Cavagnaro This article isprotected Allrights bycopyright. reserved. doi: 10.1111/plb.12549 thisversionandthe between lead todifferences been through the copyediting, typesetting, and proofreading which may pagination process, forpublicati accepted This articlehasbeen [email protected] author: Corresponding * . 4 Argentina.Corrientes, de Investigaciones Científicas y Técnicas (CONIC 3 Investigaciones Científicas y Técnicas 2 Argentina. Mendoza, (UNCuyo), deInvestigacionesNacional Cien 1 Kozub Carolina Perla grass forage of native the biology Reproductive Editor : J.Arroyo Article type : Research Paper 25-Jan-2017 : Date Accepted :07-Dec-2016 Date Revised :30-Sep-2016 Date Received Accepted Horticultura, de Ciencias de Facultad Instituto Agrarias, Nacional Universidad de Cuyo, Mendoza, (IBONE), Botánica del de Facult Nordeste Instituto Nacional Tecnología Agropecuaria de (INTA) Consulta; Consejo de E.E.A. La Nacional Instituto Biología de Agrícola de (IBAM),Mendoza Facultad CienciasInstituto de Agrarias (FCA), Consejo Article 1 , Federico Cavagnaro Pablo 1 , Karina Barboza tíficas y Técnicas (CONICET), (CONICET), Mendoza, Argentina. 2 , Florencia Galdeano 2, 4 * on and undergone full peer review buthasnot review undergone fullpeer on and ET), Universidad Nacional delNordeste (UNNE), crinita Trichloris ad deCiencias Agrarias Version of Record. PleaseVersion cite thisarticle as 3 , Camilo Luis Quarin Universidad Nacional Universidad Cuyo de (, ) (FCA), Consejo Nacional 3 , Juan Bruno , JuanBruno molecular molecular markers. Cytological analyses revealed embryo sacs with eight nucleiand of cytometric seed screen (FCSS), self-compatibility of biology the reproductive studied we work, Inthe present reproduction. of its mode on of the lack asof knowledge consequence partly water availability. Despite its im this species is widely utilized asforage andfo good to andresistance drought to forage andgrazing, areaDue its ofdistribution, quality, extensive Keywords: in aridregions. inimpact thegenetics and breeding of this specie is aself-compatible and autogamous species. Itis revealed lack of outcrossing. Altogether, these results indicate that (SSR) repeat results from markermorphological analysiswhereas and simple sequence progenies of that to seed set (%)demonstrated evaluate designed experiments and to 2C 3C contents,DNA indicatingembryo sacs of sexual pollination Controlled origin. type for all This article isprotected Allrights bycopyright. reserved. Accepted markers,molecular microsatellites Article crinita Trichloris Abstract

Trichloris T. crinita is aperennial grass forage species native accessions analyzed. FCSS histograms exhibited accessions clear corresponding analyzed. two peaks FCSShistograms exhibited , sexual reproduction, autogamy, flow cytometry, embryological analysis, analysis, autogamy, flow , sexualembryological cytometry, reproduction, portance, genetic improvement of T. crinita T. crinita r revegetation purposes in environments with low by means of embryological analyses, flow tests and progeny testing with morphological and with morphological testing progeny tests and expected that these data will have a positive s, and therefore contribute to its utilization proper to arid regions of th T. crinita T. crinita T. crinita T. crinita is self-compatible, has been very limited, reproduces sexually. It It sexually. reproduces e American continent. Polygonum

collected from Germplasm Argentina)(Mendoza, the created Bank of Grasses Native (GBNG), with accessions growing whenever soil water isavailable and the temperature is above 10° C(Seligman 2012; Peterson and north of Mexico) and ChileandArgentina) (in (Anton , andSouth , of and north Mexico) in United States south of the North America (predominantly aridandsemiaridof regions extensive availability (Cavagnaro & Trione Quiroga2007; utilized species for rangegrazing andrevegetati competing aggressiveness grasses aggressiveness other (Passeraamong competing andits aptitudes, These 1969). Wainstein (Cavagnaro& &Dalmasso Gonzalez animals 1983; and wild trampling &Cavagnaro (Greco domestic drought 2002), to andgrazingby resistance good (Cerqueira quality forage 1971), (Roig distribution area of American continent and one of the most important gr This article isprotected Allrights bycopyright. reserved. crinita Trichloris INTRODUCTION breeding programs in this species. The mode of reproduction of reproduction in this species. of The mode programs breeding Accepted and conservation of proper maintenance the resource. for Knowledge this isessential for biology itsgenetic valuable strategies on reproductive 2005). These data encouraged further investigations biomass production includingforage traits, (Cavagnaro agronomic (AFLP) analysis, as as phenotypica well polymorphism by fragment amplified length as evaluated at level, the DNA diversity broadgenetic revealed region in the west of Argentina. Characterization of the Article Due to its importance, the Instituto Argentino de Investigaciones de Zonas Áridas Investigaciones de de its to InstitutoArgentino importance, Due (IADIZA) the T.

crinita crinita T. crinita (Lag.)Parodi(Chloridoideae, Poaceae)is et al. presents disjunct dist presents an amphitropical 2007). Under natural natural as Under conditions behaves atypical 2007). it specieswarm-season natural populations throughout 350,000 km 350,000 throughout natural populations T. crinita et al et on purposeson in environments with low water . 2009). . 2009). towards conservation developing andutilization lly, for various lly, and for various physiological morphological, ass species inaridregions, due to its extensive germplasm, andis apre-requisite for initiating germplasm, T. crinita ribution, being mainly found along two found along two being ribution, mainly a perennial forage grass native aperennial forage grass to the native et al . 1992), have made have . 1992), et al germplasm collection atIADIZA T. crinita et al . 2004; Silva Colomer 1989), and and 1989), Colomer Silva . 2004; 2 of the Monte,a vastarid . 2006; Greco & Cavagnaro Cavagnaro & Greco 2006; . hasnot been T. crinita et al. a widely awidely et 1992). 1992).

al.

species of the the species of large subfamily apomictic including anunusually comprises of number taxa, Chloridoideae several This article isprotected Allrights bycopyright. reserved. Accepted andbreeding of this species. genetics biologyfloral inthisspecies areperformed. The re controlledIn addition, pollination experiments for te embryological analysis, flow cytometric seed scre (Matzk Each is pathway ploidy). characterized by a specif and (or DNA comparing content DNA- endosperm apomictic the seedembryo by from distinguished et al. accurately reconstruct the reproductive pathway of 2009; Ortiz means of distinctiveanalysis of morphological traits and/or molecular marker data (Acuña usedincombinationcommonly with controlled po Article sacs, isof embryo analysis that the anddevelopment morphology evaluate embryological origin, (Cavagnaro Cavagnaro 2002), and the lack of AFLP polymorphisms observed among of the same varieties crinita absence upon phenotypic in based data of showing segregation highlyautogamous, or apomictic demonstrated to date. However, circumstantial evidence suggests that this species may either be 2006; Dobeš Dobeš 2006; varieties cultivated during several generati et al biology of reproductive studied the we work, present In the ingrass reproduction species, available of for the assessingmethods mode Among the et al et al . 2000). . 1997;). More recently, flow cytometric seed screen (FCSS) has been used to . 2006), suggesting that these materials do not intercross. Noteworthy, the et al. 2013;Molins (Brown & Emery 1958), a close relative of et al. et 2014). FCSSanalysisallows sexual seed be to en, progeny testing andmolecular marker analysis. ploidies andendosperm embryo of ic combination ons ons pollinationunder open conditions (Greco & sults this from study willhave direct impact in the llination experiments andprogeny tests, often by seed formation in many species (Barcaccia sting anddescriptions self-compatibility of the T. crinita Trichloris by means of means by (Anderson 1974). et al.

