VIRTUALLY PAINLESS DROSOPHILA GENETICS STANDARDS A, B, C B, C C, C

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1 VIRTUALLY PAINLESS DROSOPHILA GENETICS STANDARDS 3.1.1A, 3.1.1B, 3.1.1C 3..1B, 3..1C C, 3.3.1C Wstminstr Collg INTRODUCTION Drosophila mlanogastr, th fruit fly, is an xcllnt organism for gntics studis bcaus it has simpl food rquirmnts, occupis littl spac, is hardy and complts its lif cycl in about 1 days at room tmpratur. Whil you will b using a computr program that simulats Drosophila gntic crosss, it is important to undrstand th lif cycl of this organism (Figur 1). Th Lif Cycl of Drosophila Th Eggs: Th ggs ar small, oval shapd and hav two filamnts at on nd. Thy ar usually laid on th surfac of th cultur mdium and, with practic, can b sn with th nakd y. Th ggs hatch into larva aftr about a day. Th Larval Stag: Th wormlik larva ats almost continuously, and its black mouth parts can asily b sn moving back and forth vn whn th larva itslf is lss distinct. Larva tunnl through th cultur mdium whil ating; thus, channls ar a good indication of th succssful growth of a cultur. Th larva shds its skin twic as it incrass in siz. In th last of th thr larval stags, th clls of th salivary glands contain giant chromosoms, which may b sn radily undr low-powr magnification aftr propr staining. Th Pupal Stag: Whn a matur larva in a lab cultur is about to bcom a pupa, it usually climbs up th sid of th cultur bottl or on to th strip providd in th cultur bottl. Th last larval covring thn bcoms hardr and darkr, forming th pupal cas. Through this cas th lat stags of mtamorphosis to an adult fly can b obsrvd. In particular, th ys, th wings and th lgs bcom radily visibl. Th Adult Stag: Whn mtamorphosis is complt, th adult flis mrg from th pupa cas. Thy ar fragil and light in color and thir wings ar not fully xpandd. Ths flis darkn in a fw hours and tak on th normal apparanc of an adult fly. Thy liv a month or mor and thn di. A fmal dos not mat for about tn to twlv hours aftr mrging from th pupa. Onc sh has matd, sh stors a considrabl quantity of sprm in rcptacls and frtilizs hr ggs as sh lays thm. To nsur a controlld mating, it is ncssary to us fmals that hav not matd bfor (virgins). Wstminstr Collg SIM Pag 1

2 It is important to raliz that a numbr of factors dtrmin th lngth of tim of ach stag in th lif cycl. Of ths factors, tmpratur is th most important. At room tmpratur (about 5 C), th complt cycl taks tn to twlv days. Figur 1. Drosophila Gntics Drosophila ar usful for studying gntics bcaus thy hav only 4 chromosoms, with wll studid traits that ar inhritd in a straightforward Mndlian fashion. Whil prforming this lab, it will first b ncssary to dtrmin whthr an alll, or vrsion of a gn, is dominant or rcssiv. If an alll is dominant, it will mask th phnotypic trait of othr allls of that gn. In gnral, if an alll is rcssiv, it will rquir that an offspring hav copis (on copy on ach chromosom) bfor th phnotypic trait is xprssd. It will also b important to dtrmin th mod of inhritanc. Gns that ar inhritd on th sx chromosoms ar calld sx-linkd; gns on th othr chromosoms ar considrd autosomal. Chi-squarAnalysis Th chi-squar tst is th statistical tst most frquntly usd to dtrmin whthr data obtaind xprimntally provid a good fit to th xpctd or prdictd data. To dtrmin if obsrvd data fall within accptabl limits, a chi-squar analysis is prformd to tst th validity of a null hypothsis. If thr is no statistically significant diffrnc btwn th obsrvd and xpctd data, th null hypothsis is considrd Wstminstr Collg SIM Pag

