Accepted Manuscript Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423 Beibei Liu, Shengzhong Su, Ying Wu, Ying Li, Xiaohui Shan, Shipeng Li, Hongkui Liu, Haixiao Dong, Meiqi Ding, Junyou Han, Yaping Yuan PII:

S0981-9428(15)30011-5

DOI:

10.1016/j.plaphy.2015.04.011

Reference:

PLAPHY 4183

To appear in:

Plant Physiology and Biochemistry

Received Date: 8 December 2014 Accepted Date: 11 April 2015

Please cite this article as: B. Liu, S. Su, Y. Wu, Y. Li, X. Shan, S. Li, H. Liu, H. Dong, M. Ding, J. Han, Y. Yuan, Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423, Plant Physiology et Biochemistry (2015), doi: 10.1016/j.plaphy.2015.04.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423 Beibei Liu*, Shengzhong Su*, Ying Wu, Ying Li，Xiaohui Shan, Shipeng Li, Hongkui Liu,

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Haixiao Dong, Meiqi Ding, Junyou Han, Yaping Yuan College of Plant Science, Jilin University, Changchun 130062, China * These authors contributed equally to this work.

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Corresponding author: Yaping Yuan

E-mail: [email protected] Tel: +86-431-87836266 Fax: +86-431-87836266

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Beibei Liu: [email protected]

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Address: College of Plant Science, Jilin University, Changchun 130062, China

Shengzhong Su: [email protected] Ying Wu: [email protected]

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Ying Li: [email protected]

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Xiaohui Shan: [email protected] Shipeng Li: [email protected] Hongkui Liu: [email protected] Haixiao Dong: [email protected] Meiqi Ding: [email protected] Junyou Han: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times

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subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological anal-

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ysis and scanning electron micrographs confirmed the different developmental stages of somatic

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embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were

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cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regen-

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eration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA se-

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quences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423.

Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize.

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ACCEPTED MANUSCRIPT Key Words: Maize

Intact somatic embryos

Histological analysis

cDNA-AFLP qRT-PCR

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Abbreviations: cDNA-AFLP, cDNA-amplified fragment length polymorphism; qRT-PCR, quantitative real-time PCR; TDFs, transcript-derived fragments; ISE, intact somatic embryos; NEC, non-embryonic calli; EC, embryonic calli; GO, Gene Ontology; ABA, abscisic acid; SERK, somatic embryogenesis receptor kinase; LEA, late embryogenesis abundant; PINs, proteinase inhibitor genes; CaM, calmodulin; CML, calmodulin-like protein; CDPK, calcium-dependent protein kinase; CBL, calcineurin B-like protein; N-acyl-PE, N-acylphosphatidylethanolamine; LEC, LEAFY COTYLEDON; BBM, BABY BOOM; WUS, WUSCHEL; AGL15, AGAMOUS-LIKE15.

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1. Introduction

Maize is one of the most important crops in the world and because of its wide applications

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in animal husbandry and industry, demand for maize has grown. The need to generate higher yield crops may be a long-term issue. Traditional breeding techniques have been used widely to generate high yield crops, and recently, the strategies to increase crop production, have combined traditional breeding techniques with plant transformation technologies. Maize, is one of the most

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targeted crops that have been used to generate transgenic cultivars for basic and applied purposes (Verónica et al., 2012). The transformation receptors most commonly used for maize include im-

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mature embryos, mature embryos, nodal regions, leaf tissues, anthers, tassel and ear meristems, protoplast, and shoot meristems (Sujay et al., 2010). In particular, the somatic embryo has been

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used widely to propagate transgenic organisms and to obtain genetically modified plants. After the initial report in Daucus carota (carrot) (Steward et al., 1958), somatic embryogenesis has been shown to occur in many in vitro cultures of other species including Arabidopsis (Hecht et al., 2001) and Citrus sinensis (orange) (Cardoso et al., 2011). Somatic embryogenesis is a complex process that can be divided artificially into three stages: (a) induction from immature embryos or other explants, (b) subculture of mature somatic embryos, and (c) re-differentiation into seedlings. In maize, the type of explant as well as the genetic background

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ACCEPTED MANUSCRIPT plays a determinant factor in successfully establishing embryogenic cultures (Verónica et al.,

2012). Green and Philips (1975) first reported the regeneration of maize from immature embryos. Vasil et al. (1984) found that maize calli could be subcultured long term during somatic embry-

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ogenesis. Zhao et al. (2002) found that with optimized conditions, stable callus induction frequencies from Hi-II immature embryos were a highly reproductive and reliable system for Agrobacterium tumefaciens mediated genetic transformation. In the last decade, a large number

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studies about somatic embryos and the transgenic modification of maize have been reported.

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Sujay (2010) reported the callus induction and whole plant regeneration of five elite Indian maize inbred lines, and found that genotype, medium, and type of auxin and their concentrations influenced callus induction. Frame et al. (2006), Valdez-Ortiz et al. (2007), and Takavar et al. (2010) reported improved Agrobacterium-mediated transformation systems for maize and gener-

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ated some transgenic tropical maize lines. Thus, somatic embryogenesis is a useful tool to propagate transgenic organisms that has been used widely in maize breeding. It is also a potential model system that can be used to study the regulation of gene expression required for the earliest

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developmental events in higher plants (Zimmerm, 1993). Many characteristic events occur dur-

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ing the unique developmental pathway involved in somatic embryogenesis, including dedifferentiation of cells, activation of cell division, reprogramming of cell physiology, metabolism, and gene expression patterns.

Many researchers have studied the cellular and molecular mechanisms involved in embryo induction and re-differentiation. Zhang (2010) analyzed somatic embryos in Dendrocalamus hamiltonii at different stages using histological methods, and found that somatic embryos gradually developed from an embryo-like structure to final cotyledons via a heart-shaped, torpe-

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ACCEPTED MANUSCRIPT do-shaped structure. Previously, in maize inbred line H99, the microstructure of non-embryonic

callus, embryonic callus and callus after differentiation were studied in our laboratory using paraffin section and scanning electron microscope (SEM). In this study, a histological analysis re-

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vealed that embryonic callus cells were small, regular, with dense cytoplasm, while, non-embryonic callus cells were large, irregular, and most had vacuoles and few plastids (Sun et al., 2011).

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Several genes and proteins that were found to play critical roles in embryo maturation and

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differentiation have been reported. They can be classified into the following groups (Yang et al., 2010): (a) Hormone response genes and hormones especially auxin and abscisic acid (ABA) have been used in most systems to induce somatic embryos. Auxin-responsive genes include three major gene families, the AUX/IAA, GH3 and SAUR families, as well as the proteinase in-

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hibitor genes (PINs). The ABA-inducible genes include C-ABI3 (the carrot homolog of the ABI3 transcription factor), which is involved in the seed-specific signal transduction of ABA in the early embryo development stage in carrot, while the late embryogenesis abundant (LEA) protein

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coding genes are involved in the later stages. (b) Signal transduction related genes, including the

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somatic embryogenesis receptor kinase (SERK) encoding gene family, and genes that encode proteins involved in the calcium signaling pathway, such as the calmodulin (CaM) and calmodulin-like protein (CML) families, the calcium-dependent protein kinase (CDPK) family, and the calcineurin B-like protein (CBL) family. The SERK gene was first reported by Schmidt et al. (1997) in carrot suspension cultures. SERK encodes a leucine-rich repeat containing receptor-like kinase protein and the expression of this protein was reported to cease after the globular stage during somatic embryogenesis; thus, SERK has been considered to be a specific marker of the

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ACCEPTED MANUSCRIPT development process during embryogenesis. Subsequently, paralogs of SERK have been cloned and characterized in Arabidopsis, potato, maize, and Araucaria angustifolia. (c) Genes that encode transcription factors and other proteins, such as LEC1, LEC2, FUS3, BBM, ERFs, AGL15,

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WUS , GSTs, and PICKLE. In addition, a large number of other genes should also be studied during the complex molecular events involved in somatic embryogenesis. The cDNA-amplified fragment length polymorphism (cDNA-AFLP) approach is an effective and sensitive technique