T. Mendoza, Argentina. Four plants per accession were Four accession per were used. Argentina. plants Mendoza, peatsoil: in the experimental field Facultadof de 21 selected accessions derive is presented in Table S1. Monte (Cavagnaro Monte constitute a representative sample of the broad phenotypic variation observed naturally in the andthey diverse, accessions andmorphologically genetically The study were inthis Argentina. used This article isprotected Allrights bycopyright. reserved. Accepted km 350,000 dispersed throughout populations The(IADIZA). Grasses of Native Bank (GBNG) Argentino Zonas the Áridas de at Investigaciones Instituto de Plants of Plant material Article & Rúgolo de Agrasar 1987). glume apex acuminate, 3-awned. Fruit isa one-seeded caryopsis, as in most of the Poaceae (Nicora persistent, upper compressed. pubescent, are laterally Glumes spikelets lanceolated, below, one composed of digitate andunilateral racemes, solitar cm tall in vegetative stages and 50-90 cm when flowering. They produce dense inflorescences crinita Trichloris Studied species MATERIALS AND METHODS Seeds from each accession were

T. crinita T. crinita plants decumbent are andcaespitose, approximately upright, perennial of 20-40 or used inthis from study s originated et al germplasm at GBNG collectingoriginated plantsfrom from natural 48 . 2006). The geographical location of the original populations from which the sown in 20-liter pots with 2:1 (v/v) mixture of sandy-loam sandy-loam of (v/v) mixture 2:1 pots with in sown 20-liter 2 of of Monte, anaridandsemiarid region inthe west of Ciencias Agrarias,Universidad de Nacional Cuyo, y spikelets with 1-2 sterile florets and a fertile eeds of 21 accessionseeds of21 from Germplasm the Young Young by protocol described the following solutions salicylate and methyl aseries of ethanol through at h. dissected least florets for were andpassedout 70% ethanol of to Pistils the 24 transferred formaldehy in andfixed 37% 70% ethanol, collected Two inflorescences at anthesis from 4plants per accession (8 inflorescences per accession) were inthese materials. differentiated be whenever present, were indicative of lack of ap sacs embryo Inaddition, diplosporous. sexual or sac considered were accessions sacs with asingleembryo by whereas embryo apospory), originated one embryo sacper ovule were considered as apos sexual determine the mode of reproduction. The following cr (Leica Microsystems, Wetzlar, Germany). interference contrast (DIC) system and a with a differential camera digital equipped microscope glass wasplaced over the sample and ovules were on mounted peraccession) were (70 ovules ovules Cleared in solution. each 30 for minutes ratios, and1:3 in1:1 salycilate : methyl ethanol of mixtures using concentration, in andcleared per 30 100%) 100%, minutes 90%, 80%, (70%, concentration This article isprotected Allrights bycopyright. reserved. Accepted Article analyses Embryological et al versus The number of embryo sacs per ovule, andem sacs per ovule, embryo of number The Embryo sacsEmbryo observations were performedin20 . (1979). Individual ovules were dehydrated in a series of increasing ethanol increasing ethanol inaseries of dehydrated were Individual ovules . (1979). apomictic apomictic to Accessions (2001). origin, according withLeblanc & Mazzucato than more ospory, although sexuality and diplospory could not not could sexuality anddiplospory although ospory, with multiple (more than three) antipodals, observed witha lighttransmission Leica DIASTAR porousapomictic(because the of at leastone a slide inadrop pure of methyl salicylate a cover de andglacialacetic acid(90:5:5) andthen iteria were usedto distinguish embryo sacs of bryo sacs morphologies, were evaluated to to sacsevaluated were bryo morphologies, T. crinita accessions of the GBNG-IADIZA.accessions the of This article isprotected Allrights bycopyright. reserved. Accepted in10 registered were complete flowering daysto direction of flowering andnumber the of level, flowering” was reached when only 10% of the plants remained in anthesis. At the inflorescence of “end at whereas anthesis, spikelets with 10% inflorescence plants had date inwhich one the accession, criteria per the following plants of Dafn 20 the for recorded were flowering of andend beginning the of Dates Phenological observationsfloral and morphology (Matzk pseudogamous apomixis or apomixis, parthenogenesis autonomous embryo/endosperm ratio were considered of sexual necessary. PA II´sFloMax software was used internal anadditional isnot standard cells, by andnot content, absolute the DNA and endosperm by characterized of seed formationpathways were position 100 in the linear abscissa by changing the voltage of the photomultiplier. Asthe different cells was adjusted channel nuclearto DNA of embryo 2C content representing peak position The Article per nuclei sample were measured. 3,000 nm. Approximately at 355 detector operating light the with the fluorescence intensity of DAPI-stained nucleius Precise P Staining Buffer, Partec). The DNA content of nuclei (C value) was determined by measuring 4 of mL 1.5 adding before min 1 for incubated and to a tubes sample 30 transferred through µm wasmesh, mixture filtered nylon The Germany]. extraction buffer [CyStainUV Precise P Kit Nuclei Ex dishes with 0.5 accession), bladeinPetri (20 seedsper of accession mL with were chopped arazor et al. cytometry Flow 2000) usingbulked seeds. Two samples of ten behavior of reproductive The T. crinita to process the data. Seeds with a2C:3C ′ accessions was investigated by FCSSanalysis (Matzk , 6-diamidino-2-phenylindole (DAPI; CyStain UV (DAPI; , 6-diamidino-2-phenylindole i (1992). initiation”“Flowering wasconsidered as

