Chinese Journal OF Rice Science ›› 2020, Vol. 34 ›› Issue (4): 316-324.DOI: 10.16819/j.1001-7216.2020.9134
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Guangda WANG1, Peng GAO1,2, Wenyan YANG1, Ao CUI1, Jianhua ZHAO1, Zhiming FENG1,2, Wenlei CAO1,2, Zongxiang CHEN1,2, Shimin ZUO1,2,*()
Received:
2019-12-13
Revised:
2020-02-19
Online:
2020-07-10
Published:
2020-07-10
Contact:
Shimin ZUO
王广达1, 高鹏1,2, 杨文艳1, 崔傲1, 赵剑华1, 冯志明1,2, 曹文磊1,2, 陈宗祥1,2, 左示敏1,2,*()
通讯作者:
左示敏
基金资助:
CLC Number:
Guangda WANG, Peng GAO, Wenyan YANG, Ao CUI, Jianhua ZHAO, Zhiming FENG, Wenlei CAO, Zongxiang CHEN, Shimin ZUO. Development and Utilization of Functional Markers for Imidazolinone Herbicides Resistance Gene in japonica Rice Variety Jinjing 818[J]. Chinese Journal OF Rice Science, 2020, 34(4): 316-324.
王广达, 高鹏, 杨文艳, 崔傲, 赵剑华, 冯志明, 曹文磊, 陈宗祥, 左示敏. 金粳818抗咪唑啉酮类除草剂基因的功能标记开发与应用[J]. 中国水稻科学, 2020, 34(4): 316-324.
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URL: http://www.ricesci.cn/EN/10.16819/j.1001-7216.2020.9134
标记名称 Marker name | 引物名称 Primer name | 引物序列(5'-3') Primer sequence (5'-3') |
---|---|---|
dC-ALS-627 | ALS-F ALS-R dC-ALS-627-F | CTCGCCCAAACCCAGAAACC CACATACAAACATCATAGGCATACCACTC GAGCATGTGCTGCCTATGATCCTAA |
dC-ALS-627-R | TAGAGCACATACAAACATCAT | |
K-ALS-627 | K-ALS627N-HEX | GAAGGTCGGAGTCAACGGATT ATCATGTCCTTGAATGCGCCCCCAT |
K-ALS627S-FAM | GAAGGTGACCAAGTTCATGCT ATCATGTCCTTGAATGCGCCCCCAC | |
K-ALS627S-N | TGTTGGATATCATCGTCCCGCAC |
Table 1 Primer sequences involved in this study.
标记名称 Marker name | 引物名称 Primer name | 引物序列(5'-3') Primer sequence (5'-3') |
---|---|---|
dC-ALS-627 | ALS-F ALS-R dC-ALS-627-F | CTCGCCCAAACCCAGAAACC CACATACAAACATCATAGGCATACCACTC GAGCATGTGCTGCCTATGATCCTAA |
dC-ALS-627-R | TAGAGCACATACAAACATCAT | |
K-ALS-627 | K-ALS627N-HEX | GAAGGTCGGAGTCAACGGATT ATCATGTCCTTGAATGCGCCCCCAT |
K-ALS627S-FAM | GAAGGTGACCAAGTTCATGCT ATCATGTCCTTGAATGCGCCCCCAC | |
K-ALS627S-N | TGTTGGATATCATCGTCCCGCAC |
Fig. 2. Information of the two molecular markers. The gray background part is the non-coding region sequence. The three consecutive bases in the box are the codons of amino acids at position 627 of ALS, ‘AAT’ in Jinjing 818 and ‘AGT’ in 9311 and Nipponbare, encoding amino acid ‘N’ and ‘S’ respectively. The recognition sequence of restriction endonuclease DdeⅠ is ‘CTNAG’ (‘N’ for any base). The mutant base ‘T’ was introduced into primer dC-ALS-627-F of the marker dC-ALS-627 (The base in template sequenceis ‘C’, ‘T’ and ‘C’ are highlighted with an underline). When the base ‘C’ in the template sequence of 9311 or Nipponbare is replaced with ‘T’, there is a DdeⅠ recognition sequence ‘CTAAG’ in the amplification product, which doesn’t exist in Jinjing 818. In the marker K-ALS-627, primer K-ALS627N- HEX has a base ‘T’ at the 3'end, and a HEX fluorescent label sequence (in italics) at the 5'end, and primer K-ALS627S- FAM has a base ‘C’ at the 3'end, and a FAM fluorescent label sequence (in italics) at the 5' end. The arrow at the front of primers indicates the direction of amplification.
Fig. 3. Detection results of three molecular markers. A, Detection results of dC-ALS-627; B, Detection results of K-ALS-627; C, Detection results of RM7413. M1, 20 bp DNA Ladder, M2, DL500 DNA Marker; P1, Jinjing 818, P2, Nanjing 505, P3, Huajing 0029; 1-3, Hybrid of Nanjing 505 / Jinjing 818; 4-6, Hybrid of Huajing 0029 / Jinjing 818. The diamond represents a blank control without the DNA template; the square represents the homozygous genotype of Jinjing 818 (variation site base A); the circle represents the homozygous genotypes of Nanjing 505 and Huajing 0029 (variation site base G); and the triangle represents the heterozygous type of ALS gene (with a G/A mutation site base).
Fig. 4. Genotyping of BC2F2 generation rice plants using dC-ALS-627 and K-ALS-627 markers. A, Detection results of dC-ALS-627. M, DL1000 DNA marker; P1, Jinjing 818; P2, Nanjing 505; 1-10, Partial of BC2F2 individuals of Nanjing 505 / Jinjing 818.B, Detection results of K-ALS-627. The black box represents a blank control without the DNA template; the blue box represents the homozygous genotype of Jinjing 818 (variation site base A); the yellow circle represents the homozygous genotypes of Nanjing 505 and Huajing 0029 (variation site base G); and the green triangle represents the heterozygous type of ALS gene (with a G/A mutation site base).
Fig. 5. Phenotypes of BC2F2 populations after treatment with imazethapyr. P1, Jinjing 818, P2, Nanjing 505. 1-10, Partial BC2F2 plants of Nanjing 505/ Jinjing 818.
Fig. 6. Screening herbicide resistant rice resources using dC-ALS-627 and K-ALS-627 markers. A, Detection results of dC-ALS-627. M, BM2000 DNA marker; J refers to Jinjing 818 and 1-40 refer to rice varieties sensitive to imidazolinone herbicides: Hejing 1819, Shujing 1901, Huajing 1803, Nanjing 46, Nanjing 52, Wuyunjing 23, Zhendao 11, Zhendao 14, Zhendao 14, Zhendao 18, Huaidao 5, Yangjing 103, Yangyujing 2, Yangyujing 3, Huajing 8, Huai 119, Wuyunjing 27, Wuyunjing 32, Wuyunjing 80, Yangnongdao 1, Yanjing 11, Yanjing 13, Yanjing 15, Yanjing 16, Yanjing 6, Zhendao 99, Daliang 202, Sidao 12, Sidao 15, Xinkedao 21, Xudao 5, Yuanhandao 3, Jinjing 18, Jinjing 787, Lianjing 10, Lianjing 4, Suken 118, Suxiu 867, Yangjing 818, Wujing 66, Ning 5718. B, Detection results of K-ALS-627. The black box represents a blank control without the DNA template; the blue box represents the homozygous genotype of Jinjing 818 (variation site base A); the yellow circle represents the homozygous genotypes of Nanjing 505 and Huajing 0029 (variation site base G).
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