TITLE: Creation and maintenance of Ashworth outcrossed DGRP population
AUTHORS: Fergal Waldron, katy monteith, Pedro Vale

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Infomration about the creation and maintenance of Ashworth outcrossed DGRP population</span></div></div>

[STEPS]
?. Information on creation and maintenance of Ashworth outcrossed DGRP populationFergal M Waldron, Katy M Monteith, Pedro F ValeThe Ashworth outcrossed DGRP population was founded by FMW on 15/10/14, and is derived from 113 inbred DGRP lines sampled from a wild population in Raleigh, NC, USA 1. The DGRP outcrossed population was founded with genetic contributions from the following 113 DGRP lines; RAL-28, RAL-31, RAL-48, RAL-49, RAL-57, RAL-59, RAL-69, RAL-75, RAL-83, RAL-91, RAL-93, RAL-101, RAL-129, RAL-138, RAL-149, RAL-153, RAL-158, RAL-189, RAL-195, RAL-208, RAL-217, RAL-228, RAL-237, RAL-239, RAL-280, RAL-287, RAL-288, RAL-301, RAL-303, RAL-304, RAL-306, RAL-309, RAL-310, RAL-317, RAL-321, RAL-324, RAL-348, RAL-350, RAL-352, RAL-354, RAL-358, RAL-360, RAL-361, RAL-365, RAL-366, RAL-373, RAL-375, RAL-377, RAL-379, RAL-380, RAL-381, RAL-382, RAL-386, RAL-390, RAL-392, RAL-395, RAL-397, RAL-399, RAL-405, RAL-406, RAL-409, RAL-426, RAL-427, RAL-437, RAL-439, RAL-443, RAL-486, RAL-491, RAL-492, RAL-502, RAL-508, RAL-509, RAL-517, RAL-528, RAL-530, RAL-535, RAL-555, RAL-559, RAL-563, RAL-566, RAL-575, RAL-584, RAL-589, RAL-627, RAL-630, RAL-634, RAL-703, RAL-712, RAL-716, RAL-732, RAL-765, RAL-774, RAL-776, RAL-786, RAL-796, RAL-805, RAL-808, RAL-818, RAL-820, RAL-821, RAL-822, RAL-832, RAL-852, RAL-853, RAL-855, RAL-859, RAL-879, RAL-882, RAL-884, RAL-897, RAL-907, RAL-908, RAL-913.To maximise the genetic contribution of each of the founder lines to the final outcrossed population, pairwise crosses between population founder lines were carried out to produce offspring which could then be pooled into a population cage for the 1stgeneration of outcrossing. Whilst a minimum of 57 pairwise crosses would encompass inclusion of all 113 founder lines, 100 pairwise crosses were carried out as a precautionary measure against a number of crosses failing to produce offspring (an upper limit of 100 was dictated by feasibility). Pairwise crosses were set up using 2 virgin females crossed to 2 males. All virgin females and males were age-matched controlled (1-6 days old when crosses were set up). Pairwise crosses were set up in standard Lewis medium containing vials and placed at 25°C for 5 days after which adults were removed.  For pairwise crosses, DGRP outcrossed population founder lines were randomly selected to contribute females or males for the following 100 crosses (scheme is “2 virgin females from line” x “2 males from line”): RAL-390 x RAL-381, RAL-280 x RAL-301, RAL-913 x RAL-365, RAL-796 x RAL-712, RAL-589 x RAL-49, RAL-350 x RAL-382, RAL-853 x RAL-158, RAL-288 x RAL-855, RAL-49 x RAL-366, RAL-303 x RAL-908, RAL-101 x RAL-303, RAL-712 x RAL-426, RAL-321 x RAL-732, RAL-377 x RAL-101, RAL-380 x RAL-879, RAL-820 x RAL-324, RAL-882 x RAL-535, RAL-439 x RAL-634, RAL-83 x RAL-409, RAL-28 x RAL-75, RAL-409 x RAL-832, RAL-879 x RAL-237, RAL-237 x RAL-239, RAL-443 x RAL-776, RAL-908 x RAL-627, RAL-59 x RAL-584, RAL-365 x RAL-796, RAL-634 x RAL-405, RAL-392 x RAL-852, RAL-129 x RAL-350, RAL-317 x RAL-306, RAL-427 x RAL-528, RAL-373 x RAL-502, RAL-386 x RAL-28, RAL-304 x RAL-392, RAL-774 x RAL-555, RAL-306 x RAL-386, RAL-310 x RAL-309, RAL-832 x RAL-287, RAL-405 x RAL-280, RAL-57 x RAL-774, RAL-627 x RAL-228, RAL-397 x RAL-821, RAL-348 x RAL-492, RAL-437 x RAL-443, RAL-91 x RAL-31, RAL-352 x RAL-575, RAL-301 x RAL-390, RAL-48 x RAL-897, RAL-575 x RAL-808, RAL-426 x RAL-373, RAL-375 x RAL-195, RAL-31 x RAL-59, RAL-897 x RAL-310, RAL-239 x RAL-486, RAL-287 x RAL-805, RAL-584 x RAL-765, RAL-381 x RAL-149, RAL-93 x RAL-703, RAL-379 x RAL-517, RAL-821 x RAL-630, RAL-189 x RAL-853, RAL-399 x RAL-360, RAL-907 x RAL-217, RAL-535 x RAL-786, RAL-195 x RAL-395, RAL-852 x RAL-913, RAL-502 x RAL-818, RAL-361 x RAL-375, RAL-138 x RAL-491, RAL-808 x RAL-93, RAL-517 x RAL-208, RAL-153 x