Journal of Integrative Plant Biology 2006, 48 (3): 341−347

Received 18 Aug. 2005 Accepted 5 Oct. 2005

Supported by the Hi-Tech Research and Development (863) Program of

China (2002AA221003) and the National Natural Science Foundation of

China (30425034).

*Author for correspondence. Tel: +86 (0)571 6337 0537; Fax: +86 (0)571

6337 0389; E-mail: <qianqian188@hotmail.com>.

www.blackwell-synergy.com; www.chineseplantscience.com

Genetic Analysis and Gene-Mapping of Two Reduced-Culm-Number Mutants in Rice

Hua Jiang1, 2, Long-Biao Guo1, Da-Wei Xue1, Da-Li Zeng1, Guang-Heng Zhang1,

Guo-Jun Dong1, Ming-Hong Gu2 and Qian Qian1*

(1. State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China;2. Agricultural College, Yangzhou University, Yangzhou 225006, China)

Abstract

In the present study, in order to systematically dissect the genetic mechanism of rice (Oryza sativa L.) tilling forthe super rice ideotype and the model system of branching development, two ethyl methane sulfonate-inducedrice reduced-culm-number (rcn) mutants from the progeny of Nippobare (O. sativa ssp. japonica), namely rcn8and rcn9, were used. Their maximum tillers were both less than 4. In addition, rcn9 had another major featureof rust-spotted leaves. Allelic tests between these two mutants and seven other recessive few-tiller mutantsrevealed that they were previously unknown loci. Genetic analysis showed that the rcn traits were all controlledby a pair of different recessive genes, designated as RCN8 and RCN9, respectively. Two F2 populations derivedfrom crosses between the rcn8 or rcn9 mutants and 93-11 were constructed. Linkage analysis using two rcn F2

mapping populations with published simple sequence repeat markers demonstrated that the RCN8 and RCN9genes were mapped on the long arm of chromosome 1 (119.6 cM) and the short arm of chromosome 6 (63.6 cM),respectively. The results of the present study are beneficial to map-based cloning and functional analysis of theRCN8 and RCN9 genes.

Key words: gene-mapping; reduced-culm-number; rice; simple sequence repeat marker.

Jiang H, Guo LB, Xue DW, Zeng DL, Zhang GH, Dong GJ, Gu MH, Qian Q (2006). Genetic analysis and gene-mapping of tworeduced-culm-number mutants in rice. J Integrat Plant Biol 48(3), 341−347.

Tillering in rice (Oryza sativa L.) is one of the most importantagronomic traits for grain production and has a facility for accu-mulation of information in terms of developmental biology for thestudy of branching in monocotyledonous plants (Li et al. 2003a).Many studies have found that the tiller number is widely controlledby quantitative trait loci (QTL). Xiong (1992) reported that tillersper plant held a relatively low heritability of 29.8%–49.6%. Usingtraditional genetic analysis, Li et al. (1997), Murai and Kinosita(1986), and Ahmad et al. (1986) reported that final tillers per plantwere controlled by multigenes with different additive, dominant,and epistatic effects. Xu and Shenm (1991) showed that the

additive effects for controlling tiller number with the growth ofrice plants increased gradually, but non-additive effects and en-vironmental factors decreased. Using molecular genetic analysis,many QTLs for final tiller number have been identified on 10 ricechromosomes, except on chromosomes 9 and 10 (Yan et al. 1998;Xing et al. 2002). Yan et al. (1998) found that the number of QTLsignificantly affecting tiller number was different at different growthstages. However, some researchers have confirmed that thetiller number in rice can be controlled by one single gene. Tang etal. (2001) reported on a mutant, namely G069, with few tillernumbers controlled by one recessive gene FEW-TILLING 1 (FT1).The FT1 gene was mapped to chromosome 2 between restrictionfragment length polymorphism (RFLP) markers C424 and S13984.Li et al. (2003a) screened a spontaneous monoculm 1 (moc1)mutant and isolated and characterized the MOC1 gene encodinga putative GRAS (GAI (GIBBERELLIN-INSENSITIVE), RGA(REPRESSOR of ga1–3), SCR(SCARECROW)) family nuclearprotein. However, in the rice genome, there are approximately 50000–60 000 genes (Goff et al. 2002), and less than 30% of them

342 Journal of Integrative Plant Biology Vol. 48 No. 3 2006

have been annotated and only a few have been cloned. Since the1980s, numerous rice mutants have been identified and charac-terized to investigate plant morphology and the causal genes (Itohet al. 2005). Therefore, it is still important to clone new rice genesusing different mutants to elucidate their molecular mechanism.

