Genetic Analysis and Molecular Mapping of Light-Sensitive Red-Root Mutant in Rice

Rice Science, 2009, 16(1): 27–32 Copyright © 2009, China National Rice Research Institute. Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(08)60053-0

Genetic Analysis and Molecular Mapping of Light-Sensitive Red-Root Mutant in Rice ZHANG Jun-zhi, LIU Xiao, LI Chao, XIAO Ke, DONG Yan-jun (Laboratory of Plant Genetics and Functional Genes, College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234, China)

Abstract: The light-sensitive red-root mutant, designated as HG1, was newly observed from an indica rice variety, Nankinkodo, when seedlings were grown with roots exposed to natural light. The root color of the mutant began to turn 2

slight-red when the roots were exposed to the light at the intensity of 29 μmol/(m ·s), then turned dark-red at the light intensity 2

of 180 μmol/(m ·s), suggesting that the root color of the mutant was evidently sensitive to light. Furthermore, genetic analysis showed that the character of light-sensitive red-root of the HG1 mutant was controlled by a single dominant gene, tentatively designated as Lsr. With simple sequence repeat markers, Lsr gene was located between the markers RM252 and RM303 on chromosome 4 with the genetic distances of 9.8 cM and 6.4 cM, respectively. These results could be useful for fine mapping and cloning of Lsr gene in rice. Key words: rice; light sensitivity; red root mutant; genetic analysis; gene mapping

Rice varieties with colored organs such as leaf, stem, hull and pericarp, different from normal green plants, are usually termed as color rice, resulting from the accumulation of various kinds and contents of anthocyanin pigments in vacuoles [1]. Anthocyanin pertains to flavonoid compounds and bioactive anthocyanin plays an important role in rice development. In addition, anthocyanin decreases the concentrations of serum cholesterol and serum lipids, and is physiologically antioxidant, anti-inflammatory and anti-cancer agents [2-5]. At present, the anthocyanin pigment in rice has become one of the highlights for research and development of functional foods [6-8]. However, studies on anthocyanin in rice were mainly focused on leaf, stem, hull and pericarp [9-11]. So far, to our knowledge, the rice mutant with the coloration in roots has not been reported yet. However, it is not rare case that photochromic reactions in roots are observed when the roots are exposed to natural light in some species [12], such as Impatiens spp. [13], Triticum sp. [14], Ruphanus sutivus [15], Zea mays [16] and Metrosideros excelsa [17]. However, little is known about the expression of anthocyanin Received: 26 August 2008; Accepted: 10 November 2008 Corresponding author: DONG yan-jun ([email protected]) This is an English version of the paper published in Chinese in Chinese Journal of Rice Science, Vol. 22, No. 6, 2008, Pages 578–582.

genes in rice roots. Therefore, the exploration of the genetic mechanisms and functions of the pigment genes in rice roots may provide indispensable information for profoundly understanding the expression and regulation of pigment genes on the whole rice plants. In a hydroponic culture experiment, we found an indica rice variety Nankinkodo from Japan displaying unusual red-root when the roots were exposed to natural light. Through several self-pollinations, the stably inherited light-sensitive red-root mutant was developed, designated as HG1. The objectives of this study are to identify its light sensitivity and locate the relevant gene conferring light sensitivity, thereby supply useful information for fine mapping and cloning of the gene, and for the utilization of the bio-pigment in future.

MATERIALS AND METHODS Plant materials The mutant HG1 was derived from an indica variety Nankinkodo introduced from Japan. All agronomic characters in the mutant were stably inherited through self-pollination for several years in both Shanghai and Hainan, China.

