Single-molecule real-time transcript sequencing identified flowering regulatory genes in Crocus sativus
Saffron crocus (Crocus sativus) is a valuable spice with medicinal uses in gynaecopathia and nervous system diseases. Identify flowering regulatory genes plays a vital role in increasing flower numbers, thereby resulting in high saffron yield.
Two full length transcriptome gene sets of flowering and non-flowering saffron crocus were established separately using the single-molecule real-time (SMRT) sequencing method. A total of sixteen SMRT cells generated 22.85 GB data and 75,351 full-length saffron crocus unigenes on the PacBio RS II panel and further obtained 79,028 SSRs, 72,603 lncRNAs and 25,400 alternative splicing (AS) events. Using an Illumina RNA-seq platform, an additional fifteen corms with different flower numbers were sequenced. Many differential expression unigenes (DEGs) were screened separately between flowering and matched non-flowering top buds with cold treatment (1677), flowering top buds of 20 g corms and non-flowering top buds of 6 g corms (1086), and flowering and matched non-flowering lateral buds (267). A total of 62 putative flower-related genes that played important roles in vernalization (VRNs), gibberellins (G3OX, G2OX), photoperiod (PHYB, TEM1, PIF4), autonomous (FCA) and age (SPLs) pathways were identified and a schematic representation of the flowering gene regulatory network in saffron crocus was reported for the first time. After validation by real-time qPCR in 30 samples, two novel genes, PB.20221.2 (p = 0.004, r = 0.52) and PB.38952.1 (p = 0.023, r = 0.41), showed significantly higher expression levels in flowering plants. Tissue distribution showed specifically high expression in flower organs and time course expression analysis suggested that the transcripts increasingly accumulated during the flower development period.
Full-length transcriptomes of flowering and non-flowering saffron crocus were obtained using a combined NGS short-read and SMRT long-read sequencing approach. This report is the first to describe the flowering gene regulatory network of saffron crocus and establishes a reference full-length transcriptome for future studies on saffron crocus and other Iridaceae plants.
KeywordsSaffron Flower SMRT sequencing qRT-PCR Alternative splicing
Clusters of Orthologous Groups
Duplication-Removed and Corrected Long Reads
Fragments Per Kilobase of transcript per Million mapped reads
Kyoto Encyclopedia of Genes and Genomes
Long non-coding RNA
Next Generation Sequence
Open Reading Frames
Reads of Inserts
Single Molecule Real-Time
Sequence Read Archive
Simple Sequence Repeats
Unpolished Consensus Sequences
Crocus sativus L, commonly known as saffron crocus, is prized for purple flowers that are well known for producing spice saffron from the filaments. Spice saffron is the most valuable spice used as a fabric dye and in traditional medicine with special medicinal effects of promoting blood circulation, cooling blood and detoxifying, thereby relieving depression and soothing nerves . As a valuable traditional Chinese medicine, saffron is widely used in China and Europe. Saffron crocus blooms only once a year and unlike most spring-blooming plants, saffron crocus does not blossom until autumn. In China, the daughter corms began to grow at the end of January and matured at the end of May and subsequently, entered a dormant period until mid-August. During the period, the corms were dug out from the soil when the leaves turned yellow and wilted and moved into the door to store. Experiencing the high temperature treatment in summer (ranged from 23 to 27 °C), the buds were broken up from dormancy in the middle of August and the floral primordia began to initiate. When the average room temperature fell to 15–17 °C in mid-autumn, most apical buds were in blossom . Basically, the corms had 1–3 apical buds and 6–10 lateral buds depending on their weight. Each apical bud germinated 1–3 flower primordia while lateral bud usually did not blossom. Occasionally, one or more lateral buds of corms weighing more than 30 g also blossomed. The corms weighing less than 6 g cannot blossom. As soon as all the flowers were picked up indoors, the corms were planted in the soils until the new daughter corms matured in the next May. Planting and harvesting corms as well as collecting red stigmas from flowers, is performed manually. To produce 1 kg of dry saffron, 110,000–170,000 flowers are harvested and 40 h of labour are needed to pick 150,000 flowers. Such labour-intensive cultivation practices make saffron a high expensive crop with prices ranging from $500 to $5000 per pound at wholesale and retail rates . Due to limited natural resources for saffron crocus plants, inefficient cultivation, and low yield, saffron is becoming even moreexpensive and is well known as “red gold” . It is highly important to explore comprehensive genetic information for breeding and improving its biological traits.
