Chinese Journal OF Rice Science >
Generation of Rice DMP1, DMP2 and DMP3 Mutants and Identification of Their Haploid Induction Ability
Received date: 2023-11-15
Revised date: 2023-12-12
Online published: 2025-01-14
【Objective】Exploring the haploid induction ability of rice DMP family genes to provide new gene resources for haploid breeding.【Methods】Selecting OsDMP family genes with high homology to the ZmDMP gene and high expression level in pollen as candidate genes. Using CRISPR/Cas9 multi-gene editing technology to create single-gene knockout and multi-gene combination knockout mutants of the selected OsDMP family genes and haploid induction gene OsMTL in the indica and japonica hybrid rice Chunyou 84. Conducting morphological observations and pollen fertility identification on the mutants. Investigating and statistically analyzing the seed setting rate and haploid induction rate of the T0 materials. 【Results】A total of 13 homologous genes of maize haploid induction gene ZmDMP were retrieved from the rice RAP-DB database. OsDMP1 and OsDMP2 showed the highest homology with ZmDMP, with similarity percentages of 41.24% and 37.32%, respectively; Moreover, OsDMP1 and OsDMP3 were the most highly expressed OsDMP genes in anthers. Therefore, OsDMP1, OsDMP2 and OsDMP3 were selected as candidate genes. Using CRISPR/Cas9 gene editing technology, single-gene knockout and combined knockout mutants of OsDMP1, OsDMP2, and OsDMP3 were created (osdmp1, osdmp2, osdmp3, osdmp1-osdmp2, osdmp1-osdmp3, osdmp1-osdmp2-osdmp3). Additionally, single-gene knockout mutants of OsMTL were generated (osmtl), as well as combined knockout mutants between OsDMP1, OsDMP2, OsDMP3, and OsMTL (osmtl-osdmp1, osmtl-osdmp2, osmtl-osdmp3, osmtl-osdmp1-osdmp2, osmtl-osdmp1-osdmp3, osmtl-osdmp1-osdmp2-osdmp3). Phenotypic investigation showed that compared with the wild type, plant morphology and pollen fertility of all mutants did not visibly change, but the seed setting rate decreased significantly in single gene knockout and multi-gene combination knockout mutants containing osmtl. Haploid detection results indicated that the haploid induction efficiency of the combined mutants between OsDMP1, OsDMP2, OsDMP3 and OsMTL was 0.4%±0.6%, 2.6%±2.8%, 1.4%±0.6%, 1.5%±1.3%, 2.1%±2.4%, 2.2%± 0.6%, respectively, which was not significantly different from that of osmtl mutant (1.5%±0.5%). However, no haploid was produced when OsDMP family genes were mutated individually or in combination. 【Conclusion】CRISPR/Cas9 gene editing technology was used to create single and multi-gene knockout mutants between OsDMP1, OsDMP2, OsDMP3, and OsMTL in rice. It was found that OsDMP1, OsDMP2 and OsDMP3 had no independent haploid induction ability, nor could it improve the haploid induction efficiency of OsMTL genes. These results promoted the understanding of OsDMP homologous genes in rice, and provided reference for the subsequent studies on the haploid induction gene.
Key words： rice; haploid induction; DMP; OsMTL
HU Fengyue, WANG Jian, WANG Chun, WANG Kejian, LIU Chaolei . Generation of Rice DMP1, DMP2 and DMP3 Mutants and Identification of Their Haploid Induction Ability[J]. Chinese Journal OF Rice Science, 2025 , 39(1) : 55 -66 . DOI: 10.16819/j.1001-7216.2025.231112
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