Fine mapping of a major QTL for awn length in barley using a multiparent mapping population

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1 DOI 1.17/s y ORIGINAL ARTICLE Fine mpping of mjor QTL for wn length in rley using multiprent mpping popultion Corinn B. Liller 1 Agth Wll 1,2,3 Mrtin P. Boer 4 Pete Hedley Mlolm Muly Sieglinde Effgen 1 Mri von Korff 2,3 G. Wilm vn Esse 1,3 Mrten Koornneef 1,3,6 Reeived: 4 July 216 / Aepted: 29 Septemer 216 / Pulished online: 12 Otoer 216 The Author(s) 216. This rtile is pulished with open ess t Springerlink.om Astrt Key messge Awn length ws mpped using multiprent popultion derived from v. Morex nd four wild essions. One QTL ws fine mpped nd ndidte genes were identified in NILs y RNA-seq. Astrt Brley wns re photosynthetilly tive nd ontriute to grin yield. Awn length is vrile mong oth wild nd ultivted rley genotypes nd mny mutnts with ltertions in wn length hve een identified. Here, we used multiprent mpping popultion derived from v. Morex nd four genetilly diverse wild rley lines to detet quntittive trit loi (QTLs) for wn length. Twelve QTLs, distriuted over the rley genome, were identified with the most signifint one loted on hromosome rm 7HL (QTL AL7.1). The effet of AL7.1 ws onfirmed using ner isogeni lines (NILs) nd finempped in two independent heterogeneous inred fmilies Communited y T. Komtsud. Eletroni supplementry mteril The online version of this rtile (doi:1.17/s y) ontins supplementry mteril, whih is ville to uthorized users. * Mri von Korff korff@mpipz.mpg.de * G. Wilm vn Esse vnessse@mpipz.mpg.de * Mrten Koornneef koornneef@mpipz.mpg.de 1 2 Deprtment Plnt Breeding nd Genetis, Mx Plnk Institute for Plnt Breeding Reserh, Crl von Linné Weg 1, 829 Cologne, Germny Institute of Plnt Genetis, Heinrih Heine University Duesseldorf, Universitätsstr. 1, 422 Düsseldorf, Germny to <.9 intervl. With exeption of smll effet on grin width, no other trits suh s plnt height or flowering time were ffeted y AL7.1. Vrint lling on trnsripts otined from RNA sequening reds in NILs ws used to nrrow down the list of ndidte genes loted in the intervl. This dt my e used for further hrteriztion nd unrvelling of the mehnisms underlying nturl vrition in wn length. Introdution The wn, developing s long slender extension of the lemm in grss spikelet, is redued lef (Grundher 1963). In wild speies, their funtion is thought to ssist seed dispersl nd protetion (Elum et l. 27). The wns of the erel rops rley (Hordeum vulgre), whet (Tritium estivum), rye (Sele erele) nd ot (Aven stiv) hve similr ntomy: they hve tringulr shpe with the se direted towrds the rhis; three vsulr undles; nd two long strnds of hlorenhym, whih ontriute to the net photosyntheti tivity of the 3 Cluster of Exellene in Plnt Sienes (CEPLAS), Heinrih-Heine-Universität Düsseldorf, Universitätsstr. 1, 42 Düsseldorf, Germny 4 Biometris, Plnt Reserh Interntionl, Wgeningen University, Droevendlsesteeg 1, 678 PB Wgeningen, The Netherlnds The Jmes Hutton Institute, Invergowrie, Dundee DD2 DA, Sotlnd, UK 6 Lortory of Genetis, Wgeningen University nd Reserh, Droevendlsesteeg 1, 678 PB Wgeningen, The Netherlnds

2 27 spike (Grundher 1963; Li et l. 26). A numer of loi ffeting wn length in rley re desried nd tegorised in two min phenotypi groups: (1) loi tht ffet other trits suh s plnt height, lef shpe, nd spike length; nd (2) loi tht minly ffet wn length or rittleness (Frnkowik nd Lundqvist 212; von Bothmer et l. 23). Severl genes from the first group hve reently een shown to e involved in rssinosteroid (BR) iosynthesis or signlling (Dokter et l. 214). The short wn 2 (lks2) gene, whih elongs to the seond group [lleli to reviristtum (ri) -d nd unrnhed style 4 (us4)] is loted on the long rm of hromosome 7H nd ws found to enode for SHORT INTERNODES (SHI)-type trnsription ftor (Yuo et l. 212). In generl, wned rley genotypes outyield wnless isogeni lines, espeilly under wrm nd dry onditions (Bort et l. 1994; Grundher 1963; Shren et l. 1983, Reetzke et l. 216). Within the spike the wns re the min ontriutors to the photosyntheti tivity (Aee et l. 29). Consequently, the sene of wns redues seed weight nd size euse of redution in strh ontent (Bort et l. 1994; Grundher 1963; Jing et l. 26; Li et l. 26; Shren et l. 1983). Given the potentil impt of wn length on yield in rley ultivrs, it is importnt to identify novel QTLs nd genes tht ffet this trit. Previous geneti studies desried QTLs for wn length on hromosome 3H (Chen et l. 212; Gyenis et l. 27; Wng et l. 21), possily o-lolizing with either ri- or semi-rhyti (uzu) (HvBRI1); on 6HS (Sun et l. 211); lose to the row type lous six-rowed spike 1 (Vrs1) on 2H (Smeri et l. 26), where the wnless lous Lks1 hs een mpped; on 7H (Smeri et l. 26), possily o-lolizing with either ri-f or ri-n (Druk et l. 211; Frnkowik nd Lundqvist 212); nd on 7HL whih is ssoited with DIMINUTO (HvDIM) (Dokter et l. 214). The mjority of these studies hve investigted the geneti sis of wn length differenes in ultivted rley. Sustntil vrition in wn length, however, is lso oserved in wild rley (Bdr et l. 2) whih is hrterised y higher geneti diversity thn ultivted rley nd thus represents vlule soure to improve the elite rley genepool (Russell et l. 24). Therefore, we generted multiprent interross popultion