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that has been used widely to detect differentially expressed genes; in particular, it has been used

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to identify genes involved in biotic and abiotic stress responses (Yang et al., 2010). Here, 40 maize inbred lines were used to screen for a high frequency of induction of somatic embryos and the elite inbred line Y423 was identified as a suitable model. At the anatomical level, we took both light and scanning electron microscopy images to characterize morphology

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changes that occurred during the somatic embryogenesis. The molecular mechanisms involved in somatic embryogenesis were studied using cDNA-AFLP, and 117 transcript-derived fragments (TDFs) were isolated, sequenced, and functional annotated using BLAST searches and GO

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analysis. Two full-length candidate genes that may play important roles during somatic embryo-

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genesis were cloned and characterized. 2. Materials and methods

2.1. Plant materials and calli induction In 2009, 40 maize inbred lines (listed in Supplementary Table A.1) were grown in the trial field in Jilin University in Changchun city, jilin Province, China. The whole ears were collected 12-15 days after self-pollination. Kernels were husked and sterilized three times with 75% ethyl alcohol. Immature embryos of 1.5-2.5 mm size were inoculated onto the induction medium, ac-

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ACCEPTED MANUSCRIPT cording to the procedure described as Jimenez and Bangerth (2001). For each plate, thirty embryos were used, and then were placed in the dark at 24°C. The buds generated from the imma-

ture embryos were removed every other 7 days, and the primary calli produced after 20-25 days.

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The induction medium consisted of N6 salts (Chu et al., 1975), MS inorganic salts (Murashige and Skoog, 1962) and B5 micro-elements, supplemented with 2 mg.L-1

2,4-dichlorophe-noxyacetic acid (2,4-D), 600 mg.L-1 L-proline, 500 mg.L-1 casein hydrolysate,

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200 mg.L-1 L-aspartic acid, 3% sucrose and 5.8g.L-1 agar, The pH values of the different media

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were adjusted to 5.8 prior to autoclaving at 121°C for 30 min. 2.2. Maturation and regeneration of the somatic embryos

The primary calli were transferred into subculture medium which was the induction medium adding 6.5 g.L-1 D-mannitol, and supplied with astigmatism. Calli were subcultured to the

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fresh subculture medium every 20-day, The frequency of somatic embryos induction was calculated after 4-5 times subculture.

The somatic embryos were transferred into regeneration medium, each content was half of

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that of the subculture medium, while 2,4-D was eliminated. Embryogenic cultures were incu-

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bated at 24°C under the photoperiod of 16h light and 8h dark. Then the plantlets had shoot formation and root formation at the same time after 20-30 days. The media used in the study were listed in Supplementary Table A.2. 2.3. Histomorphological analysis The ontogeny of somatic embryos was histologically analyzed at various somatic embryogenesis developmental stages. Tissues for histological observations were fixed in FAA solution (5:5:90, v/v/v, formaldehyde/acetic acid/50 % ethanol) for 48h,dehydrated through a graded eth-

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ACCEPTED MANUSCRIPT anol series (75, 85, 95, 100 and 100%) sequentially for 2h at each step. When the samples were transparent, they were incubated in xylene for 1 h, embedded in paraffin wax and sections were

cut at a 9-10µm thickness. The paraffin sections were then deparaffinized with xylene for 3 h and

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hydrolyzed in different concentrations of ethyl alcohol (100, 100, 95, 85, 75 and 65%) for 3 min per concentration. Finally, the paraffin sections were stained with 1% safranine T and 1% fast

tion) associated with the Moticam 3000C.

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green FCF. Images were obtained with a photomicroscope (Olympus BX51; Olympus Corpora-

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Samples for scanning electron microscopy were fixed in FAA solution for 48 h at room temperature . After dehydration through a graded ethanol series (75, 85, 95, 100 and 100%) for 2h per concentration, samples were dried in a vacuum dryer for 2 h, sputtered with gold using a coater (EIKO, ID-5, Japan). Finally, the samples were examined under a scanning electron mi-

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croscope (XL-30FE-ESEM FEG, USA) and photographs were taken at different magnifications. 2.4. RNA extraction and cDNA synthesis

Embryonic calli and non-embryonic calli of the elite inbred line Y423 were quickly frozen

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in liquid nitrogen and total RNA was extracted using the RNAiso Reagent kit (Takara, Japan).

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After treatment with DNase I (Promega) at 20-30°C for 15 min to digest the remaining genomic DNA, the total RNA was dissolved in 30µl of double distilled water (ddH2O) treated with diethyl pyrocarbonate (DEPC). The RNA quality and concentration were checked using sepharose gel and ultraviolet spectrophotometer. Double-stranded cDNA synthesis was performed as described by Manickavelu et al. (2007) with some modifications. All the reagents were purchased from Takara Bio Inc., Japan. First-strand cDNA was synthesized from total RNA using oligo(dT)18 primer and M-MLV ac-

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ACCEPTED MANUSCRIPT cording to the manufacturer. Second-strand cDNA was synthesized using 10U DNA polymerase I and 3U RNase H according to standard procedures. The reaction mixture was incubated at 16°C for 2.5 h and then at 80°C for 10 min; the mixture was then frozen immediately for 2min.

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Then the cDNA was stored at -20°C for future use. 2.5. cDNA-AFLP analysis

The subsequent cDNA-AFLP was performed as described by Bachem et al. (1998) with

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modifications. Briefly, double-stranded cDNA (500 ng) was double-digested with the restriction

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enzymes MseI/ PstI and MseI/ EcoRI. The digested products were ligated to MseI adaptor/ PstI adaptor and MseI adaptor/EcoRI adaptor with T4 DNA ligase (Promega) at 16°C for 4h, respectively. The ligation product was diluted tenfold directly using for pre-amplification templates, and M0, P0 and E0 were used as primers for pre-amplification. The PCR reactions conditions

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were: 94°C for 30s, 56°C for 1min and 72°C for 1min for 25 cycles. The pre-amplification PCR products were diluted 20-fold with ddH2O, MseI selective primers (M1, M2, M3, M4, M5, M6, M7, M8, M9, M10) and PstI selective primers (P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12,

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P13, P14, P15, P16) and EcoRI selective primers (E1, E2, E3, E4, E5, E6, E7, E8) were used for

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selective amplification. Selective amplification reactions were using the following touchdown PCR conditions: 94°C for 2min, 94°C for 30s, 65°C for 1min (- 0.7°C per cycle) and 72°C for 1 min for 12 cycles, followed by 94°C for 30s, 56°C for 1min and 72°C for 1min for 23 cycles. The sequences of the adaptors and the primers for the AFLP reactions are listed in Supplementary Table A.3. The selective amplification products were separated using a 4.5% polyacrylamide gel that was run at 70 W and 50°C until the bromphenol blue reached the bottom and was visualised by

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ACCEPTED MANUSCRIPT silver staining. The gel was put in a clean and ventilated area in order to dry it completely for further data analysis. 2.6. TDF sequencing and Data analysis

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Fragments with molecular weights of 100-600 bp were subjected to statistical analysis, differentially expressed transcript-derived fragments (TDFs) were extracted from the gel, eluted in 30 µl of ddH2O and amplified with the same conditions for the selective amplification of the

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cDNA-AFLP analysis. PCR products were ligated to the pUC-T vector (Beijing CoWin Biotech)

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and sequenced (BGI, China). All TDFs nucleotide sequences were searched against their homology in the database (http://www.ncbi.nlm.nih.gov/BLAST) using the BLASTX and BLASTN programs at the NCBI website (http://www.ncbi.nlm.nih.gov/). For differentially expressed genes, we performed detailed functional annotations using

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Blast2GO software v.2.7.1 (Conesa et al., 2005). The steps were followed as: BLAST searches from the NCBI NRPD (nr database), GO mapping to link all BLAST Hits to the functional information stored in the Gene Ontology database, GO annotations for the Blast matches, Inter-

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ProScan to find functional motifs and related GO terms, GO-slim to choose ‘goslim_plant.obo’

