Anatomy and transcript profiling of gynoecium development in female sterile Brassica napus mediated by one alien chromosome from Orychophragmus violaceus
© Fu et al.; licensee BioMed Central Ltd. 2014
Received: 8 August 2013
Accepted: 21 January 2014
Published: 23 January 2014
The gynoecium is one of the most complex organs of angiosperms specialized for seed production and dispersal, but only several genes important for ovule or embryo sac development were identified by using female sterile mutants. The female sterility in oilseed rape (Brassica napus) was before found to be related with one alien chromosome from another crucifer Orychophragmus violaceus. Herein, the developmental anatomy and comparative transcript profiling (RNA-seq) for the female sterility were performed to reveal the genes and possible metabolic pathways behind the formation of the damaged gynoecium.
The ovules in the female sterile Brassica napus with two copies of the alien chromosomes (S1) initiated only one short integument primordium which underwent no further development and the female gametophyte development was blocked after the tetrad stage but before megagametogenesis initiation. Using Brassica _ 95k_ unigene as the reference genome, a total of 28,065 and 27,653 unigenes were identified to be transcribed in S1 and donor B. napus (H3), respectively. Further comparison of the transcript abundance between S1 and H3 revealed that 4540 unigenes showed more than two fold expression differences. Gene ontology and pathway enrichment analysis of the Differentially Expressed Genes (DEGs) showed that a number of important genes and metabolism pathways were involved in the development of gynoecium, embryo sac, ovule, integuments as well as the interactions between pollen and pistil.
DEGs for the ovule development were detected to function in the metabolism pathways regulating brassinosteroid (BR) biosynthesis, adaxial/abaxial axis specification, auxin transport and signaling. A model was proposed to show the possible roles and interactions of these pathways for the sterile gynoecium development. The results provided new information for the molecular mechanisms behind the gynoecium development at early stage in B. napus.
The gynoecium, located in the fourth and innermost whorl of a flower, is the female reproductive organ of flowering plants which has specialized functions for successful pollination, seed maturation and seed dispersal. Although it is a highly complex organ that differs widely in form between species, most gynoecia of different angiosperms have a set of common structures: an apical stigma, a style, and a basal ovary, which encloses the ovules [1, 2]. In Brassicaceae family, including the model plant Arabidopsis thaliana and important Brassica crops, gynoecium is composed of two fused carpels and three common parts above. The stigma plays a key role in pollen binding and recognition and participates in the induction of pollen germination . The style connects the stigma with the ovary and harbors the transmitting tract essential for pollen tube growth. The ovary contains the ovules that develop into seeds after fertilization [4, 5]. The ovule contains the funiculus, the chalaza which forms outer and inner integuments, and the nucellus which is covered by the integuments and in which the embryo sac representing the megagametophyte forms [6, 7]. Incomplete or abnormal development in any part of gynoecium can cause female sterility or reduced fertility, which has been observed in various plants, including Arabidopsis and Brassica crops [8, 9]. The female sterile mutants provide the suitable materials for elucidating the genetic control of the gynoecium development.
As the gynoecium is one of the most complex and important organs of flowering plants, increasing researches focused on the genetic control of its development by using female gametophytic mutants, especially from Arabidopsis. Several genes important for ovule integument or embryo sac development have been identified, such as SIN1, BEL1, INO, ATS, ANT, TSO1, HLL, NZZ, SIN2, WUS, PFS2[7, 10], DYAD and VDD. In addition, it was proposed that adaxial–abaxial polarity mechanisms were required for integument formation [13, 14]. The auxin concentration gradient was found to determine cell fates in the embryo sac . Two genes (AGO5 and AGO9) were shown to control female gamete formation and megagametogenesis by two independent small RNA pathways [16, 17]. Brassinolide was suggested to plays a previously unrecognized role in the development of gynoecia and outer integument of the ovule . Some other genes or gene families like STY, SHI, HECATE, SHATTERPROOF, JAIBA, CLV1 and SPT were identified for gynoecium development, including stigma, style, septum, transmitting tract and carpel margin tissues [1, 2, 5, 19–22]. Recently, by applying whole genome microarray and Next Generation Sequencing (NGS) techniques, hundreds of genes were found to be specific for female gametophyte genes by comparative expression profiling between wild plants and mutants [23–25].
