Genetic Analysis and Gene Mapping of Multi-tiller and Dwarf Mutant d63 in Rice

Rice Science, 2013, 20(3): 179184 Copyright © 2013, China National Rice Research Institute Published by Elsevier BV. All rights reserved DOI: 10.1016/S1672-6308(13)60130-4

Genetic Analysis and Gene Mapping of Multi-tiller and Dwarf Mutant d63 in Rice XUE Jing-jing1, 2, WU Shao-hua3, ZHANG Hong-yu1, XU Pei-zhou1, WU Xian-jun1 (1Rice Research Institute/Key Laboratory of Southwest Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China; 2Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agriculture Sciences, Danzhou 571737, China; 3Rubber Research Institute of Chinese Academy of Tropical Agriculture Sciences, Danzhou 571737, China)

Abstract: A spontaneous mutation, tentatively named d63, was derived from the twin-seedling progenies of rice crossed by diploid SARIII and Minghui 63. Compared with wild-type plants, the d63 mutant showed multiple abnormal phenotypes, such as dwarfism, more tillers, smaller flag leaf and reduced seed-setting rate and 1000-grain weight. In this study, two F2 populations were developed by crossing between d63 and Nipponbare, d63 and 93-11. Genetic analysis indicated that d63 was controlled by a single recessive gene, which was located on the short arm of chromosome 8, within the genetic distance of 0.40 cM from RM22195. Hence, D63 might be a new gene as there are no dwarf genes reported on the short arm of chromosome 8. Key words: rice; multi-tiller and dwarf mutant; genetic analysis; gene mapping

Plant height is an important trait for plant architecture and affects grain yield in rice, which is closely related to photosynthesis and lodging resistance. In the 1960s, dwarfism gene was applied in rice breeding and increased grain yield by 20%30% (Peng et al, 1999). Dwarfism as a staple source of modern crop breeding has contributed its lodging resistance to ‘Green revolution’ since 1906 (Hedden, 2003). Several lines of evidences have proved that there is a highly negative correlation between tiller number and plant height in rice (Iwata et al, 1995; Yan et al, 1998). Tillering capacity is an important architecture trait for grain yield because tiller number per plant determines panicle number and directly affects the ultimate production (Zhang et al, 2011). A typical semidwarf gene sd1 encoding a key enzyme GA20-oxiside (GA20ox), which involves in gibberellins (GA) synthesis pathway and cell elongation, was cloned in rice plants (Monna et al, 2002). It is important to reveal the molecular mechanism of plant height, which has an important significance in rice breeding. So far, many dwarf mutants have been identified in rice, and more than 40 genes responsible for the phenotype (http://www.shigen.nig.ac.jp/rice/oryzabase) have been cloned. The cloned genes have an important

Received: 17 September 2012; Accepted: 12 December 2012 Corresponding author: WU Xian-jun ([email protected])

significance to understand the dwarf mechanism of rice. Therefore, the study about new dwarf may be necessary to elucidate the mechanism. In this study, we identified a spontaneous mutation d63 derived from the twin-seedling progenies of the cross between diploid SARIII and Minghui 63. The phenotype of dwarf mutant d63 has many differences from the dwarf mutants that have been reported (Arite et al, 2007; Gao et al, 2009; Zhang et al, 2011). Firstly, plant height is obviously dwarfed and tillers are significantly increased. Secondly, the seed-setting rate of d63 is only 42% of that of the wild type and 1000-grain weight is about 57% of that of the wild type. The results of phenotype analysis indicated that the dwarf mutant d63 was possibly controlled by a novel gene. On the basis of the phenotypic investigation and genetic analysis, the mutant d63 was fine-mapped.

MATERIALS AND METHODS Rice materials F1 and F2 populations were developed from two crosses between dwarf mutant d63 and japonica rice Nipponbare and indica rice 93-11. In 2009 and 2010, rice plants were planted at the experimental farms of Sichuan and Hainan Provinces in China.

Rice Science, Vol. 20, No. 3, 2013

180

Table 1. Comparison of agronomic traits between dwarf mutant and wild type plants.

Methods Genetic and linkage analysis The numbers of normal phenotype and dwarf plant of F2 mapping populations were investigated, and the data were used for genetic analysis. A total of 384 simple sequence repeat (SSR) markers evenly distributed on the 12 rice chromosomes were used to determine the approximate map position of the D63 locus on rice chromosome. DNA extraction and polymerase chain reaction (PCR) test were referring to Molecular Cloning 3 (Sambrook and Russell, 2011). Development of new molecular markers Based on the results of primary mapping, BLASTN (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to search for different sequences between Nipponbare and 93-11 in the rice nucleotide database (http://gramene.a grinome.org/ and http://rice.plantbiology.msu.edu/). Then, some new insertion/delection (InDel) primers were developed by Primer Primier 5.0. Construction of linkage map The genetic linkage map between the D63 locus and molecular markers was constructed with MapMaker 3.0 on the basis of the results of SSR analysis.