aspecific ratio of DNA content between embryo traction Buffer ® (Ref. 05-5002), Partec, Münster, ing aPartec PA IIflow cytometer (Partec GmbH), seeds, randomly selected from 4 plants of each origin, whereasorigin, other ratios were classified as T. crinita et al. et accessions, using 4 2000). This article isprotected Allrights bycopyright. reserved. Accepted All counts under carried a recorded. out were were threshed separately, ‘number containingwere florets’ florets and the caryopses’ of of and‘number different of plants inpots inan fieldopen and maintaining cl approximately one week after anthesis. Openpo setting, seed start at the of Allbags removed prior days to bags2 were anthesis. paper treatments. Self-pollination wasensured isolat by inflorescences different from plantsofeach acce and pollinationopen pollination conditions. For thispurpose, 19 accessions were used.Atleast 5 The breeding system of Self-compatibility tests Article florets. andsterile fertile of and number 100 spikelets per accession (~ 2,000 spikelets in total): color of awns, anthers, glumes and stigma; a under stereoscopicexamined registering floral for in the data microscope, following least traits at cleistogamy. presence of anthers in spikelets. Unopened spik the byregistering from 8inflorescences inspikelets flowers) 100 determined was unopened and presence of cleistogamy were recorded. The inflorescences per accession (200 inflorescences in total). Atthe spikelet level, the time of anthesis Mature inflorescences were harvested 2-3 week and collected inflorescences at were anthesis floral descriptions of For morphology, T. crinita accessions. T. crinita was evaluated by means of comparing seed set set self- seed under by of comparing means was evaluated elets without visible anth ose proximity among the pots with flowering plants ssion were usedinthe self andopenpollination llination conditions were ensured growingthe by presence of cleistogamy (self-pollination in ing inflorescences with moderately transparent stereoscopic microscope. Seed set percentages stereoscopic set Seed percentages microscope. s after thepollination period. Inflorescences ersare indicative of sown in sown trays andindividual seedlingswere transpla Seeds obtained from each putative cross, and seeds from the respective parental accessions were after anthesis. At maturity, inflorescences were i days 10 Bags period. were shaken Allbags daily duringthe removed anthesis gently were maturity. stage at same both forbagging were the parental accessions selected of of of the inflorescences favor pollen exchange. Emasculations were not carried out due to the small size of flowers. The two immature inflorescences, one from each parent different pairs 11 of phenotypically between attempted cross-pollination flowering season, we the During Progeny testing statisticalthe software package Infostat version 2014 Rienzo (Di treatment. Results were presented as means ±standard errors. The analyses were performed with Fisher test (p < 0.05). Contrasts were performed plants/accession(four were analyzed) was consid ‘Pollination treatment’, ‘accession’ andtheir intera Development Core Team, 2011), using the interface provided by InfoStat (Di Rienzo This article isprotected Allrights bycopyright. reserved. Accepted ArticleWe used glmer routine of lme 4 package (Bates (G linear ofgeneralized mixed models adjustment tr pollination in seedset(%)Differences among Statistical analysis treatments. were calculated as‘(fruit/flower ratio) x 100’ andused to compare open T. crinita accessions (i.e.,11 putative reciprocal crosses, totaling 22 crosses), by enclosing eatments andaccessions were analyzed by the et al. to look for intra-accession variation in each ndividually harvested to avoid mixtures of seeds. seeds. of avoid mixtures to harvested ndividually ered term. arandom Means were compared using LMM; binomial distribution, logit-link function). function). binomiallogit-link distribution, LMM; ctions were treated as fixed terms, and‘plant’ nted to the field, where comparative analysis of al accession, ina bagbefore anthesis to paper 2011) of the R statistical language (R et al. et 2014). versus

self-pollination self-pollination et al . 2014). . 2014). laboratory (Kozub (Kozub laboratory This article isprotected Allrights bycopyright. reserved. diluted afinal samples to ng.µl DNA were of ~30 concentration electrophoresis. by gel- 0.9% agarose was evaluated UK), andDNA01, integrity (PicoPet spectrophotometer Picodrop a with determined and purity were concentration to DNA (1980). & according Murray Thompson a resulting andground with The mortar. (~20 powder mg) was isolationliquid nitrogen, usedfor DNA in frozen andimmediately harvested accessionof were leaves each DNA freshyoung extraction, For DNA extractions analysis markers Molecular presence/absence of trichomes on the leaf sheath and leafblade. and nodes spike, were colorcharacters of morphological andinternodes, the evaluated: following morphological characters between progenies a (H A set of 16 simple sequence repeat (SSR) markers previously developed for developed sequenceA set (SSR) simple of 16 repeat previously markers analysis markers SSR template in PCR amplifications. two microsatellite loci with theaim detectingof hybrids. at parentals, analyzed crosses, and from respective putative were individuals two their of 78 total in cross lines each parental involved the between used and S2. presented were SSR codominant are markers polymorphic inTable that markers e

Accepted), and the inbreeding coefficient (F Article et al . 2011) were used to estimate observed (H IS ) in 8 T. crinita nd their accessions parental performed.was The were selected for analysis theirof progenies. A accessions.characteristicsthe Main SSR of o ) and expected heterozygosities heterozygosities ) andexpected T. crinita -1 and used as and used in our inour Estimation ofH (Invitrogen). ladder laterfor genotype analysis. SSRallele sizes we gels and staining photographed andthe hours.was Silver mA forperformed 40 3 60W were 1500V, usin by electrophoresis, polyacrylamide gel resolved An products further confirmed the were amplicons. of the forPCR aliquot visualization of buffer) (4 ml of TAE bromide ul/100 usingethidium electrophoresis gel by3% agarose confirmed were size sec., and 30 for1min.,72°C andafinalstep at for amplifications 72°C expected Positive 4 of min. for annealing for temperature for cycles of sec.,appropriate 94°C followed 94°C 40 45 sec., at 30 by 5 U/ polymerase buffer, 1.6 which allele has two copies. The program hastwo copies. ATETRA allele 1. The which markers showing a single allele andfour alleles hadH Observed heterozygosity (H and the maximum was 0.66, assuming AABB. For SSRs with three alleles, H This article isprotected Allrights bycopyright. reserved. markers with two alleles (e.g., A and B), the minimum H Accepted H SSRswe on estimated bandingpattern, the based Thus, unequivocally in assigned. alleles are be genotypes cannot present an SSRlocus, the three equal totheploidy level which, inthecase of marker profile show a single allele (andnullalleles are assumedabsent), theor number allelesof is tetraploid, like is allele) copies from Inaof data each for simple not polyploid species. of number molecular marker alleles two at are drawn any random identical byde Article μ l and 2.5 2.5 l and PCR reactionsPCR were performed in 20 μ T. crinita o , H l genomicof Thermocyclers DNA. were programmed as follows: initial denaturation e andF μ (Roodt & Spies 2003), exact genotypes genotypes be can only exact assigned Spies the (Roodt & 2003), when l dNTPs (2.5 mM each), 1 IS

o ) value for agivenlocus is calculated as one theminus probability that μ T. crinita re estimated by bp by comparisons with a 100 DNA re estimated l volume containing 11.6 containing 11.6 l volume μ l of 5 l of o scent. Estimation of allele dosage (i.e., the 0 was used to calculate expected heterozygosity heterozygosity used 0 calculate expected to was g denaturing polyacrylamide gels (6%) andran (6%) at gels g polyacrylamide denaturing according to Bever & Felber (1992), where the o , corresponds alleles. four to , corresponds When two or values of0.0 and1.0,respectively, whilefor o μ was assuming a0.50, genotype of AAAB, M of each primer,M ofeach 0.3 μ l water, 2 o = 0.83, regardless of μ l Taq polymerase at at polymerase l Taq μ l 10 × DNA DNA × l 10 contents of embryo and endosperm tissues, respectively, suggesting that accessions produced histograms two to clearpeakswith (Figure and 2), corresponding 2C 3C DNA accessions, FCSS was performed to complement em sexually (i.e., through (i.e., sexually coefficient (F This article isprotected Allrights bycopyright. reserved. (H type (Grossniklaustype but results innuclear (alsoentered restitution known asfirst restitution), division asin the as known diplospory), mitotic sac,formed embryo nucleate and eight unreduced classified as either sexual (also called of sacs with a cells seven antipodals), sacsand embryo mass with of embryo of complex (Figure 1 E,F). the antipodal sac appearanceofa were with or observed, per ofmass antipodals cells embryo chalazal pole (Figure 1, A-D). Occasionally, inso at the micropylar pole, the central cell (with tw cases, the sac embryo andconsisted nucleate was the seven cells: eight egg of cell synergids andtwo cytologically. Allthe accessions revealed asingle A total of 1,400 ovules from 20 screen seed cytometric flow and analyses Embryological RESULTS e