The female sterile mutants from spontaneous or artificial mutations were rarely reported in the important oilseed rape Brassica napus L. . In our pervious study, complete female sterility was observed in one Brassica napus- Orychophragmus violaceus addition line which contained all 38 chromosomes from B. napus and one or two copies of one particular chromosome from O. violaceus. It seemed that its pistils stopped to develop at early stage. So we inferred that certain genes related to early process of pistil formation on this alien chromosome silence the homologous genes in B. napus, or these alien genes interfere the normal pistil development of B. napus. In this study, we compared the developmental anatomy and transcript profiling using RNA-Seq technique of the gynoecia between the female sterile line and donor B. napus. A number of candidate genes and related pathways were revealed, which provided new insights into the genetic and biochemical controls for early stage of pistil development in B. napus. In addition, the sequence datasets serve as a valuable resource for novel gene discovery in O. violaceus.
Developmental anatomy of female sterility and donor B. napus
Illumina sequencing and mapping of transcripts
Summary of alignment statistics of RNA-Seq in H3 and S1
Total mapped reads
< = 2 bp mismatch
Screening of differentially expressed genes (DEGs)
Gene Ontology analysis and pathway analysis of DEGs
Pathway enrichment analysis of DEGs
DEGs with pathway annotation (2333)
All genes with pathway annotation (30493)
Linoleic acid metabolism
Biosynthesis of secondary metabolites
alpha-Linolenic acid metabolism
Circadian rhythm - plant
Carbon fixation in photosynthetic organisms
Valine, leucine and isoleucine degradation
Ribosome biogenesis in eukaryotes
Arginine and proline metabolism
Protein processing in endoplasmic reticulum
Glycine, serine and threonine metabolism
Starch and sucrose metabolism
Tropane, piperidine and pyridine alkaloid biosynthesis
DEGs specific to S1 and H3 plants
As S1 carried the alien O. violaceu chromosomes, it was understandable that DEGs specific to S1 (192 unigenes) were more than those of H3 (37). Significantly enriched GO terms of the two sets of DEGs were listed in Additional file 3: Table S2 and Additional file 4: Table S3. For the 192 ones, there were 51 enriched GO terms including 22 mapped to biological process ontology, 3 mapped to molecular function ontology and 26 mapped to cellular component ontology. For the 37 ones, only 2 GO terms (carbohydrate metabolic process and metabolic process) belonging to biological process ontology were significantly enriched. No GO terms were specific to H3 plants for molecular function and cellular component.
DEGs for steroid biosynthesis and metabolic process
Fifteen unigenes involved in steroid biosynthesis were differentially expressed between S1 and H3. For phytosterol, twelve of the fifteen unigenes covered brassinosteroid (BR) and stigmasterol biosynthesis, which were all down-regulated in S1 plants. These genes were identified to encode proteins LUP2 (JCVI_38125 and JCVI_42543), CAS1 (JCVI_33856 and JCVI_20625), SMO1 (JCVI_2267), C-14 Sterol Reductase (FACKEL) (JCVI_39052), SMO2 (JCVI_14504), DWARF5 (JCVI_10359 and JCVI_40245) and DWARF1 (JCVI_9676, JCVI_21095 and JCVI_7150). Down-regulation of these genes could affect the normal biosynthesis of brassinosteroid, which might have a role in gynoecium and ovule development . Furthermore, one unigene (JCVI_27911) encoding a DON-Glucosyltransferase termed UGT73C5 involved in BR metabolic process was observed to highly and only expressed in S1 plant (log2 Ratio(S1/H3) >19). In Arabidopsis, the UGT73C5 was found to regulate BR activity by catalyzing the 23-O-glucosylation of BL and castasterone, and overexpression of UGT73C5 resulted in BR-deficient phenotypes . In S1, the BR activity was likely reduced by both biosynthesis and metabolic process, and then affected the S1 gynoecium and ovule development.
