Identification and Gene Mapping of a white-stripe leaf after transplanting at low temperature Mutant in Rice

doi:10.16819/j.1001-7216.2018.8026

Abstract

Abstract:

【Objective】 Isolation and characterization of leaf-color mutation related genes lays a firm theoretical foundation for dissecting the molecular mechanism underlying chloroplast development, chlorophyll biosynthesis, and photosynthesis in rice. 【Method】A spontaneous leaf-color mutant, termed as white stripe leaf after transplanting at low-temperature (wltt), was obtained from the progeny of japonica cultivar Zhennuo 19. The main agronomic traits of the wild type and wltt were determined at maturity. The pigment contents and ultrastructure of chloroplast of newly emerged leaves were analyzed at the seedling stage, fifteen days after transplanting, at the tillering stage under direct seeding. Genetic analysis was carried out by reciprocal cross of the wild type and wltt. An F₂ population derived from the cross wltt×9311 was used for gene mapping. Quantitative RT-PCR was carried out to analyze the relative expression of genes associated with chloroplast development and chlorophyll biogenesis in the wild type and the wltt mutant. 【Result】The white-striped leaves in the wltt mutant only emerged at 15 days after transplanting at low temperature such as 20℃. No white-striped leaf was observed under direct seeding treatment. However, leaves of the mutant developed normally at the late tillering stage. Simulation experiments showed that the mutant phenotype was caused by root injury at low temperature. Compared with the wild type, the pigment contents in white-stripe leaves of the wltt mutant were significantly decreased, accompanying by reduced photosynthetic rate. Simultaneously, most of the mesophyll cells had no chloroplasts. The expression levels of genes associated with chloroplast development, chlorophyll biosynthesis, and photosynthetic system were all down-regulated in the mutant. At maturity, the mutant was featured with reduced plant height, panicle length, flag leaf length and number of spikelets per panicle relative to its wild type. Genetic analysis revealed that the mutant phenotype was controlled by a single recessive nuclear gene. Moreover, the WLTT gene was mapped within an 853 kb region near the centromere on chromosome 2, between InDel markers L22 and L26, in which no gene related to leaf color was reported. 【Conclusion】WLTT is a key gene regulating leaf color after transplanting at low temperature, which plays an important role in chloroplast development.

Key words: rice, white-stripe leaf after transplanting, chloroplast development, gene mapping

摘要：

【目的】叶色突变相关基因的鉴定与克隆为研究叶绿体发育、叶绿素合成和光合作用等分子机制提供理论基础。【方法】从常规粳稻镇糯19杂交后代中分离出一个低温移栽后叶色转成白条纹的自然变异突变体, 命名为wltt (white stripe leaf after transplanting at low temperature)。成熟期测定野生型和wltt的主要农艺性状,分别在苗期、移栽后15 d和同时期直播条件下测定新生叶片的色素含量并观察叶绿体的超微结构;将wltt和野生型正反交进行遗传分析;用wltt与籼稻9311杂交产生的F₂作为定位群体进行基因定位;采用RT-qPCR 分析叶绿体发育、叶绿素合成和光合作用相关基因在野生型和wltt中的表达水平。【结果】wltt突变体在苗期表现正常绿色,移栽15 d后心叶出现白条纹叶表型,至分蘖末期心叶叶色恢复;而不经移栽,突变体不会出现白条纹叶。人工模拟实验表明该表型是由低温条件下根损伤引起的。与野生型相比,wltt突变体移栽后的新生叶色素含量显著降低,光合速率下降;同时株高变矮,穗长、剑叶长和每穗粒数均显著降低。叶绿体的超微结构显示,突变体的叶肉细胞中,仅少数细胞含有正常的叶绿体,其余大部分叶肉细胞不含叶绿体。进一步研究发现,突变体中部分光合系统相关基因和叶绿体发育相关基因表达下调,叶绿素生物合成相关的14个基因表达也下调。遗传分析表明,该突变性状受一对隐性核基因控制。利用wltt突变体/9311的F₂群体,将该基因定位于水稻第2染色体着丝粒附近853 kb区间内。目前,该区间内没有叶色相关基因的报道。【结论】WLTT是低温条件下移栽调控叶片转色的关键基因,在叶绿体发育过程中发挥重要作用。

关键词: 水稻, 移栽叶片转色, 叶绿体发育, 基因定位

CLC Number:

Tianzi LIN, Liting SUN, Hongbin GONG, Yihua WANG, Linglong LIU, Zhigang ZHAO, Ling JIANG, Jianmin WAN. Identification and Gene Mapping of a white-stripe leaf after transplanting at low temperature Mutant in Rice[J]. Chinese Journal OF Rice Science, 2019, 33(1): 1-11.

林添资, 孙立亭, 龚红兵, 王益华, 刘玲珑, 赵志刚, 江玲, 万建民. 一个水稻低温移栽白条纹突变体wltt的鉴定和基因定位[J]. 中国水稻科学, 2019, 33(1): 1-11.

Figures/Tables 11

Table 1 Primers used for gene mapping.