RESULTS Phenotype analysis of dwarf mutant d63 The growth period of dwarf mutant d63 was similar with that of the wild type, but the agronomic traits of d63 and the wild type were significantly different (Fig. 1). Compared with the wild-type (WT) plants, the d63 mutant showed some characteristic phenotypes, such as dwarfism, increased tiller number and abnormal leaf blade morphology. The mutant reached 50% of the height of wild type (WT: 99.9 cm ± 0.6 cm; d63: 50.0 cm ± 0.3 cm) (Table 1). Internodes of the d63 mutant were shorter than those of the wild type, but

Trait

Wild type

Mutant

t-value

Plant height (cm) 99.9 ± 0.6 50.0 ± 0.3 77.36** Panicle length (cm) 24.3 ± 0.3 14.9 ± 0.3 20.82** 1000-grain weight (g) 35.8 ± 0.2 20.3 ± 0.1 66.64** No. of tillers per plant 8.1 ± 0.4 26.1 ± 1.1 15.48** No. of spikelets per panicle 85.6 ± 3.1 37.7 ± 1.5 15.10** No. of filled spikelets per panicle 40.6 ± 1.4 6.9 ± 0.6 21.62** Seed-setting rate (%) 47.9 ± 0.0 20.3 ± 0.0 15.45** Grain length (cm) 0.9 ± 0.0 0.9 ± 0.0 2.14* Grain width (cm) 0.3 ± 0.0 0.2 ± 0.0 0.06 Flag leaf length (cm) 30.2 ± 0.9 14.7 ± 0.5 14.52** Flag leaf width (cm) 1.3 ± 0.2 0.9 ± 0.0 34.07** Ratio of length to width of flag leaf 22.8 ± 0.0 21.6 ± 0.6 0.30 Top awn length (cm) 2.5 ± 0.1 0.8 ± 0.1 11.93** The results were derived from the average of 30 random samples, and were represented as mean ± SD. * and ** mean significant at 5% and 1% levels, respectively.

there were no significant differences in the length ratio of each internode and the total internodes (Table 2). Compared with the wild type, tiller numbers of the d63 mutant were significantly increased, which was 3-fold more than that of the wild type (WT: 8.1 ± 0.4; d63: 26.1 ± 1.1) (Table 1). The leaf blades of d63 mutant were shorter and thinner, and the length of flag leaf was about 48% of the wild type. The d63 mutant also showed smaller panicles and shorter rachis-branches. The length of panicles was about 61% of the wild type (WT: 24.3 cm ± 0.3 cm; d63: 14.9 cm ± 0.3 cm). The grains of d63 became shriveled and abnormal compared with those of the wild type, and decreased with seed-setting rate (WT: 47.9% ± 0.0%; d63: 20.3% ± 0.0%) and 1000-grain weight (WT: 35.8 g ± 0.2 g; d63: 20.3 g ± 0.1 g). These observations indicated multiple morphological defects in the d63 mutant (Fig. 1). Genetic analysis In order to reveal the genetic characteristic of the dwarf mutant d63, two F2 mapping populations were developed by the two crosses between d63 and Nipponbare and 93-11, respectively. The results of the

Table 2. Comparison of internode length between dwarf mutant and wild type plants. Wild type Length of internode Percentage of total internode Length of internode (cm) length (%) (cm) The first internode 31.7 ± 0.7 48.5 8.1 ± 1.8 The second internode 14.8 ± 0.3 22.7 2.4 ± 0.1 The third internode 10.2 ± 0.4 15.6 2.7 ± 0.3 1.2 ± 0.3 The fourth internode 6.5 ± 0.5 9.9 0.5 ± 0.0 The fifth internode 2.2 ± 0.3 3.3 Total 65.4 ± 1.5 14.9 ± 1.2 The results were derived from the average of five random samples, and were represented as mean ± SD. Internode

Mutant Percentage of total internode length (%) 54.3 16.1 18.1 8.2 3.3

XUE Jing-jing, et al. Genetic Analysis and Gene Mapping of Multi-tiller and Dwarf Mutant d63 in Rice