etween wild nd ultivted rley to mp nd identify the geneti sis for differenes in wn length. The dvntges of using multiprent popultions ompred to lssil i-prentl popultions re tht: (1) severl lleles nd their intertion with different kgrounds n e nlysed simultneously nd (2) QTLs n e deteted with higher preision nd resolution due to the inresed level of reomintion (Cvngh et l. 28). We generted multiprent mpping popultion y interrossing v. Morex, three ssp. spontneum from the Fertile Cresent nd one ssp. griorithon from the Tietn highlnds in Chin. After one round of krossing to the reipient Morex to redue the effet of deleterious wild lleles, the resulting BC1F1 ws interrossed in ll possile omintions to generte y single seed desent 6 supopultions with 13 1 RILs eh. The QTL nlysis resulted in the identifition of mjor QTL for wn length on 7HL where the wild rley llele deresed wn length. Fine mpping of this lous using independent heterozygous inred fmilies (HIFs) led to delimittion of the QTL lous to <.9. The position of the lous ws verified using mpping of the vrints etween RNA sequening reds otined from two ner isogeni lines differing only in the QTL region. The mjor lous on 7HL ws not ssoited with ny other trits nor did it mp to previously identified wn length genes loted in this region suh s HvLks2 or HvDIM. Mterils nd methods Plnt mteril The multiprent reominnt inred Line (RIL) mpping popultion ws onstruted through rosses etween the rley ultivr Morex nd four wild rley lines seleted for lrge geneti vrition (Bdr et l. 2). Three wild rley genotypes originted from the estern nd western Fertile Cresent, the entre of rley domestition [ssp. spontneum: HID 4 (A), Irq; HID 64 (B), Turkey; nd HID 369 (C), Isrel nd one rley genotype originted from the Tietn highlnds in Chin (ssp. griorithon: HID 382(D)]. Eh wild rley line ws rossed to v. Morex (ssp. vulgre, USA) nd the F1 hyrids were krossed to Morex one. These kross genertions were interrossed with eh other t every possile omintion, resulting in six independent supopultions. From these interrosses, 916 reominnt inred lines (RILs) with lines per supopultion were mde y single seed desent (SSD) (Online Resoure 1) for six genertions. The resulting F6 genertion ws used for genotyping nd QTL mpping. Plnt growth onditions For QTL mpping of wn length, the F6 genertion ws grown with four replite plnts per RIL under outdoor onditions. Plnts of the multiprent RIL popultion were sown mid-jnury 211 in 96-well trys, germinted under greenhouse onditions nd pled outdoors fter the first lef emerged (end of Jnury). Plnts were trnsferred (mid-fe) to 1 L pots, eh pot ontining one plnt. Pots were rrnged in 1 rows sped 1 m prt, eh row ontined 61 pots with distne of 1 m. The entire plot ws

3 divided into two loks, two replite plnts per genotype were rndomised together within eh lok. The plot ws surrounded y one row of order pots ontining elite rley ultivrs. The plnts were irrigted using sprinkler root. For QTL onfirmtion nd dditionl experiments performed in 212, 213 nd 214 the plot ws set-up using 12 L pots s desried previously (Liller et l. 21) only in this study the plot ws divided into two loks. A ustom-mde pet nd ly soil mixture (EinheitsErde ED73 Osmoote, Einheitserdewerke Werkvernd e.v., Sinntl-Altengronu, Germny) ontining long term fertilizer ws used for the outdoor nd greenhouse grown plnts. Greenhouse experiments were performed in n ir-onditioned greenhouse under long dy onditions (16 h, 22 C dy; 8 h, 18 C night) using either.3 or. L pots. In ll experiments, dditionl fertilizer nd/or pestiides were pplied to the plnts when neessry. Awn length mesurement All spikes used for wn length mesurements were olleted t mturity. The spikes were dried in drying inet (Hereus) t 37 C for t lest 3 dys. Awn length (length of the dethed longest wn of the spike) ws mesured using 2 mm digitl lliper (Mitutoyo Deutshlnd, Neuss) The popultion involves rosses etween 2-rowed HID lines nd 6-rowed Morex, nd thus lso segregtes for row-type. In the six-rowed plnts, the wn length my differ etween the entrl spikelet nd the lterl spikelet. Therefore, only the wn length of the entrl spikelet ws used for QTL mpping. At lest four independent iologil replites (n 2 tehnil replites) were used to otin n verge wn length used for QTL nlysis. DNA extrtion DNA ws extrted from fresh or freeze-dried lef mteril using the Qigen BioSprint following the mnufturer s protool (Qigen, Hilden, Germny). DNA ws eluted in 1 µl supplied uffer AE nd onentrtion ws mesured using the NnoDrop spetrophotometer (PEQLAB, Erlngen, Germny). QTL mpping One individul of eh RIL ws genotyped with seletion of 384 BOPA single-nuleotide polymorphism (SNP) mrkers from the rley Consensus Mp (Close et l. 29) t the Jmes Hutton Institute (Dundee, Sotlnd, UK) using the Illumin Golden Gte Assy system. The mrkers were ssigned to their positions on the POP- SEQ mp (Ariyds et l. 214). The SNP mrkers were used to lulte the geneti preditors, i.e. the proilities 271 tht prtiulr RIL t given lous hs the genotype of one of the five founders. The founder to offspring proilities were lulted using Hidden Mrkov Model (Riner 1989; Zheng et l. 21), n extension of the inheritne vetor pproh s desried in Hung et l. (211). The QTL-mpping ws done using liner