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Observation on the characteristics of light sensitivity in HG1 When the mutant was grown in a hydroponic culture without nutrient and treated with natural light at two levels of intensity, viz. strong (outdoors) and weak (indoor laboratory), it could be found that the young seedling roots outdoors apparently turned red, whereas those indoors displayed normal white. It could be preliminarily deduced that the root-color trait of the mutant was light sensitive. To further determine the relationship between the light intensity and root color, in December 2007, the HG1 mutant was grown in a hydroponic culture and treated with six different levels of light intensity, i.e. 29, 34, 51, 78, 112 and 180 μmol/(m2·s), at 28ºC/25ºC (day/night) in a growth chamber (GXZ300-D, Ningbo Jiangnan Instrument Factory, China). After 15 days, the root color was observed and the pigments in roots were extracted according to the method of Wu et al [18]. Then the absorption values of pigments in extraction were measured at the wavelength of 543 nm with a spectrophotometer (UV-754) with three replications. Average values for each sample were used for the characteristic analysis. Genetic analysis and development of population for mapping The HG1 mutant was crossed with an indica rice variety Khao Nok from Thailand to obtain the seeds of reciprocal hybrids and their F2 seeds through selfpollination. Then the healthy F2 seeds were germinated at 25ºC for 4 days. On 28 May 2007, the germinated seeds were planted in polystyrene foam plates in plastic containers containing only tap water at glasshouse of Shanghai Normal University, Shanghai, China. After 14 days, the root color of seedlings in F2 population was individually investigated and classified into two groups of red root and white root for genetic analysis of segregation ratio. Then all F2 seedlings were transplanted individually into the paddy fields and its progeny F3 seeds were harvested from each F2 plant. In December of the same year, 20 F3 seeds randomly selected from each F2 plant were grown individually in petri dish containing only tap water in the growth chambers at 28ºC/25ºC (day/night) and the light intensity

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of 180 μmol/(m2·s). After 20 days, the root color of each seedling was individually investigated. Then, according to the genetic analysis of segregation ratios of the root color in F3 generations, the genotypes of F2 plants were divided into three genotypes of white root (recessive homozygote), segregating red-root (heterozygote) and nonsegregating red-root (dominant homozygote). Total genomic DNA extraction Total genomic DNA of the two parents and F2 plants were extracted from fresh rice leaves following the modified protocol of McCouch et al [19], and used as templates for polymerase chain reaction (PCR) amplification. Mapping of the HG1 mutant gene In this study, a two-step analysis was used for gene mapping with simple sequence repeat (SSR) markers from Gramene (www.gramene.org). Firstly, polymorphic SSR markers on each chromosome were detected between the two parents (HG1 and Khao Nok). Secondly, the candidate SSR markers were used to genotype the F2 populations consisting of 153 plants. The PCR was performed in a volume of 50 μL, including approximately 20 ng genomic DNA, 40.5 μL ddH2O, 100 mmol/L Tris-HCl (pH 9.0), 100 mmol/L KCl, 20 mmol/L MgSO4, 80 mmol/L (NH4)2SO4, 10 mmol/L dNTP mixture, 10 μmol/L each primer and 5 U/μL Taq polymerase. PCR conditions were as follows: pre-denatured at 94ºC for 4 min followed by 34 cycles of denaturation at 94ºC for 1 min, annealing at 55ºC for 1 min, extension at 72ºC for 1 min, and finally extended at 72ºC for 10 min. The PCR products (7.0–8.0 μL) were electrophoresed on 3.5% agarose gel, stained with EB (ethidium bromide) at 120 V for 2 h in 1×TBE buffer. Electrophoresis patterns were visualized by UVP Biolmaging Systems and the bands were transformed to ‘A’, ‘B’, and ‘H’ types. ‘A’ represents the band type same to the dominant parent (HG1), ‘B’ the ones same to the recessive parent (Khao Nok), ‘H’ the heterozygous, and ‘•’ the blank band ones. Linkage analysis was performed with MAPMAKER/EXP 3.0 [20] and map distances in centiMograns (cM) were calculated by the Kosambi function.

ZHANG Jun-zhi, et al. Genetic Analysis and Molecular Mapping of Light-Sensitive Red-Root Mutant in Rice

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RESULTS Identification of the light sensitivity of root color in the HG1 mutant After the roots being exposed to light for 15 days, the root color obviously got redder along with the increase of light intensity. The roots under the light intensity of 180 μmol/(m2·s) turned dark-red, whereas there was no visible color change at the light intensity of 29 μmol/(m2·s) (Fig. 1). Likewise, the absorption values at 543 nm of root pigments in extraction from roots of the HG1 mutant were significantly enhanced with the increase of light intensity (Fig. 2). In detail, the absorption value (0.11) at the light intensity of 180 μmol/(m2·s) was 55-fold of that (0.02) at 29 μmol/(m2·s), showing that the root color of the mutant was highly light-sensitive.

