Identification and Fine Mapping of Defective Pistil and Stamens 2 in Rice

doi:10.16819/j.1001-7216.2020.9122

Abstract

Abstract:

【Objective】 Identification and cloning of organ development-related genes can lay a foundation for further study on the molecular mechanism of rice flower development.【Method】The main agronomic traits and morphological characteristics of floral organs of wild type Chunjing 06 and dps2 (Defective pistil and stamens 2) mutant were compared under conventional planting conditions in field. The structure of anther was observed under a scanning electronic microscope by paraffin sectioning, and the fertility of pollen and embryo sac was observed by staining. Map-based cloning was used for the gene localization. Quantitative RT-PCR was performed to analyze the relative expression of genes associated with flower development in the wild type and the mutant. 【Result】 Longer heading date, abnormal flowering, shriveled pistils and stamens, increased anthers and stigmas were presented in the dps2 mutant. Further study revealed that the anther chamber of the dps2 mutant collapsed, and no microspores were found. Though some anthers had their chambers, there was no starch accumulation in the shriveled pollen grains, and the embryo sac fertility of dps2 was also affected. Genetic analysis indicated that the mutant’s phenotype of dps2 was controlled by a single recessive nuclear gene. The DPS2 gene was mapped to a 91.2 kb region on the short arm of chromosome 4. The expression levels of some genes associated with class B, C and E genes in ABCDE model were up-regulated in the mutant. 【Conclusion】 The abnormal development of stamens and pistils attributed to the complete infertility of the dps2 mutant, which indicated that DPS2 play an important role in third round of stamen development and the fourth round of pistil development.

Key words: rice, dps2, floral organ development, gene mapping

摘要：

【目的】鉴定和克隆花器官发育相关基因,为进一步研究水稻花发育的分子机制奠定基础。【方法】在大田常规种植条件下比较了突变体dps2 (Defective pistil and stamens 2)和野生型春江06的主要农艺性状及花器官形态特征差异;扫描电镜及石蜡切片观察花药结构并用染色法观察花粉和胚囊的育性;利用图位克隆方法进行基因精细定位;qRT-PCR分析了花发育相关基因在野生型和突变体中的表达水平。【结果】dps2突变体抽穗期变长,不能正常扬花,雄蕊和雌蕊皱缩且花药和柱头数目增多;进一步研究发现,dps2突变体花药腔室塌陷,内无可见小孢子,即使部分花药形成腔室,花粉粒也无淀粉积累呈干瘪状。此外,突变体胚囊育性也受到影响;遗传分析表明该突变性状受一对隐性核基因控制,该基因位于第4染色体短臂上91.2 kb的区间内,区间内未见花器官发育相关基因的报道。qRT-PCR检测发现,水稻ABCDE模型中的B类、C类和E类基因的表达在突变体中显著升高。【结论】dps2突变体的雄蕊及雌蕊均发育异常,最终导致完全不育,推测DPS2可能在水稻第3轮雄蕊发育和第4轮雌蕊发育调控中发挥重要作用。

关键词: 水稻, dps2, 花器官发育, 基因定位

CLC Number:

Q755
S511.01

Changjian WANG, Long CHEN, Liping DAI, Xueli LU, Jinli HE, Long YANG, Jiang HU, Li ZHU, Guojun DONG, Guangheng ZHANG, Zhenyu GAO, Deyong REN, Guang CHEN, Lan SHEN, Qiang ZHANG, Longbiao GUO, Qian QIAN, Dali ZENG. Identification and Fine Mapping of Defective Pistil and Stamens 2 in Rice[J]. Chinese Journal OF Rice Science, 2020, 34(2): 115-124.

王昌健, 陈龙, 代丽萍, 路雪丽, 贺金立, 杨龙, 胡江, 朱丽, 董国军, 张光恒, 高振宇, 任德勇, 陈光, 沈兰, 张强, 郭龙彪, 钱前, 曾大力. 水稻花器官发育基因DPS2的鉴定和精细定位[J]. 中国水稻科学, 2020, 34(2): 115-124.

Figures/Tables 9

Table 1 Part of primers used in the research.

引物名称	前引物	后引物
Primer name	Forward primer(5'-3')	Reverse primer(5'-3')
BY-4	GGAGGTGACTACGAAGGCATT	GAAGCTTATCCTTTTCACTTTCG
BY19	GAACTCCAAGTCCACCCTGAT	GATAAATTCTGTTGGTGGGAGATTG
BY5	GTTCACCAATAATCACCACAAGAGC	TAGCACCCCAGCAAGGACTCG
BY6-2	CTCGGCTCTCTTTGCTCGG	TGCCGACGAGGTCACCGCC
BY7-3	TATTTAGCCCTGGTTGAATGGT	TACCTAAGCTAAGTGGAAAGA
BY20	GACCAGAGGTGGACCAAGGA	GTGAATGGAAAGTGTGTGAAAGATC
BY10-1	GACTATATAGATATGTCCTCGGAAG	TTACCTATAACGAACCAAAGGC
BY13	GGAAACAATATACTATGGTGTGGAT	CGTGGGTGGAGTTTAGAATG

Fig. 1. Phenotypic comparison of dps2 mutant and wild type CJ06 at heading and maturity stage. A and B, Plants of Chunjiang 06(CJ06) and dps2 mutant at heading date; C and D, Panicle of CJ06 and dps2 at heading date; E, Heading date of CJ06 and dps2 in Lingshui, Hainan and Hangzhou, Zhejiang; F and G, Plants of CJ06 and dps2 at maturity stage; H and I, Panicle of CJ06 and dps2 at maturity stage; J, Seed-setting rate of CJ06 and dps2 in Lingshui and Hangzhou. Mean±SD(n=3). * and ** The difference between the wild type and mutant is significant at 0.05 and 0.01 level, respectively according to Student’s t test. In A, B, F, G, Bar=10 cm; In C, D, H, I, Bar=2 cm.