A

B

C

181

D

MT-4

WT-4

MT-3

WT-3

MT-2

WT-2

E

Fig. 1. Morphological comparison between wild type (WT) and d63 (MT). A, The plant architecture; B, Flag leaves; C, Seeds; D, Panicles; E, Different internodal length and the numbers indicate the internodes from the top to the stem base.

two crosses showed that the agronomic traits of F1 were in accordance with those of the wild type, which illustrated that the mutant phenotype is a recessive character. Further observation on the phenotypes of F2 mapping populations showed that two different types of phenotype changes in F2 populations. One type was in accordance with the dwarf mutant and the other type was normal and in accordance with Nipponbare or 93-11. The phenotypic ratio of mutant to wild type plants was 1:3 (Table 3), which confirmed that the d63 phenotype was controlled by a single recessive gene.

Primary mapping A total of 384 SSR markers were used to amplify the DNAs of the two parents. There were 124 polymorphism markers between the dwarf mutant d63 and Nipponbare, and 84 polymorphism markers between d63 and 93-11. We used the 124 polymorphism markers between d63 and Nipponbare to determine the approximate map position of the D63 locus on rice chromosome by means of detecting 45 mutant plants of F2 populations. Finally, two markers RM25 and RM310, which located

Rice Science, Vol. 20, No. 3, 2013

182 Table 3. Genetic analysis of rice multi-tiller and dwarf mutant d63 in F2 populations. Cross d63 / 93-11 d63 / Nipponbare

No. of normal plants

No. of dwarf plants

Total

c2 (3:1)

P value

2 526 2 141

875 751

3 401 2 892

0.96 1.45

0.400.60 0.100.60

on chromosome 8, were obviously associated with the tagged traits. Then more SSR markers in the flanks of RM25 and RM310 (http://gramene.agrinome.org/) were used to score the F2 individual plants, including RM152, RM408, RM126 and RM72, etc. Fine mapping To further determine the locus of D63 gene on chromosome 8, 751 mutant plants in the F2 population derived from the cross between d63 and Nipponbare were used for fine mapping. We found ten recombinant plants in this group by using the marker RM152, with the genetic distance approximately 0.67 cM, and nine recombinant plants by using the marker RM408, with the genetic distance about 0.60 cM. Moreover, the nine recombinants of RM408 were contained in the ten recombinants of RM152. Hence, the two markers RM152 and RM408 located in the same side of the dwarf gene and RM408 was more closed to the dwarf gene. The results showed that the physical location of the marker RM408 on chromosome 8 was 125 297 bp to 125 487 bp, and was in the BAC contig, AP005406. By map-based cloning, the D63 gene was located on the short arm of chromosome 8 near the telomere. To further narrow the locus, a few additional markers were developed based on the genome sequence of the short arm of chromosome 8. Two markers RM22195 and Indel9 (upstream primer: 5-GCCATTATCATCTTG ACCTA-3; downstream primer: 5-GGAACGACGAC TGCTCCATC-3) each could reveal six recombinants, whereas Indel1 (upstream primer: 5-GGAATTATCC GTTAACAACC-3; downstream primer: 5-AGTCCG ATTCTTATCAGGTC-3) near the telomere revealed no recombinant in the 751 mutant plants. Finally, D63 was located on the short arm of chromosome 8 within the genetic distance of 0.40 cM from RM22195 (Fig. 2).

mutants have been cloned in recent years. In this study, genetic analysis and molecular location of the dwarf mutant d63 was conducted and the D63 gene was located on the short arm of chromosome 8 in rice. More than 80 dwarfism genes have been found in rice (Chen et al, 2011). Several dwarf rice phenotypes may result from the changes in various mechanisms involving synthesis, regulation and signal transduction of gibberellin acid (GA) and brassinosteroids (BR), such as d1 (Ashikari et al, 1999) and d18 (Aoki et al, 2002) involved in GA synthetic pathway, and d2 (Hong et al, 2003) and d61 (Yamamuro et al, 2000) involved in BR pathway. In addition, there are many other synthetic pathways regulating plant height. It is reported that D3 (Ishikawa et al, 2005), D17/HTD1 (Zou et al, 2005, 2006) and D10 (Arite et al, 2007) have similar functions in the growth process of plants and are generally recognized in MAX/RMS/D pathway (Table 4). D27 which encodes a novel iron-containing protein is involved in the MAX/RMS/D pathway, participating in the biosynthesis of strigolactones (Lin et al, 2009). D88/D14 encodes a novel putative rice esterase, which regulates cell growth and organ development and may be a new pathway regulating plant development (Gao et al, 2009a, b). In this study, the dwarf mutant d63 was a spontaneous mutation

DISCUSSION Plant height is an important trait for plant architecture and affects biomass and grain yield in rice, which is closely related to photosynthesis and lodging resistance. Lots of rice dwarfism mutants have been found since ‘Green revolution’, and more genes regulating the dwarf

Fig. 2. Location of D63 gene on short arm of chromosome 8 (Chr. 8).

