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Scientia Horticulturae 167 (2014) 76–83

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h om epa ge: www.elsev ier .com/ locate /sc ihor t i

nreduced embryo sacs escape fertilization via a ‘female-late-on-date’trategy to produce clonal seeds in apomictic crabapples

an-Dan Liua,1, Mou-Jing Fanga,1, Qing-Long Dongb, Da-Gang Hua, Li-Jie Zhoua,uang-Li Shac, Zhong-Wu Jiangd, Zhi Liue, Yu-Jin Haoa,∗

National Key Laboratory of Crop Biology, National Research Center for Apple Engineering and Technology, College of Horticulture Science and Engineering,handong Agricultural University, Tai-An, Shandong 271018, ChinaInstitute of Pomology of CAAS, Xingcheng, Liaoning 125100, ChinaQingdao Agricultural Academy, Qingdao, Shandong 266100, ChinaYantai Agricultural Academy, Yantai, Shandong 264100, ChinaLiaoning Agricultural Academy, Xiongyue, Liaoning 115009, China

r t i c l e i n f o

rticle history:eceived 25 September 2013eceived in revised form0 December 2013ccepted 30 December 2013

a b s t r a c t

Tea crabapple (Malus hupehensis Redh. var. Pingyiensis) is a typical facultative apomictic apple root-stock. Its asexual seed formation is affected by environmental temperature; however, little is knownregarding the mechanisms underlying this phenomenon. In this study, we found that unreduced embryosacs escaped fertilization due to a developmental delay, which is referred to as the female-late-on-date(FLD) strategy. The percentage of FLD was positively correlated with the apomictic capacity and wasgreatly affected by the environmental temperature during blossoming and pollination. Temperature also

eywords:ea crabapplepomixispomeiosisarthenogenesismbryo sac

affected the FLD and apomictic capacity by altering pistil longevity. Taken together, FLD is a crucial devel-opmental event for apomixis in apomictic crabapples, which prevents the unreduced embryo sacs frompollination and fertilization. Artificial manipulation of FLD by shortening pistil longevity may be used tocontrol the percentage of apomictic seeds, which is helpful not only for uniform seedling production butalso for the apomictic breeding program.

elay

. Introduction

Reproduction is a dynamic process in flowering plants that reliesn the formation of inflorescences, fertilization, and subsequenteed formation. Most flowering plants undergo sexual repro-uction to produce hybrid seeds and offspring. However, somengiosperms have evolved an alternative form of reproductionnown as apomixis, which is a form of asexual reproduction thatnables the formation of genetically uniform maternal populationsBicknell and Koltunow, 2004). In nature, there are approximately00 flowering plant taxa, including members of 35 diverse plantamilies of apomictic plants (Carman, 1997). Within the past fewecades, Hieracium, Tripsacum, Boechera have been used as models

or herbaceous plants to study the developmental events and genet-cs of gametophytic apomixes (Bicknell and Borst, 1994; Koltunowt al., 2000). Typically, nucellus, integument, and epidermis cells

Abbreviations: FLD, female-late-on-date; LSCM, laser scanning confocal micro-cope.∗ Corresponding author. Tel.: +86 538 824 6692.

E-mail address: haoyujin@sdau.edu.cn (Y.-J. Hao).1 1These authors contributed equally to this work.

304-4238/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.scienta.2013.12.035

© 2014 Elsevier B.V. All rights reserved.

from the ovule can initiate apomixis in plants (Crane, 2001).Cells, which initiate early in ovule development, generally undergogametophytic apomixis and finally form unreduced embryo sacs.According to the origin of the cell that initiates unreduced embryosac formation; gametophytic apomixis is usually subdivided intotwo forms, diplospory and apospory. When embryo sac formationinitiates from the MMC (megaspore mother cell) cells, it is known asdiplospory. In contrast, if an unreduced embryo sac formation ini-tiates from a position other than the MMC, it is known as apospory.In addition, more than one cell can form multiple embryo sacsin an individual ovule (Koltunow and Grossniklaus, 2003). How-ever, both types of apomixis can and do exist in the apomicticspecies of Beta and Rosaceae (Nybom, 1988; Jassem, 1990). In someaposporous grasses, citrus, and specific Hieracium, apospory andadventitious embryony coexist (Koltunow and Grossniklaus, 2003).