DEGs involved in floral organ, embryo sac or ovule development
In the flower development GO terms, 35 unigenes showed different expression levels. Among 9 unigenes for floral organ or whorl development, 5 encoding ABCB19 (JCVI_15407 and JCVI_35748), SHP2 (JCVI_13562), HEC1 (JCVI_35218) and SPT (JCVI_28865) were involved in carpel development [20, 28–30], 3 encoding SRS5 (JCVI_29726) and TAA1 (JCVI_9912 and JCVI_18521) in gynoecium development [19, 31], only 1 in sepal development instead of carpel or gynoecium development. All 8 female organ-related genes were down-regulated. In the DEGs, some others for embryo sac or ovule development were observed. For example, the genes encoding VDD (JCVI_5595 and JCVI_25612) were found to be a direct target of the MADS domain ovule identity complex and affect embryo sac differentiation in Arabidopsis; the gene encoding AGO5 (JCVI_17792) was a putative effector of small RNA (sRNA) silencing pathways, an insertion of which inhibited the initiation of megagametogenesis ; the gene which encoded a protein disulfide isomerase, PDIL2-1 (JCVI_23062), its truncation would function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance . In addition, the genes which encoded SEP2 (JCVI_18581), PI (JCVI_17089), AP3 (JCVI_7877), EDA14 (JCVI_13115), EDA17 (JCVI_16034) were also involved in female gametophyte or ovule development [28, 29, 33]. Most of these genes were down-regulated except AGO5, PI and EDA14.
Verification of DEGs by qRT-PCR
The corresponding primers of qRT-PCR
GGATGGAATAGCTGGAATCATT (F) TCATAACTGGATACCTGACTGTTGG (R)
CGTGAACGAGTCCAATTACCT (F) CGTACTCCAAGACAGGCCATA (R)
TCCTTCTCACATCAGCTCACAAGT (F) AACCAATGTAGTCTCAAGCATGTC (R)
TTCCGCTGAAGCTCTCACTCT (F) CTGCATGATCTTAGCGAACTCAG (R)
CAGTATCTGAAGGAGAACGGTAGC (F) AAGAACCGATGGAGTGAAGCTC(R)
CTACACAATCATAACAAGCCAATGC (F) TTGAAGATAGGAAGACTAGGACCAC (R)
ACGCAAGCCTCGTCCTCCTT(F) AAGCTCTGCCACATGAACCG (R)
AGCGACTGCCTCTAAGTGACG (F) TGCCACACGATGTTCTCTGG (R)
GGTGACAGTCCTGGATTCTTCA (F) TAAGCATCTCCGTACCATCTGG (R)
TTGTGGACGATAATCTGGTGAACAG (F) TCTTCGCTAGTAGCCTGAATCACAT (R)
CGTAGCAAGTGGTCAGCAAC (F) CCAGTCAACAGCAGTTCCTG (R)
TTGGACTATGGTAGCTGGAGGAG (F) CCATTGAAGGTAGCAGCAACATC (R)
GACCGTTACTCCATCCGCAT (F) TCTCGCAGAAGCACTCTCGT (R)
TAACATTGTGCTTAGTGGTGGAACC (F) GTCCAGATTCGTCATACTCAGCCTT (R)
TAGTGACCTTGCTGCATCTGGAG (F) CCAACAGAATCCTTATGACAGCCT (R)
AACGAGACTTACCAGCTTCAGG (F) TGGAGAGAATGCTGATGCAG (R)
GGTGAGATCAACGAGGACAACGT (F) CTTCCACATGCGCCTCTTCTTC (R)
CACTTACGCCGACGAGCTTC (F) GAGTAACGGAACCGCCACAC (R)
GATCCTCTTCGTCTCTTCTTCAT (F) TTGGCACCATGTGATCGTAG (R)
CGAAGCCTTCTGTCTCATCG (F) ACACCGTCTCCGTCTCTGTC (R)
ATAATGGAGCCGTGGAGGTG (F) AATGGCGACGAGAAGACGAA (R)
TCCATCCATCGTCCACAG (F) GCATCATCACAAGCATCCTT (R)
Actin of B. napus
In this study, complete and stable female sterility in B. napus mediated by one alien chromosome was characterized for its anatomy of gynoecium development and gene expressions. The female sterility was insensitive to the dosage of the alien chromosome from O. violaceus, because the gynoecium development was nearly the same in the plants with one or two copies of the alien chromosome. This chromosome also caused complete female sterility in the nullisomic line with one chromosome pair lost (2n = 36) of B. napus (data unshown), suggesting that the genes on this alien chromosome for female sterility were still active in the altered genetic background of B. napus. These results indicated that the female sterile lines were valuable for elucidating the genetic mechanisms behind the gynoecium development for Brassica crops and other crucifers.