3 valid. If a chi-squar analysis indicats that th data vary too much from th xpctd, an altrnativ hypothsis is accptd. Th formula for chi-squar is: = ( o ) χ whr o = obsrvd numbr of individuals = xpctd numbr of individuals = th sum of th valus (in this cas, th diffrncs, squard, dividd by th numbr xpctd Th χ valu is thn compard to a Critical Valus Tabl (Tabl 1). Th two componnts of this tabl ar Dgrs of Frdom (df) and Probability (p). Dgrs of frdom rfr to th numbr of phnotypic classs minus 1. For xampl, in a monohybrid cross, you will s two diffrnt phnotyps, wild-typ and mutant. This would giv 1 df, or ( phnotyps 1). Th probability, or p valu, sts a minimum thrshold, or critical valu, for th statistical significanc of th rsults obtaind for an xprimnt. For gntic crosss, th p valu usd is usually If your χ valu for a givn df is blow th valu in th tabl, thn you ar 95% sur that th null hypothsis is valid. For xampl, if χ =.16 for 1 df, thn 3.84>.16, indicating that your obsrvd data is not statistically diffrnt than th xpctd data, and any variation from your prdiction is du to random chanc. Tabl 1. Critical Valus of th Chi Squar Distribution Probability Dgrs of Frdom (df) (p) In this lab you will us a computr program, DrosophiLab 1.0, to produc virtual gntic crosss of fruit flis. This program allows you to collct data from F1 and F gnrations, and analyz th phnotypic rsults from: A monohybrid cross: In ths xprimnts, th mod of inhritanc is dtrmind whn a singl contrasting pair of charactristics is involvd. You will b xpctd to dtrmin th mod of inhritanc and th particular mutation involvd. A dihybrid cross: In ths xprimnts, th mod of inhritanc is dtrmind whn two pairs of contrasting charactristics ar considrd simultanously. Th mutations may not b radily apparnt, and you will b xpctd to mak prdictions (null hypothsis) about th gnotyp of th parntal gnration (P1), Wstminstr Collg SIM Pag 3

4 idntify th mutations and th mod of inhritanc, and dsign back-crosss to tst ths prdictions. Chi-squar analysis will b usd to support your prdictions or to hlp you slct an altrnativ hypothsis. GUIDING QUESTIONS How do th rsults from gntic crosss allow for dtrmination of gnotyp of th parnt flis? How ar you abl to us gntic crosss to dtrmin if a gn is autosomal or sx-linkd? How ar you abl to us gntic crosss to illustrat th principl of indpndnt assortmnt of two gns? What dos Chi-squar analysis of th data tll you about th rsults of a gntic cross? MATERIALS DrosophiLab 1.0 program Laptop computr Drosophila chromosom map PROCEDURE Part I A Monohybrid Cross 1. Turn on th laptop computr. Doubl click on th DrosophiLab icon. Clos th Tip of th Day window. A blank program window will appar. Figur. Nw Exprimnt window. Slct Fil>Nw Exprimnt from th mnus at th top lft of th window. Th following Crat Nw Exprimnt window will appar (Figur ). Crat an xprimnt nam and typ it in th Exprimnt Nam box. 3. To start an xprimnt, you must choos a mal and fmal to b th parnt gnration (P1). Your instructor will assign an Exprimnt # to your group. You will slct both th mal and fmal with th corrsponding numbr for your first trial. For xampl, if you ar assignd Exprimnt #, you will choos th fils Exptm for th mal and Exptf for th fmal. Wstminstr Collg SIM Pag 4

5 4. Click on th? icon nxt to th Mal parnt. A window showing a sris of.fly fils will appar. Slct th mal fly fil with th appropriat Exprimnt #, thn click Opn. If you hav slctd a parnt, a fly icon will appar whr th? usd to b, and th nam of th Exprimnt fil will b writtn to th right of th fly icon. 5. Rpat Stp 4 to choos a fmal fly with th appropriat Exprimnt #. 6. Slct OK whn you hav chosn th appropriat mal and fmal P1 gnration. Th main xprimnt scrn will appar (Figur 3). At th top lft portion of th window, thr is a microscop window which should b mpty. Blow this is a box labld P1, showing th two parnt fly icons. To xamin on of th parnt flis, click on on of th P1 fly icons just blow th microscop and drag th fly to th microscop box. A 3D modl of th fly should appar as shown in Figur 3. Figur 3. Main xprimnt scrn 7. Thr ar svral ways in which you can manipulat th fly to xamin it mor closly. You should xprimnt with ths until you ar comfortabl using th virtual microscop. a. Shift + Right click allows you to cntr th fly in th fild of viw. b. Ctrl + Right click allows you to rotat th fly so that you can s crtain sctions of th body mor clarly. c. Th sliding scal to th right of th microscop lts you incras magnification. This is particularly usful for mutations involving th antnna and bristls. 8. Examin th first fly undr th microscop and dtrmin whthr it is a mal or fmal (Figur 4). In gnral, mals ar slightly smallr than fmals. Th most distinguishing faturs ar found on th abdomn of th flis. Th fmal has svral transvrs strips, and an ovipositor which givs th fly a mor pointd abdomn. Th mal abdomn is darkr, and has a much bluntr tip. In liv flis, thr would also b a sx combs, groups of black bristls on th upprmost joint front lgs of th mal. This dtail is not prsnt in th virtual flis. Wstminstr Collg SIM Pag 5

