Tracing key genes associated with the Pinctada margaritifera albino phenotype from juvenile to cultured pearl harvest stages using multiple whole transcriptome sequencing

- Background: Albino mutations are commonly observed in the animal kingdom, including in bivalves.
- In the black- lipped pearl oyster Pinctada margaritifera, albino specimens are characterized by total or partial absence of.
- Additionally, significant phenotype- associated SNPs were selected (N = 159), including two located in the Pif biomineralization gene, which codes for nacre formation.
- Interestingly, significantly different transcript splicing was detected between juvenile (N = 1366) and adult mantle tissue (N = 313) in, e.g., the tyrosinase Tyr-1 gene, which showed more complex regulation in mantle, and the Notch1 encoding gene, which was upregulated in albino juveniles..
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- Mutations affecting pigmentation were among the first in the animal kingdom to be studied for Mendelian inherit- ance.
- In the aquaculture industry, body pig- mentation can be an economically important trait selected to directly enhance the commercial value of a given species, for example, the red Nile tilapia (Oreochromis niloticus) compared with the wild type [2], or the majority of fresh- water/marine ornamental species such as clownfishes [3]..
- Albino individuals have also been observed among aquacultured Russian sturgeons, one of the most valuable freshwater fish species worldwide [6].
- In bivalves, white specimens of the Akoya pearl oyster Pinctada fucata are already used in selective breeding programs [7, 8].
- Recently, the Notch signalling path- way was found to play a crucial role in shell pigmenta- tion in the clam Meretrix meretrix, following a gene- dosage dependent pattern [15].
- The calcium signalling pathway may also be implicated through activation of the Notch pathway in this species.
- Other studies impli- cated the Wnt signalling pathway in the maintenance of.
- melanocyte and keratinocyte homeostasis and, therefore, its impact on pigmentation in the sea cucumber Aposti- chopus japonicus [18].
- It is worth mentioning that the majority of the previous studies working on pigmenta- tion in bivalves focused on mantle tissue samples..
- Marine aquaculture of the black-lipped pearl oyster Pinctada margaritifera var.
- Nacre layers secreted by the mineralizing ac- tivity of the pearl sac (tissue which originated from the graft mantle) are progressively recovering the nucleus dur- ing culture time, forming the cultured pearl [21].
- margaritifera produces the largest range of cultured pearl colour in the genus [22, 23], including white pearls pro- duced by the rare white albino shell morphotype, when these individuals are used as donors [24].
- In the present study, we investigated the multiple al- bino phenotype expression in P.
- Three distinctive RNA-seq ap- proaches were then used to compare gene expression between the white shell phenotype (albino) and the black shell phenotype (wild type) in the same cohort.
- We identified new candidate genes for white-shelled molluscs, together with genes already known to be expressed in the mantle tissue of P.
- The raw data of the three datasets were submitted to the National Center for Biotechnology (NCBI.
- There were notably fewer DEGs in the PS.
- J and M datasets PCAs were able to discriminate the albino phenotype from black wild-type samples along the first principal compo- nent, accounting for 36.91% (J) and 31.91% (M) of the total variance.
- In the PS dataset PCA, no clustering of samples according to phenotype can be observed (Fig.
- Out of the total number of DEGs, 79.70% (J), 83.33%.
- Interestingly, a high proportion of common DEGs in pairwise compari- sons of the three datasets were not regulated in a con- sistent way: 62.82% (M &.
- Only four DEGs over- lapped the three datasets, of which two were consistently up-regulated in the albino phenotype and only one had a predicted function (endonuclease enzyme) in the avail- able annotation (Additional file 2)..
- Table 1 General statistics of sequencing reads used in the three P.
- Two DEGs in the J dataset were involved in ABC transporters, as was one DEG in the M dataset.
- The table (d) summarises the up- and down-regulated genes in the P.
- No Tyrosinase-related genes were present in the PS DEGs dataset.
- In J samples, up-regulation of DEGs in the albino phenotype was observed for one Tyr-1 (log2FC = 1.62) and one Tyr-2 (log2FC = 1.76) homolog.
