Development and Application of Functional Markers for Purity Identification of Wild-abortive Three-line Hybrid Rice

doi:10.16819/j.1001-7216.2026.250204

Abstract

Abstract:

【Objective】 Developing stable, efficient, and reliable markers for purity testing of three-line hybrid seeds is crucial for current hybrid rice seed production. 【Method】 Based on the specific sequences of the wild abortive-type sterility gene WA352, the major fertility-restoring gene Rf4, and the internal reference gene Actin, multiple single-gene detection markers were designed. PCR amplification was then performed by optimizing the primer mixture ratio. 【Result】 A mixture of primers WA352-2, Rf4-3, and Actin-3 in a ratio of 1:11:3 enabled simultaneous detection of the genotypes of WA352, Rf4, and Actin in a single PCR reaction, allowing differentiation among wild abortive-type CMS lines, restorer lines, maintainer lines, and hybrids. 【Conclusion】 The detection method established in this study is simple to perform and yields stable and reliable results. It can not only identify genotypes in wild-abortive three-line rice but also serve as an effective tool for molecular marker-assisted breeding. By improving the accuracy of purity assessment during breeding, this method provides strong support for achieving high-yield and high-quality rice breeding objectives in China.

Key words: rice, wild-abortive, three-line, detection marker, PCR

摘要：

【目的】开发稳定、高效、可靠的三系杂交种纯度检测标记，对于当前三系水稻制种而言至关重要。【方法】根据野败型不育基因WA352、主效恢复基因Rf4和内参基因Actin的特异性序列，设计了多对单基因检测标记，并通过调整引物混合比例，进行PCR扩增。【结果】引物WA352-2、Rf4-3和Actin-3按照1∶11∶3的比例混合可实现单次PCR扩增同时检测WA352、Rf4和Actin的基因类型，区分野败型不育系、恢复系、保持系和杂交种。【结论】本研究开发的检测标记操作方法简便，结果稳定可靠，不仅能够检测野败型三系水稻的基因型，还可以作为分子标记辅助育种的有效工具，提高育种过程中纯度检测结果的准确性，从而为我国水稻高产优质育种目标的实现提供有力支持。

关键词: 水稻, 野败, 三系, 检测标记, PCR

ZHANG Mengke, LU Jiayu, HE Jin, XU Xue, WU Shuang, WANG Peiran, CHEN Ruofan, JIN Qing, WANG Xiufeng. Development and Application of Functional Markers for Purity Identification of Wild-abortive Three-line Hybrid Rice[J]. Chinese Journal OF Rice Science, 2026, 40(2): 244-252.

张梦柯, 陆佳雨, 何金, 许学, 吴爽, 王沛然, 陈若凡, 金青, 汪秀峰. 水稻野败型三系杂交种纯度检测功能标记的开发与应用[J]. 中国水稻科学, 2026, 40(2): 244-252.

Figures/Tables 8

Table 1. Sequence of PCR amplification primers

引物名称 Primer name	引物序列 Primer sequence	目标基因 Target gene	目的条带大小 Target band size(bp)
WA352-1	F: GTGCTTAAATCACTACAGGAGCG R: CCCTGGAAGCGGTTGATTGA	WA352	528
WA352-2	F: GAGGATTTTGCCTCCCCTCC R: GGTGCAACCTAGCCAAGTCT	WA352	604
WA352-3	F: GCTTACGCTATGGAGAACCCT R: TTCCCCCGTGCTTTCTTGA	WA352	93
Rf4-1	F: CTTTAGTTCAATAATTAGCGATCT R: TGACATTGGGCTTCACACCA	Rf4	101
Rf4-2	F: CTTTAGTTCAATAATTAGCGATCT R: ACCAGCTAAGCAGCATCCAT	Rf4	142
Rf4-3	F: CTTTAGTTCAATAATTAGCGATCT R: ACTAACGCGTCTTCCATCCT	Rf4	267
Actin-1	F: GCTCCTGAGGAACATCCAATTT R: AGCCACATACATTGCTGGTG	Actin	117
Actin-2	F: GGTTTGATTCTTATACCATTGTCCA R: GGGCATCATCACCAGCAAAA	Actin	115
Actin-3	F: GAGGAACATCCAATTTTGCTGA R: CACATACATTGCTGGTGCATT	Actin	108

Fig. 1. Schematic diagram of primer binding sites on the target gene WA352 and WA352 primer amplification results A, Primer binding sites on the target gene WA352. The numbers in parentheses represent the coordinates of the primers on the genomic sequence, with the first base at the 5' end of the DNA sequence as the starting point; B, Amplification results of WA352 primers. 1, CMS line Hengfeng A; 2, Restorer line Yinzhan 3; 3, Hybrid variety Quanyousimiao; 4, Maintainer line Huhan7 B; PN, Primer name; M, DL2000 DNA marker (100-2000 bp).