XUE Jing-jing, et al. Genetic Analysis and Gene Mapping of Multi-tiller and Dwarf Mutant d63 in Rice

183

Table 4. Multi-tiller and dwarf mutants reported. Gene name

Chromosome

Mutagenesis

Phenotypic description

Function

Shorter culm, more tillers, slender leaves, shorter panicles and reduced seed-setting rate Shorter culm and more tillers

Encodes OsCCD7, involves in the outgrowth of auxiliary buds, and belongs to MAX/RMS/D pathway Encodes an F-box LRR protein and involves in MAX/RMS/D pathway Encodes OsCCD8, involves in the outgrowth of auxiliary branches, and belongs to MAX/RMS/D pathway Regulates the biosynthesis of strigolactones Encodes a novel putative rice esterase and regulates cell growth and organ development No cloning

D17/HTD1

4

Slender dwarf

D3

6

Bunketsu-waito

D10

1

Kikeibanshinriki

More tillers and apical dominance evidence

D27

11

Bunketsuto

More tillers and rapid growth of auxiliary buds Shorter culm, more tillers and reduced seed-setting rate

D88/D14

HTD3

3

12

Kamikawabunwai

SIL046

Shorter culm and more tillers

derived from the twin-seedling progenies, and had obvious diversity. Genetic analysis indicated that the D63 gene was located on the short arm of chromosome 8. Using Rice Genome Automated Annotation System (http://rice.plantbiology.msu.edu/index.shtml), 14 open reading frames were predicted between telomere and RM22195. Therefore, D63 might be a new gene as there are no dwarf genes reported on the short arm of chromosome 8. On average, a genetic distance of 1 cM on the rice molecular map corresponds to approximately 240 kb (Li et al, 2004). However, some concrete evidences have proved that the ratio between genetic distance and physical distance varies with the change of the location on the chromosome (Zhang et al, 2011). Telomere is consist of short repetitive G-rich double-stranded sequences, and plays a key role in stabilizing and keeping the chromosome integrity, suppressing different chromosome recombination and degradation of exonuclease (Blackburn, 1991; Greider, 1996). In rice, D88, D3 and Hd1 are located on different chromosomes within about 1020 kb, by using 700 to 1 500 F2 mutant plants, respectively (Yano et al, 2000; Ishikawa et al, 2005; Gao et al, 2009b). The target gene may be located in a 20 kb region without recombination suppression by using about 1 000 mapping population. D62 is located on the short arm of chromosome 6 within about 131 kb region by using 546 F2 mutant plants, and number of plants in which the recombination is closed to the telomere is fewer (Li et al, 2010). In this study, the molecular marker InDel1 closed to the telomere had no recombination in the 751 mutant plants, and the marker RM22195 was far away from the telomere within the genetic distance of 0.4 cM. Compared to the mapping results of mutant d62, the D63 gene was located on the short arm of chromosome

Reference Zou et al (2005, 2006)

Ishikawa et al (2005) Arite et al (2007)

Lin et al (2009) Gao et al (2009a, b)

Zhang et al (2011)

8 and the recombination of this region was suppressed, which resulted lower recombination frequency in this region. Therefore, it is very difficult to clone the D63 gene in the region closed to the telomere. In addition, we will also construct a larger segregating population to increase recombination frequency for identification of the D63 gene.

REFERENCES Aoki T, Kitano H, Kameya N, Nakamura I. 2002. Accelerated shoot overgrowth of rice mutant ao-1 is epistatic to gibberellinsensitive and -insensitive dwarf mutants. J Plant Res, 115(3): 195202. Arite T, Iwata H, Ohshima K, Maekawa M, Nakajima M, Kojima M, Sakakibara H, Kyozuka J. 2007. DWARF10, an RMS1/ MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice. Plant J, 51(6): 10191029. Ashikari M, Wu J Z, Yano M, Sasaki T, Yoshimura A. 1999. Rice gibberellin-insensitive dwarf mutant gene Dwarf1 encodes the -subunit of GTP-binding protein. Proc Natl Acad Sci USA, 96(18): 1028410289. Blackburn E H. 1991. Structure and function of telomeres. Nature, 350: 569573. Chen H X, Zhou C B, Xing Y Z. 2011. A new rice dwarf1 mutant caused by a frame-shift mutation. Hereditas, 33(4): 397403. (in Chinese with English abstract) Gao Z Y, Liu X H, Guo L B, Liu J, Dong G J, Hu J, Han B, Qian Q. 2009a. Identification of a novel tillering dwarf mutant and fine mapping of the TDDL(T) gene in rice (Oryza sativa L.). Chin Sci Bull, 54: 20622068. Gao Z Y, Qian Q, Liu X H, Yan M X, Feng Q, Dong G J, Liu J, Han B. 2009b. Dwarf88, a novel putative esterase gene affecting architecture of rice plant. Plant Mol Biol, 71: 265276. Greider C W. 1996. Telomere length regulation. Annu Rev Biochem, 65: 337365. Hedden P. 2003. The genes of the green revolution. Trends Genet, 19: 59.

