Natural variation in ARF18 gene simultaneously affectsseed weight and silique length in polyploid rapeseedJing Liua,1, Wei Huaa,1, Zhiyong Hua, Hongli Yanga, Liang Zhanga, Rongjun Lib, Linbin Denga, Xingchao Suna,Xinfa Wanga, and Hanzhong Wanga,2

aOil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministryof Agriculture, Wuhan 430062, China; and bKey Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, ChineseAcademy of Sciences, Wuhan 430062, China

Edited by Qifa Zhang, Huazhong Agricultural University, Wuhan, China, and approved August 6, 2015 (received for review February 3, 2015)

Seed weight (SW), which is one of the three major factors influencinggrain yield, has been widely accepted as a complex trait that iscontrolled by polygenes, particularly in polyploid crops. Brassicanapus L., which is the second leading crop source for vegetable oilaround the world, is a tetraploid (4×) species. In the present study,we identified a major quantitative trait locus (QTL) on chromosomeA9 of rapeseed in which the genes for SW and silique length (SL)were colocated. By fine mapping and association analysis, we uncov-ered a 165-bp deletion in the auxin-response factor 18 (ARF18) geneassociated with increased SW and SL. ARF18 encodes an auxin-response factor and shows inhibitory activity on downstream auxingenes. This 55-aa deletion prevents ARF18 from forming homodimers,in turn resulting in the loss of binding activity. Furthermore, reciprocalcrossing has shown that this QTL affects SW by maternal effects.Transcription analysis has shown that ARF18 regulates cell growthin the siliquewall by acting via an auxin-response pathway. Together,our results suggest that ARF18 regulates silique wall developmentand determines SW via maternal regulation. In addition, our studyreveals the first (to our knowledge) QTL in rapeseed and may provideinsights into gene cloning involving polyploid crops.

seed weight | silique length | ARF18 | cell growth | maternal effect

The rapid growth of the world population has increased theglobal requirement for food, which in turn warrants signifi-

cant improvement in crop grain yield. As one of the three directfactors influencing crop grain yield, seed weight (SW) has beenwidely accepted as a complex trait that is controlled by poly-genes. Therefore understanding the genetic and molecular basisof SW is extremely important for crop-improvement programs.The size of seeds is influenced by a variety of cellular processes

(1). In Arabidopsis, some mutants such as ap2, arf2, da1, eod3,ttg2, and klu control seed size mainly by regulating cell elonga-tion in the integument surrounding the seed (1–5). In mini3,iku1, iku2, and shb1 mutants, premature cellularization or pro-liferation of the endosperm in the early phase of seed devel-opment affects seed mass (6–10). The met1 gene has beendetermined to have parent-of-origin effects on seed size becauseof the loss of methylation in cytosine residues in CG islands (11).In rice, a total of 47 quantitative trait loci (QTLs) for grainlength and 48 for grain width have been identified (12). Recentstudies have shown that certain genes such as GW2, GIF1, qSW5,GS3, GS5, GW8, and qGL3 regulate grain size (13–20). Amongthese, GW2 and qSW5 were determined to regulate grain weightby increasing cell number in the outer glume, whereas the othersaffected grain weight by directly regulating cell division and/orcell expansion of grain. Despite this progress, no genes res-ponsible for SW have been identified in polyploid crops.Polyploidy is produced by the multiplication of a single ge-

nome (autopolyploid) or the combination of two or more di-vergent genomes (allopolyploid), which commonly occurs inflowering plants, including many important agricultural crops(21). The complexity of polyploid genomes results in difficultiesin QTL localization such as the inaccuracies caused by homol-

ogous sequences from different chromosomes and the inter-actions between homolog genes. For example, Brassica napusL. (AACC), the world’s second leading crop source of vegetableoil following soybean, is a tetraploid (4×) species containing twoancestors, namely, Brassica rapa and Brassica oleracea, both ofwhich underwent whole-genome triplication (22). B. rapa har-bors the Brassica A genome and is closely related to B. oleracea,which contains the Brassica C genome (23). Rapeseed is animportant global agricultural crop that has recently gained at-tention in the field of plant genomics. Despite the identificationof more than 80 QTLs for SW across the 19 chromosomes of theB. napus genome (24–27), no genes associated with these QTLshave been identified.We previously examined the F2 populations derived from a

cross between the rapeseed lines zy72360 and R1 and detected amajor QTL for SW. This QTL was localized to chromosome A09and explained ∼30% of the phenotypic variation observed in SW.Interestingly, other rapeseed lines also harbored this QTL, asdetected by Yang et al. (26) and Li et al. (27). In the presentstudy, we used map-based cloning and targeted-region associa-tion to clone and characterize the gene related to the majorQTL for SW. We further elucidated the possible mechanism

Significance

Seed weight is a complex trait controlled by polygenes, and itsunderlying regulatory mechanisms, especially those involvingpolyploidy crops, remain elusive. Brassica napus L., which is thesecond leading crop source of vegetable oil around the world,is an important tetraploid (4×) crop. Our results have gener-ated three significant findings. (i) By combining the linkageand associated analysis, this study revealed the first (to ourknowledge) quantitative trait locus (QTL) in rapeseed, whichwill provide insights for QTL cloning in polyploidy crops. (ii) Thefunctional gene andmarker could be useful in rapeseed breeding.(iii) We revealed a maternal regulatory pathway affectingseed weight that differs from the mechanisms described inprevious reports.

