Creation of Rice Mutant by Gamma-ray and Its Molecular Identification

doi:10.13560/j.cnki.biotech.bull.1985.2024-0667

Abstract

Abstract:

Objective Gamma-ray mutagenesis has been widely used in rice breeding because of its high efficiency and simple method. OsGRF4 is an important and rare pleiotropic gene in rice, which can significantly improve nitrogen use efficiency, yield and cold tolerance, while reduce seed shattering, this work aims to obtain different types of mutant resources and mine the regulatory genes of OsGRF4. Method The rice material NIL-GRF4 was mutated by γ rays in this study, the authenticity and genetic background of the mutants were identified by functional marker and SSR markers. Result By traits investigating, 15 mutants of grain shape, 6 mutants of zebra leaf, 4 mutants of tiller and 3 mutants of sterile were obtained. Among them, an important grain type mutant was obtained. Its grain length reached 14.10 mm, and its grain length and 1 000-grain weight increased by 1.50 mm and 8.85 g respectively compared with the wild type, with an increase of 11.90% and 23.95% respectively. The authenticity and genetic background of the mutants were identified by using the functional markers of OsGRF4 and 48 pairs of SSR markers. The results showed that the mutants were all derived from wild-type NIL-GRF4, and the genetic background of the mutants was very similar to that of wild-type NIL-GRF4. Conclusion The F₂ populations have been constructed by crossbreeding the mutants of grain shape, zebra leaf and sterile with the wild type, providing important gene resources for rice breeding.

Key words: gamma-ray, OsGRF4, mutation breeding, BSA-Seq, map-based cloning, rice

LI Xin-peng, ZHANG Wu-han, ZHANG Li, SHU Fu, HE Qiang, GUO Yang, DENG Hua-feng, WANG Yue, SUN Ping-yong. Creation of Rice Mutant by Gamma-ray and Its Molecular Identification[J]. Biotechnology Bulletin, 2025, 41(3): 35-43.

Figures/Tables 6

Table 1 Comparison of phenotype between wild-type and grain shape mutants

水稻材料 Rice material	粒长 Grain length/mm	粒宽 Grain width/mm	长宽比 Length-width ratio	千粒重 Thousand-seed weight/g
NIL-GRF4	12.60±0.46	2.69±0.20	4.68±0.27	36.95
23cs3	11.98±0.73	3.30±0.20	3.63±0.33	44.30
23cs4	12.29±0.51	2.73±0.17	4.50±0.12	39.34
23cs5	11.07±0.43	2.80±0.19	3.95±0.45	37.98
23cs6	9.94±0.69	3.11±0.19	3.20±0.03	35.71
23cs7	11.05±0.62	3.41±0.20	3.24±0.05	36.06
23cs8	11.22±0.54	2.69±0.24	4.17±0.21	36.89
23cs9	11.04±0.63	2.78±0.20	3.97±0.10	32.07
23cs10	9.00±0.54	2.16±0.19	4.17±0.14	24.40
23cs11	10.86±0.46	2.75±0.19	3.95±0.12	36.80
23cs22	10.43±0.53	2.59±0.19	4.03±0.11	29.29
23cs24	10.57±0.46	2.66±0.19	3.97±0.26	28.10
23cs25	11.04±0.53	2.66±0.25	4.15±0.24	35.28
23cs31	10.95±0.52	2.67±0.20	4.10±0.49	32.06
23cs32	14.10±0.69	2.77±0.22	5.09±0.19	45.80
23cs33	9.68±0.53	2.98±0.15	3.25±0.04	36.97

Fig. 1 Comparison of grain shape and leaf color between wild type NIL-GRF4 and mutantsA: Comparison of grain shape between wild-type NIL-GRF4 and mutants; B: leaf color comparison between wild-type NIL-GRF4 and mutants, the one on the left is the zebra leaf mutant, and the one on the right is the wild type; C: leaf color phenotype of mutant 23cs20 in field

Table 2 Comparison of panicle traits between wild type and sterile mutants

材料 Rice material	穗长 Spike length/cm	总粒数 Total grains	实粒数 Filled grain number	一次枝梗 Number of primary branches	二次枝梗 Number of secondary branches	结实率 Seed setting rate/%
NIL-GRF4	32.9±0.35	207±12.70	128±5.58	14±1.00	33±3.06	62.01±2.42
23cs13	33.3±1.06	186±19.00	5±0.58	13±0.58	35±4.04	2.50±0.43
23cs14	32.8±0.36	186±4.93	6±1.53	12±0	32±1.00	3.05±0.75
23cs29	32.4±0.47	232±2.64	14±1.73	12±0	44±1.73	6.03±0.68

Fig. 2 Phenotypes of sterile and tillering mutantsA: Microscopic examination of wild type fertility. B: Microscopic examination of mutant fertility. C: Comparison of seed setting rates between wild type NIL-GRF4 and sterile mutant 23cs29. D: Comparison of sterility between wild type NIL-GRF4 and mutant 23cs29. E: Comparison of tiller and plant height between wild type NIL-GRF4 and mutant 23cs26. Wild type on the left and mutant on the right

Fig. 3 Genotypic identification of wild type and mutants by functional markersM: 250 bp maker; A and C: PF+DMR+PR primer combination; B and D: PF+XMR+PR primer combination; 1-26: mutants; 27-33: NIL-GRF4, Huahang 32, Xiangzaoxian 6, Ganninggeng 1, Nongxiang 42, Chuan 7 and Zhuxiangzhan, respectively

Fig. 4 Amplification of wild-type NIL-GRF4 and mutants by SSR primersM: 100 bp maker; 1-16: primer RM471, RM72, RM19, RM119, RM339, RM17, RM551, RM331, RM3331, RM443, RM85, RM336, RM209, RM232, RM481, RM224, respectively; DNA sequence is wild type, mutant 23cs13 and 23cs22

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