Comprehensive analysis of microRNAs in the mantle central and mantle edge provide insights into shell formation in pearl oyster Pinctada fucata martensii.

MicroRNAs (miRNAs) are a class of non-coding RNA molecules with post-transcriptional regulatory activity in various biological processes. Pearl oyster Pinctada fucata martensii is one of the main species cultured for marine pearl production in China and Japan. In this study, we constructed two small RNA libraries of mantle central (MC) and mantle edge (ME) from P. f. martensii and obtained 24,175,537 and 21,593,898 clean reads, respectively. A total of 258 miRNAs of P. f. martensii (Pm-miRNA) were identified, and 93 differentially expressed miRNAs (DEMs) including 49 known Pm-miRNAs and 44 novel Pm-miRNAs were obtained from the MC and ME. The target transcripts of these DEMs were obviously enriched in neuroactive ligand-receptor interaction pathway, and others. After over-expression of Pm-miR-124 and Pm-miR-9a-5p in the MC by mimic injection into the muscle of P. f. martensii, nacre exhibited a disorderly growth as detected by scanning electron microscopy. Pm-nicotinic acetylcholine receptor alpha subunit, Pm-neuropeptide Y and Pm-chitin synthase were investigated as the targets of Pm-miR-124; and Pm-tumor necrosis factor receptor associated factor 2 and Pm-chitin synthase were investigated as the targets of Pm-miR-9a-5p. These predicted target transcripts were down-regulated after the over-expression of Pm-miR-124 and Pm-miR-9a-5p in MC. This study comprehensively analyzed the miRNAs in mantle tissues to enhance our understanding of the regulatory mechanism underlying shell formation.

Computational prediction of candidate miRNAs and their potential functions in biomineralization in pearl oyster Pinctada martensii.

MicroRNAs (miRNAs) are a class of non-coding RNA molecules with presumed post-transcriptional regulatory activity in various biological processes, such as development and biomineralization. Pinctada martensii is one of the main species cultured for marine pearl production in China and Japan. In our previous research, 258 pm-miRNAs had been identified by solexa deep sequencing in P. martensii, while it is far from the number of miRNAs found in other species. In this study, based on the transcriptome database of pearl sac, we identified 30 candidate pm-miRNAs by computational prediction. Among the obtained 30 pm-miRNAs, 13 pm-miRNAs were generated from the complementary strand of protein-coding mRNAs, and 17 pm-miRNAs could not be annotated using blastx and tblastn analysis. Notably, 10 of the 30 pm-miRNAs, such as pm-miR-1b, pm-miR-205b and pm-miR-375b, were homologous with the reported pm-miRNAs, respectively. To validate the existence of the identified pm-miRNAs, eight randomly selected pm-miRNAs were tested by stem loop quantitative RT-PCR analyses using 5.8S as the internal reference gene. Target prediction between the obtained pm-miRNAs and biomineralization-related genes by microTar, miRanda and RNA22 indicated pm-miR-2386 and pm-miR-13b may be the key factors in the regulation network by regulating the formation of organic matrix or the differentiation of mineralogenic cell during shell formation. Thus, this study enriched miRNA databases of pearl oyster and provided a new way to understand biomineralization.

De novo assembly, gene annotation, and simple sequence repeat marker development using Illumina paired-end transcriptome sequences in the pearl oyster Pinctada maxima.

We analyzed the mantle transcriptome of pearl oyster Pinctada maxima and developed EST-SSR markers using Illumina HiSeq 2000 paired-end sequencing technology. A total of 49,500,748 raw reads were generated. De novo assembly generated 108,704 unigenes with an average length of 407 bp. Sequence similarity search with known proteins or nucleotides revealed that 30,200 (27.78%) and 25,824 (23.76%) consensus sequences were homologous with the sequences in the non-redundant protein and Swiss-Prot databases, respectively, and that 19,701 (18.12%) of these unigenes were possibly involved in approximately 234 known signaling pathways in the Kyoto Encyclopedia of Genes and Genomes database. Ninety one biomineralization-related unigenes were detected. In a cultured stock, 1764 simple sequence repeats were identified and 56 primer pairs were randomly selected and tested. The rate of successful amplification was 68.3%. The developed molecular markers are helpful for further studies on genetic linkage analysis, gene localization, and quantitative trait loci mapping.