Figure 2

Positional cloning of the cot locus.

(A) Physical map showing the outcome of the linkage analysis using 1211 BC₁ individuals. The cot locus was narrowed to the genomic region flanked by the SNP markers chr15_Bm_scaf3_1271214 and chr15_Bm_scaf3_1002769, as indicated by the red arrows. Putative genes predicted with SilkDB are shown below the map and Bmsei (BGIBMGA007794) is shown with the orange arrow. (B) Three isoforms were detected in the head of each strain with genotype cot/cot, cot/+ and +/+ on day 5 of fifth instar with RT–PCR using Bmsei primers. Bmsei-p7 was based on the predicted sequence of Bmsei. Bmsei-q9, which was not detected in cot, was based on the mRNA sequence of WT. Bmsei-q8, which was not detected in WT, was based on the sequence of isoform II. (C) Relative expression of normal mRNA of Bmsei was verified with qPCR in the head of three genotypes (cot/cot, cot/+ and +/+) on day 5 of fifth instar. (D) The 15-bp Bmsei deletion in the fifth intron splicing region and the abnormal Bmsei transcripts in the cot mutant were detected by sequencing the genome and coding sequence (CDS). Isoform I lacks the entire fifth exon and causes a premature stop codon at nucleotide 733 of the CDS and encodes an aberrant protein lacking the fourth, fifth and sixth transmembrane domains, the pore (P) region and the cyclic nucleotide-binding domain (cNBD). Isoform II has a 36-bp deletion at the 3′ end of the fifth exon, creating a 12-amino-acid deletion in the fifth transmembrane domain of the protein. Isoform III is not spliced in the fifth intron, resulting in a 54-bp insertion in the fifth intron at the 3′ end of the fifth exon, producing a stop codon at nucleotide 853 of the CDS. Purple arrow, green arrow and blue arrow indicate the position of three primers (Bmsei-p7, Bmsei-q9 and Bmsei-q8), respectively. Black arrows in isoforms I and III denote premature stop codons. (E) Predicted structures and domains of Bmsei protein and isoforms.