6 Figur 4. Fmal and Mal Drosophila Abdomn with Svral Transvrs Strips Havily Pigmntd Tip of Abdomn Pointd Ovipositor Blunt Tip of Abdomn 9. Also not if th fly has any mutation associatd with it. Common mutations ar in y color or shap, wing shap, body color, and bristl or antnna morphology. Your instructor will dmonstrat th charactristics of a wild-typ fly so that you may idntify a mutation. Rcord th sx and crat a nam for any phnotypic mutations on th Data Sht undr Part I, P1 Data. Slct Empty Microscop whn you ar don. 10. Drag th scond parnt fly to th microscop and follow stps 8 & 9. Slct Empty Microscop whn you ar finishd rcording th sx and mutation information. Figur 5. Nw Gnration window 11. To crat an F1 gnration from ths two flis, slct Exprimnt>Nw Gnration from th mnus. A Nw Gnration window will appar (Figur 5). Undr Gnration nam, kp th F1 and add any mutation nam you hav givn th flis. St 00 as th Numbr of offspring, and slct th Icons button. Slct OK whn finishd. Not: Any fwr than 00 offspring will rsult in problms prforming a Chi-squar tst latr. 1. Th Main Exprimnt scrn will appar again, this tim with an F1 panl displaying 00 fruit fly icons (Figur 6). Just as you would with liv flis, you will b rquird to drag ach fly to th microscop and sort it basd on sx and phnotyp. Basd on your initial obsrvations, prdict th typs and/or numbrs of sorting jars you will nd (s Data Sht, Part I). For xampl, in almost all cass, you will nd a wild-typ fmal and wild-typ mal jar. Nw jars can b cratd at any point during th sorting procss should you discovr a mutation you had not prdictd. Wstminstr Collg SIM Pag 6

7 Figur 6. F1 Gnration displaying offspring icons and counting jars. 13. To crat sorting jars, slct th Exprimnt mnu>add counting jar or Ctrl J. A window will appar. Entr a short jar labl that is dscriptiv and clar to you and your lab mats. Click OK. A jar with that labl will appar as a jar icon in th Counting Jars panl of th Main Scrn (Figur 5). Whn you drop flis into that containr, th computr will kp count for you and post th total numbr of flis with that phnotyp undr th jar labl. 14. Bfor you start counting your flis and placing thm in th counting jars, you should b awar of svral quirks in th computr program that can mak crating an F gnration difficult. a. Th counting jars ar ssntially a fly morgu. Onc you plac an icon in th jar, that fly is no longr availabl to b usd to crat futur gnrations. b. It is bst if you sav a mal and fmal of ach phnotyp, so that if ncssary, you can st thm as parnts for th F gnration. To sav thm, slct Empty Microscop aftr idntifying thm, rathr than dragging thm to th sorting jars. This rturns thm to th F1 icon panl. c. Each fly has its own idntifying numbr. To s this numbr, hold th mous arrow ovr a fly icon. It should b writtn as Gnration (#), i.. F1(). Whn saving flis to crat a nw gnration, you might want to rcord this numbr, sx of th fly and phnotyp to rmmbr which flis you want to st as parnts for th F gnration (s Data Sht, Part I). Wstminstr Collg SIM Pag 7

