Computational protocol: Population Genetics of Duplicated Alternatively Spliced Exons of the Dscam Gene in Daphnia and Drosophila

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Protocol publication

[…] In Da. magna the entire Dscam protein, depending on exon usage, is composed of approximately 1960 amino acids and the whole locus is 31 Kb long . For the present study, we analyzed three regions of the Dscam gene: two regions containing alternatively spliced, duplicated exons belonging to arrays 4 and arrays 6 (and, for comparison, one region containing the constitutive exon 10, which was chosen because it codes for Ig6, which is structurally similar to the Igs 2 and 3, coded for by arrays 4 and 6 (data not shown).In Da. magna, array 4 consists of eight paralogous exons, (named 4.1 to 4.8, covering around 3390 bp in total) and array 6 contains 24 paralogous exons (6.1 to 6.24, around 6100 bp in total). We obtained sequence data on all exons of array 4, except exon 4.5 (3200 bp in total, accession numbers JN977549 to JN977579)), exons 6.5 to 6.7 and 6.10 to 6.14 (1683 bp in total, accession numbers JQ037914 to JQ037973), and 327 bp of the constitutive exon 10 (the total length of which is 423 bp, accession numbers JQ037974 to JQ037993). Part of the intron sequences (mostly from array 4) had to be excluded from the analysis due to alignment ambiguities, repetitive sequences, and insertion/deletion polymorphisms. Thus, only 1759 bp of array 4 sequences and 1679 bp of array 6 sequences were retained for analysis (). All exons sampled are known to be expressed . The same sequence data was also obtained for one genotype of Da. lumholtzi. We were unable to obtain array 6 sequence from Da. similis, thus we restrict the analysis of between-species divergence mostly to divergence between Da. magna and Da. lumholtzi which is the closest known species to Da. magnaInsects have three other Dscam paralogs that have been named Dscam-like (Dscam-L) , , and we have found orthologues of these Dscam-L genes in the genome of Daphnia pulex (unpublished data). The distinction between the variable Dscam and the Dscam-L genes is very clear and we are confident that we have amplified only the variable Dscam in Daphnia.The Dscam sequence data from Dr. melanogaster comprises almost the entire Dscam coding region (22795 bp). For the interspecific comparisons of the six Drosophila species from the melanogaster group, we used all orthologous exons of arrays 4 (12 exons, 1950 bp in total). For array 6, 43 orthologous exons were used, 32 occurring in all six species and eleven in five of them (5205 bp in total). Exons that confidently (>60% of 100 bootstrap replicates) shared a common ancestor in a maximum likelihood tree were considered orthologous . Trees were built with RAxML trough the Cipres Portal . […]

Pipeline specifications

Software tools RAxML, CIPRES Science Gateway
Application Phylogenetics
Organisms Drosophila melanogaster, Daphnia magna