生物技术通报 ›› 2022, Vol. 38 ›› Issue (7): 153-159.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1228
李白1(), 蔡之军1, 王蕾1, 陈婕1, 曹奎荣1, 李军2, 种高军1()
收稿日期:
2021-09-24
出版日期:
2022-07-26
发布日期:
2022-08-09
作者简介:
李白,男,硕士,农艺师,研究方向:分子辅助育种;E-mail: 基金资助:
LI Bai1(), CAI Zhi-jun1, WANG Lei1, CHEN Jie1, CAO Kui-rong1, LI Jun2, CHONG Gao-jun1()
Received:
2021-09-24
Published:
2022-07-26
Online:
2022-08-09
摘要:
Pigm是水稻抗稻瘟病基因之一,抗性广且持久。针对Pigm基因,已开发多种分子标记,但检测特异性或辨识度尚存不足。通过NCBI对7个Pigm标记进行序列比对分析,包括Pigm-4、T9E4、DG-2、M80410、R060939、GMR-3和T-Pigm,并对40个水稻品种开展检测验证。根据序列分析与检测结果开发了一种组合标记,应用于镇糯19×空育131(Pigm)分子标记辅助选择育种。序列分析结果表明,M80410标记特异性强,但不能区分杂合基因型;Pigm-4、T-Pigm和T9E4等标记能区分杂合基因型,但特异性略有不足。结合水稻品种检测结果,筛选了Pigm-4与M80410作为组合标记,并用于辅助育种,选育了糯稻品系NK29,具有较好稻瘟病抗性。组合标记能快速准确地鉴定Pigm 的纯合显性、纯合隐性和杂合基因型,为Pigm分子育种奠定基础。
李白, 蔡之军, 王蕾, 陈婕, 曹奎荣, 李军, 种高军. 抗稻瘟病基因Pigm组合标记的开发及应用[J]. 生物技术通报, 2022, 38(7): 153-159.
LI Bai, CAI Zhi-jun, WANG Lei, CHEN Jie, CAO Kui-rong, LI Jun, CHONG Gao-jun. Development and Application of the Combinatorial Marker for the Rice Blast Resistance Gene Pigm[J]. Biotechnology Bulletin, 2022, 38(7): 153-159.
标记名称 Marker name | 正向引物序列 Forward primer sequence(5'-3') | 反向引物序列 Reverse primer sequence(5'-3') |
---|---|---|
M80410[ | GGATTGTCTTGTCTCTCTCGC | CAGGACTTAGGGTTTCTCTCTTT |
Pigm-4[ | ATGCTCGATTCGTTACATTT | CGTCCCACACTTTCTTTTT |
T9E4[ | CAGAGCAGTAACAAACCCTA | TCCGCAAGATCAACATTC |
DG-2[ | GAGACGCAGGAACAGGGTG | CCGAGTGGCTGAGCAACAA |
R060939[ | CTCAGCAAACCATGTTCAGT | AAACAGCTCGTGGTTTCGCT |
GMR-3[ | AGTTCTACTTACGGAGGAGC | AGAATTATGATAAAGAGAAAGGAA |
T-Pigm[ (ARMS-PCR) | TAAGAATTGAGGTGGTTAGTTGAACGGAGA | TTGCATGGCTCCACTACCCACTATAAG |
TGAAAATAAAAATGGTATGATGGTTACG | TAGGGATGAAACGGCTCGAAAACGATCG |
表1 用于检测Pigm基因的分子标记
Table 1 Molecular markers used to detect Pigm
标记名称 Marker name | 正向引物序列 Forward primer sequence(5'-3') | 反向引物序列 Reverse primer sequence(5'-3') |
---|---|---|
M80410[ | GGATTGTCTTGTCTCTCTCGC | CAGGACTTAGGGTTTCTCTCTTT |
Pigm-4[ | ATGCTCGATTCGTTACATTT | CGTCCCACACTTTCTTTTT |
T9E4[ | CAGAGCAGTAACAAACCCTA | TCCGCAAGATCAACATTC |
DG-2[ | GAGACGCAGGAACAGGGTG | CCGAGTGGCTGAGCAACAA |
R060939[ | CTCAGCAAACCATGTTCAGT | AAACAGCTCGTGGTTTCGCT |
GMR-3[ | AGTTCTACTTACGGAGGAGC | AGAATTATGATAAAGAGAAAGGAA |
T-Pigm[ (ARMS-PCR) | TAAGAATTGAGGTGGTTAGTTGAACGGAGA | TTGCATGGCTCCACTACCCACTATAAG |