8 15. Sort th rmaining flis by phnotyp into th counting jars, and rcord th information in Tabl 1 in Part I of th Data Sht. Dpnding on th gntic cross, you may not nd to us all th rows providd. 16. Th nxt stp is to crat an F gnration by prforming an F1 x F1 cross. To st th savd flis as parnts for th nxt gnration, right click on on of fly icons in th F1 panl. Choos St as parnt from th mnu that appars. B sur to st both a mal and a fmal parnt. If you hav don this succssfully, th icon will turn light pink. 17. Follow stps 9-13 to sort and rcord th rsults of th F gnration. Thr is no nd to sav any flis for furthr gnrations unlss your instructor indicats othrwis. 18. Two Important Nots: a. It is vry, vry important to rcord your data prcisly. Unlss you hav accss to a printr, all this data WILL BE LOST whn you clos th program. Whil it is not as tim consuming as losing a vial of liv flis, you will still hav to rpat th gntic crosss and sort all th flis again! b. If you do not hav tim to prform th F cross and collct data, you must sav th flis to a fly fil! To sav th slctd flis for th nxt gnration, right click on th pink highlightd fly icon. Choos Sav fly to fil from th drop-down mnu. Whn giving th fil a nam, labl it with your initials, mutation nam and indicat whthr th fly is mal or fmal. You will b abl to rtriv ths fils to continu with your F cross during your nxt lab priod, but you and your lab group will nd to us th sam computr throughout th xprimnt. Part II Dihybrid Cross and Indpndnt Assortmnt 19. In this part of th lab, you will xamin th indpndnt assortmnt of allls using a dihybrid cross. Your instructor will assign an xprimnt numbr as bfor, i.. if you ar assignd Dihybrid Exprimnt #, you will choos th fil Dihm for th mal and Dihf for th fmal. 0. Examin th first fly undr th microscop and dtrmin whthr it is a mal or fmal. Not if th fly has any phnotypic mutation associatd with it. Mutations for this part of th lab will b radily visibl, and will involv th ys, body and wings. Rcord th sx and phnotypic mutations of th parnts in Part II of th Data Sht. 1. NOTE: As this is dihybrid cross, som mutations may not b visibl until th F1 gnration is producd! If mutations ar not vidnt in th parnts, b alrt for thm to appar in th F1 offspring. Also, thr ar no tricks to ths flis. Th xprimntal flis will NOT hav linkd gns (gns clos togthr on sam chromosom).. Onc you hav rcordd th sx and phnotyp of th P1 gnration, follow stps to crat F1 offspring. It is spcially important to sav flis with various mutations of both sxs. Rcord th sx and phnotyp of th savd flis in th appropriat spot in Wstminstr Collg SIM Pag 8

9 Part II of th Data Sht. In this part of th lab you ar xpctd to mak prdictions about th gnotyps of th parnt flis and dsign a back-cross to tst thos prdictions. This may involv a cross btwn F1 flis or a cross with on of th parnts. 3. Rcord th rsults of th F1 gnration in Tabl 4 on th Data Sht. Rmmbr to add in th savd fly totals to th totals from th sorting jars. 4. Whn planning a cross to dtrmin th gnotyp of th P1 gnration, us th data from th F1 gnration to mak a prdiction. Qustions that might hlp you with this prdiction ar: Wr any mutations visibl in th P1 gnration? Did any nw mutations appar in th F1 gnration? What do you think th mod of inhritanc is? Dominant? Rcssiv? Sxlinkd? Do th rsults from th F1 cross lad you to bliv that th parnts wr homozygous or htrozygous? Ar thr any flis that you ar sur of th gnotyp? Would using on of ths in a cross hlp dtrmin if thr ar any hiddn mutations in on of th parnts? 5. It may tak mor than an F gnration to lucidat th gntic background of th parnt flis. Crating Punntt squars to modl your prdictions may hlp you dcid which savd flis to us for furthr crosss. REFERENCES Th Collg Board Advancd Placmnt Program. Biology Lab Manual for Studnts. Lab Svn: Gntics of Organisms. 001 by th Collg Examination Board. Pp Th Collg Board Advancd Placmnt Program. Biology Lab Manual for Tachrs. Lab Svn: Gntics of Organisms. 001 by th Collg Examination Board. Pp DrosophiLab 1.0, Hanns Jnsn, Educational links to othr Drosophila wbsits ar includd in this wbsit. Drosophila Lif Cycl imag obtaind from: CREDITS This lab dsign was rvisd and adaptd by Dr. Stphani Corrtt-Bnntt using th DrosophiLab 1.0 computr download and th AP Biology laboratory manuals. Wstminstr Collg SIM Pag 9

10 DATA SHEET & ANALYSIS Nam: Group: Dat: Data for Part I P1 Data: Sx Phnotyp Mutation nam? Sx Phnotyp Mutation nam? Prdiction for phnotyps of F1 gnration (stimat # s mal & fmal offspring and phnotyp using symbols cratd to idntify th mutation): Mals (xpctd): Fmals (xpctd): Punntt Squar for F1 prdiction: Fmal Mal Phnotyp Ratio: Gnotyp Ratio: Fmal Mal Savd Flis F1(#)/Phnotyp Wstminstr Collg SIM Pag 10

11 Tabl 1: F1 Gnration Data Phnotyp & Symbol Fmals Mals F1 Obsrvations: Did your obsrvations/ rsults match your prdictions? Prform a Chi-squar tst on your rsults and compar thm to th Critical Valu Tabl in th introduction. Chi-squar analysis for F1 Gnration Sx/Phnotyp Obsrvd Expctd (o ) (o-) (o - ) χ ( o = ) = What do ths data tll you about th mod of inhritanc of this trait? What is th dominant alll? What is th rcssiv alll? What ar th phnotyps of th parnts that will b usd to crat th F gnration? F1 Parnt Data: Sx Phnotyp Sx Phnotyp Mutation nam Wstminstr Collg SIM Pag 11