Compared to the sexual process, apomixis demonstrates threecritical components, e.g., apomeiosis (meiosis is avoided priorto embryo sac formation), parthenogenesis and endosperm for-mation, which requires specific developmental adaptations and

distinguishes apomictic reproduction from the sexual pathway.The reproductive developmental processes between sexual repro-duction and apomixis are not mutually exclusive. Partial apomictsretain the capacity to sexually reproduce and are thus defined as

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D.-D. Liu et al. / Scientia H

acultative apomicts (Koltunow and Grossniklaus, 2003). In Pen-isetum, Crataegus, and Hieracium, the initiation of apospory results

n the abortion of the concurrent sexual process. However, inrachiaria, both sexual and aposporous embryo sacs exist in theame ovule. As a result, successfully initiating embryogenesis inmbryo sacs derived from both pathways can result in a polyem-ryonic seed (Araujo et al., 2000; Koltunow et al., 2001).

In Rosaceae, particularly the polyploidy species of the genusalus, such as the tea crabapple (Malus hupehensis var. Pingyiensis),

pomixis in the form of apospory is widespread (Schmidt, 1977).heir seedlings are genetically uniform with maternal plants, andave thus been used as apple rootstock in the past half century

n China. It is a major concern for these apomictic crabapple root-tocks to produce uniform seedlings by promoting the formation ofpomictic seeds. However, Rosaceae plants perform typical faculta-ive apomixes; thus, additional fertilization also occurs (Sax, 1959).ecombinant progeny can be generated in varying amounts byrossings or open pollination (Bisognin et al., 2009). Furthermore,nvironmental cues can affect apomictic reproduction (Burton,982; Houliston et al., 2006). However, it is largely unknownhy and how this occurs. In this study, following the identifica-

ion of apomeiosis and parthenogenesis in facultative apomicticea crabapple, the developmental and genetic features of thenreduced embryo sacs were investigated using microscopic obser-ation and genetic segregation. Factors that affect pistil longevitynd apomixis were also identified. Finally, the use of the develop-ental feature identified in this study was discussed in a seedling

roduction and breeding program of apomictic apple rootstocks.

. Materials and methods

.1. Plant materials

The apomictic crabapple apple species used were the triploidea crabapple (Malus hupehensis Redh. var. Pingyiensis; 3X = 51),etraploid M. Sargentii and M. Xiaojinensis (4X = 68). Triploid culti-ar ‘Jonagold’ (3X = 51) performs sexual reproduction was used asontrol. A hybrid population was obtained from a sexual cross withrtificially hand-made pollination using apomictic tea crabapples maternal plant and sexual ‘Maypole’ apple (diploid; 2X = 34) asollen donor. All plant materials were grown in the experimentalrchard of Shandong Agricultural University.

.2. Floral stages, pistil decapitation, and apomixes evaluation

Floral development was divided into 6 stages for convenience.tages 1–4 refer to flowers before blossom. The flowers began tolossom from stage 5 and were then ready to receive pollens forollination. Subsequently, the petals fell down at stage 6, indicatinghat the pistils turned senescent and had lost their capacity to com-lete pollination. From year 2007 to 2009, pistil decapitation wasrtificially performed at stage 4 with scissors to avoid pollination.pomictic seed settings were surveyed at autumn.

Apomixes comprises two components, e.g., parthenogenesisnd apomeiosis, in apomictic crabapples. Parthenogenesis includesertilization-independent embryogenesis and endosperm forma-ion. To examine parthenogenesis percentage, pistil decapitationas performed at spring at stage 4. Compared with the total num-

er of decapitated flowers, numbers of parthenocarpic fruits whichould produce seeds in the absence of fertilization were calculated

s parthenogenesis percentage.

To examine apomeiosis percentage, the asexual seeds were har-ested from parthenocarpic fruits and used for ploidy analysissing a flow cytometry assay. The percentage of triploid seeds was

lturae 167 (2014) 76–83 77

calculated as apomeiosis percentage, compared with the total num-ber of seeds tested.

2.3. Ploidy determination using flow cytometry

Ploidy of plants was determined with flow cytometry accord-ing to the instructions of Partec CyStain UV Precise T reagent kit(PARTEC, Cod. 05-5003). Firstly, Young leaves or embryos frommature seeds were collected and chopped with a razor bladein nuclei extraction buffer, following by the addition of stainingbuffer with 1/5 volume of nuclei extraction buffer according toinstructions. Subsequently, nuclear suspensions were filtered twicethrough a 20-um nylon mesh to remove debris and to collect nuclei.At last, the fluorescence intensity of the nuclei was measured usingan arc lamp-based flow cytometer (PARTEC PA, Germany). A total of30,000 nuclei were measured for each cytogram. At least three dif-ferent plants for each line were measured, and each sample analysiswas repeated three times.

