 盐粳11号 Yangeng 11 G 1
3 黄华占 Huanghuazhan X 1 42 镇稻99 Zhendao 99 G 1
4 IR36 X 1 43 扬粳113 Yanggeng 113 G 1
5 玉针香 Yuzhenxiang X 1 44 WP16 G 1
6 南京16号 Nanjing 16 X 1 45 WP59 G 1
7 Kasalath X 1 46 泗稻15号 Sidao 15 G 1
8 连粳7号 Liangeng 7 G 1 47 连糯1号 Liannuo 1 G 1
9 南粳9108 Nangeng 9108 G 1 48 皖粳糯1号 Liangengnuo 1 G 1
10 武运粳24号 Wuyungeng 24 G 1 49 圣稻19 Shengdao 19 G 1
11 华粳5号 Huageng 5 G 1 50 JX14-3 G 1
12 扬育粳2号 Yangyugeng 2 G 1 51 JX14-4 G 1
13 南粳44 Nangeng 44 G 1 52 K11 G 1
14 南粳45 Nangeng 45 G 1 53 N14-109 G 1
15 南粳46 Nangeng 46 G 1 54 宁粳7号 Ninggeng 7 G 1
16 南粳5055 Nangeng 5055 G 1 55 YJN1 G 1
17 金粳818 Jingeng 818 G 1 56 常粳144 Changgeng 144 G 1
18 苏秀867 Suxiu 867 G 1 57 常农粳4号 Changnonggeng 4 G 1
19 武运粳21号 Wuyungeng 21 G 1 58 常农粳6号 Changnonggeng 6 G 1
20 武运粳27号 Wuyungeng 27 G 1 59 常农粳7号 Changnonggeng 7 G 1
21 淮稻5号 Huaidao 5 G 1 60 Feng1326 G 1
22 南粳53045 Nangeng53045 G 1 61 华粳3号 Huageng 3 G 1
23 武运粳29号 Wuyungeng 29 G 1 62 华粳4号 Huageng 4 G 1
24 楚粳39 Chugeng 39 G 1 63 华粳6号 Huageng 6 G 1
25 丰粳3227 Fenggeng 3227 G 1 64 华瑞稻1号 Huaruidao 1 G 1
26 华粳7号 Huageng 7 G 1 65 淮1188 Huai 1188 G 1
27 淮稻7号 Huaidao 7 G 1 66 淮208 Huai 208 G 1
28 淮糯11号 Huainuo 11 G 1 67 淮稻10号 Huaidao 10 G 1
29 淮优粳2号 Huaiyougeng 2 G 1 68 淮稻11号 Huaidao 11 G 1
30 连粳11号 Liangeng 11 G 1 69 淮稻13号 Huaidao 13 G 1
31 连粳12号 Liangeng 12 G 1 70 淮稻8号 Huaidao 8 G 1
32 连粳4号 Liangeng 4 G 1 71 淮稻9号 Huaidao 9 G 1
33 连粳6号 Liangeng 6 G 1 72 淮粳096 Huaigeng 096 G 1
34 连粳147729 Liangeng 147729 G 1 73 淮香稻15号 Huaixiangdao 15 G 1
35 农香软米 Nongxiangruanmi G 1 74 津稻263 Jindao 263 G 1
36 泗稻785 Sidao 785 G 1 75 连粳9号 Liangeng 9 G 1
37 苏秀326 Suxiu 326 G 1 76 连粳10号 Liangeng 10 G 1
38 徐稻3号 Xudao 3 G 1 77 南粳40 Nangeng 40 G 1
39 盐稻11号 Yandao 11 G 1 78 南粳41 Nangeng 41 G 1
79 南粳42 Nangeng 42 G 1 119 镇稻7号 Zhendao 7 G 1
80 南粳49 Nangeng 49 G 1 120 镇稻88 Zhendao 88 G 1
81 宁2600 Ning 2600 G 1 121 镇稻9424 Zhendao 9424 G 1
82 宁粳2号 Ninggeng 2 G 1 122 宁粳1号 Ninggeng 1 G 1
83 宁粳4号 Ninggeng 4 G 1 123 宁粳3号 Ninggeng 3 G 1
84 宁粳5号 Ninggeng 5 G 1 124 南粳47 Nangeng 47 G 1
85 迁稻11-72 Qiandao 11-72 G 1 125 通粳981 Tonggeng 981 G 1
86 泗稻12号 Sidao 12 G 1 126 武运粳7号 Wuyungeng 7 G 1
87 苏沪香粳 Suhuxianggeng G 1 127 武运粳19号 Wuyungeng 19 G 1
88 苏粳5号 Sugeng 5 G 1 128 武运粳23号 Wuyungeng 23 G 1
89 苏秀10号 Suxiu 10 G 1 129 常农粳3号 Changnonggeng 3 G 1
90 武粳13号 Wugeng 13 G 1 130 常农粳5号 Changnonggeng 5 G 1
91 武粳15号 Wugeng 15 G 1 131 苏粳8号 Sugeng 8 G 1
92 武粳4号 Wugeng 4 G 1 132 苏香粳2号 Suxianggeng 2 G 1
93 武陵粳1号 Wulinggeng 1 G 1 133 嘉33 Jia 33 G 1
94 武香粳14号 Wuxianggeng 14 G 1 134 嘉991 Jia 991 G 1