Accepted Article (Van Carlo simulations Puyvelde Monte ) using10,000 With the aim of further characterizing the type of embryo sacs present in On the basis of these two anatomical phenotypes observed cytologically (i.e., embryo sacs IS ) was estimated as F ) wasestimatedas et al et double fertilization). . 2001; Ortiz Ortiz . 2001; T. crinita Antennaria IS = 1- (H et al Polygonum accessions (70 ovules per accession) were examined . 2013)]. o /H type; or or type; e ) for each locus and averaged across allloci. o polar nuclei) andthree antipodal cellsat the me cases (accessions 6, 17 and 21) more antipodals megagametophyte per ovule (Figure 1). In nearly all Innearly (Figure 1). perovule megagametophyte either without entering meiosis (i.e., through type) or apomictic diplosporous [i.e., an [i.e., diplosporous or apomictic type) bryological analyses. Seed samples from all the via meioticdiplospory, in which meiosisis et al . 2010). In addition, Inbreeding . Inbreeding Inaddition, 2010). T. crinita T. crinita T. crinita seeds developed developed seeds could be could Taraxacum

This article isprotected Allrights bycopyright. reserved. increase) than in open-pollinated higherinself-posignificantly ‘accession’ seed seton (Table Inaverage found were 1). for accessions, allthe (%) seed set was accessions are self-compatible. Significanteffe All theaccessions produced seedsunder conditions,self-pollination indicatingthat seedsetvariation tests and Self-compatibility accessions was observed for color awns,of stig (Figure H).Glumes pubescent awns 4 (Figure G).ClearD, were phenotypic among 4 variation the lateral the than always each awn and the were floret central longer B, C).Lemmas was 3-awned of Cleistogamous spikelets were not observ inflorescence. entire anthesis inthe complete before required was Two days were a.m. from the inflorescence apex to the basal spikelets (Figure 3).Anthesis occurred between 7 and 11 wasperiod homogeneous in all theaccessions. In ingeneraland began inNovember time Flowering Phenological observationsfloral and morphology system. reproductive Acceptedinteraction was detected (P<0.0001). Thus,pollinatio conditions, respectively (Table andFigure S4 Significant5). ‘pollinationtreatment xaccession’ accessions, with ranges of % 67-95.6 and 47-9 Article All the spikelets analyzed (n=1900) had a sterile floret and a fertile one underneath (Figure 4 (Figure underneath floret hadasterile andafertile one analyzed spikelets (n=1900) All the a are sexual with observations consistent data FCSSandembryological In combination, llinated inflorescences, which produced 17.1% more seeds (relative florescences (Table 1). Seed seflorescencesSeed (Table1). ed inany of the studiedaccessions. 5.5 % observed under self- and open-pollination open-pollination self- and under %observed 5.5 cts (P<0.0001) for treatment’‘pollination and ma andma glumes (Table S3andFigure 4A-L). all cases the bloom occurred downwards, starting lasted untilAprilof thefollowing Flowering year. n treatments affected seed setdifferentially t varied significantly amongthe significantly t varied T. crinita

theresultsconfirming from analysis. morphological asgenotype the line,maternal even inreciprocal same SSR individuals hadthe used. All progeny lines parental the the between polymorphic were crosses, putative including cr reciprocal three their S4). seed set, respectively, and accessions and11 7 were seed13 set was significantly reduced underself-po increase was highest for accession T. crinita accession was used as the mother. These data clea regardless phenotype progenieshadthe of which maternal the i.e., were results observed, same the same maternal phenotype for allanalyzed trai these attempted crosses, were analyzed with the the which and parental canclearly accessions differentiate in used expressivity, high penetrance different phenotypically and genetically 14 crosses between putative from 11 obtained individuals ofwere 1,084 A total Progeny testing by morphological and molecular marker analysis This article isprotected Allrights bycopyright. reserved. Accepted Article significant increase inseed set across T. crinita Progeny testingProgeny usingSSR markers led tothe sa parental lines. accessions and S4). 5 Although(Figure fourTable accessions of allbut the evidenced T. crinita under self-pollination compared to accessions (Table S5). Four qualitative morphological traits traits with morphological accessions qualitative (Table S5).Four 4 (52.6 % increase) and 6(43.3 %). In contrast, in accessions 8 and crosses, indicatingabsence cross-pollination and of aim of detecting putative hybrids. All progeny had hybrids.Allprogeny putative aim detecting of ts. When reciprocalts. When cro osses, were analyzed with two SSRmarkers that rly indicate absence cross-pollinationof between llination, revealing and 12.7% llination, reduction8.5% in not affected by the pollination treatment (Table me results.me Atotal of 78 individuals from open pollinationopen conditions, this sses were analyzed the an average of 0.56.average of Eight an 8) (Table 2). The average H (H the accessions were homozygous andshowed asingle allele for these loci. Estimates F of For example, in example, For and andthat is sexually species. it aself-compatible reproduces autogamous and morphologi markers cytology, molecular sacs (namely, ovules, inflorescences and seeds from 20 accessions) analyzed by flow cytometry, embryo of approaches methodological different study, In this DISCUSSION heterozygosity exists at some SSR loci, lomost although acertain that accession loci broadly, of indicating of degree each individual varied This article isprotected Allrights bycopyright. reserved. A total of 16 microsatellite loci were usedto estimate observed (H Analysis of heterozygosity and inbreeding Hardy-Weinberg expectations. Multilocus F Multilocus expectations. Hardy-Weinberg in positive locilocus analyzed,mostwere of indicatingthe compared adeficit of heterozygotes with (H from results tests embryological analysisfrom andprogeny withof characters morphological (Brown & Emery 1958; Connor 1979; Voigt & Bashaw 1972). Conversely, in in Conversely, 1972). &Bashaw Voigt 1979; Connor 1958; Emery o e T. crinita ) andtheinbreeding coefficient (F

Accepted Article) for all the accessions was 0.209 and ranged from 0.119 (in accession 17) to 0.250 (in accession The positiveThe F values of The Chloridoideae subfamily presents high diversity of reproductive systems (Connor 1981). (Connor 1981). systems highdiversity of reproductive presents subfamily Chloridoideae The . Altogether, our results from a large number of plant materials and reproductive from aorgans number of. Altogether, andreproductive plant results large our materials Eragrostis curvula Eragrostis o the 16 SSR locianalyzed hadH per locus was 0.463. H IS suggest that andC IS ) in eight hloris IS was also positive with a mean value of 0.284. apomictic and sexual individuals coexist, as evidenced as evidenced coexist, individuals andsexual apomictic T. crinita ci duewere to self-fertilization. homozygous T. crinita cal characters, conclusively indicate that e were used to determine the reproductive biology biology reproductive the determine to used were for each locus ranged from 0.219 to 0.785 with accessions are H autogamous. accessions. Meanobservedheterozygosity o =0, H e =0 andone allele perlocus, i.e., all Cynodon o ) and expected heterozygosities heterozygosities ) andexpected and Eleusine o only sexual values for IS for each