Increasing the flower number of saffron crocus is a viable way to produce more saffron to meet the ever-increasing demand in the market [4, 5]. Research has been conducted to investigate the factors that affect floral development including temperature, photoperiod, corm size, and bud position [2, 6]. We can obtain samples of different flowering quantities by controlling these factors artificially. Therefore, C. sativa is a good material for studying the development of flowering. Many genes related to plant floral development have been discovered along with the rapid development of technology in molecular biology. For example, long-day conditions can promote Arabidopsis flowering through the function of FLOWERING LOCUS T (FT) protein, which is considered to be the main component of “florigen” [7, 8]. The transcription factor Flowering Locus C (FLC) is a key regulator of the vernalization process of Arabidopsis thaliana. The transcription factor PIF4 is a major regulator of high temperature-induced flowering . Using the FT gene in Arabidopsis thaliana as a reference, Tsaftaris et al. cloned a CENTRORADIALIS/TERMINAL FLOWER1 (CsatCEN/TFL1) like gene  and three FT-like genes  from the flowers, flower buds, leaves, and corms of saffron crocus, respectively, and further proved that their expression patterns were tissue-specific and depended on the flower developmental stage. Other studies found a serial potential flower-related gene in saffron crocus, for instance, B-class paleo AP3-like genes (CsatAP3-like) , AP1-like MADS-box genes , B-class floral homeotic genes PISTILLATA/GLOBOSA , E-class SEPALLATA3-like MADS-box genes , and CsMYB1, a transcription factor belonging to the R2R3 family . Later, NGS-based RNA-seq technology was widely used for gene discovery, which led to the identification and functional characterization of flowering genes in various species. For example, trehalose 6-phosphate synthase and squamosa promoter-binding protein-like genes promoted the floral induction of apple trees . A series of genes related to the circadian clock are important key regulators for the flower development of hibiscus . Using NGS-based RNA-seq technology, Baba et al.  and Jain et al.  discovered the gene expression of saffron crocus involved in apocarotenoid biosynthesis and further explored the expression profiling of zinc-finger transcription factors . However, the underlying molecular mechanism controlling and/or affecting the number of flowers of saffron crocus has not been determined. The genome has not been fully elucidated to date, even in the whole Iridaceae family, only de novo assembly based short-fragment transcriptome of saffron crocus was provided by Illumina RNA-seq sequencing [19, 20, 21].
Recently, the third-generation sequencing platform, SMRT sequencing, developed by PACBIO RS (Pacific Biosciences of California, Menlo Park, CA, USA), was used in transcript sequencing. The sequencing platform is good for long reads with an averaged read length of > 10 kb, and real length can reach 60 kb (http://www.pacb.com/smrt-science/smrt-sequencing/read-lengths/). After correction by next generation sequence (NGS) reads and self-correction via circular-consensus sequence (CCS) reads, the error rate of SMRT sequencing is expected to be 1% . This technology has been applied to access complete transcriptome data of a few plants, including Carthamus tinctorius (safflower) , Cassia obtusifolia (Jue-ming-zi) , Panax ginseng (Korean ginseng) , Salvia miltiorrhiza (danshen) , Sorghum bicolor (sorghum)  and Zea mays (maize) .
Compared with the NGS platform, PacBio Iso-Seq can obtain a collection of high quality full-length transcripts without assembly, which is especially important for species without reference genome sequences. Some transcripts might contain repeat regions, whereas transcripts of different gene isoforms show high sequence similarity. The assemblies of short sequencing reads often encounter complications without reference genome sequences. The problem seems more severe for saffron crocus, because of its relatively larger genome size  (greater than 10 Gb) and polyploid characteristics  (2n = 3x = 24). Saffron crocus consists mainly of repetitive DNA sequences, such as retrotransposon and satellite DNA , resulting in particular challenges for the accuracy of short-read assembly. The PacBio Iso-Seq technology can overcome these difficulties by generating sequence information for the full length sequence as a single sequence read without further assembly.
In this paper, NGS and SMRT sequencing were combined to generate two sets of full-length transcriptomes of flowering and non-flowering saffron crocus. Moreover, differentially expressed full-length transcripts of flowering and non-flowering saffron crocus were identified and characterized.
Materials and methods
Saffron crocus plants were cultivated at a research farm at South Tai Lake Agricultural Park, Huzhou (longitude 120.6° E, latitude: 30.52° N, elevation 0 m), using a two-stage cultivation method: corms planted in soil to allow them to grow outdoors and be cultivated indoors without soil . In May 2016, dormant corms were excavated from the field and stored indoors for approximately half a year until flowering.
Two sample pools were set up to establish the PacBio Iso-seq libraries of flowering saffron crocus and non-flowering saffron crocus separately. One sample pool was constructed for the full-length transcript set of flowering saffron crocus, which included 1) top bud tissues, 2) tuber tissues of flowering corms (5–7 mm, ≈20 g) (recently differentiated flower primordia and leaf primordia), 3) pistils, 4) stamens of flowering corms (≈20 g) when colours turned from yellow to red, and 5) leaves of flowering corms (≈20 g) when colours turned from white to green), and 6) purple petals of flowering corms (≈20 g). The other sample pool was constructed for the full-length transcript set of non-flowering saffron crocus, which included 1) top bud tissues, 2) lateral bud tissues, 3) tuber tissues of non-flowering corms (5–7 mm, ≈20 g), 4) leaves of non-flowering corms (≈20 g) when turned from white to green, and 5) top bud tissues of non-flowering corms (5–7 mm, ≈6 g) (Additional file 1: Figure S1).
Meanwhile, an additional five groups of saffron crocus corms were prepared to construct higher-accuracy short-read libraries using an Illumina RNA-seq method. The sample pools included 1) top buds of flowering saffron crocus corms, 2) paired top buds of non-flowering saffron crocus corms (≈20 g) that were split into two parts and cultivated at room temperature (20–25 °C, flowering phenotype) and 10 °C (non-flowering phenotype) for 15 days, 3) lateral buds of flowering saffron crocus corms, 4) paired lateral buds of non-flowering saffron crocus corms (≈30 g), and 5) top buds of non-flowering saffron crocus corms (≈6 g) (Additional file 1: Figure S1). All five bud samples were collected when they were 5–7 mm long. A total of 15 plants, (three plants per group) were harvested to construct 15 Illumina RNA-seq libraries.
All the samples prepared for both PacBio Iso-seq and Illumina RNA-seq sequencing were immediately frozen in liquid nitrogen until RNA was isolated.