Phenotype of aberrant gynoecium
In crucifers, the pollen tube must breach the stigma surface and burrow through the extracellular matrix of the stigma epidermal cells and transmitting tract cells before reaching its ovule targets . It grows typically through the foot to penetrate the stigmatic cuticle and then enters the outer layer of the stigmatic cell wall, which would require cutinase or esterase to break down the stigmatic cuticle and modify the cell walls . In our S1 line, the failure of the pollen tubes to penetrate the stigma epidermal cells on the defective stigmatic papillae may be caused by the aberrant structure and component of the abortive papillae cell walls or the enzyme activities. On the other hand, two differentially expressed unigenes encoded the S locus glycoprotein (SLG) (JCVI_18221 and JCVI_22894) were observed, which were specific to H3 and showed large schange of gene expression level (log2 Ratio(H3/S1) >16). The SLG was expressed specifically in the stigma epidermal cell wall and played a role in pollen-pistil interaction and pollen adhesion . Down-regulated expression of SLG was in conformity with that the fewer pollen grains adhered on S1 stigma.
In Arabidopsis thaliana, one of several ovule-defective mutants, the ant showed ovules without integument development and blocked megasporogenesis at tetrad stage . In ovules of S1 plant, the integuments arrested at the initiative stage and presented a small protuberance at the chalaza region, which indicated that the inner integument could initiate but not grow and the outer integument did not developed. So the abortive stage of S1 was a little later than the ant mutant. Consistently, the megasporogenesis was blocked after the tetrad stage and the megagametogenesis did not initiate in S1, as the tetrad and degraded megaspore were observed but the functional megaspore never discovered. Additionally, expression of ANT gene showed no difference between S1 and H3, suggesting that defective ovules in S1 may be caused by downstream genes of ANT or other metabolic pathways.
Global gene transcription changes related with female sterility
The fact that more unigenes appeared in S1 (28,065) than H3 (27,653) was obviously connected with the alien chromosomes from O. violaceus, suggesting that newly initiated transcription occurred in S1. Furthermore, 4540 DEGs were induced or repressed by more than two folds in S1 plants compared with H3 plants, some of them were confirmed by the qRT-PCR analysis. As there were too many GO enrichment terms when using hypergeometric test for the 4540 unigenes, we chose the DEGs with RPKM ≥ 50 and fold change ≥ 2 (1987) for the GO functional categorization. There were several processes related to female sterile traits, including flower development, embryo sac development, gametophyte development, cell growth, cell differentiation, cell death, cell cycle, pollen-pistil interaction. One inexplicable phenomenon was that, in the ontology of biological process, the most over-represented GO terms were responses to stress or stimulus, suggesting that significant changes of the stress-resistant reaction in female sterile plants happened. More than 200 differentially expressed unigenes without any annotation might have some novel functions.
Genes and metabolism pathways for female sterility
The second group of genes was those involved in adaxial/abaxial axis specification. Recent studies have shown that adaxial–abaxial polarity mechanisms were required for integument formation [14, 38, 39]. At present, eight genes from three gene families were confirmed to determine adaxial/abaxial axis specification during the inner and outer integuments development: INO from YABBY gene family, KAN1, KAN2 and ABERRANT TESTA SHAPE (ATS, also referred to as KANADI4, KAN4) from KANADI gene family, and four Class III Homeodomain-leucine zipper (HD-ZipIII) genes (CNV, PHB, PHV and REV) . A balance model for the adaxial/abaxial determinants underlying integument morphogenesis was also proposed: in the inner integument, ATS and a proposed additional abaxial factor acted in balanced opposition to CNA/PHB/PHV and REV to promote inner integument growth; in the outer integument, the abaxial activities of INO, KAN1 and KAN2 acted in balance with REV to control morphogenesis . Thus, HD-ZIPIII transcription factors acted in both the outer and inner integuments as adaxial determinants. In our study, thirteen unigenes with differential expression were included in polarity specification of adaxial/abaxial axis. Six of them encoding four kinds of protein: IAMT1 (JCVI_39696), ERECTA (JCVI_34288), HD2 (JCVI_28751, JCVI_5685 and JCVI_29422) and MYB91 (JCVI_11157), were only found to control the development of leaf polarity [40–42], and seven encoding the four HD-ZIPIII transcription factors: PHB (JCVI_16242 and JCVI_36221), PHV (JCVI_19406 and JCVI_29851), CNA (JCVI_25718 and JCVI_10400) and REV (JCVI_32385), had roles in both leaf and integument adaxial/abaxial polarity formation . Mutation of these genes would cause injured ovule integuments and abortive embryo sac. All of the seven unigenes were down-regulated in S1 plants, conforming to the ovule phenotype in S1. This group of genes was possibly the other important factors affecting gynoecium or ovule development in S1. In addition, it has been demonstrated that overexpression of miR165 led to the down-regulation of all five HD-ZIP III genes, and concomitantly recapitulated the phenotypes of simultaneous loss-of-function mutation of REV, PHV and PHB. It was possible that the miR165 was overexpressed in gynoecia of S1 and caused the integuments damage indirectly, because the four genes were all down-regulated.