2.4. Laser scanning confocal microscope (LSCM) observation ofembryo sacs

Embryo sacs were observed using a modified whole stain-clearing technique with LSCM (Yang, 1986; Herr, 1971). Ovules atdifferent stages were dissected from the ovary and immediatelyfixed overnight in solution of formalin-acetic acid-alcohol (FAA;10% ethanol, glacial acetic acid, and 37% formaldehyde at a ratioof 18:1:1) at 4 ◦C. The fixed samples were hydrated by passingthe samples through an alcohol series (70%, 50%, 30%, and 10%).The samples were then washed 3 times using distilled water for20 min, and stained with a 0.5% sucrose eosine solution. The sam-ples were then dehydrated through a graded alcohol series (10%,30%, 50%, 70%, 90%, and 100%) and cleared in a mixed solution ofmethyl salicylate and alcohol (1:1). The samples were then clearedin 100% methyl salicylate for 20 min. The embryo sac samples wereobserved using a laser scanning microscope (Zeiss LSM510 META,Jena, Germany) with a 488 nm argon laser. Image analysis wasperformed using a Zeiss Axio Vision, Zeiss CLSM-5, and Adobe Pho-toshop 7.0.

2.5. Determination on maintaining time of pistil longevity

To artificially manipulate pistil longevity, in vitro and in vivoexperiments were conducted. In experiment in vitro, branches thatbear flower buds ready to bloom were detached from the same treeand placed to growth chambers at different temperatures including16 ◦C, 21 ◦C, and 25 ◦C, respectively. In experiment in vivo, flowersjust blossoming were sprayed with H2O, anti-staling agent (30 g/Lsucrose, 2 g/L CaCl2, 100 mg/L borax, 100 mg/L GA, 200 mg/L 8-hydroxyquinoline), and 1 baume lime-sulfur solution (23 baumesolution is composed of quicklime, sulfur power and water at aratio of 1:2:10).

The pistil longevity of flowers was determined by observing ifthe pollens germinate and produce pollen tube on the pistil. Fluo-rescence staining of pollen tubes was performed as described by Shiet al., (2005). The pistils were cleared in 10% chloral hydrate at 65 ◦Cfor 5 min, washed with distilled H2O, softened with 5 M NaOH at65 ◦C for 5 min, and then washed again with distilled H2O. The pis-tils were treated with 0.1% Aniline Blue in 0.1 M K3PO4 buffer pH 8.3

for 3 h in darkness and washed with 0.1 M K3PO4 buffer. The pistilswere then mounted onto a microscope slide using a drop of glyceroland carefully pressed under a cover slip. The pistils were observedusing a fluorescence microscope (Olympus; CKX41-F32FL; Japan).

78 D.-D. Liu et al. / Scientia Horticulturae 167 (2014) 76–83

Table 1Number of seeds per fruit and percentages of parthenogenesis and apomeiosis in2007, 2008, and 2009.

Year Percentage ofparthenocarpy (%)

Number ofseeds per fruit

Percentage ofapomeiosis (%)

2007 94.4 ± 5.8 1.9 ± 0.2 94.6 ± 4.82008 92.5 ± 3.5 2.0 ± 0.3 95.5 ± 3.52009 94.2 ± 3.6 3.0 ± 0.2 96.5 ± 1.4

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Table 2Percentages of FLD (percentage of immature embryo sac) in apomictic crabapplesand sexgual ‘Jonagold’ apple in 2009.

Species FLD (%) DAB (stigma lost the viability toaccept the pollen)

Jonagold 3.3 ± 0.8 3 ± 1Tea crabapple 87.9 ± 4.2 4 ± 1M. Sargentii 74.1 ± 2.4 3 ± 1M. Xiaojinensis 75.4 ± 3.5 3 ± 1

he data represents the mean ±SD of three independent experiments each with 200owers, fruits, and seedlings.

. Results

.1. Tea crabapple undergoes facultative apomixes

Tea crabapple performs facultative apomixes under natural con-itions and produces no pollen in the stamen. To obtain sexualybrids, artificial pollinations to tea crabapple pistils were per-

ormed with another wild apple (M. pumila Mill. var. ‘Maypole’)s the pollen donor. Its red leaf, which is controlled by a domi-ant heterozygous locus, was used as a morphological indicatoro identify hybrids in the progeny population (Fig. 1A). Within theast seasons, a total of 125 red-leaf hybrids were obtained from700 offspring, indicating approximately 14.7% hybrids (two timeshe red-leaf offspring due to the dominant heterozygosis of theed-leaf phenotype) in tea crabapple progeny populations. Thus,he apomictic percentage of the tea crabapple was approximately5.3%.