All tissues were ground in liquid nitrogen and total RNA was extracted using an RNeasy@Plant Mini Kit (Qiagen Corporation, Hilden, Germany) according to the manufacturer’s protocol. The isolated RNA samples were detected using 1% agarose electrophoresis to avoid degradation and genomic DNA contamination. RNA purity (OD 260/280 = 2.0–2.2, A260/A280 = 1.8–2.1) was quantified using a Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA), and the concentration of RNA samples was quantified using a Qubit 2.0 Fluorometer (Thermo Scientific, MA, USA). RIN Integrity Number (RIN) values and 28S/18S (28 s: 18 s > = 1.5, RIN > = 8) were measured using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).
PacBio Iso-Seq library preparation and sequencing
Illumina RNA-seq library preparation, sequencing, and Contigs assembly
Fifteen RNA samples from saffron crocus buds were used for Illumina RNA-seq library construction and sequencing. Total RNA was enriched using Oligo (dT) magnetic beads and randomly broken into short fragments that were further used as a template to synthesize cDNA with random hexamer-primers. The cDNA products were end-repaired, A-tailed, and added with Illumina paired-end adapters. The fragments were selected using AMPure XP beads and PCR amplified to obtain sequencing libraries that were qualified and paired-end sequenced with an Illumina Hiseq 2000 (Illumina, San Diego, CA, USA).
The raw reads of the sequences were obtained by removing adapter reads, reads with length of < 100 bp, and reads with content of ambiguous bases ‘N’ > 5%. De novo assembly of transcriptome sequencing without reference genome, including steps of Inchworm, Chrysalis, and Butterfly with default parameters was conducted using Trinity software.
Quality control, error correction of PacBio reads and Contigs mapping between corrected PacBio reads and Contigs from RNA-seq
The raw data from the PacBio RS II platform were filtered using SMRTLink software (version 4.0) to obtain Post-Filter Polymerase reads, namely, CCS, when the adaptors, subreads < 50 bp, polymerase reads < 50 bp and accuracy of polymerase reads < 0.75 were deleted. CCS were further self-corrected and filtered with the criterion of full passes > 1 and the predicted consensus accuracy > 0.8 toobtain high-quality reads of inserts (ROIs). ROIs were classified into non-full-length reads and full-length reads (including full-length non-chimeric reads and full-length chimeric reads) based on the presence and location of 3′ primer, 5′ primer and polyA. Full-length non-chimeric reads were corrected by the CEC algorithm and produced Unpolished Consensus Sequences (UCS). The UCS and the remaining ROIs were further corrected using Quiver software to obtain polished high-quality isoforms (accuracy > 0.99) and polished low-quality isoforms.
Subsequently, all Quiver-polished isoforms were mapped to Trinity-assembled contigs from RNA-seq to produce Trinity-corrected Pacbio Isoforms using LoRDEC software . By aligning the Trinity-corrected Pacbio Isoforms to contigs assembled by Trinity with a high level of similarity (> 99% threshold), the longest contigs were assigned to the duplication-removed and corrected long reads (DRCLR). The DRCLR was corrected to remove redundant information using CD-HIT software (version 4.6) and regarded as Unigene.
To predict unigene function, unigenes were searched against five databases, including Cluster of Orthologous Groups of proteins (COG), SwissProt, NCBI non-redundant (NR), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Functional annotation of unigenes was obtained from sequence similarity alignment using the BLAST algorithm with a criterion of E-value <1e-10.
Prediction of coding DNA sequence and protein
All the isoforms were used to predict the coding sequences (CDS) and protein sequences using ANGEL software with Arabidopsis thaliana and Phalaenopsis equestris (orchid) genomes as the reference genomes. The genome of the Iridaceae family has not been fully elucidated to date . Among all species with known genomes released recently, Phalaenopsis equestris has the most homology with saffron crocus .
SSR annotation and long non-coding RNA identification
SSRs (simple sequence repeats) were searched using MISA software (version 1.0) . Long non-coding RNAs (lncRNA) were predicted according to the guiding principles of lncRNAs pipeline (https://bitbucket.org/arrigonialberto/lncrnas-pipeline) with PLEK (an improved k-mer scheme tool) as the core algorithm . PLEK is widely used to discriminate protein-coding mRNAs and non-coding RNAs and has the ability of predict all possible open reading frames (ORFs) and translate the sequences into peptides.
Alternative splicing analysis and validation
The alternative splicing (AS) events were predicted based on the BLAST alignment of DRCLR to the Trinity-assembled contigs from RNA-seq sequencing using default parameters. AS events were defined when the alignment gaps were longer than 50 bp and were at least 100 bp from the 3′ and 5′ ends . The specific AS presented in only the PacBio Iso-seq library of flowering or non-flowering saffron crocus were screened separately. To validate the accuracy of the AS detected with PacBio Sequencing, RT-PCR of three randomly selected unigenes, PB.174, PB.313 and PB.988,was performed. Total RNA of saffron crocus buds was extracted as described above. The PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan) and SYBR Premix Ex Taq II (TaKaRa, Japan) were used for reverse transcription reaction and PCR assay. Specific primers (Additional file 2:Table S1) of the chosen genes were designed using Primer Premier 5.0 software (Premier, Vancouver, British Columbia, Canada) according to the homologous sequences at the upstream and downstream ends of all the different alternative splicing fragments. The PCR amplification procedure included 98 °C 10 s, 56 °C 30 s, 72 °C 3 min for 30 cycles and then 72 °C extended for 5 mins. PCR products were monitored by 1% agarose gel electrophoresis. Sequencing of the PCR products further confirmed the correctness of the amplification.