- In M tissue, up-regulation of DEGs in the albino phenotype was observed for one Tyr-1 (log2FC = 3.32) and one Tyr-3 (log2FC = 2.00) homolog, whereas down-regulated DEGs included four Tyr-1 (log2FC ranging from − 3.48 to − 2.26) homologs, none of which corresponded to those DEGs found in the M dataset (Additional file 1)..
- Moreover, in the M dataset, one P protein-related gene was down-regulated in the albino phenotype (log2FC.
- Two calmodulin-like proteins were up-regulated in al- bino phenotype in the M dataset (log2FC = 3.22 and 2.49).
- One was found down-regulated in the J dataset (log2FC.
- One Notch-related gene was found up-regulated in the albino phenotype in the J dataset (log2FC = 3.06) and one Frizzled-related gene was down-regulated in the M dataset (log2FC.
- One abcb gene was found up-regulated in the albino phenotype in the M dataset (log2FC.
- 1.66) and one down-regulated in the J dataset (log2FC = 2.15).
- Two abcc genes were found down-regulated in the M dataset (log2FC.
- 3.01) and one up- regulated in the J dataset (log2FC.
- The number of total DEGs in the J, M and PS datasets are represented on the left (J total, M total, PS total) as are the number of up-regulated (J up, M up, PS up) and down- regulated genes (J down, M down, PS down) in the albino phenotype, as separate gene sets.
- The sizes of the intersections between these different sets are represented on the top barplot.
- One Pif gene was down-regulated in the M tissue (log2FC.
- Population genetics analysis showed a strong family ef- fect in the J and M datasets with maximal group assign- ment probability (Additional file 4: Fig.
- Only one common transcript was annotated (probable RNA- directed DNA polymerase from transposon BS reverse transcriptase) in the reference transcriptome..
- In this study, we used RNA-seq to explore the molecular bases of the albino phenotype and to identify genes and molecular pathways associated with colour mutation across culture stages, from juvenile to adult, and tissue types (whole juvenile, mantle graft and pearl sac).
- The roles of the Notch signalling system have been well described in de- velopment [17] and mammalian hair pigmentation, where it is involved in the maintenance of melanoblasts [28].
- In the clam M.
- meretrix, it was suggested that the Notch signalling pathway plays an important role in shell pig- mentation following a gene dosage-dependent pattern and was also potentially involved in the shell colour.
- The Notch signalling pathway has also been reported to play a role in the pigmentation process in Pacific oyster C.
- Tyrosinase-1 in the mantle (a) and Notch homolog protein 1 in juvenile (b).
- Values in the box plot represent p-values (Fisher ’ s test) for each gene section.
- Finally, in the sea cucumber Apostichopus japonicus, the Notch signalling pathway might be an upstream component of the pigmentation process by determining the location and boundaries of pigment occurrence [18].
- These results strongly support the early role of Notch signalling regulation, which most likely re- sults in the inhibition of shell pigmentation.
- We further show differen- tial exon usage of the same Notch1 gene, but no related SNPs in coding regions.
- Putative splice variants of Notch were also identified in the butterfly Vanessa cardui and suggested to be part of a conserved Notch signalling cas- sette.
- Admittedly, further studies will be needed to assess the precise role of alter- native splicing in the Notch signalling pathway and any subsequent impact on shell pigmentation in P.
- Here, the inclusion of juvenile specimens in our design allowed us to see the specific impact of the Notch signalling pathway in earlier shell formation and pigmentation..
- Tyrosinases are, for example, essential in the melanogenesis pathway as key regulators of melanin bio- synthesis [33].
- Tyrosinase deregulation has been re- ported in the bivalves C.
- In bivalve molluscs, mantle tis- sue secretes proteins responsible for shell calcification and pigmentation, and pigments are probably formed in the secretory cells in the mantle and incorporated into.
- margar- itifera occurs predominantly in the mantle tissue [32].