.2. Tea crabapple displays a high capacity to performarthenogenesis and apomeiosis

It has been well documented that apomixis has three com-onents, e.g., apomeiosis (meiosis is either suppressed or imper-ect), fertilization-independent embryogenesis, and endospermormation. To examine parthenogenesis, including fertilization-ndependent embryogenesis and endosperm formation, pistilecapitation was performed from 2007 to 2009. We found thathe tea crabapple established approximately 94% of parthenocarpicruits, which produced seeds in the absence of fertilization, indicat-ng that it has a high capacity to perform parthenogenesis (Table 1).

Subsequently, the asexual seeds harvested from parthenocarpicruits obtained from 2007 to 2009 were examined for ploidy using

flow cytometry assay to verify if the apomeiotic embryo sacad formed and subsequently produced an unreduced egg fromuppressed or imperfect meiosis. All of the tested asexual seedsriginated from unfertilized eggs, such that each seedling exhib-ted ploidy that was precisely identical to its original egg. Theseesults showed that approximately 95% of offspring seedlings wereriploid similar to the tea crabapple (Table 1), suggesting that theea crabapple performed apomeiosis. Thus, the tea crabapple had aigh capacity to perform parthenogenesis and apomeiosis.

.3. 2n embryo sacs are capable to normally fertilize in apomicticea crabapple

After several seasons of artificial pollination, a total of 125ybrids were obtained. Subsequently, tea crabapples were exam-

ned for ploidy using the flow cytometry assay. These resultshowed that 120 hybrids were tetraploid, indicating that they hadriginated from fertilized 2n triploid embryo sacs. In addition, only

hybrids exhibited cell DNA content (Fig. 1E) between triploid

Fig. 1C) and tetraploid (Fig. 1D) in a flow cytometry assay, indicat-ng that they were from meiotic embryo sacs. Thus, the 2n embryoac tends to undergo normal fertilization and subsequently devel-ps into a sexual seed in the tea crabapple. However, most of these

The data represents the mean ±SD of three independent experiments each with 200embryo sacs. FLD, female-late-on-date; DAB, days after blooming.

sacs formed asexual seeds via the apomictic pathway, indicatingthat the most 2n embryo sacs are capable to escape fertilizationduring apomixes in apomictic crabapples.

3.4. Embryo sacs exhibit a developmental delay in apomicticcrabapples

To examine how the 2n embryo sacs escape fertilization, adevelopmental observation was performed during the embryo sacformation of the tea crabapple using the whole-mount transparentmethod with laser scanning confocal microscopy. A triploid applecultivar ‘Jonagold’ was used as a sexual control.

Embryo sac development was observed in the tea crabapple andJonagold according to different stages of floral morphology. In Jon-agold, ovule differentiation began at stage 1 and the megasporemother cell (MMC) differentiated near the tip of the ovule pri-mordium (Fig. 2B). During stage 2, the MMC had completed meiosisto form three megaspores (Fig. 2C). Functional megaspores under-went three cycles of mitosis to form a binucleate, four-nucleateand eight-nucleate embryo sac, respectively (Fig. 2D–G). Nearly allof the embryo sacs were mature at stage 3 and 4 of the flower.However, in the tea crabapple, sexual reproduction initiated firstand four megaspores formed at stage 3 (Fig. 2I). In contrast fromsexual embryo sac development, one or more large aposporousinitial (AI) cells, which differentiated closely to megaspore cells,were completely developed (Fig. 2I). Subsequently, four megas-pores were all degraded (Fig. 2J). A binucleate, four-nucleate andeight-nucleate embryo sac was formed after three cycles of mitosisin AI cells (Fig. 2K–M). Interestingly, two developing embryo sacswere found in one ovule (Fig. S1A–B) and dual-embryo seedlingswere also present in the tea crabapple (Fig. S1C).

Correspondingly, sexual ‘Jonagold’ produced 96.7% matureembryo sacs at the end of the pollination stage. In contrast, only12.1% of the embryo sacs matured at the same stage in the teacrabapple (Table 2), indicating that these embryo sacs should be fer-tilized and form sexual seeds. In fact, the percentage of the matureembryo sac was very close to the hybrid percentage of 14.7% inthe artificial pollination experiment in the tea crabapple. Gener-ally, the pollination stage spans from 3 to 4 days after blooming(DAB) under natural conditions. Further observations revealed thatthe tea crabapple produced approximately 50% mature embryosacs at 6 DAB when the pistils had already turned senescent andhad expired for pollination. The results suggested that the femaleembryo sac of apomictic crabapples was late on the date with themale sperm from pollen donor. Thus, the developmental delay inembryo sac maturation was hereinto referred to as “female-late-on-date” (FLD).