mixed models in Genstt 17 (VSN interntionl). In the first step of the QTL nlysis we used genome sn, known s simple intervl mpping (SIM) (Lnder nd Botstein 1989). Let y ik e the wn length for RIL i in ross k, then the following model n e given: y ik = µ k + x T ikl β l + ε ik where µ k is fixed effet, the vetor x ikl = (x ikl1, x ikl2,..., x ijl ) T ontins the proilities tht RIL i in ross k for lous l hs the genotype of wild rley line f (f = 1,,4). The vetor β l is 4-dimensionl vetor of rndom founder effets orresponding to lous l. Finlly, ε ik is the residul error for RIL i in ross k, with ross-speifi vrine V ( ε ik ) = σ 2 ε,k. In the seond step, we rn genome sn using multi- QTL model djusting for kground QTLs. The kground QTL with the strongest effet ws first dded, then new QTL-sn ws performed to detet the seond strongest QTL, nd this proedure ws repeted until no new QTLs were deteted ove the QTL signifine threshold of log1(p) = 3.2 (Hung et l. 211). Eh kground QTL ws modelled s rndom effet, nd the model is n extension of Eq. (1): y ik = µ k + xik T β + xt ikl β + ε l ik C where C is set of o-ftors to orret for QTLs elsewhere in the genome, nd l is the puttive QTL. Co-ftors within 1 of the puttive QTL were exluded. In the finl step of the QTL-nlyses ll QTLs deteted with model (2) were inluded in the model: y ik = µ k + q Q x T ikq β q + ε ik where Q is the set of seleted QTLs. For eh QTL the min log1(p)-vlues, the founder effets nd the stndrd errors were estimted. Epistti intertions etween the 12 QTL were tested pirwise ording to Hung et l. (211). QTL onfirmtion nd fine mpping For QTL onfirmtion, offspring of RILs heterozygous t the most signifint QTL (QTL AL7.1) in the F 6 genertion ws evluted. These RILs represent Heterogeneous Inred Fmilies (HIFs) s defined y Tuinstr et l. (1997) nd were genotyped with mrkers losest to the puttive (1) (2) (3)

4 272 QTL. For the mjor wn length QTL on hromosome rm 7HL (AL7.1) HIFs were grown under outdoor onditions in 212. Genotyping ws rried out with the mrkers 3_1489 nd 2_117 whih re loted lose to the puttive wn length QTL. Supopultions were revited sed on the prents segregting t AL7.1, defining Morex (M) nd lleles of the wild rleys HID 4 (A), HID 64 (B), HID 369 (C) nd HID 382 (D) resulting in the possile llele omintions AM, BM, CM nd DM. The selfed progeny of, respetively, 47 heterozygous plnts of the HIFs BM1 nd 42 of DM1 were used for fine mpping. Reominnts were seleted using the flnking mrkers 3_93/1_999 for BM1 nd 2_483/1_999 for DM1 resulting in 47 nd 13 reominnt lines, respetively. All reominnt nd seven non-reominnt plnts per genotype were trnsplnted to lrge pots, genotyped with dditionl newly developed CAPS mrkers (Figs. 4, ) loted within the region of AL7.1 nd grown to mturity. The HIFs were fixed for the other QTLs, thus the BM1 nd DM1 HIFs used for fine mpping only segregted t AL7.1. For onfirmtion nd further delimittion of the QTL intervl, homozygous lines were seleted from the (heterozygous) reominnt plnts of popultions BM1 nd DM1 nd phenotyped for wn length under outdoor onditions (Online Resoure 2). Linkge mps for hromosome 7HL in the BM1 nd DM1 popultions were onstruted using JoinMp 4 (Kyzm B.V., Wgeningen, The Netherlnds, Polymorphi SNP mrkers were designed s CAPS/dCAPS mrkers (Koniezny nd Ausuel 1993; Neff et l. 1998) sed on SNPs desried y Close et l. (29), the Brley Consensus Mp nd the iselet hip ( CAPS/dCAPS sites were identified using dcaps finder ( html) (Neff et l. 1998). A list with primer sequenes used for fine mpping n e found in Online Resoure 3. PCR ws performed in totl retion volume of 1 µl ontining 2 4 ng genomi DNA, 1 supplemented uffer,. µm forwrd nd reverse primer, 1 µm dntps,.2 U Tq polymerse (Ampliqon, Odense, Denmrk) nd % glyerol or 2 mg BSA if neessry. Amplifition ws done using touhdown (TD) PCR with n initil 3 min denturtion t 9 C, 1 TD yles (3 s t 9 C, 3 s TD ( 1 C per yle) from 67 C to 2 C nd 3 s t 72 C) nd 2 yles with the 2 C nneling temperture. PCR produts were digested with the respetive restrition enzyme (New Englnd Biols, Ipswih, MA, USA) ording to mnufturer s instrutions. Bnds were visulised on 2. or 4% grose gel ontining 4 1 vol/vol % ethidium romide. RNA extrtion nd sequening Homozygous ner isogeni lines (NILs) derived from the (reominnt) DM1 HIF line -e3r hroring either the HID 382 (D) or Morex (M) llele t the QTL position were grown in the greenhouse under long dy onditions (16 h, 22 C dy; 8 h, 18 C night). Shoot pies were stged ording to the Wddington sle (Wddington et l. 1983) nd pex tissue ws olleted t stge. y pooling ten pies from homozygous D or M plnts nd immedite freezing in liquid nitrogen. RNA ws extrted using the Qigen RNesy Plnt Mini Kit (Qigen, Hilden, Germny) following the mnufturers protool nd stored t 8 C fter DNse tretment (Amion, Crlsd, USA). The qulity of the RNA ws evluted efore lirry preprtion using ionlyzer (Agilent). The Illumin TruSeq lirries were prepred using the mnufture s protool (version 2, Illumin). Single end sequening ws performed on the HiSeq 2 (Illumin ) pltform. For eh lirry minimum of 1 million reds were generted y multiplexing eight lirries. The initil qulity ontrol of the rw reds ws performed using the FstQC softwre. Reds were trimmed using the Trimmomti