95 武香粳9号 Wuxianggeng 9 G 1 135 镇稻10号 Zhendao 10 G 1
96 武育粳18号 Wuyugeng 18 G 1 136 镇稻16号 Zhendao 16 G 1
97 武育粳3号 Wuyugeng 3 G 1 137 镇稻17号 Zhendao 17 G 1
98 武运粳11号 Wuyungeng 11 G 1 138 镇稻18号 Zhendao 18 G 1
99 兴化紫稻 Xinghuazidao G 1 139 苏粳9号 Sugeng 9 G 1
100 徐稻8号 Xudao 8 G 1 140 宁2602 Ning 2602 G 1
101 徐稻9号 Xudao 9 G 1 141 宁2604 Ning 2604 G 1
102 盐稻8号 Yandao 8 G 1 142 嘉58 Jia 58 G 1
103 盐稻9号 Yandao 9 G 1 143 苏05-1176 Su 05-1176 G 1
104 盐粳16号 Yangeng 16 G 1 144 苏12-130 Su 12-130 G 1
105 盐粳9号 Yangeng 9 G 1 145 沪香软268 Huxiangruan 268 G 1
106 扬9709 Yang9700 G 1 146 沪香软386 Huxiangruan 386 G 1
107 扬辐粳1号 Yangfugeng 1 G 1 147 软大穗 Ruandaoli G 1
108 扬辐粳8号 Yangfugeng 8 G 1 148 大粒糯 Dalinuo G 1
109 扬粳4227 Yanggeng 4227 G 1 149 太湖香粳稻 Taihuxianggengdao G 1
110 扬粳4308 Yanggeng 4308 G 1 150 JX14-5 G 1
111 扬粳805 Yanggeng 805 G 1 151 南粳43 Nangeng 43 G 1
112 扬农210 Yangnong210 G 1 152 农垦57 Nongken 57 G 1
113 扬中稻1号 Yangzhongdao 1 G 1 153 农垦58 Nongken 58 G 1
114 镇糯19号 Zhennuo 19 G 1 154 秀水63 Xiushui 63 G 1
115 镇稻12号 Zhendao 12 G 1 155 徐81698 Xu 81698 G 1
116 镇稻13号 Zhendao 13 G 1 156 徐稻4号 Xudao 4 G 1
117 镇稻15号 Zhendao 15 G 1 157 盐稻980 Yandao 980 G 1
118 镇稻1号 Zhendao 1 G 1 158 盐丰2号 Yanfeng 2 G 1
159 扬粳9224 Yanggeng 9224 G 1 195 14CZY43 G 1
160 扬育粳116 Yangyugeng 116 G 1 196 14CZY47 G 1
161 银玉2239 Yinyu 2239 G 1 197 14CZY49 G 1
162 镇稻615 Zhendao 615 G 1 198 14CZY53 G 1
163 隆粳968 Longgeng 968 G 1 199 14CZY60 G 1
164 大华香糯 Dahuaxiangnuo G 1 200 14CZY65 G 1
165 华粳8号 Huageng 8 G 1 201 14CZY71 G 1
166 盐粳13号 Yangeng 13 G 1 202 14CZY73 G 1
167 盐粳15号 Yangeng 15 G 1 203 14CZY83 G 1
168 南粳505 Nangeng 505 G 1 204 14CZY84 G 1
169 盐丰稻2号 Yanfengdao 2 G 1 205 14CZY85 G 1
170 软香玉 Ruanxiangyu G 1 206 14CZY86 G 1
171 苏香粳100 Suxianggeng 100 G 1 207 14CZY87 G 1
172 苏2100 Su 2100 G 1 208 15ZZY2 G 1
173 沪粳137 Hugeng 137 G 1 209 15ZZY7 G 1
174 沪香粳165 Huxianggeng 165 G 1 210 15ZZY8 G 1
175 松粳9号 Songgeng 9 G 1 211 15ZZY79 G 1
176 14CZY1 G 1 212 15ZZY89 G 1
177 14CZY8 G 1 213 15ZZY16 G 1
178 14CZY11 G 1 214 15ZZY48 G 1
179 14CZY13 G 1 215 15ZZY53 G 1
180 14CZY15 G 1 216 15ZZY58 G 1
181 14CZY16 G 1 217 15ZZY73 G 1
182 14CZY17 G 1 218 15ZZY90 G 1
183 14CZY21 G 1 219 15ZZQ6 G 1
184 14CZY22 G 1 220 15ZZQ12 G 1
185 14CZY23 G 1 221 15CZY3 G 1
186 14CZY28 G 1 222 15CZY31 G 1
187 14CZY30 G 1 223 15CZY65 G 1
188 14CZY31 G 1 224 15CZY70 G 1
189 14CZY36 G 1 225 15CZY71 G 1
190 14CZY37 G 1 226 15CZY77 G 1
191 14CZY38 G 1 227 15CZY84 G 1
192 14CZY39 G 1 228 15CZY86 G 1
193 14CZY40 G 1 229 15CZY91 G 1
194 14CZY41 G 1