In addition to the tea crabapple, apomictic crabapples Malus sar-gentii (4X) and M. xiaojinensis (4X) were also observed during theirembryo sac formation. We found that both of these species exhib-

ited a developmental delay in embryo sac maturation. M. sargentiiand M. xiaojinensis produced 25.9% and 24.6% mature embryo sacsat the end of the blooming stage (Table 2). Thus, the developmentaldelay in the embryo sac was a ubiquitous phenomenon in apomictic

D.-D. Liu et al. / Scientia Horticulturae 167 (2014) 76–83 79

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ig. 1. Progeny of apomictic tea crabapple (3x) and sexual ‘Maypole’ apple (2x).

epresent the red-leaf offspring. (B–E) Ploidy determination. (B) Diploid Maypole. (C

rabapples. As a result, the pollination stage was expired when theost embryo sacs matured in the apomictic crabapples, thereby

scaping fertilization.

.5. Apomictic seed formation is promoted by the shortened pistilongevity

Under natural conditions, it was found that the percentages ofybrids were varied with environmental temperature in artificialollination studies (Table 3). Generally, the average temperaturef the pollination stage was approximately 14 ◦C; for example,4.2 ◦C in 1999, in our experimental orchard at Qingdao of Shan-ong Province in China. After artificial pollination by hands, theybrid percentage was 9.5% (8/84) in 1999. However, when theverage temperature was 12.5 ◦C in 2001, the hybrid percentageas increased up to 21.96% (47/214). Thus, the environmental tem-erature at the pollination stage was negatively correlated with thepomictic capacity in the tea crabapple.

On the basis of this phenomenon, we proposed the hypothe-is that pistil longevity varied with environmental temperature,nd was positively related with the number of mature embryo sacst the pollination stage. Branches that bear flower buds ready toloom were detached from the same tree and used to verify thisypothesis in vitro. Pollens dropped to the stigma of the fresh pis-il, and then produced pollen tubes. In contrast, pollens failed toerminate in the senescent pistil (Fig. S2). To extend the time formbryo sac development, in vitro flowering branches were placednto incubators set at different temperatures, including 16 ◦C, 21 ◦C,nd 25 ◦C. We found that the pistil exhibited the greatest longevityf up to 7 ± 1 days at 16 ◦C. The pistil longevities were 4 ± 1 dayst 21 ◦C and 2 ± 1 days at 25 ◦C, respectively. In addition, the corre-ponding percentages of the mature embryo sacs were 65.2%, 43.5%,

nd 11.7%, respectively, prior to when the stigma turned senescentTable 4). Furthermore, low temperature extended the longevity ofhe pistil and stigma, thereby making more embryo sacs mature athe pollination stage compared to high temperatures. In addition,

enotype separation in the progeny of tea crabapple and ‘Maypole’ apple. Arrowsloid tea crabapple. (D) Tetraploid hybrid. (E) 3x+ Aneuploid hybrid.

to the tea crabapple, M. sargentti exhibited a similar phenomenon,indicating that it was ubiquitous in apomictic crabapples.

However, the in vitro experiment failed to calculate the hybridpercentage because detached flowering branches could not gener-ate seeds. To artificially manipulate the longevity of the pistil ina tree under natural conditions, the anti-staling agent, lime sulfurand H2O were sprayed on to the pistils of apomictic crabapples,such as the tea crabapple and M. sargentti. We demonstrated thatH2O-treated pistils showed an intermediate longevity of 5 ± 1 daysin both tea and M. sargentti crabapples. Their pistils exhibited amaximum longevity of up to 6 ± 1 days when treated with the anti-staling agent spray, and a minimum longevity of 2 ± 1 days whentreated with the lime-sulfur spray. Furthermore, tea/M. sargentticrabapples produced 40 ± 2.9/65.1 ± 3.6 mature embryo sacs priorto pistil senescence and 21.5 ± 4.9/23.6 ±2.3 hybrids in H2O spraytreatment. Moreover, 57.3 ± 3.5/68.2 ± 2.7 mature embryo sacs and37.2 ±3.6/52.3 ± 5.1 hybrids were observed after the anti-stalingagent spray treatment, while 16.4 ± 4.2/17.3 ± 3.4 mature embryosacs and 9.7 ± 2.7/8.9 ± 2.8 hybrids were found when treated withthe lime-sulfur spray. The longer the pistil longevity, the moreembryo sacs matured prior to the pistil senescence, and conse-quently, more hybrids were generated (Table 5).

