A de novo transcriptome of the noble scallop, Chlamys nobilis, focusing on mining transcripts for carotenoid-based coloration
© Liu et al.; licensee BioMed Central. 2015
Received: 30 May 2014
Accepted: 13 January 2015
Published: 5 February 2015
The noble scallop Chlamys nobilis Reeve displays polymorphism in shell and muscle colors. Previous research showed that the orange scallops with orange shell and muscle had a significantly higher carotenoid content than the brown ones with brown shell and white muscle. There is currently a need to identify candidate genes associated with carotenoid-based coloration.
In the present study, 454 GS-FLX sequencing of noble scallop transcriptome yielded 1,181,060 clean sequence reads, which were assembled into 49,717 isotigs, leaving 110,158 reads as the singletons. Of the 159,875 unique sequences, 11.84% isotigs and 9.35% singletons were annotated. Moreover, 3,844 SSRs and over 120,000 high confidence variants (SNPs and INDELs) were identified. Especially, one class B scavenge receptor termed SRB-like-3 was discovered to express only in orange scallops and absent in brown ones, suggesting a significant association with high carotenoid content. Down-regulation of SRB-like-3 mRNA by RNA interference remarkably decreased blood carotenoid, providing compelling evidence that SRB-like-3 is an ideal candidate gene controlling carotenoid deposition and determining orange coloration.
Transcriptome analysis of noble scallop reveals a novel scavenger receptor significantly associated with orange scallop rich in carotenoid content. Our findings pave the way for further functional elucidation of this gene and molecular basis of carotenoid deposition in orange scallop.
Carotenoids are bright yellow and red pigments that are responsible for some coloration found in animals . Carotenoids also play important physiological roles such as acting as antioxidants in the immune system [2,3]. Unlike other pigments types such as melanins, carotenoids cannot be synthesized by animals and must be acquired through diet . There are a number of factors (such as food source, seasonal change) that potentially limit the ability of animals to deposit carotenoids in their body tissues [5,6]. Although carotenoid traits have often been shown to be condition-dependent, carotenoid coloration and accumulation is also dependent on underlying genetic mechanisms. Animals preferentially deposit certain carotenoids over others, and are able to enzymatically convert and cleave dietary carotenoids into other derived forms , implying strongly the involvement of genes encoding appropriate carotenoid-binding and transport proteins or enzymes participating carotenoid metabolism.
The SRB (scavenger receptor class B) is first identified as playing a role in the uptake of lutein , carotene , zeaxanthin and xanthophylls , and lycopene . A SRB homologue, ninaD, is essential for cellular uptake of carotenoids in Drosophila and a mutation in this gene results in carotenoid-free and thus a vitamin A deficient phenotype . Two recently cloned genes, Cameo2 and SCRB15 of CD36 (Cluster Determinant 36), which are homologous to SRB, have been shown to be involved in the selective transport of lutein and β-carotene, respectively, into the silk gland of Bombyx mori [13,14]. StAR (steroidogenic acute regulatory)/MLN64 (metastatic lymph node 64) are members of the StAR domain family that are involved in the intracellular transport of cholesterol for the initiation of steroidogenesis . StAR isolated in the macula of primate retina could selectively bind lutein with high affinity . The B. mori carotenoid-binding protein (CBP) is an orthologue of vertebrate MLN64, and is involved in the transport of lutein . The BCMO (β,β-carotene-15,15’-monooxygenase)/BCDO(β,β-carotene-9,9’-oxygenase) is involved in the enzymatic cleavage of carotenoids . Loss-of-function mutation in BCMO results in hypercarotenemia . Carotenoids are cleaved to form colorless apo-carotenoid derivatives in chickens with white skin, while yellow-skinned chickens presumably have one or more cis-acting regulatory mutations in BCDO, resulting in a yellow coloration in the skin because of deposition of uncleaved carotenoids . Other genes involved in the transport and binding of carotenoids are Niemann Pick C1-like 1 (NPC1L1) , ATP-binding cassette sub-family G member 5 (ABCG5) , Glutathione S-transferase Pi1 (GST)  and crustacyanin . Lastly, intestinal transcription factor (ISX)  and retinoic acid receptor (RAR)/retinoid X receptor (RXR)  are important transcription factors that regulate the expression of genes (such as BCMO and SRB) involved in carotenoid deposition.