The third related pathway was auxin transport and signaling pathway. As the first plant hormone studied, auxin had wide-ranging effects on growth and development throughout the plant, including gynoecium and ovule morphogenesis [44–46]. Auxin can be distributed by passive diffusion through the mature phloem or active polar auxin transport (called PAT) that mediates cell-to-cell movement of auxin through two different types of proteins, efflux and influx carriers . Transport proteins, best represented by PIN-FORMED1 (PIN1) and P-GLYCOPROTEINS19 (PGP19/ABCB19), have been shown to coordinately regulate auxin efflux, and AUXIN1 (AUX1) and its paralogs LIKE-AUX1 (LAX1-3) are the auxin influx carriers . In our study, 21 unigenes related with auxin transport or signaling pathway showed different expression levels, some encoded the efflux and influx carriers, such as PIN1 (JCVI_31884), PIN3 (JCVI_7893, JCVI_6698 and JCVI_31660), ABCB19 (JCVI_15407 and JCVI_35748) and LAX3 (JCVI_32855), and two PIN phosphorylation regulators PID (JCVI_34187) and PP2A (JCVI_14923). PIN1, the first gene in PIN gene family, had a role in basipetal auxin transport in stem as a catalytic auxin efflux carrier [3, 47]. PIN3, another member of PIN gene family, was a component of the lateral auxin transport system regulating tropic growth and essentially involved in mediating differential shoot growth . ABCB19 encoded one of the PGP proteins (PGP19) that belonged to ATP-binding cassette (ABC) transporter superfamily and had an important role in stabilizing PIN1 localization at the plasma membrane microdomains . LAX3, one of the paralogs of AUX1, might function in concentrating auxin in the cytoplasm of cells of L1 layer and preventing auxin diffusion in the apoplast, inducing auxin to flow into the neighboring cells . PID encoded a Ser/Thr protein kinase and functioned on PIN phosphorylation that caused the preferential location of PIN in the apical side. But the phosphatase PP2A acted antagonistically to PID on phosphorylation of PIN proteins. Here, all of these genes participating in mediation of auxin fluxes directly or indirectly were down-regulated expressed at different levels. Alteration of these genes probably disturbed the auxin flux and distribution in the S1 gynoecia, which would arrest the normal development of gynoecia and ovules (Figure 7).
The last group of genes was those with important role in gynoecium or ovule development, such as SHP2, HEC1, SPT, SRS5, CKRC1, VDD, AGO5, PDIL2-1, SEP2, PI, AP3, EDA14 and EDA17. All of these genes were down-regulated except AGO5, PI and EDA14, which was generally in accorded with female sterility. Among these differently expressed genes between S1 and H3, the two MADS box genes, SHP2 and SEP2 were reported to be ovule identity factors, controlling ovule integuments identity with other genes .
Interactions between BR, auxin and HD-ZipIII genes on gynoecium development
Up to now, numerous studies have addressed that there are interactions and crosstalks between brassinosteroids (BRs) and auxins. It was demonstrated that the IAA genes were induced by brassinolide (BL) via activating the auxin response elements . In the growth of Arabidopsis hypocotyl, either application of BRs or disruption of BR synthesis would alter auxin response, presumably by affecting auxin transport . It was confirmed that the gene BRX acted at the nexus of a feedback loop that mediates threshold brassinosteroid levels to permit optimal auxin action . A recent study showed that a crucial gene for BR biosynthesis, DWF4, played a novel role in the BR-auxin crosstalk . The PP2A protein mediating auxin fluxes mentioned above also had a dual role in the shift between inhibition and activation of BR signaling . Besides BRs, HD-ZipIII genes also had the link with auxin. It has been proposed that IFL1/REV could influence auxin polar transport and cell differentiation and morphology . The loss of Class III HD-Zip gene activity resulted in a loss of bilateral symmetry during embryogenesis by altering the PIN1 localization and mediating auxin signal transduction . Overall, the brassinosteroid (BR), auxin and HD-ZipIII genes likely affected the formation of ovule integuments and growth of gynoecia of S1 plants, respectively and corporately. It has been proposed that the enhanced seu cyp85A2 double mutant phenotypes in ovule and gynoecium maybe result from the combination of brassinosteroid- and auxin-dependent signaling pathways . Here, a model of BR-, auxin- and HD-ZipIII genes-dependent pathways and their interactions for gynoecium development in the female sterile plant was proposed (Figure 7). In addition, the DEGs and related pathways involved in gynoecium development mentioned above were also listed (Additional file 5: Table S4).