pltform, emedded within the trinity pipeline (Grherr et l. 211; using the following defult riteri: phred 33, leding nd triling 3, sliding window 4:1 nd minimum red length of 36. Sequene lignments were performed y Bowtie2 (version 2.1.; owtie2/index.shtml) using merged dtset of High Confidene (HC) nd Low Confidene (LC) predited rley genes (The Interntionl Brley Genome Sequening Consortium 212) s referene. SAMtools (Li et l. 29, version 1.2; ws used for BAM formt onversion, sorting nd indexing nd red duplite removl ws onduted using the Pird ommnd-line tool MrkDuplite (version 1.11; ommnd-line-overview.html). To orret mislignments, the Genome Anlysis ToolKit (GATK, version 3.1) religner ws used with reommended settings. Vrints were otined using the GATK UnifiedGenotyper pltform (minimum phred sore of 3). Refinement of the vrints ws performed with the GATK Vrint Filtrtion tool (Fisher Strnd vlues FS >3.; Qul By Depth vlues QD <2.) to redue flse positive SNPs. Resulting SNP lls were kept for further nlysis if they pssed the filtrtion step nd their red overge exeeded four reds. Trnsripts ontining filtered homozygous SNPs were mpped to their respetive position long the POPSEQ mp of rley with R (The Interntionl Brley Genome Sequening Consortium 212). For expression nlysis, the reds were ligned to the high onfidene

5 273 Tle 1 Awn length of prentl lines nd ll RILs of the multiprent RIL popultion Prentl lines All RILs H 2 Morex HID 4 HID 64 HID 369 HID 382 Averge Rnge Awn length (mm) 164 ± ± ± ± ± ± Letters nd indite signifint differenes (p.) etween the prentl lines, determined with One-wy ANOVA using Tukey HSD s post ho test Averge wn length vlues mm ± STDEV, for prentl lines n 6 wns (HC) nd low onfidene (LC) gene set s desried ove nd only in this se, the red duplites were not removed from the BAM file. Rw ounts were extrted from the BAM file using Slmon (Ptro et l. 21). Differentilly regulted reds were lled with flse disovery rte of. using the R ioondutor pkge limm-vroom (Rithie et l. 21). All RNA sequening dt hs een sumitted to the GEO dtse (GSE8338). For phylogeneti studies, the mino id sequene of ndidte MLOC_ ws used s query string to perform sequene lst ginst the NCBI nr dtse of following orgnisms: Hordeum vulgre (txid:413), Oryz stiv (txid:43), Brhypodium disthyon (txid:1368), Sorghum iolor (txid:48), Aridopsis thlin (txid:372). Puttive orthologues proteins were identified with utoff of e vlue e 1. The multiple lignment of hosen sequenes ws performed with CLUSTAL O (1.2.2) nd phylogeneti tree ws inferred using the Neighor-Joining method in MEGA. Effet of wn length on grin filling Plnts of v. Morex were grown under outdoor onditions. Upon heding, the plnts were rndomly ssigned to three groups of equl size (n = 7) nd ll emerging spikes of these plnts were treted s follows (ltest 3 dys fter heding): (1) only the very tips of the wns were removed with sissors (full wn), (2) the wns were ut in hlf (hlf wn), or (3) the wns were ut off ompletely (no wn). This tretment ws repeted with every emerging spike until the lst spike hd emerged. The plnts were then llowed to reh full mturity without further disturne nd hrvested individully. The lened seed (three tehnil replites of ml per iologil replite) ws mesured with the MARVIN Seed Anlyser (GTA Sensorik, Neurndenurg, Germny) to otin seed width, length nd re nd thousnd grin weight (TGW). To ssess the effet of the differenes in wn length oserved in the HIFs, seed from homozygous individuls from two mpping fmilies (BM1 nd DM1) otined in the 212 nd 214 outdoor experiments ws lened nd seed trits were lso mesured with the MARVIN Seed Anlyser. Results frequeny 2 A 1 1 B D M C Awn length [mm] Fig. 1 Distriutions of wn length in the mpping popultion. Histogrms of verge wn length of RILs from the mpping popultion. The entire mpping popultion onsists of 916 RILs. Brs mrk verges of prentl lines (A HID 4, B HID 64, C HID 369, D HID 382, M morex) QTL mpping of wn length in the multiprent RIL popultion The multiprent RIL popultion used in this study ws onstruted from three lines of ssp. spontneum originting from different prts round the Fertile Cresent (Irq, Turkey nd Isrel) nd one line of ssp. griorithon from the Tietn highlnds (Chin) rossed with the v. Morex. To evlute if the prents of this popultion differ in wn length we mesured wn length of plnts grown under outdoor onditions. The prents of the multiprent mpping popultion differed from one nother in wn length, with HID 4 (Irq), HID 64 (Turkey) nd HID 382 (Chin) hving signifintly shorter wns when ompred to v. Morex (USA) nd HID 369 (Isrel) (Tle 1). In the whole mpping popultion the trit showed norml distriution rnging from 73.4 to 23. mm per RIL, inluding trnsgressive segregtion in oth diretions nd hd rod sense heritility of H 2 =.7 (Figs. 1, 2; Tle 1, Online Resoure 4). Genotyping with 384 BOPA single-nuleotide polymorphism (SNP) mrkers resulted in totl of 36 polymorphi mrkers in the entire popultion, with out 2 23 polymorphi mrkers in eh of the supopultions (Online Resoure ). A totl of 12 signifint QTLs (LOD >3) for