Fig. 2. Effects of light intensity on the absorbance of root pigments in the HG1 mutant.

Genetic analysis of root color The root color of all F1 seedlings from reciprocal crosses (Khao Nok/HG1 and HG1/Khao Nok) was red, and the segregation ratios of red root to white root in F2 populations derived from both reciprocal crosses fit 3:1 by Ȥ2 test, showing that the light-sensitive photochromic red-root trait in HG1 was controlled by a single dominant gene (Table 1). Likewise, in F3 generation derived from 100 random F2 plants, the segregation ratio of homozygote recessive white root (22 F2 plants), heterozygous red root (56 F2 plants) and homozygote dominant red root (22 F2 plants) fit 1:2:1 by Ȥ2 test. All these results showed that the trait of light-sensitive red-root in HG1 was controlled by a single dominant gene, and tentatively designated as Lsr. Primary mapping for Lsr gene

Fig. 1. Performances of the root color of the HG1 mutant at different light intensities. A, Root color of the HG1 mutant at the light intensity of 180 μmol/(m2·s); B, Root color of the HG1 mutant at the light intensity of 29 μmol/(m2·s).

In this study, the F2 population derived from the cross between Khao Nok/HG1 were used for the primary mapping of Lsr gene. Two SSR primers showing polymorphisms between the two parents were detected on each of chromosomes 1, 2, 3, 4, 6, 7, and 8, respectively. The linkage relationship among 36 recessive white-root F2 plants was analyzed using the 14 SSR markers. The two SSR markers, RM303 and RM252 on chromosome 4, were detected to be closely

Table 1. Segregation of red-root in F2 population derived from the cross of Khao Nok and HG1. No. of red-root plants

No. of white-root plants

Ȥ2 (3:1)

P

153

112

41

0.066

0.70–0.80

181

135

46

0.004

0.95–0.99

334

247

87

0.049

0.80–0.90

Cross

No. of total plants

Khao Nok/HG1 HG1/Khao Nok Total

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Fig. 3. Segregation of the SSR markers RM303 and RM252 among the two parents and some F2 plants with white roots. P1 and P2 represent the segregation of the SSR markers RM303 and RM252 in the parents Khao Nok and HG1, respectively; The others are the segregation of the SSR markers RM303 and RM252 in recessive F2 plants with white roots.

linked with the gene conditioning the trait of lightsensitive red-root in HG1 (Fig. 3). Furthermore, five other polymorphic SSR markers, namely RM348, RM317, RM348, RM456B and RM564, were detectable on chromosome 4. Finally, three pairs of primers (RM303, RM252 and RM348) with distinct polymorphisms were selected for mapping the target gene Lsr in the F2 population derived from Khao Nok/HG1. In addition, the linkage analysis for the type of SSR bands and the segregation of the target trait in F2 population were performed with the MAPMAKER/ EXP 3.0 software [20] to construct a molecular linkage map covering the target gene (Lsr). The results showed that the dominant light-sensitive red-root gene Lsr was located between RM252 and RM303 on chromosome 4 with the genetic distances of 9.8 cM and 6.4 cM, respectively (Fig. 4).

DISCUSSION It is clear that genotypic and environmental factors have a combinative influence on the expression of anthocyanin genes in rice, however, the study involved these fields is still limited [11]. In 1996, Reddy [21] reviewed that the occurrence of tissue-specific pigmentation is determined by the C-A-P system: The C gene, mapped into a 59.3 kb region on the short arm of chromosome 6 by Fan et al [22], is an essential gene for the production of chromogen; A gene (activator) on chromosome 1 activates C gene, turning the chromogen into anthocyanin; and P gene (distributor) is responsible for the tissue-specific distribution and accumulation of anthocyanin pigments. The different tissue-specific regulations of P gene are classified as P (apiculus), Pl (leaf), Pn (node), Prp (pericarp) and so on. In addition, Singh et al [23] reported that anthocyanin genes had