TGAAAATAAAAATGGTATGATGGTTACG | TAGGGATGAAACGGCTCGAAAACGATCG |
标记名称 Marker name | 谷梅4号 Pigm扩增片段 Gumei 4 Pigmamplified fragments | 相同或相似扩增片段Identical or similar amplified fragments | |||||||
---|---|---|---|---|---|---|---|---|---|
起始 Beginning | 终止 Termination | 长度 Length/bp | 片段名称,NCBI登记号 Fragment name,NCBI accession | 起始 Beginning | 终止 Termination | 长度 Length/bp | |||
Pigm-4 | 143 136 | 143 216 | 81 | Pi50,KP985760.1 | 44 033 | 44 113 | 81 | ||
Digu Pid4,MG839283.1 | 821 | 901 | 81 | ||||||
T9E4 | 115 153 | 115 945 | 793 | Pi50,KP985760.1 | 16 082 | 16 874 | 793 | ||
Shuhui498,CP018162.1 | 10 198 813 | 10 199 605 | 793 | ||||||
Zhonghua 11 Pizh,MH807580.1 | 114 411 | 115 202 | 792 | ||||||
Nipponbare,AP014962.1 | 10 408 011 | 10 408 803 | 793 | ||||||
DG-2 | 22 033 | 22 780 | 748 | Pi50,KP985759.1 | 21 992 | 22 739 | 748 | ||
Shuhui498,CP018162.1 | 10 136 210 | 10 136 990 | 781 | ||||||
Zhonghua 11 Pizh,MH807580.1 | 73 816 | 74 596 | 781 | ||||||
Pi9,DQ285630.1 | 66 719 | 67 468 | 750 | ||||||
M80410 | 91 983 | 92 499 | 517 | Null | - | - | - | ||
R060939 | -897 333 | -896 515 | 819 | Shuhui498,CP018162.1 | 9 343 478 | 9 344 296 | 819 | ||
Nipponbare,AP014962.1 | 9 395 525 | 9 396 343 | 819 | ||||||
RP Bio-226,CP012614.1 | 9 782 855 | 9 783 673 | 819 | ||||||
Zhenshan 97,CP056057.1 | 9 146 431 | 9 147 249 | 819 | ||||||
Minghui 63,CP054681.1 | 9 252 055 | 9 252 873 | 819 | ||||||
GMR-3 | 63 184 | 63 281 | 98 | Pi50,KP985759.1 | 6 509 | 6 606 | 98 | ||
RP Bio-226,CP012614.1 | 10 611 454 | 10 611 551 | 98 | ||||||
Piz(t),HE580189.1 | 2 382 | 2 479 | 98 | ||||||
T-Pigm | 11 149 | 11 443 | 295 | Pi50,KP985759.1 | 11 108 | 11 402 | 295 |
表2 Pigm标记引物NCBI比对分析
Table 2 Alignment and analysis of Pigm marker primers by NCBI
标记名称 Marker name | 谷梅4号 Pigm扩增片段 Gumei 4 Pigmamplified fragments | 相同或相似扩增片段Identical or similar amplified fragments | |||||||
---|---|---|---|---|---|---|---|---|---|
起始 Beginning | 终止 Termination | 长度 Length/bp | 片段名称,NCBI登记号 Fragment name,NCBI accession | 起始 Beginning | 终止 Termination | 长度 Length/bp | |||
Pigm-4 | 143 136 | 143 216 | 81 | Pi50,KP985760.1 | 44 033 | 44 113 | 81 | ||
Digu Pid4,MG839283.1 | 821 | 901 | 81 | ||||||
T9E4 | 115 153 | 115 945 | 793 | Pi50,KP985760.1 | 16 082 | 16 874 | 793 | ||
Shuhui498,CP018162.1 | 10 198 813 | 10 199 605 | 793 | ||||||