RAL-189, RAL-149 x RAL-352, RAL-732 x RAL-509, RAL-818 x RAL-563, RAL-630 x RAL-57, RAL-395 x RAL-380, RAL-358 x RAL-822, RAL-765 x RAL-406, RAL-703 x RAL-859, RAL-406 x RAL-153, RAL-508 x RAL-379, RAL-716 x RAL-427, RAL-509 x RAL-358, RAL-555 x RAL-48, RAL-360 x RAL-321, RAL-786 x RAL-69, RAL-855 x RAL-354, RAL-559 x RAL-437, RAL-563 x RAL-361, RAL-158 x RAL-559, RAL-805 x RAL-884, RAL-208 x RAL-566, RAL-492 x RAL-397, RAL-382 x RAL-399, RAL-75 x RAL-508, RAL-884 x RAL-138, RAL-530 x RAL-83, RAL-69 x RAL-348. Offspring from pairwise crosses were collected 28 days after parents were removed and pooled into a large Drosophila population cage, for the 1st generation of outcrossing and subsequent embryo collection. For this, and each subsequent generation of outcrossing, the outcrossed DGRP population was maintained employing a method used to maintain constant larval densities (223 ± 14.3 (95% CI)) in stock bottles 2. Briefly this method involves populating a large Drosophila cage with thousands of flies on the day 1, providing these with fruit juice (grape/apple) agar plates for embryo laying. After a 24 hr habituation period, agar plates are replaced (day 2). On the day 3, agar plates are recovered and embryos are collected from the surface. Using PBS and a brush, concentrated egg/PBS solutions are prepared, and these are squirted on the surface of Lewis media in bottles. This process is typically carried out every 20-25 days. The outcrossed DGRP populations is maintained at a density of 20-25 bottles (20 bottles maintains the population at >4000 individuals).Below is a table outlining details of the outcrossed DGRP population foundation and maintenance.  ABCDE1  Date    Action    Temp    No. Bottles    Worker  2  18/10/2014    Tipped DGRP onto new food, and placed at 18C for virgin collection in
  mid-Oct    18C    NA    FMW/WHP  3  07/10/2014    Tipped out adult DGRPs from vials for subsequent virgin collection    25C    NA    FMW  4  8/102014 - 14/10/2014    Virgins and males collected     25C    NA    FMW  5  15/10/2014    Pairwise crosses (n=100)set up (virgin male and female flies from 113
  lines)    25C    NA    FMW  6  20/10/2014    Pairwise crosses parents removed from vials    25C    NA    FMW  7  19/11/2014    Egg squirts from offspring of pairwise crosses: 1st generation of
  complete outcrossing    25C    30    FMW  8  18/12/2014    Tipped flies from old bottles into fresh bottles    25C    30    FMW  9  30/01/2015    Egg squirts: 2nd generation of complete outcrossing    25C    30    FMW  10  06/03/2015    Tipped flies from old bottles into fresh bottles    25C    30    FMW  11  09/04/2015    Egg squirts: 3rd generation of complete outcrossing    25C    25    FMW  12  19/05/2015    Egg squirts: 4th generation of complete outcrossing    25C    21    FMW  13  19/06/2015    Egg squirts: 5th generation of complete outcrossing    25C    25    FMW  14  30/07/2015    Egg squirts: 6th generation of complete outcrossing    25C    25    FMW  15  01/10/2015    Egg squirts: 7th generation of complete outcrossing    25C    25    FMW  16  29/10/2015    Egg squirts: 8th generation of complete outcrossing    25C    20    KMM  17  02/12/2015    Tipped flies from old bottles into fresh bottles    25C    20    KMM  18  06/01/2016    Egg squirts: 9th generation of complete outcrossing    25C    20    KMM  19  10/02/2016    Egg squirts: 10th generation of complete outcrossing    25C    20    KMM  20  22/02/2016    Tipped flies from old bottles into fresh bottles    25C    20    KMM  21  25/03/2016    Egg squirts: 11th generation of complete outcrossing    25C    24    KMM  22  26/04/2016    Egg squirts: 12th generation of complete outcrossing    25C    22    KMM  23  18/05/2016    Egg squirts: 13th generation of complete outcrossing    25C    20    KMM  24  14/06/2016    Egg squirts: 14th generation of complete outcrossing    25C    22    KMM  25  14/07/2016    Egg squirts: 15th generation of complete outcrossing    25C    22    KMM  26  10/08/2016    Egg squirts: 16th generation of complete outcrossing    25C    25    KMM  27  07/09/2016    Egg squirts: 