In summary, the percentages of hybrids were increased withpistil longevity in apomictic crabapples. Lime-sulfur spraying effec-tively shortened the pistil longevity and promoted the formationof apomictic seeds.

3.6. FLD is positively related with apomictic capacity which isgenetically determined by apomeiosis and parthenogenesis

In tea crabapple, the apomictic percentage, e.g., 85.3%, wasnearly equal to the product of the parthenogenesis percentage

(average 93.7% from 2007 to 2009) multiplied by the apomeio-sis percentage (average 95.5% from 2007 to 2009). Thus, thehypothesis that the apomictic capacity is attributable to twoseparate development events or genetic loci, e.g., apomeiosis

80 D.-D. Liu et al. / Scientia Horticulturae 167 (2014) 76–83

Fig. 2. Embryo sac development in apomictic tea crabapple and sexual ‘Jonagold’ apple. (A) Six development stages of apple flowers. (B–G) Female gametophyte and embryosac development in sexual ‘Jonagold’ apple. (B) MMC initiated in the ovule as indicated by the arrow. (C) One megaspore developed and the other three megaspores wereabolished. (D) Binucleate embryo sac. (E) Four-nucleate embryo sac. (F) Eight-nucleate embryo sac. (G) Mature embryo sac in ‘Jonagold’ apple. (H–M) Female gametophyteand embryo sac development in apomictic tea crabapple. (H) MMC initiated stage in the ovule. (I–J) Apospory initiation (AI) cells from ovule tissue. (K) Dual-nucleate embryosac. (L) Four-nucleate embryo sac. (M) Immature embryo sac in tea crabapple flower that was out of pollination stage. Scale bars represent 50 �m.

Table 3Effect of temperature on hybrid seed obtained from cross combination of tea crabapple and Maypole after artificial pollination in 1999 and 2001.

Pollination period (month/date/year) 4/24–26/1999 4/20–27/2001

Percentage of hybrid seed (hybrid seed number/total seed number) 9.5% (8/84) 21.96% (47/214)Average temperatures of early, middle, and late April (◦C) 10/11.5/14.2 8.7/12.6/12.5

Table 4Effect of temperature on FLD percentage in apomictic crabapples.

Tea crabapple M. Sargentii

Temperature (◦C) 16 21 25 16 21 25

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Pistil longevity (day) 7 ± 1 4 ± 1

FLD percentage (%) 34.8 ± 2.6 56.5 ± 3.1

he data represents the mean ±SD of three independent experiments each with 20

nd parthenogenesis was examined. To confirm this hypothe-is, the apomeiosis and parthenogenesis percentages of a hybridopulation containing 34 hybrids that were randomly chosenrom the 125-hybrid population were analysed. The resultshowed that both the apomeiosis and parthenogenesis capac-ties were completely separated (Fig. 3A). In the population,he lowest apomeiosis and parthenogenesis percentages were6.9 ± 3.4% and 21.3 ± 1.6%, respectively, while the highest was

p to 74.3 ± 4.9% and 76.7 ± 5.3%, respectively. Furthermore, theesults also indicated that the genetic separation of the apomeiosisnd parthenogenesis capacities was not synchronously separated.hus, the ability of parthenogenesis uncoupled with apomeiosis

able 5ffect of pistil longevity on FLD and hybrid percentages in apomictic crabapples.

Tea crabapple

Treatment Anti-staling agent Lime sulfur H2O

Pistil longevity (day) 6 ± 1 3 ± 1 5FLD percentage (%) 42.8 ± 2.6 83.6 ± 5.1 59.9Hybrid seeds (%) 37.3 ± 3.6 9.7 ± 2.7 21.5

he data represents the mean ±SD of three independent experiments each 200 embryo s

± 1 6 ± 1 4 ± 1 2 ± 1 ± 2.8 26.6 ± 2.5 33.7 ± 2.7 81.5 ± 3.8

ryo sacs. FLD, female-late-on-date.

(Fig. 3A), indicating that they were two separate developmentalevents or genetic loci crucial for apomictic seed formation in teacrabapple.