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to reduce plant-specific version of the Gene Ontology, make combined Graph based on the pre-established relationships. Finally, functional categorization was described by biological process, cellular component and molecular function. 2.7. Quantitative real-time PCR analysis There were nine TDFs to be selected for quantitative real-time PCR(qRT-PCR). Primers (Supplementary Table A.4) for qRT-PCR were designed and synthesized based on the TDFs’ sequences. Six samples were immature embryos, calli of 20d, 40d and 60d after induction, the em-

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ACCEPTED MANUSCRIPT bryonic calli and the non-embryonic calli respectively, and three biological replicates were performed. Total RNA extractions and cDNA first-strand syntheses were carried out according to

the methods described previously. The quantitative RT-PCR was performed on the ABI 7500 re-

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al-time PCR system using the 2 × SYBR Green Master Mix with ROX (Takara, Japan). Each 20µl reaction mixture contained 10µM each primers 0.4µl, 2 × SYBR Premix Ex Taq 10µl, 50 × ROX Reference Dye II 0.4µl and cDNA template 2µl. Reaction conditions were as follows: 95°C

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for 30s, 95°C for 5s, 60°C for 34s for 40 cycles. Melting curve analysis was performed to ex-

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clude the occurrence of primer dimers and unspecific PCR products. The threshold cycle (Ct) values were generated by the instrument automatically, and the amount of the transcripts of each gene normalised to the internal control actin1 was analysed using the 2-△△Ct method (Livak and Schmittgen, 2001).

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2.8. Cloning and sequence analysis

Full length of two TDFs designated ZmSUF4 and ZmDRP3A were obtained from the maize genetics and genomics database (maizegdb, http://www.maizesequence.org), and validated

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through cloning and sequencing using the following methods. The total RNA extraction from

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somatic embryos and the cDNA first-strand synthesis were as mentioned above. Each RT-PCR reaction was performed in a final volume of 25µl containing 2.5µl 10 × PCR buffer, 2µl of the cDNA, 2µl dNTPs (2.5mM each), 0.5 µl of each specific primer (10mM) and 0.3 µl LA Taq (5 U/µl). The PCR reactions conditions were as follows: 94°C for 3min, 56°C (ZmSUF4) or 54°C (ZmDRP3A) for 1 min and 72°C for 2 min for 35 cycles. The amplified DNA sequences were ligated to pMD18-T vector (TaKaRa, Japan) and sequenced (BGI, China). The primers for the RT-PCR reactions were listed in the Supplementary Table A.6.

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The gene sequences and protein structures were analyzed by DNAStar software and the net service ExPASy Proteomics Server (http://www.expasy.ch/ tools/protparam.html). The orthologs and paralogs of the candidate genes and their phylogenetic trees were built in the maizegdb gene

3. Results 3.1. Induction and regeneration of intact somatic embryos

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(http://www.smart.embl-heidelberg.de/).

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model, as well as the domains were identified with the SMART software

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The intrinsic embryogenic potential of the explant used as starting material for plant in vitro cultures varies depending on the genotype of the plant species. In maize as well as the species genetic background, genotype has been often considered an important factor in determining the response in successfully inducting somatic embryogenesis and regeneration (Verónica et al.,

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2012). In this study, 40 maize inbred lines were chosen and induced to calli; however, intact somatic embryos (ISE) were obtained only in the elite inbred line Y423 (Supplementary Table A.1).

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Immature embryos were collected from the whole ears of the elite inbred line Y423 after 13

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days self-pollination and placed on the induction medium (Fig. 1a). After 20 days of cultivation in the dark, pale yellow, translucent and compact primary embryonic calli could be seen (Fig. 1b). The primary embryonic calli were transferred to the subculture medium and, after subculturing 4-5 times, somatic embryos that were yellow, loose, and small granular were observed on the embryonic calli surface (Fig. 1c). The ISE were either reserved through subculture or shifted onto differentiation medium, and after 7-10 days, green buds protruded (Fig. 1d). Then the root and shoot both formed at 20-25 days (Fig. 1e-f). Finally, the well-grown plantlets regenerated

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ACCEPTED MANUSCRIPT (Fig. 1g-i). 3.2. Histology of intact somatic embryogenesis To illustrate the morpho-anatomical character of the intact somatic embryogenesis process

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in monocots, especially maize, we characterized the microstructure of ISE at different stages using a light microscope and SEM.

The results showed a difference between the non-embryonic calli (NEC) and embryonic

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calli (EC). The NEC had more large vacuoles without plastids than the EC, and a loose cell ar-

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rangement without rules (Fig. 2a). SEM analysis revealed that NEC had more elongated cells with larger intracellular spaces than EC (Fig. 3a). The proembryos that were formed on the surfaces of the EC had, organized structures with a thickened cell wall similar to dicots (Fig. 2b). The appearance of these early stages of ISE was soon followed by the formation of globu-

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lar-shaped embryos (Fig. 2c). At the same time, the SEM revealed that the globular-shaped embryos were bigger than the early stages of ISE (Fig. 3b -c). Then structural differences in the follow stages between monocots and dicots. ISE were grown to the pear-shaped embryos, scuti-

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form embryos, and mature embryos stages (Fig. 2d-f). The shoot apical meristem formed at the

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late stage of somatic embryogenesis (Fig. 2e-f). The pear-shaped embryos and scutiform embryos were also tracked using SEM (Fig. 3d-e). Single well-developed ISE were found with shoot apical meristem, coleorhizae, coleoptiles, and scutellum (Fig. 3f). 3.3. cDNA-AFLP analysis of embryonic calli and non-embryonic calli After testing several restriction enzyme combinations, the combination of MseI/EcoRI and MseI/PstI were found to produce an acceptable range of fragment sizes for cDNA-AFLP analysis. Around 20 TDFs per primer pair were obtained depending up on primer combinations and dif-

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ACCEPTED MANUSCRIPT ferent treatments. A total of 240 selective amplification primer combinations were used in this study, including 80 MseI/EcoRI (10 × 8) and 160 MseI/PstI (10 × 16) primer combinations (Supplementary Table A.3). Products ranging in size from approximately 50 to 1,000 bp, but

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mostly from 100 to 500 bp were obtained. A representative polyacrylamide gel is shown in Fig.4. A total of approximately 6583 TDFs were produced, 2297 of them were differentially expressed, including 1023 bands for EC (Fig. 4B-1) and 1274 bands for NEC (Fig. 4B-2). To vali-

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date the differentially expressed TDFs, 192 were isolated from the gel and reamplified using the

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original selective primers, 147 of them were selected for sequencing, and 143 TDFs were sequenced successfully. Finally, 117 sequences with insertions longer than 100 bp were used for further analysis. Each sequence was identified by similarity searches against the GenBank nonredundant public sequence (nr) database using the BLAST program (Altschul et al., 1997).

A.5 for details).

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Based on the results, 66 of the 117 TDFs were assigned putative functions (Supplementary Table

Functional categorization was done using the Blast2Go program. Sequences were analyzed

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based on the three Gene Ontology (GO) categories; biological process, cellular component and

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molecular function (Fig. 5). Under biological process, the cellular process category had the most number of annotated TDF sequences (25%), followed by metabolic process (24%), response to stimulus (11%), cellular component organization or biogenesis (7%), single-organism process (6%), multicellular organismal process (6%), developmental process (6%), reproduction (5%), localization (4%), biological regulation (3%), signaling (2%) and growth (1%)(Fig. 5A). Under cellular component, the plastid category had the most number of annotated TDF sequences (24%), followed by nucleus (21%), mitochondrion (15%), cytosol (13%), intracellular organelle

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lumen (11%), ribosome (4%), vacuole (3%), Golgi apparatus (3%), endoplasmic reticulum (2%), endosome (2%) and microbody (2%) (Fig. 5B). Under molecular function, the related to organic cyclic compound binding, and heterocyclic compound binding categories both had the same

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number of annotated TDF sequences (26%), followed by small molecule binding (14%), transferase activity (11%), hydrolase activity (11%), protein binding (7%), sequence-specific DNA binding transcription factor activity (2%), lipid binding (2%) and chromatin binding (1%) (Fig.

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5C).