Table 3

Identification of partial monogenic lines of Lijiangxintuanheigu by GMRA"

编号No. 名称Designation 目标基因Target gene 带型Type
1 IRBLa-A Pia 1
2 IRBLa-C Pia 1
3 IRBLi-F5 Pii 1
4 IRBLks-F5 Pik-s 1
5 IRBLks-S Pik-s 1
6 IRBLk-ka Pik 1
7 IRBLkp-K60 Pik-p 1
8 IRBLkh-K3 Pik-h 1
9 IRBLz-Fu Piz 1
10 IRBLzt-T Piz-t 1
11 IRBLta-K1 Pita 1
12 IRBLta-CT2 Pita 1
13 IRBLb-B Pib 1
14 IRBLt-K59 Pit 1
15 IRBLsh-S Pish 1
16 IRBLsh-B Pish 1
17 IRBL1-CL Pi1 1
18 IRBL3-CP4 Pi3 1
19 IRBL5-M Pi5(t) 1
20 IRBL7-M Pi7(t) 1
21 IRBL9-W Pi9 1
22 IRBL12-M Pi12(t) 1
23 IRBL19-A Pi19 1
24 IRBLkm-Ts Pik-m 1
25 IRBL20-IR24 Pi20 1
26 IRBLta2-Pi Pita2 1
27 IRBLta2-Re Pita2 1
28 IRBLta-CP1 Pita 1
29 IRBL11-Zh Pi11(t) 1
30 丽江新团黑谷 Lijiangxintuanheigu - 1
31 谷梅4号 Gumei 4 Pigm 2

Fig. 6

The molecular detection of BC1F2 of Nangeng53045/Gumei 4 by GMRA M: DL2000 marker; 1-24: Represent partial BC1F2 plants of Nangeng53045/Gumei 4"

Fig. 7

The molecular detection of BC1F3 of Nangeng53045/Gumei 4 by GMRA M: DL2000 marker; 1-20: Represent partial BC1F3 plants of Nangeng53045/Gumei 4"

Fig. 8

Infection of rice panicle blast in BC1F3 plants of Nangeng53045/Gumei 4 PigmR-: The plants without PigmR; PigmR+: The plants with PigmR"

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