T. crinita This article isprotected Allrights bycopyright. reserved. Accepted 1999). (Naumova ovaries sectioned to compared improved, images the are clarity andcontrast of Additionally, processing samples. inovules more the allows is it It and, quicker sac dissection technique. time-wise, the for embryo analysis over advantages used has andfinallystained study inthis (Feder clearing & technique microtome O´Brien The 1968). are dehydrated, inflorescences cleared inanalcoholwhich sliced embedded, series, with a (Bhanwra 1988). Arundinoideae and Pooideae inChloridoideae, hasbeen behavior observed This commonly 4). insome alateral to have displacement position antipodals observed We structure (Holloway2008).Friedman & proliferationlevels of antipodal are adaptive areor aneutral variation of female gametophyte unclear modest it However, remains whether Bhatnagar (Kapil 1981). & taxa certain of antipodals sporophytic tissue is certainly congruent with this view, asis the presence of wall ingrowths in the (Johri and endosperm gametophyte female the Articlehypothesis is that antipodals function as transfer but demonstrated, accepted the widely more conclusively has antipodals been not of role this name to proposed (1984) species of Chloridoideae (Bhanwra 1988; Strydo formation of a mass antipodalsof cells, ananti or accessions (6, antipodal cells numerous underwent and21), 17 divisions inthe resulted which FCSS. Themorphology of embryo sacs inall 20 2010). reported (Rachie & hasbeen Pe reproduction Embryological analyses have been traditionally performed using the dissection method, in usingthe method, dissection performed have been analyses traditionally Embryological analyses along with embryological through sexuality study, wasdetermined present In the Polygonum type of embryo sac as ‘Poaceae variant’. The physiological ters 1977; Strydom & Spies 1994; Subba Reddi Spies 1994; & Strydom 1977; ters accessions analyzed was very similar. Insome et al. m & Spies1994). Furthermore, Anton & Cocucci cells to transport nutrients from the sporophyte to podal complex, asit was demonstrated in others 1992). The position of antipodals near the the near antipodals of position The 1992). T. crinita accessions (3 and et al . This article isprotected Allrights bycopyright. reserved. Mendoza, like in many arid regions, brief high-intensity rainfalls are frequent (Hudson 1987). rainfallsare arid (Hudson inmany frequent high-intensity like Thus, 1987). brief regions, Mendoza, conditions)pollination could bepartially explainedby environmental factors. During in summer pollination both treatments, whereas for allogamous plants similar for seed set autogamous (because results under were yields unexpected expected wasconditions significantly higher(P<0.05) andautogamous.compatible Inaverage for a showed exclusively 2C/3C histogram peaks for each one of the 21 analyzed accessions. nucleus apomict in Our results of male withmeiosis restitution microsporocytes. pseudogamous to 6C DNA content when pseudogamy involves an unreduced sperm cell, as isthe casefor a correspond peak would endosperm the However, pseudogamy. upon depend does not development cell peak should (2n sperm + andareduced endosperm + ifits the be nuclei 2n n).However, 4C polar unreduced two syngamy of involving by pseudogamy be develops could 5C ifit endosperm the by develop to is expected embryo the diplospory because in should 2C always be embryo Inaddition, or 2C/6C. the 2C/4C been 2C/5C, have should peaks the embryo/endosperm namely be expected, would histogram features other formation, discardedseed was should pathway be Ifdiplospory inthis diplospory of the material. development, classifiedusually asthe sacs embryo regular consistent meiotic was with cytologically observed of allmegagametophytes feature the distinguishing confirmation that allowed inthis study performed FCSSanalysis The complementary in bethe same to ovules. mature expected unreduced sacs from diplosporous embryo sexual Accepted andseedflowers set under pollination open conditions Article Results from controlled pollination experiments demonstrated that demonstrated pollination experiments from controlled Results diplosporous to also sacs correspond fact the may embryo that 8-nucleate Despite differentiate inanthesis, not could reduced sacs observations we performed embryo We Polygonum type. than under open-pollination than under open-pollination conditions. These ll the accessions, seed set underself-pollination parthenogenesis from parthenogenesis from cell,an unreduced but egg that are typical grasses, reproducing sexual of sacs, because their sacs, are their features morphological because higher seedset were expected under open are likelymore to benegatively-affected by T. crinita is self- under open-pollination conditions),under open-pollination indicating higher seedset under self-pollination condition (o efficiency)pollination flowers andseeds. systems (Charlesworth remain unbagged. ifinflorescences behigher would treatment’ interaction suggeststhat for some ac seed production can be achieved. Conversely, in if bagged, for seedproduction, andsuggest are that the yields higher inflorescences important areflowers exclusively exposed to –andfertilized autogamous autogamous tetraploid species, such as This article isprotected Allrights bycopyright. reserved. (0.284) and H andprofuse flowering, inflorescences millets (Gupta observed in development floral pattern of general The Sicard 2011). & Lenhard &Rausher 2013; (Duncan lifefloral spans,small flowers, andsmall anther increased seed set, as compared in self-pollinated inflorescences. ofrainstorms, causing loss andseeds becaus flowers Accepted that evidence additional provide 1992). Schoen & Lloyd 2003; (Barrett spontaneously favor selfingandprobablyto cause acertai Article Regardless of the underlying cause, the vast majority of the accessions had significantly significantly had accessions the of majority vast the cause, underlying the of Regardless Floral morphology and temporal development have a breeding on have influence plant strong development andtemporal Floral morphology Our results analysis from of microsatellite loci T. crinita T. crinita o et al. (0.463) values, which are similar toprevious estimates F of native (Whalley Australian 2012) grassspecies and many was similar to that described for other et al et . 2006;. 1992). Dafni The ob Presumably, the bagging inthese T. crinita to open pollination, reducinglo by as observed in this for study Elymus alaskanus Elymus alaskanus is autogamous, as indicated by the obtained mean F mean by obtained as isautogamous, the indicated n amount byof self-pollination geitonogamy the observed significant‘genotype xpollination that higher seed se -stigma separation, are typical of selfing species cessions, namely accessions 8 and 13, seed yield by- by- their ownpollen. These results may be nly nly of2 the accessions 20 had higher seedset e inflorescences were not inside a paper bag, not inside were apaper e as inflorescences for estimating heterozygosity and inbreeding served served floral traits in (Sun self-pollination grasses,self-pollination includingsmall et al t is obtained whenis obtained t lf-pollination treatment promoted T. crinita . 2002)and ss pollen (andof thereby favoring IS andH T. crinita accessions, are believed et al Agave lechuguilla o . 2013). Dense in other T. crinita , suchasa short

IS

tectorum This article isprotected Allrights bycopyright. reserved. Poaceae, such as (Silva-Montellano & Eguiarte 2003). When comparin through emasculation in controlled crosses. crosses. incontrolled emasculation through pollination experimentsof at testingaimed the degree of geitonogamy andallogamy, for example, possible. Knowledge on the floral biology of (Shivanna completely & and,2014) Tandon outcrossing degree acertain is thus, of outcrossing rates. higher had outcrossing significantly autopolyploids whereas selfing, predominantly were allopolyploids polyploids, species. Among diploid corresponding heterozygous (Table 3). Inthe present st -ifnotpredominantly exclusively- via self-fert whereas the remaining 10 loci were homozygous. Thus, even though Schemske 1997). (Husband& diploids issl rise inhomozygosity Hence, the 1938). Wright (Haldane segregation) 1930; of pattern the by (depending increases on 17-21%, homozygosity a heterozygous at diploid homozygosity selfing, of nature polyploid by the explained be H may occur (Siol species. However, as observed in other autogamous andmolecular 1084) marker analysis (N= 78), suggest the fact progenies were evaluated number that despite alarge of found, o

