A) Graphical Illustration of BLAST results in the Eimeria nieschulzi genome data. Contig Enie_470 encodes the complete gam82 ortholog, whereas contigs Enie_5532, Enie_5533, and Enie_23299 encode only 5' parts and contig Enie_10086 encodes a 3' area of gam56. Dashed areas of the boxes indicate the missed sequences of the corresponding genes. Sequences were completed, ordered and manually assembled, resulting in the map shown in B2).

B1-2) Mapping of contigs for final assembly. Based on the initial BLAST analysis (A) PCR experiments (B1) with potential primer pairs were performed on genomic DNA (1*-3*). 1*: PCR product (lane 2) at ca. 2,100 bp (primer #3/#7) show that contig Enie_5533 is adjacent to contig Enie­_16402. 2*: PCR products at 1,800 bp (primer #4/#5) and 2,500 bp (primer #4/#6) show vicinity of contig Enie_5532 and contig Enie_16403. 3*: PCR products at ∼8 kb (primer #8/#10 (lane 2), primer #8/#9 (lane 3) respectively, show interconnection between contig Enie_5532 and contig Enie_470 (B2). PCR products were verified by sequencing. Several other low-coverage contigs (Enie_23088 cov. 10.19; Enie_18448 cov. 13.62; Enie_17906 cov. 15.53; Enie_9794 cov. 15.48) map to different areas of the gam locus, predominantly outside of open reading frame-containing regions (B2). DNA size standard: 1 kb ladder.

C) The expression of gam genes in gametocytes was confirmed by amplification from gametocyte cDNA with gene-specific primers (lane 2: reaction control (ribosomal subunit); lane 3: gam56_1; lane 4: gam56_2; lane 5: gam82; size standard lane 1: 1 kb ladder). Gam transcripts were absent in sporozoite cDNA (control).