Screening differentially expressed Unigenes and GO and KEGG enrichment analyses
The expression levels of all the unigenes in fifteen samples were assayed based on the Illumina short reads dataset, and reference sequences were the unigene libraries. Relative gene expression levels of each unigene were determined by FPKM (fragments per kilobase of transcript per million mapped reads) and differentially expressed unigenes were screened using DESeq2 R with parameter cutoff p-value < 0.05, FDR < 0.01 and fold change ratio > 2.
Differentially expressed unigenes were also employed for the enrichment analyses of GO and KEGG pathway with adjusted p-value (q-value) < 0.05 serving as the standard for significantly enriched pathway.
Validation of differentially expressed Unigenes using real-time qRT-PCR
Twenty (8 flowering and 12 non-flowering) top buds and ten (4 flowering and 6 non-flowering) lateral buds of saffron crocus with various corm weighst and bud lengths were used to validate differentially expressed unigenes using real-time quantitative reverse transcription PCR (qRT-PCR). Eleven differentially expressed unigenes between flowering and non-flowering samples were selected for validating key flower unigenes. All buds were ground in liquid nitrogen, and total RNA was prepared using an RNeasy@Plant Mini Kit. The PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan) and SYBR Premix Ex Taq II (TaKaRa, Japan) were used for reverse transcription reaction and qRT-PCR assay. Specific primers of the chosen genes were designed using Primer Premier 5.0 software (Additional file 2: Table S2). PCR products were verified by dissociation curves, and data were normalized with endogenous reference tubulin gene to obtain ΔCt values. Water was used as a negative and quality control, and each sample was measured in triplicate.
Expression analysis of the flower-related genes in tissues and organs
The expression analysis of the flower-related genes in different tissues and organs was performed with qRT-PCR. Total RNA from the top and lateral buds (0.5–1 cm in length), the inner immature flowers (obtained from top bud when it grew to 1.5–3 cm in length), the corms, leaves, petals, stigmas, stamens and the remaining protective sheath of the full-bloom flowers, were extracted using an RNeasy@Plant Mini Kit and the following reverse transcription reaction and qRT-PCR assays were conducted according to the above description. The expression levels of flower-related genes in each sample were normalized to the tubulin gene to obtain ΔCt values. The top bud was used as a control sample, and the relative expression levels of target genes in the other samples were analysed using the 2-ΔΔCt method: ΔΔCt = ΔCt other sample (Ct target gene- Ct tublin)- ΔCt control sample (Ct target gene- Ct tubulin).
Time course expression analysis of flower-related genes during the flower development
Total RNA from four different stages of top buds from 20 g corms, including resting bud (1–2 mm in length), early stage of shoot growth (2–5 mm in length), late stage of shoot growth (5–10 mm in length), and stage of visually distinguishable flower organ formation (10–15 mm), were extracted using an RNeasy@Plant Mini Kit and the following reverse transcription reaction and qRT-PCR assays were conducted according to the above description.
The raw data were uploaded to Sequence Read Archive (SRA) (http://www.ncbi.nlm.-nih.gov/) with a reference of PRJNA528829.
Long-length Transcriptome of saffron Crocus from PacBio Iso-seq
High-quality RNAs from top buds, tubers, pistils, stamens, petals and leaves of flowering saffron crocus were combined to acquire the PacBio Iso-seq libraries. Meanwhile, PacBio Iso-seq libraries of non-flowering saffron crocus were constructed using leaves, lateral buds, tubers, and top buds of non-flowering corms (20 g and 6 g). Multiple size-fractionated cDNA and cells (3 cells for 1–2 kb, 3 cells for 2–3 kb, 2 cells for > 3 kb) were prepared to construct flowering/non-flowering Iso-seq libraries separately. This approch avoids loading bias and obtaining more RNA sequences representing the gene expression profiles in flowering and non-flowering saffron crocus.
A total of 22.85 Gb of clean data were obtained from all sixteen cells with 1,325,207 raw polymerase reads and 23.9 billion nucleotides. After the adaptor and low-quality sequences were filtered, a total of 12,433,006 subreads were obtained, among which 7,178,336 and 5,254,670 subreads were in the libraries of flowering and non-flowering saffron crocus, respectively (Additional file 2: Table S3). High quality ROIs were further generated from CCS after filtering with full passes and accuracy. The numbers of ROIs from the flowering saffron crocus libraries were 224,710 for 1–2 kb, 199,782 for 2–3 kb, and 106,171 for 3–6 kb, respectively, which were more than those of the corresponding non-flowering saffron crocus libraries (179,712 for 1–2 kb, 73,160 for 2–3 kb, 52,904 for 3–6 kb) (Additional file 2: Table S4). In total, 394,653 (74.4%) and 252,850 (82.7%) full-length non-chimaera reads (FL non-chimaera, full-length reads with 3′ primer, 5′ primer and polyA reads after chimaera was filtered) were produced from ROIs of flowering and non-flowering saffron crocus libraries, respectively, with average lengths of 1223 bp, 2333 bp and 3512 bp in corresponding flowering saffron crocus libraries and 1188 bp, 2236 bp and 3322 bp in that of non-flowering saffron crocus libraries (Fig. 1b, Additional file 2: Table S4)).