Accepted Article ge andhigher less higher, inbreeding are suggesting An important feature of autogamous plants is that their floral morphologies do not prevent According to Husband (Ramakrishnan et al. Eleusine indica 2008). 2008). et al . 2006), we noted that F et al (Chong (Chong . (2008) polyploids have lower outcrossing rates lower than have outcrossing polyploids their . (2008) Six of the 16 SSR loci displayed high values of heterozygosity, heterozygosity, high of displayed SSRvalues 16 loci the Six of . et al udy, no udy, no evidence for cross fertilization in T. crinita . 2011), ilization, a small fraction theof lociremained locus increases by 50%, ower andinbreedingislower in tetraploids thanin grass species, outcrossing events, although rare, g our results with those obtained in diploid Setaria italica Setaria generated in this study in this generated IS ing thatcross-fertilization rareis very inthis ne diversity than diploids. This behavior may behavior diploids. This than diversity ne values in values In polyploids, after of In polyploids, one generation T. crinita (Kumari (Kumari T. crinita while intetraploids via are lower, whereas H et al morphological (N = (N morphological will facilitate the design accessions reproduces . 2011), T. crinita Bromus was e and REFERENCES deInvestigacionesNacional Cien is of the afellow Consejo Carolina Perla Kozub and06A/569. 06A/476 de Cuyo, Nacional Proyectos from by Secretaría deCiencia, was work supported grants Técnica y The Universidad Posgrado, ACKNOWLEDGEMENTS a inthis broad studies genetic allow range species. of for particular traitspopulations (e.g.,biomass directionallypossibility of intercrossing interesting facilitatenewtolerance,development the will va of based on desi characterized previously genotypes This article isprotected Allrights bycopyright. reserved. Accepted implications. possible phylogenetic andits A.M, The CocucciA.E. grass Anton (1984) megagametophyte Argentina Anton A.,Zuloaga F.O.,Rúgolo Bulletin BiologicalSeries genus the of (1974) D.E. Anderson 581–588. andcharacterization of progeny. agronomic segregating reproductive germplasm: A.R., Quesenberry K.H., Acuña C.A.,Blount Kenworth Article Most important, from a genetics and breeding perspective, the intercross of intercross the from agenetics perspective, andbreeding important, Most . Graficamente Ediciones, Cordoba, Argentina. Plant Systematics andEvolution, Systematics Plant , 29 , 1–133. 1–133. , de Agrasar Z.E.(2012) tíficas y Técnicas (CONICET).

Chloris 146 production, forage quality, drought resistance) will rable traits, such asbiomass drought rable traits, or yield , 117–121. T. crinita rieties with combined tr (Gramineae). (Gramineae). y y HannaW.W.K.E., (2009) Bahiagrass tetraploid Flora Argentina: flora delaRepúblicaFlora vascular Argentina: genotypes to develop segregating segregating develop to genotypes Brigham YoungBrigham Science University aits ofinterest. The Crop science Crop T. crinita

, 49 , Bates D,Maechler M., MatrixL.T. (2011) http://mirrors.dotsrc.org/pub/pub/cran/web/packages/lme4/lme4.pdf http://mirrors.dotsrc.org/pub/pub/cran/web/packages/lme4/lme4.pdf This article isprotected Allrights bycopyright. reserved. Mendoza. grasses of innative andfrequency of cutting intensity different to Response A. J.B.,Dalmasso (1983) Cavagnaro botany Brown W.V., Emery W.H.P. (1958) Apomixis inthe Gramineae 233. Bhanwra R.K. (1988) Embryology in re biology evolutionary autopolyploidy. of population genetics theoretical F.(1992) Felber The J.D., Bever beyond. Barrett S.C.H.(2003. Mating strategies inflow 322–334. reproductive biology in ecotypes of the facultative apomict Barcaccia F.,SharbelG., Arzenton S., Varotto T.F., Genetic Lucchin M. Parrini and P., diversity (2006) characters, andquantitative agronomic traits. native of varieties forage the among grass Cavagnaro P.F., Cavagnaro J.L.,J.B., Lemes Masue 337–347. of shade to responses and biochemical morphological Physiological, S.O. J.B.,Trione (2007) Cavagnaro Cerqueira E.D., Sáenz A.M., Rabotnikof, C.M. (2004) Seasonal nutritive value of native grasses nutritive value of E.D., A.M., Seasonal Sáenz Cerqueira C.M. Rabotnikof, (2004)

AcceptedTrichloris crinita Article , 45 Philosophical Transactions ofthe B RoyalSociety Series Transactions ofLondon Philosophical , 253–263. , 253–263. , a grassfrom the forage aridzone Argentina. of , Pappophorum caespitosum Pappophorum 8 , 185–185. 185–185. , lation to systematicslationGramineae. to of “Package ‘lme4’.” /packages/lme4/lme4. Trichloris crinita ering plants: the and paradigm plants: outcrossing-selfing ering Genome, and lli R.W., Passera C.B. (2006) Genetic diversity Trichloris crinitaTrichloris

49 , 906–918. based on AFLP markers, morphological on based AFLP morphological markers, Hypericum perforatum Hypericum

