Rare allele of OsPPKL1 associated with grainlength causes extra-large grain and a significantyield increase in riceXiaojun Zhanga,1, Jianfei Wanga,1, Ji Huanga,1, Hongxia Lana, Cailin Wangb, Congfei Yina, Yunyu Wua, Haijuan Tanga,Qian Qianc,2, Jiayang Lid,2, and Hongsheng Zhanga,2

aState Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China; bInstitute of Food Crops,Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; cState Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou310006, China; and dState Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology,Chinese Academy of Sciences, Beijing 100101, China

Contributed by Jiayang Li, November 13, 2012 (sent for review November 2, 2012)

Grain size and shape are important components determining ricegrain yield, and they are controlled by quantitative trait loci (QTLs).Here, we report the cloning and functional characterization of amajor grain length QTL, qGL3, which encodes a putative proteinphosphatase with Kelch-like repeat domain (OsPPKL1). We founda rare allele qgl3 that leads to a long grain phenotype by an aspar-tate-to-glutamate transition in a conserved AVLDT motif of thesecond Kelch domain in OsPPKL1. The rice genome has other twoOsPPKL1 homologs, OsPPKL2 and OsPPKL3. Transgenic studiesshowed that OsPPKL1 and OsPPKL3 function as negative regula-tors of grain length, whereas OsPPKL2 as a positive regulator. TheKelch domains are essential for the OsPPKL1 biological function.Field trials showed that the application of the qgl3 allele couldsignificantly increase grain yield in both inbred and hybrid ricevarieties, due to its favorable effect on grain length, filling,and weight.

QTL mapping | positional cloning | Oryza sativa L.

Grain number, panicle number, and grain weight are threeimportant components of grain yield of rice (Oryza sativa

L.). When grain number per panicle and panicle number perplant reach an ideal level, improvement of grain weight plays akey role in further yield increase in rice-breeding programs (1).Grain weight is largely determined by grain size, which is spec-ified by its three dimensions (length, width, and thickness) andthe degree of filling. Currently, 1,000-grain weight of commercialrice varieties is usually 25∼35 g. To date, several quantitative traitloci (QTLs) that affect rice grain size and/or shape have beencloned from different rice germplasms. GS3 (2–4) and DEP1(5, 6), two QTLs affecting rice grain length, function as negativefactors; however, their homolog in Arabidopsis, AGG3, has beenfound to be an atypical heterotrimeric G protein γ-subunit thatpositively regulates organ size (7, 8). For grain width, GW2 (9)(encoding a RING-type E3 ubiquitin ligase) and qSW5/GW5 (10,11) have been reported as negative regulators, whereas GS5 (12)(encoding a putative serine carboxypeptidase) and GW8 (13)(encoding a transcription factor with SBP domain) are positiveregulators. These loci, except GW2, have been artificially se-lected in rice-breeding programs. To realize the goal of breedingsuperrice varieties, it is well worth it to explore novel genetic locithat regulate grain shape and weight in rice germplasms (14).Here we report the QTL mapping, positional cloning, and func-tional characterization of qgl3, a rare allele that shows an ex-traordinary effect on rice grain length and grain weight, which canbe applied in breeding new elite varieties to significantly improverice grain yield.

Results and DiscussionMap-Based Cloning of qGL3. By screening more than 7,000 germ-plasms, we identified an extra large-grain japonica accession

