BACKGROUND
carotenoids are bright yellow and red pigments that are responsible for some coloration found in animals  <cit> .
carotenoids also play important physiological roles such as acting as antioxidants in the immune system  <cit> .
unlike other pigments types such as melanins, carotenoids cannot be synthesized by animals and must be acquired through diet  <cit> .
there are a number of factors  that potentially limit the ability of animals to deposit carotenoids in their body tissues  <cit> .
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  <cit> , implying strongly the involvement of genes encoding appropriate carotenoid-binding and transport proteins or enzymes participating carotenoid metabolism.
the srb  is first identified as playing a role in the uptake of lutein  <cit> , carotene  <cit> , zeaxanthin and xanthophylls  <cit> , and lycopene  <cit> .
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  <cit> .
two recently cloned genes, cameo <dig> and scrb <dig> of cd <dig> , 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  <cit> .
star /mln <dig>  are members of the star domain family that are involved in the intracellular transport of cholesterol for the initiation of steroidogenesis  <cit> .
star isolated in the macula of primate retina could selectively bind lutein with high affinity  <cit> .
the b. mori carotenoid-binding protein  is an orthologue of vertebrate mln <dig>  and is involved in the transport of lutein  <cit> .
the bcmo /bcdo is involved in the enzymatic cleavage of carotenoids  <cit> .
loss-of-function mutation in bcmo results in hypercarotenemia  <cit> .
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  <cit> .
other genes involved in the transport and binding of carotenoids are niemann pick c1-like  <dig>   <cit> , atp-binding cassette sub-family g member  <dig>   <cit> , glutathione s-transferase pi <dig>   <cit>  and crustacyanin  <cit> .
lastly, intestinal transcription factor   <cit>  and retinoic acid receptor /retinoid x receptor   <cit>  are important transcription factors that regulate the expression of genes  involved in carotenoid deposition.
the noble scallop chlamys nobilis reeve, an important aquaculture bivalve in china, displays conspicuous polymorphism in shell color  and difference in muscle color .
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  <cit> .
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  <cit> .
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  <dig> 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.
methods
ethics statement
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
in noble scallop, orange color was dominant to brown color.
color segregation occurred when crossing two orange scallops  <cit> .
both orange and brown scallops used in the present study were from a line of f <dig> generation produced by continuous crossing orange parental scallops .
a total of  <dig> orange scallops  and  <dig> brown scallops  at 14-month old were randomly chosen.
average shell size in length, height and width for the orange was  <dig>  ±  <dig>  mm,  <dig>  ±  <dig>  mm and  <dig>  ±  <dig>  mm, and for the brown was  <dig>  ±  <dig>  mm,  <dig>  ±  <dig>  mm and  <dig>  ±  <dig>  mm, respectively.
tissues including the gonad, mantle, gill and adductor muscle  were sampled and homogenized with the qiashredder  for total rna extraction using the qiagen rneasy  kit.
mrna was then purified by using the qiagen oligotex mrna purification kit.
equal amounts of mrna from four tissues were pooled for either an “orange” or a “brown” scallop sample.
from the two pooled samples, about 600 ng mrna was used for cdna generation with the smart cdna synthesis kit .
quality control in each extraction step was investigated using gel electrophoresis and nanodrop spectrophotometry .
both orange and brown scallop cdnas were further checked with a bioanalyzer  <dig> .
as a result, two cdna libraries  with an average length of 400 bp were generated according to the manufacturers’ protocol and sequenced on a  <dig> genome sequencer system  with flx and titanium chemistry.figure  <dig>
line of the
c. nobilis
was used in transcriptome sequencing.
both parents are orange scallop.
offspring with orange and brown coloration segregation obtained by crossing two orange scallops.
four kinds of tissues: gonad , mantle , gill , and adductor muscle .
male scallop has a white or lighter orange color gonad, while female scallop has a heavier orange color gonad.
sequence assembly and functional annotation
all sequence reads taken directly from the  <dig> gs-flx sequencer were run through the sff file program  to remove sequencing adapters a and b. barcodes were removed by seqclean  program and poor sequence data were further cleaned by lucy v <dig>  program .
sequences with homopolymers of a single nucleotide occupying 60% of the read and those less than  <dig> 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 v <dig>  .
all c. nobilis est  sequences were submitted to ncbi sequence read archive under accession no.
srx <dig>  ests that did not form isotigs  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  and swiss-prot database .
all unique sequences were sequentially compared using blastx  with the sequences in two public protein databases .
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  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  program .
default settings were employed to detect perfect di-, tri-, tetra-, penta-, and hexa-nucleotide motifs .
to be assigned, di-nucleotide ssrs  required a minimum of  <dig> repeats, and all other ssr types needed a minimum of  <dig> repeats.
two neighboring ssrs with the maximum interruption no more than  <dig> 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  <dig> or more reads contained polymorphisms at the variant allele.
snps were identified using default parameters in gsmapper v <dig>   to align isotigs from two color datasets.
in addition, only an overall transition vs transversion  ratio was calculated across the dataset.
perl script modules linked to the primer modeling software primer <dig> were used to design pcr primers flanking for each unique snp region identified.
data mining of transcripts with putative function involved in carotenoid deposition
from public databases, we compiled a dataset of the  <dig> known gene involved in carotenoid deposition were collected .
the amino-acid sequences of the known carotenoid deposition genes, covering carotenoid absorption, transport and cleavage, were used to search  for homologues in 454-derived sequences.
those sequences with scores more than or equal to  <dig> and e values less than or equal to 1e- <dig> were clustered to develop unigenes, and all of the unigenes were considered as candidate transcripts.
the resulting unigenes were in turn used to search the genbank databases by blastx to confirm their putative carotenoid-related functions.table  <dig>
carotenoid-related candidate gene
name
evidence for potential role in carotenoid deposition
protein id
reference
mrna expression of selected candidate transcripts in orange and brown scallop
expression of selected transcripts was investigated in adductor muscle from  <dig> orange scallop or  <dig> brown scallops at 14-month old, and two technical replicates were performed for each scallop.
all scallops used in this experiment were from a f <dig> 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 .
quantitative real-time rt-pcr was conducted in a lightcycler® <dig> system using the sybr premix ex taq ii qrt-pcr kit .
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  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  <dig>  were considered statistically significant.
detecting presence of srb -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,  <dig> scallops , derived from  <dig> lines produced by crossing two orange scallops in the spring of  <dig>  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.
<cit> .
dsrna synthesis
srb-like- <dig> gene was amplified through pcr with noble scallop cdna as template and 1fi and 1ri as primers .
the pcr products were separated, purified, ligated with vector pmd-18 t , and transformed into dh5α e. coli cell.
the plasmid was extracted using minibest plasmid purification kit ver.
<dig>   according to the manufacturer’s protocol.table  <dig>
primer sequences used for dsrna synthesis
primer
sequence
note: the lower case is t <dig> promoter sequence.
for dsrna synthesis, srb-like- <dig> was amplified by pcr with the primers 2fi and 2ri  using the recombinant plasmid pmd-18 t-srb as the template and .
similarly, for dsrna synthesis of egfp gene  <cit> , plasmid pegfp-n <dig> 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  <dig> % agarose gel.
dsrna was synthesized in vitro using megascript rnai kit  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  assay
forty orange scallops were used, and each of them was injected with 40 μg dsrna of srb-like- <dig> or egfp gene  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  <dig>   <dig>   <dig>  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  <dig> technological replicates for each sample.
effect of dsrna on carotenoid deposition in the blood and adductor muscle
orange scallops were randomly chosen, and  <dig> of them were injected with dsrna of srb-like- <dig> or egfp gene,  <dig> 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,  <dig> scallops from rnase-free water group,  <dig> scallops from dssrb-like- <dig> group, and  <dig> scallops from dsegfp group were sampled.
1 ml blood from each scallop was freeze-dried and added with  <dig>  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.
<cit> .
the samples were always under n <dig> until measurement of absorption at 480 nm to determine their carotenoid content.
RESULTS
roche  <dig> gs-flx sequencing and isotigs assembly
a total of  <dig> , <dig> raw reads were obtained from the  <dig> gs-flx sequencer, including  <dig> , <dig> clean reads  after adaptor trimming, size-selection and quality control .
the size distribution of raw reads and clean reads are shown in figure 2a and b.table  <dig>
summary statistics for est and de novo assembly
total
brown
orange
no.
of raw reads
average length
% reads removed
no.
of reads after cleaning
average length of cleaned reads
no.
of reads assembled as isotigs
no.
of isotigs
average length of isotigs
range of isotig lengths
isotigs above 200 bp
no.
of singletons
average length of singletons
range of singletons lengths
singletons above 200 bp
no.
of unique sequences
a
no.
of unique sequences
unique sequences above  <dig> bases
athe total number of isotigs and singletons.
overview of the
c. nobilis
transcriptome sequencing and assembly.
size distribution of  <dig> raw reads.
size distribution of  <dig> reads after removal of adaptor and short sequences.
log-log plot showing the dependence of isotigs lengths on the number of reads assembled into each isotigs.
size distribution of isotigs.
sequences that passed basic quality standards were clustered and assembled de novo .
overall, approximately 91%  reads were assembled into  <dig>  isotigs, and the others  remained as singletons .
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  <dig> to  <dig>  bp with an average of 580 bp.
the percentage of reads assembled de novo is similar to that found in other studies  <cit> .
the large numbers of unique sequences  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  <cit> .
annotation of the transcriptome
all isotigs and singletons were subjected to cd-hit program  to remove redundant sequences, leaving  <dig>  unique sequences .
the annotation for unique sequences from c. nobiliswas based on sequence similarity searches against public databases.
these databases included ncbi nr and swissprot.
about  <dig> % of all sequences  identified a homologue mostly in both or at least one of the databases at e-value 1e- <dig> .
because the significance of sequence similarity depends in part on the length of the query sequence, short unique sequences frequently cannot be matched to known genes  <cit> .
the proportion of sequences with matches in public databases was greater for the longer assembled sequences.
namely,  <dig> % matches were recorded for sequences ≥ 300 bp,  <dig> % for those longer than 1 kb, but only  <dig> % for those short sequences  .
the percentage of sequences with annotation information in this study was considerably low .
the poor annotation efficiency may be due to the insufficient sequences in public databases for phylogenetically close species to date  <cit> .table  <dig>
summary of annotation of the
c. nobilis
transcriptome
isotigs
≥300 bp
≥1000 bp
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
gene ontology   <cit>  could provide a dynamic, controlled vocabulary and hierarchical relationships for the representation of information on molecular function, cellular component and biological process, allowing a coherent annotation of gene products.
of annotated unique sequences in nr or swissprot database,  <dig>  unique sequences were assigned to one or more go terms .
a total of  <dig>  go terms were obtained, with  <dig>  unique sequences for biological processes,  <dig>  unique sequences for cellular components and  <dig>  unique sequences for molecular function .
of those sequences for biological processes, the major ones were cellular process  and metabolic process .
for cellular component, the most represented categories were cell  and cell part .
regarding molecular functions, binding  was the most represented sequences according to go terms, followed by catalytic activity .
similar results were also found in other species such as the yesso scallop   <cit>  and the freshwater prawn   <cit> .
these go annotations can provide a comprehensive information on transcript functions of c. nobilis.figure  <dig>
functional annotation of assembled sequences based on gene ontology  categorization.
go analysis was performed at the level  <dig> for three main categories .
putative molecular markers
selection based on molecular markers is well known to be vital in shellfish aquaculture industry  <cit> .
<dig> sequencing may provide an excellent source for mining and development of these markers for c. nobilis, because few genetic markers are currently available.
a total of  <dig>  unique sequences were found to possess  <dig>  ssrs, of which  <dig>  unique sequences annotated in nr and/or swissport database were considered as priority candidates for maker development .
the most frequent repeat motifs were di-nucleotide repeats , followed by tri-nucleotide repeats  and tetra-nucleotide repeats .
penta-nucleotides repeats and hexa-nucleotides repeats only accounted for  <dig> %.
among the di-nucleotide repeat classes, ta was the most frequent dimer motif .
with regard to tri-nucleotide repeats, tgg  was the most common motif, followed by gat  and tga .table  <dig>
summary of simple sequence repeat  nucleotide classes among different nucleotide types found in
c. nobilis
sequences
ssr type
no.
of ssr- containing ests
no.
of ssrs
% of total ssrs
di-nucleotides
tri-nucleotides
tetra-nucleotides
penta-nucleotides
hexa-nucleotides
total
note: both isotigs and singletons sequences are used to predict the ssr loci.
snps in c. nobilis est isotigs were identified using the ssahasnp program .
of the  <dig>  snps detected,  <dig>  were putative transitions  and  <dig>  were putative transversions , giving a mean ts: tv ratio of  <dig> :  <dig>  across the transcriptome .
the ts: tv ratio can help to identify genes affected by selection.
a total of  <dig>  indels across the transcriptome were detected.
however, much caution must be paid to those indels because of technical problems associated with  <dig> pyrosequencing  <cit> .figure  <dig>
classification of single nucleotide polymorphisms  identified from  <dig> sequences.
the overall frequency of these snp types in c. nobilis transcriptome is one per 278 bp.
the overall frequency of all types of snps in the transcriptome, including indels, was one per 278 bp.
of the predicted snps, including indels,  <dig>   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  <cit> .
among the snps,  <dig>   were identified from isotigs with annotation information.
twenty five of these predicted snps were randomly selected for validation using pcr and sanger sequencing, and  <dig> of these tests  were successful .
the result here confirmed that the majority of computationally predicted snps from the  <dig> transcriptome sequences would benefit us in our future genetic markers development.
identification of carotenoid-based coloration transcripts from  <dig> sequences
full length protein sequences of the  <dig> known genes responsible for carotenoid absorption, binding or carotenoids cleavage were used to perform tblastn searches against 454-derived sequences.
a total of  <dig> isotigs and  <dig> singletons from 454-derived sequences were identified with scores more than or equal to  <dig> and e values less than or equal to 1e- <dig>  and  <dig> non-redundant sequences were developed and used to identify their putative functions by blastx searches against the genbank databases .
after blastx searches against nr database,  <dig> transcripts were considered as tentative c. nobilis carotenoid-related transcripts .
quantitative real-time reverse transcription pcr was performed for each of the  <dig> candidate transcripts to determine their relative levels of expression in the adductor muscle taken from orange scallop and brown scallop.
however, we failed to find any transcripts with significant difference in expression between the orange and white adductor muscle .
we speculated sequence variations might exist in these transcripts.figure  <dig>
comparison of the expression level of  <dig> selected tentative carotenoid deposition transcripts in orange and brown scallop adductor muscle.
<dig> scallops were used in the experiment and each expression analysis was also performed in two independent experiments.
significant difference was performed by one-way anova test .
members of three gene families, srb/cd <dig>  star/mln <dig>  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  <dig> srb-like genes,  <dig> star-like genes and  <dig> bcmo-like genes based on transcriptome data by race pcr , and screened possible mutation sites of cds.
several missense mutations were found, which, however, showed no correlation with carotenoids accumulation .
nonetheless, a scavenge receptor gene termed srb-like- <dig> was identified, which showed a high sequence similarity with srb-like- <dig> .
pcr was performed using primers  for srb-like- <dig>  interestingly, srb-like- <dig> was only detected in orange scallop and absent in brown scallop, suggesting that srb-like- <dig> is significantly associated with high carotenoid content and possibly an important candidate gene in carotenoid deposition .
tissue expression profile  showed that srb-like- <dig> was highly expressed in the gonad  and intestine , indicating that srb-like- <dig> mainly functions in these two tissues.figure  <dig>
alignment of srb-like- <dig> and srb-like- <dig>  primers s3f <dig> and s3r <dig> give special pcr amplification of srb-like- <dig>
progeny testing of the four lines in scallop
c. nobilis
no.
carotenoid content of adductor muscle  and srb-like- <dig> detected
line1
line2
line3
line4
orange
brown
orange
brown
orange
brown
orange
brown
dsrb-like- <dig> was detected; ndsrb-like- <dig> was not detected.
**indicate very significant differences  in carotenoid content between orange and brown scallops in the same line.
tissue expression profile of srb-like- <dig> in orange scallop.
different letter means significant difference by one-way anova test .
mrna expression of srb-lile- <dig> after rnai
to find out whether srb-like- <dig> was involved in carotenoid deposition, dsrna of srb-like- <dig>  was synthesized and injected into orange scallop adductor muscle.
results of real-time pcr showed that the mrna level of srb-like- <dig> in tested tissue was down-regulated by dssrb-like- <dig> .
the expression level of srb-like- <dig> mrna was significantly suppressed by 67%, 48% and 45% in the adductor muscle, blood and intestine, respectively, when compared to that of the dsegfp injected group 24 h after injection.figure  <dig>
mrna expression of srb-like- <dig> after injection of dsrna.
letter indicates comparison of the same tissue.
different letter means significant difference by one-way anova test .
carotenoid content in the blood and adductor muscle after rnai
total caronoid content in the blood was measured after rnai.
color density of the dssrb-like- <dig> group is lighter than that of dsegfp group , implying that the dssrb-like- <dig> group had a relative low carotenoid content.
carotenoid content in the blood was measured, showing that the dssrb-like- <dig> group indeed had a significantly lower carotenoid content when compared to that of the dsegfp group or blank group , while there was no statistical difference between the dsegfp group and the rnase-free water group.
carotenoid content in the adductor muscle showed no remarkable difference among the dssrb-like- <dig>  dsegfp and rnase-free water group .
anyhow, our result providing compelling evidence that srb-like- <dig> was a candidate gene that was at least responsible for blood carotenoid content, implying that srb-like- <dig> might take part in absorption of carotenoid to blood.figure  <dig>
carotenoids extration from the blood .
carotenoid content  of blood
dssrb-like-3
dsegfp
rnase-free water
id
cc
id
cc
id
cc
note: means with different sub letter indicate significant difference .
carotenoid content  of adductor
dssrb-like-3
dsegfp
rnase-free water
id
cc
id
id
cc
id
note: no significant difference was detected among the three groups.
CONCLUSIONS
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.
<dig>  ssrs and over  <dig>  high confidence variants  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- <dig> is only expressed in orange scallop but absent in brown scallop, significantly associated with high carotenoid content, suggesting srb-like- <dig> 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- <dig> is involved in carotenoid deposition in blood.
additional files
additional file 1: table s <dig>  sequences with significant blastx matches against swiss-prot and nr database.
additional file 2: table s <dig>  gene ontology of c. nobilis unique sequences.
additional file 3: table s <dig>  details of ssr motifs in c. nobilis unique sequences.
additional file 4: table s <dig>  details of snps and indels in c. nobilis isotigs.
additional file 5: table s <dig>  validation of predicted snps by pcr and re-sequencing.
additional file 6: table s <dig>  candidate transcripts.
additional file 7: table s <dig>  information of  <dig> candidate carotenoid deposition genes.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
conceived and designed the experiments: hl, hzheng.
performed the experiments: hl, hzhang.
analyzed the data: hl, ld, fm, yw.
contributed reagents/materials/analysis tools: hl, hzhang, zg, gz.
wrote the paper: hl, hzheng, wl, sw, sl.
all authors read and approved the final manuscript.
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 , ministry of education of p.r.
china , china modern agro-industry technology research system , national basic research program of china , knowledge innovation program of deep sea science and engineering, chinese academy of sciences , department of education , department of science & technology  and oceanic and fisheries administrator  of guangdong province, china.

 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. 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 that potentially limit the ability of animals to deposit carotenoids in their body tissues. 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 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, cameo and scrb of cd, 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. star /mln 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 is an orthologue of vertebrate mln and is involved in the transport of lutein. the bcmo /bcdo 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, atp-binding cassette sub-family g member, glutathione s-transferase pi and crustacyanin. lastly, intestinal transcription factor and retinoic acid receptor /retinoid x receptor are important transcription factors that regulate the expression of genes involved in carotenoid deposition. the noble scallop chlamys nobilis reeve, an important aquaculture bivalve in china, displays conspicuous polymorphism in shell color and difference in muscle color. 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. 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 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. methods ethics statement 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 in noble scallop, orange color was dominant to brown color. color segregation occurred when crossing two orange scallops. both orange and brown scallops used in the present study were from a line of f generation produced by continuous crossing orange parental scallops. a total of orange scallops and brown scallops at 14-month old were randomly chosen. average shell size in length, height and width for the orange was  ±   mm,  ±   mm and  ±   mm, and for the brown was  ±   mm,  ±   mm and  ±   mm, respectively. tissues including the gonad, mantle, gill and adductor muscle were sampled and homogenized with the qiashredder for total rna extraction using the qiagen rneasy kit. mrna was then purified by using the qiagen oligotex mrna purification kit. equal amounts of mrna from four tissues were pooled for either an “orange” or a “brown” scallop sample. from the two pooled samples, about 600 ng mrna was used for cdna generation with the smart cdna synthesis kit. quality control in each extraction step was investigated using gel electrophoresis and nanodrop spectrophotometry. both orange and brown scallop cdnas were further checked with a bioanalyzer. as a result, two cdna libraries with an average length of 400 bp were generated according to the manufacturers’ protocol and sequenced on a genome sequencer system with flx and titanium chemistry.figure line of the c. nobilis was used in transcriptome sequencing. both parents are orange scallop. offspring with orange and brown coloration segregation obtained by crossing two orange scallops. four kinds of tissues: gonad, mantle, gill, and adductor muscle. male scallop has a white or lighter orange color gonad, while female scallop has a heavier orange color gonad. sequence assembly and functional annotation all sequence reads taken directly from the gs-flx sequencer were run through the sff file program to remove sequencing adapters a and b. barcodes were removed by seqclean program and poor sequence data were further cleaned by lucy v program. sequences with homopolymers of a single nucleotide occupying 60% of the read and those less than 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 v. all c. nobilis est sequences were submitted to ncbi sequence read archive under accession no. srx ests that did not form isotigs 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 and swiss-prot database. all unique sequences were sequentially compared using blastx with the sequences in two public protein databases. 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 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 program. default settings were employed to detect perfect di-, tri-, tetra-, penta-, and hexa-nucleotide motifs. to be assigned, di-nucleotide ssrs required a minimum of repeats, and all other ssr types needed a minimum of repeats. two neighboring ssrs with the maximum interruption no more than 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 or more reads contained polymorphisms at the variant allele. snps were identified using default parameters in gsmapper v to align isotigs from two color datasets. in addition, only an overall transition vs transversion ratio was calculated across the dataset. perl script modules linked to the primer modeling software primer were used to design pcr primers flanking for each unique snp region identified. data mining of transcripts with putative function involved in carotenoid deposition from public databases, we compiled a dataset of the known gene involved in carotenoid deposition were collected. the amino-acid sequences of the known carotenoid deposition genes, covering carotenoid absorption, transport and cleavage, were used to search for homologues in 454-derived sequences. those sequences with scores more than or equal to and e values less than or equal to 1e- were clustered to develop unigenes, and all of the unigenes were considered as candidate transcripts. the resulting unigenes were in turn used to search the genbank databases by blastx to confirm their putative carotenoid-related functions.table carotenoid-related candidate gene name evidence for potential role in carotenoid deposition protein id reference mrna expression of selected candidate transcripts in orange and brown scallop expression of selected transcripts was investigated in adductor muscle from orange scallop or brown scallops at 14-month old, and two technical replicates were performed for each scallop. all scallops used in this experiment were from a f 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. quantitative real-time rt-pcr was conducted in a lightcycler® system using the sybr premix ex taq ii qrt-pcr kit. 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 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 were considered statistically significant. detecting presence of srb -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, scallops, derived from lines produced by crossing two orange scallops in the spring of 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.. dsrna synthesis srb-like- gene was amplified through pcr with noble scallop cdna as template and 1fi and 1ri as primers. the pcr products were separated, purified, ligated with vector pmd-18 t, and transformed into dh5α e. coli cell. the plasmid was extracted using minibest plasmid purification kit ver. according to the manufacturer’s protocol.table primer sequences used for dsrna synthesis primer sequence note: the lower case is t promoter sequence. for dsrna synthesis, srb-like- was amplified by pcr with the primers 2fi and 2ri using the recombinant plasmid pmd-18 t-srb as the template and. similarly, for dsrna synthesis of egfp gene, plasmid pegfp-n 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 % agarose gel. dsrna was synthesized in vitro using megascript rnai kit 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 assay forty orange scallops were used, and each of them was injected with 40 μg dsrna of srb-like- or egfp gene 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 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 technological replicates for each sample. effect of dsrna on carotenoid deposition in the blood and adductor muscle orange scallops were randomly chosen, and of them were injected with dsrna of srb-like- or egfp gene, 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, scallops from rnase-free water group, scallops from dssrb-like- group, and scallops from dsegfp group were sampled. 1 ml blood from each scallop was freeze-dried and added with  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 n until measurement of absorption at 480 nm to determine their carotenoid content.  roche gs-flx sequencing and isotigs assembly a total of, raw reads were obtained from the gs-flx sequencer, including, clean reads after adaptor trimming, size-selection and quality control. the size distribution of raw reads and clean reads are shown in figure 2a and b.table summary statistics for est and de novo assembly total brown orange no. of raw reads average length % reads removed no. of reads after cleaning average length of cleaned reads no. of reads assembled as isotigs no. of isotigs average length of isotigs range of isotig lengths isotigs above 200 bp no. of singletons average length of singletons range of singletons lengths singletons above 200 bp no. of unique sequences a no. of unique sequences unique sequences above bases athe total number of isotigs and singletons. overview of the c. nobilis transcriptome sequencing and assembly. size distribution of raw reads. size distribution of reads after removal of adaptor and short sequences. log-log plot showing the dependence of isotigs lengths on the number of reads assembled into each isotigs. size distribution of isotigs. sequences that passed basic quality standards were clustered and assembled de novo. overall, approximately 91% reads were assembled into isotigs, and the others remained as singletons. 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 to  bp with an average of 580 bp. the percentage of reads assembled de novo is similar to that found in other studies. the large numbers of unique sequences 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 all isotigs and singletons were subjected to cd-hit program to remove redundant sequences, leaving unique sequences. the annotation for unique sequences from c. nobiliswas based on sequence similarity searches against public databases. these databases included ncbi nr and swissprot. about % of all sequences identified a homologue mostly in both or at least one of the databases at e-value 1e-. because the significance of sequence similarity depends in part on the length of the query sequence, short unique sequences frequently cannot be matched to known genes. the proportion of sequences with matches in public databases was greater for the longer assembled sequences. namely, % matches were recorded for sequences ≥ 300 bp, % for those longer than 1 kb, but only % for those short sequences. the percentage of sequences with annotation information in this study was considerably low. the poor annotation efficiency may be due to the insufficient sequences in public databases for phylogenetically close species to date.table summary of annotation of the c. nobilis transcriptome isotigs ≥300 bp ≥1000 bp 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 gene ontology could provide a dynamic, controlled vocabulary and hierarchical relationships for the representation of information on molecular function, cellular component and biological process, allowing a coherent annotation of gene products. of annotated unique sequences in nr or swissprot database, unique sequences were assigned to one or more go terms. a total of go terms were obtained, with unique sequences for biological processes, unique sequences for cellular components and unique sequences for molecular function. of those sequences for biological processes, the major ones were cellular process and metabolic process. for cellular component, the most represented categories were cell and cell part. regarding molecular functions, binding was the most represented sequences according to go terms, followed by catalytic activity. similar results were also found in other species such as the yesso scallop and the freshwater prawn. these go annotations can provide a comprehensive information on transcript functions of c. nobilis.figure functional annotation of assembled sequences based on gene ontology categorization. go analysis was performed at the level for three main categories. putative molecular markers selection based on molecular markers is well known to be vital in shellfish aquaculture industry. sequencing may provide an excellent source for mining and development of these markers for c. nobilis, because few genetic markers are currently available. a total of unique sequences were found to possess ssrs, of which unique sequences annotated in nr and/or swissport database were considered as priority candidates for maker development. the most frequent repeat motifs were di-nucleotide repeats, followed by tri-nucleotide repeats and tetra-nucleotide repeats. penta-nucleotides repeats and hexa-nucleotides repeats only accounted for %. among the di-nucleotide repeat classes, ta was the most frequent dimer motif. with regard to tri-nucleotide repeats, tgg was the most common motif, followed by gat and tga.table summary of simple sequence repeat nucleotide classes among different nucleotide types found in c. nobilis sequences ssr type no. of ssr- containing ests no. of ssrs % of total ssrs di-nucleotides tri-nucleotides tetra-nucleotides penta-nucleotides hexa-nucleotides total note: both isotigs and singletons sequences are used to predict the ssr loci. snps in c. nobilis est isotigs were identified using the ssahasnp program. of the snps detected, were putative transitions and were putative transversions, giving a mean ts: tv ratio of: across the transcriptome. the ts: tv ratio can help to identify genes affected by selection. a total of indels across the transcriptome were detected. however, much caution must be paid to those indels because of technical problems associated with pyrosequencing.figure classification of single nucleotide polymorphisms identified from sequences. the overall frequency of these snp types in c. nobilis transcriptome is one per 278 bp. the overall frequency of all types of snps in the transcriptome, including indels, was one per 278 bp. of the predicted snps, including indels, 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, were identified from isotigs with annotation information. twenty five of these predicted snps were randomly selected for validation using pcr and sanger sequencing, and of these tests were successful. the result here confirmed that the majority of computationally predicted snps from the transcriptome sequences would benefit us in our future genetic markers development. identification of carotenoid-based coloration transcripts from sequences full length protein sequences of the known genes responsible for carotenoid absorption, binding or carotenoids cleavage were used to perform tblastn searches against 454-derived sequences. a total of isotigs and singletons from 454-derived sequences were identified with scores more than or equal to and e values less than or equal to 1e- and non-redundant sequences were developed and used to identify their putative functions by blastx searches against the genbank databases. after blastx searches against nr database, transcripts were considered as tentative c. nobilis carotenoid-related transcripts. quantitative real-time reverse transcription pcr was performed for each of the candidate transcripts to determine their relative levels of expression in the adductor muscle taken from orange scallop and brown scallop. however, we failed to find any transcripts with significant difference in expression between the orange and white adductor muscle. we speculated sequence variations might exist in these transcripts.figure comparison of the expression level of selected tentative carotenoid deposition transcripts in orange and brown scallop adductor muscle. scallops were used in the experiment and each expression analysis was also performed in two independent experiments. significant difference was performed by one-way anova test. members of three gene families, srb/cd star/mln 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 srb-like genes, star-like genes and bcmo-like genes based on transcriptome data by race pcr, and screened possible mutation sites of cds. several missense mutations were found, which, however, showed no correlation with carotenoids accumulation. nonetheless, a scavenge receptor gene termed srb-like- was identified, which showed a high sequence similarity with srb-like-. pcr was performed using primers for srb-like- interestingly, srb-like- was only detected in orange scallop and absent in brown scallop, suggesting that srb-like- is significantly associated with high carotenoid content and possibly an important candidate gene in carotenoid deposition. tissue expression profile showed that srb-like- was highly expressed in the gonad and intestine, indicating that srb-like- mainly functions in these two tissues.figure alignment of srb-like- and srb-like- primers s3f and s3r give special pcr amplification of srb-like- progeny testing of the four lines in scallop c. nobilis no. carotenoid content of adductor muscle and srb-like- detected line1 line2 line3 line4 orange brown orange brown orange brown orange brown dsrb-like- was detected; ndsrb-like- was not detected. **indicate very significant differences in carotenoid content between orange and brown scallops in the same line. tissue expression profile of srb-like- in orange scallop. different letter means significant difference by one-way anova test. mrna expression of srb-lile- after rnai to find out whether srb-like- was involved in carotenoid deposition, dsrna of srb-like- was synthesized and injected into orange scallop adductor muscle. results of real-time pcr showed that the mrna level of srb-like- in tested tissue was down-regulated by dssrb-like-. the expression level of srb-like- mrna was significantly suppressed by 67%, 48% and 45% in the adductor muscle, blood and intestine, respectively, when compared to that of the dsegfp injected group 24 h after injection.figure mrna expression of srb-like- after injection of dsrna. letter indicates comparison of the same tissue. different letter means significant difference by one-way anova test. carotenoid content in the blood and adductor muscle after rnai total caronoid content in the blood was measured after rnai. color density of the dssrb-like- group is lighter than that of dsegfp group, implying that the dssrb-like- group had a relative low carotenoid content. carotenoid content in the blood was measured, showing that the dssrb-like- group indeed had a significantly lower carotenoid content when compared to that of the dsegfp group or blank group, while there was no statistical difference between the dsegfp group and the rnase-free water group. carotenoid content in the adductor muscle showed no remarkable difference among the dssrb-like- dsegfp and rnase-free water group. anyhow, our result providing compelling evidence that srb-like- was a candidate gene that was at least responsible for blood carotenoid content, implying that srb-like- might take part in absorption of carotenoid to blood.figure carotenoids extration from the blood. carotenoid content of blood dssrb-like-3 dsegfp rnase-free water id cc id cc id cc note: means with different sub letter indicate significant difference. carotenoid content of adductor dssrb-like-3 dsegfp rnase-free water id cc id id cc id note: no significant difference was detected among the three groups.  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. ssrs and over high confidence variants 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- is only expressed in orange scallop but absent in brown scallop, significantly associated with high carotenoid content, suggesting srb-like- 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- is involved in carotenoid deposition in blood. additional files additional file 1: table s sequences with significant blastx matches against swiss-prot and nr database. additional file 2: table s gene ontology of c. nobilis unique sequences. additional file 3: table s details of ssr motifs in c. nobilis unique sequences. additional file 4: table s details of snps and indels in c. nobilis isotigs. additional file 5: table s validation of predicted snps by pcr and re-sequencing. additional file 6: table s candidate transcripts. additional file 7: table s information of candidate carotenoid deposition genes. competing interests the authors declare that they have no competing interests. authors’ contributions conceived and designed the experiments: hl, hzheng. performed the experiments: hl, hzhang. analyzed the data: hl, ld, fm, yw. contributed reagents/materials/analysis tools: hl, hzhang, zg, gz. wrote the paper: hl, hzheng, wl, sw, sl. all authors read and approved the final manuscript. 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, ministry of education of p.r. china, china modern agro-industry technology research system, national basic research program of china, knowledge innovation program of deep sea science and engineering, chinese academy of sciences, department of education, department of science & technology and oceanic and fisheries administrator of guangdong province, china.
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. especially, one class b scavenge receptor termed srb-like- was discovered to express only in orange scallops and absent in brown ones, suggesting a significant association with high carotenoid content. 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. there is currently a need to identify candidate genes associated with carotenoid-based coloration. down-regulation of srb-like- mrna by rna interference remarkably decreased blood carotenoid, providing compelling evidence that srb-like- is an ideal candidate gene controlling carotenoid deposition and determining orange coloration. of the unique sequences, % isotigs and % singletons were annotated. in the present study, gs-flx sequencing of noble scallop transcriptome yielded, clean sequence reads, which were assembled into isotigs, leaving reads as the singletons.
 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, gs-flx sequencing of noble scallop transcriptome yielded, clean sequence reads, which were assembled into isotigs, leaving reads as the singletons. of the unique sequences, % isotigs and % singletons were annotated. moreover, ssrs and over high confidence variants were identified. especially, one class b scavenge receptor termed srb-like- 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- mrna by rna interference remarkably decreased blood carotenoid, providing compelling evidence that srb-like- 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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords chlamys nobilistranscriptome sequencingcarotenoid colorationcandidate genesissue-copyright-statement© the author 2015 