Chromosome Segregation

Genomic DNA was extracted with various standard procedures, and specimens were identified to species and molecular forms by PCR-RFLP [38 (link),39 (link)]. SINE200 elements were located in silico by BLASTN searches on the genome sequence of the A. gambiae PEST genome using the obtained SINE200 consensus sequence as a query. Thirteen SINE200 insertions lying within the A. gambiae molecular form speciation islands (sensu Turner [11 (link)]) on X, 2L and 2R chromosomes, and characterized by the presence of 500 bp flanking regions showing a single hit in the genome, were selected. Primers were designed to amplify across the element using Primer 3 software [40 (link)]. The selected loci were named 'S200' followed by the abbreviation of the chromosomal arm (2L, 2R, X), by a number/letter corresponding to the chromosomal location on the cytogenetic map [4 (link)] and by an additional number aimed to distinguish primer sets positioned on the same chromosome division. Genes annotated within a 20 Kb genome sequence including SINE200 insertions for each locus were retrieved from the PEST genome ver. Agam P3 Feb. 2006 (Table 2).
PCR reactions were carried out in a 25 μl reaction which contained 1 pmol of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2, 2.5 U Taq polymerase, and 0.5 μl of template DNA extracted from a single mosquito. Thermocycler conditions were 94°C for 10 min followed by thirty-five cycles of 94°C for 30 s, 54°C for 30 s and 72°C for 1 min., with a final elongation at 72°C for 10 min, and a 4°C hold. The resulting products were analysed on 1.5% agarose gels stained with ethidium bromide, with low and high molecular weight bands corresponding to fragments containing or lacking the targeted SINE200, respectively.
PCR products representing 'filled' and 'empty' sites of S200 X6.1 locus on X chromosome were sequenced on both strands using ABI Big Dye Terminator v.2 chemistry and an ABI Prism 3700 DNA Analyser. Multiple alignments were performed using ClustalX [37 (link)]. All sequences were deposited in GenBank under accession numbers EU881868–EU881887.
Indices of polymorphism (i.e. SINE200 insertion frequency and heterozygosity) and differentiation (Fst) at polymorphic loci were computed using Fstat 2.9.3.2 [41 ]. Significance was tested with Bonferroni-adjusted P-values, using the randomization approach implemented in Fstat.

To produce the distributions used to generate synthetic mutations, we identified a set of sites heterozygous for natural polymorphisms, regardless of the genotypes called from the sequencing data, taking advantage of the lack of recombination in Heliconius females. F₂ offspring receive whole chromosomes from the F₁ mother, so SNPs from the same chromosome have identical segregation patterns in the offspring, and segregation patterns for each of the 21 H. melpomene chromosomes for this cross are known (Heliconius Genome Consortium 2012 (link)). We identified SNPs from each chromosome by compiling sites called as heterozygous in the F1 mother, homozygous in the F1 father, and matching one of the chromosome segregation patterns for the bait offspring. Heterozygous focal offspring could then be identified based on segregation pattern, without reference to their sequenced genotype. We used these heterozygous focal offspring to tabulate frequency distributions of numbers of nonreference base calls for read depths 1, … 100 (see fig. 1).

The ABC superfamily of transporters is an ancient, diverse and ubiquitous group of transporters, present throughout all kingdoms of life [36 (link)]. This superfamily, which performs both uptake and efflux duties, comprises a large number of families and subfamilies. These are defined based primarily on the significant evolutionary and structural variation in the membrane component, as these are highly conserved, comprising a number of motifs and regions that are well characterized [37–39 (link)].The Pfam family ABC_tran PF00005 (https://www.ebi.ac.uk/interpro/entry/pfam/PF00005/) identifies the highly conserved ATP-binding cassette, which binds and hydrolyses ATP, thereby coupling transport to ATP hydrolysis in a large number of biological processes. Besides transporter ATP-binding cassettes, PF00005 also identifies a group of ATPases, e.g. excinuclease ATPase (UvrA), chromosome segregation ATPase (Smc), recombinational DNA repair ATPase (RecF) and ATPases involved in chromosome partitioning (Soj, Mrp), as well as a group of CBS-domain-containing transcriptional regulators and signal transduction proteins. To remove these false positives, we set up a series of negative rules whereby all putative ABC transporters, based on an inclusive and promiscuous search with PF00005, that also have positive hits to the COGs representing these non-transporter ATPases and CBS-domain-containing proteins (shown in Table 1) are deleted. Furthermore, a text search of the annotation of blastp matches to the NR database is also conducted, and where the top hits are found to contain keywords shown in Table 1, these entries are also removed. These rules assist in the specific identification of ATP-binding cassette transporters.
The submission database is polled every 24 h and new submissions are automatically processed via the TransAAP pipeline on a dedicated Linux cluster, running the relevant searches and predicting the complete transporter contents, then uploading all the results into the TransportDB MySQL database. Users can check the status of the annotation of their submitted genomes and once complete can access a list of predicted transporters, or individual transporter annotation pages. Users can view annotation, supporting evidence, and curate the annotation on each individual transporter annotation page, or download the result as a tab delimited text file (Fig. 1).