184 Hong Z, Ueguchi-Tanaka M, Umemura K, Uozu S, Fujioka S, Takatsuto S, Yoshida S, Ashikari M, Kitano H, Matsuoka M. 2003. A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450. Plant Cell, 15: 29002910. Ishikawa S, Maekawa M, Arite T, Onishi K, Takamure I, Kyozuka J. 2005. Suppression of tiller bud activity in tillering dwarf mutants of rice. Plant Cell Physiol, 46: 7986. Iwata N, Takamure I, Wu H K, Siddinq E A, Rutger J N. 1995. List of genes for various traits (with chromosome and main literature). Rice Genet Newsl, 12: 6193. Li J M, Thomson M, McCouch S R. 2004. Fine mapping of a grain-weight quantitative trait locus in the pericentromeric region of rice chromosome 3. Genetics, 168(4): 21872195. Li W Q, Wu J G, Weng S L, Zhang Y J, Zhang D P, Shi C H. 2010. Identification and characterization of dwarf62, a loss-of-function mutation in DLT/OsGRAS-32 affecting gibberellin metabolism in rice. Planta, 232: 13831396. Lin H, Wang R X, Qian Q, Yan M X, Meng X B, Fu Z M, Yan C Y, Jiang B, Su Z, Li J Y, Wang Y H. 2009. DWARF27, an iron-containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth. Plant Cell, 5(11): 15121525. Monna L, Kitazawa N, Yoshino R, Suzuki J, Masuda H, Maehara Y, Tanji M, Sato M, Nasu S, Minobe Y. 2002. Positional cloning of rice semidwarfing gene, sd-1: Rice ‘green revolution gene’ encodes a mutant enzyme involved in gibberellin synthesis. DNA Res, 9(1): 1117. Peng J, Richards D E, Hartley N M, Murphy G P, Devos K M, Flintham J E, Beales J, Fish L J, Worland A J, Pelica F, Sudhakar

Rice Science, Vol. 20, No. 3, 2013 D, Christou P, Snape J W, Gale M D, Harberd N P. 1999. ‘Green revolution’ genes encode mutant gibberellin response modulators. Nature, 400: 256261. Sambrook J, Russell D W. 2001. Molecular Cloning: A Laboratory Manual. 3rd edn. New York: Cold Spring Harbor Laboratory Press. Yamamuro C, Ihara Y, Wu X, Noguchi T, Fujioka S, Takatsuto S, Ashikari M, Kitano H, Matsuoka M. 2000. Loss of function of a rice brassinosteroid insensitive 1 homolog prevents internode elongation and bending of the lamina joint. Plant Cell, 12(9): 15911605. Yan J Q, Zhu J, He C X, Benmoussa M, Wu P. 1998. Quantitative trait loci analysis for the developmental behavior of tiller number in rice (Oryza sativa L.). Theor Appl Genet, 97(1/2): 267274. Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T, Baba T, Yamamoto K, Umehara Y, Nagamura Y, Sasaki T. 2000. Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell, 12(12): 24732483. Zhang B S, Tian F, Tan L B, Xie D X, Sun C Q. 2011. Characterization of a novel high-tillering dwarf3 mutant in rice. J Genet Genom, 38(9): 411418. Zou J H, Chen Z X, Zhang S Y, Zhang W P, Jiang G H, Zhao X F, Zhai W X, Pan X B, Zhu L H. 2005. Characterizations and fine mapping of a mutant gene for high tillering and dwarf in rice (Oryza sativa). Planta, 222(4): 604612. Zou J H, Zhang S Y, Zhang W P, Li G, Chen Z X, Zhai W X, Zhao X F, Pan X B, Xie Q, Zhu L H. 2006. The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds. Plant J, 48(5): 687696.