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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (11): 3029-3041.doi: 10.3724/SP.J.1006.2023.22061

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Identification of a rolling leaf allelic mutant e202 and fine mapping of E202 gene in rice

ZHOU Wen-Qi1,2(), QIANG Xiao-Xia3, LI Si-Yu1, WANG Sen4, WEI Wan-Rong1,*()   

  1. 1Key Laboratory of Southwest China Wildlife Resources Conservation / College of Life Sciences, China West Normal University, Nanchong 637000, Sichuan, China
    2Crops Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China
    3Lanzhou No. 4 High School, Lanzhou 730050, Gansu, China
    4Shaanxi Province Animal Husbandry Industry Experimental Demonstration Center, Xianyang 713702, Shaanxi, China
  • Received:2022-10-28 Accepted:2023-05-24 Online:2023-11-12 Published:2023-06-15
  • Supported by:
    Young Scientific and Technological Talents Support Project of Gansu Association for Science and Technology in 2020

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Abstract:

Good leaf morphology is one of the factors for ensuring crop yield. A certain degree of curling can keep rice leaves standing and not drooping, which is beneficial to improve the light area of the population, enabling it to receive more light energy and improve the photosynthetic efficiency. In this study, the mature seeds of ZH11 in rice were mutated by EMS, and a mutant named e202 with curly and straight leaves was screened from M2 progeny. Phenotypic identification showed that e202 exhibited a variety of defective phenotypes. Compared with the wild type Zhonghua 11 (ZH11), the length and number of adventitious roots were decreased, and the panicle development was abnormal. The morphology of floret, anther, and pollen was deformed, the fertility was significantly reduced, the chlorophyll content was significantly increased, and the stomatal density was decreased. The structure of the leaves was observed by resin sectioning. Compared with the control, the number of vesicular cells increased and the volume decreased, resulting in leaf curling. Meanwhile, the anthers were sliced and observed. Normal pollen could not be formed in e202 or only very few pollen grains were formed, resulting in reduced fertility. The candidate genes were located in the physical region of 440 kb on chromosome 10 by map-based cloning. Gene sequencing analysis within the interval revealed that one base G was missing in the 1935 position of the fourth exon of Os10g0562700 (LOC_Os10g41310) gene, which resulted in the subsequent amino acid confusion. Protein translation is terminated prematurely, and e202 is a novel allelic mutant of REL2. This study provides a theoretical basis for revealing the involvement of REL2 in leaf rolling and flower development in rice.

Key words: rice, rolled-leaf, drought resistance, flower development, fine mapping

Table 1

Part of the primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 用途
Use
e202-F ATGGGGTGCACGGCGTCGAAGG 基因克隆Gene cloning
e202-R CTACCGCACCGATCCAGCTCGCCT
InDel-1F TTATGTCAACCTTACAATT 基因定位Fine mapping
InDel-1R GCATACTGTATTCCTATATC
InDel-2F TTGCTTAGTGGTAAGCTATG
InDel-2R GGAGTTCTTGAAGAAACTCC
InDel-3F GTATGTTTGTGGAGTAATAT
InDel-3R CATGCAATGTGCCAAGAACC
InDel-4F GCATACGAATGTACTTGTGT
InDel-4R TGGAAGTATGCGTGGAATAT
InDel-5F CTCAAATCACTTATATTATG
InDel-5R GTGTAACCAGTAGAAGAATG
InDel-6F TGTATAATTTGTTGAGATGAG
InDel-6R GTAGATCTTGTGATATTGGAG
C10-1-F GATTCACGGGTCGATCTACT 基因定位Fine mapping
C10-1-R GTGTGGTGCCATGCCTAATG
C10-22-F CAAGTATAATAATACTTAGAG
C10-22-R GAGCAAGGCTAATAATACATC
C10-38-F GTTGAAGTGCTACAGGTATG
C10-38-R GCTTCCTCAGCCTTGATCTC
C10-45-F GTATTCGCACTCTCAAATGAG
C10-45-R CAAATTAAGTCACAGATAAGTG
C10-57-F CAAGAATTCTAAGAGCTCG
C10-57-R CGAGCTTATGCATAAGCTTTG
C10-72-F TAGGAGGAGCAGAAGGAATAG
C10-72-R CGATCGATCGTCTTCTTCCAC
TUB-F TTTCACTCTTGGTGTGAAGCAGAT 实时荧光定量PCR技术RT-PCR
TUB-R GACTTCCTTCACGATTTCATCGTAA
REL2-F TGATCATCGTGACTTCACAGGC 实时荧光定量PCR技术RT-PCR
REL2-R TCTACCAGACCACGGACTTGC