Author contributions: H.W. designed research; J.L., Z.H., H.Y., L.Z., R.L., L.D., and X.S.performed research; X.W. contributed new reagents/analytic tools; J.L., W.H., Z.H., andH.Y. analyzed data; and J.L. and W.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the NCBIdatabase [accession nos. BnaA09g55520D cDNA from zy72360 (KT000600); BnaA09g55520DcDNA from R1 (KT000602); BnaA09g55570D promoter from zy72360 (KT000601);BnaA09g55570D promoter from R1 (KT000605); BnaA09g55560D from ZY72360(KT000603); BnaA09g55560D from R1 (KT000604); BnaA09g55580D from zy72360(KT000607); BnaA09g55580D from R1 (KT000608); and BnaA09g55580D promoter from R1(KT000606)]. The transcriptome data for silique wall and seed has been deposited in ocri-genomics.org/RNA-seq/.1J.L. and W.H. contributed equally to this work.2To whom correspondence should be addressed. Email: wanghz@oilcrops.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1502160112/-/DCSupplemental.

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regulating SW to provide critical information for breeding high-yield crops.

ResultsFine Mapping of the Major SW-Regulating Gene on Chromosome A9.In the present study we used B. napus lines zy72360 and R1,which showed significant differences in SW (zy72360: 5.53 ± 0.43 g;R1: 3.86 ± 0.25 g) (Fig. 1A) and silique length (SL) (zy72360:85 ± 5.4 mm; R1: 60 ± 6.5 mm) (Fig. 1B). First, we mapped theSW QTL between markers TSNP08 and TSNP149 within the F2population. With newly developed markers based on insertionsand deletions (indels) and SNPs in the region of A09 betweenzy72360 and R1 (Fig. 1C and Table S1), we localized the SWQTL to a high-resolution linkage map by progeny testing ofhomozygous recombinant plants (BC4F2) and narrowed the SWlocus to a 147-kb region between markers TSNP13 and TSNP16(Fig. 1D). Based on the SNP information on the 147-kb regionbetween zy72360 and R1, we further narrowed the distance to120 kb by PCR confirmation of the recombinant lines (Fig. 1E).To narrow the distance of the SW QTL further, association

analysis was conducted using 380 lines that showed a large rangeof phenotypic variation for SW. Scanning of the association ofSW with 11 QTL-linked markers on the A09 linkage group

generally displayed an obvious peak between SSR-72 and SSR-89 (Fig. 1F). Using the results of fine mapping and targeted-region association, we narrowed the distance to 50 kb andidentified seven putative ORFs in this region. Irrespective of thenear isogenic line (NIL) or association population, the SW andthe SL genes were colocated within the QTL region; The phe-nomenon might be induced by pleiotropism in this QTL.

ARF18 Decreases SW and SL in Rapeseed. Based on the genomesequence of rapeseed (www.ncbi.nlm.nih.gov/assembly/GCA_000686985.1/), we cloned the seven genes includingthe upstream regulatory and coding regions from the parentszy72360 and R1 (Table S2). Sequence comparison showed thatthree genes, namely, BnaA09g55580D, BnaA09g55560D, andBnaA09g55520D, showed differences in amino acids, and theupstream regulation sequence of BnaA09g55570D showed SNPsand indels between the parents. These four genes were chosenfor functional identification by overexpression in Arabidopsisunder the control of the Cauliflower mosaic virus (CaMV)35S promoter. Except for the BnaA09g55580D-overexpressingtransgenic lines, these genes showed no change in SW (Fig. S1).Therefore, BnaA09g55580D was implicated as a candidate genefor SW. Genome and cDNA sequence analysis of R1 showed

Fig. 1. Fine mapping of the SW QTL in rapeseed using NILs and association populations. (A) SW in parents of zy72360 and R1. (Scale bar, 0.5 cm.) **P = 0.01.(B) SL in parents of zy72360 and R1. (Scale bar, 0.5 cm.) **P = 0.01. (C) The SW locus was detected on chromosome A09 in the F2 population. Positional cloningnarrowed the SW locus to a 1.1-Mb region between P534 and BrBAC248. (D) Testing of six recombinant plants (BC3F4) narrowed the SW locus to the regionbetween markers TSNP13 and TSNP16 (147 kb). The seeds from the main inflorescence were used to determine the SW for each plant. Open bars indicate theS1 homozygous regions, gray bars indicate the S2 homozygous regions, and black bars indicate the heterozygous regions. The SWs of the progenies weresignificantly different. (E) PCR identification narrowed the SW locus to the region between SNP3 and SNP5 (120 kb). (F) Scanning of the association of SWwith11 marker loci on the A09 linkage group in a rapeseed association population. Eleven maker loci are arranged on the horizontal axis according to theirphysical positions on chromosome A9.

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that the BnaA09g55580D gene comprised 12 exons and 11 in-trons. BnaA09g55580D homologs from B. rapa and Arabidopsisthaliana were detected in the National Center for BiotechnologyInformation (NCBI) database and were annotated as auxin-response factor 18 (ARF18) proteins; therefore, we named theBnaA09g55580D gene “BnaA.ARF18.a” based on the standardnomenclature of Østergaard and King (28). Comparison of theARF18 coding sequences in zy72360 and R1 showed that, inaddition to differences in a 6-bp indel site and four SNPs in-ducing changes in four amino acids, the seventh exon (165 bp)in zy72360 was lost (Fig. 2A and Fig. S2).A total of 30 independent homogenous T3 transgenic Arabi-

dopsis lines with overexpression of ARF18 from R1 and zy72360,respectively, were analyzed. Among these, 10 ARF18-over-expressing lines from R1 showed phenotypes distinct from thatof the WT line. For example, in the two lines with high ARF18expression levels, R1-ARF18-5 and R1-ARF18-8, seed sizeswere smaller (thousand kernel weight: 17.6 ± 0.38 mg and 17 ±0.4 mg, respectively) than in the WT line (thousand kernelweight: 19.6 ± 0.34 mg) (Fig. 2B and Fig. S3A). On the otherhand, overexpression of ARF18 in zy72360 did not affect seedsize (thousand kernel weight: 19.3 ± 0.41 mg and 19.7 ± 0.5 mg,respectively), as compared with that of WT plants (Fig. 2B andFig. S3A). The transgenic R1-ARF18-5 and R1-ARF18-8 plants