Breeders and scientists have paid increasing attention to ricetillering studies due to “few-tiller” being one of the main targettraits for the super-rice ideotype (Khush 2000). Thus, in the presentstudy, we used two ethyl methane sulfonate (EMS)-induced ricereduced-culm-number (rcn) mutants, namely rcn8 and rcn9, fromthe progeny of Nippobare (O. sativa ssp. japonica). In addition toa maximum tiller number of 4, the rcn9 mutant has another muta-tion of rust-spotted leaves. Allelic tests between these two mu-tants and other known recessive tillering mutants (Iwata et al.1995) revealed that both mutant genes for rcn8 and rcn9 werelocated at previously unknown loci. In the present study, we re-port on the genetic analysis and primary mapping of these genes,designated RCN8 and RCN9, respectively, controlling the rcn traitsusing F2 mapping populations derived from crosses between rcn8or rcn9 and 93-11.

Results

Phenotypes of the mutants under different cultivation con-ditions

Field phenotyping showed that the maximum number of tillers ofthe two rcn mutants rcn8 and rcn9 was less than 4 for both. Themutants had strong culm and the plant height of both mutants wasas high as that of the wild-type plant. The hull of the rcn8 mutantwas smooth at the mature stage and rust spots appeared on theleaves of the rcn9 mutant.

As indicated in Table 1, differences in the tiller number of mu-tants under different cultivation conditions, specifically three plantdensities and three nitrogen levels in Hangzhou and Hainan, wererather small (less than 0.8). In contrast, the tiller number ofNipponbare varied more than 5 under the different cultivationconditions. The results show that the rcn traits are mainly con-trolled by heredity and are less influenced by environmentalfactors.

Allelic tests

Allelic tests between the two rice rcn mutants and seven otherrecessive few-tiller mutants (rcn1-6 and moc1) indicated that tillernumber in individual F1 plants was similar to that in Nipponbare,ranging from 8 to 14. The tiller number of F2 plants was segregated,including the parents’ phenotypes. The results demonstrate thatboth RCN8 and RCN9 were previously unknown loci.

Genetic analysis of the rcn traits

The tiller number of the F1 plants of the rcn mutant/93-11 crosswas as many as that of 93-11, but the tiller number of the indi-vidual plants in the F2 populations was segregated into two groupswith different peaks (Figure 1A, B).

The individuals of the two F2 populations could be classifiedinto two groups according to tiller number, specifically plants inthe few-tillering group (0–4 tillers) and the high-tillering group (>5tillers) based on the minimum tiller number between the two peaksin the distribution profile. In the F2 population of the rcn8/93-11cross, there were 168 rcn plants and 527 high-tillering plants(Figure 1A), whereas, there were 212 rcn plants and 693 high-tillering plants with rust-spotted leaves in the F2 population of thercn9/93-11 cross (Figure 1B).

The proportion of normal and mutant plants for these two popu-lations was fit to the segregation ratio of 3 : 1 (F2 population ofrcn8/93-11: χ2

0.05 = 0.211 5 < χ20.05, 1= 3.84, 0.95 < P < 1; F2 popu-

lation of rcn9/93-11: χ20.05 = 1.975 5 < χ2

0.05,1 = 3.84, 0.95 < P < 1),indicating that the two rcn traits were independently controlled bydifferent single recessive genes (Table 1).

Molecular marker mapping of the genes for the two rcnmutants

The F2 populations of the rcn8/93-11 and rcn9/93-11 crosses wereused as mapping populations. The total DNA of the two mutantsand 93-11 was amplified with SSR primers well-distributed on 12chromosomes in rice (Figure 2A). The 162 SSR markers withpolymorphisms between the mutant and 93-11 were used to de-tect marker genotypes of 168 rcn8 plants or 212 rcn9 plants in therespective F2 populations (Figure 2B).

Table 1. Tiller number of mutant plants under the different environmental conditions

Location Plant materialPlanting density Nitrogen levels (kg/hm2)

16.6 cm × 16.6 cm 26.6 cm × 16.6 cm 43.2 cm × 16.6 cm 0 225 450Hangzhou rcn8 2.4 ± 0.1 2.8 ± 0.1 2.9 ± 0.3 2.6 ± 0.3 2.9 ± 0.1 3.1 ± 0.2

rcn9 2.6 ± 0.2 2.7 ± 0.1 2.9 ± 0.2 2.5 ± 0.2 2.8 ± 0.2 3.2 ± 0.1Nipponbare 8.2 ± 1.0 13.4 ± 2.1 19.1 ± 3.2 7.8 ± 1.1 12.9 ± 3.2 20.4 ± 2.0

Hainan rcn8 2.4 ± 0.3 2.6 ± 0.2 2.9 ± 0.2 2.5 ± 0.1 2.9 ± 0.3 3.0 ± 0.4rcn9 2.4 ± 0.1 2.9 ± 0.1 3.1 ± 0.2 2.5 ± 0.4 3.0 ± 0.3 3.2 ± 0.3

Nipponbare 8.2 ± 1.4 14.2 ± 2.9 20.6 ± 2.3 8.1 ± 0.9 13.7 ± 2.4 19.7 ± 3.0rcn8, rcn9, reduced-culm-number (rcn) mutants.