6 H 2 2H Fine mpping of the mjor QTL on 7H -log1(pvlue) -log1(pvlue) -log1(pvlue) -log1(pvlue) AL AL3.1 3H 1 1 H AL H AL.2 AL7.1 wn length were identified using the whole mpping popultion (Tle 2, Online Resoure 6). The mjor QTL for wn length is loted t position on hromosome 7H ( log1(p) = 71.2). The llele effets of the four wild rley prents when ompred to Morex were determined for eh QTL. From eh prent, QTL with positive s well s negtive effets were identified (Tle 2). No intertions etween ny of the 12 QTL were signifint fter multiple testing orretion nd we onlude tht the loi t in n dditive wy. -log1(pvlue) -log1(pvlue) AL2.1 AL2.1 AL2.3 4H AL H Fig. 2 QTL mpping for wn length. Results of multiple-qtl mpping (MQM) of wn length on the multiprent RIL popultion. Lines show p vlues over the seven rley hromosomes. The horizontl line indites the onfidene threshold ( log1(p) = 3.2) -log1(pvlue) AL6.1 AL6.2 AL4.2 AL7.1 on hromosome rm 7HL ws the most signifint QTL, therefore, it ws seleted for fine mpping. The mjor dvntge of heterozygous inred fmilies (HIFs) is tht it enles fine mpping of the QTL while ompring the trit in n otherwise isogeni kground (Tuinstr et l. 1997). Two HIFs segregting for ultivted rley (Morex, M) nd lleles of the wild rleys HID 4 (A), HID 64 (B) nd HID 382 (D) were seleted for eh llele omintion (AM, BM, DM). HID 369 (C) ws not inluded in fine mpping, s it ws not signifintly different in wn length from Morex (Tle 1). The HIFs were fixed for the wild llele of vrs1 resulting in 2-rowed spike. The segregting fmilies were grown under outdoor onditions nd genotyped with mrker 3_1489 loted lose to the pek of AL7.1. The two fmilies segregting for the AM llele omintion did not exhiit signifint differenes in wn length etween the genotypes, whih is onsistent with the reltively smll llele effet of prent HID 4 (Tle 2). In ll tested fmilies segregting for the B nd M, or D nd M lleles t 3_1489, the homozygous HIFs differed signifintly (p.) in wn length from one nother (Fig. 3). In eh se, the Morex llele inresed wn length ompred to the wild rley llele, whih ws onsistent with the llele effet estimted for this QTL (Tle 2). In the BM nd DM popultions the heterozygotes were intermedite to the homozygote genotypes inditing tht the two lleles hve inomplete dominne (Fig. 3). One fmily from eh of the onfirmed llele omintions ws seleted for fine mpping (BM1 nd DM1). Both fmilies segregted for the wild rley nd Morex lleles ording to Mendelin rtios nd exhiited reomintion events etween the flnking mrkers used (Online Resoure 7). Reominnt plnts were genotyped with dditionl mrkers to determine the reomintion rekpoints (Online Resoure 8) nd the genotype dt were used to onstrut linkge mps of the QTL region for oth fmilies (Fig. 4). All reominnt plnts nd seleted non-reominnt plnts were grown to mturity nd phenotyped for wn length. The non-reominnt plnts exhiited similr differenes in wn length s oserved in the previous genertion (Online Resoure 9). The NIL progenies of ll reominnts (Online Resoure 8) were genotyped for mrkers etween the two mrkers flnking the QTL region nd wn length ws mesured for ll plnts llowing omprison of the effet of Morex, HID 64 (B) nd HID 382 (D) for eh mrker. Similr to erlier mpping experiments wn length inherited in n inomplete dominnt mnner with the Morex llele inresing wn length ompred to the

7 27 Tle 2 Positions of QTLs for wn length on the rley geneti mp nd their lleli effets (in mm) QTL hr pos Mrker log1 (p) Allele effet ± SE Pek Flnking 1 Flnking 2 HID 4 HID 64 HID 369 HID 382 AL1.1 1H _798 2_617 2_ ± ± ± ± 1. AL2.1 2H 2. 2_1261 1_18 2_ ± ± ± ± 1.6 AL2.2 2H 3.7 1_32 1_1 2_ ± ± ± ± 1.6 AL2.3 2H _214 1_786 1_ ± ± 1.7. ± ± 1.4 AL3.H 2. 1_46 1_81 1_ ± ± ± ± 1. AL4.1 4H 9.6 2_924 1_142 1_ ± ± ± ± 1.6 AL4.2 4H _166 1_334 2_ ± 1..1 ± ± ± 1.2 AL.1 H _11 1_167 2_ ± ± ± ± 1.4 AL.2 H _94 2_8 2_ ± ± ± ± 2.1 AL6.1 6H 4.8 2_882 2_292 2_ ± ± 1.7. ±. 1.1 ± 1.6 AL6.2 6H 1.8 2_36 1_1 1_ ± ± ± 3.3. ± 1.4 AL7.1 7H _962 2_114 End ± ± ± ± 1.3 Chromosome numer QTL position () of the pek mrker Additive effets (in mm) of the wild rley lleles ompred to Morex (±stndrd error). The positive or negtive vlue indites tht the wild rley llele inreses or redues the trit, respetively Awn length [mm] llele A B D H M AM1 AM2 BM1 BM2 DM1 DM2 Fig. 3 Boxplots of wn length of six HIFs segregting for the QTL on hromosome rm 7HL. For eh llele omintion (AM, BM, DM) two HIFs were seleted tht segregte for the ultivted rley (Morex) nd lleles of the wild rleys. A HID 4, B HID 64, D HID 382, M Morex, H heterozygous. Different smll letters indite signifint differenes etween genotypes (one-wy ANOVA, p., n 1) wild rley lleles (Online Resoure 1,11 nd 12). Fine mpping showed tht AL7.1 is loted etween mrkers 2_483 nd 2_117 for popultion BM1 s well s for DM1 (Fig., Online Resoure 8). This redued the QTL intervl to less thn 1 (oserved mp distne:.9 m in BM1 nd.4 in DM1, respetively) nd did not reomine with mrker 3_1489 in ll tested individuls. Awn length lleli vrints re often ssoited with pleiotropi effets on other trits suh s tillering nd or plnt height. Therefore, tiller numer, spike length, plnt height nd flowering time were mesured in the HIFs exhiiting the lerest differenes in wn length (BM1, DM1 nd DM2). The homozygous HIFs did not exhiit signifint differene in ny of these trits (Online Resoure 13). To evlute the novelty of AL7.1 we performed n in silio mpping using mrkers tht were previously ssoited with