Zhonghua 11 Pizh,MH807580.1 | 114 411 | 115 202 | 792 | ||||||
Nipponbare,AP014962.1 | 10 408 011 | 10 408 803 | 793 | ||||||
DG-2 | 22 033 | 22 780 | 748 | Pi50,KP985759.1 | 21 992 | 22 739 | 748 | ||
Shuhui498,CP018162.1 | 10 136 210 | 10 136 990 | 781 | ||||||
Zhonghua 11 Pizh,MH807580.1 | 73 816 | 74 596 | 781 | ||||||
Pi9,DQ285630.1 | 66 719 | 67 468 | 750 | ||||||
M80410 | 91 983 | 92 499 | 517 | Null | - | - | - | ||
R060939 | -897 333 | -896 515 | 819 | Shuhui498,CP018162.1 | 9 343 478 | 9 344 296 | 819 | ||
Nipponbare,AP014962.1 | 9 395 525 | 9 396 343 | 819 | ||||||
RP Bio-226,CP012614.1 | 9 782 855 | 9 783 673 | 819 | ||||||
Zhenshan 97,CP056057.1 | 9 146 431 | 9 147 249 | 819 | ||||||
Minghui 63,CP054681.1 | 9 252 055 | 9 252 873 | 819 | ||||||
GMR-3 | 63 184 | 63 281 | 98 | Pi50,KP985759.1 | 6 509 | 6 606 | 98 | ||
RP Bio-226,CP012614.1 | 10 611 454 | 10 611 551 | 98 | ||||||
Piz(t),HE580189.1 | 2 382 | 2 479 | 98 | ||||||
T-Pigm | 11 149 | 11 443 | 295 | Pi50,KP985759.1 | 11 108 | 11 402 | 295 |
品种 Variety | T9E4 | DG-2 | Pigm-4 | M80410 | R060939 | GMR-3 | T-Pigm |
---|---|---|---|---|---|---|---|
9311 | - | - | - | - | + | - | - |
IR64 | - | - | - | - | - | + | - |
常农粳8号 Changnongjing 8 | - | - | - | - | + | - | - |
谷梅4号 Gumei 4 | + | + | + | + | + | + | + |
淮稻5号 Huaidao 5 | - | - | - | - | + | - | - |
黄华占 Huanghuazhan | - | - | - | - | + | - | - |
嘉58 Jia 58 | + | + | - | - | + | - | - |
嘉丰优2号 Jiafengyou 2 | - | - | - | - | ± | - | - |
嘉禾218 Jiahe 218 | - | - | - | - | + | - | - |
嘉禾优001 Jiaheyou 001 | - | - | - | - | ± | - | - |
嘉花1号 Jiahua 1 | + | + | - | - | + | - | - |
嘉科优11 Jiakeyou 11 | + | + | - | - | ± | - | - |
嘉优中科1号 Jiayouzhongke 1 | - | - | - | - | ± | + | - |
嘉育938 Jiayu 938 | + | + | - | - | + | - | - |
空育131(Pigm)Kongyu131(Pigm) | + | + | + | + | + | + | + |
连粳11号 Lianjing 11 | - | - | - | - | + | - | - |
南粳46 Nanjing 46 | - | - | - | - | + | - | - |
南粳9108 Nanjing 9108 | - | - | - | - | + | - | - |
宁84 Ning 84 | + | + | - | - | + | - | - |
日本晴 Nipponbare | + | - | - | - | + | - | - |
苏香粳100 Suxiangjing 100 | - | - | - | - | + | - | - |
苏秀867 Suxiu 867 | + | + | - | - | + | - | - |
太湖糯 Taihunuo | - | - | - | - | + | - | - |
特青 Teqing | - | - | - | - | + | - | - |
天优华占 Tianyouhuazhan | + | + | - | - | ± | - | - |