12 Prdiction for phnotyps of F gnration (stimat # s mal & fmal offspring and phnotyp using symbols cratd to idntify th mutation): Mals (xpctd): Fmals (xpctd): Punntt Squar for F prdiction: Fmal Mal Phnotyp Ratio: Gnotyp Ratio: Tabl : F Gnration Data Phnotyp Fmals Mals F Obsrvations: Did your obsrvations/ rsults match your prdictions? Prform a Chi-squar tst on your rsults and compar thm to th Critical Valu Tabl in th introduction. Wstminstr Collg SIM Pag 1

13 Chi-squar analysis for F Gnration Sx/Phnotyp Obsrvd Expctd (o ) (o-) (o - ) = ( o ) χ = What is th gnotyp of th parnt flis? If you still ar unsur of th gnotyp of on of th parnts, can you dsign a (thortical) F3 cross that would dfinitivly dtrmin th gnotyp? P1 Parnt Data: Mal gnotyp Fmal gnotyp Data for Part II Dihybrid P1 Data: Sx Sx Phnotyp Phnotyp Prdiction for phnotyps of F1 gnration (stimat # s mal & fmal offspring and phnotyp using symbols cratd to idntify th mutation): Mals: Fmals: Wstminstr Collg SIM Pag 13

14 Punntt Squar for Dihybrid F1 prdiction: Fmal Mal Phnotyp Ratio: Gnotyp Ratio: Fmal Mal Savd Flis F1(#)/Phnotyp Tabl 4: Dihybrid F1 Gnration Data Phnotyp & Symbol Fmals Mals Dihybrid F1 Obsrvations: Did your obsrvations/ rsults match your prdictions? Prform a Chi-squar tst on your rsults and compar thm to th Critical Valu Tabl in th introduction. Wstminstr Collg SIM Pag 14

15 Chi-squar analysis for Dihybrid F1 Gnration Sx/Phnotyp Obsrvd Expctd (o ) (o-) (o - ) = ( o ) χ = If not, how was it diffrnt? Dos this tll you somthing about th gnotyp of on of th parnt flis? What ar th phnotyps of th parnts that will b usd to crat th dihybrid F gnration? F1 Parnt Data: Sx Phnotyp Sx Phnotyp Mutation nam Prdiction for phnotyps of F gnration (stimat # s mal & fmal offspring and phnotyp using symbols cratd to idntify th mutation): Mals (xpctd): Fmals (xpctd): Wstminstr Collg SIM Pag 15

16 Punntt Squar for Dihybrid F prdiction: Fmal Mal Phnotyp Ratio: Gnotyp Ratio: Tabl : F Gnration Data Phnotyp Fmals Mals F Obsrvations: Did your obsrvations/ rsults match your prdictions? Prform a Chi-squar tst on your rsults and compar thm to th Critical Valu Tabl in th introduction. Wstminstr Collg SIM Pag 16

17 Chi-squar analysis for Dihybrid F Gnration Sx/Phnotyp Obsrvd Expctd (o ) (o-) (o - ) χ ( o = ) = What is th gnotyp of th parnt flis? If you still ar unsur of th gnotyp of on of th parnts, can you dsign an F3 cross that would dfinitivly dtrmin th gnotyp? Why ar you choosing ths particular flis as parnts? P1 Parnt Data: Mal gnotyp Fmal gnotyp QUESTIONS Givn th following gntic cross, answr th following qustions: An invstigator obsrvs that whn pur-brding, long-wing Drosophila ar matd with pur-brding, short-wing flis, th F1 offspring hav an intrmdiat wing lngth. Whn svral of th intrmdiat-wing lngth flis ar allowd to intrbrd, th following rsults ar obtaind: Obsrvd 30 long wings 510 intrmdiat-lngth wings 60 short wings Wstminstr Collg SIM Pag 17

18 1. What is th gnotyp of th F1 intrmdiat-wing lngth flis?. Writ a hypothsis dscribing th mod of inhritanc of wing lngth in Drosophila (this is your null hypothsis). 3. Complt th following tabl. Chi-squar analysis for Wing-lngth Cross Phnotyp Obsrvd Expctd (o ) (o-) (o - ) ( o = ) χ 4. Calculat th Chi-squar valu for ths data. a. How many dgrs of frdom (df) ar thr? = b. What is th Chi-squar (from tabl abov)? c. Rfrring to th critical valus chart, what is th probability valu for ths data? 5. According to th critical valu of χ, can you accpt or rjct th null hypothsis? Explain why. Wstminstr Collg SIM Pag 18