wn length genes (Online Resoure 14). Two wn length relted QTLs re loted on 7HL, the SHORT INTERNODES (SHI)-fmily trnsription ftor enoded y the HvLks2 gene, nd the rss GATK VrintFiltrtion tool gene. AL7.1 is loted 3 distl of HvLks2, lose to HvDIM whih mps to 14.3 on the POPSEQ mp. Neither HvLks2 nor HvDIM re loted in etween the mrkers flnking AL7.1. Tken together, we hve fine mpped n wn length QTL in the distl region of hromosome rm 7HL tht does not mp to ny previously desried wn length lous. In ddition, AL7.1 did not exhiit ny pleiotropi effet on the other mesured trits. RNA sequening of NILs orroortes fine mpping To verify the fine mpping of AL7.1 we performed RNA sequening, using next genertion sequening on the homozygous NIL pir derived from the heterozygous reominnt DM1-e3R HIF line. RNA ws otined from shoot pil meristem tissue t Wddington stge (W). (Wddington et l. 1983), t this time point the stylr nl strts to lose nd the first wns re emerging. Trnsripts exhiiting SNP when ompred to the referene sequene (v. Morex) were mpped to the rley POPSEQ mp. A totl of 129 genes n e ssigned to derive from

8 276 Fig. 4 Fine mpping of BM1 nd DM1. Mini mp derived from the BM1 nd DM1 segregting fmilies, nd position of the mrkers on the POPSEQ Mp (Ariyds et l. 214) tht were used to onstrut linkge mps of the QTL region in BM1 nd DM1. Mrker 3_1489 (purple) ompletely o-segregtes with AL7.1. From fmily BM1, mp etween mrkers 3_93 nd 1_999 ould e reonstruted orresponding to.4. Fmily DM1 segregted only etween mrkers 2_483 nd 1_999 with geneti distne of POPSEQ 3_93 1_13 1_127 2_12 2_483 3_1489 2_ BM1 3_93 1_13 1_139 1_127 2_12 2_483 3_1489/ AL7.1 2_ _4 DM _483 3_1489/AL7.1 2_ _4 1_ _ _999 the introgressed wild rleys, while 344 genes show the Morex llele. The numer of Morex lleles ounts for 72%, refleting the expeted vlue of 7% of the Morex genome in the HIF progenies. Diret omprison of the SNPs etween the DM1- e3r NILs onfirmed the position of the segregting region on hromosome rm 7HL, s well s the introgression pttern derived from the initil genotyping dt (Fig. 6). RNA sequening n lso e used to nrrow down the numer of ndidte genes, whih omprise differentilly regulted genes, s well s trnsripts with nonsynonymous SNPs. The segregting region on hromosome 7HL identified with RNA sequening spns n intervl of 2.2 ( ) ording to the POP- SEQ mp (Online resoure 1). This intervl ontins 123 genes (43 HC nd 8 LC genes). The.9 intervl identified through fine mpping is ompletely loted within this 2.2 intervl nd ontins 66 genes (26 HC, 4 LC). Within this intervl, trnsripts were deteted for 19 out of the 66 genes nd only eight trnsripts exhiited polymorphisms etween the DM1-e3R NILs (Online resoure 1). Anlysis of trnsripts with no ssigned position ording to the POPSEQ mp reveled three dditionl genes ontining polymorphisms. Expression nlysis performed on the DM1-e3R NILs indited tht two genes (MLOC_ nd MLOC_4434.1), oth loted within the introgression, were differentilly regulted etween the DM1-e3R NILs (djusted p vlue.) (Online resoure 1). The low numer of differentilly regulted genes identified etween the two NILs my e used y the high vrition oserved etween the individul iologil replites (Online resoure 16). Beuse MLOC_4434.1, enoding zin finger protein 3, lso reveled polymorphism in the vrint lling, we performed Snger sequening of MLOC_ in the BM1 nd DM1 NILs. The NILs tht exhiited redued wn length ontined n AG insertion t position 37. This insertion uses frmeshift in the protein sequene nd introdues premture stop odon. The mrker developed sed on the deteted vrint in MLOC_4434.1, whih is

9 277 Fig. Seletion of HIFs used for QTL onfirmtion. Grphi representtion of reominnts used for QTL fine-mpping. Blk horizontl rs represent positions of tested mrkers. Purple dshed line position of the most signifint QTL mrker; lk dshed lines positions of mrkers just outside the QTL region. Colour ode red Morex, lue wild rley, nd green heterozygous. (B) Boxplots of wn lengths olleted from reominnt homozygous NILs (B HID 64, D HID 382, M morex.). Different smll letters indite signifint differenes etween the genotypes (one-wy ANOVA, p., n 6) wn length [mm] v44 e24 j17 y26 u24 l4 n1 41 s7 d18 e3 e3-r BM1 llele HID 64 Morex 2_12 2_483 3_1489 2_117 wn length [mm] DM1 llele HID 382 Morex v44 e24 j17 y26 u24 l4 n1 41 s7 d18 e3 e3-r loted t in the POPSEQ mp, o-segregted with the phenotype of the reominnt homozygous NILs from the two mpping popultions BM1 nd DM1 (Online resoure 1). Assuming tht the ustive gene is expressed in the seleted developmentl stge, we onsider this zinfinger TF s prime ndidte gene for the wn length phenotype. Phylogeneti nlysis reveled no homology with known zin-finger domin ontining wn length genes HvLks2 nd OsDL (Online resoure 17). Tken together, RNA sequening of the NILs improved the fine-mpping nd signifintly redued the numer of ndidte genes. Awns signifintly ontriute to grin filling To study the ontriution of wns to grin filling under our experimentl onditions, we studied the effet of wn removl on seed width, length nd weight. Plnts of v. Morex were grown under outdoor onditions nd when spikes of these plnts emerged the wns were removed. Three groups were mde: in the first group, wns were ompletely removed (no