Subsequently, 15 hybrids were randomly chosen from these 34plants to detect the products of parthenogenesis percentage mul-tiplied by apomeiosis percentage. Meanwhile, the FLD percentagesof these 15 hybrids were also examined. The results showed thatthe FLD percentages were positively correlated with the products of

the parthenogenesis percentage multiplied by the apomeiosis per-centage (Fig. 3B). Thus, the FLD percentage was positively linked tothe apomictic capacity, both of which were genetically determinedby apomeiosis and parthenogenesis.

M. Sargentii

Anti-staling agent Lime sulfur H2O

± 1 6 ± 1 3 ± 1 5 ± 1 ± 3.2 31.8 ± 2.4 82.7 ± 4.3 34.9 ± 3.7 ± 4.9 52.3 ± 5.1 8.9 ± 2.8 23.6 ± 2.3

acs and seedlings. FLD, female-late-on-date.

D.-D. Liu et al. / Scientia Horticulturae 167 (2014) 76–83 81

Fig. 3. Genetic analysis of parthenogenesis, apomeiosis, and FLD. (A) Genetic segregation of parthenogenesis and apomeiosis in a progeny population of 34 hybrids from ac mberT sac)

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ross between tea crabapple and ‘Maypole’ apple. Abscissa represents the hybrid nuhe relationship between parthenogenesis, apomeiosis, and FLD (immature embryohile the ordinate represents the percentage of FLD and multiplied product of the p

. Discussion

Apomixis is a form of asexual reproduction in angiosperms,hich results in the production of viable seeds from maternal tis-

ues of the ovule in the absence of meiosis and fertilization (Bicknellnd Koltunow, 2004). During the past decades, several herbaceouspomictic plants, such as Hieracium, Tripsacum, Boechera, have beenntensively studied (Koltunow et al., 2000; Bicknell et al., 2001;aumova et al., 2001). In woody Malus plant species, apomixis wasiscovered in several wild crabapple species, such as the M. hupe-ensis, M. sargentii, M. xiaojinensis, M. sieboldii (Ohen et al., 1986).early all of the apomictic plants characterized thus far were poly-loidy, independent of whether they were herbaceous or woodypecies (Sax, 1959). However, it is still unclear why apomixis onlyccurs in polyploidy. Within the past few decades, FIS class genesave been intensively characterized with functions in apomic-ic components. Their expressions were regulated by genomicmprinting (Grossniklaus et al., 2001a; Baroux et al., 2006). Themprinted expression of the FIS1 gene serves as a ploidy sensor andesults in seed developmental aberrations in triploid Arabidopsiseeds with increased paternal genome contributions (Erilova et al.,009). In addition, it has been well established that polyploidy for-ation triggers epigenetic changes in various plants (Chen, 2007).

ncreasing evidence indicates that epigenetic control is involved inhe regulation of apomixis reproduction or in apomixis-like pheno-ypes (Aguilar et al., 2010; Singh et al., 2011; Verhoeven and Preite,013). Thus, it is reasonable to draw a hypothesis that polyploidy is

inked to apomixis perhaps by regulating genomic imprinting andpigenetic modification.

Most apomictic plant species identified to date display facul-ative apomixis (Sax, 1959; Schmidt, 1977; Kartte and Seemüller,991; Seemüller et al., 1991). As a result, sexual reproduction, oromponents of it, is not completely excluded in apomictic plantsKoltunow et al., 2011). In this study, a fraction of the seedlingsere different from the maternal genotype, as indicated by the

xistence of tetraploidy and aneuploidy in the hybrid populationf tea crabapple, which permitted the apomictic crabapples to besed in the apple rootstock breeding program.

In facultative apomictic plant species, both sexual and apomicticeproductions can simultaneously occur. Two reproductive path-ays may share common elements, but there are also numerous

bnormalities (Koltunow et al., 1998). In Hieracium, which is aodel plant for the study of aposporous apomixis, sexual processes

nitiate first, and then one or more aposporous initial (AI) cells dif-erentiate close to the sexual initial cells (Koltunow, 1993). Usually,

he sexual process ceases when apospory initiates. Thus, it is easyo explore the signaling between apomictic and sexual pathwaysnd to examine the mechanisms that mediate aposporous embryoac and one or more embryo formation in Hieracium (Koltunow

, while ordinate represents the percentage of parthenogenesis and apomeiosis. (B)in a progeny population of 15 hybrids. The abscissa represents the hybrid number,nogenesis and apomeiosis percentages. FLD, female-late-on-date.

et al., 1998, 2000; Bicknell et al., 2003). The tea crabapple also per-forms aposporous apomixis and forms one or more embryo sacs,suggesting that they may share a common mechanism to regulatethe initiation of apomixes in plants, such as in Hieracium (Koltunowet al., 1998).