: Panicoideae. Journal of Journal Arid Environments . Deserta Annals of Botany of Annals , 358 American journal of journal American , 7 , 991–1004. , 203–218. Oxford surveysin L. Heredity , 62 , , 215– 96 , 68 , , Greco S.A., Cavagnaro J.B. (2002) Effects of drought in biomass production and allocation in three This article isprotected Allrights bycopyright. reserved. ofBotany journal methods. principles andnew some (1968) microtechnique: N.,O'brien Plant T.P. Feder malaysia. and glyphosate-susceptible J.L.,WickneswariChong R., S. Ismail B.S., Salmijah System. Breeding of Plant Evolution (2006) D. Charlesworth Garden Botanical Graminae. Systems inthe H.E. of Reproductive Evolution (1981) Connor 547–574. H.E.Connor (1979) Breeding systems inthe grasses: a survey. Argentine Calden Forest Range. AcceptedScience in selfing T.M., Rausher Evolution of the M.D. syndrome Duncan (2013) Rosaceae). in polyploid cytotypes sexual into and apomictic differentiation DobešCh., MilosevicA., Prohaska Scheffknecht D., http://www.infostat.com.ar Nacional de FCA, URL GrupoInfoStat, Córdoba, Universidad Argentina. (2014) Rienzo J.A.,Casanoves F.,Di BalzariniM.G., M., Robledo L., Tablada Gonzalez C.W.versión InfoStat pp. Dafni A. (1992) Article , 4 , 1–8. Malaysian Applied BiologyJournal Malaysian Annals ofBotanyAnnals Pollination ecology: a practical approach apractical ecology: Pollination , , 55 68 , 123–142. , 123–142. , 48–74. Eleusine indica Eleusine , 112 Journal of Arid Environments ofArid Journal , 1159–68. 1159–68. , (l.) from populations peninsular Gaertn (Poaceae) , 40 , 27–36. (2011). Genetic diversity of glyphosate-resistant glyphosate-resistant of diversity Genetic (2011). S., Sharbel T.F., Hülber K. (2013) Reproductive K. S., Hülber (2013) T.F., Sharbel . Oxford University. Oxford Press, New York, US: 250 Current Biology Current , New ZealandJournal ofBotany 59 Potentilla puberula Potentilla , 645–656. Ipomoea Annals ofAnnals the Missouri , 16 , 727–735. . Frontiers in Plant in Plant Frontiers (Potentilleae, American , 17 , This article isprotected Allrights bycopyright. reserved. pp. GmbH: Heidelberg 1174 Berlin Springer-Verlag, Ambegaokar B.M., K.B., (1992) Johri Srivastava P. neopolyploids. andinsights patterns from current duplication: of consequences genome (2008) Mating S.L.,L. Pollock B., Husband B.C.,Ozimec Martin Evolution Epilobium angustifolium of (Onagraceae): implicatio populations andtetraploid indiploid inbreeding The (1997) D.W. effect Husband B.C.,Schemske of theof United Nations, Rome, Italy, FAOSoils Bulletin 57. Hudson N. (1987) plants. diversification of early monocotyledonous the on S.J.,Friedman Holloway W.E. Embryological (2008) features of Millets. S.,AgrawalGupta A.,Sood BhattP.K., J.C.(2012) apomixis. gametophytic U.,Nogler G.A.,Grossniklaus sex to avoid (2001) genetic How van Dijk the P.J. control of three varieties of J.B.(2005)Greco S.A.,Cavagnaro Growth characte Ecology varieties of Accepted Theoretica (1930) J.B.S. Haldane Article Journal rangeland The , The European Journal of Plant andBiotechnology EuropeanThe Science JournalofPlant 164, , 51, 737–746. Trichloris crinita Trichloris 125–135. Trichloris crinita Trichloris Soil and water conservation in semi-arid areas. areas. insemi-arid conservation and Soil water , 27 The cell plant , 135–142. (Poaceae) a forage grass from the arid Monte region of Argentina, l genetics of autopolyploids. (Poaceae), aforage grass native to the aridregions Argentina. of , 13 , 1491–1498. , 1491–1498. Floral biolog inSmall System and Pollination Floral biolog Comparative embryology ofangiosperms International Journal Plant Sciences Plant Journal International ristics associated with biomass inproduction n for the genetic basis of inbreeding depression. basis inbreeding depression. genetic of n for the Annals ofbotany Annals Journal ofGenetics Journal Tofieldia glutinosa Tofieldia , Food and Agriculture Organization 6 , 80–86. , 102 , 167–182. , 22 and their bearing bearing and their , 359–372. , 169 , 195–206. , 195–206. . Plant Plant Argentina de Biología, San de Juan, Argentina. Argentina This article isprotected Allrights bycopyright. reserved. tetraploid accessions of and indiploid andsexuality (1999) Apomixis M. Wagenvoort T.N, M.D., Hayward Naumova acids research plant DNA. of Rapidisolation highmolecular weight W.F.Murray M.G., Thompson (1980) ( variationBiogeographic in genetic apom variability, Molins M.P., Corral J.M., Aliyu O.M., Koch M.A., Betzin A., Maron J.L., Sharbel T.F. (2014) regionsIndia. of representative collection of foxtail [ millet Kumari R., Dikshit N.,Sharma D., of Journal Plant Sciences International D.G., (1992) SchoenD.J. Lloyd Selfandcross- engineering. Y., Carman J.G.,Dresselhaus T.(Eds), procedures A. O., Screening (2001) Mazzucato Leblanc para C.,Cavagnaro P.F., Desarrollo J.B.(2011) de Cavagnaro microsatélites moleculares marcadores Kozub plants. Kapil R.N., Bhatnagar A.K. (1981) Ultrastructure a seeds of monocots and dicots. dicots. and monocots of seeds using pathways screen Anefficient mature for I.(2000) reproductive F., A.,Schubert Meister Matzk Accepted perforatum Hypericum Article Trichloris crinita Trichloris International Review ofCytology International CIMMYT,IRD, EuropeanCommission DGMexico: VI 121–134. , 8 , 4321–4326. Physiology and Molecular Biologyof Plants andMolecular Physiology , una forrajeragramínea del Monte argentino. XIIIJornadas delaSociedad ) across its and introduced native range. Brachiaria decumbens Brachiaria Plant Journal Plant Bhat, K.V.(2011) Analysis of geneticmolecular ina diversity The flowering of apomixis: from geneticThe of mechanisms to flowering apomixis: , 153 , 70 Setaria italica Setaria , 358–369. , 358–369. , 291 –337., 291 , fertilization inplants.I.Functional dimensions. 21 . Sexual Plant Reproduction Sexual Plant , 97–108. nd biology of the female gametophyte in flowering inflowering gametophyte female the of nd biology ixis expression and ploidy wort of St. John's to identify and quantify apomixis. In: Savidan In: apomixis. and quantify identify to (L.) Beauv.]P. from different agro-ecological , 17 , 363–374. Annals of Annals Botany , 12 , 43–52. , 43–52. , 113 , 417–27. Nucleic Sur, Buenos Aires. Argentina: pp. Sur, Buenos 611 This article isprotected Allrights bycopyright. reserved. 201–239. Roig F.A. (1971) Flora y vegetación de la Reserva Forestal de Ñacuñán. La vegetación. Biology microsatellite in variation western North Americanpopulations of Ramakrishnan A.P., Meyer ColemanS.E., FairbanksD.J., C.E.(2006) Ecological significance of pp. (ICRISAT), India: Tropics Semi-arid 187 for the Hyderabad, Research Institute Rachie K.O., Peters L.V. (1977) áridos. E.,Quiroga Blanco L., Orionte E. inArgentina.grazing capacity C.B., Borsetto O., Passera on influences Candia andseedling C.R.(1992) R.J.,Stasi Shrub control from Cáceres M.E., F.(2013) Pupilli, Harnessingapomicti QuarinC.L., J.P.A., Ortiz S.C.,AcuñaC.,Pessino Martínez F., Sartor E.J.,Espinoza Hojsgaard D., M.E., grass. in reproduction of the mode determining S.C., J.P.A., Ortiz Pessino Leblanc O.,Hayward M.D., Quarin (1997) fingerprinting for Genetic C.L. E.G.,Nicora de Rúgolo AgrasarZ.E.(1987) AcceptedGrasses: Chloridoideae. New Classification S.J.(2007) of andBiogeography World J.T., Columbus Pennington P.M., Peterson Article Paspalum Theoretical andappliedgenetics Theoretical , Ecología Austral 21 , 61–73. . Annals of Annals botany , Aliso (A Journal of Aliso (AJournal Botany) Systematic and Evolutionary 19 , 107–117. Journal ofRangeManagement Journal The Eleusines: A review ofthe world literature. (2009) Evaluación de estrategias derehabilitación depastizales , 112 , 767–787. , 95 Paspalum notatum Paspalum , 850–856. 850–856. , Los géneros austral deAmérica de gramíneas c reproduction ingrasses: what we havelearned , , a forage apomictic subtropical 45 , 480–482. 480–482. , Bromus tectorum Bromus International International Crops , 23 , 580–594. . Plant Species Species Plant Deserta . Hemisferio , 1 , This article isprotected Allrights bycopyright. reserved. B.,Von Sun G.L.,Salomon Bothmer R. (2002) Microsatellite andgenetic polymorphism grasses. (201Subba ReddiC.,RajuN.S.N.,Rao M.V. (Poaceae). tribe of the representatives sacin some development Embryo A.,Spies J.J.(1994) Strydom 517–525. a in casestudy history selfing populations: the of understand to pattern helps genotypic How Bonnin J.(2008) J.M., M.,multilocus Prosperi I.,Ronfort Siol inbreeding estimates”. lechuguilla (Agavaceae) inthe Chihuahuandesert Agave in ecology reproductive of A.,Eguiarte L.E.the patterns “Geographic (2003) Silva-Montellano Congress forage grass Argentina. arid of of the zones native of ecotypes inthree values andnutritive Productivity J.(1989) Colomer Silva basis inplants. of morphological adaptation evolutionary a and genetic for studying the syndrome: The selfing model (2011) Sicard A.,Lenhard M. pp. India:170 Delhi, R. Tandon K.R., (2014) Shivanna chromosome numbers. Roodt R.,Spies J.J.(2003) Chromosome studies Accepted Article grassland. perennial-species of semiarid a warm-season, production N.G.,Cavagnaro J.B.,Seligman M.E. Horno Simulation(1992) defoliationof effects primary on Nordic Journal ofBotany, Nordic , Montpellier, France, 815–816. 815–816. France, , Montpellier, American ofBotany Journal Taxon Bothalia, , 52 Reproductive ecology of flowering plants: A plants: Reproductive manualecology offlowering , 557–583. , 557–583.