引物名称 Primer name	前引物 Forward sequence (5′-3′)	后引物 Reverse sequence (5′-3′)	BAC克隆 BAC clone
L1	ATTCAGTAAGACTACACGCAT	AATGACAGATTACTTGTTCCA	OJ1756_H07
L6	CTAACATAATGGGTAAAGAGG	TTAGTTGGTTGCCGTGT	OJ1124_E11
L8	ATAGTTTAGGGAGTTATGTGCT	CGTGTGCCTATTGACTTCTC	OSJNBa0030M21
L11	ACAGAACGGAACGGGATA	CTCACAATCTTTTATCACCCA	OSJNBa0078K05
L14	AACCAAGAATCGGAAAGAA	ATCCCATTTCCATTTCTCT	OSJNBa0008C07
L16	TTTCCTGAGCGAATCCA	AAAAGGCACTTATGAGACACT	OSJNBb0080M22
L18	TAGGTGGTTGAATGGTGC	TATGCTTCTTTTGGGTTG	P0543C11
L20	TGAGATACGCAGAATGGG	GAGGAGGATGCAGGGAC	P0705A04
L22	GTTCTTTTGTCTTCCCTCA	ATTATCCTTGGTCTTGGTAT	OJ1134_B09
L26	TTGGAGAATGAAGTTGCTAA	TTACCAAGCAGGACTAAAGAT	OSJNBb0037J12
I2-7	GAACCAGTCCGCTCTCTGAC	TACGCGTCGTGTATCGTAGC	OSJNBa0035A24

Fig. 1. Phenotypic characterization of the wltt mutant and its wild type(WT). A, Seedling stage; B, Fifteen days after transplanting, at the tillering stage; The insert represents the new fully-expanded leaf blades of the wild type (left) and the mutant (right); C, Fifteen days after transplanting at the heading stage. The inserts represents the leaves of regenerated tillers of the wild type (left) and the mutant (right); D, Plants at the mature stage (transplanting at the tillering stage). E, Wild-type and mutant plants at the tillering stage under direct seeding. F, Net photosynthetic rate of new fully expanded leaf blades of the wild type and wltt mutant at the tillering stage. WT, Wild type. ** P<0.01(Student’s t test).

Table 2 Comparison of major agronomic traits between the wltt mutant and its wild type(WT).

材料 Material	株高 Plant height /cm	有效穗数 No. of effective panicles	剑叶长 Flag leaf length /cm	穗长 Panicle length /cm		每穗总粒数 No. of spikelets per panicle	结实率 Seed-setting rate /%	千粒重 1000-grain weight /g
WT	86.7±0.2	9.2±0.4	22.28±0.53		20.44±0.46	173.6±5.7	95.8±0.4	29.18±0.26
wltt	75.8±0.5^**	8.2±0.4	18.10±0.37^**		18.40±0.41^**	136.2±6.9^**	95.6±1.7	28.46±0.32

Fig. 2. Pigment contents in leaves of wltt mutant and its wild type(WT). Chla, Chlorophyll a; Chlb, Chlorophyll b; Car, Carotenoids; Total, Total pigment contents. Values are presented as mean±SD. **The difference between the wild type and wltt is significant at 0.01 level according to Student’s t test.

Fig. 3. Transmission electron microscopic (TEM) images of chloroplast ultrastructure in the wild type and the wltt mutant. A-C, Transmission electron microscopic (TEM) images of chloroplast ultrastructure in wild type; D-F, Two types of chloroplasts with (E, F) or without (D) a normal ultrastructure in the wltt mutant; Cp, Chloroplast; Thy, Thylakoid lamellae; OB, Osmiophilic body.

Fig. 4. Expression analysis of genes associated with chloroplast development and photosynthetic system in the wild type(WT) and wltt mutant. Mean±SD (n=3). **The difference between the wild type and wltt is significant at 0.01 level according to Student’s t test.

Fig. 5. Expression analysis of genes associated with chlorophyll biogenesis in the wild type(WT) and wltt mutant. Means ± SD (n=3). * and **The difference between the wild type and wltt is significant at 0.05 and 0.01 level, respectively according to Student’s t test.

Fig. 6. Effects of environmental conditions on leaf color variation. A, The seedlings of the wild type and wltt with non-injured roots; B, C, The seedlings of wild type and wltt 10 days after root cutting treatment at 20℃ at the light intensity of 25 000 lx(B) and 6250 lx(C), respectively. D, E, The seedlings of wild type and wltt 10 days after root cutting treatment at 30℃ at the light intensity of 25 000 lx(D) and 6250 lx(E), respectively.

Fig. 7. Effects of environmental conditions on pigment contents after root cutting treatment. Means±SD (n=3). **The difference between the wild type and wltt is significant at 0.01 level according to Student’s t test.

Table 3 Segregation of F2 population from wltt mutant and its wild type(WT).

杂交组合 Cross	正常株数 No. of normal plants	白条纹株数 No. of white stripe plants	实际分离比 Segregation ratio	χ²_(3:1)
WT×wltt	375	127	2.95:1	0.02
wltt×WT	368	130	2.83:1	0.06

Fig. 8. Location of WLTT on rice chromosome 2. A, The gene is mapped to the centromeric region of chromosome 2 between the InDel markers I2-5 and I2-8; B, Mapping of the gene locus between markers L22 and L26 within an 853 kb region. CEN, Centromere; n, Number of individuals with white stripe leaf after transplanting.

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