also showed significantly shorter siliques (13.62 ± 0.4 mm and13.38 ± 0.34 mm, respectively) compared with the plants con-taining ARF18 from zy72360 (15.35 ± 0.39 mm and 15.54 ±0.35 mm, respectively), which yielded siliques similar in lengthto those of WT plants (15.4 ± 0.41 mm) (Fig. 2C). The ArabidopsisARF18 mutant was planted also. Compared with WT Arabidopsis,the mutant generated larger seeds (21.2 ± 0.52 mg) and longersiliques (17 ± 0.4 mm) (Fig. S3 C and D).Based on these findings, ARF18 and its upstream regulatory

region (the 1.6 kb that included the 5′ noncoding region) thatoriginated from R1 was transfected into rapeseed line zy72360 bya hypocotyl transgenic system of Agrobacterium. A total of 25independent homogenous T2 transgenic lines were used foranalysis of phenotypes, including SW and SL. The SW in morethan 30% of these transgenic lines decreased by at least 15%(Fig. S4A). For example, in the two transgenic lines R1-ARF18-BnT5 and R1-ARF18-BnT11, which had high expression levelsof R1-ARF18, the SW decreased (4.61 ± 0.3 g and 4.81 ± 0.22 g,respectively) compared with that of the zy72360 control (5.50 ±0.38 g) (Fig. 2D and Fig. S3B). The SL also shortened in thesetwo lines (70 ± 4 mm and 72 ± 3 mm, respectively) comparedwith the zy72360 control (86 ± 6 mm) (Fig. 2E).

Incorrect Splicing of the Sixth Intron Induces Nonfunctional ARF18That Originated from zy72360. A search inquiry of the NCBI da-tabase identified two BnaA09g55580D homologs in B. rapa andone in Arabidopsis. A hypothetical protein in Capsella rubella wasidentified also. These proteins have high amino acid sequenceidentities (B. rapa 1: 86%; B. rapa 2: 99%; A. thaliana: 81%;C. rubella: 80%) with ARF18 of B. napus (rapeseed) (Fig. 3A).Thus far, none of these homologous genes has been character-ized functionally. Previous studies have shown that ARFs act astranscription factors and comprise three domains: an N-terminalDNA-binding domain (DBD), a C-terminal dimerization domain(CTD) similar to domains III and IV of the Aux/IAA family ofproteins, and the middle region (MR), which determines that theARF protein functions as an transcriptional activator/repressorof auxin-responsive genes (Fig. 3A). Subcellular localization intobacco showed that R1-ARF18 localized to the nucleus(Fig. 3B).Comparison of the parental ARF18 proteins showed that the

55-aa deletion in ARF18 of zy72360 was located between theDBD and the MR (Fig. 3 A and C). We deleted the 165-bpsegment in ARF18 from R1 by fusion PCR and overexpressed itin Arabidopsis. The transgenic lines showed normal phenotypes,suggesting that the 55-aa deletion in ARF18 generated a non-functional protein (Fig. S4B). To corroborate that the 165-bpdeletion in zy72360 was induced by sequence variation, two GFPreporter vectors driven by a CaMV 35S promoter were con-structed. The sixth intron of R1 (69 bp) or the sequence fromzy72360 corresponding to the sixth intron site in R1 (80 bp) wasinserted into the middle of GFP gene (Fig. 4 A and B). Detectionof transient expression in tobacco leaf (Nicotiana tabacum L.)showed that only the vector inserted with the intron of R1 dis-played green fluorescence, indicating that the intron was shared(Fig. 4B).Previous reports have shown that Q-rich MRs in ARFs func-

tion as activation domains (29). This finding suggests thatARF18 might function as a repressor because it does not possessa Q-rich MR. To confirm our inference, we detected the tran-scriptional activity of ARF18 by using a protoplast assay system.In this system, ARF18 genes from zy72360 and R1 were fusedwith the CaMV 35S promoter as effector plasmids (Fig. 4C), andthe β-glucuronidase (GUS) gene under the control of auxin-responsive direct repeat DR5(7×) was used as reporter. Luciferase(LUC) driven by the CaMV 35S promoter was used as internalreference. After the plasmids were introduced into the protoplasts,GUS activity was measured to determine ARF18 transcriptional

Fig. 2. Structural characterization and functional identification of ARF18.(A) ARF18 structure and mutation sites, including nucleotide substitutionsand deletions, in zy72360 and R1. (B and C) Comparison of SW (B) and the SL(C) in Arabidopsis (vector control) and ARF18 transgenic lines in Arabidopsis.(Scale bars, 0.5 mm in B and 1 mm in C.) All data are expressed as mean ± SD.(D and E), Comparison of SW (D) and SL (E) in the zy72360 line with apromoter-driven expression of R1-ARF18. (Scale bars, 0.5 cm.) All data areexpressed as mean ± SD. **P = 0.01.

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activity. Fig. 4C shows that under the N-acetyl-aspartate (NAA)treatment the effector gene encoding the full-length ARF18 re-pressed the transcription of GUS, whereas the effector gene fromzy72360 did not differ significantly from that of the control withoutan effector.

ARF18 from zy72360 Lost Its Binding Activity Because of Failure inForming a Homodimer. Because the 55 amino acids were sharedbetween the DBD and the MR, it was essential to determinewhich domain had lost its activity. To identify the inhibitionactivity of the ARF18 proteins, the allelic genes were constructedinto PGBKT7 as bait vectors and were cotransformed withPGADT7 into yeast. PGBKT7-VP16 was used as positive con-trol. The empty vectors PGBKT7 and PGADT7 were used asnegative control. The results showed that the ARF18 proteinsfrom the two parents had nearly the same inhibition activity,suggesting that the partial deletion in the MR region did notaffect the inhibition activity of ARF18 from zy72360 (Fig. 5A).