28 24 , 354–365. 354–365. , , 101–105. , 101–105. in the grass subfamily Chloridoideae. I.Basic 0) Pollination andseed0) Pollination set intropical wetland . II.Genetic differentiation,variation, and Proceedings of the XVI International Grassland Proceedings ofthe InternationalXVI Grassland , 90 ,

700–706. 700–706. Annals ofBotany Annals Medicago truncatula Ecological Modelling , 107, Trichloris crinita Trichloris 1433–1443. . Heredity . Springer,New , 60 , , a , a , 45–61. 100 , Voigt Voigt BashawP.W., E.C. (197 This article isprotected Allrights bycopyright. reserved. ofSciences Academy National polyploids. inof populations frequencies gene distribution of The S.(1938) Wright the usinglocal importance provenances. reassessment of of I. Chivers R.D.B., Whalley (reserva ecológicaMendoza Ñacuñán). de plantas Valor del S.(1969) nutritivo forrajeras Gonzalez deP., este Wainstein la de provincia Resources tetraploid microsatellite data: comparison with TETRA and TETRASAT. Van K., ATETRA, programGeert analyse L. to software (2010) A.,Triest anew Van Puyvelde Evolution Norwegian populations of inthree differentiation Young B.A.,Sherwood R.T.

Accepteddetecting aposporous ingrasses. apomixis Article , , 234 10 , 331–334. , 101–110. , 101–110. H., Waters C.M. (2013) Revegetation with Australian native grasses–a , Bashaw E.C. (1979) Cleared-pistil 6) Facultative6) Apomixis in , 24 , 372–377. Revista delaFacultad Agrarias deCiencias Canadian Journal ofBotany Canadian Journal Elymus alaskanus Eragrostis curvula Eragrostis The RangelandJournal and thick-sectioning techniques for techniques and thick-sectioning (Poaceae). , Molecular Ecology 57 . , 1668–1672. , 1668–1672. Crop Science Crop Plant Systematics and Systematics Plant , Proceedings of the 15 , 35 , 133–142. , , 155–166. , 155–166. 16 , 803–806. 803–806. , difference at P<0.05. Seed set was calcul was set Seed at P<0.05. difference χ

². Chi-square test; df. Degrees of freedom; SE.Standa freedom; of df.Degrees test; ². Chi-square

This article isprotected Allrights bycopyright. reserved. Accepted Article conditions pollination Table 1. Source Source Self-pollination 1.53 0.16 82.2 ± 82.2 2.4 a 72.0 ± 3.3 b 0.16 0.16 0.94 1.53 <0.0001 <0.0001 Open pollination 1 <0.0001 Self-pollination 18 Pollination treatment 1596.9 1515.3 18 treatmentPollination x Accession Accession 5159.1 treatment Pollination Statistical analysis fixedeffect (testof

ated as (fruit/flower ratio) x 100. χ β from GLMM)from for seedsetunder selfandopen- ²df p-value rd error; * Different letters indicate significant SE Mean ± SE Mean ±

*

This article isprotected Allrights bycopyright. reserved. Table 2. number of loci evaluated. loci evaluated. H of number Accepted Article Observed heterozygosity (H heterozygosity Observed o values are means for all the lo ceso Ho Accession N Mean 41 0.192 0.119 24 13 0.194 17 14 12 12 60.240 0.250 9 16 0.194 8 14 0.244 7 12 0.242 4 13 3 11 o ) asrevealed in8 by SSRmarkers, 31 0.209 13.13 ci evaluated ineach accession. T. crinita T. crinita accessions. N. This article isprotected Allrights bycopyright. reserved. Table 3. Accepted Article N: of accessions number SSRloci. analyzed. individual Observed (H S ou N H N Locus SSR Mean 8 0.463 0.56 0.284 0.284 0.56 80.463 Mean 0 .81 0.48 5 0 100 380320510.443 -0.029 0.561 0.486 8 0.312 -0.189 60.5 0.077 1 0.701 93 0.662 -0.274 60.833 90 0.219 3 0.611 0.785 80 77 71 72 53 11 .4 .8 0.243 0.587 6 0.447 8 o ) and expected(H e ) heterozygosities and inbreeding andinbreeding (F coefficient ) heterozygosities o H e F IS

IS ) for Figure 3. 3. Figure D. analyzed had histograms with 2 clear peaks, corresponding to 2C (embryo) and 3C (endosperm) and3C(endosperm) 2CDNA clear (embryo) peaks, 2 hadhistogramswith to corresponding analyzed 2. Figure 25 µm a inaccessions Bar antipodals complex and17. with mass of cells 6 Megagametophyte or antipodal Mature megagametophyte with antipodals ( fertile (ff) one below. fertile (ff)one 4. Figure of flowering inflorescence. contents. This article isprotected Allrights bycopyright. reserved. Central awn (ca) longer than laterals (la). stigma andawns. Purple with 1. Figure FIGURE CAPTIONS indicate nosignificantdifferences (GLMM co (OP)open conditions. pollination pointsData repr 5. Figure spike.

Accepted Article Accession 24 (sexual 2C/3C)

synergids ( Floral morphology in morphology Floral Embryo sac morphologies of sac morphologies Embryo Flowering characteristics of FCSS histograms from cell bulked nuclei samples seed of of Seed set percentage variation among 19 A. A. Accession 3 (sexual 2C/3C) C. sy Anthesis in half of the inflorescence (or middle of anthesis). ), egg cell ( D. 3-awned lemma, central lemma, (ca) the than 3-awned awn (la). islonger the laterals G. Purple stigma and purple-green, pubescent glumes. glumes. pubescent stigma andpurple-green, Purple ec T. crinita ), polar nuclei ( Trichloris crinita. Trichloris T. crinita accessions. B. I. I. Accession 8 (sexual 2C/3C) Purple stigma.Purple an ntrast, P>0.05) between treatments. pollination pn ) in lateral position inaccessions 3 and 4. accessions. ) and antipodals ( ) and antipodals esent means±SE of seed set percentage. Asterisks Trichloris crinita A. Purple awns.

A, B. J. White stigma. White Beginning of flowering in the apex of the A, B. Polygonum an accessions under self accessions self under (AP) and B, C. ) in accessions 1and 22. T. crinita. C. Accession 21 (sexual 2C/3C) One sterile floret (sf) and a K. type megagametophyte megagametophyte type D. D. Green spike. All the accessionsAll the Full flowering. H. White awns. E, F. L. Purple C,D. E, F. E. End

This article isprotected bycopyri Accepted Article g g ht. Allrights reserved.

This article isprotected Allrights bycopyright. reserved. Accepted Article

This article isprotected bycopyri Accepted Article g g ht. Allrights reserved.

This article isprotected bycopyri Accepted Article g g ht. Allrights reserved.

This article isprotected Allrights bycopyright. reserved. Accepted Article