The noble scallop Chlamys nobilis Reeve, an important aquaculture bivalve in China, displays conspicuous polymorphism in shell color (such as orange, orange-purple, brown, etc.) and difference in muscle color (such as orange and white). The orange scallops have carotenoid-based orange mantle and adductor muscle due to high presence of carotenoids. Our previous work showed that the orange scallops with orange shell and muscle had a significantly higher carotenoid content than the brown ones with brown shell and white muscle . By establishing different scallop lines, both shell color and muscle color have been confirmed to be control by at least two loci, with one locus showing dominance epistasis to the other [28,29]. Therefore, the carotenoid-based orange coloration in muscle is likely due to differential expression of one or a few genes at the site of carotenoid deposition.
In recent years, transcriptome analysis has been widely recognized as a very useful tool to identify candidate genes underlying molecular mechanisms. In the present study, we first sequenced and assembled the transcriptome of noble scallop C. nobilis using a GS-FLX 454 platform. Second, we quantified the expression of genes that are homologous to known carotenoid candidate genes in the adductor muscle, which actively deposits carotenoids in scallop, and investigated whether differential expression was associated with carotenoid content variation in orange scallops versus brown scallops. Our goals were to 1) generate a transcriptome database useful for functional genetic studies of C. nobilis; and 2) identify candidate transcripts involved in carotenoid-based coloration or carotenoid deposition.
The scallops used in this study were taken from Nan’ao Marine Biology Station of Shantou University, located at Nan’ao island of Shantou, Guangdong, China. No specific permits were required for the described field studies, as the sampling locations were not privately owned or protected in any way. These field studies also did not include endangered or protected species. The animals were processed according to “the Regulations for the Administration of Affairs Concerning Experimental Animals” established by the Guangdong Provincial Department of Science and Technology on the Use and Care of Animals.
Sample collection and preparation
Sequence assembly and functional annotation
All sequence reads taken directly from the 454 GS-FLX sequencer were run through the sff file program (Newbler v2.6, Roche) to remove sequencing adapters A and B. Barcodes were removed by Seqclean (Lastest86_64) program and poor sequence data were further cleaned by Lucy v1.20 program (–m 50 –e 0.03 0.03 –w 30 0.03 10 0.1 –b 4 0.03). Sequences with homopolymers of a single nucleotide occupying 60% of the read and those less than 50 nucleotides in length were discarded. Trimmed sequences from orange or brown scallop were mixed and then assembled de novo using the default parameters of Newbler v2.6 (Roche). All C. nobilis EST (expressed sequence tags) sequences were submitted to NCBI Sequence Read Archive under Accession No. SRX253988. ESTs that did not form isotigs (singletons) and isotigs resulting from the assembly of multiple sequences were referred to as unique sequences. These unique sequences were translated into six reading frames and used as a query to search the public databases including Non-redundant protein database (Nr) and Swiss-Prot database (Swiss-Prot). All unique sequences were sequentially compared using BlastX (cut-off E-value of 1e-5) with the sequences in two public protein databases (Nr and Swiss-Prot). Once a sequence had a blast hit in one of the databases, a description was built from the description of that hit. Additionally, Gene Ontology (GO) terms were deduced from the blast results using Blast2GO, and sorted into the immediate subcategories for ‘molecular function’, ‘cellular component’ and ‘biological process’.
Identification of EST-SSR motifs and EST-SNPs
All EST sequences were searched for SSR motifs using the MISA (MIcroSAtellite identification) program (http://pgrc.ipk-gatersleben.de/misa/). Default settings were employed to detect perfect di-, tri-, tetra-, penta-, and hexa-nucleotide motifs (including compound motifs). To be assigned, di-nucleotide SSRs (Simple Sequence Repeats) required a minimum of 6 repeats, and all other SSR types needed a minimum of 5 repeats. Two neighboring SSRs with the maximum interruption no more than 100 nucleotides were considered as a compound SSR.
