Development and Utilization of Functional Markers for Imidazolinone Herbicides Resistance Gene in japonica Rice Variety Jinjing 818

doi:10.16819/j.1001-7216.2020.9134

Abstract

Abstract:

【Objective】Cultivating herbicide resistant rice varieties is an important direction in rice breeding. Developing efficient functional markers can accelerate the screening of herbicide resistance trait. 【Method】dCAPS and KASP techniques were used to analyze the genotypes of the experimental materials. In addition, the phenotypes were identified by herbicide spraying test. 【Result】Through sequence alignment analysis, we further confirmed that the mutation of 627 amino acid of acetolactate synthase (ALS) gene from serine (S) into asparagine (N) in Jinjing 818 (here after named ALS^627N) produces strong resistance to imidazolinone herbicides. According to the DNA sequence flanking the mutation site, we developed two novel functional markers, dC-ALS-627 and K-ALS-627, for detecting this resistant allele ALS^627N. The two markers can effectively distinguish homozygous ALS^627N type, heterozygous type and homozygous wild type. Combined with herbicide spraying test, we confirmed that ALS^627N is a dominant herbicide resistant gene. 【Conclusion】 We validated that the two markers have great potential in marker-assisted breeding for ALS^627N gene. Totally, this study will accelerate the development of new varieties with resistance to imidazolinone herbicides, and have significance in breeding practice.

Key words: rice, imidazolinone herbicide, acetolactate synthase, functional marker, assisted selection

摘要：

【目的】培育抗除草剂品种是水稻育种中的一个重要方向,开发高效的功能标记可以加快抗除草剂性状的筛选工作。【方法】利用开发的dCAPS和KASP分子标记对试验材料的基因型分型,另结合除草剂喷施试验进行表型鉴定。【结果】测序结果验证了前人发现的金粳818中乙酰乳酸合成酶（acetolactate synthase,ALS）基因第627位氨基酸由丝氨酸（serine,S）突变为天冬酰胺（asparagine,N）是其抗咪唑啉酮类除草剂的主要原因,本文将其命名为ALS^627N。根据此变异位点的DNA序列差异,开发了两个新的功能标记dC-ALS-627和K-ALS-627,可有效区分ALS^627N纯合型、杂合型及非突变纯合型。【结论】两个标记均可以广泛用于ALS^627N基因的标记辅助选择育种,有助于加快培育抗咪唑啉酮类除草剂新品种,具有重要的实际应用价值。

关键词: 水稻, 咪唑啉酮类除草剂, 乙酰乳酸合成酶, 功能标记, 辅助选择

CLC Number:

S511.032

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.

Figures/Tables 7

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	dC-ALS-627-R	TAGAGCACATACAAACATCAT
K-ALS-627	K-ALS^627N-HEX	GAAGGTCGGAGTCAACGGATT ATCATGTCCTTGAATGCGCCCCCAT
	K-ALS^627S-FAM	GAAGGTGACCAAGTTCATGCT ATCATGTCCTTGAATGCGCCCCCAC
	K-ALS^627S-N	TGTTGGATATCATCGTCCCGCAC

Fig. 1. ALS gene sequence alignment results.

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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