The structures of the ARF18 proteins were predicted byusing SWISS-MODEL. The results showed that ARF18 fromR1 could form homodimers, whereas the protein from zy72360could form only monomers (Fig. S4C). Yeast two-hybridizationshowed that R1-ARF18 interacted with itself, but zy72360-ARF18 did not self-interact (Fig. 5B). The revised R1-ARF18(R1-ARF18r) in which the 55 amino acids were deleted alsofailed to interact with itself. To detect further differences inbinding activity between the two ARF18 proteins, gel mo-bility shift assays using a labeled DR5(7×) probe were per-formed. The results showed that under the same conditionsR1-ARF18 bound the DR5(7×) probe as expected, butzy72360-ARF18 failed to bind the sequence (Fig. 5C). Based onthese results, we concluded that the full-length ARF18 proteinfrom R1 acts as a repressor in regulating transcription, andARF18 with the exon deletion from zy72360 lost that activitybecause of its failure to form dimers.

Fig. 3. Comparative analysis of ARF18 proteins from zy72360 and R1. (A) Amino acid alignments of ARF18 proteins from other species with rapeseed ARF18.The black lines indicate the three conserved domains. (B) Nuclear localization of the ARF18-GFP fusion protein in tobacco epidermal cells. Cells transformedwith the plasmid were viewed under a fluorescent filter to show GFP (Left), under a bright field for cell morphology (Center), and in combination (Right).(Scale bar, 50 μm.) (C) Putative conserved domains detected in ARF18 proteins from the alleles of zy72360 and R1.

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ARF18 Is Expressed Differentially in Various Tissues and Is ExpressedPredominantly in the Silique Wall. Quantitative RT-PCR (qRT-PCR) analysis of various organs of the NIL (SW) and R1 lines

indicated that ARF18 was expressed differentially in the root, leaf,stem, bud, silique wall, and ovule. At 25 d after flowering (daf), theexpression levels of ARF18 were significantly higher in the silique

Fig. 4. Identification of incorrect splicing in ARF18 from zy72360 and comparison of inhibitory activity in ARF18 alleles. (A) Comparison of the ARF18 partialCDS and genomic sequences originating from zy72360 and R1. (B) The sixth intron from R1 and the corresponding sequence from the zy72360 gene wereinserted into the GFP gene, which was driven by the CaMV 35S promoter and transformed into tobacco leaves. A is the intron from zy72360, and B is theintron from R1. (Scale bar, 100 μm.) (C) Effector genes that consisted of the CaMV 35S promoter encoding full-length ARF18 proteins were cotransfected intoArabidopsis suspension cell protoplasts with a DR5(7×)-GUS reporter gene. GUS activities were measured from protoplasts treated with 10 μM NAA. The dataare representative of three independent experiments. All data are expressed as mean ± SD. **P = 0.01.

Fig. 5. Comparison of inhibitory and binding activity of two ARF18 proteins. (A) Detection of ARF18 transcriptional activity in a yeast one-hybrid system.ARF18 genes from zy72360 and R1 were constructed as bait vector. +, positive control; −, empty control vector. (B) Detection of interaction in ARF18 proteinsby yeast two-hybridization. (C) EMSA indicating that ARF18 from R1 binds to the biotin-labeled DR5 probe. The free probe is visible at the bottom of the gel,and complexes with the ARF18 protein are shifted upward.

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walls than in the other tissues; the stems and seeds showed verylow transcript levels of ARF18 (Fig. 6A). No differences in ARF18expression level in the two lines were observed, suggesting that thesequence change in the coding region accounted for the functionalvariation in the two alleles. Further expression analysis was per-formed using GUS fusion studies in which the ARF18 promoter(promoter length, 1.6 kb) was ligated into pCXGUS-P andtransformed in Arabidopsis. The results of ARF18 promoter-GUS expression analysis are shown in Fig. 6B. The seedlings,older leaves, and silique walls of transgenic plants showed highlevels of GUS expression. Moderate expression levels were de-tected in the root, stem, and floral organs, including the stamenand pistil. In the developing seeds, moderate levels of ARF18expression were observed in the embryo but not in the seed coat.GUS expression driven by the ARF18 promoter basically isconsistent with the results of RT-qPCR analysis.

ARF18 Might Affect SW by Regulating the Development of the SiliqueWall. To investigate further the mechanism by which ARF18 in-fluences SW, reciprocal cross experiments between NIL (SW)(ARF18−) and R1 (ARF18+) were performed. Pollinating NIL(SW) plants with R1 pollen led to the development of ARF18−/+

embryos within an ARF18−/− seed coat. Similarly, pollinating R1plants with NIL (SW) pollen led to the development of ARF18+/−

embryos within an ARF18+/+ seed coat. No obvious differencesexisted in the weight between the resulting seeds and the corre-

sponding self-pollinated seeds; this result was suggestive of ma-ternal control of SW in NIL (SW) plants (Fig. S4D).Phenotype comparison between the NIL (SW) and R1 lines

showed differences in plant height and leaf size of the seedlings,but no differences in the other characters such as the number ofbranches or the number of siliques per branch were observed(Fig. S5). SL increased by >30% in the NIL (SW) line (80.8 ± 9.6mm) as compared with the R1 line (61.2 ± 7.1 mm), but thenumber of seeds in siliques did not differ (Fig. 7A). Microscopicobservation showed that the longer siliques in the NIL (SW) linemight result from the increased length of the cells (Fig. 7B).The silique wall is an important source organ that providesphotosynthates to the developing seeds. Therefore, by deter-mining the surface area of the silique wall, SL may affect seedfilling as a maternal organ by regulating the accumulationof photosynthates.To understand better the mechanism underlying the regula-

tion of SW in ARF18, we compared the differentially expressedgenes (DEGs) in the silique wall and seed in the NIL (SW) and R1lines. In the silique wall in NIL (SW), 2,178 genes were up-regulated,