Fig. 2. Stamens phenotypic observation of wild type Chunjiang 06(CJ06) and dps2 mutant. A, Glumous flower of CJ06; B to D, Glumous flower of dps2 mutant; E, F, Anthers of the wild type and dps2; G, H, The outmost surface on epidermis anthers of the wild type and dps2; I, J, Pollens of CJ06 and dps2 mutant. Lo, Lodicule; An, Anther; F, Filament. From A to D, Bar=1 mm; In E, F, Bar=25 μm; In G, H, Bar=10 μm; In I, J, Bar=50 μm.

Fig. 3. Transverse section of anthers of dps2 mutant and wild type Chunjiang 06(CJ06) at different stages. A to C, CJ06; D to I, dps2 mutant; A, D, G, Cross-section of anther at the stage 9; B, E, H, Cross-section of anther at the stage 10; C, F, I, Cross-section of anther at the stage 12; Ep, Epidermis; En, Endothecium; T, Tapetum; DT, Degeneration tapetum; Ms, Microsporocyte; Msp, Microspore; Mp, Mature pollen. Bar=10 μm.

Fig. 4. Pistils phenotypic observation of wild type Chunjiang 06(CJ06) and dps2 mutant. A, Pistils of wild type; B, C, Pistils of dps2 mutant; D, Embryo sac of wild type; E, F, Embryo sac of dps2 mutant. Sti, Stigma; ov, Ovary. Scale bars: 1 mm in A, B and C, 50 μm in D to F.

Table 2 Genetic analysis of the dps2 locus

杂交组合 Hybridized combination	F₁				χ²_{(3: 1)}	χ²_(0.05)
杂交组合 Hybridized combination	正常表型 Wild-type phenotype	突变表型 dps2 phenotype	正常表型 Wild-type phenotype	突变表型 dps2 phenotype	χ²_{(3: 1)}	χ²_(0.05)
DPS2dps2/日本晴 DPS2dps2/Nipponbare	7	0	236	79	0.001	3.84
日本晴/DPS2dps2 Nipponbare/DPS2dps2	4	0	133	43	0.030	3.84
DPS2dps2/9311	5	0	160	42	1.908	3.84
9311/ DPS2dps2	4	0	127	35	0.996	3.84

Fig. 5. Location of DPS2 on rice chromosome 4. A, Preliminary localization of DPS2 gene; B, Fine mapping of DPS2 gene; C, ORFs between markers BY6-2 and BY7-3.

Table 3 The 12 genes in the candidate interval on chromosome 4.

登录号	位置	功能注释
Gene accession	Location/bp	Functional annotation
LOC_Os04g08290	4 427 683-4 424 830	ZOS4-04-C₂H₂锌指蛋白 ZOS4-04-C₂H₂ zinc finger protein
LOC_Os04g08300	4 433 624-4 433 839	反转录转座子蛋白,Ty3-gypsy亚类 Retrotransposon protein, putative, Ty3-gypsy subclass
LOC_Os04g08310	4 436 856-4 441 657	表达蛋白 Expressed protein
LOC_Os04g08320	4 444 004-4 441 881	假定蛋白YIF1B YIF1B, putative
LOC_Os04g08330	4 446 960-4 446 649	表达蛋白 Expressed protein
LOC_Os04g08340	4 452 696-4 455 350	OsSigP3-假定的Ⅰ型信号肽酶同系物;采用假定的Ser/Lys催化二元体 OsSigP3-putative Type I signal peptidase homologue, employs a putative Ser/Lys catalytic dyad
LOC_Os04g08350	4 461 845-4 457 458	半胱氨酸合酶,叶绿体/染色质前体 Cysteine synthase, chloroplast/chromoplast precursor
LOC_Os04g08360	4 463 046-4 463 552	表达蛋白 Expressed protein
LOC_Os04g08370	4 473 512-4 470 642	富含亮氨酸的重复家族蛋白 Leucine rich repeat family protein
LOC_Os04g08390	4 490 101-4 484 997	富含亮氨酸的重复家族蛋白 Leucine rich repeat family protein
LOC_Os04g08400	4 497 789-4 503 741	反转录转座子蛋白 Retrotransposon protein
LOC_Os04g08410	4 513 883-4 508 804	含有ELM2结构域蛋白 ELM2 domain containing protein

Fig. 6. Expression profile of the genes involved in the number and development of flower organs of dps2 mutant and wild type Chunjiang 06. Mean±SD(n=3). * and ** indicate the difference between the wild type Chunjiang 06 and mutant dps2 is significant at 0.05 and 0.01 levels, respectively according to Student’s t test.

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