After classification and correction by Clustering for Error Correction (CEC) and Quiver programs, 79,841 high-quality (Accuracy > 0.99) and 219,720 low-quality polished CCS were generated from ROIs. CCS were further corrected using the de novo assembly reads derived from Illumina RNA-seq. Ultimately, a total of 216,419 isoform level transcripts and 75,351 unigene transcripts were obtained after two-step CD-HIT classification of both flowering and non-flowering PacBio libraries. The length distribution of polished CCS, isoform and unigene is shown in Fig. 1c, with a majority of sequences ranging from 1 kb to 4 kb. The libraries of flowering and non-flowering saffron crocus were constructed separately, and the specific isoforms in each library and the differential expression profiles between flowering and non-flowering saffron crocus plants were obtained. The number of isoforms that expressed in both flowering and non-flowering saffron crocus was 174,369, while the number of isoforms that only expressed in flowering saffron crocus (30,188) were considerably more than those in non-flowering saffron crocus (11,862). These isoforms may provide a novel avenue to clarify the underlying molecular mechanism of floral development of saffron crocus.
Total 125 mRNAs derived from saffron crocus were reported on NCBI database at present. All the 75,351 full-length unigene transcripts were homologously aligned with them using BLAST. The results showed total 108 previously reported mRNAs were identified and matched with their highly homologous sequences in our data, with 86.4% coverage rate (Additional file 2: Table S5). Among them, 44 unigenes have a sequence identity of 99% or more and the identity of 88 unigenes were more than 95%, which suggested a full-length unigene database of saffron crocus with satisfactory coverage and accuracy was obtained in this study.
Saffron Transcriptome of short-reads from Illumina RNA-seq
Fifteen Illumina RNA-seq libraries constructed from saffron crocus with different numbers of flowers (0–3) were sequenced to correct the polished CCS of PacBio Iso-seq and to quantify full length transcripts obtained from PacBio Iso-seq. After trimming process and screening with a high quality score, a total of 745 million clean reads were produced from all samples. Over 575 million short reads were successfully mapped back to the full-length of PacBio Iso-seq with an average mapping ratio of 77.2% (Additional file 2: Table S6), which suggested that the full-length transcripts derived from PacBio Iso-seq data method represented the majority of the genetic information of both flowering and non-flowering saffron crocus.
Databases such as NR, Swiss-Prot, KEGG (Additional file 3: Figure S2a), COG (Additional file 3: Figure S2b), and GO (Additional file 3: Figure S2c) were used to perform functional annotations to the 75,351 unigenes.
A total of 14,159 (21.9% of annotated unigenes) unigenes were associated with 34 pathways in KEGG pathway analysis. A high percentage of unigenes were assigned to “Translation” (10.3%) and “folding, sorting and degradation” (9.3%) of the genetic information process as well as “signal transduction” of the environmental information process (9.7%) (Additional file 3: Figure S2a).
A total of 64,562 unigenes (85.7%) were successfully matched to known sequences in at least one database. There were 99.5% matched unigenes in the NR database, 82.0% in SwissProt, and 72.0% in COG (Additional file 3: Figure S2d).
A total of 1193 GO terms were assigned to 33,117 unigenes (51.3% of annotated unigenes) with 454 biological processes, 159 cellular components and 580 molecular functions. In the class of biological processes, the top three GO terms were “metabolic process”, “cellular process”, and “single-organism process”. In the cellular component, “cell” was dominant and then “cell part” and “organelle”. In the class of molecular functions, a high percentage of the unigenes were enriched in “binding”, “catalytic activity” and “molecular function regulator” (Additional file 3: Figure S2c).
CDS, SSR, and LncRNA prediction
SSRs, also known as microsatellite DNAs, have a tandem repeat motif of 1–6 bp in length. The most common motifs are dinucleotide repeats, such as (CA) n and (TG) n. The characters of high polymorphism (mainly due to the difference in the number of tandem motifs), stability, and reliability enable it to be an ideal molecular marker that is widely used in such applications as genetic map construction, quantitative trait locus (QTL) mapping and genetic diversity assessment. A total of 79,028 SSRs were identified in 34,895 unigenes (46.3% of total unigenes), including six types of SSR: mono-nucleotide (56,262, 71.2% of all SSRs), di-nucleotide (12,397, 15.7%), tri-nucleotide (9411, 11.9%), tetra-nucleotide (548, 0.7%), penta-nucleotide (165, 0.2%), and hexa-nucleotide (245, 0.3%) (Fig. 2b); among them, 28,993 SSRs present in compound formation.
The PLEK workflow of lncRNA-pipeline was used to discriminate between coding and non-coding transcripts and then identify lncRNAs using PacBio data from species with no reference genome. To obtain more putative lncRNA candidates for saffron crocus, 216,419 isoform transcripts were used to predict lncRNAs in this study. A total of 72,603 (33.5%) PacBio non-coding transcripts were obtained and the length ranged from 194 bp to 6860 bp with an average length of 1367 bp. Similar to other species, the length abundance is concentrated at 500–1500 bp (54,296, 74.8%) (Fig. 2c).
Alternative splicing analysis and validation
Furthermore, different splicing events between flowering and non-flowering saffron crocus were screened in PacBio-Seq libraries because AS is highly tissue-specific. A total of 42 AS events were identified in the PacBio-Seq libraries of flowering saffron crocus only, while 28 AS events were found in those libraries of non-flowering one (Fig. 3d, Additional file 2: Table S8). The AS events of three randomly selected unigenes, PB.174, PB.313 and PB.988, were validated using RT-PCR. Four, three, and two AS events were shown in the gel electropherogram (Fig. 3e), and the correctness of the amplification was proved by sequencing of the PCR products.