皖稻68 Wandao 68 | - | - | - | - | + | - | - |
武运粳23 Wuyunjing 23 | - | - | - | - | + | - | - |
湘晚籼17 Xiangwanxian 17 | - | - | - | - | + | - | - |
祥湖84 Xianhu 84 | - | - | - | - | + | - | - |
秀水134 Xiushui 134 | + | + | - | - | + | - | - |
秀优7113 Xiuyou 7113 | + | + | - | - | ± | + | - |
徐稻3号 Huaidao 3 | - | - | - | - | + | - | - |
甬优9 Yongyou 9 | - | - | - | - | ± | + | - |
玉针香 Yuzhenxiang | - | - | - | - | + | - | - |
浙粳88 Zhejing 88 | + | + | - | - | + | - | - |
镇稻11 Zhendao 11 | - | - | - | - | + | - | - |
镇糯19 Zhennuo 19 | - | - | - | - | + | - | - |
中禾优1号 Zhongheyou 1 | - | - | - | - | ± | - | - |
中花11 Zhonghua 11 | + | + | - | - | + | - | - |
中嘉8号 Zhongjia 8 | - | - | - | - | + | - | - |
表3 水稻样品Pigm标记检测
Table 3 Detection of Pigm markers in rice varieties
品种 Variety | T9E4 | DG-2 | Pigm-4 | M80410 | R060939 | GMR-3 | T-Pigm |
---|---|---|---|---|---|---|---|
9311 | - | - | - | - | + | - | - |
IR64 | - | - | - | - | - | + | - |
常农粳8号 Changnongjing 8 | - | - | - | - | + | - | - |
谷梅4号 Gumei 4 | + | + | + | + | + | + | + |
淮稻5号 Huaidao 5 | - | - | - | - | + | - | - |
黄华占 Huanghuazhan | - | - | - | - | + | - | - |
嘉58 Jia 58 | + | + | - | - | + | - | - |
嘉丰优2号 Jiafengyou 2 | - | - | - | - | ± | - | - |
嘉禾218 Jiahe 218 | - | - | - | - | + | - | - |
嘉禾优001 Jiaheyou 001 | - | - | - | - | ± | - | - |
嘉花1号 Jiahua 1 | + | + | - | - | + | - | - |
嘉科优11 Jiakeyou 11 | + | + | - | - | ± | - | - |
嘉优中科1号 Jiayouzhongke 1 | - | - | - | - | ± | + | - |
嘉育938 Jiayu 938 | + | + | - | - | + | - | - |
空育131(Pigm)Kongyu131(Pigm) | + | + | + | + | + | + | + |
连粳11号 Lianjing 11 | - | - | - | - | + | - | - |
南粳46 Nanjing 46 | - | - | - | - | + | - | - |
南粳9108 Nanjing 9108 | - | - | - | - | + | - | - |
宁84 Ning 84 | + | + | - | - | + | - | - |
日本晴 Nipponbare | + | - | - | - | + | - | - |
苏香粳100 Suxiangjing 100 | - | - | - | - | + | - | - |
苏秀867 Suxiu 867 | + | + | - | - | + | - | - |
太湖糯 Taihunuo | - | - | - | - | + | - | - |
特青 Teqing | - | - | - | - | + | - | - |
天优华占 Tianyouhuazhan | + | + | - | - | ± | - | - |
皖稻68 Wandao 68 | - | - | - | - | + | - | - |
武运粳23 Wuyunjing 23 | - | - | - | - | + | - | - |
湘晚籼17 Xiangwanxian 17 | - | - | - | - | + | - | - |
祥湖84 Xianhu 84 | - | - | - | - | + | - | - |
秀水134 Xiushui 134 | + | + | - | - | + | - | - |
秀优7113 Xiuyou 7113 | + | + | - | - | ± | + | - |
