Coleoptile Purple Line Regulated by A-P Gene System Is a Valuable Marker Trait for Seed Purity Identification in Hybrid Rice

doi:10.1016/j.rsci.2022.07.005

Abstract

Abstract:

In plants, a large number of anthocyanin biosynthetic genes encoding enzymes and regulatory genes encoding transcription factors are required for anthocyanin synthesis. Coleoptile purple lines are two purple lines on both sides of coleoptiles after seed germination. However, the molecular mechanism of coleoptile purple line is not clear in rice so far. In this study, two major dominant genes, coleoptile purple line 1 (OsCPL1, also known as OsC1) and coleoptile purple line 2 (OsCPL2), were isolated via map-based cloning, and both of them were required for anthocyanin biosynthesis of coleoptile purple line in rice. The knockout and complementation experiments confirmed that OsC1 was required for purple color in most organs, such as coleoptile line, sheath, auricle, stigma and apiculus, whereas OsCPL2 was just required for coleoptile purple line. OsC1 was predominantly expressed in coleoptiles, flag leaves, and green panicles, and highly expressed in young leaves, whereas OsCPL2 was predominantly expressed in coleoptiles, and extremely lowly expressed in the other tested organs. Loss-of-function of either OsC1 or OsCPL2 resulted in significant reduction of transcript levels of multiple anthocyanin biosynthesis genes in coleoptiles. Coleoptile purple line was further used as a marker trait in hybrid rice. Purity identification in hybrid rice seeds via coleoptile purple line just needed a little water, soil and a small plate and could be completed within 5 d. Molecular marker and field identification analyses indicated that coleoptile purple line was reliable for the hybrid seed purity identification. Our findings disclosed that coleoptile purple line in rice was regulated by two major dominant genes, OsC1 and OsCPL2, and can be used as a simple, rapid, accurate and economic marker trait for seed purity identification in hybrid rice.

Key words: OsC1, OsCPL2, coleoptile purple line, anthocyanin, seed purity identification, marker trait

Du Shuanglin, Wang Zhongwei, Chen Yun, Tan Yao, Li Xiang, Zhu Wenping, He Guanghua, Lei Kairong, Guo Longbiao, Zhang Yi. Coleoptile Purple Line Regulated by A-P Gene System Is a Valuable Marker Trait for Seed Purity Identification in Hybrid Rice[J]. Rice Science, 2022, 29(5): 451-461.

Figures/Tables 6

Fig. 1. Phenotypic characteristics of coleoptile purple line and coleoptile non-purple line on R25 and YR25 at the germination stage. A and C, Phenotypes of coleoptile purple line in R25. B and D, Phenotypes of coleoptile non-purple line in YR25. Coleoptile purple line and non-purple line are indicated by black and white arrows, respectively. Scale bars, 1 mm.

Table 1. Purple organs in different rice materials used in this study.

Line	Cl	Sh	In	Au	L	St	Ap
R25	+	+	-	+	-	+	+
YR25	-	-	-	-	-	-	-
II-32 A	+	+	-	+	-	+	+
II-32 B	+	+	-	+	-	+	+
YII-32 A	-	+	-	+	-	+	+
YII-32 B	-	+	-	+	-	+	+
Zhongjiu A	-	-	-	-	-	-	-
Zhongjiu B	-	-	-	-	-	-	-
NJ7B	-	+	-	+	-	+	+
2081A	-	+	-	+	-	+	+
2081B	-	+	-	+	-	+	+
06A2066	-	-	-	-	-	-	-
Luhui 17	-	-	-	-	-	-	-
R30	-	-	-	-	-	-	-
R287	-	-	-	-	-	-	-
R725	-	-	-	-	-	-	-

Fig. 2. Map-based cloning of OsCPL1. A, OsCPL1 locus is mapped to chromosome 6 (Chr. 6). B, OsCPL1 locus is narrowed down between markers RM5754 and RM19665. =C, Open reading frame (ORF) of candidate gene OsCPL1 in the mapped region. The mutation site is indicated by a red upright triangle, and the premature stop codon is indicated by a black triangle. D, Independent T1 mutants generated using a CRISPR/Cas9 system. The mutants (Cas9-OsC1-1 and -2) display non-purple color in coleoptile. R25, Wild type plants. Scale bar, 0.5 cm. E, Independent T1 complementary plants (Com-OsC1-1 and -2). The positive plants display purple color in coleoptile. Zhongjiu B is a Osc1 mutant. Scale bar, 0.5 cm.

Fig. 3. Map-based cloning of OsCPL2. A, OsCPL2 locus was mapped to chromosome 11 (Chr. 11). B, OsCPL2 locus was narrowed down to a 51.2-kb interval. C, Open reading frames (ORFs) of the candidate gene LOC_Os11g15210 in the mapped region. The sense mutation sites are indicated by red upright triangles. D and E, Independent T1 mutants of R25 (D) and II-32B (E) generated using the CRISPR/Cas9 system. The mutants have non-purple line in the coleoptile. R25 and II-32B are wild type plants. Scale bars, 0.5 cm.

Fig. 4. Expression patterns of OsC1 and OsCPL2 and transcript expression analysis of anthocyanin biosynthesis genes in coleoptiles. A, Expression pattern analysis of OsC1 and OsCPL2 in various organs of wild type R25. B, Transcript expression analysis of anthocyanin biosynthesis genes in the coleoptiles of R25, the Osc1 mutant Zhongjiu B and the Oscpl2 mutant YII-32B. OsUbiquitin1 (Os03g0234200) was used as the internal control. Values are Mean ± SD with three biological replicates. The asterisks indicate statistical significance between R25 and the Osc1 or Oscpl2 mutant, as determined by the Student’s t-test (**, P < 0.01).

Table 2. Seed purity identification of hybrid rice seeds via coleoptile purple line.

Cross combination	Total number	Number of off-type seeds	Hybrid seed purity (%)
2081A × 06A2066	1 000	26	97.4
2081A × R30	1 000	12	98.8
2081A × R287	1 000	12	98.8
2081A × R725	1 000	7	99.3

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