Fig. 4. Location of Lsr gene on chromosome 4.

pleiotropy effects and could express in multi-tissues of rice plants. To date, any pigment genes expressing in rice roots have not been reported yet. In this paper, we report that the pigment gene (Lsr gene) could express in rice roots and the red-root trait is light-sensitive in rice. Specifically, the root color of the HG1 mutant turned slight-red when the roots were exposed to light at the intensity of 29 μmol/(m2·s). Lsr gene was located between the SSR markers RM252 and RM303 on chromosome 4 with the genetic distances of 9.8 cM and 6.4 cM, respectively. The rough mapping in this study can be useful for fine mapping and cloning of Lsr gene, and further understanding for the spatial expression of pigment gene in rice plants. In rice, studies on root are far behind those on aerial-part. Although it is one of important approaches to explore safe and edible pigments by using the colored rice-tissues, such as root, stem, leaf and kernels, the related works barely started out. In 1996, Zhuang et al [24] mapped a major gene controlling purple-black rice between the RFLP markers RG329 and RG314 on chromosome 4. In addition, Phoka et al [25] detected two QTLs, which were located between RM314 and

ZHANG Jun-zhi, et al. Genetic Analysis and Molecular Mapping of Light-Sensitive Red-Root Mutant in Rice

RM241, and RM252 and RM214 on chromosome 4, respectively. Interestingly, all genomic regions of the gene/QTLs mentioned above are located near the Lrs gene between RM252 and RM30, which well coincide with our results that the light-sensitive red-root trait in the HG1 mutant is tightly linked to the black-rice trait in the same F2 population (data not shown). More interestingly, we found that another black-rice variety ‘HB-1’ exhibited white root and was not lightsensitive for root color under all the same conditions as the HG1 mutant (data not shown), which suggests that the Lrs gene might be different from the blackrice gene in the HG1 mutant. From these results, it can be presumed that there might be a functional domain on chromosome 4 that controls the expression(s) of the pigment gene(s) in rice, which might provide a new approach for further studying the spatial expression of the pigment genes in rice plants.

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protective effect of cyaniding 3-O-beta-D-glucoside. J Nutr Biochem, 2005, 16(1): 30–37. 6

Chung H S, Woo W S. A quinoline alkaloid with antioxidant activity from the aleurone layer of anthocyanin-pigmented rice. J Nat Prod, 2001, 64(12): 1579–1580.

7

Hu P S. Research and utilization of functional nutritional rice. China Rice, 2003(5): 3–5. (in Chinese)

8

Zhang M W, Guo B J. Summary on nutritional features and health-care function of pigmented rice grain and its processing techniques. Acta Agric Shanghai, 2002, 18(suppl): 18–24. (in Chinese with English abstract)

9

Hadagal B N, Manjunath A, Gouo V J. Linkage of genes for anthocyanin pigmentation in rice (Oryza sativa L.). Euphytica, 1980, 30: 747–754.

10

Wataru S, Taku O, Keisuke K, Chikara M, Akira S, Minoru M, Kazuhiko N, Masahiko M. The purple leaf locus of rice: The plw allele has a complex organization and includes two genes encoding basic helix-loop-helix proteins involved in anthocyanin biosynthesis. Plant Cell Physiol, 2001, 42(9): 982–991.

11

Sun M M, Han L Z, Li G X, Hong X T, Yu Y J. Advances in genetic research of gain anthocyanins content in rice. J Plant Genet Res, 2006, 7(2): 239–245. (in Chinese with English

ACKNOWLEDGEMENTS

abstract)

We sincerely thank Dr. XU Jian-long at the Chinese Academy of Agricultural Sciences, China for his generous helps. This research was partly supported by the Shanghai Municipal Education Commission of China (Grant No. 06ZZ21), Shanghai Municipal Science and Technology Commission of China (Grant Nos. 06PJ14074, 075405117 and 08PJ14085) and the 948 Program from Ministry of Agriculture, China (Grant No. 2006-G1).