徐稻3号 Huaidao 3 | - | - | - | - | + | - | - |
甬优9 Yongyou 9 | - | - | - | - | ± | + | - |
玉针香 Yuzhenxiang | - | - | - | - | + | - | - |
浙粳88 Zhejing 88 | + | + | - | - | + | - | - |
镇稻11 Zhendao 11 | - | - | - | - | + | - | - |
镇糯19 Zhennuo 19 | - | - | - | - | + | - | - |
中禾优1号 Zhongheyou 1 | - | - | - | - | ± | - | - |
中花11 Zhonghua 11 | + | + | - | - | + | - | - |
中嘉8号 Zhongjia 8 | - | - | - | - | + | - | - |
图2 M80410、Pigm-4扩增条带类型 1:谷梅4号M80410扩增片段;2:日本晴 Pigm-4扩增片段;3:9311 Pigm-4扩增片段;4:谷梅4号 Pigm-4扩增片段
Fig.2 Types of M80410 and Pigm-4 amplified bands 1:M80410 amplified fragment of Gumei 4;2:Pigm-4 amplified fragment of Nipponbare;3:Pigm-4 amplified fragment of 9311;4:Pigm-4 amplified fragment of Gumei 4
图3 M80410和Pigm-4组合标记扩增条带结果 1:空育131(Pigm);2:镇糯19;3:杂交F1;4:NK29;5-31:部分高世代材料
Fig.3 Amplification results of M80410 and Pigm-4 combi-ned marker 1:Kongyu 131(Pigm);2:Zhennuo 19;3:hybrid F1;4:Nk29;5-31:part of high generation materials
材料名称 Material name | 基因型 Genotype | 叶瘟发病指数 Leaf blast incidence/(±s) | 抗病性 Blast resistance |
---|---|---|---|
镇糯19 | - | 5.5±0.27 | MS |
空育131(Pigm) | + | 1.71±0.13 | R |
F1 | ± | 1.23±0.34 | R |
BC1F1 | + | 0.85±0.47 | R |
BC2F6-1 | + | 1.69±0.28 | R |
BC2F6-2 | + | 2.10±0.39 | MR |
BC2F6-3 | ± | 1.14±0.26 | R |
BC2F6-4 | + | 0.92±0.46 | R |
BC2F6-5 | + | 2.33±0.52 | MR |
BC2F6-6 | ± | 1.14±0.63 | R |
BC2F6-7 | + | 1.35±0.49 | R |
BC2F6-8 | + | 1.61±0.25 | R |
BC2F6-9(NK29) | + | 1.78±0.54 | R |
BC2F6-10 | ± | 3.18±0.15 | MR |
BC2F6-11 | + | 0.91±0.38 | R |
BC2F6-12 | + | 1.82±0.12 | R |
表4 Pigm导入系高世代稻瘟病抗性鉴定
Table 4 Identification of blast resistance of Pigm introgre-ssion line in high generation
材料名称 Material name | 基因型 Genotype | 叶瘟发病指数 Leaf blast incidence/(±s) | 抗病性 Blast resistance |
---|---|---|---|
镇糯19 | - | 5.5±0.27 | MS |
空育131(Pigm) | + | 1.71±0.13 | R |
F1 | ± | 1.23±0.34 | R |
BC1F1 | + | 0.85±0.47 | R |
BC2F6-1 | + | 1.69±0.28 | R |
BC2F6-2 | + | 2.10±0.39 | MR |
BC2F6-3 | ± | 1.14±0.26 | R |
BC2F6-4 | + | 0.92±0.46 | R |
BC2F6-5 | + | 2.33±0.52 | MR |
BC2F6-6 | ± | 1.14±0.63 | R |
BC2F6-7 | + | 1.35±0.49 | R |
BC2F6-8 | + | 1.61±0.25 | R |
BC2F6-9(NK29) | + | 1.78±0.54 | R |
BC2F6-10 | ± | 3.18±0.15 | MR |
BC2F6-11 | + | 0.91±0.38 | R |
BC2F6-12 | + | 1.82±0.12 | R |
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