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3.4. Quantitative real-time PCR analysis

Nine candidate genes (TDF-173, TDF-130, TDF-135, TDF-185, TDF-2-6, TDF-2-68, TDF-117, TDF-2-57 and TDF-137) were selected to validate the reliability of the cDNA-AFLP results using qRT-PCR. Some of these candidate genes were functionally similar to Oryza

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brachyantha and Setaria italica genes and the products of these genes in these two plant species were reported to be involved in biosynthetic process, cellular process, cell cycle, flower development, post-embryonic development, and transferase activity; however, some of the candidate

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genes remained unannotated in Zea mays (Table 1).

TDFs 173

130

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Table 1 Homologous analysis of ninet candidate genes involved in somatic embryogenesis in maize. Primer combination

Length (bp)

Protein

Homology

E-value

M9/P6

158

Calmodulin-binding family protein

NM_001176053.1

2e-59

XM_006660915.1

9e-62

XM_004971021.1

2e-67

XM_004963347.1

3e-65

M7/P13

318

（Zea mays） Suppressor of fir 4-like protein （Oryza brachyantha）

135

M7/P15

296

Dynamin-related protein （Setaria italica）

185

M9/P13

281

N-acylphosphatidylethanolamine syn-

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M1/P13

386

GRIP-like protein

XM_004957613.1

3e-146

XM_004983479.1

1e-145

（Setaria italica） 2-68

M8/P11

429

RCD1-like

117

M7/P6

232

Translational activator GCN1-like （Setaria italica）

M7/P11

185

Glutathione S-transferase GSTU6 （Zea mays）

137

M8/E2

247

AP2 domain transcription factor

5e-72

EU966792.1

2e-80

EF659468.1

5e-107

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（Zea mays）

XM_004981894.1

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2-57

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（Setaria italica）

To examine whether these genes were associated with the induction of ISE, samples were collected from calli at different stages throughout the induction process. The samples included

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immature embryos as a control, the calli 20, 40, 60 days after induction, EC and NEC. As shown in Fig. 6, our data suggest that the induction phase of ISE is associated with changes in gene ex-

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pression. After the immature embryos were induced to ISE, the expression levels of the nine candidate genes in the EC were higher than in the NEC, which is consistent with the

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cDNA-AFLP profiles. Changes in the expression levels of TDF-173, TDF-130, and TDF-135 were negligible during the induction by qRT-PCR, but their expression levels increased in the formation of embryonic calli. The expression patterns of TDF-185, TDF-2-57, and TDF-137 showed a strong regularity during the induction, suggesting that they may play roles in the development of immature embryos to ISE. The TDF-2-6, TDF-2-68, and TDF-117 genes were expressed inconsistently during the intact somatic embryogenesis. The qRT-PCR results showed that the candidate genes TDF-173, TDF-130 and TDF-135 may play important roles in the for-

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ACCEPTED MANUSCRIPT mation of ISE. 3.5. Cloning and sequence analysis of ZmSUF4 and ZmDRP3A

The cDNA full-length sequences of two candidate genes (TDF-130 and TDF-135) were as-

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sembled from the GenBank nr database and the Maize Genome Sequence database. TDF-130 was part of an uncharacterized Z. mays sequence (GRMZM2G081812) located in chromosome 2, which may have four transcripts. The phytozome annotation showed that the

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GRMZM2G081812 sequence was similar to a C2H2 zinc finger protein family from both Ara-

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bidopsis (best hit: AT1G30970.1) and rice (best hit : LOC_Os09g38790.1). The open reading frame of this gene is 1080-bp long encoding a 358-amino acid protein with the molecular weight of 38 KDa, and a theoretical pI of 7.69. This translated protein was named ZmSUF4 (suppressor of fir 4-like protein). Domain analysis of ZmSUF4 using the SMART database showed that it

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had two ZnF_C2H2 (C2H2-type [classical] zinc fingers [Znf] ) domains (Fig. 7A). Based on the Kyte–Doolittle scale, ZmSUF4 was predicted to be hydrophilic with a grand average of hydropathicity of -0.233 (Supplementary Fig. A.1A). The gene tree view showed that ZmSUF4 was

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closely related to proteins from Sorghum bicolor (ortholog: Sb02g032810), Setaria italic

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(ortholog: Si030356m.g), and Oryza sativa japonica (ortholog: LOC_Os09g38790) (Supplementary Fig. A.2A), all of which are zinc finger-like proteins. TDF-135 was similar to a hypothetical Z. mays sequence (GRMZM2G180335) located in chromosome 8 and has three transcripts. Phytozome annotation showed that the GRMZM2G180335 sequence was similar to dynamin-related protein 3A from both Arabidopsis (best hit: AT4G33650.1) and rice (best hit: LOC_Os01g69130.1). The translated protein was named as ZmDRP3A (dynamin-related protein). GRMZM2G180335_T01 had a complete coding region

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KDa and the theoretical pI was 6.60. This protein has a DYNc (dynamin GTPase) domain and a GED (dynamin GTPase effector) domain (Fig. 7B). The grand average of hydropathicity

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(GRAVY) was -0.300, which may indicate that ZmDRP3A is hydrophilic (Supplementary Fig. A.1B). A gene tree view revealed that ZmDRP3A was closely related to Sorghum bicolor (ortholog: Sb03g044010), Setaria italica (ortholog: Si000322m.g), Oryza sativa japonica

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(ortholog: LOC_Os01g69130) (Supplementary Fig. A.2B), all of which are similar to putative dy-

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namin like protein 2a. 4. Discussion

Green and Phillips (1975) first reported the use of somatic embryogenesis for maize tissue culture. Over the years, researchers have found that the genetic diversity of maize made it was

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difficult to obtain somatic embryos for maize elite inbred lines. However, A188, HiII, H99, and some other inbred lines have all been inducted to embryonic calli and are well know receptors for genetic transformation with high efficiency (Ishida et al., 1996; Zhao et al., 2001; Sun et al.,

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2011). Nevertheless, future demands require that new and improved high-yield cultivars are

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generated. A biotechnological approach combining tissue culture and genetic transformation with genes of interest can generate genetically transformed germplasm; however, because of their weak combining ability, many of these inbred lines were not suitable for productive applications. In this study, we found an elite inbred line, Y423, which can be inducted easily into ISE and can be used in both production and scientific research. Using its immature embryos as explants for induction, we successfully produced ISE. Some of the features of the ISE can be described as follows: (1) Cell totipotency and easy differentiation, excellent bipolarity with shoot and root

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Strikingly, the developmental histology of intact somatic embryogenesis in dicots has been studied exclusively (Kärkönen, 2000; Sun et al., 2003), while there are few similar studies in monocots, especially maize. Vasil et al. (1985) described the globular and mature embryos dur-

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ing somatic embryo formation in cultured immature embryos of hybrid maize cultivars. In our

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study, we performed a detailed tracking of the somatic embryogenesis using paraffin and scanning electron microscopy methods. For monocots, somatic embryogenesis can be divided into globular-shaped embryo, pear-shaped embryo, scutiform embryo, mature embryo, and finally seedling generation.

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The identification of the important genes involved in the complex process of somatic embryogenesis is required to help understand the process of somatic embryogenesis. The SERK gene was thought to be a major gene in this process in many species, and the involvement of this

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gene has also been reported in maize (Zhang et al., 2011). AGL15 encodes a MADS-domain

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protein that plays an important role in somatic embryogenesis because it has been shown to interact with LEC2, FUS3, ABI3 and SERK1, and to participate in the gibberellin metabolic pathway (Zheng et al., 2009). In our study, we identified nine candidate genes that were differentially expressed between embryonic calli and non- embryonic calli using cDNA-AFLP. These genes were validated by qRT-PCR. A translated protein annotated as calmodulin-binding (TDF-173) has low expression levels at the early stage of embryo induction; however, it was reported previously that a calmodulin-binding protein as present only in the apical meristematic regions, and

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member of the glutathione S-transferase family, and is involved in defense reactions, senescence, and response to stress in plants. It has an important function in the induction from immature em-

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bryo using auxin (Galland et al., 2001). Our qRT-PCR results showed that the expression of TDF2-57 increased gradually during the induction process. The AP2 domain transcription factor (TDF137) belongs to a very large family that contains about 150 members, and plays an im-

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portant role during plant growth. During embryogenesis, AP2 regulates embryo size by nega-

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tively controlling cell proliferation during seed development (Ohto et al., 2005). TDF-185 may encode a N-acylphosphatidylethanolamine synthase-like protein.

