Map-based Cloning of Light-responsive Gene ELM1 in Rice

doi:10.13560/j.cnki.biotech.bull.1985.2024-1246

Abstract

Abstract:

Objective Exploring the regulatory genes of rice flowering date and conducting positional cloning research, the genetic and molecular mechanisms of rice flowering can be clarified, the regulatory network of rice heading date genes can be improved, and certain theoretical and practical significance can be provided for rice production and breeding. Method Using the late-flowering mutant elm1 under long-day conditions in rice as the material, we analyzed agronomic traits such as flowering time. Then we constructed a population through reciprocal crosses and analyzed the genetic patterns by phenotyping the F₂ population. Further we also developed a mapping population with the indica rice Dular for positional cloning and gene localization. Finally, we sequenced the candidate genes within the fine-mapped interval, predicted and analyzed the candidate genes using bioinformatics tools, and predicted the changes in protein structure using AlphaFold2 software. Result The elm1 mutant demonstrated a significantly prolonged flowering time under long-day conditions, which was controlled by a single recessive nuclear gene. The ELM1 gene through positional cloning was cloned. The ELM1 gene encoded a SET-domain histone methyltransferase and was allelic to the previously reported lvp1 mutant. Sequencing results showed that a point mutation (G to T) in the elm1 mutant occurred in the 5th exon of the LOC_Os09g13740 gene (3 293 bp downstream of the ATG start codon), resulting in a change from glycine (hydrophilic) to valine (hydrophobic) at this site. Protein structure prediction using AlphaFold2 indicated that this mutation caused a conformational change in the protein, which may affect its function. Field experiments showed that it showed a phenotype with significantly increased number of grains per panicle and higher yield when the elm1 mutant was grown in suitable latitudinal regions. Conclusion The mutation site in the elm1 mutant is a weak allele. Breeding this allelic mutant in suitable latitudinal regions may enhance rice yield. Further studies have demonstrated that the elm1 mutant is an excellent allelic variation material.

Key words: rice, heading date, elm1 mutant, map-based cloning, weak mutation, protein structure

DU Liang-heng, TANG Huang-lei, ZHANG Zhi-guo. Map-based Cloning of Light-responsive Gene ELM1 in Rice[J]. Biotechnology Bulletin, 2025, 41(5): 82-89.

Figures/Tables 7

Fig. 1 Phenotypic of the flowering period between WT and elm1A: Comparison of wild type (WT) and elm1 mutant at the flowering stage in the experimental field in Beijing (Bar=20 cm). B: Comparison of WT and elm1 mutant at the flowering stage in Sanya, Hainan (Bar=20 cm). C: Comparison of heading date between WT and elm1 mutant in the experimental field in Beijing (long-day), *** indicates a significant difference at P<0.001. D: Comparison of heading date between WT and elm1 mutant in the experimental field in Sanya, Hainan (short-day), ns stands for not significant

Table 1 Comparison of agronomic traits between WT and elm1 mutant (Hainan)

性状类别

Trait category

旗叶叶长

Flag leaf length （cm）

旗叶叶宽

Flag leaf width （cm）

株高

Plant height （cm）

分蘖数

Tiller number

千粒重

Thousand-grain weight （g）

每穗粒数

Grain number per panicle

elm1

Table 2 Statistical results of F2 generation segregation population

统计类别 Statistical category	日本晴× elm1 Nipponbare×elm1	elm1×日本晴 elm1×Nipponbare
植株总数 Total number of plants	396	374
正常植株数 Number of normal plants	288	276
不开花植株数 Number of non-flowering plants	108	98
分离比 Segregation ratio	3∶1	1∶3
χ²	0.066 7	0.051 2

Fig. 2 Gel electrophoresis of gene linkage analysis for the mutant elm1A: Linkage analysis of the Indel-3 marker in individual F2 plants (1‒32: Electrophoresis banding results of 32 individual plants). B: Linkage analysis of the Indel-5 marker in individual F2 plants (1‒37: Electrophoresis banding results of 37 individual plants). Nip: Nipponbare. Du: Dular

Fig. 3 Map-based cloning of gene ELM1A: Preliminary gene localization of elm1 mutant. The distance between each marker is represented by the recombination frequency, measured in centimorgans (cM). B: The fine mapping of the gene in the mutant elm1. C: Candidate region. D: The mutation in mutant elm1 occurs in the fifth exon of the LOC_Os09g13740, where the lvp1-1 mutation was a deletion of 79 base pairs in the fifth exon, and a mutation involving the insertion of 115 base pairs between the eighth and ninth exons

Fig. 4 Prediction of the ELM1 protein structure in WT and mutantA: Prediction of the ELM1 protein structure in the wild type. B: Prediction of the ELM1 protein structure after mutation

Fig. 5 Agronomic traits comparison between wild type and elm1 mutantA: Panicle comparison between WT and elm1 mutant (Bar=5 cm). B: Comparison of grain number per panicle between WT and elm1 mutant. ** indicates a significant difference at P<0.001. C: Comparison of plant height between WT and elm1 mutant. D: Comparison of thousand-grain weight between WT and elm1 mutant. E: Comparison of seed setting rate between WT and elm1 mutant. F: Comparison of tiller number between WT and elm1 mutant

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