Mapping of rcn8, 9 in Rice 343

Thirty and 32 recombinants were found among the F2 mutantplants from rcn8/93-11 and rcn9/93-11, respectively. Linkage analy-sis indicated that RCN8 was mapped at a position 8.9 cM from theSSR marker RM6836, on the short arm of rice chromosome 6, andapproximately 1.7 cM from the centromere (Figure 3A). The othergene, RCN9, was located at a position 7.5 cM from RM3411 on thelong arm of rice chromosome 1 (Figure 3B).

Discussion

A series of mutants for various traits in rice forms the basis ofgenetic analysis and functional genomics (Li et al. 2003a, 2003b).Until now, seven rcn mutants in rice have been registered on theGramene web (http://www.gramene.org/db/mutant/search_core).Three of the mutants, namely rcn1, rcn2, and rcn5, had been

Figure 1. Tiller number distribution in the F2 segregating populations.

(A) rcn8/93-11.(B) rcn9/93-11.rcn8, rcn9, reduced-culm-number (rcn) mutants.

located to chromosomes 6, 4 and 6, respectively. In addition, Tanget al. (2001) mapped the rice FT1 gene to chromosome 2 and Li etal. (2003a) cloned the MOC1 gene located on chromosome 6. Inthe present study, RCN8 was also located on chromosome 6, butat a different position to that of RCN1, RCN5, and MOC1, indicat-ing that there are at least three RCN genes and one MONOCULMgene on the same chromosome 6. In contrast with the other RCNgenes, the RCN9 gene was mapped to chromosome 1. Geneticmapping reconfirmed the results of allelic tests between the rcn8/rcn9 mutants and seven other recessive few-tiller mutants (rcn1-6 and moc1), indicating that RCN8 and RCN9 are two previouslyunknown loci.

Two major hypotheses, quantitative traits versus qualitativetraits, have been proposed to explain the control of rice tilleringbased on different mapping materials and methods. Most of therecent studies have reported that rice tillering is a complex

344 Journal of Integrative Plant Biology Vol. 48 No. 3 2006

process and is controlled by QTL (Xiao et al. 1996; Li et al. 1997;Yan et al. 1998; Wu et al. 1999; Miyamoto et al. 2004), whichseems to indicate that tillering-related QTLs are more difficult tomap and clone than qualitative/main-effect genes. The isolationand use of special mutants will be a better way in which theseproblems can be solved. For example, the moc1 mutant has beenidentified as a single recessive nuclear locus involved in a mem-ber of the plant-specific GRAS family proteins and successfullyapplied in map-based cloning. Li et al. (2003a) have also foundthat moc1 has another distinct function of a negative effect onplant height. Wu (1996) and Yan et al. (1998) have reported thatsome QTLs for tillering could simultaneously affect plant height. Inthe present study, we reported that RCN9 was located on thelong arm of rice chromosome 1 near the SD-1 region, but it needsto be determined whether the RCN9 gene can affect plant height.

The tiller number per plant of the mutants did not differ signifi-cantly under different cultivation conditions, but the tiller numberof regenerated rcn8 plants increased to 6–8, especially underconditions of high nitrogen or low plant density (data not shown).This may indicate that the expression of RCN varies at differentdevelopment stages and results in changes in phenotype. Li et al.(2003a) reported that tillering is a complex process in which theexpression of many genes must be fine tuned. The influence maycome from redundant genes, modifying genes and/or interactivegenes. The phenomenon of attenuation of the mutant phenotype

Figure 2. Linkage analysis of REDUCED-CULM-NUMBER (RCN) genes and simple sequence repeat (SSR) markers.

(A) Polymorphisms of the SSR primers between the two parents rcn8 and 93-11.(B) Segregation of the SSR marker RM212 in the F2 population of rcn9/93-11.P1, rcn9; P2, 93-11; lanes 1–21, mutant plants in the F2 population.

Figure 3. Linkage maps of the genes REDUCED-CULM-NUMBER(RCN8 and RCN9) on rice chromosomes.

(A) RCN8 on the short arm of rice chromosome 6.(B) RCN9 on the long arm of rice chromosome 1.

