Development and application of an allele-specific PCR assay for detecting T409C mutation of cyp51 gene linked with tebuconazole resistance in Villosiclava virens (rice false smut)

Figures & data

Table 1. Fifteen wild-type isolates and five tebuconazole-resistant mutants of Villosiclava virens used in this study.

Number	Name	Reaction to tebuconazole
1	F10-338	Sensitive
2	JY13107	Sensitive
3	JY13041	Sensitive
4	JY1601	Sensitive
5	JY1602	Sensitive
6	NH13018	Sensitive
7	NH13052	Sensitive
8	NH13047	Sensitive
9	NH13045	Sensitive
10	SX13023	Sensitive
11	SX13033	Sensitive
12	SX13035	Sensitive
13	SH13001	Sensitive
14	SH13002	Sensitive
15	SH13003	Sensitive
16	JY13107C	Resistant
17	JY13107D	Resistant
18	F10-338B	Resistant
19	F10-338C	Resistant
20	F10-338D	Resistant

Fig. 1 (Colour online) Symptoms of rice false smut caused by Villosiclava virens. (a) typical symptoms observed on rice panicles in the greenhouse. (b) close-up of galls.

Table 2. Primers used in this study.

Primer	DNA sequence (5ʹ-3ʹ)	Purpose
S-4	AACCTTCCTTTTCCCCAATAGC	cyp51 genomic DNA
A-3	GCCGTTACCCGCTCTATTATT	cyp51 genomic DNA
S-2	ATGGGCGTCCTTCAAGACGTTG	cyp51 cDNA
A-2	CTAATCTCGTCGTTCCCAGAAAATG	cyp51 cDNA
Uv137F1	TTCACTGGTTCCCCTTTATCGG	Allele-specific PCR
Uv137AR	GGCGTTGGGGCAATCGTG	Allele-specific PCR
Uv137BR	GGCGTTGGGGCAATCGAG	Allele-specific PCR
Uv137CR	GGCGTTGGGGCAATCGGG	Allele-specific PCR
Uv137DR	GGCGTTGGGGCAATCGCG	Allele-specific PCR

Fig. 2 (Colour online) Sequence alignment of the polymorphic cyp51 coding region from tebuconazole-sensitive isolates and resistant mutants of V. virens.

Fig. 3 Sensitivity test of allele-specific PCR for detection of T409C mutation in tebuconazole-resistant mutant F10-338D of V. virens. Lanes 1–7, genomic DNA templates of F10-338D at concentrations of 600, 60, 6, 0.6, 0.06, 0.006 and 0.0006 ng µL⁻¹, respectively. M, 2-kb DNA ladder (Takara Bio).

Fig. 4 Allele-specific PCR amplification products from genomic DNA extracted from eight tebuconazole-sensitive isolates and five tebuconazole-resistant mutants of V. virens. M, 2-kb DNA ladder (Takara Bio); lane 1, negative control; lanes 2–9, isolates F10-338, JY13107, JY1601, NH13018, NH13047, SX13023, SH13002 and SH13003; Lanes 10–14, isolates F10-338B, F10-338C, F10-338D, JY13107C and JY13107D.

Fig. 5 Allele-specific PCR amplification products from genomic DNA extracted from false smut balls. M, 2-kb DNA ladder (Takara Bio); lane 1, negative control; lanes 2–6, false smut balls derived from tebuconazole-resistant mutants; lanes 7–18, false smut balls derived from tebuconazole-sensitive isolates.