水稻颖壳异常突变体ah1的鉴定与候选基因分析

doi:10.3864/j.issn.0578-1752.2024.03.001

Abstract

Abstract:

【Objective】Rice is the staple grain crop worldwide, and the morphology of its grains directly influences its ultimate yield, nutritional excellence, and economic significance. Moreover, the intricate interplay between floral development and grain morphology adds further significance to this relationship. Thus, exploring novel rice floral development regulatory genes and molecular regulatory mechanisms lays the foundation for larger and more plump grains rice varieties. 【Method】Ethyl methyl sulfonate (EMS) was used to mutate XD1B (xian-type maintainer line), and a dwarf mutant abnormal hull 1 (ah1) with abnormal formation of glume and lodicule was identified. The agronomic traits of both the wild-type and mutant were observed and recorded. Spikelets from various flowering stage were collected to histological and morphological analysis. The F₂ segregating population was established by ah1 and 56S (xian-type thermo-sensitive sterility line), and utlized for genetic analysis and gene mapping. RNA was isolated from young panicles of both the wild-type and mutant, then reverse transcribed into cDNA. The RT-qPCR analysis was performed to analyze the relative expression levels of the genes regulating floral development and the key genes in the ABA synthesis pathway. 【Result】The observation of agronomic traits revealed that the dwarfed plant was caused by the dramatic shortening of the internodes. At the same time, the mutant is also accompanied by severe spikelet abnormalities and low fruit setting rate. Histological and morphological analysis revealed that the ah1 mutant spikelets exhibited varying degrees of degeneration in floral organs such as palea, lemma, lodicules, and stamens. Some severely affected spikelets displayed altered floral organ characteristics and determinacy of floral meristems, often accompanied by extensive whitening. Based on the extent of degeneration, these spikelets could be classified as slight or severe mutant phenotypes. Genetic analysis showed a segregation ratio of 3﹕1 for the wild-type and mutant within the segregating population, indicating that the mutant traits of ah1 were controlled by a single recessive locus. The AH1 was mapped between the molecular markers RM6716 and RM128 on the chromosome 1, with a physical distance of approximately 8 Mb. Resequencing analysis of the mutant revealed that the LOC_Os01g53450 and LOC_Os01g51860 within this interval showed variation between wild-type and mutant, thus these two genes were provisionally identified as candidate genes. RT-qPCR analysis revealed significant alterations in the relative expression levels of floral organ development regulatory genes during the early developmental stages of mutant panicles; meanwhile, the relative expression levels of OsNCED1/OsNCED2/ OsNCED3/OsNCED4/OsNCED5, the ABA synthesis pathway key genes, were severe inhibited.【Conclusion】AH1 plays a crucial role in the morphological formation of floral organs, such as palea and lemma in rice. LOC_Os01g53450 and LOC_Os01g51860 were provisionally identified as candidate genes in this work.

Key words: rice (Oryza sativa L.), glume, floral development, candidate genes

ZHANG BiDong, LIN Hong, ZHU SiYing, LI ZhongCheng, ZHUANG Hui, LI YunFeng. Identification and Candidate Gene Analysis of the ABNORMAL HULL 1 (ah1) Mutant in Rice (Oryza sativa L.)[J].Scientia Agricultura Sinica, 2024, 57(3): 429-441.

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Figures/Tables 7

Table 1

Fig. 1

Phenotype analysis of spikelets in wild-type (WT) and ah1 mutant A1: WT spikelet; A2: WT spikelet from which the palea and lemma have been removed; A3: WT floral primordium in stag Sp6; A4: WT floral primordium in stag Sp8; A5: Transverse section of WT spikelet; B1: Spikelet of ah1 with slight mutant phenotype; B2: Spikelet of ah1 in B1 from which the palea and lemma have been removed; B3: Floral primordium of ah1(with slight mutant phenotype) in stag Sp6; B4: Floral primordium of ah1(with slight mutant phenotype) in stag Sp8 ; B5: Transverse section of spikelet of ah1 with slight mutant phenotype; C1: Spikelet of ah1 with severe mutant phenotype; C2: Spikelet of ah1 in C1 from which the palea and lemma have been removed; C3: Floral primordium of ah1(with severe mutant phenotype)in stag Sp6; C4: Floral primordium of ah1(with severe mutant phenotype) in stag Sp8; C5: Transverse section of spikelet of ah1 with severe mutant phenotype; D1 and D2: Spikelets of ah1 with elongated palea, degraded lemma, and abnormal lodicule (Type Ⅰ); D3: The degraded palea, the detail of red frame in D2; D4: Abnormal development of lodicule of spikelet in D2; D5: Transverse section of vestigial palea of spikelet in D1; D6: Transverse section of the abormal lodicule of the spikelet in D1; E1 and E2: Spikelets of ah1 with pistilloid-stamens, degraded palea and lemma, and elongated lodicule (Type Ⅱ); E3: The vestigial lemma, the detail of red frame in E2; E4: The elongated lodicule of spikelets in E2; E5: Double-flowered spikelet of ah1 (Type Ⅲ). Abbreviation: le: Lemma; pa: Palea; lo: Lodicule; st: Stamen; pi: Pistil; sl: Sterile lemma; rg: Rudimentary glume; mrp: Marginal region of palea; dl: Degraded lemma; ele: Elongated lemma; alo: Abnormal lodicule; elo: Elongated lodicule; ex-floret: Extra floret; *: Stamen. Bars: 5 000 µm in A1, and A2; 1 000 µm in B1, B2, C1, C2, D1, E1, and E5; 500 µm in A4, A5, B4, B5, C4, C5, D4, D5, and D6; 2 000 µm in D2, and E2; 100 µm in A3, B3, C3, D3, E3, and E4"