N-acylphosphatidylethanolamine (N-acyl-PE) is an anionic minor membrane glycerophospholipid and is involved in a variety of physiological processes, such as response to pathogens, plant

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development, and germination in plants (Evgeny and Teresa, 2011). The RCD1 protein (TDF2-68) is a regulator of both developmental and stress responses in Arabidopsis thaliana where it interacts with several transcription factors. RCD1 could act as a scaffold protein to as-

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semble transcription factors for their post-translational modification, relocalization, or degrada-

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tion (Pinja et al., 2010). GRIP-like protein (TDF-2-6) may be a useful Golgi marker for immunolocalization studies because it has been reported to target the Golgi apparatus in mammalian cells (Paul et al., 2004).

The expression levels of ZmSUF4 (TDF-130; GRMZM2G081812) and ZmDRP3A (TDF-135; GRMZM2G180335) increased during intact somatic embryogenesis, and their expression levels were higher in EC than in NEC. ZmSUF4 has two ZnF_C2H2 domains at the N-terminal end. Znf domains are relatively small protein motifs containing multiple finger-like

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protrusions that make tandem contacts with their target molecules. They were first identified as a DNA-binding motif in the transcription factor TFIIIA from Xenopus laevis (African clawed frog), and are now known to bind DNA, RNA, protein, and/or lipid substrates. Znf C2H2 motifs are the

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most common DNA-binding motifs found in eukaryotic transcription factors. Our GO analysis of the paralogs of ZmSUF4 under the molecular function category showed enrichment in binding (GO:0005488), not only DNA binding (GO:0003677), but also ion binding (GO:0043167), and

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cation binding (GO:0043169). Under biological process, ZmSUF4 was predicted to be involved

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in protein homodimerization activity (GO:0042803) and the regulation of post-embryonic development (GO:0048580). In Arabidopsis, SUF4 is required for the upregulation of FLOWERING LOCUS C (FLC) by FRIGIDA (FRI). SUF4 mutations strongly suppress the late-flowering phenotype of FRI. Early in development, SUF4 was expressed at higher levels in the shoot and

(Sang and Scott, 2006).

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root apex, and later in development it was expressed in the apical regions, leaves, and flowers

ZmDRP3A has a GTPase effector domain (GED) domain at the C-terminal and a dynamin

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GTPase (DYNc) domain at the N-terminal. Dynamin is a GTP-hydrolyzing protein that is an es-

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sential participant in clathrin-mediated endocytosis by cells. Mutation studies indicated that dynamin functions as a molecular regulator of receptor-mediated endocytosis. Large GTPases mediate vesicle trafficking. Dynamin participates in the endocytic uptake of receptors, associated ligands, and plasma membrane following an exocytic event. Under molecular function, our GO analysis showed enrichment in purine nucleoside binding (GO:0001883), hydrolase activity, and acting on acid anhydrides in phosphorus-containing anhydrides (GO:0016818). Correspondingly, ZmDRP3A has been shown to be involved in the assembly process in purine ribonucleoside tri-

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phosphate metabolic process (GO:0009205). The Arabidopsis dynamin-related proteins DRP3A is localized to mitochondria and peroxisomes where it controls their division, and is co-localized with DRP3B in leaf epidermal cells (Masaru et al., 2009; Shoji et al., 2004). These analyses

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suggest that ZmSUF4 and ZmDRP3A function as important regulation factors in intact somatic embryogenesis.

Somatic embryogenesis is a remarkable and intricate process that includes transformation in

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morphology, histology, and gene expression. Here, we conducted intact somatic embryogenesis

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and for the first time observed the globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo in an elite maize Y423. We then revealed the pattern of gene expression in these different developmental stages. In addition, we verified nine candidate genes related to intact somatic embryogenesis using qRT-PCR, and obtained two full-length sequences of

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genes which may play important roles during the formation of ISE. Thus, we have identified a new receptor of an elite maize (Zea mays L.) inbred line for genetic transformation with high

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ISE in more detail.

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efficiency via intact somatic embryogenesis. Our finding will help understand the formation of

Fig. 1. Morphological stages of somatic embryogenesis in maize (Zea mays L.) from immature zygotic embryos. (a) the immature embryos, after 12–14 days of pollination; (b) the primary embryonic calli after 20 days of cultivation in the dark; (c) somatic embryos after 60 days of culture on subculture medium; (d) the green buds protruding from somatic embryos in differentiation medium; (e) and (f) root and shoot formed simultaneously on the surface of somatic embryos; (g) and (h) maize plants regenerated from somatic embryogenesis. (i) well-developed plantlets. Bars: a-f = 0.5cm, g-i = 1cm. Fig. 2. Histological analysis of somatic embryos. (a) non-embryonic callus (bar:50µm); (b) proembryos with thickened cell wall (bar: 50µm); (c) globular-shaped embryo ( bar: 200µm); (d) pear-shaped embryo (bar: 200 µm); (e) scutiform embryo (bar: 200µm); (f) mature embryo (bar: 200µm). Col, coleoptiles; SM, shoot apical meristem; Scu, scutellum. Fig. 3. Scanning electron microscope analysis of somatic embryos. (a) non-embryonic calli (scale bar: 50µm); ( b) the early stages of somatic embryos (scale bar: 200µm); (c) globular-shaped embryos (scale bar:200µm);(d)

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pear-shaped embryos (scale bar:200 µm);(e) scutiform embryos (scale bar: 200µm); (f) the single well-developed somatic embryos (scale bar: 50µm). Col, coleoptiles; SM, shoot apical meristem; Scu, scutellum; Cor, coleorhizae. Fig. 4. A cDNA-AFLP display of gene expression among different primer combinations for embryonic calli and non- embryonic calli. (A) Each group was amplified with a different pair of selective AFLP primers: M3/E1-E8 and M3/P1-P16, M: DNA marker pBR322 DNA/MspI. (B) Enlarged view of the boxed area in (A). Lanes are in groups of two, lane E: embryonic calli , lane N: non-embryonic calli. The arrows indicate three differential expression patterns, 1. upregulated expression; 2. downregulated expression; 3. co-expression. Fig. 5. Functional categorization of cDNA-AFLP TDFs sequences based on (A) biological process, (B) cellular component and (C) molecular function. Fig. 6. Quantitative analyses of ninet selected genes with real-time qRT-PCR.

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Fig.7. Domains of two candidate genes were detected using smart. (A) TDF-130: ZmSUF4; (B) TDF-135: ZmDRP3A.

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Acknowledgment

This research was supported by the National S&T Major Projects—Breeding of New Varieties for Transgenic Biology of China (2009ZX08003-024B, 2014ZX0800305B)

Contributions

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Conceived and designed the experiments: Beibei Liu, Shengzhong Su, Yaping Yuan. Performed the experiments: Beibei Liu, Ying Li, Meiqi Ding.

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Analyzed the data: Beibei Liu, Shengzhong Su, Xiaohui Shan, Haixiao Dong. Contributed reagents/materials/analysis tools: Ying Wu, Shipeng Li, Hongkui Liu, Junyou Han.

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Wrote the paper: Beibei Liu, Shengzhong Su. All authors have contributed significantly, and all authors are in agreement with the content of the manuscript.

Supporting Information Additional Supporting Information may be found in the online version of this article: Table A.1：Influence of different basal media on embryogenic callus induction from immature embryos in selected elite maize inbred lines

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Table A.6：Primers used for the RT-PCR reactions

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Table A.5：Homology analysis of TDFs

scale. (A) TDF-130: ZmSUF4; (B) TDF-135: ZmDRP3A.

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Fig. A.1 Hydrophobic character prediction of two deduced proteins based on the Kyte-Doolittle

Fig. A.2 Gene tree view of GRMZM2G081812 and GRMZM2G180335 with their paralogs. (A)

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TDF-130: ZmSUF4; (B) TDF-135: ZmDRP3A.