The complete female sterility in Brassica napus mediated by one alien chromosome was caused by the lack of formation of inner and outer integuments and the blocked megasporogenesis. Among the 4540 DEGs detected by RNA-seq in the female sterility, a number of important genes and metabolism pathways were involved in the development of gynoecium, ovule, integuments as well as the interactions between pollen and pistil. Particularly, the genes for the pathways related with BR, auxin and adaxial/abaxial axis specification were most likely responsible for the abortive development of female organs.
Plant materials and RNA preparation
The monosomic or disomic additional plants with a single, or one pair of the Orychophragmus violaceus chromosomes and all 38 chromosomes of Brassica napus (2n = 39, AACC + 1O; 2n = 40, AACC + 2O) were identified among the successive backcrossing progenies of the intergeneric somatic hybrid (2n = 62, AACCOO) with B. napus L. cv. Huashuang 3  (Additional file 6: Figure S2, and the method of GISH for Figure S2 was the same as previously described ). One of the addition line used in this study was female sterile but male fertile. After the line pollinated B. napus, the progenies were found to be male and female fertile B. napus, or female sterile B. napus with the O. violaceus chromosome(s) which were selected for study. The female sterile disomic addition line with one pair of the O. violaceus chromosomes (designated as S1) and donor B. napus cv. Huashuang No. 3 (H3) were grown in the experimental field of Qinghai University in Xining, Qinghai Province, and gynoecium samples in 1.5 to 3 mm long flower buds were collected. All samples were immediately conserved in RNAfixer Stabilization Reagent (BioTeke Corporation) for one week, then transferred to liquid nitrogen and kept at -80°C until use. Total RNA from the two samples was extracted using the Polysaccharide and Polyphenol Total RNA Isolation Kit (spin column; Bioteke Corporation). The quality of the RNA was analyzed by agarose gel (1.5%) electrophoresis and the total RNA content was assessed by spectroscopy at 260/280 nm (GeneQuant II; Pharmacia Biotech). Finally, the RIN (RNA integrity number) was evaluated using Agilent 2100 by BGI-Shenzhen, and the value of S1 and H3 were 9.2 and 9.1 respectively.
Pollen germination, pollen tube growth
About 10 pollinated pistils were collected 2, 6, 24, 48 and 72 h after artificial pollinations, and fixed in FAA solution (50% ethanol, 5% acetic acid, 3.7% formaldehyde) at 4°C overnight. Before observation, the pistils were soften with 6 mol/L NaOH for 12 h, then rinsed with distilled water for three times, and dipped in 0.1% water soluble aniline blue solution (aniline blue was diffused in 0.1 mol/l K3PO4 solution) for 24 h . Then the pistils were mounted on slides and covered lightly with coverslips, and observed under the fluorescence microscopy (Nikon Eclipse 80i).
Scanning electron microscopy
Fresh gynoecia in flower buds with different sizes and opening flowers of S1 and H3 were collected at the same time, and fixed overnight in 2% glutaraldehyde, and dehydrated through graded ethanol. All samples were dried, sputter-coated and analyzed as previously described .
Fresh gynoecia in flower buds with different size range and opening flowers of S1 and H3 were collected and fixed in FAA solution at 4°C overnight. After dehydrated through graded ethanol, cleared whole-mount tissues were prepared by dissecting ovules from carpels using needles, and cleared in the mixture with 1/2 Chloral hydrate solution (chloral hydrate:glycerol:water = 8:1:2 )  and 1/2 absolute alcohol for 2 h, and then in Chloral hydrate solution for 3 times (12 h each time). For those too small to dissect ovules, the whole pistils were cleared directly. All samples were observed using a Nikon DS-Ri 1 microscope equipped with differential interface contrast (DIC) optics.