N411 (1,000-grain weight = 72.13 ± 2.32 g; Fig. 1A). To dissectthe genetic basis of the large grain size of N411, we carried outa QTL analysis in an F2 population derived from a cross betweenN411 and a polymorphic small-grain indica variety N643 (1,000-grain weight = 17.80 ± 0.82 g; Fig. 1A). Twenty-three QTLs af-fecting grain size and/or weight were mapped in this population,including four for grain length, four for width, seven for thickness,and eight for weight (Table S1). Among them, three major loci,qGL3a, qGW2.1, and qGW5, were mapped at the same locationsof four reported genes, GS3, GW2, qSW5/GW5, and GS5, re-spectively (Fig. 1B; Table S1). The allelic sequencing data ofthese four genes confirmed allele variances in N411 and N643(Table S2). Importantly, we identified a major grain length QTL,qGL3, on the long arm of chromosome 3 (Fig. 1B), explaining38.38% phenotypic variation of grain length, 27.99% of grainweight, 11.89% of grain thickness, and 5.96% of grain width(Table S1). The recessive allele from N411, qgl3, contributespositively to these traits. The result indicates that N411 pyr-amids at least four known positive grain-size loci (gw2, gs3,gw5, and GS5) and a major grain length locus (qgl3) to formextra-large grains.A high-quality elite indica variety, 93-11 (1,000-grain weight =

30.08 ± 1.34 g; Fig. 1A), was used as a recurrent parent to furthervalidate and disassemble qGL3. qGL3 was confirmed and map-ped to an interval between simple sequence repeat (SSR) markersRM15551 and RM15578 with a high log-likelihood value (limitof detection = 68.8) and explained 95.57% phenotypic variationin a N411 × 93-11 BC2F2 population (Fig. S1). A total of 2,968BC2F3 individuals derived from four heterozygous N411 × 93-11BC2F2 plants were screened for recombinants within the targetregion defined by SSR markers RM15548 and RM3513 (Fig. 1C),outward to markers RM15551 and RM15578, respectively. High-resolution linkage analysis narrowed qGL3 to a 46.6-kb regionbetween insertion and deletion (InDel) markers XJ39 and XJ26(Fig. 1D; Table S3). In this region, there are five predicted ORFsaccording to the Nipponbare genome sequence (annotated byMichigan State University Rice Genome Annotation ProjectRelease 7.0; Fig. 1E).Based on RT-PCR analysis and expressed sequence tag (EST)

information, we found that only ORF3 and ORF4 were expressed,

Author contributions: Q.Q., J.L., and H.Z. designed research; X.Z., J.W., J.H., H.L., C.W.,C.Y., Y.W., and H.T. performed research; X.Z. and H.Z. analyzed data; and J.L. and H.Z.wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1X.Z., J.W., and J.H. contributed equally to this work.2To whom correspondence may be addressed. E-mail: hszhang@njau.edu.cn, jyli@genetics.ac.cn, or qianqian188@hotmail.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1219776110/-/DCSupplemental.

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whereas ORF1 and ORF2 encoding retrotransposon proteinsand ORF5 encoding a transposon were not expressed in rice.Sequence comparison of these two expressed ORFs between93-11 and N411 revealed four SNPs in the ORF4. SNP1, a singlenucleotide transition from C (93-11) to A (N411) (c.+1092C→A),is present in the 10th exon of the ORF4, and SNP2, a singlenucleotide transition from C (93-11) to T (N411) (c.+1495C→T),in the 11th exon (Fig. 1F). These two transitions cause amino acidresidue changes from aspartate to glutamate (Asp364Glu) andhistidine to tyrosine (His499Tyr), respectively (Fig. 1G). SNP3(c.+2643A→G, in the 18th exon) and SNP4 (c.+2838T→C, in the21st exon) do not cause amino acid residue changes (Fig. 1F). Nodifference in the ORF3 sequence was identified between 93-11and N411. Furthermore, RT-PCR analyses showed no expres-sional difference in young panicles between 93-11 and its near-isogenic line (NIL)-qgl3 (Fig. S2) that carries a ∼113-kb segment,including the N411 qgl3 allele in the 93-11 background. There-fore, ORF4 was most likely the candidate gene for qGL3. TheORF4 encodes a putative type 2A phosphatase (PP2A), and it istherefore designated as OsPPKL1, which belongs to a small genefamily because there are three protein phosphatases with Kelch-like repeat domain (PPKL) members in O. sativa, four in Ara-bidopsis thaliana, two in Brachypodium distachyon, three in Zeamays, three in Sorghum bicolor, and three in Setaria italica.Sequence alignment of these plant homologs showed that theamino acid transition (Asp364Glu) caused by SNP1 inOsPPKL1N411

occurs in a conserved AVLDT motif of the second Kelch domain(Fig. 1G), whereas the transition (His499Tyr) caused by SNP2occurs in a nonconserved region (Fig. S3A).

Confirmation of OsPPKL1 as qGL3. To confirm that OsPPKL1N411 isqgl3 and to understand its functional nature, we overexpressed(OE) OsPPKL193-11 and OsPPKL1N411, respectively, in a japonicavariety, Zhonghua 11 (ZH11), which has a relatively short grain.We observed a decrease in grain length in the OE-OsPPKL193-11

transgenic plants (T1) compared with the control ZH11 (Fig. 2 Aand B), whereas no difference in grain length was observed betweenthe OE-OsPPKL1N411 (T1) and ZH11 (Fig. 2 A and B), suggestingthat OsPPKL193-11 plays an inhibitory function in regulating thegrain length, and the amino acid transition in OsPPKL1N411 abol-ishes its inhibitory role. Accordingly, the transfer DNA (T-DNA)insertion mutant of OsPPKL1, osppkl1 (3D-50056.R, created usingan O. sativa L. ssp. japonica variety, Dongjin) (15, 16), exhibitslonger grains compared with its wild type (Fig. 2 C and D). Tofurther understand the inhibitory function of OsPPKL1, weoverexpressed the Kelch domains and the PP2A domainof OsPPKL193-11 in ZH11, respectively (Fig. 2E). The OE-Kelch93-11 produced shorter grains compared with ZH11, and evenshorter than the OE-OsPPKL193-11, whereas the OE-PP2A93-11

produced similar grains to ZH11 (Fig. 2 A and B). These resultssuggest that the inhibitory regulation effect of OsPPKL193-11

results from its Kelch domains. The Kelch domain has been

Fig. 1. Map-based cloning of qGL3. (A) Parentalgrains used for QTL analysis and fine-mapping.(Scale bar: 10 mm.) (B) QTLs affecting grain lengthand width identified in the N411 × N643 F2 pop-ulation. Red and blue circles show grain length andwidth, respectively. Circle sizes reflect their effectson phenotypic variations. The major loci, includingthree known loci (shown in bracket) and the qGL3locus, are indicated by arrows. (C) A fine-linkagemap generated by analyzing 2,968 N411 × 93-11BC2F3 segregating plants. The recombinant numbersare given between markers. (D) High-resolutionlinkage analysis of phenotypes and marker geno-types. White bars represent chromosomal segmentsfor 93-11 homozygote, black with spots for N411 ho-mozygote, and grille for heterozygote, respectively.Progeny testing was used to confirm the genotypesat the qGL3 locus. S, segregation; D, desegregation.(E) Predicted ORFs based on the Nipponbare ge-nome sequence. The horizontal arrows representpredicted five ORFs (ORF1, LOC_Os03g44460; ORF2,LOC_Os03g44470; ORF3, LOC_Os03g44484; ORF4,LOC_Os03g44500; and ORF5, LOC_Os03g44510). (F)The gene structure of ORF4. Empty boxes refer to 5′and 3′ UTRs, gray boxes to exons, and the lines be-tween boxes to introns. The SNPs in the OsPPKL1N411

are shown by dashed lines; SNP1, c.+1092C→A;SNP2, c.+1495C→T; SNP3, c.+2643A→G; SNP4,c.+2838T→C. (G) The Kelch domains and thePP2A domain predicted in the protein encodedby ORF4. Solid lines show the positions of twoamino acid transitions.

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reported to represent one β-sheet blade, and several repeats areable to interact to form a β-propeller (17). In rice, LARGERPANICLE (LP) encoding a Kelch repeat-containing F-box pro-tein was reported to play a negative role in regulating plant ar-chitecture, particularly panicle architecture (18). In this study, wealso found that the Kelch domains in OsPPKL193-11 play a negativeregulation role in forming grain length.

Sequence Polymorphisms of qGL3 in Rice Germplasms. Ninety-fourrice germplasms with abundant diversity in grain size were se-lected for sequencing a 3.6-kb genomic DNA fragment thatcovers the four mutation sites of the qgl3. We found only onevariety, DT108 (grain length = 12.08 mm), with the same SNP1as N411, whereas polymorphisms of SNP2, SNP3, and SNP4 arewidely distributed in either japonica or indica varieties (TableS4). By association analysis of the SNPs and InDel markers inthe 3.6-kb region with grain length of the 94 germplasms (19), wefound that SNP1 had a high contribution to grain length, whereasother polymorphic sites had no significant contributions to grainlength (Table S5). These results indicate that the qgl3 is a rareallele in rice, and SNP1 is a functional mutation for long grain.The positive alleles of GS3 (2–4), qSW5/GW5 (10, 11), GS5 (12),and GW8 (13) for grain size are common in modern rice varie-ties, and the beneficial allele (qgl3) of qGL3 is rare, which is similarto GW2 (20). As a major pleitrophic QTL that has beneficialeffects on grain length, grain thickness, grain width, and thou-sand-grain weight, qgl3 should be selected in rice-breeding.However, the rareness of the qgl3 allele in the collected acces-sions may reflect its recent occurrence, which needs to beaddressed in the future.

Functions of Two OsPPKL1 Homologs. The rice genome encodesother two OsPPKL1 homologs, OsPPKL2 (LOC_Os05g05240,identity = 57%) and OsPPKL3 (LOC_Os12g42310, identity =89%). To understand whether they have similar functions toOsPPKL1, we first examined their expression profiles and levels.As shown in Fig. S3 B and C, all three OsPPKLs exhibit similarexpression profiles in various rice organs, but OsPPKL2 ex-presses at a lower level than OsPPKL1 and OsPPKL3. We thenoverexpressed OsPPKL293-11 and OsPPKL393-11 in ZH11, re-spectively. Like OE-OsPPKL193-11, OE-OsPPKL393-11 transgeniclines also produced short grains, whereas OE-OsPPKL293-11 lineshad long grains (Fig. 2 A and B). As expected, the T-DNA in-sertion mutants, osppkl3 (2C-10162.L) and osppkl2 (3A-06810.L)(15, 16), produce longer and shorter grains, respectively (Fig. 2 Cand D). The phylogenetic analysis showed that OsPPKL2 andtwo Arabidopsis homologs AtBSU1 and AtBSL1 belong to onesubgroup, and OsPPKL1 and OsPPKL3 to another subgroup(Fig. S3A). AtBSU1 and AtBSL1 were reported to be involved inbrassinosteroid signaling to enhance cell elongation and division(21, 22). It is possible that the OsPPKL2 subgroup may also pos-itively regulate cell elongation or division, whereas the OsPPKL1/3subgroup plays a negative role.

Biological Roles of qgl3. With a scanning electron microscope, weobserved a higher cell density in the outer surface of glumes inNIL-qgl3 (OsPPKL1N411; Fig. 3 A–E) than that in 93-11(OsPPKL193-11) at the early developing stages (spikelet = 2, and5 mm) of spikelet, but no significant difference at the maturitystage (Fig. 3 D and E). Therefore, it is very likely that the longglumes of NIL-qgl3 result from an increase in cell numbers

Fig. 2. Functional analysis of OsPPKLs. (A) Grainsof the transgenic plants (T1) and ZH11. (Scale bar:10 mm.) (B) Comparison of the grain length be-tween ZH11 and the transgenic plants in A. P valuesfrom a t test of the transgenic plants against ZH11were indicated. (C) The grains of OsPPKL knockoutmutants in the Dongjin background. (D) Compar-isons of the grain length of the mutant plants. Pvalues from a t test of the mutant against Dongjinwere indicated. (E) Schematic representation ofconstructs for overexpressing different domains.

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longitudinally, which is consistent with the observation that theNIL-qgl3 ovaries and grains are longer than those of 93-11 (Fig.S4). Comparison of the grain-filling rates between NIL-qgl3 and93-11 by measuring dry weight of grains during grain-fillingrevealed that the dry weight of NIL-qgl3 was significantly higherthan that of 93-11 from 8 d after fertilization (Fig. 3F), sug-gesting that the qgl3 allele has a great potential in breeding eliterice varieties.

qgl3 Enhances Rice Yield Significantly. The grain yield and otheragronomic traits of NIL-qgl3 were evaluated under field con-ditions. Compared with 93-11, NIL-qgl3 showed an increase of16.20% in yield, resulting from increases of 19.68% in grainlength, 1.15% in grain width, 8.25% in grain thickness, 37.03% infilled-grain weight, and 11.76% in panicle length (Table 1). No

significant differences in grain number per panicle, tiller numberper plant, days to heading, and plant height were observed be-tween 93-11 and NIL-qgl3 (Fig. 3 A–C and Table 1). Therefore,the increase of grain yield in the NIL-qgl3 resulted from the im-provement in grain weight. The chalk rate of white rice of NIL-qgl3 (23.14 ± 1.62, mean ± SD) was very similar to that of 93-11(22.33 ± 1.32, mean ± SD). To explore the application of NIL-qgl3 in hybrid rice-breeding, we crossed 93-11 and NIL-qgl3 withthree commercial photothermosensitive male sterile lines, P88S,Peiai64S (PA64S), and Guangzhan63S (GZ63S) to generate F1seeds. Compared with P88S/93-11, PA64S/93-11, and GZ63S/93-11, the P88S/NIL-qgl3, PA64S/NIL-qgl3, and GZ63S/NIL-qgl3produced significantly longer grains (Fig. 4A), resulting in anincrease of 10.12∼13.48% grain yield in field trials (Fig. 4B).Except P88S/NIL-qgl3, PA64S/NIL-qgl3 and GZ63S/NIL-qgl3

Fig. 3. Comparison of different traits between 93-11 and NIL-qgl3. (A) Grains of 93-11 and NIL-qgl3.(Scale bar: 10 mm.) (B) Panicles of 93-11 and NIL-qgl3. (Scale bar: 2 cm.) (C) Plants of 93-11 and NIL-qgl3. (Scale bar: 10 cm.) (D) Scanning electron andlight microscope photos of glume outer surfaces of93-11 and NIL-qgl3 spikelets at three stages. Stage 1,spikelet = 2 mm; stage 2, spikelet = 5 mm; stage 3,maturity. (Scale bars: yellow, 50 μm; blue, 1.0 mm.)(E) Cell density of glume outer surfaces of 93-11 andNIL-qgl3 spikelets at three stages. (F) Grain fillingrate of 93-11 and NIL-qgl3 after fertilization, in-dicated by dry weight of 1,000 grains.

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showed similar chalk rates of white rice to PA64S/93-11 andGZ63S/93-11 (Fig. 4C). Therefore, the qgl3 allele can be appliedin breeding elite varieties to improve rice grain productionwithout sacrificing grain quality. In fact, the goal of breedingsuperelite rice with high yield and good quality could be realizedby pyramiding production- and quality-related genes, such as ipa1

(23, 24), dep1 (5, 6), gs3 (2–4), gw2 (9), qsw5/gw5 (10, 11), GS5(12), GW8 (13), wx (25), or alk1 (26), through molecular marker-assisted selection and genetic engineering technologies.

MethodsPlant Materials. The extra large-grain rice accession N411 (Oryza sativaL. japonica) was screened from more than 7,000 germplasms and used asa desirable allele donor parent. The small-grain indica rice variety N643 wascrossed with N411 to create an F2 population for primary QTL analysis. Ahigh-quality elite variety 93-11 (indica), was used as the recurrent parent tofurther validate and disassemble qGL3 by the backcrossing method. NIL forqgl3 (NIL-qgl3), which carries a ∼113-kb segment, including the N411 qgl3allele (from XJ19 to RM15575) in 93-11 background, was developed bybackcrossing and molecular marker analysis. A japonica cultivar, ZH11, wasused in the genetic transformation. Three T-DNA insertion mutants forOsPPKLs 3D-50056.R, 3A-06810.L, and 2C-10162.L in the japonica Dongjinbackground were obtained from Kyung Hee University, Korea (http://cbi.khu.ac.kr/RISD_DB.html) (15, 16). Three photothermosensitive male sterilelines, P88S, Peiai64S, and Guangzhan63S, were crossed, respectively, with93-11 and NIL-qgl3 to produce six combinations, P88S/93-11, PA64S/93-11,GZ63S/93-11, P88S/NIL-qgl3, PA64S/NIL-qgl3, and GZ63S/NIL-qgl3 for yieldtrials. The 94 rice varieties used in sequence analysis of qGL3 are listed inTable S4.

Primers. Primers used for QTL mapping are listed in Table S3 and those forgene cloning, expression analysis and vectors construction are given inTable S6.

QTL Mapping of qGL3. An F2 population of 182 plants, derived from a crossbetween N411 and N643, was used to construct a genetic map with 108

Table 1. Comparison of grain yield and other agronomic traitsbetween 93-11 and NIL-qgl3

Traits 93-11 NIL-qgl3 P value

GL, mm 10.21 ± 0.14 12.22 ± 0.23 1.21 × 10−15

GW, mm 2.62 ± 0.06 2.65 ± 0.07 0.015GT, mm 2.18 ± 0.05 2.36 ± 0.04 0.034TGW, g 30.08 ± 1.34 41.22 ± 1.42 3.10 × 10−19

PL, cm 24.50 ± 1.63 27.38 ± 2.32 1.38 × 10−08

GPP 216.60 ± 13.16 212.31 ± 17.64 0.71PH, cm 113.60 ± 5.61 116.32 ± 4.34 0.37TN 7.69 ± 1.54 7.87 ± 2.32 0.66DTH, d 156.30 ± 3.50 158.50 ± 4.20 0.89YPP, kg 12.84 ± 0.46 14.92 ± 0.38 8.63 × 10−12

All traits data are given as mean ± SD. Each P value for each trait wasobtained from a t test between NIL-qgl3 and 93-11. DTH, days to heading(summer 2012, Nanjing, Jiangsu, China); GL, grain length; GPP, grains perpanicle; GT, grain thickness; GW, grain width; PH, plant height; PL, paniclelength; TGW, thousand filled-grain weight; TN, tiller number per plant; YPP,yield per plot.

Fig. 4. Evaluation of the qgl3 effects on hybrid rice. (A) Comparison of grains and brown rice of three pairs of hybrids. (Scale bar: 10 mm.) (B) Actual grainyields of different hybrids in plots. (C) Chalk rates of white rice of different hybrids. P values from a t test were indicated.

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polymorphic SSR markers distributing averagely in the rice genome for pri-mary QTL analysis. To fine-map the qGL3 locus, additional SSR markers in thetarget region were selected from the previously published SSRs (27), andeight InDel markers (Table S3) were developed in this study. The geneticmap was constructed using MapMaker3.0/EXP version 3.0 (28). QTL analysiswas performed by IciMapping3.1 (www.isbreeding.net/) along with thecomposite interval mapping method (29), and the threshold was obtainedby 1,000 permutations. The distribution of grain length of 206 random BC2F3plants confirmed that the qGL3 locus was disassembled to a single geneticfactor for grain length (Fig. S1). The genotype of individuals could be de-termined by the phenotype of grain length (9.6–10.3 mm for the 93-11homozygote, 10.6–11.5 mm for the heterozygote, and 11.8–12.8 mm for theN411 homozygote).

Gene Cloning, Construct Generation, and Transformation. The full-lengthOsPPKL1N411 was amplified from cDNA samples of N411, and OsPPKL193-11,OsPPKL2, and OsPPKL3 were amplified from 93-11. These four full-lengthgenes were cloned into the plant binary vector pCAMBIA1300S (30) togenerate OE-OsPPKL1N411, OE-OsPPKL193-11, OE-OsPPKL293-11, and OE-OsPPKL393-11, respectively. The truncated OsPPLK193-11 constructs lackingeither PP2A or Kelch domains were amplified from 93-11 and cloned intovector pCAMBIA1300S to generate constructs OE-Kelch93-11 or OE-PP2A93-11

(Fig. 2E). All of the genes or gene fragments were controlled by the CaMV35S promoter and introduced into ZH11 by Agrobacterium-mediatedtransformation as reported previously (31).

Expression Analysis. Total RNA was extracted from various plant tissues of93-11 and NIL-qgl3 using a RNA extraction kit (RNAiso Plus; TaKaRa Bio, Inc.).The first-strand cDNA was synthesized using 6 μg RNA and 4 μg reverse-transcriptase mix (PrimeScript RT Master Mix Perfect Real Time; TaKaRa Bio,Inc.) in a volume of 40 μL. Semiquantitative PCR was performed in a totalvolume of 25 μL containing 2 μL of cDNA, 0.2 mM gene-specific primers,2.5 μL 1× RT buffer, 10 μM dNTP mix. The rice OsRac1 gene was used as the

internal control. Real-time PCR was carried out in a total volume of 25 μLcontaining 2 μL of cDNA, 0.2 mM gene-specific primers, 12.5 μL SYBR PremixEx Taq II, and 0.5 μL of Rox Reference Dye II (TaKaRa Bio, Inc.), using an ABI7500 Fast Real-Time PCR System according to the manufacturer’s instruc-tions. A rice 18S rRNA gene was used as the internal control. The relativequantification of the transcript levels was performed using the comparativeCt method (32).

Histological Observation. The spikelets of 93-11 and NIL-qgl3 at three variousdeveloping stages (stage 1, 2 mm; stage 2, 5 mm; stage 3, maturity) werecollected and treated with 2.5% (vol/vol) glutaraldehyde, vacuumed threetimes, and fixed for 24 h as described previously (33). The outer surfaces ofglumes of the spikelets were observed with a scanning electron microscopeat an accelerating voltage of 15 kV, and cell density of the glume was cal-culated as cell number mm−1 in longitude.

Plant Growth Conditions. To evaluate the effect of qgl3, the yields and otheragronomic traits of 93-11 and NIL-qgl3 were compared by using three pairs(P88S/93-11 and P88S/NIL-qgl3; PA64S/93-11 and PA64S/NIL-qgl3; and GZ63S/93-11 and GZ63S/NIL-qgl3) of rice materials, which were grown in theJiangpu Experiment Station of Nanjing Agricultural University. A total of 320plants for each material were grown in a plot of 13.4 m2 with three repli-cates. For each plot, the grain yield and other agronomic traits, includingdays to heading, plant height, tiller number, panicle length, grain number,grain length, grain width, grain thickness, filled-grain weight, and chalk rateof white rice, were measured and analyzed.

ACKNOWLEDGMENTS. This work was supported by Natural Science Foun-dation of China Grant 31071386, National High Technology Research andDevelopment Program of China Grant 2006AA10A102, National Science andTechnology Support Plan of China Grant 2006BAD01A10-3, and OpenSubject of State Key Laboratory of Rice Biology Grant 110101.

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