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ACCEPTED MANUSCRIPT Authors’ Contributions Conceived and designed the experiments: Beibei Liu, Shengzhong Su, Yaping Yuan. Performed the experiments: Beibei Liu, Ying Li, Meiqi Ding. Analyzed the data: Beibei Liu, Shengzhong Su, Xiaohui Shan, Haixiao Dong.

Wrote the paper: Beibei Liu, Shengzhong Su.

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Contributed reagents/materials/analysis tools: Ying Wu, Shipeng Li, Hongkui Liu, Junyou Han.

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All authors have contributed significantly, and all authors are in agreement with the content of the manuscript.

ACCEPTED MANUSCRIPT Table A.1 Influence of different basal media on embryogenic callus induction from immature embryos in selected elite maize inbred lines Embryogenic callus induction frequency (%) N6+B5 88 80 75 75 60 60.5 66 43 20 14 19 25 22 19 18 19 18 15 20.5 18 11 28 27 26.5 10 20 24 6 10 17 12 30 24 17 18 34 24 20.5 25 20

MS+N6 69 75 60.5 69 64 53 50 30 21 10 20 22.5 20 18 19.5 14 15 20 25 15.5 10 30 24 23 10.5 16 20 5 8 11.5 10 25.5 20.5 19 18 28 20 17 18 20.5

MS+N6+B5 a 94 b 96 b 83 b 89.5 c 81 c 72.5 c 70 c 62.5 24 19 20 25 22.5 19.5 19 20 30 33 30.5 20 15 41 30 29 12 20 30 6.5 14 20 16 33 29 20 19 35 25 27 21 29

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MS+B5 70.5 71 70 77 60 55 55 40 18 10 13 21 16 10 10.5 14 11 17 19 0 0 30 20 24 0 11 27 0 5 0 14 19 19.5 19 5 20 13 16.5 11 13

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MS+N6+B5 188 192 166 179 162 145 140 125 48 38 40 50 45 39 38 40 60 66 61 40 30 82 60 58 24 40 60 13 28 40 32 66 58 40 38 70 50 54 42 58

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MS+N6 138 150 121 138 128 106 100 60 42 20 40 45 40 36 39 28 30 40 50 31 20 60 48 46 21 32 40 10 16 23 20 51 41 38 36 56 40 34 36 41

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N6+B5 176 160 150 150 120 121 132 86 40 28 38 50 44 38 36 38 36 30 41 36 22 56 54 53 20 40 48 12 20 43 24 60 48 34 36 68 48 41 50 40

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Y423 Y412 Y425 Y715 YY1 YY7 YY8 E28 137 JI853 Si287 7922 478 Dan1324 Dan988 Y853 Dan599 4112 Mo17 Chang7-2 Dan598 4462 Y287 DH8237 Si144 Y251 YY2 YY3 YY4 YY5 YY6 YY9 YY10 YY11 YY12 Y713 Y718 Y5108 PH4CV PH6WC

Embryogenic callus-forming embryos MS+B5 141 142 140 154 120 110 110 80 36 20 26 42 32 20 21 28 22 34 38 0 0 60 40 48 0 22 54 0 10 0 28 38 39 38 10 40 26 33 22 26

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Genotypes

For each line, 200 immature embryos were placed onto each medium and incubated under the same conditions. a

Plantlet regeneration via embryogenesis

b

Plantlet regeneration via organogenesis

c

Embryogenic calli were dead during subculturing

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N6 salts MS inorganic salts B5 micro-elements 2,4-D (mg.L-1) L-proline (mg.L-1) casein hydrolysate (mg.L-1) L-aspartic acid (mg.L-1) Sucrose (g.L-1) Agar (g.L-1) D-mannitol (g.L-1) pH

Induction medium + + + 2 600 500 300 30 5.8 5.8

Regeneration medium 1/2 1/2 1/2 300 250 150 15 5.8 6.5 5.8

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Organic/inorganic supplements + vitamins

ACCEPTED MANUSCRIPT Table A.3： ：Primers used in cDNA-AFLP The sequences of the adaptors: MseI adaptor-I: 5′-GACGATGAGTCCTGAG-3′ MseI adaptor-II: 5′-TACTCAGGACTCAT-3′

EcoRI adaptor-II: 5′-AATTGGTACGCAGTCTAC-3′

PstI adaptor-II: 5′-TGTACGCAGTCTAC-3′

The primers for pre-amplification:

P0:5′-GACTGCGTACATGCAG-3′

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E0:5′-GACTGCGTACCAATTC-3′

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M0:5′-GATGAGTCCTGAGTAA-3′

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PstI adaptor-I: 5′-CTCGTAGACTGCGTACATGCA-3′

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EcoRI adaptor-I: 5′-CTCGTAGACTGCGTACC-3′

ACCEPTED MANUSCRIPT The primers for selective amplification:

EcoRI primers(8)

MseI primers(10)

PstI primers(16)

M1: GATGAGTCCTGAGTAAGGA

P1: GACTGCGTACATGCAGAA

E2: GACTGCGTACCAATTCAAG

M2: GATGAGTCCTGAGTAAGGC

P2: GACTGCGTACATGCAGAC

E3: GACTGCGTACCAATTCACA

M3: GATGAGTCCTGAGTAACAG

P3: GACTGCGTACATGCAGAG

E4: GACTGCGTACCAATTCACC

M4: GATGAGTCCTGAGTAACAT

P4: GACTGCGTACATGCAGAT

E5: GACTGCGTACCAATTCACG

M5: GATGAGTCCTGAGTAAGGG

P5: GACTGCGTACATGCAGCA

E6: GACTGCGTACCAATTCACT

M6: GATGAGTCCTGAGTAACTC

P6: GACTGCGTACATGCAGCC

E7: GACTGCGTACCAATTCAGC

M7: GATGAGTCCTGAGTAACTG

P7: GACTGCGTACATGCAGCG

E8: GACTGCGTACCAATTCAGG

M8: GATGAGTCCTGAGTAAGGT

P8: GACTGCGTACATGCAGCT

M9: GATGAGTCCTGAGTAACGA

P9: GACTGCGTACATGCAGGA

M10: GATGAGTCCTGAGTAACGC

P10: GACTGCGTACATGCAGGC

M AN U

SC

RI PT

E1: GACTGCGTACCAATTCAAC

AC C

EP

TE D

P11: GACTGCGTACATGCAGGG P12: GACTGCGTACATGCAGGT P13: GACTGCGTACATGCAGTA P14: GACTGCGTACATGCAGTC P15: GACTGCGTACATGCAGTG P16: GACTGCGTACATGCAGTT

ACCEPTED MANUSCRIPT Table A.4： ：Primers used in qRT-PCR RT-PCR forward primer(5′-3′)

RT-PCR reverse primer(5′-3′)

173

CCCGTCCCCGCTCTAAACAC

CGCCTGACAAGGTCCTCTGGT

130

GGGATAATCTACCGAGCAAAGCC

TGAAGCAATGTCAATGGAGGAAAG

135

ATCAGATTAGGGGCAGGCATA

CCAGCAGTGGTAGTAAGAAAGGA

185

GTTGGTAACTGTTTTCCACATTCTC

GTACCCCATGACTCATCCTCTG

2-6

ATTGGCTCAGTCACAGAGGCATA

TAAACTGTAGTAACGTCCCAACCAC

2-68

CTTACACCAGCAAATCGTTCG

AAGGTCGTCTACTCCACTATCAAAA

117

CGTTCGCAGCTAGACGTACAGG

GCTTATCTTGGCCTTGCGTGA

2-57

GTTAATTGGCCCAGTTTGTCTAGCT

ACCCGCAGGGACGAACTTTAT

137

TGCGTCAACAGCAACATTAGC

TATTAGGCATCAAAGAAGAAGAGGG

Actin1

CGATTGAGCATGGCATTGTCA

CCCACTAGCGTACAACGAA

α-tubulin

ACTTCATGCTTTCGTCCTACGCTCCA

CTGGGAGGCTGGTAGTTGATTC

AC C

EP

TE D

M AN U

SC

RI PT

TDFs

ACCEPTED MANUSCRIPT Table A.5： ：Homology analysis of TDFs No.