Mapping of rcn8, 9 in Rice 345

was also found in rcn-like mutatnts reported by Hu (1961) and Itohet al. (1998), the changes in which are similar to the concept thatthe development of the terminal and lateral spikelits may be con-trolled by different genetic programs (Purugganan et al. 1995;Bonhomme et al. 1997; Komatsu et al. 2001). All these studieshave suggested that some key genes controlling developmenttraits in plants could function at different developmental stages,different positions and in different organs. However, in the presentstudy, the change in tiller number in regenerated plants is differ-ent from the results reported by tillering QTL (Xiao et al. 1996; Li etal. 1997), in which changes in tiller number are mainly significantduring the same vegetative stage. Further studies are needed todissect the differential expression of tillering mutants in the earlyand mature stages.

Super high-yielding rice will be the next target of rice breedingin the 21st century and few tiller is one of the main target traits forthe breeding of super-rice (Khush 2000). Many component analy-ses have been undertaken to enhance yield potential by improvingindividual component traits since Donald (1968) advocated theideotype concept (Gravois and Helms 1992; Sparnaaij and Bos1993; Piepho 1995; Wu et al. 1998; Florent et al. 2001; Guo et al.2002). Except for MOC1, other tillering genes, such as RCN8 andRCN9, and multi-tiller genes will be isolated continuously and char-acterized using the map-based cloning approach. In the future,we will transfer these genes controlling different tiller numbersinto commercial cultivars to construct rice tiller gradient materialswith the same genetic background using traditional and molecularbreeding methods. Then, we could use this series of tiller gradientmaterials to predict the change in yield under different cultivationenvironments and to propose “a quantitative ideotype” in rice forthe improvement of super-rice.

Materials and Methods

Plant materials and growth conditions

Two rice (Oryza sativa L.) rcn mutants, namely rcn8 and rcn9,were isolated from the progeny of Nipponbare (O. sativa ssp.japonica) following induction by EMS. To detect environmentaleffects on the rcn traits, these two mutant rice plants were testedat three different planting densities ((16.6 ± 16.6) cm, (26.6 ±16.6) cm, and (43.2 ± 16.6) cm) or with three different nitrogen

levels (0, 225, and 450 kg/hm2 as top dressing) in the fields ofHangzhou and Hainan, China. In addition, Nipponbare was plantedas a control. Final tiller number per plant was measured at themature stage (Table 2).

The rcn8 or rcn9 mutants were crossed with seven other re-cessive few-tiller mutants (rcn1-6 and moc1; Iwata et al. 1995;Tang et al. 2001; Li et al. 2003a) to analyze their allelism. Two F2

segregation populations (168 individuals and 212 individuals) de-rived from crosses between rcn8 or rcn9 and 93-11 were usedfor gene mapping. The two F2 segregation populations and theirparents were cultivated in an experimental field at China NationalRice Research Institute during the natural growing season.

Identification of the rcn traits in segregation populations

From the 695 F2 plants of the rcn8 mutant/93-11 cross, 168 plantsshowing the rcn8 mutant phenotype were selected for genemapping. Similarly, 212 plants showing the rcn9 mutant pheno-type were selected from 924 F2 plants of the rcn9 mutant/93-11cross.

Extraction of total genomic DNA with RCN genes and simplesequence repeat analysis

The DNA was extracted from fresh leaves of each individualselected from the F2 populations constructed for gene mapping.Total DNA was isolated using the cetyltrimethylammonium bromide(CTAB) method with some minor modifications (Murray and Th-ompson 1980).

Simple sequence repeat (SSR) analysis and PCR protocols wereperformed according to the methods described by Akagi et al.(2001). The PCR products were separated on a 3.0%–5.0% aga-rose gel according to the lengths of the amplified fragments andstained using ethidium bromide.

Gene mapping of RCN8 and RCN9

Based on the results of the SSR analysis, the band pattern ofeach F2 individual that was identical with rcn8 or rcn9 and 93-11 was marked as 1 and 2, respectively; a mixed parentalpattern was marked as 3. A linkage map was constructed withMapmaker 3.0 (Lander et al. 1987) according to the linkage dataof the rcn loci and polymorphic SSR markers in the F2 mapping

Table 2. Segregation of tiller numbers in rcn8/93-11 and rcn9/93-11 populationsF2 population Total No. of plants No. of normal plants No. of mutant plants Expected ratio χ2 Prcn8/93-11 695 527 168 3 : 1 0.211 5 0.75–0.90

rcn9/93-11 924 693 212 3 : 1 1.975 5 0.10–0.25rcn8, rcn9, reduced-culm-number (rcn) mutants.

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populations.

Acknowledgments

The authors thank Dr Takamure I (Plant Breeding Institute, Facultyof Agriculture, Hokkaido University, Sapporo, Japan) for providingthe rcn1-6 mutants.

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