wn), in the seond group the wns were ut in hlf (hlf wn) nd in the third group wns were only ropped t the very tips (full wn) to ount for effets y wounding. As n dditionl ontrol, the thousnd grin weight (TGW) nd seed width of Morex free from physil injury were lso inluded. Averge width of seeds nd TGW otined from Morex spikes with no wn ws signifintly redued to, respetively 9.3 nd 83.9% when ompred to the ontrol (Tle 3). These experiments onfirm tht wn length indeed influenes grin filling in rley. Sine this experiment involved very severe redution in wn length, we investigted whether the rther moderte hnges in wn length oserved in our mpping popultions lso signifintly ffeted grin filling. For this, seeds from homozygous NILs from the mpping popultions BM1 nd DM1 grown under outdoors onditions in two growing sesons (212 nd 214) were tested. In oth growing sesons, these NILs exhiited signifint differenes in wn length ompred to eh other, the B nd D llele redued

10 278 Fig. 6 Comprison etween the DM1-e3R NILs. The mjor QTL on 7HL ssoiting with wn length is mrked with n rrow hed. Left pnel shows the introgressions of the wild rley into the v. Morex. Right pnel shows expressed vrints etween wild HID lines (lk) nd Morex (red). Green rs indite the vrints polymorphi etween the two DM1-e3R NILs, homozygous for, respetively, the Morex nd HID382 (D) llele Morex HID 382 HID 4 HID 4, 64 or 382 1H 2H 3H 4H H 6H 7H QTL intervl 1H 2H 3H 4H H 6H 7H Tle 3 Averge seed width nd thousnd grin weight mesured for plnts of v. Morex or homozygous NILs Genotype Awn length (mm) Seed width (mm) (g) Growing seson Avg ± SD % 4 Avg ± SD % d Avg ± SD % d Morex Control n.d. 3.6 ± ± Morex Full wn [1] 3.6 ± ± Morex Hlf wn [] 3. ± ± Morex No wn [] 3.4 ± ± BM1 M 11 ± ±. 1 ± BM1 B 13 ± ± ± DM1 M 16 ± ± ± DM1 D 17 ± ± ± BM1-j17 M 189 ± ± ± BM1-j17 B 161 ± ± ± DM1-d18 M 188 ± ± ± DM1-d18 D 134 ± ± ± Thousnd grin weight Averge of iologil replites (n 7) Stndrd devition d Perentge ompred to ontrol (=1). Different smll letters indite signifint differenes etween genotypes (one-wy ANOVA, p.) wn length ompred to the M llele to pproximtely 86 nd 7%, respetively. In the DM1 popultion, signifint redution in seed width to 97.2% ws oserved only in the 214 growing seson (Tle 3), ut no signifint differenes were found for TGW. The BM1 popultion exhiited no signifint redutions in seed width nd TGW. Disussion Although the importne of wn length for grin yield is widely reognised (Bort et l. 1994; Grundher 1963; Shren et l. 1983), little is known out the genes involved in this trit. In this work we used multiprent

11 mpping popultion, onsisting of six supopultions to identify wn-length relted QTLs. We oserved signifint vrition in wn length, inluding trnsgressive segregtion in oth diretions, whih rgues for the presene of oth positive nd negtive lleles in the prents nd/or intertions etween loi. Awn length distriution did not differ signifintly from normlity in the six individul supopultions, suggesting the involvement of multiple loi. The reltively high rod sense heritility of the trit (H 2 =.7) ws in ordne with previous findings tht wn length is highly heritle trit (Theri et l. 211). Most QTL studies in rops hve een onduted in iprentl popultions. However, this pproh is limited s the smll numer of prentl lines represent only smll portion of the geneti diversity for the trit of interest. Multiprent mpping popultions re more genetilly diverse thn i-prentl popultions nd llow to sreen for effets of multiple lleles per lous. The high geneti diversity omined with inresed levels of reomintion due to interrossing inrese the resolution nd preision of the QTL detetion (Cvngh et l. 28; Psul et l. 216). To dte, only few multiprent mpping popultions using minly elite ultivrs, hve een desried in whet nd rley (Hung et l. 212; Mky et l. 214; Snnemnn et l. 21). These studies use, respetively, four (Hung et l. 212) nd eight (Mky et l. 214; Snnemnn et l. 21) different prentl lines s founders for the multiprent mpping popultion. In our study, we used multiprent mpping popultion sed on five prentl lines; three wild rley genotypes from the estern nd western Fertile Cresent; one rley genotype from the Tietn highlnds in Chin; nd the ultivr Morex. The wild rley prents were seleted to mximise geneti diversity in the mpping popultion. All wild rley lines were rossed with Morex fter whih one round of krossing to the elite prent Morex ws used to redue the proportion of unfvourle wild lleles. As suh this interross popultion represents vlule resoure for QTL detetion, gene identifition nd s pre-reeding mteril. Due to the rossing nd krossing with Morex, on verge 7% of the genome of the RILs originte from the reurrent prent Morex. Suh symmetri popultion struture is not optiml to onstrut linkge mp s reomintion events nnot e oserved in lrge prts of the genome. We, therefore, used the redily ville POPSEQ mp, whih is supported y the genome ssemly, s sis for the QTL mpping. We were le to detet 12 signifint wn length QTLs from whih AL1.1, AL3.1, AL4.1 nd AL.1 o-lolise with regions of previously desried short wn (lks) or reviristtum (ri) loi, whih re known to exhiit wn length phenotypes (Frnkowik nd Lundqvist 212; Druk et l. 211). In ddition, Morex is smooth wned rley vriety rrying