12

Harbome J B, Grayer R J. The anthocyanins. In: Harborne J B. The Flavonoids. London: Chapman and Hall, 1988: 1–20.

13

Thakur M, Nozzolillo C. Anthocyanin pigmentation in roots of Impatiens species. Can J Bot, 1978, 56: 2898–2903.

14

Stenlid G. On the effects of some flavonoid pigments upon growth and ion absorption of wheat roots. Physiol Plant, 1961, 14: 659–670.

15

Ishikura N, Hayashi K. Anthocyanins in red roots of a radish: Studies on anthocyanins. XXXVII. Bot Mag Tokyo, 1962, 75: 28–36.

16

Duke S O, Naylor A W. Light control of anthocyanin biosynthesis in Zea seedlings. Physiol Plant, 1976, 37: 6248.

REFERENCES

17

Solangaarachchi S M, Kevins G. Anthocyanin pigmentation in the adventitious roots of Metrosideros excelsa (Myrtaceae).

1

New Zealand J Bot, 2001, 39: 161–166.

Wu J, Cheng J H, Yang F C. Transcriptional regulation of anthocyanin biosynthesis in plants. Chinese J Cell Biol, 2006,

18

method of anthocyanin of black rice. Amino Acids & Biotic

28: 453–456. (in Chinese with English abstract) 2 3

Resour, 2001, 23(4): 44–66. (in Chinese with English abstract)

Tang C H. Plant Bioactive Substances. Beijing: Chemical Industry Publishing House, 2004: 218–231. (in Chinese)

19

chromosome. Theor Appl Genet, 1988, 76: 815–829.

L, LiVolti G. Cyanidin and cyaniding 3-O-beta-D-glucoside

4

20

Lincoln S E, Daly M J, Lander E S. Constructing genetic

Toxicol, 2003, 19(4): 243–252.

linkage maps with MAPMAKER/Exp 3.0. 3rd edn. Whitehead

Lazzé M C, Pizzala R, Savio M, Stivala L A, Prosperi E,

Institute Technical Report. Cambridge MA: Whitehead Institute, 1992.

Bianchi L. Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth and 5

McCouch S R, Kotcket G, Yu Z H, Wang Z Y, Khush G S, Coffman W R, Tanksley S D. Molecular mapping of rice

Acquaviva R, Russo A, Galvano F, Galvano G, Barcellona M as DNA cleavage protectors and antioxidants. Cell Biol

Wu S Q, Min S T, Li X S, Deng W H, Study on determination

21

Reddy A R. Genetic and molecular analysis of the

hepatoma cells. Mutat Res, 2003, 535(1): 103–115.

anthocyanin pigmentation pathway in rice. In: Khush G S.

Russo A, La F L, Acquaviva R, Campisi A, Raciti G, Scifo C.

Rice Genetics III. Proceedings of the Third International Rice

Ochratoxin A-induced DNA damage in human fibroblast:

Genetics Symposium, 16–20 Oct 1995. Manila, Philippines:

32

22

Rice Science, Vol. 16, No. 1, 2009 International Rice Research Institute, 1996: 341–352.

Linkage analysis of RFLP markers and the gene for purple.

Fan F J, Fan Y Y, Du J H, Zhuang J Y. Fine mapping of C

Acta Genet Sin, 1996, 23(5): 372–375. (in Chinese with English abstract)

(chromogen for anthocyanin) gene in rice. Rice Sci, 2008, 15(1): 1-6. 23

24

25

Phoka N, Tragonrung S, Vanavichit A. Anthocyanin intensity

Singh A P, Prasad S C, Tomar J B, Paul A. Expressivity of

in rice grain is regulated by splicing efficiency of

pleiotropic genes governing anthocyanin pigmentation in

dihydroflavono1-4-reductase and is temperature sensitive.

male sterile line of rice. J Res Birsa Agric Univ, 1997, 9(2):

[2008-02-28]. http://dna.kps.ku.ac.th/rice_files/rice for future/

123–127.

poster_rice_anthocyanin.htm.

Zhuang J Y, Yang C D, Qian H R, Zhao C Z, Zheng K L.