Transcriptome Analysis of Maternal Gene Transcripts in Unfertilized Eggs of Misgurnus anguillicaudatus and Identification of Immune-Related Maternal Genes

Abstract

Maternal genes are important in directing early development and determining egg quality in fish. We here report the de novo transcriptome from four tissue libraries of the cyprinid loach, Misgurnus anguillicaudatus, and for the first time identified maternal gene transcripts in unfertilized eggs and suggest their immune system involvement. Expression profiles and functional enrichment revealed a total 24,116 transcripts were expressed as maternal transcripts in unfertilized eggs, which were involved in a wide range of biological functions and pathways. Comparison expression profiles and analysis of tissue specificity revealed that the large numbers of maternal transcripts were stored in unfertilized eggs near the late phase of ovarian maturation and before ovulation. Functional classification showed a total of 279 maternal immune-related transcripts classified with immune system process GO term and immune system KEGG pathway. qPCR analysis showed that transcript levels of identified maternal immune-related candidate genes were dynamically modulated during development and early ontogeny of M. anguillicaudatus. Taken together, this study could not only provide knowledge on the protective roles of maternal immune-related genes during early life stage of M. anguillicaudatus but could also be a valuable transcriptomic/genomic resource for further analysis of maternally provisioned genes in M. anguillicaudatus and other related teleost fishes.

Keywords: Misgurnus anguillicaudatus; de novo transcriptome; immune-related gene; maternal gene transcript; unfertilized egg.

Figure 1

Functional annotation of transcripts from the de novo assembled M. anguillicaudatus transcriptome: (a) Venn diagram of all annotated transcripts from the reference transcriptome against non-redundant (NR), Swiss-Prot, Pfam, non-supervised orthologous groups (eggNOG), gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; (b) E-value distribution; (c) similarity distribution; and (d) species distribution.

Figure 2

GO classification for the reference transcriptome and enrichment analysis of maternal gene transcripts using the Pearson Chi-Square test. Horizontal axis displays the percentage of significant transcripts in each column of which the number at the end indicates the number of transcripts. Vertical axis displays the detailed GO term corresponding to each functional type. Star marks in front of terms indicate significantly enriched GO terms (p < 0.05).

Figure 3

KEGG pathway annotation for the reference transcriptome and enrichment analysis of maternal gene transcripts using the Pearson Chi-Square test. Star marks in front of pathways indicate significantly enriched KEGG pathway (p < 0.05).

Figure 4

Expression analysis of transcripts in different tissues of M. anguillicaudatus and characterization of maternal transcripts: (a) overlapping comparison of expressed transcripts with a cut-off trimmed mean normalization of M-values (TMM) value < 1 in each tissue; (b) the number of transcripts with different expression abundances in each tissue; (c) heatmap of maternal transcripts with substantial expression differences across tissues; and (d) the number and abundance of transcripts with tissue-specificity by the TissueEnrich tool, which were coexpressed in more than two tissues. “Maternal” in the horizontal axis represented transcripts that were coexpressed in the unfertilized eggs with other tissues; (e) GO terms (p < 0.05) that contained significantly different numbers of maternal transcript with tissue-specificity.

Figure 5

Identification of maternal immune-related gene transcripts based on GO and KEGG pathway annotations: (a) Distribution of maternal gene transcripts in the immune system process (GO:0002367) at GO level 3 and (b) distribution of maternal gene transcripts in the immune system pathway subcategories.

Figure 6

Transcriptional levels of identified maternal immune-related candidate genes during ontogeny of M. anguillicaudatus: (a) the ontogeny of M. anguillicaudatus, time for each developmental stage was represented by HPF (hours post-fertilization) and DPH (days post-hatching) and transcriptional levels of (b) RAC1; (c) MAPK3; (d) MAPK14; (e) IL1B; (f) CTNB1; (g) FADD; and (h) ITB1. The results analyzed in statistics are shown in Table S6.