Primer

Size

Sequence similarity

Accession no.

E value

Setaria italica transcription factor

XM_004966189.1

2e-71

comb. (bp) 12

M1/P10

257

19

M1/P16

314

Setaria italica DDB1- and CUL4-associated factor homolog 1-like

M2/E3

235

retrotransposon protein, putative, Ty3-gypsy subclass [Oryza sativa Japonica Group]

M2/P5

311

phosphopantothenate--cysteine

2e-102

ABA94234.1

3e-34

ACG36148.1

2e-39

AF090447.2

1e-32

XM_004970794.1

5e-53

AF546188.1

3e-04

M AN U

32

XM_004959894.1

SC

21

RI PT

IIIB 90 kDa subunit-like

ligase [Zea mays] 46

M3/P2

168

Zea mays 22 kDa alpha zein gene cluster

66

M4/P5

254

Setaria italica DNA-directed RNA polymerase III subunit

107

M6/P12

85

TE D

RPC4-like

Contiguous genomic DNA

sequence comprising the 19-

kDa-zein gene family from Zea

EP

mays

M7/P1

107

plectin-like [Setaria italica]

XP_004956734.1

0.012

123

M7/P8

122

Zea mays putative zinc finger

AY530950.1

7e-38

AF546188.1

1e-51

XM_004980913.1

1e-57

XM_006660915.1

9e-62

AC C

112

125

M7/P10

153

protein Contiguous genomic DNA sequence comprising the 19kDa-zein gene family from Zea mays

128

M7/P11

198

Setaria italica transducin betalike protein 2-like

130

M7/P13

318

Oryza brachyantha suppressor of fir 4-like protein

ACCEPTED MANUSCRIPT 132

M7/P14

374

Zea mays transcription factor

NM_001158175.1

4e-175

XM_004971021.1

2e-67

DQ417752.1

5e-83

PosF21 135

M7/P15

296

Setaria italica dynamin-related protein 3A-like

141

M8/E5

256

Zea mays B73 pathogenesis-

RI PT

related protein 2 and GASA-like

142

M8/E6

178

AY574035.1

3e-58

Zea mays clone 1476912 40S

EU954690.1

4e-154

ribosomal protein S21 mRNA

SC

protein genes Zea mays rust resistance protein rp3-1 (rp3-1) gene 145

152

M8/P1

M8/P4

335

217

Setaria italica myosin-J heavy

153

M8/P6

M AN U

chain-like

XM_004969680.1

184

Setaria italica pre-mRNA-

1e-68

XM_004982751.1

9e-64

NM_001157071.1

7e-116

XM_004951840.1

3e-74

AF090447.2

3e-89

NM_001175842.1

3e-120

NM_001176854.1

2e-38

processing protein 40C-like 161

M8/P16

249

Zea mays acid

phosphatase/vanadium-

162

M8/P16

225

TE D

dependent haloperoxidase related Setaria italica sporulationspecific protein 15-like

170

M9/P3

229

Zea mays 22 kDa alpha zein

175

EP

gene cluster

M9/P8

259

Zea mays putative MYB DNA-

AC C

binding domain superfamily

2-1

M1/P2

307

protein

Zea mays putative oxysterol binding domain family protein

2-2

M1/P10

208

Zea mays RNA-binding protein

NM_001154153.1

3e-49

2-6

M1/P13

386

Setaria italica protein GRIP-like

XM_004957613.1

3e-146

2-8

M2/E3

174

Zea mays clone 263959 protein

EU963548.1

6e-70

FJ444997.1

1e-108

farnesyltransferase/geranylgeran yltransferase type I alphasubunit 2-19

M3/P2

277

Zea mays autophagy-related 3

ACCEPTED MANUSCRIPT variant 2 (Atg3) mRNA 2-21

M3/P3

156

Zea mays succinate

NM_001156945.1

3e-57

NM_001112164.1

0.0

AJ307886.1

2e-136

XM_004983479.1

1e-145

semialdehyde dehydrogenase 2-52

M7/P2

599

Zea mays ribosomal protein s6 RPS6-2 (rps6-2)

M7/P7

286

Zea mays partial mRNA for

RI PT

2-56

pollen signalling protein with adenylyl cyclase activity (psiP gene) M8/P11

429

Setaria italica inactive poly [ADP-ribose] polymerase RCD1-like

M9/P8

460

Setaria italica ABC transporter B

XM_004967560.1

2e-148

XM_004979246.1

2e-60

HQ234502.1

8e-85

XM_004962744.1

3e-126

NM_001176845.1

8e-111

NM_001196798.1

2e-81

NM_001152569.1

1e-35

EF659468.1

5e-107

EU956232.1

5e-107

M AN U

2-76

SC

2-68

family member 21-like 7

M1/P2

182

Setaria italica disease resistance protein RGA2-like

9

M1/P4

222

Zea mays clone BAC

ZMMBBb0342E21 DNA-

31

M2/P4

342

TE D

binding protein

Setaria italica structural

maintenance of chromosomes protein 3-like

M3/P1

287

Zea mays putative RING zinc

EP

44

AC C

finger domain superfamily

52

118

137

M3/P7

M7/P6

M8/E2

207

115

247

protein

Zea mays cyclin dependent kinase inhibitor Zea mays tetratricopeptide repeat protein Zea mays clone BAC b0288K09 AP2 domain transcription factor

151

M8/P3

241

Zea mays clone 1559345 phosphoglycerate mutase-like protein

ACCEPTED MANUSCRIPT 157

M8/P9

185

Zea mays protein phosphatase

NM_001112434.1

1e-61

X56877.1

7e-82

XM_004963347.1

3e-65

2A regulatory subunit B' (PPP2R5B) 172

M9/P5

325

Z.mays transposable element Bg sequence

M9/P13

281

Setaria italica N-

RI PT

185

acylphosphatidylethanolamine synthase-like 186

M9/P13

332

Zea mays rust resistance protein

179

M9/P10

235

maize sucrose synthetase gene (shrunken) 3' end

M4/P1

288

Setaria italica helicase SRCAP-

J01241.1

5e-82

XM_004953444.1

7e-52

NM_001174379.1

4e-85

GQ905618.1

1e-124

EU963753.1

1e-97

EU966792.1

2e-80

XM_004978783.1

4e-124

AF334758.1

2e-04

NM_001154476.1

1e-153

EU964030.1

1e-138

NM_001177005.1

2e-43

M AN U

2-30

3e-11

SC

rp3-1 (rp3-1) gene

AY574035.1

like

2-36

M4/P9

341

Zea mays putative argonaute family protein

2-81

M9/P12

360

Zea mays clone zma-miR408-3

precursor miRNA zma-miR408,

2-17

M3/P1

556

TE D

precursor RNA

Zea mays clone 272368 glycerophosphodiester

phosphodiesterase mRNA

M7/P11

185

Zea mays clone 297455

EP

2-57

glutathione S-transferase GSTU6

M7/P15

327

Setaria italica superkiller

AC C

2-60

Hordeum vulgare homeodomain

M2/P1

Zea mays beta-catenin-like

24

26

M2/E6

151

235

viralicidic activity 2-like 2-like

protein JUBEL1 (JuBel1) gene

protein 1 M2/P5

294

33

Zea mays clone 275336 phosphopantothenate--cysteine ligase mRNA

42

M3/E8

146

Zea mays ZIP-like protein 1

ACCEPTED MANUSCRIPT (ZLP1) 45

M3/P1

171

Zea mays clone 490587 60S

EU975447.1

5e-71

XR_215307.1

3e-95

ribosomal protein L17 M3/P4

255

Setaria italica UDP-

48

glucuronate:xylan alpha-

91

92

167

173 117

Setaria italica auxilin-related protein 1-like

XM_004962756.1

1e-67

M4/P2

161

Zea mays ATPP2-A13

NM_001159065.1

1e-56

M4/P10

112

Zea mays tartrate-resistant acid

NM_001279571.1

2e-13

AF053468.1

2e-72

phosphatase type 5 precursor M5/P12

301

Zea mays DnaJ-related protein

SC

71

177

M AN U

62

M3/P5

ZMDJ1 (mdJ1) gene M6/E2

330

Hordeum vulgare subsp. vulgare

AK366529.1

9e-156

EU968540.1

1e-93

NM_001176053.1

2e-59

XM_004981894.1

5e-72

mRNA for predicted protein M9/P2

212

Zea mays clone 322004 cell wall integrity protein scw1

M9P6

158

Zea mays putative calmodulin-

TE D

50

RI PT

glucuronosyltransferase 1-like

binding family protein

M7/P6

232

Setaria italica translational

EP

activator GCN1-like

Function Unknown Protein, Hypothetical and Unclassified Protein M1/E8

124

AC C

5

16

131

M1/P13

M7/P13

257

212

Zea mays subsp. mays genotype

DQ490951

1e-39

NM_001195891.1

1e-48

NM_001152235.1

6e-96

XM_002465932.1

9e-69

CMS-S mitochondrion

Zea mays uncharacterized LOC100501052 (LOC100501052), mRNA Zea mays uncharacterized LOC100279211 (LOC100279211), mRNA