Fig. 6. ARF18 expression patterns by RT-qPCR and GUS assays. (A) Analysisof ARF18 expression in selected tissues in R1 using RT-qPCR. ENTH was usedas the reference. Data are expressed as the means of three biological rep-licates; error bars indicate SDs. (B) GUS expression patterns(blue staining) atvarious stages in the ARF18 promoter-GUS transgenic line. GUS was stronglyexpressed in the cotyledon (cn), seedling (sl), older leaves (lf), and silique wall(sw). Moderate expression levels were detected in the bud (bd) and embryo(sd). No expression was observed in the seed coat (sd). (Scale bars, 5 mm forcotyledon, seedling, and older leaves; 0.8 mm for silique wall and bud; 0.1 mmfor embryo.)

Fig. 7. Analysis of silique phenotype and DEGs in the silique wall and seedof the NIL (SW) and R1 lines. (A) Comparisons of seed number per silique andsilique length. (Scale bar, 0.5 cm.) **P = 0.01. (B) Comparison of cell length.(Scale bar, 50 μm.) **P = 0.01. (C) Differentially expressed genes analyses inthe silique wall and seed between the NIL (SW) and R1 lines. (D) KEGG en-richment analysis of the regulated genes in the silique wall and seed.

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and 1,774 genes were down-regulated (Fig. 7C). An overview ofDEGs in the seed showed that expression levels of fewer geneswere affected in the seed (702 up-regulated and 625 down-reg-ulated) than in the silique wall (Fig. 7C). DEGs between thesilique wall and seeds were detected also: 158 genes were up-regulated, and 69 genes were down-regulated in the silique walland seed (Fig. 7C).Kyoto Encyclopedia of Genes and Genomes (KEGG) path-

way enrichment analysis for DEGs showed the top 10 pathwaysaffected in the silique wall and seed, respectively (Fig. 7D).Comparative analysis showed that except for the commonpathways including cell wall transport and hormone metabolismthat occur in both the silique wall and the seed, some specialpathways such as nitrogen metabolism, carbohydrate metabo-lism, and development that might influence silique developmentwere affected in the silique wall. In the seed, pathways that couldaffect seed filling (e.g., glycolysis, lipid metabolism, and aminoacid metabolism, among others) were affected. Evidently, genesin the plant hormone metabolism were enriched in both the si-lique wall and seed (Table S3). Among these genes, in additionto the other ARF genes, three different kinds of early auxin-response genes such as Aux/IAA, small auxin up-regulated RNA(SAUR), and GH3, were differentially expressed in the NIL (SW)and R1 lines. However, significantly fewer auxin-response geneswere detected in the seed than in the silique wall (Table 1).

DiscussionVariation in the Region Upstream of the 3′ Splice Site InducesNonfunctionalization of the Sixth Intron in zy72360. In the presentstudy, comparison of ARF18 cDNAs and genome sequencesfrom zy72360 and R1 indicated skipping of the seventh exon inARF18 from zy72360. By comparing the sixth intron of theARF18 alleles, extensive variations, including six SNPs and an11-bp insertion, were detected in the region upstream of the 3′splicing site in the nonfunctional sixth intron. In plants, althoughindividual introns exhibit extensive variation around highlyconserved dinucleotides (GU and AG) at the 5′ and 3′ splicesites (29), branchpoint sequences (YUNAN consensus) thatusually are located 18–40 nt upstream of the 3′ splice site ofintrons play an important role in splicing efficiency (30). Intronsare removed via a two-step cleavage–ligation reaction in whichthe first step involves cleavage at the 5′ splice site with formationof an intron lariat at the branchpoint (31). Because dinucleotides(AG) at the 3′ splice site in the sixth intron show no change, wededuced that the skipping of the seventh exon might have beeninduced by the variation in the upstream sequence of the 3′splicing site in the sixth intron. Comparative analysis showed thatthe possible branchpoints differed between the ARF18 genesfrom zy72360 and R1, suggesting that the disruption of thebranchpoint in the sixth intron induced the loss of splicingfunction at the 3′ site in zy72360. In fact, exon skipping caused by

Table 1. Auxin-response genes including ARF, Aux/IAA, SAUR, and GH3 were regulated in NIL (SW) and R1

Gene ID NIL (SW) RPKM R1 RPKM Log2 ratio, NIL (SW)/R1 P value Tissue KEGG

Fna_59492 0.15657358 1.661168129 3.407285394 2.50E-11 Silique wall Auxin-response factorFna_70218 0.094105012 0.685181657 2.864143069 0.000106615 Silique wall Auxin-response factorFna_20825 3.337202011 12.77690534 1.936827523 1.91E-19 Silique wall Auxin-response factorFna_31741 1.331146322 4.087832828 1.618667034 1.53E-16 Silique wall Auxin-response factorFna_56595 3.535266632 8.669274747 1.294092278 5.43E-24 Silique wall Auxin-response factorFna_37605 3.460516441 1.642247956 −1.075315389 4.85E-08 Silique wall Auxin-response factorFna_74610 1.043493546 10.00968212 3.261902577 4.85E-17 Silique wall Auxin-responsive protein IAAFna_71197 4.669576781 11.58219301 1.310544739 5.09E-05 Silique wall Auxin-responsive protein IAAFna_15898 5.899374056 12.99909607 1.139777512 4.36E-09 Silique wall Auxin-responsive protein IAAFna_87374 1.750631568 0.001 −10.77365978 6.56E-09 Silique wall Auxin-responsive protein IAAFna_37713 2.682714694 0.079726258 −5.07249487 1.09E-09 Silique wall Auxin-responsive protein IAAFna_34616 76.66316166 29.54259831 −1.375736779 1.09E-61 Silique wall Auxin-responsive protein IAAFna_10794 10.18816752 4.279637301 −1.251334148 7.17E-07 Silique wall Auxin-responsive protein IAAFna_78680 21.84578229 9.782028116 −1.159149252 7.42E-12 Silique wall Auxin-responsive protein IAAFna_29766 14.05158922 6.495878046 −1.113136855 2.58E-07 Silique wall Auxin-responsive protein IAAFna_73932 10.4476168 5.061371644 −1.045573571 4.25E-10 Silique wall Auxin-responsive protein IAAFna_70892 1.099146535 5.335281168 2.279180568 7.32E-05 Silique wall SAUR family proteinFna_00244 6.846535654 15.01269593 1.132737 5.00E-05 Silique wall SAUR family proteinFna_10378 6.628624935 1.62966765 −2.024131849 3.92E-07 Silique wall SAUR family proteinFna_61491 12.17256579 3.13397625 −1.957567146 1.40E-11 Silique wall SAUR family proteinFna_23302 37.89295131 16.18333818 −1.227420282 7.11E-17 Silique wall SAUR family proteinFna_50955 15.95535292 7.375964749 −1.113136855 6.86E-05 Silique wall SAUR family proteinFna_74090 0.001 0.866117073 9.758418237 2.09E-09 Silique wall Auxin responsive GH3 gene familyFna_86647 0.08921644 4.083123343 5.516219766 7.25E-37 Silique wall Auxin responsive GH3 gene familyFna_02366 0.193511714 1.207684369 2.641750648 8.76E-07 Silique wall Auxin responsive GH3 gene familyFna_54159 9.730243207 52.38524289 2.428612686 1.74E-243 Silique wall Auxin responsive GH3 gene familyFna_61300 0.328727892 1.740711637 2.40471145 5.05E-09 Silique wall Auxin responsive GH3 gene familyFna_72281 0.627571431 2.14480541 1.772995181 9.26E-08 Silique wall Auxin responsive GH3 gene familyFna_74219 6.106369637 19.58386937 1.681279012 1.87E-20 Silique wall Auxin responsive GH3 gene familyFna_30176 2.085362661 0.166852676 −3.643651571 3.91E-14 Silique wall Auxin responsive GH3 gene familyFna_72776 2.073861386 0.359520294 −2.528174354 6.07E-08 Silique wall Auxin responsive GH3 gene familyFna_59492 8.306 3.469 −1.260 1.41E-17 Seed Auxin-response factorFna_17490 0.001 1.996 10.963 2.44E-09 Seed Auxin-response factorFna_77259 1.739 0.584 −1.574 5.80E-06 Seed Auxin responsive GH3 gene familyFna_02366 2.068 0.715 −1.533 1.35E-06 Seed Auxin responsive GH3 gene familyFna_55303 0.747 0.058 −3.689 9.63E-07 Seed Auxin responsive GH3 gene familyFna_72776 1.558 0.458 −1.767 6.92E-05 Seed Auxin responsive GH3 gene family

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an insertion in the sequence upstream of the 3′ splice site of theintron has been reported previously (32, 33).

ARF18 Acts as a Repressor of Auxin-Responsive Genes by FormingHomodimers. By affecting cell division, cell growth, or cell dif-ferentiation, auxins are involved in controlling virtually all as-pects of plant growth and development (34, 35). Auxin-responsefactors as transcriptional regulators mediate auxin responses bybinding to the auxin-response elements in the promoter region ofearly auxin-response genes and mediate auxin gene-inductionresponses (36, 37). In Arabidopsis, the ARF proteins are encodedby a large family of genes that includes 23 members, and eachmember is thought to play a central role in various auxin-medi-ated developmental processes (38). The MR determines theARF protein functions as an activator or repressor of auxin-responsive genes (39). Phylogenetic analysis of Arabidopsis ARFgenes has revealed that ARF18 belongs to the class that alsoincludes ARF1, ARF2, ARF9, and ARF11 (39). Of these, ARF1and ARF2 function as transcription repressors (40). The presentstudy has determined that ARF18 also functions as a repressor.Although the MR is partially deleted in zy72360-ARF18, its in-hibitory activity does not decrease significantly compared withthat observed in R1-ARF18.Transcription factor dimerization is a common element of the

transcriptional control mechanism. Most of the ARF genes areno exception, and homodimerization also is required for its bi-ological function (29). Dimer interface contacts include hydro-phobic interactions between several highly conserved residues.Based on the reports on ARF1 and ARF5, ARF dimerization isinduced through the DBD (41). The present study performedyeast hybridization assays to determine that R1-ARF18 interactswith itself, suggesting that ARF18 functions as a homodimer.On the other hand, in zy72360-ARF18 some highly conservedresidues were located within the 55 deleted amino acids, re-sulting in a failure in dimerization and subsequently loss ofbinding activity.

Functional Differentiation of BnaA.ARF18.a and BnaC.ARF18.a in B.napus. As a tetraploid species, the ARF18 gene of B. napus oc-curs as two homologs, namely BnaA.ARF18.a and BnaC.ARF18.a(BnaC08g31690D), which are located on chromosomes A9 andC8, respectively. It is assumed that when a gene is duplicated, thetwo duplicates are almost identical in sequence and have fullredundancy (functional overlap) (42). In B. napus, based ontranscriptome sequencing data, we observed a higher level ofBnaA.ARF18.a expression than of BnaC.ARF18.a in the siliquewall. The opposite trend was observed in the seed (Fig. S6A).Promoter sequence comparison also indicated major differencesbetween BnaA.ARF18.a and BnaC.ARF18.a (Fig. S6B). InA. thaliana, more than half of the duplicates were found to havedivergence in expression levels (43). Divergence in the expres-sion states may lead to new expression states (neofunctionalization),partitioning of ancestral functions (subfunctionalization), or loss ofexpression state that leads to pseudogenization (44). Therefore, webelieve that subfunctionalization might have been generated be-tween BnaA.ARF18.a and BnaC.ARF18.a. As expected, based onthe transcriptome data, the expression level of BnaC.ARF18.a in C8did differ in the NIL (SW) and R1 lines. In general, BnaA.ARF18.aplays indispensable roles in the development of the silique wallthat could not be replaced by BnaC.ARF18.a in rapeseed.

Silique Wall Genes Exert Maternal Control of SW in B. napus. Thepresent study showed that the ARF18 gene regulated SW byaffecting the development of the silique wall. In fact, severalgenes have been reported to affect SW by maternal organs. InArabidopsis, the most commonly reported genes, e.g., TTG2,AP2, ARF2, and DA1, influence seed size by affecting cellexpansion and/or cell proliferation in the integuments or seed

coat (1). Therefore the integument or seed coat plays a keyrole in the maternal control of seed size. In rice, the loss ofGW2 function increases cell number, resulting in a largerspikelet hull, and increases the grain milk-filling rate, which inturn enhances grain weight (13).In rapeseed, the silique wall not only is an important sink

organ that assimilates the carbohydrates synthesized in the veg-etative parts of the plant such as leaves and stem but also is animportant source organ that provides photosynthates to the de-veloping seeds (45). Because the functional leaf area declinesrapidly during seed filling, silique wall photosynthesis is the mainsource of nutrition for seed growth (46). Moreover, signalsoriginating from the silique wall coordinate seed filling andregulate reallocation of reserves (45). SL has been significantlycorrelated with SW variation (47). By determining the surfacearea of the silique wall, SL may affect SW by regulating theaccumulation of photosynthates.The present study has determined that ARF18 is up-regulated

in the silique wall and down-regulated in the seed, suggestingthat this gene might regulate SW indirectly by affecting siliquedevelopment. Further studies showed that the loss of ARF18activity induced the development of a longer silique by acceler-ating cell expansion in the silique wall. This change has resultedin more photosynthates in the longer silique which then enter theseed, generating larger seeds.

MethodsPlant Materials. Rapeseed line zy72360 was crossed with line R1. The resultantF1 plants were self-pollinated to produce F2 seeds, which were backcrossedwith R1 plants to produce BC4F1 seeds. We selected several plants in whichthe region around the SW locus was heterozygous and almost all other re-gions were homozygous for R1. These plants were used to develop segre-gating populations for fine mapping and high-resolution mapping of SW byrepetitive backcrossing and marker-assisted selection. From the BC4F2 gen-eration, we developed a nearly isogenic line with a small zy72360 chromo-somal region containing the SW locus in the R1 genetic background (4,000lines). The association population consisted of a panel of 380 inbred rapeseedlines. The association population was arranged in a randomized completeblock design with three replicates.

Trait Measurement. Seeds from themain inflorescence were used to representSW in each plant. The average SW of 10 plants from the center of each plotwas used to represent SW in the association population.

Fine Mapping and High-Resolution Mapping. A BC4F2 population was used forfine mapping of SW, which was based on rough mapping the F2 population.To perform SW QTL analysis, six molecular markers were developed in thetarget region containing the SW locus to detect recombinants in the 4,000BC4F2 plants. To determine further the location of the recombination nearestto the SW locus, we developed 11 markers on the basis of the parental se-quence and determined the genotypes of the recombinants using thesemarkers. The BC4F2 progeny derived from recombinant plants were used toscreen for recombination products. Themolecularmarker primers are listed inTable S1.

Gene Cloning and Transgenic Analysis. Total RNA was extracted from variousplant tissues in NILs and transgenic lines. Seven candidate genes were namedaccording to Chalhoub et al. (22). The upstream regulatory and coding re-gions of seven genes were cloned with the cDNAs originating from the R1and zy72360 lines and were sequenced. Genes with existing SNPs and indelsbetween the parents were ligated into the TOPO vector (Invitrogen) andtransferred into the plant binary vector pEarleyGate-100 (Invitrogen). Allconstructs were introduced into Agrobacterium tumefaciens strain EHA105and were transferred into Col-0 Arabidopsis by floral dip. The emptypEarleyGate-100 vector also was transformed into Arabidopsis as a control.The genome sequence including the ARF18 gene and its upstream promotersequence originating from the R1 line was cloned and ligated into thePbi121 vector (Invitrogen). The construct was transferred into A. tumefaciensstrain EHA105 and was transformed into the rapeseed line zy72360 by thehypocotyl transgenic system. The primers used in this experiment are listedin Table S2.

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Protein Subcellular Localization and Intron Splicing Identification. To in-vestigate the cellular localization of ARF18, a 35S GFP-ARF18 fusion construct(ARF18 from R1) was transfected into the tobacco leaves based on themethod of Sparkes et al. (48). The sixth intron in the ARF18 genome se-quence from R1 and the corresponding sequence from zy72360 were clonedand inserted into the GFP gene by fusion PCR. The two reconstructive GFPgenes were ligated into the TOPO vector and then were transferred into theplant binary vector, pEarleyGate-100. The splicing function of introns wasdetected by using a tobacco transient expression system. Primers used in thisexperiment are listed in Table S4.

Detection of Inhibitory Activity and Dimers by Using a Yeast Hybrid System.The inhibitory activity of ARF18 was assessed using a yeast hybrid system. TheARF18 CDS from zy72360 and R1 and VP16 were constructed into PGBKT7 asbait vectors and were cotransformed with PGADT7 into yeast strain MaV203(Invitrogen). VP16 was used as positive control, and the empty vectorsPGBKT7 and PGADT7 were used as negative control. Yeast growth was an-alyzed at 30 °C on the SD agar plates lacking Leu, Trp, and His and con-taining various concentrations of 3-amino-1,2,4-triazole as indicated. Fordimer detection, the ARF18 CDs from zy72360 and R1 were fused to theBD (PGBKT7) and AD (PGADT7), respectively. pGBKT7-53 and pGADT7-Twere used as positive controls. pGBKT7-lam and pGADT7-T were used asa negative controls. All the vectors were transformed into yeast stainAH109 (Clontech).

Detection of Binding Activity by Using Electrophoretic Mobility Shift Assays.zy72360-ARF18 and R1-ARF18 CDS were subcloned into the expression vectorpGEX6P-1 (Amersham Pharmacia Biotech). The GST fusion protein wasextracted from bacteria using glutathione-tagged Sepharose (GE Health-care). The eluted GST fusion protein was assayed by SDS/PAGE. To test ARF18binding activity, a 5′ biotin-labeled DR5(7×) probe used in the electropho-retic mobility shift assay (EMSA) was synthesized (Genecreate). EMSA wasperformed with a LightShift Chemiluminescent EMSA kit (Pierce). Briefly,protein extracts in 20 μL of a 1× binding buffer with 2.5% (vol/vol) glycerol,5 mMMgCl2, 50 ng/L poly(dI-dC), 0.05% Nonidet P-40, 1 mMDTT, and 20 fmolprobe were incubated at room temperature for 15 min. For STAT3 supershiftanalysis, an anti-STAT3 polyclonal antibody (Santa Cruz; 1 g per reaction)was incubated with the nuclear proteins on ice for 20 min before the ad-dition of the labeled oligonucleotides. Reaction products were separated byelectrophoresis [5% acrylamide (29:1 acryl/bis)] in 0.5 Tris/borate/EDTA. Afterelectrophoresis, the protein–DNA complexes were transferred onto nylonmembranes and detected by using chemiluminescence.

Expression Pattern Analysis. Total RNA was extracted from rapeseed tissues,including seed, root, stem, leaf, bud, and the silique wall, and from theArabidopsis silique at 3 daf using the RNeasy Plant Mini kit (Qiagen). Thereverse transcription reaction was performed using the First Strand cDNASynthesis Kit for RT-PCR (Takara). Epsin N-terminal homology (ENTH) andArabidopsis β-actin1 (At2g37620) were used as references (49, 50). Datawere expressed as the mean of three biological replicates ± SD. The ARF18

promoter was ligated into pCXGUS-P. The construct was introduced into A.tumefaciens strain EHA105 and transferred into Col-0 Arabidopsis. GUS ac-tivity was visualized by staining all tissues from homozygous transgenic linesovernight in an X-Gluc solution (Biosharp), and the tissues then were clearedin 75% (vol/vol) ethanol. The tissues were imaged directly or captured undera microscope. The primers used are listed in Table S4.

Cell Length Observation. Twenty daf rapeseed siliques were sampled and fixedin 50% ethanol, 5% glacial acetic acid, and 5% formaldehyde for 16 h,dehydrated in an ethanol series, and photographed with an Olympus com-pound microscope.

Protoplast Transformation and Transient Expression Assays. Protoplast fromleaves was isolated according to the tape-Arabidopsis sandwich methoddescribed by Wu et al. (51). Plasmid DNA was prepared using a QIAfilterPlasmid Midi Kit (Qiagen). The coding sequence of ARF18 under the controlof a 35S promoter was amplified from the pEarleyGate-100 vector andsubcloned into a PUC19 vector. The DR5GUS reporter construct comprisedseven copies of DR5 cloned upstream of a −46 CaMV 35S promoter with aTMV 5′ leader (40). The efficiency of transfection was standardized using aCaMV 35S promoter–LUC construct, which shows no response to auxin (52).Three plasmids expressing a regulatory effector, a specific reporter, and atransfection control reporter were used at a ratio of 5:4:1. Ten microgramsof the plasmid were introduced into Arabidopsis protoplasts by PEG/calcium-mediated transformation. After the protoplasts were cultured and har-vested, LUC and GUS assays were performed (53). All transfections wereperformed in triplicate, and at least three independent transfection assayswere performed with protoplasts.

Transcriptome Analysis. Total RNAs from siliquewall (16 daf) and seed (25 daf)were sent to Beijing Genomics Institute, and the transcriptomes were se-quenced on an Illumina HiSeqTM 2000 platform. Gene-expression levels werecalculated using the reads per kilobase per million (RPKM) method (54). Toscreen for DEGs, a P value corresponding to a differential gene-expressiontest based on the method introduced by Audic and Claverie was used (55).False-positive (type I errors) and false-negative (type II) errors were correctedusing the false-discovery rate (FDR) method (55). Finally, we chose FDR≤0.001 and the absolute value of log2 ratio ≥1 as the thresholds to judge thesignificance of the differences in gene expression in the silique wall and seedbetween the NIL (SW) and R1 lines. The DEGs were submitted to the websiteof Bio-Analytic Resource for Plant Biology program (bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi) for MapMan analysis.

ACKNOWLEDGMENTS. This study was supported by Grants 2015CB150200and 2011CB109300 from the National Key Basic Research Program of China,Grant 2013AA102602 from the National High Technology Research and De-velopment Program of China, Grant 31201241 from the National NaturalScience Foundation of China, and Grant 2014020101010066 from the Ap-plied Basic Research Project in Wuhan.

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