- A complex evolutionary history has been described for tyrosinase genes in bivalves, in which tyrosinase duplicates may have been retained because of their functional diversification in the mantle [32].
- We also observed down- regulation of the P protein-encoding gene, a key gene in- volved in melanogenesis, in the albino phenotype mantle of P.
- P protein, known as the pink-eyed dilution protein encoded by the OCA2 gene, could be in- volved in the transport of tyrosine, the precursor to mel- anin synthesis, within human melanocytes.
- Moreover, frizzled-3 was also found to be down-regulated in the mantle of the P.
- Frizzled is an essential receptor associ- ated with the Wnt signalling pathway, which is involved in the maintenance of melanocyte and keratinocyte homeostasis [18].
- We also observed deregulation of members of the ATP-binding cassette (ABC) transporter family, which is a large trans- porter family related to cellular detoxification, observed in all kingdoms of life [13, 38].
- Moreover, in human, cal- cium signalling plays a role in the co-regulation of retinal pigment epithelial cells [40].
- In the present study, we showed that the calcium signalling pathway, espe- cially Calmodulin gene expression, is associated with the albino phenotype in adult and juvenile P.
- An understanding of the molecular and genetic basis of the P.
- Results of the transcriptome analysis in three different datasets: juvenile whole flesh (J), adult mantle (M) and pearl sac (PS) revealed quite specific transcrip- tomic signatures with some related functions.
- The study also showed the putative association of significantly different exon usage and SNPs in the albino phenotype.
- margaritifera, the involvement of the Notch signalling pathway in pigmentation was highlighted.
- Notch1 was specifically deregulated and showed alternative splicing in the J dataset, suggesting an early role of this gene in shell formation and pigmen- tation.
- In the J and M datasets, we investigated sites that were variable between the white albino and black wild-type populations.
- We did not include the PS dataset in this part of the study because of possible contamination with re- cipient RNA during pearl sac sampling.
- To detect differential splicing events in the three RNA- seq datasets, the filtered reads were mapped on the draft genome of P.
- Lists of enriched GO terms in the P.
- margaritifera albino phenotype compared with the black wild-type phenotype in the three datasets (Juvenile, Mantle and Pearl Sac)..
- The role of the funders consisted on a financial support for all experiment realisations..
- The full name of the data is.
- The full names of the data are: 1) SUBID: SUB .
- The study was approved by the ethics committee of the French institute of research and exploitation of the sea (Ifremer).
- Color and weight of pearls produced by grafting the mantle tissue from a selected population for white shell color of the Japanese pearl oyster Pinctada fucata martensii (dunker).
- Targeted mutagenesis in Atlantic salmon (Salmo salar L.) using the CRISPR/Cas9 system induces complete knockout individuals in the F0 generation.
- Comparative Transcriptome analysis of the Pacific oyster Crassostrea gigas characterized by Shell colors:.
- Identification of genes associated with shell color in the black-lipped pearl oyster, Pinctada margaritifera BMC Genomics .
- Transcriptome analysis of shell color-related genes in the clam Meretrix meretrix.
- A detailed description of pearl-sac development in the black-lip pearl oyster, Pinctada margaritifera (Linnaeus 1758).
- The Mendelian inheritance of rare flesh and shell colour variants in the black-lipped pearl oyster (Pinctada margaritifera).
- Whole transcriptome sequencing and biomineralization gene architecture associated with cultured pearl quality traits in the pearl oyster, Pinctada margaritifera.
- Cultured pearl surface quality profiling by the Shell matrix protein gene expression in the Biomineralised pearl sac tissue of Pinctada margaritifera.
- Transcriptome analysis of the painted lady butterfly, Vanessa cardui during wing color pattern development.
- Evolution of the tyrosinase gene family in bivalve molluscs: independent expansion of the mantle gene repertoire.
- Characterization of the mantle transcriptome of yesso scallop (Patinopecten yessoensis): identification of genes potentially involved in biomineralization and pigmentation.
- genepop ’ 007: a complete re-implementation of the genepop software for windows and Linux

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