Multiple nucleotide sequence alignments of isotigs identified among the EST libraries were undertaken to identify putative SNPs. Since few reference sequences were available, SNPs were identified as superimposed nucleotide peaks where 2 or more reads contained polymorphisms at the variant allele. SNPs were identified using default parameters in gsMapper v2.3 (Roche) to align isotigs from two color datasets. In addition, only an overall transition vs transversion (Ts/Tv) ratio was calculated across the dataset. Perl script modules linked to the primer modeling software Primer3 were used to design PCR primers flanking for each unique SNP region identified.
Data mining of transcripts with putative function involved in carotenoid Deposition
Carotenoid-related candidate gene
Evidence for potential role in carotenoid deposition
Responsible for carotenoid uptake in Drosophila and mutation leads to carotenoid deficient
SRB type I
Involved in the uptake of carotenoids; homologous to ninaD in Drosophila
Involved in selective absorption of lutein
Homologous to carotenoid-uptake gene Cameo2 and SCRB15 in Bombyx mori
Carotenoid binding protein (CBP)
Involved in lutein binding and transportation in B. mori
Orthologue of B. mori CBP
CBP in the carapace of crustaceans and binding astaxanthin
Lower expression levels lead to the retention of carotenoids and a yellow skin phenotype
Lower activity leads to hypercarotenemia in human being
Involved in intocopherol intestinal absorption using Caco-2 cells and in situ perfusions in rats. Lower expression levels inhibit the uptake of several carotenoids in Caco-2 cells.
Binding carotenoid in the mammalian retina
A genetic variant in ABCG5 associate with plasma lutein concentration
Form heterodimers of RXR-RAR and regulate retinoid-responsive elements
Gatekeeper that controls intestinal β, β-carotene absorption
mRNA expression of selected candidate transcripts in orange and brown scallop
Expression of selected transcripts was investigated in adductor muscle from 6 orange scallop or 6 brown scallops at 14-month old, and two technical replicates were performed for each scallop. All scallops used in this experiment were from a F2 generation as described above, and cultured in the same cage. Total RNA was extracted and quality and quantity determined using a nanodrop spectrophotometer. 1 μg mRNA was used to synthesize cDNA by PrimeScript RT reagent kit with gDNA Eraser (TaKaRa). Quantitative real-time RT-PCR was conducted in a LightCycler®480 System using the SYBR Premix Ex Taq II qRT-PCR Kit (TaKaRa). Each assay was performed with β-actin mRNA as the internal control. The real-time PCR program was 95°C for 30s, followed by 40 cycles of 95°C for 5 s, and 60°C for 30s according to the instructions of the manufacturer. Dissociation analysis of amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. The comparative CT method (2-ΔΔCT method) was used to analyze the expression level of each candidate genes. All data were given in terms of relative mRNA expressed as means ± SE. The data were subjected to analysis of one-way ANOVA, and p-values smaller than 0.05 were considered statistically significant.
Detecting presence of SRB (scavenger receptor class B)-3-like and measurement of total carotenoid content in scallops
Four scallop lines derived from orange parents, which have color segregation of orange and brown, were chosen to performed this experiment. In total, 80 scallops (40 orange and 40 brown), derived from 4 lines produced by crossing two orange scallops in the Spring of 2012, were used to detect the presence of SRB-3-like in the blood and determine total carotenoid content in the adductor muscle. Presence of SRB-3-like in the blood was detected using primers S3F1: CGATTTTGGAACGGTAACAGTAACTTGGA and S3R1: ATGGATTGACTGATGTGAGATGT. PCR amplification product was confirmed by sequencing. Total carotenoid content in the adductor muscle was determined using the method of Zheng et al. .