the smooth wn 1 (rw1) gene whih is loted 279 lose to AL.2. To our knowledge, none of the other wn length QTLs inluding AL7.1, whih hs the lrgest effet on wn length, mp to previously desried loi. The phenotypi vrition in oth wild nd ultivted speies suggests tht the trit hs not een under stringent seletion during domestition (Ao et l. 214). Three out of four wild rley lleles redued wn length when ompred to Morex, while ll essions rry QTL tht hve positive s well s negtive ontriution, explining the oserved trnsgression. To refine the mpping of AL7.1 we performed RNA sequening of two ner isogeni lines differing only in the QTL region for HID 382 (DM1-e3). Using this pproh, we were le to preisely mp the position of the introgression on hromosome rm 7HL. No vrints were oserved in HvLks2 or HvDIM, where oth re loted outside of the introgression. In ddition, no diret orthologues of known wn-length relted genes were identified within the QTL region. Awn length relted genes in rie identified so fr inlude: the zin finger DROOPING LEAF (OsDL); the uxin response ftor ETTIN2 (OsETT2); wn-1 (n-1) nd n-2 whih enode, respetively si helix-loophelix protein nd the LONELY GUY LIKE PROTEIN 6 (OsLOGL6), whih tlyses the finl step of ytokinin synthesis; nd LONG AND BARBED AWN1 (OsLABA1), whih enodes ytokinin-tivting enzyme (Luo et l. 213; Tori nd Hirno 214; Gu et l. 21, Hu et l. 21). Seletion of the ndidte genes ws done sed on the vrint lling nd expression nlysis of the RNA sequening. At the seleted developmentl stges (W.) reltively low numer of differentilly regulted genes were identified in the expression nlysis. This n e due to the seleted tissue type (entire pex inluding florl primordi) nd/or the reltively erly stge of development of the wn. In the vrint lling, only MLOC_4434.1, whih enodes for zin finger (ZF) protein 3 gene, ontined smll deletion of 2 nuleotides nd o-segregted with the phenotypes of NILs derived from the HIFs DM1 nd BM1. Tken together, this MLOC_ ppers to e promising ndidte gene for this mjor wn length QTL. Noteworthy, two wn length relted genes Lks2 nd DL lso ontin zin-finger domin (Yuo et l. 212; Tori nd Hirno 214). However, phylogeneti nlysis indites tht there is no strong homology with MLOC_ nd these previously desried wn length relted genes. Previous studies hve shown tht the photosyntheti tivity of the spike in generl nd the wn in prtiulr re importnt for grin filling in rley (Aee et l. 29; Bort et l. 1994; Shren et l. 1983; Xue et l. 1997). We hve shown tht de-wning Morex plnts shortly fter heding resulted in signifint redution in seed width nd TGW, suggesting tht wn length signifintly ontriutes to grin yield. Only

12 28 sutle non-signifint effet on seed width nd TGW ws oserved DM1 nd BM1 NILs. On one hnd, this ould e due to the rther moderte redution in wn length in these lines when ompred to de-wning of Morex. On the other hnd, vrition in wn length nd orrelted differenes in net photosyntheti rte of the spike might only eome more pprent in stress prone environments with wter limittion, high ir tempertures, lef diseses, nd other uses of premture lef senesene (Mrtin et l. 1993). Severl nturl mutnt lleles of lks2 re present in the Est Asin rley gene pool (Yuo et l. 212). Long wns might e seen s hindrne in hrvest or storge y the frmer (Yuo et l. 212). In modern ultivrs, the presene of long wn redues the vlue of the rley strw used for feed (Tkhshi 19). However, Morex is known s smooth-wn mlting vriety derived from Mnhurin rley imported in the erly 192s from Northest Asi. Morex ws hrterised y longer wns thn three out of the four seleted donor rley genotypes. Aordingly, the AL7.1 llele from Morex strongly inresed wn length nd represents previously unhrterised QTL. However, under fvourle onditions, only moderte effets on grin size were oserved for this QTL. Tken together, these dt exemplify how multiprent mpping popultion in rley onsisting of wild essions krossed into ommonly used ultivr inresed the geneti diversity. Eventully, suh pprohes my e employed s tool to introdue novel nd enefiil trits into the elite rley germplsm. Author ontriution sttement CL, WvE nd MK plnned the experiments. CL performed experiments nd wn length mesurements. SE ontriuted to the development of the mpping popultion. MM nd PH performed SNP dt nlysis. MB performed QTL nlysis. CL, WvE, AW, MvK nd MK wrote the mnusript. WvE nd AW nlysed the RNAseq dt. All uthors red nd pproved the finl mnusript. Aknowledgements Open ess funding provided y Mx Plnk Soiety. We would like to thnk Sheil Adimrgono for her ontriution to the development of the mpping popultion nd Mrinne Hrpersheidt, Cren Dwidson, Kurt Stueer, Andre Lossow nd Kerstin Lux for exellent tehnil ssistne in lortory, greenhouse nd outdoors. This reserh ws funded y the Germn Cluster of Exellene on Plnt Sienes (CEPLAS) EXC128 nd sholrship wrded to the first uthor y the Interntionl Mx Plnk Reserh Shool (IMPRS). Compline with ethil stndrds Conflit of interest The uthors delre tht they hve no onflit of interest. Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. Referenes Ao S, Vn-Oss RP, Gopher A, Srng Y, Ofner R, Peleg Z (214) Plnt domestition versus rop evolution: oneptul frmework for erels nd grin legumes. 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