Fig. 1

Phenotypic analysis of e202 and ZH11 plants A: at the early growth stage, e202 was shorter than ZH11, and leaves were curled. B: the height of e202 was the same as that of the control at anthesis stage. C: the angle between stem and leaf of ZH11 seedlings was relatively larger; D: the angle between stem and leaf of e202 was smaller than that of ZH11. E: at seedling stage, the root of e202 was underdeveloped, and the length and number of adventitious roots was decreased. F: left: ZH11 blade, middle: e202 micro-coil blade, right: e202 was reeled blade; G: F2 generation, dwarfed by e202, leaf rolling. A, B, G: bar, 10 cm; C, D, E, F: bar, 1 cm."

Fig. 2

Mutant phenotype of e202 rice panicle A, E: ZH11 complete spike flower structure; B-D, F-L: e202; A: spikelet structure of ZH11 toggle lemma and palea; B-D: e202 glume was removed, and the number of lemma and palea increased; F-H: the multiple aberrant phenotypes of e202 rice husk; I-L: the aberrant structures of e202 deleted stamens and pistils, marked by asterisks. Bar: 0.1 cm."

Fig. 3

Mutant phenotypes of some flowers and pollen of e202 A, I: ZH11; B-H, J-L: e202; A: anatomical diagram of flowers of the ZH11 lemma and palea; B-D: e202, 2 pistils; E-H: distorted structure of stamens, stigma and paddles of e202; I: anther structure of ZH11; J-L: the distorted structure of e202 pollen, the different morphology of e202 anthers, and the decreased fertility. A-H: bar, 0.05 cm; I-L: bar, 0.01 cm."

Fig. 4

The number of various flower organs A: stamens; B: pistil; C: stigma; D: blades; E: lemma; F: palea. The control group were 50 flowers, and the e202 were 150 flowers. n = 50/150."

Fig. 5

Pollen staining and transection of anthers in ZH11 and e202 A: ZH11 flowering; B: e202 was unable to bloom, and the glume did not open at the pollinating stage; C: the anthers and pollen grains of ZH11; D: the anthers and pollen of e202, a small amount of pollen: E: ZH11 ripe and full seeds; F: most of the e202 seeds obtained were shrivelled and empty; G, H: pollen of ZH11 and e202 stained with 0.1% iodide and potassium iodide; e202 pollen could be stained very little; I-J: ZH11 anther cross section, K-L: anther cross section of e202. There were almost no plump pollen grains in e202. A-D: bar, 1 cm; E-H: bar, 100 μm."

Fig. 6

Transverse sections of e202 and ZH11 blades A-C: ZH11; D-F: e202. A, D: the large vascular bundle structures in the flag leaf, arrows indicate parenchyma cells. The small triangle represents the absence or reduction of the sclerenchyma cell layer in e202. B, E: the small vascular bundle structure in the flag leaf, and arrow represents vascular bundle; C, F: vesicular cells, compared with the control, the number of cells in e202 increased and the volume decreased."

Fig. 7

Measurement of chlorophyll content in ZH11 and e202 leaves * indicates significant difference compared with the control at P < 0.05; ** indicates extremely significant difference compared with the control at P < 0.01."

Table 2

Genetic separation ratio test between normal and roll leaf plants in F2 population"

群体
Population		 总株数
Total plant number		 正常表型
Normal phenotype		 矮化表型
Dwarfing phenotype		 期望分离比
Segregation ratio		 P值
P-value		 χ2
(ZH11×e202) F2 560 433 127 3﹕1 0.21 1.731

Fig. 8

Preliminary mapping and fine mapping of the candidate genes A: the genetic map of the candidate gene by preliminary mapping and fine mapping; B: the structure of the candidate gene; C: the relative expression of REL2 gene in ZH11 and e202. ‘n’ refers to the number of F2 mutants used for mapping."

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