Differentially expressed Unigenes between flowering and non-flowering saffron Crocus corms
The expression levels of all unigenes from PacBio-Seq libraries in five groups of saffron crocus corms with different flowering phenotypes were quantified using an FPKM method based on the results of Illumina RNA-seq. In general, when FPKM < 0.1, the gene does not express; 0.1 ≤ FPKM < 3.75, the gene is considered to be expressed at a low level; 3.75 ≤ FPKM < 15, the gene is at a median level; FPKM ≥15, the gene is expressed at a high level. RNA-Seq2 software was used to screen differentially expressed unigenes between flowering and non-flowering saffron crocus corms, and the threshold of significance was determined as p-value < 0.05, false discovery rate (FDR) < 0.01 and log2 fold-change < − 1 or > 1.
A total of 1086 significant DEGs (Additional file 2: Table S9), 81.7% (887) of which were up-regulated, were identified in top buds between flowering saffron crocus and non-flowering saffron crocus of 6 g corms, with log2 fold-changes ranging from − 14.2 to 10.6. A total of 141 GO terms were clustered, and the top 5 pathways in each main ontology are shown in Fig. 4b. Within the cellular component ontology, the DEG-coding proteins were mainly located in the “macromolecular complex”, “intracellular part” and “cytoplasm”. For the biological process ontology, DEG-coding proteins were mainly involved in the transcription processes, such as “regulation of transcription, DNA-templated”, “regulation of RNA metabolic process” and “regulation of nucleic acid-templated transcription”. In terms of molecular function ontology, DEG-coding proteins were binded with nucleic acid and functioned as transcriptional regulation factors (Fig. 4b). In addition, 70 and 32 unigenes were exclusively highly expressed in all samples of large corms of flowering saffron crocus and small corms of non-flowering saffron crocus, respectively (Additional file 2: Table S11).
hree paired samples of lateral buds from the same corms of flowering and non-flowering saffron crocus were collected to screen key genes that regulate the flowering of lateral buds. A total of 267 DEGs (Additional file 2: Table S11), 83.5% (223) of which were down-regulated, were identified with log2 fold-changes ranging from − 8.5 to 9.4. All 267 DEGs were assigned to 75 GO terms. The only GO terms in the cell component ontology was “nucleus”. The most highly enriched pathway in biological progress ontology was “microtubule- based movement” (GO level 5), which was in the sub-grade GO level of both top 2 (“movement of cell or subcellular component”, GO level 4) and top3 (“microtubule-based process”, GO level 4) pathways, followed by the pathway of “DNA replication”. In the molecular function ontology, most unigenes were highly enriched in a series of pathways from GO level 4 (hydrolase activity, acting on acid anhydrides) to GO level 7 (Nucleoside-triphosphatase (NTPase) activity). NTPases activation may regulate the nuclear export of mRNA and are known to be involved in defense signaling pathways and apoptosis regulation  (Fig. 4c).. In addition, 15 unigenes (Additional file 2: Table S11) were highly expressed in three lateral buds of non-flowering saffron crocus specifically but were not expressed in lateral buds of flowering saffron crocus. Only 7 unigenes were exclusively highly expressed in lateral buds of flowering saffron crocus (Additional file 2: Table S12). These 22 unigenes could be preferentially used to explore the molecular mechanism of the flowering process of lateral buds.
As for KEGG pathway analysis, although the DEGs involved in each experiment are different, “plant hormone signal transduction” was assigned by all the three comparable experiments, which even is the only pathway enriched in flowering vs. non-flower lateral buds (Fig. 4c). The changes of flowering phenotype caused by nutritional status (normal vs. small top buds), low temperature stress (normal vs. cold treatment) or natural variation (flowering vs. non-flower lateral buds) may be involved in different pathways, but they shared the same regulation network of plant hormones, including the response to hormone auxin, abscisic acid and cytokinin (for example, auxin-responsive protein IAA10-like, probable indole-3-acetic acid-amido synthetase GH3.5, auxin transporter-like protein 3, abscisic acid receptor PYL8-like, two-component response regulator ARR8).
Both DEGs of normal vs. small top buds and normal vs. cold treatment were assigned to the KEGG pathways of “starch and sucrose metabolism” and “linoleic acid metabolism”. It has been recognized for long time that temperature could modify carbohydrate metabolism of some plants by regulating starch and sugar levels . Furthermore, altered carbohydrate metabolism impacted plant biomass production, as well as flower development. Actually, altering the expression of multiple sucrose metabolism-related genes in tobacco, such as UGPase, SuSy and SPS which were also found in DEGs of saffron crocus, not only enhanced primary growth but also altered flower morphology . Based on the fact that flower numbers of saffron crocus were heavily depended on temperature and corm weight, the results of KEGG enrichment suggested that carbohydrate metabolism may play important roles in flower development of saffron crocus.
Identification and expression analysis of flower-related DEGs in saffron Crocus
A total of 62 DEGs were functionally annotated as putative flower-related genes (Additional file 2: Table S13) and we analysed the expression patterns of all the transcripts using RNA-seq data (Fig. 5c). We identified 39 flower-related genes (assigned with predicted gene names) in the normal vs cold treatment group, 24 of which were significantly highly expressed in flowering corms. CsVRN (B3 domain-containing transcription factor) is a key gene involved in the vernalization pathway, which may affect flower development when the saffron crocus was treated with cold.