RNA sequencing library construction, Illumina sequencing, and data processing
Approximately 40 μg total RNA of each sample (S1 and H3) was sent to BGI-Shenzhen where the RNA-seq libraries were constructed and sequenced by using Illumina HiSeq™ 2000. The mRNA enrichment, RNA fragmentation, the first and second strand cDNA synthesis and purifying, sequencing adaptors ligation and PCR amplification were performed as previously described . After sequencing, the preliminary data processing was also carried out by BGI-Shenzhen, according to the procedure. Briefly, the original image data was transferred into sequence data by base calling, which was defined as raw data or raw reads and saved as fastq files. The dirty raw reads including those with adaptors, containing more than 10% of unknown bases, and low quality reads (the percentage of the low quality bases of quality value ≤ 5 is more than 50% in a read) were filtered, then clean reads were obtained for further analysis. Brassica _95k_unigene (http://brassica.nbi.ac.uk/) was used as the reference genome, and clean reads of each sample were mapped to reference sequences using SOAP2 , allowing no more than two mismatched bases. The gene expression level was calculated by using RPKM method (Reads Per kb per Million reads) . If there were more than one transcript for a gene, the longest one was used to calculate its expression level and coverage.
Screening of differentially expressed genes (DEGs)
Referring to “The significance of digital gene expression profiles ”, BGI have developed a strict algorithm to identify differentially expressed genes between two samples using the Poisson model. Denoted the number of unambiguous clean tags from gene A as x, given that every gene's expression occupies only a small part of the library, p(x) will closely follow the Poisson distribution: p(x) = e-λλx / x! (λ is the real transcripts of the gene). Then, P-value corresponds to differential gene expression test. FDR (False Discovery Rate) is a method to determine the threshold of P-value in multiple tests. More stringent criteria with smaller FDR and bigger fold-change value can be used to identify DEGs. We used “FDR ≤ 0.001 and the absolute value of log2Ratio ≥ 1” as the threshold to judge the significance of gene expression difference.
GO and pathway enrichment analysis of DEGs
GO enrichment analysis provides all GO terms that are significantly enriched in DEGs comparing to the genome background, and filter the DEGs that correspond to biological functions. This method firstly maps all DEGs to GO terms in the database, calculating gene numbers for every term, then using hypergeometric test to find significantly enriched GO terms in DEGs comparing to the genome background. The calculating formula used was the same as previously described . The calculated p-value goes through Bonferroni Correction, taking corrected p-value ≤ 0.05 as a threshold. GO terms fulfilling this condition are defined as significantly enriched GO terms in DEGs. Pathway enrichment analysis also applies a hypergeometric test to identify significantly enriched metabolic pathways or signal transduction pathways in DEGs comparing with the whole genome background, using the major public pathway-related database: KEGG . The calculating formula is the same as that in GO analysis, the pathways with a Q value of ≤ 0.05 are defined as those with significantly differentially expressed (enriched) genes . Additionally, GO function analysis of DEGs with the criteria of RPKM ≥ 50 and fold change ≥ 2, DEGs specific to S1 and specific to H3 plants were performed using blast2Go (http://www.blast2go.com/b2ghome), respectively. GO enrichment analysis of these genes was performed based on the TAIR GO slim provided by blast2GO, filtered by Seq: cutoff = 5.0, as well as the soft agriGO (http://bioinfo.cau.edu.cn/agriGO/).
Real-time Quantitative RT-PCR (qRT-PCR) analysis
The RNA samples used for the qRT-PCR assays were the same as for the RNA-seq experiments. First-strand cDNA synthesis was performed with 1500 ng of total RNA using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas), total RNA (0.5 μg) was reverse-transcribed with oligo (dT)18 primer (0.5 μg/μl) using RevertAid™ Reverse Transcriptase according to the described protocol. Gene-specific primers were designed according to the reference unigene sequences using the Primer 3.0, all primer sequences are listed in Table 3. A primer was also designed for B. napus actin gene to normalize the amplification efficiency. QRT-PCR assays in triplicate were performed using THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) with a Bio-Rad CFX96 Real-Time Detection System. The actin gene was used as an internal control for data normalization, and quantitative variation in the different replicates was calculated using the delta-delta threshold cycle relative quantification method.
Availability of supporting data
The data set supporting the results of this article is available in NCBI’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE49606 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE49606). Other supporting data are included within the article and its additional files.
This work was funded by Natural Science Foundation of China (Grant No. 30900903) and Ministry of Science and Technology of China (2012BAD49G00).
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