Fig. 2. Schematic diagram of primer binding sites on the target gene Rf4 and Rf4 primer amplification results A, Primer binding sites on the target gene Rf4. The numbers in parentheses represent the coordinates of the primers on the genomic sequence, with the first base at the 5' end of the DNA sequence as the starting point. The three specific primers for Rf4 adopt the same F-primer; B, Amplification results of Rf4 primers; 1, CMS line Hengfeng A; 2, Restorer line Yinzhan 3; 3, Hybrid variety Quanyousimiao; 4, Maintainer line Huhan 7B; PN, Primer name;M, DL2000 DNA marker (100-2000 bp).

Fig. 3. Schematic diagram of primer binding sites on the target gene Actin and amplification results of Actin primers A, Primer binding sites on the target gene Rf4. The numbers in parentheses represent the coordinates of the primers on the genomic sequence, with the first base at the 5' end of the DNA sequence as the starting point; B, Amplification results of Actin primers; 1, CMS line Hengfeng A; 2, Restorer line Yinzhan 3; 3, Hybrid variety Quanyousimiao; 4, Maintainer line Huhan 7B; PN, Primer name; M, DL2000 DNA marker (100-2000 bp).

Fig. 4. Initial screening results of primer combinations 1, CMS line Hengfeng A; 2, Restorer line Yinzhan 3; 3, Hybrid Quanyousimiao; 4, Maintainer line Huhan 7B; PR, Primer ratio of WA352-2, Rf4-3 and Actin-3; M, DL2000 DNA marker (100-2000 bp).

Fig. 5. Optimization results of primer combinations A, First primer combination ratio optimization results; B, Second primer combination ratio optimization results, 1, CMS line Hengfeng A; 2, Restorer line Yinzhan 3; 3, Hybrid Quanyousimiao; 4, Maintainer line Huhan 7B; PR, Primer ratio of WA352-2, Rf4-3 and Actin-3; M, DL2000 DNA marker (100-2000 bp).

Fig. 6. Amplification results of WRA-F/R primers in different rice materials A, Using genomic DNA from restorer line rice as the template. Lane 1, Yizhan 3; Lane 2, Yuehui 9712; Lane 3, Yuezhan; Lane 4, Meixiangxinzhan; Lane 5, R2117; Lane 6, Lvsimiao; Lane 7, Yuehesimiao; B: Using genomic DNA from CMS line rice as the template. Lane 8 is Hengfeng A ; Lane 9, Huhan7A; Lane 10, Longtefu A; Lane 11, Hefeng A; Lane 12, Mingtai A；Lane 13,WufengA; Lane 14, Guang8A; C, Using genomic DNA from hybrid rice as the template, lane Lane 15, Quanguangyou 4; Lane 16, Quanyousimiao; Lane 17, Shenyouyuehesimiao; Lane 18, Wuyoujingsimiao; Lane 19, Yiyouyuehesimiao；Lane 20, Zhuyou 110; Lane 21, Quanzaoyousimiao; D, Using genomic DNA from conventional rice as the template, Lane 22 is Zhonghua11; Lane 23, Guangluai4; Lane 24, Shuhui498; Lane 25, Nipponbare; Lane 26, 9311；Lane 27, Taizhong 65; Lane 28, Nanjing 11, where Shuhui 498 is the restorer line, M, DL2000 DNA marker (100-2000 bp).

Fig. 7. Detection results of WRA-F/R primers on single-plant agarose gel electrophoresis of the wild-abortive three-line hybrid rice samples A, DNA profile of Sample #1, with each lane corresponding to DNA extracted from one of 192 randomly selected rice seedlings post-nursery cultivation. B, DNA profile of Sample #2, with each lane corresponding to DNA extracted from one of 192 randomly selected rice seedlings. M, DL2000 DNA marker (100-2000 bp).

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