Primer sequences used for dsRNA synthesis
For dsRNA synthesis, SRB-like-3 was amplified by PCR with the primers 2Fi and 2Ri (containing T7 promoter) using the recombinant plasmid pMD-18 T-SRB as the template and (Table 2). Similarly, for dsRNA synthesis of EGFP gene , plasmid pEGFP-N1 was used as the template for PCR using EGFPF and EGFPR as the primers. Quantity and quality of the DNA fragments were assessed by nanodrop spectrophotometry and electrophoresis in 1.0% agarose gel. dsRNA was synthesized in vitro using MEGAscript RNAi Kit (Life Technology) following the manufacturer’s protocol. After being incubated at 75°C for 5 min, dsRNA was cooled to room temperature, digested with DNase and RNase, and purified.
RNAi (RNA interference) assay
Forty orange scallops were used, and each of them was injected with 40 μg dsRNA of SRB-like-3 or EGFP gene (as a control) into the adductor muscle. Scallops were labeled and placed in a cage. The blank group was injected with Rnase-free water. Five individuals were sampled at 3, 6, 12, and 24 h for each group. Adductor muscle muscle, blood and intestine were subjected to total RNA extraction. Real-Time PCR was performed as described above with 2 technological replicates for each sample.
Effect of dsRNA on carotenoid deposition in the blood and adductor muscle
Orange scallops were randomly chosen, and 20 of them were injected with dsRNA of SRB-like-3 or EGFP gene, 5 of them were injected with RNA-free water as the blank group. 24 h later, they were injected again. 12 h after the second injection, 5 scallops from Rnase-free water group, 10 scallops from dsSRB-like-3 group, and 10 scallops from dsEGFP group were sampled. 1 ml blood from each scallop was freeze-dried and added with 0.5 ml acetone to extract caroteoid for about 2-4 h at darkness. Caroteoids from adductor muscle were extracted according to method by Zheng et al. . The samples were always under N2 until measurement of absorption at 480 nm to determine their carotenoid content.
Results and discussion
Roche 454 GS-FLX sequencing and isotigs assembly
Summary statistics for EST and de novo assembly
NO. of raw reads
Average length (bp)
% reads removed
NO. of reads after cleaning
Average length of cleaned reads (bp)
NO. of reads assembled as isotigs
NO. of isotigs
Average length of isotigs
Range of isotig lengths
50 - 7,102 bp
Isotigs above 200 bp
NO. of singletons
Average length of singletons
Range of singletons lengths
50 - 654 bp
Singletons above 200 bp
NO. of unique sequences a
NO. of unique sequences (left after CD-hit)
Unique sequences above 200 bases
Sequences that passed basic quality standards were clustered and assembled de novo (Newbler v2.60; Roche). Overall, approximately 91% (1,070,902) reads were assembled into 49,717 isotigs, and the others (110,158) remained as singletons (Table 1). Sequencing coverage of isotigs is shown in Figure 2C with an average 7-fold coverage. The size distribution of isotigs is shown in Figure 2D, which ranges from 50 to 7,102 bp with an average of 580 bp. The percentage of reads assembled de novo is similar to that found in other studies [31-33]. The large numbers of unique sequences (singletons and isotigs) in this study are likely due to the extensive diversity in the initial RNA samples as mentioned above. Different organs and sexes, and sequence variants in individuals are known to produce extensive alternatively spliced transcripts, resulting in misalignments and incorrect assembly between reads arising from the same genomic region .
Annotation of the transcriptome
Summary of annotation of the C. nobilis transcriptome
Total number of sequences
Sequences with Blast matches against Nr database
Sequences with Blast matches against SwissProt database
Sequences assigned GO terms
ESTs assigned with EC numbers
Gene ontology assignments
Putative molecular markers
Summary of simple sequence repeat (SSR) nucleotide classes among different nucleotide types found in C. nobilis sequences
No. of SSR- containing ESTs
NO. of SSRs
% of total SSRs
The overall frequency of all types of SNPs in the transcriptome, including INDELs, was one per 278 bp. Of the predicted SNPs, including INDELs, 122,927 (73.15%) were identified from isotigs covered by ten or more reads, suggesting the majority of SNPs identified in this study were covered at sufficient sequencing depth and more likely represent ‘true’ SNPs . Among the SNPs, 53,831 (32.03%) were identified from isotigs with annotation information.