In the normal vs. small top buds group, 33 flower-related genes were identified and interesting, putative flowering time control protein (CsFCA) and squamosa promoter-binding-like protein 12 (CsSPL12), which are involved in autonomous and age pathways, were significantly highly expressed in the larger corms. The decreasing expression levels of CsFCA and CsSPLs may partly explain why the small corms (weight less than 6 g) cannot bloom. Furthermore, SPL12, strongly regulated together with CONSTANS (CO), were negatively regulated by miR156b and miR156h, suggesting microRNAs biogenesis or microRNA alteration maybe involved in the process of floral initiation .
We identified fourteen shared flower-related DEGs in normal vs cold treatment and normal vs. small top bud groups. Most of them (6/14) were involved in the gibberellin pathway, including putative gibberellin 3-beta-dioxygenase 1-like (CsG3OX), gibberellin-regulated protein 6-like (CsGASA6) and gibberellin-regulated protein 14 (CsGASAE) .
Only five flower-related DEGs were identified in the flowering vs. non-flowering lateral bud groups and all of them were significantly lower in the flowering samples. There may be some novel genes, and even new regulatory pathways trigger the lateral bud bloom.
Validation of novel flower-related genes in larger-scale samples using real-time RT-PCR
Ten DEGs related to the flowering process were selected to validate the key flowering genes using an additional 30 saffron crocus buds. The biological parameters of saffron crocus buds were collected, including the type of bud (top or lateral) number of flowers (0–2), length of bud (2 mm - 5 cm), and fresh corm weight. Real-time RT-PCR was used to assay the expression levels of 10 unigenes in the 30 buds of saffron crocus and the correlation between relative expression levels of each gene (ΔCT) and biological parameters of saffron crocus buds were analysed using a Pearson (length of bud, fresh corm weight) or a Spearman method (type of bud, number of flower).
Correlation analyses between expression levels of flower-related candidate genes and biological parameters of saffron crocus buds
Expression analysis of the novel flower-related genes in tissues and organs
The analysis of PB.20221.2 and PB.38952.1 was performed on the buds, corms, immature flowers, leaves, petals, stigmas, stamens and the remaining protective sheath (Fig. 7b). Compared to top buds, both PB.20221.2 and PB.38952.1 were highly expressed in the immature flowers (mean fold change = 3.13 and 2.64, respectively). PB.20221.2 and PB.38952.1 were relatively less expressed in lateral buds (mean fold change = 0.10 and 0.08, separately), petals (mean fold change = 0.17 and 0.15, separately), stigmas (mean fold change = 0.18 and 0.11, separately) and stamens (mean fold change = 0.38 and 0.29, separately). PB.38952.1 was weakly expressed in corms (mean fold change = 0.01), leaves (mean fold change = 0.002) and protective sheath (mean fold change = 0.003), while PB.20221.2 could be detected in leaves (mean fold change = 0.01) and corms (mean fold change = 0.08) with notable low expression levels but not in protective sheath (Ct > 35).
Both genes were relatively highly expressed in immature flowers, which was similar to the expression pattern of previously isolated flower-related genes in saffron-CsatFT2, CsatFT3, CsatCEN/TFL1 [10, 13] and AP1/FUL In addition, PB.20221.2 and PB.38952.1 were relatively more highly expressed in the various tissues of flower organs (petals, stigmas, stamens) than in other organs (corms, leaves, sheath), which suggested a potential function in flower development.
Time course expression analysis of novel flower-related genes during flower development
To understand the variations of PB.20221.2 and PB.38952.1 expression at different stages of floral organ development, we performed a quantitation of the transcripts in top buds from the dormant period to the start of the flowering season (Additional file 5: Figure S4). As shown in Fig. 7c, PB.20221.2 showed a fluctuation in the expression levels with the maximum expression during the stage of visually distinguishable flower organ formation (mean fold change = 8.62). The transcripts of PB.38952.1 remained constant from the stage of resting bud to the early stage of shoot growth (mean fold change = 1.31), began to accumulate at the late stage of shoot growth (mean fold change = 17.60) and largely increased at the stage of visually distinguishable flower organ formation (mean fold change = 23.79).
Taken together, the expression data in different tissues, organs and different floral development stages suggest that PB.20221.2 and PB.38952.1, probably had the function of promoting floral organ formation and development and were new flower-related genes worthy of further study in the regulatory network of the flowering pathway. The nucleotide sequences of PB20221.2 and PB38952.1 were carried out alignment with Web BLAST-blastX on NCBI database to characterize them and the listing protein sequences were downloaded and screened for Phylogenetic analysis. The deduced amino acid sequences of PB20221.2 and PB38952.1 were aligned with the selected listing protein sequences using the multiple sequence alignment program ClustalX and phylogenetic tree were constructed with MEGA-X software using neighborjoining-method with 1000 bootstrap replicates. Aligning with homologous sequences from 38 different species, Phylogenetic studies revealed that PB20221.2 and PB38952.1 belonged to heat stress transcription factor family (HSF) and clustered in HSF-A2b sub-family (Additional file 6: Figure S5), indicating they were probably associated with the regulation of heat stress in flowering process of saffron crocus and further studies were needed to investigate their specific functions.