133

M7/P14

198

Sorghum bicolor hypothetical protein, mRNA

ACCEPTED MANUSCRIPT 148

M8/P2

272

Zea mays clone 207833 mRNA

EU958773.1

0.0

BT084791.2

1e-76

EU949499.1

4e-123

sequence 155

M8/P8

185

Zea mays full-length cDNA clone ZM_BFb0181G07 mRNA, complete cds

M8/P9

274

Zea mays clone 421746 mRNA

RI PT

156

sequence 158

M8/P10

266

Zea mays clone 286073 mRNA sequence

M9/P7

199

Zea mays hypothetical protein (LOC100383414), mRNA

178

M9/P9

224

Zea mays clone 1633822 mRNA

181

M9/P11

NM_001176067.1

3e-88

EU943598.1

M AN U

sequence

3e-125

SC

174

EU945807.1

275

Zea mays LOC100383598

3e-94

NM_001176246.1

3e-124

NM_001151672.1

2e-121

XM_002460768.1

7e-111

AC197049.5

3e-104

EU959614.1

0.0

BT067719.1

0.0

NM_001143544.1

5e-98

NM_001196072.1

9e-110

(umc2224), mRNA 182

M9/P11

256

Zea mays uncharacterized LOC100278375

184

M9/P12

259

TE D

(LOC100278375), mRNA

Sorghum bicolor hypothetical protein, mRNA

187

M9/P14

239

Zea mays BAC clone CH20153G1 from chromosome 5,

M1/P11

426

AC C

2-3

EP

complete sequence

2-10

2-11

M2/P3

M2/P3

393

293

Zea mays clone 218874 enolase mRNA, complete cds

Zea mays full-length cDNA clone ZM_BFc0144L23 mRNA, complete cds Zea mays uncharacterized LOC100217184 (LOC100217184), mRNA

2-16

M2/P16

258

Zea mays uncharacterized LOC100501321 (LOC100501321), mRNA

ACCEPTED MANUSCRIPT 2-18

M3/P1

180

Zea mays hypothetical protein

NM_001176954.1

4e-72

BT018457.1

3e-52

XM_002450245.1

2e-59

(LOC100384414), mRNA 2-20

M3/P2

138

Zea mays clone EL01N0412H01.d mRNA

M3/P4

178

Sorghum bicolor hypothetical protein, mRNA

2-25

M3/P7

181

Zea mays clone ZMMBBb37E5, complete sequence

2-27

M3/P8

255

Zea mays uncharacterized LOC100274956 (LOC100274956), mRNA

M4/P2

340

Zea mays clone 325600

5e-56

NM_001149192.1

3e-115

EU969018.1

1e-134

NM_001174930.1

0.0

NM_001148674.1

0.0

XM_002443044.1

7e-106

EU947204.1

4e-67

NM_001174853.1

1e-93

NM_001148820.1

7e-172

NM_001176543.1

0.0

BT018617.1

6e-35

BT037588.1

1e-61

M AN U

2-31

AC165179.2

SC

2-23

RI PT

sequence

hypothetical protein mRNA, complete cds 2-32

M4/P4

374

Zea mays uncharacterized LOC100382170

(LOC100382170), mRNA M4/P5

393

Zea mays LOC100274311

TE D

2-33

(pco061337), mRNA

2-42

M6/E7

264

Sorghum bicolor hypothetical protein, mRNA

199

Zea mays clone 332432 mRNA

M7/E7

214

sequence

Zea mays hypothetical protein

AC C

2-51

M6/P6

EP

2-44

Zea mays hypothetical protein

2-69

Zea mays hypothetical protein

2-67

M8/P10

M9/E6

354

532

(LOC100382091), mRNA

(LOC100274461), mRNA

(LOC100383923), mRNA 4

M1/E8

166

Zea mays clone EL01N0501E07.d mRNA sequence

17

M1/P13

152

Zea mays full-length cDNA

ACCEPTED MANUSCRIPT clone ZM_BFb0180P02 mRNA, complete cds 53

M3/P7

189

Zea mays BAC clone CH201-

AC194907.5

4e-62

EU976843.1

1e-29

215L17 from chromosome 5, complete sequence M4/E7

242

Zea mays clone 991618

RI PT

60

hypothetical protein mRNA, complete cds 122

M7/P8

339

Zea mays uncharacterized

NM_001151772.1

LOC100278526

149

M8/P2

399

Sorghum propinquum locus

150

M8/P2

DQ429696.1

M AN U

pSB1307 genomic sequence

SC

(LOC100278526), mRNA

395

Zea mays clone 207833 mRNA

3e-155

1e-65

EU958773.1

3e-57

EU953863.1

3e-104

NM_001050508.1

9e-13

BT017158.1

2e-143

EU964030.1

5e-138

NM_001174886.1

0.0

AJ309824.2

2e-142

AC202177.4

8e-60

sequence 177

M9/P9

236

Zea mays clone 1447947

hypothetical protein mRNA, complete cds M3/P5

148

Oryza sativa Japonica Group

TE D

2-24

Os01g0698100 (Os01g0698100) mRNA, complete cds

61

M10/E8

346

Zea mays clone

M10/P3

291

AC C

63

EP

EL01N0368A12, mRNA

2-35

22

M4/P9

M2/E4

310

Zea mays clone 275336

phosphopantothenate-cysteine ligase mRNA Zea mays hypothetical protein (LOC100382125), mRNA Zea mays 25S rRNA gene and transposon-like sequence

M2/E6 23

425

sequence

211

Zea mays BAC clone ZMMBBb279L24 from chromosome 5, complete sequence

ACCEPTED MANUSCRIPT M3/P8

254

Zea mays uncharacterized

54

NM_001149192.1

1e-113

NM_001175799.1

4e-139

NM_001152089.1

9e-89

LOC100274956 (LOC100274956), mRNA M3/P9

307

Zea mays hypothetical protein

M3/P10

206

Zea mays uncharacterized

56

LOC100279021 (LOC100279021), mRNA M4/P9

250

Setaria italica uncharacterized

69

LOC101755212 (LOC101755212), mRNA M4/P12

270

Zea mays uncharacterized

73

LOC100192596

M7/E6

XM_004976850.1

NM_001137811.1

M AN U

(LOC100192596), mRNA

RI PT

(LOC100383133), mRNA

SC

55

316

Zea mays uncharacterized

111

LOC100192866

AC C

EP

TE D

(LOC100192866), mRNA

NM_001138055.1

2e-46

2e-107

4e-154

ACCEPTED MANUSCRIPT Table A.6： ：Primers used for the RT-PCR reactions The primers for ZmSUF4:

upstream primer: 5′- TCGGCGAAACCCTAACC-3′ downstream primer: 5′- TCCCAATGAGGCGACAGT-3′

upstream primer: 5′-CTCCTCGTCCAAAACCCT-3′

AC C

EP

TE D

M AN U

SC

downstream primer: 5′-CAAATCCGTGGCAAGTCA-3′

RI PT

The primers for ZmDRP3A:

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT