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A carotenoid oxygenase is responsible for muscle coloration in scallop a,1
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Xue Li , Shuyue Wang , Xiaogang Xun , Mengran Zhang , Shi Wang , Hengde Li , Liang Zhaoa,b, Qiang Fua, Huizhen Wanga, Tingting Lia, Shanshan Liana,b, Qiang Xinga, Xu Lia, ⁎ ⁎ Wei Wua, Lingling Zhanga,b, Xiaoli Hua,b, , Zhenmin Baoa,b, a
MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China c Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China d Ministry of Agriculture Key Laboratory of Aquatic Genomics, CAFS Key Laboratory of Aquatic Genomics and Beijing Key Laboratory of Fishery Biotechnology, Center for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences, Beijing 100141, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Scallop Carotenoid coloration Muscle Fine mapping PyBCO-like 1
As lipid microconstituents mainly of plant origin, carotenoids are essential nutrients for humans and animals, and carotenoid coloration represents an important meat quality parameter for many farmed animals. Currently, the mechanism of carotenoid bioavailability in animals is largely unknown mainly due to the limited approaches applied, the shortage of suitable model systems and the restricted taxonomic focus. The mollusk Yesso scallop (Patinopecten yessoensis) possessing orange adductor muscle with carotenoid deposition, provides a unique opportunity to research the mechanism underlying carotenoid utilization in animals. Herein, through family construction and analysis, we found that carotenoid coloration in scallop muscle is inherited as a recessive Mendelian trait. Using a combination of genomic approaches, we mapped this trait onto chromosome 8, where PyBCO-like 1 encoding carotenoid oxygenase was the only differentially expressed gene between the white and orange muscles (FDR = 2.75E-21), with 11.28-fold downregulation in the orange muscle. Further functional assays showed that PyBCO-like 1 is capable of degrading β-carotene, and inhibiting PyBCO-like 1 expression in the white muscle resulted in muscle coloration and carotenoid deposition. In the hepatopancreas, which is the organ for digestion and absorption, neither the scallop carotenoid concentration nor PyBCO-like 1 expression were significantly different between the two scallops. These results indicate that carotenoids could be taken up in both white- and orange-muscle scallops and then degraded by PyBCO-like 1 in the white muscle. Our data suggest that PyBCO-like 1 is the essential gene for carotenoid metabolism in scallop muscle, and its downregulation leads to carotenoid deposition and muscle coloration.
1. Introduction As dietary lipids acquired solely from food, carotenoids have long been recognized as essential nutrients for humans and most animals, with positive effects on antioxidation and preventing vitamin A deficiency [1]. In meat-producing animals, such as cattle, sheep, chicken, and especially salmonid fishes, flesh yellowness or redness determined by carotenoid coloration is an important parameter to assess meat quality, with higher values being preferred. Additionally, carotenoidbased coloration in animals has also become a focus of evolutionary studies regarding the establishment and maintenance of this trait [2–6]. A better understanding of the molecular mechanisms underlying
carotenoid deposition has been of the interest from both animal breeding and evolutionary perspectives [7,8]; however, these mechanisms currently remain largely unknown mainly due to the limited approaches applied, the shortage of suitable model systems and the restricted taxonomic focus [2]. Bivalve scallops are widely distributed throughout the world and are also of worldwide economic importance for their delicious and remarkably large adductor muscle. Similar to that in most other bivalve species, the adductor muscle of the scallop is completely white, without coloration. In recent years, orange muscle arising from carotenoid pigmentation by accumulating pectenolone and pectenoxanthin was reported in the Yesso scallop (Patinopecten yessoensis) [9,10].
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Corresponding authors at: MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. E-mail addresses: [email protected] (X. Hu), [email protected] (Z. Bao). 1 Xue Li and Shuyue Wang contributed equally to this work. https://doi.org/10.1016/j.bbalip.2019.03.003 Received 2 December 2018; Received in revised form 28 February 2019; Accepted 6 March 2019 Available online 09 March 2019 1388-1981/ © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.2. Construction of white-muscle and orange-muscle scallop crosses
Subsequently, carotenoid coloration of muscle was also found in other scallop species, including the Noble scallop (Chlamys nobilis) and Bay scallop (Argopecten irradians) [11,12]. Due to the commercial potential of this trait for improving carotenoid supply, a strain of Yesso scallop named ‘Haida golden scallop’, which is characterized by an orange adductor muscle with carotenoid deposition, was developed and massively farmed. These characteristics, together with the well-assembled and annotated genome of the Yesso scallop [13], make this bivalve species a potential model for studies on the molecular mechanism underlying carotenoid utilization in animal muscle. Recent progress has been made in understanding carotenoid coloration in scallop muscle mostly based on the hypothesis that increased carotenoid absorption or transportation was the causation. Then, several genes potentially related to carotenoid intake and exhibiting higher expression in orange muscle than in white muscle were identified [14–16]. For example, in the Yesso scallop, a stearoyl-CoA desaturase (SCD) gene encoding the key enzyme in the biosynthesis of monounsaturated fatty acids which could enhance carotenoid absorption [14], and a lysophosphatidylcholine acyltransferase 1 (LPCAT1) gene, which might be involved in carotenoid storage in the cell [15], were found to be upregulated in orange muscle; proteomic analysis revealed expression enhancement in orange muscle of several proteins possibly in relation to lipid-associated carotenoid absorption or transportation [16]. However, without functional validation, whether the upregulation of these genes is the causal factor of or the induced reaction to carotenoid deposition is unknown. Meanwhile, for the expression of carotenoid coloration, in addition to carotenoid uptake and transport steps, which have been the focus of previous studies on scallops, another physiological step, carotenoid metabolism or cleavage, is also involved [17] but was not considered in reported studies on scallop muscle coloration. Recently, an association analysis of muscle coloration in the Bay scallop identified hundreds of associated SNPs and genes throughout the genome; however, which gene is responsible for this trait was not revealed [12]. Therefore, the key regulator of carotenoid pigmentation in scallop muscle is still unknown. To reveal the genetic mechanism of orange muscle in scallops, in the present study, through family construction and analysis together with a combination of genomic approaches, we found that carotenoid coloration in scallop muscle is recessively inherited as a Mendelian trait, and the downregulation of the gene PyBCO-like 1 encoding carotenoid oxygenase was responsible for muscle carotenoid coloration, which was further supported by functional assays.
Yesso scallop family founders were collected at the hatchery of Zhangzidao Group Co., Ltd. (Dalian, China) and a series of F1 and F2 families were constructed (Fig. 2A and B). F1 families were established through crossing between white-muscle scallops, between orangemuscle scallops, and between white-muscle and orange-muscle scallops. For crosses between white-muscle and orange-muscle scallops, three female orange-muscle scallops were mated with one male white-muscle scallop to produce three F1 families. Then, three F2 families were constructed, among which family 13-4 was constructed by crossing one male and one female offspring within the first F1 family; family 15-1 was produced by crossing within the third F1 family; and family 13-2 was constructed by mating one female in the first F1 family with one male in the second F1 family. More than two hundred scallops from each F1 and F2 family were collected for muscle color observation. 2.3. Genome-wide genotyping and genome-wide association study (GWAS) The four F0 progenitors, six F1 progenies and 116 F2 progenies, including 38 (13 orange and 25 white) from family F13-2, 38 (17 orange and 21 white) from F13-4, and 40 (19 orange and 21 white) from F15-1, were used for GWAS. Genomic DNA was extracted from the adductor muscle using the traditional phenol/chloroform extraction method [18]. 2b-RAD libraries (using the endonucleases Bsa-XI) were constructed following the protocol developed by Wang et al. [19], with the library of each individual being assigned a unique barcode. The libraries were then pooled for single end sequencing by HiSeq 2000 (Illumina, America). Raw reads were trimmed to remove adaptors and low-quality sequences as described by Jiao et al. [20]. Then, the remaining highquality reads were mapped to the genome reference of the Yesso scallop using SOAPaligner [21]. Single nucleotide polymorphisms (SNPs) were called by RADtyping v1.3 under default parameters [22]. SNPs that could be genotyped in at least 60% of the individuals were considered high-quality markers and retained for further analysis. Association analyses between genotypes and muscle colors were conducted using Plink 1.9 [23] by comparing allele frequencies between orange and white muscle. Bonferroni correction was performed for multiple testing. The confidence interval was calculated using Li’s method [24]. 2.4. Genome resequencing and bulk segregant analysis (BSA)
2. Materials and methods Yesso scallops used for genome resequencing and BSA were collected near Zhangzidao Island of the north Yellow Sea. Genomic DNA from 50 white-muscle scallops and 50 orange-muscle scallops were pooled separately. For each of the two DNA pools, a paired-end sequencing library with insert size of 180 bp was constructed following the Illumina's standard DNA library preparation protocol and was then sequenced to a coverage of ~50× using the Illumina HiSeq 2000 platform. The raw data were trimmed to remove low-quality sequences, and the resulting clean reads of the two libraries were separately mapped against the reference genome assembly of the Yesso scallop using BWAMEM alignment algorithms to obtain the SAM format files [25]. After being converted to BAM format and then sorted and deduplicated using SAMtools [26] and Picard pipelines (http://broadinstitute.github.io/ picard/), the resulting sorted BAM files were converted to pileup files for SNP identification and allele counting. Before BSA analysis, reads were screened with the following filters: 1) a minimum mapping quality of 20; 2) a minimum coverage of 10× and a maximum coverage of 500 per position; and 3) at least 1% reads supporting the minor allele in polymorphic sites. Genetic differentiation across Chr 8 between white and orange muscle was summarized in windows of 20 kb moved in steps of 15 kb using the fixation index (FST),
2.1. High performance liquid chromatography (HPLC) analysis of scallop carotenoids The Animal Care and Use Committee of Ocean University of China approved all the scallop procedures performed in this work. The main carotenoids deposited in the orange muscle of Yesso scallop were pectenolone and pectenoxanthin (Fig. 1A and B), which were detected in scallop muscle and hepatopancreas using reversed-phase HPLC, as described in [10], with minor modification. In short, 0.5 g adductor muscle was lyophilized and extracted three times with 50 ml ethyl acetate, which contained 0.1% butylated hydroxytoluene (BHT). After solvent evaporation, the residues were dissolved in 2 ml mobile phase solution and then filtered. Carotenoids were separated and quantified using an analytical-scale LaChrom C18 (4.6 × 250 mm, inner diameter 5 μm) (HITACHI, Japan) with the following parameters: 10 μl injection volume; 25 °C column temperature; acetonitrile, methanol, and methylene chloride mobile phase (50:46:4, v/v/v) with 0.1% BHT; 1.0 ml per minute flow rate; 450 nm UV–Vis detector; and 10 min separation time. The carotenoids pectenolone and pectenoxanthin were identified and quantified by comparison with the standards prepared by the methods described in [10]. 967
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Fig. 1. The orange muscle of the Yesso scallop accumulates carotenoids. A, An orange-muscle scallop (left) and a white-muscle scallop (right). The adductor muscle is indicated with a black arrow. B, Structure of the carotenoids pectenolone and pectenoxanthin deposited in the orange muscle [10]. C, Concentrations of pectenolone and pectenoxanthin in scallop adductor muscle and hepatopancreas.
as implemented in the PoPoolation2 package [27].
2.5. Transcriptome sequencing and expression profiling Total mRNA was extracted from the adductor muscles of 16 whiteand 16 orange-muscle Yesso scallops using the conventional 968
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Fig. 2. Pedigree analysis of the crosses between orange-muscle scallops (A, left), between white-muscle scallops (A, right), and between orange-muscle and whitemuscle scallops (B). The orange color marks an adductor muscle with carotenoids accumulation. The ratios of the orange- and white-muscle scallops in the F2 families 13-2, 13-4, and 15-1 are shown in B.
2.6. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis
guanidinium isothiocyanate method [28]. RNA-Seq libraries were constructed individually according to the standard Illumina protocols and were subject to Illumina PE100 sequencing. Reads containing bases ‘N’ or excessive numbers of low-quality bases (> 30% bases with quality scores < 10) were removed. After trimming, the high-quality reads from multiple RNA-Seq datasets were aligned to the P. yessoensis genome [13] using STAR 2.4.1d [29], allowing two mismatches. The total number of reads matching the gene regions was counted by script ‘HTSeq-count’ and then calculated for digital expression values as RPKM (Reads Per Kilobase per Million mapped reads) [30,31]. Reads that were not uniquely aligned to the genome were discarded. Genes with RPKM > 0.5 in at least three of the 32 muscle samples were subjected to subsequent expression profiling. The R (version 2.15.2) Bioconductor package EdgeR (v2.4.6) [32] was applied to identify significantly differentially expressed genes between orange and white muscle. Differentially expressed genes were defined as having a fold change > 2.5 in the average RPKM between orange and white muscle, and a Benjamini and Hochberg corrected FDR of < 0.01 [33].
Total RNA was extracted from the adductor muscle and hepatopancreas of the white- and orange-muscle scallop following the method described in [28]. The first-strand cDNA was synthesized according to the manufacturer's instruction of M-MLV Reverse Transcriptase (Invitrogen, CA, USA), and then diluted to 1:30 to be used as the template for qRT-PCR in analyzing PyBCO-like 1 expression. Primers for qRT-PCR were listed in Additional file 1: Table S1. The gene encoding DEAD-box RNA helicase-like protein (HELI) was used as the reference gene for normalization [34]. The qRT-PCR results were analyzed using Realtime PCR Miner (http://www.miner.ewindup.info/). 2.7. Verification of PyBCO-like 1 function in E. coli The pORANGE plasmid [35] containing β-carotene synthesis genes was transformed into E. coli JM109 (DE3) (Beijing Dingguo Changsheng Biotech, China). The CDS of PyBCO-like 1 was amplified with the primers listed in Additional file 1: Table S1, and then ligated into the pET969
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28a (+) vector (Novagen, Germany) using T4 DNA Ligase (New England Biolabs, USA), followed by transformation into the E. coli strain containing pORANGE plasmid. Colonies with both β-carotene synthesis genes and PyBCO-like 1 were induced by IPTG (1 mM). The color of E. coli with only the pORANGE plasmid, with both pORANGE and PyBCOlike 1 containing plasmids, and with only the vector pET-28a (+) was examined.
carotenoids in preventing oxidative damage stress [37–41]. The ratio of white-muscle to orange-muscle scallops observed in the F2 families suggests that carotenoid coloration of scallop muscle is likely controlled by a single Mendelian factor.
2.8. In vivo verification of PyBCO-like 1 function by RNA interference (RNAi)
To determine the genomic region underlying carotenoid coloration in scallop muscle, GWAS was performed using a total of 126 individuals (52 orange-muscle and 74 white-muscle scallops) in the families constructed (Fig. 2), including the four progenitors (F0), six F1 progenies and 116 F2 progenies, with 2b-RAD methods for genome-wide marker genotyping [19]. The statistical sequencing depth corresponded to ~20× in the F0, ~30× in the F1 and ~7× in the F2 progenies. A total of 29,674 polymorphic SNPs were obtained through mapping the reads to the P. yessoensis reference genome (Assembly: ASM211388v2). Association between SNP genotypes and muscle color was performed by Plink 1.9 [23] using a case/control method. After Bonferroni correction and confidence interval estimation, a major genome region of ~19 Mb (P < 3.37E-07, 23,888,002 to 42,852,177 bp) with a strong association signal was detected on chromosome 8 (Chr 8) (Fig. 3A, Additional file 2: Table S2). Considering the limited recombination events that occurred in family samples, for further fine mapping, we performed genome resequencing using population samples through pooling DNA for 50 orange muscles and 50 white muscles. A total of 39,937 SNPs located on Chr 8 were obtained from the ~50× sequencing data. Allele frequency differentiation across Chr 8 between orange muscle and white muscle was analyzed using the fixation index (FST) with sliding window method. One region that contained all values above the 99th percentile (FST ≥ 0.376) clearly stood out, providing an excellent candidate region harboring the orange-muscle gene. This region spanned ~2.5 Mb (25,154,020–27,593,320 bp), which is within the significant interval detected by GWAS (Fig. 3B). An examination of the genome annotation of the Yesso scallop revealed 79 protein-coding genes in this interval, with 42 expressed in scallop muscle (RPKM > 0.5 in at least three of the 32 muscle samples subjected to the subsequent RNA-Seq analysis) (Additional file 3: Table S3); the causal gene for orange muscle is expected to be among these genes.
3.2. Identification of the genomic region associated with carotenoid coloration
White-muscle scallops that were two years of age were subjected to double-stranded RNA (dsRNA) mediated silencing of PyBCO-like 1. Before and during the experiments, all scallops were acclimated in aerated tanks at 8 °C and fed with golden algae (Isochrysis galbana) as described in [14]. PyBCO-like 1 dsRNA was synthesized using the MEGAscript® RNAi Kit (Thermo, USA) following the manufacturer's protocol, with the primers listed in Additional file 1: Table S1. After being purified, the synthesized dsRNA was diluted to 1 μg/μl with nuclease-free 1 × PBS (Sangon, China). For each scallop, 100 μl PyBCOlike 1 dsRNA or 100 μl 1 × PBS was injected into the muscle once per week for four weeks. Then, the adductor muscle was dissected for PyBCO-like 1 expression analysis by qRT-PCR and carotenoid detection using reverse-phase HPLC by analyzing the peak areas of the two main carotenoids, pectenolone and pectenoxanthin. 3. Results 3.1. Carotenoid coloration in scallop muscle is a recessive trait Previous studies revealed that the orange muscle of the Yesso scallop contained two carotenoids, pectenolone and pectenoxanthin (Fig. 1A, B) [9,10]. To better understand the difference in carotenoid deposition between orange- and white-muscle scallops, the concentrations of the two carotenoids were analyzed in the adductor muscle and the hepatopancreas, an organ responsible for digestion and absorption of food in bivalves [36]. In orange muscle, 16.35 ± 2.31 and 17.80 ± 2.55 μg/g pectenolone and pectenoxanthin were detected, respectively, while no carotenoid was detected in white muscle (Fig. 1C), indicating that carotenoid deposition and coloration in scallop muscle was a discrete character. For the hepatopancreas, the concentrations of the two carotenoids were not significantly different between orange- and white-muscle scallops (Fig. 1C), which implies a similar ability of the two scallops to absorb carotenoids from foods. To validate the hereditary pattern of carotenoid coloration in scallop muscle, a series of families were constructed, including crosses between white-muscle scallops, between orange-muscle scallops, and between white-muscle and orange-muscle scallops. All F1 offspring derived from the crosses between white-muscle scallops and between orange-muscle scallops resembled the muscle color of their parents (Fig. 2A). However, for the crosses between white-muscle and orangemuscle scallops (Fig. 2B), the F1 individuals all possessed white muscle, indicating that white color is dominant to orange. Moreover, in the F2 families, segregation of the trait was observed with a ratio of 2.28:1 (153 white and 67 orange), 1.92:1 (171 white and 89 orange) and 2.64:1 (174 white and 66 orange), in families F13-2, F13-4 and F15-1, respectively. The ratios in family F13-2 (χ2 = 1.63, P = 0.20) and F151 (χ2 = 0.39, P = 0.53) followed Mendelian ratios (3:1) for single-gene segregation, while family F13-4 showed distortion away from 3:1, with a higher proportion of orange muscle scallops than the expected Mendelian ratio 1/4 (χ2 = 5.31, P = 0.02). In addition, families F13-2 and F15-1 also exhibited a proportion of orange-muscle scallops higher than 1/4, probably due to the slightly higher viability of orange-muscle scallops under farming conditions. The positive effect of carotenoid accumulation on individual viability and fitness has been reported in various species, including scallop, largely attributed to the function of
3.3. Downregulation of PyBCO-like 1 explains carotenoid deposition in muscle We then analyzed the gene expression difference between orange and white muscle, especially for the genes in the associated genomic interval. RNA-Seq was conducted for 32 muscle samples (16 orangemuscle and 16 white-muscle scallops), and 450 Mb sequencing data covering 11,807 expressed genes (RPKM > 0.5 in at least three of the 32 samples) were generated. A total of 109 differentially expressed genes were found between orange and white muscle (FDR < 0.01, −2.5 > fold change > 2.5), with 48 upregulated and 61 downregulated in the orange muscle (Additional file 4: Table S4). Among the 42 expressed genes in the 2.5 Mb candidate genomic region, only one gene, PY716271.18, was significantly differentially expressed between the two muscles (FDR: 2.75E-21) (Fig. 3C). PY716271.18 also showed the lowest FDR value among all the 109 differentially expressed genes, with 11.28-fold downregulation in orange muscle (Additional file 4: Table S4). Further validation with qRT-PCR also revealed a significantly reduced expression of PY716271.18 in orange muscle compared to that in white muscle (Fig. 3D). BLAST analysis indicated that PY716271.18 (named PyBCO-like 1 in Yesso scallop) encoded carotenoid oxygenase, the enzyme known to catalyze carotenoid cleavage in a variety of organisms [42–46]. Phylogenetic analysis also supported PyBCO-like 1 belonging to the carotenoid oxygenase family, with the close relative being the invertebrate homologs (Additional file 5: Fig. S1). Further 970
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Fig. 3. Identification of the PyBCO-like 1 gene by GWAS, BSA and transcriptome sequencing. A, GWAS in scallop families revealed that the genomic region associated with carotenoid coloration in scallop muscle was located on Chr 8 (P < 3.37E-07). B, FST between orange- and white-muscle populations was assessed by BSA based on genome resequencing. An ~2.5 Mb candidate region underlying muscle coloration was identified on Chr 8, which contains all the values above the 99th percentile (FST > 0.376) (up panel). Blue boxes in the lower panel represent the genes harbored in this region. C, Volcano plot of gene expression comparison between white and orange muscle. Genes located in the 2.5 Mb candidate region that was identified by GWAS and FST analysis are indicated by triangles, while dots represent genes located in the other region of the genome. Differentially expressed genes are marked in orange. PyBCO-like 1 was the most significantly expressed gene (FDR: 2.75E-21) in the whole transcriptome analysis, and the only differentially expressed gene in the 2.5 Mb candidate region. D, qRT-PCR analysis of PyBCO-like 1 expression in the adductor muscle and hepatopancreas of orange- and white-muscle scallops.
carotenoid in E. coli. The coding sequence of PyBCO-like 1 was cloned into the expression vector pET-28a (+) to express PyBCO-like 1 protein. This recombinant plasmid was transformed into a bacterium strain that accumulates β-carotene and exhibits orange color. Evident decoloration was observed in the β-carotene-accumulating bacteria in which PyBCO-like 1 was expressed (Fig. 4A), indicating that PyBCO-like 1 is a functional enzyme in degrading carotenoid.
genome and transcriptome analysis indicated that, PyBCO-like 1 was the only carotenoid oxygenase gene that was differentially expressed between white and orange muscle, albeit there were other carotenoid oxygenase encoding genes in Yesso scallops. Therefore, the coloration of orange muscle is supposed to be due to the downregulation of PyBCO-like 1, which further reduces the degradation of carotenoids. We also analyzed PyBCO-like 1 expression in the hepatopancreas of orange-muscle and white-muscle scallops by qRT-PCR. Unlike the results obtained in muscle, no significant difference in PyBCO-like 1 expression was found in the hepatopancreas between orange- and whitemuscle scallops (Fig. 3D). Therefore, the downregulation of PyBCO-like 1 in the orange muscle of scallops might be tissue-specific. The carotenoids are assumed to be absorbed in both scallops but are degraded to colorless metabolites in the white muscle by PyBCO-like 1.
3.5. Inhibition of PyBCO-like 1 expression in white muscle of scallop results in carotenoid coloration RNA interference (RNAi) was performed to examine the role of PyBCO-like 1 in carotenoid coloration in scallop muscle. Doublestranded RNA (dsRNA) for PyBCO-like 1, which precluded homolog sequences to the other genes in the genome, was synthesized and injected into white muscle at 100 μg once a week for each individual. The white muscle turned orange after inhibiting PyBCO-like 1 expression for four weeks (Fig. 4B, D), with accumulation of the carotenoids
3.4. PyBCO-like 1 is capable of degrading carotenoid We further verified the function of PyBCO-like 1 in cleaving 971
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Fig. 4. Validation of PyBCO-like 1 function. A, PyBCO-like 1 expressed in E. coli could degrade βcarotene. The E. coli cells in the tube marked by (C−) contain pORANGE plasmid carrying β-carotene synthesis genes and exhibit orange color, and cells in (C+) tube contain both pORANGE plasmid and the pET-28a (+) plasmid with PyBCO-like 1 gene. Decoloration occurred in (C+) tube after PyBCO-like 1 expression was induced. E. coli cells without any plasmid are marked by (−). B, In vivo verification of PyBCO-like 1 function by RNAi. White muscle shifted to orange after injection of PyBCO-like 1 dsRNA. C, Carotenoids pectenolone and pectenoxanthin were detected in the muscle injected with PyBCO-like 1 dsRNA, while they were not detected in the control muscle injected with PBS. D, PyBCO-like 1 expression was inhibited by dsRNA, as revealed by qRT-PCR.
pectenolone and pectenoxanthin (Fig. 4C, Additional file 6: Fig. S2). This result supports the hypothesis that downregulation of PyBCO-like 1 leads to the reduction of carotenoid degradation, which is responsible for carotenoid accumulation and coloration in scallop muscle.
three physiological steps: absorbing, transporting, and then metabolizing to apocarotenoids or depositing in the target tissues [17]. Previous investigation of the mechanism regulating scallop carotenoid coloration mainly focused on the steps of carotenoid absorption and transportation, and several potentially related genes that exhibited higher expression in orange muscle than in white muscle were identified through candidate gene analysis or transcriptomic/proteomic data mining [14–16]. In contrast to various genes having been considered to
4. Discussion For animals, carotenoid coloration primarily relies on the following 972
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A carotenoids and nonprovitamin A carotenoids [58,64]. Here, scallop PyBCO-like 1 expressed in E. coli showed cleavage activity toward provitamin A carotenoid β, β-carotene. Meanwhile, inhibition of the PyBCO-like 1 gene resulted in the deposition of scallop carotenoids, pectenolone and pectenoxanthin, the nonprovitamin A carotenoids, meaning that PyBCO-like 1 has a broad substrate spectrum similar to BCO2. However, in D. melanogaster, Nina B, which is similar to BCO1, cleaves carotenoids symmetrically and can convert provitamin A and nonprovitamin A carotenoids, zeaxanthin and lutein [65]. Whether PyBCO-like 1 cleaves carotenoids symmetrically or asymmetrically is still unknown. Phylogenetic analysis suggests that the divergence of BCO1 and BCO2 occurs in the common ancestor of chordates (Additional file 5: Fig. S1); therefore, BCOs in most invertebrates, such as scallops and fruit flies, might remain the characters of the ancestors of animal carotenoid oxygenases, which probably could cleave diverse carotenoid substrates. Future evaluation of the substrate specificity and the identification of cleaving products of PyBCO-like 1 will be helpful for understanding the characteristics of this enzyme and the evolution of carotenoid oxygenase. In summary, we show that carotenoid coloration in Yesso scallop muscle is recessively inherited as a Mendelian trait, and the downregulation of muscle PyBCO-like 1 expression leads to carotenoid deposition due to impaired carotenoid degradation. PyBCO-like 1 is the key gene for carotenoid metabolism in scallops, with diverse carotenoid substrates. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbalip.2019.03.003.
regulate carotenoid coloration in scallop muscle, our F1 and F2 family analysis suggests that this trait was controlled by a single Mendelian factor. Inhibiting the expression of the SRB (scavenger receptor class B) gene, which is essential for cellular uptake of carotenoids [47,48], did not change carotenoid content in scallop muscle, although the carotenoid content in blood decreased greatly [49], further implying that carotenoid uptake might not be the causal process for carotenoid deposition in scallop muscle. In this study, after multiple genomic analyses and functional verifications, PyBCO-like 1 was revealed as the potential factor responsible for scallop muscle coloration. These results mean that the reduction in carotenoid metabolism due to PyBCO-like 1 downregulation led to the orange pigmentation in scallop muscle. Therefore, the upregulation of the genes in orange muscle, as described in previous studies and detected in the transcriptome analysis in this study, was largely the induction associated with carotenoid deposition. Our observations contribute to the accumulating evidence supporting carotenoid oxygenase as a pivotal regulator in carotenoid coloration in animals. The animal carotenoid oxygenase gene was first identified in Drosophila melanogaster, where the Nina B (carotenoid oxygenase gene in fruit fly) mutation resulted in carotenoid accumulation [35,50]. Subsequently, three carotenoid oxygenases were characterized in several vertebrates: BCO1 (β-carotene-15, 15′-oxygenase) and BCO2 (β-carotene-9′, 10′-oxygenase), which cleave carotenoid symmetrically and asymmetrically, respectively, and RPE65 (retinal pigment epithelium-specific 65 kDa protein), an isomerohydrolase of retinoid. Then, BCO1 and BCO2 mutations affecting carotenoid conversion efficiency and carotenoid content were identified in humans, chickens, cows and sheep, as well as in BCO1 and BCO2 knockout mice [51–58]. For aquatic species, the characterization of carotenoid oxygenase is very limited. Currently in Atlantic salmon, carotenoid oxygenase genes were described in terms of sequence and tissue expression pattern [59], although whether they are involved in the regulation of carotenoid deposition is unknown. Our identification of PyBCO-like 1 as the key gene for carotenoid coloration in scallop muscle suggests that PyBCO-like 1 is an excellent candidate to initiate studies on the regulation of carotenoid bioavailability in scallops and even other aquaculture animals. Carotenoids from diets are mainly absorbed and metabolized in the digestive system, which is the major organ for carotenoid oxygenase expression and function in animals [60–62]. We also detected PyBCOlike 1 expression in scallop hepatopancreas, the main organ for food digestion and absorption. Unlike the downregulation of PyBCO-like 1 in muscle, no significant difference of PyBCO-like 1 expression in hepatopancreas was found between the white- and orange-muscle scallop (Fig. 3D), which is consistent with the similar concentration of carotenoids (both pectenolone and pectenoxanthin) in the hepatopancreas between the two scallops (Fig. 1C). This result suggests that the decreased expression of PyBCO-like 1 is tissue-specific. The downregulation of PyBCO-like 1 in muscle without affecting carotenoid availability in the digestive system might also be the reason for the high survival rate of orange-muscle scallops in the families constructed in this study and the mass production of the ‘Haida golden scallop’. Therefore, PyBCO-like 1 is a promising gene to be used for improving muscle carotenoid accumulation in scallop aquaculture. We failed to reveal the causative mutation leading to PyBCO-like 1 downregulation in the orange muscle mainly due to the insufficient chromosome recombination that occurred in the populations we examined, as the Yesso scallop was originally introduced to China by a limited number of individuals from Japan in 1980s [63]. Further efforts broadly collecting and analyzing wild Yesso scallops would aid to refine the associated region and determine the mutations inhibiting PyBCO-like 1 expression. As the enzyme catalyzing carotenoid cleavage, BCO1 in humans is considered to cleave substrates mainly limited to provitamin A carotenoids, such as β, β-carotene, α-carotene, and β-cryptoxanthin, but fails to convert nonprovitamin A carotenoids such as lycopene and zeaxanthin; BCO2 has broader substrate specificity for both provitamin
Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements We sincerely thank Dr. Johannes von Lintig for kindly providing us the pORANGE plasmid, and Zhangzidao Group Co., Ltd. (Dalian, China) for providing scallop populations and aiding in scallop family construction. Funding This work was supported by the National Key Research and Development Project (2018YFD0900104), National Natural Science Foundation of China (31630081 and 31472276), the Major basic research projects of Natural Science Foundation (ZR2018ZA0748), the Fundamental Research Funds for the Central Universities (201762001), and Taishan Scholar Project Fund of Shandong Province of China. Availability of data Raw sequencing data have been submitted to the NABI Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra) under the project number of PRJNA407738 (2b-rad), PRJNA407739 (RNAseq) and PRJNA407358 (Pooled Genome re-sequencing). Competing interest The authors have declared that no competing interests exist. References [1] W. Miki, Biological functions and activities of animal carotenoids, Pure Appl. Chem. 63 (1) (1991) 141–146. [2] D.P. Toews, N.R. Hofmeister, S.A. Taylor, The evolution and genetics of carotenoid processing in animals, Trends Genet. 33 (3) (2017) 171–182.
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BBA - Molecular and Cell Biology of Lipids journal homepage: www.elsevier.com/locate/bbalip
A carotenoid oxygenase is responsible for muscle coloration in scallop a,1
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a,b
a
a,c
T
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Xue Li , Shuyue Wang , Xiaogang Xun , Mengran Zhang , Shi Wang , Hengde Li , Liang Zhaoa,b, Qiang Fua, Huizhen Wanga, Tingting Lia, Shanshan Liana,b, Qiang Xinga, Xu Lia, ⁎ ⁎ Wei Wua, Lingling Zhanga,b, Xiaoli Hua,b, , Zhenmin Baoa,b, a
MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China c Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China d Ministry of Agriculture Key Laboratory of Aquatic Genomics, CAFS Key Laboratory of Aquatic Genomics and Beijing Key Laboratory of Fishery Biotechnology, Center for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences, Beijing 100141, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Scallop Carotenoid coloration Muscle Fine mapping PyBCO-like 1
As lipid microconstituents mainly of plant origin, carotenoids are essential nutrients for humans and animals, and carotenoid coloration represents an important meat quality parameter for many farmed animals. Currently, the mechanism of carotenoid bioavailability in animals is largely unknown mainly due to the limited approaches applied, the shortage of suitable model systems and the restricted taxonomic focus. The mollusk Yesso scallop (Patinopecten yessoensis) possessing orange adductor muscle with carotenoid deposition, provides a unique opportunity to research the mechanism underlying carotenoid utilization in animals. Herein, through family construction and analysis, we found that carotenoid coloration in scallop muscle is inherited as a recessive Mendelian trait. Using a combination of genomic approaches, we mapped this trait onto chromosome 8, where PyBCO-like 1 encoding carotenoid oxygenase was the only differentially expressed gene between the white and orange muscles (FDR = 2.75E-21), with 11.28-fold downregulation in the orange muscle. Further functional assays showed that PyBCO-like 1 is capable of degrading β-carotene, and inhibiting PyBCO-like 1 expression in the white muscle resulted in muscle coloration and carotenoid deposition. In the hepatopancreas, which is the organ for digestion and absorption, neither the scallop carotenoid concentration nor PyBCO-like 1 expression were significantly different between the two scallops. These results indicate that carotenoids could be taken up in both white- and orange-muscle scallops and then degraded by PyBCO-like 1 in the white muscle. Our data suggest that PyBCO-like 1 is the essential gene for carotenoid metabolism in scallop muscle, and its downregulation leads to carotenoid deposition and muscle coloration.
1. Introduction As dietary lipids acquired solely from food, carotenoids have long been recognized as essential nutrients for humans and most animals, with positive effects on antioxidation and preventing vitamin A deficiency [1]. In meat-producing animals, such as cattle, sheep, chicken, and especially salmonid fishes, flesh yellowness or redness determined by carotenoid coloration is an important parameter to assess meat quality, with higher values being preferred. Additionally, carotenoidbased coloration in animals has also become a focus of evolutionary studies regarding the establishment and maintenance of this trait [2–6]. A better understanding of the molecular mechanisms underlying
carotenoid deposition has been of the interest from both animal breeding and evolutionary perspectives [7,8]; however, these mechanisms currently remain largely unknown mainly due to the limited approaches applied, the shortage of suitable model systems and the restricted taxonomic focus [2]. Bivalve scallops are widely distributed throughout the world and are also of worldwide economic importance for their delicious and remarkably large adductor muscle. Similar to that in most other bivalve species, the adductor muscle of the scallop is completely white, without coloration. In recent years, orange muscle arising from carotenoid pigmentation by accumulating pectenolone and pectenoxanthin was reported in the Yesso scallop (Patinopecten yessoensis) [9,10].
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Corresponding authors at: MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China. E-mail addresses: [email protected] (X. Hu), [email protected] (Z. Bao). 1 Xue Li and Shuyue Wang contributed equally to this work. https://doi.org/10.1016/j.bbalip.2019.03.003 Received 2 December 2018; Received in revised form 28 February 2019; Accepted 6 March 2019 Available online 09 March 2019 1388-1981/ © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.2. Construction of white-muscle and orange-muscle scallop crosses
Subsequently, carotenoid coloration of muscle was also found in other scallop species, including the Noble scallop (Chlamys nobilis) and Bay scallop (Argopecten irradians) [11,12]. Due to the commercial potential of this trait for improving carotenoid supply, a strain of Yesso scallop named ‘Haida golden scallop’, which is characterized by an orange adductor muscle with carotenoid deposition, was developed and massively farmed. These characteristics, together with the well-assembled and annotated genome of the Yesso scallop [13], make this bivalve species a potential model for studies on the molecular mechanism underlying carotenoid utilization in animal muscle. Recent progress has been made in understanding carotenoid coloration in scallop muscle mostly based on the hypothesis that increased carotenoid absorption or transportation was the causation. Then, several genes potentially related to carotenoid intake and exhibiting higher expression in orange muscle than in white muscle were identified [14–16]. For example, in the Yesso scallop, a stearoyl-CoA desaturase (SCD) gene encoding the key enzyme in the biosynthesis of monounsaturated fatty acids which could enhance carotenoid absorption [14], and a lysophosphatidylcholine acyltransferase 1 (LPCAT1) gene, which might be involved in carotenoid storage in the cell [15], were found to be upregulated in orange muscle; proteomic analysis revealed expression enhancement in orange muscle of several proteins possibly in relation to lipid-associated carotenoid absorption or transportation [16]. However, without functional validation, whether the upregulation of these genes is the causal factor of or the induced reaction to carotenoid deposition is unknown. Meanwhile, for the expression of carotenoid coloration, in addition to carotenoid uptake and transport steps, which have been the focus of previous studies on scallops, another physiological step, carotenoid metabolism or cleavage, is also involved [17] but was not considered in reported studies on scallop muscle coloration. Recently, an association analysis of muscle coloration in the Bay scallop identified hundreds of associated SNPs and genes throughout the genome; however, which gene is responsible for this trait was not revealed [12]. Therefore, the key regulator of carotenoid pigmentation in scallop muscle is still unknown. To reveal the genetic mechanism of orange muscle in scallops, in the present study, through family construction and analysis together with a combination of genomic approaches, we found that carotenoid coloration in scallop muscle is recessively inherited as a Mendelian trait, and the downregulation of the gene PyBCO-like 1 encoding carotenoid oxygenase was responsible for muscle carotenoid coloration, which was further supported by functional assays.
Yesso scallop family founders were collected at the hatchery of Zhangzidao Group Co., Ltd. (Dalian, China) and a series of F1 and F2 families were constructed (Fig. 2A and B). F1 families were established through crossing between white-muscle scallops, between orangemuscle scallops, and between white-muscle and orange-muscle scallops. For crosses between white-muscle and orange-muscle scallops, three female orange-muscle scallops were mated with one male white-muscle scallop to produce three F1 families. Then, three F2 families were constructed, among which family 13-4 was constructed by crossing one male and one female offspring within the first F1 family; family 15-1 was produced by crossing within the third F1 family; and family 13-2 was constructed by mating one female in the first F1 family with one male in the second F1 family. More than two hundred scallops from each F1 and F2 family were collected for muscle color observation. 2.3. Genome-wide genotyping and genome-wide association study (GWAS) The four F0 progenitors, six F1 progenies and 116 F2 progenies, including 38 (13 orange and 25 white) from family F13-2, 38 (17 orange and 21 white) from F13-4, and 40 (19 orange and 21 white) from F15-1, were used for GWAS. Genomic DNA was extracted from the adductor muscle using the traditional phenol/chloroform extraction method [18]. 2b-RAD libraries (using the endonucleases Bsa-XI) were constructed following the protocol developed by Wang et al. [19], with the library of each individual being assigned a unique barcode. The libraries were then pooled for single end sequencing by HiSeq 2000 (Illumina, America). Raw reads were trimmed to remove adaptors and low-quality sequences as described by Jiao et al. [20]. Then, the remaining highquality reads were mapped to the genome reference of the Yesso scallop using SOAPaligner [21]. Single nucleotide polymorphisms (SNPs) were called by RADtyping v1.3 under default parameters [22]. SNPs that could be genotyped in at least 60% of the individuals were considered high-quality markers and retained for further analysis. Association analyses between genotypes and muscle colors were conducted using Plink 1.9 [23] by comparing allele frequencies between orange and white muscle. Bonferroni correction was performed for multiple testing. The confidence interval was calculated using Li’s method [24]. 2.4. Genome resequencing and bulk segregant analysis (BSA)
2. Materials and methods Yesso scallops used for genome resequencing and BSA were collected near Zhangzidao Island of the north Yellow Sea. Genomic DNA from 50 white-muscle scallops and 50 orange-muscle scallops were pooled separately. For each of the two DNA pools, a paired-end sequencing library with insert size of 180 bp was constructed following the Illumina's standard DNA library preparation protocol and was then sequenced to a coverage of ~50× using the Illumina HiSeq 2000 platform. The raw data were trimmed to remove low-quality sequences, and the resulting clean reads of the two libraries were separately mapped against the reference genome assembly of the Yesso scallop using BWAMEM alignment algorithms to obtain the SAM format files [25]. After being converted to BAM format and then sorted and deduplicated using SAMtools [26] and Picard pipelines (http://broadinstitute.github.io/ picard/), the resulting sorted BAM files were converted to pileup files for SNP identification and allele counting. Before BSA analysis, reads were screened with the following filters: 1) a minimum mapping quality of 20; 2) a minimum coverage of 10× and a maximum coverage of 500 per position; and 3) at least 1% reads supporting the minor allele in polymorphic sites. Genetic differentiation across Chr 8 between white and orange muscle was summarized in windows of 20 kb moved in steps of 15 kb using the fixation index (FST),
2.1. High performance liquid chromatography (HPLC) analysis of scallop carotenoids The Animal Care and Use Committee of Ocean University of China approved all the scallop procedures performed in this work. The main carotenoids deposited in the orange muscle of Yesso scallop were pectenolone and pectenoxanthin (Fig. 1A and B), which were detected in scallop muscle and hepatopancreas using reversed-phase HPLC, as described in [10], with minor modification. In short, 0.5 g adductor muscle was lyophilized and extracted three times with 50 ml ethyl acetate, which contained 0.1% butylated hydroxytoluene (BHT). After solvent evaporation, the residues were dissolved in 2 ml mobile phase solution and then filtered. Carotenoids were separated and quantified using an analytical-scale LaChrom C18 (4.6 × 250 mm, inner diameter 5 μm) (HITACHI, Japan) with the following parameters: 10 μl injection volume; 25 °C column temperature; acetonitrile, methanol, and methylene chloride mobile phase (50:46:4, v/v/v) with 0.1% BHT; 1.0 ml per minute flow rate; 450 nm UV–Vis detector; and 10 min separation time. The carotenoids pectenolone and pectenoxanthin were identified and quantified by comparison with the standards prepared by the methods described in [10]. 967
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Fig. 1. The orange muscle of the Yesso scallop accumulates carotenoids. A, An orange-muscle scallop (left) and a white-muscle scallop (right). The adductor muscle is indicated with a black arrow. B, Structure of the carotenoids pectenolone and pectenoxanthin deposited in the orange muscle [10]. C, Concentrations of pectenolone and pectenoxanthin in scallop adductor muscle and hepatopancreas.
as implemented in the PoPoolation2 package [27].
2.5. Transcriptome sequencing and expression profiling Total mRNA was extracted from the adductor muscles of 16 whiteand 16 orange-muscle Yesso scallops using the conventional 968
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Fig. 2. Pedigree analysis of the crosses between orange-muscle scallops (A, left), between white-muscle scallops (A, right), and between orange-muscle and whitemuscle scallops (B). The orange color marks an adductor muscle with carotenoids accumulation. The ratios of the orange- and white-muscle scallops in the F2 families 13-2, 13-4, and 15-1 are shown in B.
2.6. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis
guanidinium isothiocyanate method [28]. RNA-Seq libraries were constructed individually according to the standard Illumina protocols and were subject to Illumina PE100 sequencing. Reads containing bases ‘N’ or excessive numbers of low-quality bases (> 30% bases with quality scores < 10) were removed. After trimming, the high-quality reads from multiple RNA-Seq datasets were aligned to the P. yessoensis genome [13] using STAR 2.4.1d [29], allowing two mismatches. The total number of reads matching the gene regions was counted by script ‘HTSeq-count’ and then calculated for digital expression values as RPKM (Reads Per Kilobase per Million mapped reads) [30,31]. Reads that were not uniquely aligned to the genome were discarded. Genes with RPKM > 0.5 in at least three of the 32 muscle samples were subjected to subsequent expression profiling. The R (version 2.15.2) Bioconductor package EdgeR (v2.4.6) [32] was applied to identify significantly differentially expressed genes between orange and white muscle. Differentially expressed genes were defined as having a fold change > 2.5 in the average RPKM between orange and white muscle, and a Benjamini and Hochberg corrected FDR of < 0.01 [33].
Total RNA was extracted from the adductor muscle and hepatopancreas of the white- and orange-muscle scallop following the method described in [28]. The first-strand cDNA was synthesized according to the manufacturer's instruction of M-MLV Reverse Transcriptase (Invitrogen, CA, USA), and then diluted to 1:30 to be used as the template for qRT-PCR in analyzing PyBCO-like 1 expression. Primers for qRT-PCR were listed in Additional file 1: Table S1. The gene encoding DEAD-box RNA helicase-like protein (HELI) was used as the reference gene for normalization [34]. The qRT-PCR results were analyzed using Realtime PCR Miner (http://www.miner.ewindup.info/). 2.7. Verification of PyBCO-like 1 function in E. coli The pORANGE plasmid [35] containing β-carotene synthesis genes was transformed into E. coli JM109 (DE3) (Beijing Dingguo Changsheng Biotech, China). The CDS of PyBCO-like 1 was amplified with the primers listed in Additional file 1: Table S1, and then ligated into the pET969
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28a (+) vector (Novagen, Germany) using T4 DNA Ligase (New England Biolabs, USA), followed by transformation into the E. coli strain containing pORANGE plasmid. Colonies with both β-carotene synthesis genes and PyBCO-like 1 were induced by IPTG (1 mM). The color of E. coli with only the pORANGE plasmid, with both pORANGE and PyBCOlike 1 containing plasmids, and with only the vector pET-28a (+) was examined.
carotenoids in preventing oxidative damage stress [37–41]. The ratio of white-muscle to orange-muscle scallops observed in the F2 families suggests that carotenoid coloration of scallop muscle is likely controlled by a single Mendelian factor.
2.8. In vivo verification of PyBCO-like 1 function by RNA interference (RNAi)
To determine the genomic region underlying carotenoid coloration in scallop muscle, GWAS was performed using a total of 126 individuals (52 orange-muscle and 74 white-muscle scallops) in the families constructed (Fig. 2), including the four progenitors (F0), six F1 progenies and 116 F2 progenies, with 2b-RAD methods for genome-wide marker genotyping [19]. The statistical sequencing depth corresponded to ~20× in the F0, ~30× in the F1 and ~7× in the F2 progenies. A total of 29,674 polymorphic SNPs were obtained through mapping the reads to the P. yessoensis reference genome (Assembly: ASM211388v2). Association between SNP genotypes and muscle color was performed by Plink 1.9 [23] using a case/control method. After Bonferroni correction and confidence interval estimation, a major genome region of ~19 Mb (P < 3.37E-07, 23,888,002 to 42,852,177 bp) with a strong association signal was detected on chromosome 8 (Chr 8) (Fig. 3A, Additional file 2: Table S2). Considering the limited recombination events that occurred in family samples, for further fine mapping, we performed genome resequencing using population samples through pooling DNA for 50 orange muscles and 50 white muscles. A total of 39,937 SNPs located on Chr 8 were obtained from the ~50× sequencing data. Allele frequency differentiation across Chr 8 between orange muscle and white muscle was analyzed using the fixation index (FST) with sliding window method. One region that contained all values above the 99th percentile (FST ≥ 0.376) clearly stood out, providing an excellent candidate region harboring the orange-muscle gene. This region spanned ~2.5 Mb (25,154,020–27,593,320 bp), which is within the significant interval detected by GWAS (Fig. 3B). An examination of the genome annotation of the Yesso scallop revealed 79 protein-coding genes in this interval, with 42 expressed in scallop muscle (RPKM > 0.5 in at least three of the 32 muscle samples subjected to the subsequent RNA-Seq analysis) (Additional file 3: Table S3); the causal gene for orange muscle is expected to be among these genes.
3.2. Identification of the genomic region associated with carotenoid coloration
White-muscle scallops that were two years of age were subjected to double-stranded RNA (dsRNA) mediated silencing of PyBCO-like 1. Before and during the experiments, all scallops were acclimated in aerated tanks at 8 °C and fed with golden algae (Isochrysis galbana) as described in [14]. PyBCO-like 1 dsRNA was synthesized using the MEGAscript® RNAi Kit (Thermo, USA) following the manufacturer's protocol, with the primers listed in Additional file 1: Table S1. After being purified, the synthesized dsRNA was diluted to 1 μg/μl with nuclease-free 1 × PBS (Sangon, China). For each scallop, 100 μl PyBCOlike 1 dsRNA or 100 μl 1 × PBS was injected into the muscle once per week for four weeks. Then, the adductor muscle was dissected for PyBCO-like 1 expression analysis by qRT-PCR and carotenoid detection using reverse-phase HPLC by analyzing the peak areas of the two main carotenoids, pectenolone and pectenoxanthin. 3. Results 3.1. Carotenoid coloration in scallop muscle is a recessive trait Previous studies revealed that the orange muscle of the Yesso scallop contained two carotenoids, pectenolone and pectenoxanthin (Fig. 1A, B) [9,10]. To better understand the difference in carotenoid deposition between orange- and white-muscle scallops, the concentrations of the two carotenoids were analyzed in the adductor muscle and the hepatopancreas, an organ responsible for digestion and absorption of food in bivalves [36]. In orange muscle, 16.35 ± 2.31 and 17.80 ± 2.55 μg/g pectenolone and pectenoxanthin were detected, respectively, while no carotenoid was detected in white muscle (Fig. 1C), indicating that carotenoid deposition and coloration in scallop muscle was a discrete character. For the hepatopancreas, the concentrations of the two carotenoids were not significantly different between orange- and white-muscle scallops (Fig. 1C), which implies a similar ability of the two scallops to absorb carotenoids from foods. To validate the hereditary pattern of carotenoid coloration in scallop muscle, a series of families were constructed, including crosses between white-muscle scallops, between orange-muscle scallops, and between white-muscle and orange-muscle scallops. All F1 offspring derived from the crosses between white-muscle scallops and between orange-muscle scallops resembled the muscle color of their parents (Fig. 2A). However, for the crosses between white-muscle and orangemuscle scallops (Fig. 2B), the F1 individuals all possessed white muscle, indicating that white color is dominant to orange. Moreover, in the F2 families, segregation of the trait was observed with a ratio of 2.28:1 (153 white and 67 orange), 1.92:1 (171 white and 89 orange) and 2.64:1 (174 white and 66 orange), in families F13-2, F13-4 and F15-1, respectively. The ratios in family F13-2 (χ2 = 1.63, P = 0.20) and F151 (χ2 = 0.39, P = 0.53) followed Mendelian ratios (3:1) for single-gene segregation, while family F13-4 showed distortion away from 3:1, with a higher proportion of orange muscle scallops than the expected Mendelian ratio 1/4 (χ2 = 5.31, P = 0.02). In addition, families F13-2 and F15-1 also exhibited a proportion of orange-muscle scallops higher than 1/4, probably due to the slightly higher viability of orange-muscle scallops under farming conditions. The positive effect of carotenoid accumulation on individual viability and fitness has been reported in various species, including scallop, largely attributed to the function of
3.3. Downregulation of PyBCO-like 1 explains carotenoid deposition in muscle We then analyzed the gene expression difference between orange and white muscle, especially for the genes in the associated genomic interval. RNA-Seq was conducted for 32 muscle samples (16 orangemuscle and 16 white-muscle scallops), and 450 Mb sequencing data covering 11,807 expressed genes (RPKM > 0.5 in at least three of the 32 samples) were generated. A total of 109 differentially expressed genes were found between orange and white muscle (FDR < 0.01, −2.5 > fold change > 2.5), with 48 upregulated and 61 downregulated in the orange muscle (Additional file 4: Table S4). Among the 42 expressed genes in the 2.5 Mb candidate genomic region, only one gene, PY716271.18, was significantly differentially expressed between the two muscles (FDR: 2.75E-21) (Fig. 3C). PY716271.18 also showed the lowest FDR value among all the 109 differentially expressed genes, with 11.28-fold downregulation in orange muscle (Additional file 4: Table S4). Further validation with qRT-PCR also revealed a significantly reduced expression of PY716271.18 in orange muscle compared to that in white muscle (Fig. 3D). BLAST analysis indicated that PY716271.18 (named PyBCO-like 1 in Yesso scallop) encoded carotenoid oxygenase, the enzyme known to catalyze carotenoid cleavage in a variety of organisms [42–46]. Phylogenetic analysis also supported PyBCO-like 1 belonging to the carotenoid oxygenase family, with the close relative being the invertebrate homologs (Additional file 5: Fig. S1). Further 970
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Fig. 3. Identification of the PyBCO-like 1 gene by GWAS, BSA and transcriptome sequencing. A, GWAS in scallop families revealed that the genomic region associated with carotenoid coloration in scallop muscle was located on Chr 8 (P < 3.37E-07). B, FST between orange- and white-muscle populations was assessed by BSA based on genome resequencing. An ~2.5 Mb candidate region underlying muscle coloration was identified on Chr 8, which contains all the values above the 99th percentile (FST > 0.376) (up panel). Blue boxes in the lower panel represent the genes harbored in this region. C, Volcano plot of gene expression comparison between white and orange muscle. Genes located in the 2.5 Mb candidate region that was identified by GWAS and FST analysis are indicated by triangles, while dots represent genes located in the other region of the genome. Differentially expressed genes are marked in orange. PyBCO-like 1 was the most significantly expressed gene (FDR: 2.75E-21) in the whole transcriptome analysis, and the only differentially expressed gene in the 2.5 Mb candidate region. D, qRT-PCR analysis of PyBCO-like 1 expression in the adductor muscle and hepatopancreas of orange- and white-muscle scallops.
carotenoid in E. coli. The coding sequence of PyBCO-like 1 was cloned into the expression vector pET-28a (+) to express PyBCO-like 1 protein. This recombinant plasmid was transformed into a bacterium strain that accumulates β-carotene and exhibits orange color. Evident decoloration was observed in the β-carotene-accumulating bacteria in which PyBCO-like 1 was expressed (Fig. 4A), indicating that PyBCO-like 1 is a functional enzyme in degrading carotenoid.
genome and transcriptome analysis indicated that, PyBCO-like 1 was the only carotenoid oxygenase gene that was differentially expressed between white and orange muscle, albeit there were other carotenoid oxygenase encoding genes in Yesso scallops. Therefore, the coloration of orange muscle is supposed to be due to the downregulation of PyBCO-like 1, which further reduces the degradation of carotenoids. We also analyzed PyBCO-like 1 expression in the hepatopancreas of orange-muscle and white-muscle scallops by qRT-PCR. Unlike the results obtained in muscle, no significant difference in PyBCO-like 1 expression was found in the hepatopancreas between orange- and whitemuscle scallops (Fig. 3D). Therefore, the downregulation of PyBCO-like 1 in the orange muscle of scallops might be tissue-specific. The carotenoids are assumed to be absorbed in both scallops but are degraded to colorless metabolites in the white muscle by PyBCO-like 1.
3.5. Inhibition of PyBCO-like 1 expression in white muscle of scallop results in carotenoid coloration RNA interference (RNAi) was performed to examine the role of PyBCO-like 1 in carotenoid coloration in scallop muscle. Doublestranded RNA (dsRNA) for PyBCO-like 1, which precluded homolog sequences to the other genes in the genome, was synthesized and injected into white muscle at 100 μg once a week for each individual. The white muscle turned orange after inhibiting PyBCO-like 1 expression for four weeks (Fig. 4B, D), with accumulation of the carotenoids
3.4. PyBCO-like 1 is capable of degrading carotenoid We further verified the function of PyBCO-like 1 in cleaving 971
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Fig. 4. Validation of PyBCO-like 1 function. A, PyBCO-like 1 expressed in E. coli could degrade βcarotene. The E. coli cells in the tube marked by (C−) contain pORANGE plasmid carrying β-carotene synthesis genes and exhibit orange color, and cells in (C+) tube contain both pORANGE plasmid and the pET-28a (+) plasmid with PyBCO-like 1 gene. Decoloration occurred in (C+) tube after PyBCO-like 1 expression was induced. E. coli cells without any plasmid are marked by (−). B, In vivo verification of PyBCO-like 1 function by RNAi. White muscle shifted to orange after injection of PyBCO-like 1 dsRNA. C, Carotenoids pectenolone and pectenoxanthin were detected in the muscle injected with PyBCO-like 1 dsRNA, while they were not detected in the control muscle injected with PBS. D, PyBCO-like 1 expression was inhibited by dsRNA, as revealed by qRT-PCR.
pectenolone and pectenoxanthin (Fig. 4C, Additional file 6: Fig. S2). This result supports the hypothesis that downregulation of PyBCO-like 1 leads to the reduction of carotenoid degradation, which is responsible for carotenoid accumulation and coloration in scallop muscle.
three physiological steps: absorbing, transporting, and then metabolizing to apocarotenoids or depositing in the target tissues [17]. Previous investigation of the mechanism regulating scallop carotenoid coloration mainly focused on the steps of carotenoid absorption and transportation, and several potentially related genes that exhibited higher expression in orange muscle than in white muscle were identified through candidate gene analysis or transcriptomic/proteomic data mining [14–16]. In contrast to various genes having been considered to
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A carotenoids and nonprovitamin A carotenoids [58,64]. Here, scallop PyBCO-like 1 expressed in E. coli showed cleavage activity toward provitamin A carotenoid β, β-carotene. Meanwhile, inhibition of the PyBCO-like 1 gene resulted in the deposition of scallop carotenoids, pectenolone and pectenoxanthin, the nonprovitamin A carotenoids, meaning that PyBCO-like 1 has a broad substrate spectrum similar to BCO2. However, in D. melanogaster, Nina B, which is similar to BCO1, cleaves carotenoids symmetrically and can convert provitamin A and nonprovitamin A carotenoids, zeaxanthin and lutein [65]. Whether PyBCO-like 1 cleaves carotenoids symmetrically or asymmetrically is still unknown. Phylogenetic analysis suggests that the divergence of BCO1 and BCO2 occurs in the common ancestor of chordates (Additional file 5: Fig. S1); therefore, BCOs in most invertebrates, such as scallops and fruit flies, might remain the characters of the ancestors of animal carotenoid oxygenases, which probably could cleave diverse carotenoid substrates. Future evaluation of the substrate specificity and the identification of cleaving products of PyBCO-like 1 will be helpful for understanding the characteristics of this enzyme and the evolution of carotenoid oxygenase. In summary, we show that carotenoid coloration in Yesso scallop muscle is recessively inherited as a Mendelian trait, and the downregulation of muscle PyBCO-like 1 expression leads to carotenoid deposition due to impaired carotenoid degradation. PyBCO-like 1 is the key gene for carotenoid metabolism in scallops, with diverse carotenoid substrates. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bbalip.2019.03.003.
regulate carotenoid coloration in scallop muscle, our F1 and F2 family analysis suggests that this trait was controlled by a single Mendelian factor. Inhibiting the expression of the SRB (scavenger receptor class B) gene, which is essential for cellular uptake of carotenoids [47,48], did not change carotenoid content in scallop muscle, although the carotenoid content in blood decreased greatly [49], further implying that carotenoid uptake might not be the causal process for carotenoid deposition in scallop muscle. In this study, after multiple genomic analyses and functional verifications, PyBCO-like 1 was revealed as the potential factor responsible for scallop muscle coloration. These results mean that the reduction in carotenoid metabolism due to PyBCO-like 1 downregulation led to the orange pigmentation in scallop muscle. Therefore, the upregulation of the genes in orange muscle, as described in previous studies and detected in the transcriptome analysis in this study, was largely the induction associated with carotenoid deposition. Our observations contribute to the accumulating evidence supporting carotenoid oxygenase as a pivotal regulator in carotenoid coloration in animals. The animal carotenoid oxygenase gene was first identified in Drosophila melanogaster, where the Nina B (carotenoid oxygenase gene in fruit fly) mutation resulted in carotenoid accumulation [35,50]. Subsequently, three carotenoid oxygenases were characterized in several vertebrates: BCO1 (β-carotene-15, 15′-oxygenase) and BCO2 (β-carotene-9′, 10′-oxygenase), which cleave carotenoid symmetrically and asymmetrically, respectively, and RPE65 (retinal pigment epithelium-specific 65 kDa protein), an isomerohydrolase of retinoid. Then, BCO1 and BCO2 mutations affecting carotenoid conversion efficiency and carotenoid content were identified in humans, chickens, cows and sheep, as well as in BCO1 and BCO2 knockout mice [51–58]. For aquatic species, the characterization of carotenoid oxygenase is very limited. Currently in Atlantic salmon, carotenoid oxygenase genes were described in terms of sequence and tissue expression pattern [59], although whether they are involved in the regulation of carotenoid deposition is unknown. Our identification of PyBCO-like 1 as the key gene for carotenoid coloration in scallop muscle suggests that PyBCO-like 1 is an excellent candidate to initiate studies on the regulation of carotenoid bioavailability in scallops and even other aquaculture animals. Carotenoids from diets are mainly absorbed and metabolized in the digestive system, which is the major organ for carotenoid oxygenase expression and function in animals [60–62]. We also detected PyBCOlike 1 expression in scallop hepatopancreas, the main organ for food digestion and absorption. Unlike the downregulation of PyBCO-like 1 in muscle, no significant difference of PyBCO-like 1 expression in hepatopancreas was found between the white- and orange-muscle scallop (Fig. 3D), which is consistent with the similar concentration of carotenoids (both pectenolone and pectenoxanthin) in the hepatopancreas between the two scallops (Fig. 1C). This result suggests that the decreased expression of PyBCO-like 1 is tissue-specific. The downregulation of PyBCO-like 1 in muscle without affecting carotenoid availability in the digestive system might also be the reason for the high survival rate of orange-muscle scallops in the families constructed in this study and the mass production of the ‘Haida golden scallop’. Therefore, PyBCO-like 1 is a promising gene to be used for improving muscle carotenoid accumulation in scallop aquaculture. We failed to reveal the causative mutation leading to PyBCO-like 1 downregulation in the orange muscle mainly due to the insufficient chromosome recombination that occurred in the populations we examined, as the Yesso scallop was originally introduced to China by a limited number of individuals from Japan in 1980s [63]. Further efforts broadly collecting and analyzing wild Yesso scallops would aid to refine the associated region and determine the mutations inhibiting PyBCO-like 1 expression. As the enzyme catalyzing carotenoid cleavage, BCO1 in humans is considered to cleave substrates mainly limited to provitamin A carotenoids, such as β, β-carotene, α-carotene, and β-cryptoxanthin, but fails to convert nonprovitamin A carotenoids such as lycopene and zeaxanthin; BCO2 has broader substrate specificity for both provitamin
Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements We sincerely thank Dr. Johannes von Lintig for kindly providing us the pORANGE plasmid, and Zhangzidao Group Co., Ltd. (Dalian, China) for providing scallop populations and aiding in scallop family construction. Funding This work was supported by the National Key Research and Development Project (2018YFD0900104), National Natural Science Foundation of China (31630081 and 31472276), the Major basic research projects of Natural Science Foundation (ZR2018ZA0748), the Fundamental Research Funds for the Central Universities (201762001), and Taishan Scholar Project Fund of Shandong Province of China. Availability of data Raw sequencing data have been submitted to the NABI Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra) under the project number of PRJNA407738 (2b-rad), PRJNA407739 (RNAseq) and PRJNA407358 (Pooled Genome re-sequencing). Competing interest The authors have declared that no competing interests exist. References [1] W. Miki, Biological functions and activities of animal carotenoids, Pure Appl. Chem. 63 (1) (1991) 141–146. [2] D.P. Toews, N.R. Hofmeister, S.A. Taylor, The evolution and genetics of carotenoid processing in animals, Trends Genet. 33 (3) (2017) 171–182.
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