Protein-coding genes in B chromosomes of the grasshopper Eyprepocnemis plorans

Abstract

For many years, parasitic B chromosomes have been considered genetically inert elements. Here we show the presence of ten protein-coding genes in the B chromosome of the grasshopper Eyprepocnemis plorans. Four of these genes (CIP2A, GTPB6, KIF20A, and MTG1) were complete in the B chromosome whereas the six remaining (CKAP2, CAP-G, HYI, MYCB2, SLIT and TOP2A) were truncated. Five of these genes (CIP2A, CKAP2, CAP-G, KIF20A, and MYCB2) were significantly up-regulated in B-carrying individuals, as expected if they were actively transcribed from the B chromosome. This conclusion is supported by three truncated genes (CKAP2, CAP-G and MYCB2) which showed up-regulation only in the regions being present in the B chromosome. Our results indicate that B chromosomes are not so silenced as was hitherto believed. Interestingly, the five active genes in the B chromosome code for functions related with cell division, which is the main arena where B chromosome destiny is played. This suggests that B chromosome evolutionary success can lie on its gene content.

Figure 1. Identification of protein-coding genes located in B chromosomes of the grasshopper E. plorans, using the number of mapped reads that map to the CDSs found in the de novo assembled transcriptome, in the 0B (X axis) and 4B (Y axis) libraries of genomic DNA.

Red triangles represent those sequences that code for known proteins, blue diamonds represent those sequences that code for transposable elements and grey circles represent non-annotated CDSs. The CDSs corresponding to the 10 genes found in the B chromosome are labelled.

Figure 2

Coverage for the CIP2A transcript in the gDNA (0B and 4B) and RNA (0B and 1B) Illumina reads (A), and qPCR on gDNA (B) and cDNA (C). Note that coverage was higher in the 4B library across the complete sequence length, including the full CDS (delimited by the dotted vertical lines), the 5′ UTR (from the 5′ end to the first dotted line) and 3′ UTR (from the second dotted line to the 3′ end). Likewise, note the higher coverage for this transcript in the B-carrying RNA library. The shaded zone in A marks the region amplified by qPCR. qPCR on gDNA (B) revealed that genomic copy number for the CIP2A gene increases with B chromosome number, following a dose-dependent pattern, thus supporting its presence in the B chromosome. qPCR on cDNA (C) revealed that CIP2A is expressed in all tissues and sexes analyzed, also following a dose-depending pattern and suggesting the active transcription of B chromosome gene copies. RQ = Relative quantity. NREQ = Normalized relative expression quantity. P = P-value and Pb = Sequential Bonferroni P-value for Spearman rank correlation (B) and Kruskal-Wallis (C) analyses.

Figure 3

Coverage for the MYCB2 transcript in the gDNA (0B and 4B) and RNA (0B and 1B) Illumina reads (A), and qPCR on gDNA (B) and cDNA (C). Note that less than half of MYCB2 CDS (between the dotted vertical lines) showed high coverage in the 4B library, specifically from position 8961 to the 3′ end (A), suggesting that this B chromosome gene is truncated. Two regions were selected for qPCR amplification of this gene, one within the region being apparently absent in the B chromosome (shaded zone 1) and the other within the region being present in it (shaded zone 2). qPCR on gDNA with zone 1 primers showed that copy number for this gene region was independent on the number of B chromosomes (B1). However, qPCR on gDNA with zone 2 primers showed that abundance of this MYCB2 gene region increased with B chromosome number in a dose-dependent pattern (B2). Likewise, qPCR on cDNA showed that MYCB2 expression was independent of B chromosome number when probed with zone 1 primers (C1) but it increased in a dosage-dependent pattern with zone 2 primers (C2), suggesting the active transcription of B chromosome truncated gene copies. RQ = Relative quantity. NREQ = Normalized relative expression quantity. P = P-value and Pb = Sequential Bonferroni P-value for Spearman rank correlation (B) and Kruskal-Wallis (C) analyses.