In aposporous plants, the AI cells developed into unreducedembryo sacs, and then formed clonal seeds independent of fer-tilization (Koltunow, 1993). However, our findings showed thatthe unreduced embryo sacs were completely capable to receivepollens, become fertilized and form sexual seeds in apomictic teacrabapples, which raises a question concerning how they escapefertilization to form clonal seeds in apomictic plants. The answerto this question is that the developmental delay of the unre-duced embryo sacs, known as FLD in this study, prevents thesacs from fertilization by escaping the pollination stage. The FLDphenomenon also occurs in Arabidopsis, swi1, jason, and pdil2-1mutants, which exhibit defects in gametophyte development andpollen tube guidance (Juan et al., 1999; Wang et al., 2008; Erilovaet al., 2009). Our results further indicated that it was positivelycorrelated with apomixis in apomictic crabapples. Thus, it was rea-sonable to propose that FLD is a crucial developmental event forapomictic reproduction in Malus and even in other plant species,although the mechanism underlying this phenomenon remainslargely unknown.

Because the environmental temperature greatly affects FLDthe apomictic capacity and hybrid percentage vary with theatmospheric temperature during blossoming and pollination(McWilliam et al., 1970). In apple production, lime sulfur is widelyused for flowering and fruit thinning (McArtney et al., 2006; Schuppet al., 2008). Our results indicated that lime sulfur could also beused to enhance the clonal seed percentage and to improve seedlinggrowth uniformity in apple rootstock production.

Apomixis is known to occur via several different pathways,all of which share the general features of avoidance meiosis anddevelopment of an embryo without fertilization. Apomeiosis andparthenogenesis are two important events in asexual reproduction,which are genetically linked in Tripsacum (Leblanc et al., 2009). Incontrast, genetic loci controlling apomeiosis and parthenogenesisare separated from each other in some apomictic plants, such asHieracium subgenus Pilosella (Grimanelli et al., 2001; Grossniklauset al., 2001b). In Pilosella, apomixis is controlled by two domi-nant independent genetic loci, e.g., LOSS OF APOMEIOSIS (LOA) andLOSS OF PARTHENOGENESIS (LOP) (Catanach et al., 2006; Okadaet al., 2007). Loss-of-function in both LOA and LOP loci resulted ina total reversion to sexual reproduction (Koltunow et al., 2011).

In this study, it was found that parthenogenesis and apomeiosiswere genetically uncoupled and that their genetic recombinationremarkably decreased the apomictic capacity in the hybrid popula-tion. Interestingly, FLD was positively correlated with the apomictic

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apacity, which was genetically determined by parthenogenesisnd apomeiosis, thereby genetically proving that it is a crucialevelopmental event for apomixis.

Apomixis mainly exists in non-agronomic plants. If apomixisould be harnessed in crops, it would indicate the beginning of

new era for plant breeding and seed production (Grossniklausnd Koltunow, 1998). Many attempts have been made to intro-uce apomixis into crop plants during the past decades, yet theyere proven unsuccessful (Leblanc et al., 2009; Rodriguez-Leal andielle-Calzada, 2012). A major reason is that apomixis is a complex

rait consisting of three components, each of which is controlled byumerous genes in most plant species (Koltunow and Grossniklaus,003). In this case, there should be a large hybrid population forcreening candidate individuals with acceptable apomictic capac-ty in agricultural production. However, it is difficult to distinguishetween hybrids in the apomictic breeding program because aew of them are mixed in numerous apomictic seeds (Ozias-Akinst al., 1993; Hanna, 1995). Our results showed that the applicationf an effective anti-staling agent increases the hybrid percentage.hus, it can be used in the apomictic breeding program to enhancecreening efficiency.

. Conclusions

FLD is a crucial developmental event for apomixis in apomic-ic crabapples, which prevents the unreduced embryo sacs fromollination and fertilization. Artificial manipulation of the FLD byhortening pistil longevity can be used to control the percentagef apomictic seeds, which are helpful not only for uniform seedlingroduction but also for the apomictic breeding program. Thus, arti-cial manipulation of FLD might be useful in uniform seedlingroduction and the apomictic breeding program.

cknowledgements

This work was supported by the Ministry of Science and Tech-ology of China (2013CB945103), the Ministry of Agriculture ofhina (201203075-3), and the Ministry of Education of ChinaIRT1155), and the Shandong Province (SDAIT-03-022-03).

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.scienta.013.12.035.

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