Generation of a new thermo-sensitive genic male sterile rice line by targeted mutagenesis of TMS5 gene through CRISPR/Cas9 system

Abstract

Background: Two-line hybrid rice with high yield potential is increasingly popular and the photo- and temperature-sensitive male sterile line is one of the basic components for two-line hybrid rice breeding. The development of male sterile lines through conventional breeding is a lengthy and laborious process, whereas developing thermo-sensitive genic male sterile (TGMS) lines for two-line hybrid breeding by editing a temperature-sensitivity gene by CRISPR/Cas9 is efficient and convenient.

Results: Here, thermo-sensitive genic male sterility (TGMS) was induced by employing the CRISPR/Cas9 gene editing technology to modify the gene TMS5. Two TGMS mutants, tms5-1 and tms5-2, both lacking any residual T-DNA, were generated in the indica rice cultivar Zhongjiazao17 (cv. YK17) background. When grown at a sub-optimal temperature (22 °C), both mutants produced viable pollen and successfully produced grain through self-fertilization, but at temperatures 24 and 26 °C, their pollen was sterile and no grain was set. F₁ hybrids derived from the crosses between YK17S (tms5-1) and three different restorer lines outperformed both parental lines with respect to grain yield and related traits.

Conclusion: The YK17S generated by CRISPR/Cas9 system was proved to be a new TGMS line with superior yield potential and can be widely utilized in two-line hybrid breeding of indica rice.

Keywords: Gene editing; Heterosis; TMS5 gene; Thermo-sensitive genic male sterility (TGMS); Two-line hybrid rice system.

Fig. 1

CRISPR/Cas9-mediated editing of TMS5. a Vector structure of Cas9/gRNA (VK005–01). b Structure of TMS5. c The TMS5 target site aligned with the tms5–1 (a 2 nucleotides deletion) and the tms5–2 (1 nucleotide insertion) mutant sequences. LB: vector left border, Hyg: Hygromycin, 35S: CaMV 35S promoter, rU6: rice U6 promoter, gRNA: guide RNA, mpCas9: Cas9 protein, Ubi: Ubiqutin promoter, RB: vector right border, PAM: protospacer adjacent motif

Fig. 2

PCR-based identification of T-DNA-free T₁ segregants using primers directed at the hpt sequence. Segregants among (a) tms5–1 plants, and (b) tms5–2 plants. VK005–01 was used as the positive control (CK+) and cv. YK17 as the negative control (CK-). M: 2 kbp DNA ladder

Fig. 3

Phenotype of cv. YK17 and T₂ generation of tms5–1 and tms5–2 grown in the field during the normal rice growing season. a-c Appearance of the whole plant. d-f Spikelets after removal of the lemma and palea. g-i Pollen grains stained with I₂-KI. j Panicles of cv. YK17, tms5–1 and tms5–2 from left to right, respectively. k Grain set by tms5–1 (left) and tms5–2 (right) after manual pollination. Bar in A-C: 15 cm, D-F: 0.5 cm, G-I: 100 μm, J: 5 cm, K: 3 cm

Fig. 4

Pollen fertility and grain set of cv. YK17 and T₂ generation tms5–1 and tms5–2 plants grown under various daily average regimes. a [a-f] cv. YK17, [g-l] tms5–1, [m-r] tms5–2 plants exposed to 22 °C, 24 °C or 26 °C. b Appearance of the whole plant of (a) cv. YK17, (b) tms5–1, (c) tms5–2 grown at 22 °C. c-e Grain set at 22 °C, 24 °C or 26 °C. Values are shown as mean ± SD (n = 20). Asterisks (* P < 0.05; ** P < 0.01) indicate statistically significant differences between means of a mutant and cv. YK17, as determined by a student’s t-test. Bars in (A) [a, c, e, g, i, k, m, o, q]: 100 μm; [b, d, f, h, j, l, n, p, r]: 5 cm; in B: 15 cm

Fig. 5

TMS5 expression in cv. YK17 and T₂ generation of tms5–1 and tms5–2 plants. a Transcript abundance as estimated by qRT-PCR in the leaves and anthers of plants grown at either 22 °C or 26 °C. Values shown in the form mean ± SD (n = 3). Columns marked with a different letter indicate statistically significant differences between means (P < 0.05). b Western blot assay of TMS5 in the panicle of plants grown at 26 °C

Fig. 6

Phenotype of cv. YK17, YK17S1, R101 and the F₁ hybrid of YK17S1 x R101. Appearance of the (a-d) whole plant and (e-h) panicle of cv. YK17, YK17S1, R101 and the F₁ hybrid of YK17S1 X R101 from left to right, respectively. Bar in A-D: 15 cm, E-H: 5 cm

Fig. 7

Grain yield performance and yield components of cv. YK17, YK17S1, R101 and the F₁ hybrid YK17S1 x R101. a Number of panicles per plant, b number of grains set on the main panicle, c thousand grain weight (g), d grain set (%), e yield per plant (g), f yield per plot (g), g plant height (cm) and h number of days to flowering. Values shown in the form mean ± SD (a, b, d, g: n = 20; c: n = 4, e: n = 6, f, h: n = 2). Asterisks (* P < 0.05; ** P < 0.01) indicate statistically significant differences between the mean performances of either cv. YK17 and YK17S1 x R101, or R101 and YK17S1 x R101, as determined by a student’s t-test