Twenty five of these predicted SNPs were randomly selected for validation using PCR and Sanger sequencing, and 17 of these tests (68%) were successful (Additional file 5: Table S5). The result here confirmed that the majority of computationally predicted SNPs from the 454 transcriptome sequences would benefit us in our future genetic markers development.
Identification of carotenoid-based coloration transcripts from 454 sequences
Full length protein sequences of the 15 known genes responsible for carotenoid absorption, binding or carotenoids cleavage were used to perform tBlastn searches against 454-derived sequences. A total of 44 isotigs and 20 singletons from 454-derived sequences were identified with scores more than or equal to 100 and E values less than or equal to 1e-10, and 48 non-redundant sequences were developed and used to identify their putative functions by BlastX searches against the GenBank databases (Additional file 6: Table S6). After BlastX searches against Nr database, 26 transcripts were considered as tentative C. nobilis carotenoid-related transcripts (Additional file 6: Table S6).
Members of three gene families, SRB/CD36, StAR/MLN64, and BCMO/BCDO have been implicated in uptake and deposition of carotenoids in animal tissues, providing plausible candidates for carotenoids accumulation. To find out whether sequence mutations existed in transcripts that belonged to these three families, we cloned 4 SRB-like genes, 2 STAR-like genes and 2 BCMO-like genes based on transcriptome data by RACE PCR (Additional file 7: Table S7), and screened possible mutation sites of CDS. Several missense mutations were found, which, however, showed no correlation with carotenoids accumulation (data not shown).
Progeny testing of the four lines in scallop C. nobilis
Carotenoid content of adductor muscle (μg/g dry weight) and SRB-like-3 detected
Mean ± SD
90.96 ± 4.69**
3.10 ± 0.55
95.96 ± 8.13**
5.40 ± 0.68
98.21 ± 7.97**
5.84 ± 1.25
94.72 ± 4.54**
5.64 ± 1.00
mRNA expression of SRB-lile-3 after RNAi
Carotenoid content in the blood and adductor muscle after RNAi
Carotenoid content (CC) of blood
Mean ± SE
0.43b ± 0.13
Mean ± SE
1.05a ± 0.12
Mean ± SE
1.15a ± 0.15
Carotenoid content (CC) of adductor
Mean ± SE
98.96 ± 7.21
Mean ± SE
97.23 ± 12.03
Mean ± SE
97.99 ± 6.05
Here we documented a large-scale, multi-organ transcriptome for the noble scallop C. nobilis, which has the unique characterization of carotenoid accumulation but few molecular knowledge has been available. Our findings provide a nearly complete description of the expressed genes, which is a substantial contribution to the existing sequence resources for this species. Application of these resources will greatly enhance future genetic and genomic studies on scallop and other mollusks. The description of the expressed genes and their functions was illustrated according to annotation and GO assignment. 3,844 SSRs and over 120,000 high confidence variants (SNPs and INDELs) were identified that can be useful for mapping and QTLs in this scallop and related species. The most important point is that a scavenge receptor termed SRB-like-3 is only expressed in orange scallop but absent in brown scallop, significantly associated with high carotenoid content, suggesting SRB-like-3 is possibly a candidate gene responsible for carotenoid deposition in orange scallop. Results from RNAi study of this gene provides convincing evidence that SRB-like-3 is involved in carotenoid deposition in blood.
We are grateful to Dr. Chiju Wei for his careful revision and many constructive comments. Funding for this research was provided by National Natural Science Foundation of China (41076107, 31372528), Ministry of Education of P.R. China (20114402110001), China Modern Agro-industry Technology Research System (CARS-48), National Basic Research Program of China (973 Program, No. 2010CB126402), Knowledge Innovation Program of Deep Sea Science and Engineering, Chinese Academy of Sciences (SIDSSE-QN-201407), Department of Education (2050205-95), Department of Science & Technology (2013B020503061) and Oceanic and Fisheries Administrator (B201300B06) of Guangdong Province, China.
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