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引物 Primer	正向序列 Forward sequence (5′-3′)	反向序列 Reverse sequence (5′-3′)	用途 Purpose
RM6716	CCTATGCGATCTCCTATTTGG	CGAGTCAGACACGTACTACTGC	定位 Mapping
RM128	TGATTTCTTGGAAGCGAAGAGTGAGG	CCTCCTTGTGCTCAGCCATGC
RM1268	GCTGTCACTGACCGAGCGTAGG	TCGAGAGATCCAATCCAGTTTGC
ACTIN	GACCCAGATCATGTTTGAGACCT	CAGTGTGGCTGACACCATCAC	RT-qPCR
OsMADS14	CTCATCATCCTCCTTCAT	GCTACATCCTCTATCCTT
OsMADS15	GCTGAATCCGAGAGTGAG	TGAGGTGTTTGTGACATTTG
OsMADS18	GGTGATGAATATGGAATT	CTTATATGCTTCAGAGAAT
OsMADS2	ATGATTGAGGAGGCACTT	TCGTCTTCCAGCATCTTAT
OsMADS4	CCAATCTGCGGGACAAGA	AGCCAAATTGGCAGTGCTC
OsMADS16	TTCGCCACAGGAAGTATCA	CCTCGTAGGAGTGCTTCA
OsMADS3	CGATCATATGGTTAATAACCCA	AGACGAGTTGCCAAAGTTCGA
OsMADS58	ATATCACCACCATGAACCA	TTCTTCCGTGCTCTAATCT
OsMADS13	AGAGCCAGGAAGAATGAA	ATGTTGTCGTTCTGAAGC
OsMADS1	GCTGCAACTACAACTCACAGG	TGATGGTGAGCATGAGGGTG
OsMADS5	ACTGAACTAAGCAATTACCAAGA	CAAGTCCTCGCCAAGAAG
OsMADS7	ATCTTGATTCATTAGGCATAA	TTGTCCTTGTAGTTCTGA
OsMADS8	GCACATTAGATCCACAAG	TTCTTCTGAGGCACTTAT
OsMADS34	AGGAATATGTGAACTTGAA	TTCTGACTACTTGACTCT
DL	CCCATCTGCTTACAACCGCTT	GTTGGAGGTGGAAACCGTCG
OsMADS6	TGATGATGGAACAAGTGGAG	TTGTGCTTGAGTTGCCTAT
OsMADS32	GGAGTTTGTCGAGAGGTACGA	CTGAACGTCAAGCGTCCATC
SL1	AAGCATCCAGGGTTACTAA	GCAGAACCTACACTCGTA
FON1	TGCGTGAAGGAGGACAACATCATC	AGCAGCAGGTTCGTCTCCCTGTT
OsNCED1	AAGCAGACCGAGCAAGAG	ATTCGTCGTAGCAATAACAGTTC
OsNCED2	AGCCAATGTTATGACTCT	TCTGTAGATTACGGTGAG
OsNCED3	AGGATATGCTCACATACAG	AGAATCTCACCGAATTGG
OsNCED4	ACGATGATCCACGACTTC	ATCTCCTGGAGCTTGAAC
OsNCED5	ATCAAGAAGCCATACCTGAA	CTGCTCAAGCTCGATCTC
LOC_Os01g51860	TGAAGGAATCTGCTGAAGT	AGTATCTGAGGACCACAATT
LOC_Os01g53450	ACGGAATGGCACAGTGGGAC	GGTCCCATGCCGTACTCTTGAG

序号 Number	基因名称 Gene name	注释 Annotation	位置 Position (Mb)
1	LOC_Os01g51860	紫黄素脱环氧化酶，推测，表达 Violaxanthin de-epoxidase, putative, expressed	28.44
2	LOC_Os01g53450	转氨酶，Ⅰ类和Ⅱ类，结构域蛋白，表达 Aminotransferase, classesⅠandⅡ, domain containing protein, expressed	29.29

Identification and Candidate Gene Analysis of the ABNORMAL HULL 1 (ah1) Mutant in Rice (Oryza sativa L.)

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