17th generation of complete outcrossing    25C    25    KMM  28  04/10/2016    Egg squirts: 18th generation of complete outcrossing    25C    25    KMM  29  02/11/2016    Egg squirts: 19th generation of complete outcrossing    25C    25    KMM  30  30/11/2016    Egg squirts: 20th generation of complete outcrossing    25C    25    KMM  31  05/01/2017    Egg squirts: 21st generation of complete outcrossing    25C    25    KMM  32  09/02/2017    Egg squirts: 22nd generation of complete outcrossing    25C    25    KMM  33  01/03/2017    Egg squirts: 23rd generation of complete outcrossing    25C    25    KMM  34  05/04/2017    Egg squirts: 24th generation of complete outcrossing    25C    25    KMM  References1. Mackay, T. F. C. et al. The Drosophila melanogaster Genetic Reference Panel. Nature 482, 173–178 (2012).2. Clancy, D. J. & Kennington, J. W. A simple method to achieve consistent larval density in bottle cultures. Drosoph. Inf. Serv. 84, 168–169 (2001).
ABCDE1  Date    Action    Temp    No. Bottles    Worker  2  18/10/2014    Tipped DGRP onto new food, and placed at 18C for virgin collection in
  mid-Oct    18C    NA    FMW/WHP  3  07/10/2014    Tipped out adult DGRPs from vials for subsequent virgin collection    25C    NA    FMW  4  8/102014 - 14/10/2014    Virgins and males collected     25C    NA    FMW  5  15/10/2014    Pairwise crosses (n=100)set up (virgin male and female flies from 113
  lines)    25C    NA    FMW  6  20/10/2014    Pairwise crosses parents removed from vials    25C    NA    FMW  7  19/11/2014    Egg squirts from offspring of pairwise crosses: 1st generation of
  complete outcrossing    25C    30    FMW  8  18/12/2014    Tipped flies from old bottles into fresh bottles    25C    30    FMW  9  30/01/2015    Egg squirts: 2nd generation of complete outcrossing    25C    30    FMW  10  06/03/2015    Tipped flies from old bottles into fresh bottles    25C    30    FMW  11  09/04/2015    Egg squirts: 3rd generation of complete outcrossing    25C    25    FMW  12  19/05/2015    Egg squirts: 4th generation of complete outcrossing    25C    21    FMW  13  19/06/2015    Egg squirts: 5th generation of complete outcrossing    25C    25    FMW  14  30/07/2015    Egg squirts: 6th generation of complete outcrossing    25C    25    FMW  15  01/10/2015    Egg squirts: 7th generation of complete outcrossing    25C    25    FMW  16  29/10/2015    Egg squirts: 8th generation of complete outcrossing    25C    20    KMM  17  02/12/2015    Tipped flies from old bottles into fresh bottles    25C    20    KMM  18  06/01/2016    Egg squirts: 9th generation of complete outcrossing    25C    20    KMM  19  10/02/2016    Egg squirts: 10th generation of complete outcrossing    25C    20    KMM  20  22/02/2016    Tipped flies from old bottles into fresh bottles    25C    20    KMM  21  25/03/2016    Egg squirts: 11th generation of complete outcrossing    25C    24    KMM  22  26/04/2016    Egg squirts: 12th generation of complete outcrossing    25C    22    KMM  23  18/05/2016    Egg squirts: 13th generation of complete outcrossing    25C    20    KMM  24  14/06/2016    Egg squirts: 14th generation of complete outcrossing    25C    22    KMM  25  14/07/2016    Egg squirts: 15th generation of complete outcrossing    25C    22    KMM  26  10/08/2016    Egg squirts: 16th generation of complete outcrossing    25C    25    KMM  27  07/09/2016    Egg squirts: 17th generation of complete outcrossing    25C    25    KMM  28  04/10/2016    Egg squirts: 18th generation of complete outcrossing    25C    25    KMM  29  02/11/2016    Egg squirts: 19th generation of complete outcrossing    25C    25    KMM  30  30/11/2016    Egg squirts: 20th generation of complete outcrossing    25C    25    KMM  31  05/01/2017    Egg squirts: 21st generation of complete outcrossing    25C    25    KMM  32  09/02/2017    Egg squirts: 22nd generation of complete outcrossing    25C    25    KMM  33  01/03/2017    Egg squirts: 23rd generation of complete outcrossing    25C    25    KMM  34  05/04/2017    Egg squirts: 24th generation of complete outcrossing    25C    25    KMM