Comprehensive, high-quality, full-length transcriptome sequences were presented for saffron crocus using SMRT technology in this study. A total of 75,351 full-length saffron crocus unigenes were identified with an average length of 2049 bp in the flowering saffron crocus library and 1802 bp in the non-flowering library. Among these unigenes, 64,562 (85.7%) were functionally annotated. Based on RNA-seq data, Baba et al. identified 64,438 transcripts with an average length of 609 bp and a functional annotation ratio of 58.5% (37,696) . Subsequently, Jain et al. found 105,269 transcripts with an average length of 1047 bp , and 54% of them were functionally annotated. Basically, illumina RNA-seq can obtain higher coverage of genome and more transcripts than SMRT-Seq due to relatively higher sequencing depth. Our results further confirmed that SMRT-seq was more effective in recovering full-length transcripts . The higher functional annotation ratio of unigenes in our study suggested that SMRT-seq data could provide higher accuracy and more effective information on saffron crocus transcriptomes.
Based on the PacBio-seq platform, 79,028 SSRs were identified in 34,895 unigenes (46.3% of total unigenes) in this study, which was considerably more than that predicted in the Illumina platform (16,721 SSRs identified in 13,407 transcripts). As popular molecular markers, SSRs are widely used for determining genetic variations, and the most important advantage is locus specificity, which is highly suitable for allopolyploid species, such as saffron crocus . It should be noted that the status of genetic variation in saffron crocus is still controversial since several studies provided contrasting results . Thus, the novel candidate SSRs of saffron crocus may provide pools to explore new alternative SSRs. In addition, the comprehensive CDS, lncRNAs and AS prediction of saffron crocus were reported for the first time in this study.
PacBio Iso-Seq libraries of flowering and non-flowering saffron crocus were constructed separately to identify as many candidate genes as possible without missing flowering suppressor genes that are expressed in non-flowering corms only. More unigenes were identified exclusively in flowering saffron crocus (30,188) than in non-flowering saffron crocus (11,862). This result suggested that saffron crocus plants might have additional genes to regulate floral development. The result might also indicate that suppressor genes for floral development may exist in some tissues of non-flowering saffron crocus only. The individual PacBio Iso-Seq library of flowering and non-flowering saffron crocus facilitated the clarification of specific AS in each library. AS functioned to regulate gene expression by introducing a premature termination codon andled to special RNA splicing isoform degradation. Increased AS was found in various plants with the application of high-throughput sequencing while the biological significance of most AS events was not determined. Recent research has demonstrated the direct function of AS in controlling the initiation and timing of flowering . A total of 25,400 AS events in the full-length transcriptome were identified, and 42 and 28 AS events were identified in the flowering and non-flowering libraries, respectively.
The underlying molecular mechanism for lateral buds of saffron crocus to flower was possibly different from top buds. The GO and KEGG pathway analyses of DEGs in lateral buds suggested potential genetic variation of flowering lateral buds, based on the enriched pathways of “cell cycle”, “meiosis”, “mismatch repair”, “DNA replication” and “nucleoside-triphosphatase activity”. The DEGs in top buds were found to function at transcriptional levels as well as in the signal pathways responding to hormones, nutrients, and other environments. Furthermore, the DEGs in lateral buds were completely different from top buds. The six shared DEGs were expressed in opposite trends between lateral and top buds. Saffron crocus multiplies by means of corms, but corm multiplication does not generate genome variation, except for occasional somatic mutations. However, phenotypic variations of flowers have been frequently observed in saffron crocus, as demonstrated by different numbers of stigmas, different aspects of tepals or blooming in lateral buds [30, 46]. As the genetic variability in saffron crocus is still under debate, whether the phenotype variations are influenced by genetic variability or the environment warrants further investigation .
In this study, PB.20221.2 and PB.38952.1 were significantly correlated with flowering quantities and were suggested as flower-related genes. Phylogenetic analysis showed both genes encoded for HSF-A2b. Heat stress transcription factor family are the terminal components of a signal transduction chain mediating the activation of genes responsive to heat stress, including more than twenty members and classified as A, B and C in plants. HSF-A2 is a strong transcription activator through interacting with heat shock proteins (HSPs) during long-term heat stress . Heat stress-regulated flowering was recently recognized as a floral transition pathway , together with the fact that HSP family was required for floral meristem formation . Thus, as the central regulator of HSP expression, HSF-A2 may play important roles in the induction of inflorescence meristem formation. In fact, all the corms of saffron crocus can blossom only after enough heat accumulation of high temperature higher than 23 °C, which also consistent with our results.
In conclusion, full-length transcriptomes of flowering and non-flowering saffron crocus were obtained using a combined NGS short-read and SMRT long-read sequencing approach. This method enables the generation of predicted comprehensive databases of AS, lncRNA, SSR, CDS, and DEGs in samples with phenotypic differences in flowering traits. These results were used to further identify genes related to flowering, including 62 genes homologous to known flowering gene sequences in public databases and novel flower-related genes,such as PB.20221.2 and PB.38952.1. Our study represents a first step to establish a reference full-length transcriptome for future studies of the gene atlas of saffron crocus and other species in the Iridaceae family. In the future, these genes, which are closely related to the number of flowers selected out in this study, maybe used as biomarkers for screening multi-flowering varieties and monitoring the optimal environmental conditions in field production.
LLQ conceptualized and designed experiments and wrote the manuscript; QXD, SYP, ZGF and YYM performed experiments; LJ and HHL contributed to data analysis; XLM contributed to the material planting and sample collection. All authors read and approved the final manuscript.
This research was supported by the National Natural Science Foundation of China [No.31600255].
Ethics approval and consent to participate
This study has not directly involved humans, animals or plants.
Consent for publication
The authors declare that they have no competing interests.
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