A study of the IGS/ETS region in octocorals and black corals

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(1)A STUDY OF THE IGS/ETS REGION IN OCTOCORALS AND BLACK CORALS. SONIA ELEONORA HERNÁNDEZ DURÁ N. UNIVERSIDA D DE LOS ANDES SCIENCE FACULTY BIOLOGICAL SCIENCE DEPARTM ENT BOG OTA 2008.

(2) A STUDY OF THE IGS/ETS REGION IN OCTOCORALS AND BLACK CORALS. SONIA ELEONORA HERNÁNDEZ DURÁ N. MICROBIOLOGY UNDERGRA DUATE THESIS. Director JUAN ARM ANDO SÁNCHEZ M arine Biologist Ph.D. Co- Director GLORIA INÉS URIBE Bacteriologist M. Sc. UNIVERSIDA D DE LOS ANDES SCIENCE FACULTY BIOLOGICAL SCIENCE DEPARTM ENT BOG OTA 2008.

(3) CONTENTS. FIG URE LIST ACKNOW LEDGMENTS Page SUMMARY.......................................................................................................................6 INTRO DUCTION ............................................................................................................6 MA TERIA LS AND METHO DS ......................................................................................8 Sample collection........................................................................................................8 DNA Extraction............................................................................................................8 Confirmation of DNA extr action ................................................................................9 IGS/ETS PCR standardization .................................................................................9 Standar dization of DNA conc entration................................................................9 Standar dization of PCR conditions ....................................................................10 Res tric tion and ligation assay - RFLPs ................................................................. 11 A mplif ic ation of restric tion fragments..................................................................... 11 MseI- M13 PCR......................................................................................................11 M13 PCR................................................................................................................12 RESULTS AND DIS CUSSION ...................................................................................12 Standar diz ation of DNA conc entration .................................................................. 13 Standar diz ation of PCR conditions ........................................................................ 14 IGS/ETS PCR Pr oducts ......................................................................................... 16 RFL Ps ( Restric tion Fr agment Length Poly morphisms) ...................................... 18 A mplif ic ation of restric tion fragments..................................................................... 19 CO NCLUSIO NS ............................................................................................................20. REFERENCES ANNEX ES. 3.

(4) FIGURE LIST. Page Figure 1 Gener al str uctur e of IGS/ETS r egion in eukaryotes…………..………..7 Figure 2 Standar diz ation of DNA dilutions for Pacifigor gia spp. and Leptogor gia sp.…………………………………………………………………………..13 Figure 3 Temper atur e gradient for oc toc oral s amples and black cor al samples……………………………..…………………………………….14 Figure 4 PCR pr oducts of octocorals and black corals …………………...…….16 Figure 5 RFL Ps of blac k corals and octocorals…………………………………..18 Figure 6 A mplific ation of restriction fragments using M13 tailed primers……...19. 4.

(5) ACKNOW LEDGMENTS. First of all, I w ould like to thank my family for being the incr edible suppor t that they ar e and for alw ays keeping me grounded. I know that no matter w hat I c an alw ays count on you and that nothing of this w ould ev er be poss ible w ithout you.. I w ould als o like to thank Juan Ar mando Sánchez, for his guidance, his w ords of wisdom and for letting me into the w onderful group that is the BIOMMA R lab.. Gloria Ur ibe, for being ther e w henever I needed a little guidance, a pep talk or a friendly fac e on a rough day. The w onderful people at BIOMMAR, it w as such a pleas ure to be part of this lab. I w ould s pec ially like to thank Martic a Cár denas for being w illing to answ er my ev ery question, Camila Gr anados and Nelson Manr ique for their support, their friendship and their help and Carlos Umaña and Daniel Dorado for all the helpful infor mation and support. Also, Néstor Ardila, Vic tor Piñer os, Luisa Dueñas , Claudia Agudelo and Pedr o Castr o for their help and for alw ays making me laugh no matter w hat.. Adriana Bernal, for alw ays having a solution at hand and for helping me solve many doubts.. And of cours e, my friends : Elis a Chaparr o, Humberto Rodr íguez, Pipe Serrano and Jimena V illegas for being there w henev er I needed them, life just w ouldn’t be as fun w ithout you guys !. 5.

(6) SUMMARY The IGS/ETS region has been vir tually unexplored in cnidar ians, w hich makes for an exc iting s tudy alternative for developing new molecular mar kers in these organis ms . In this study, the siz e and repetitive natur e of the IGS/ETS of sever al oc tor als and black cor als w ere analyzed for inters pecif ic var ia tion us ing a RFLP appr oach. All the samples included in this study pr esented a multicopy region w ith different number and sizes of copies as w ell as different numbers of MseI r estr iction sites at different positions w hich implies a high v ar iability betw een c or al s pecies. All this makes for an ideal molecular mar ker in future studies inv olv ing corals. How ever, futur e w ork should continue standar ization of these methods and inc lude a mor e diverse gr oup of c or al spec ies so as to be able to gener aliz e thes e findings and construct a more s pecif ic pic tur e for the IGS/ETS region in c orals.. INTRODUCT ION Very little is know n about the inter genic spacer and exter nal transcribed s pacer (IGS/ETS) region in cor als. The study of this region is partic ularly complicated and tedious due to its large siz e and high number of tandem r epeats in higher organis ms (Coté et al, 2001), and it is not c lear w hether the exis ting models for more advanc ed organis ms apply for low er organis ms suc h as c nidarians ( Chen et al, 2000). In general, r ibos omal DNA (r DNA) is arr anged in transcr ip tion units w hich are separ ated by an intergenic spac er ( IGS) ranging from 2.5 to 30 Kb in animals (Sollner-Webb and Mougey 1991). Each tr anscription unit consis ts of 3 rib osomal subunits ( 18s, 5.8s and 28s) s epar ated by an inter nal tr anscribed spacer, ITS1 or ITS2, w hich c ontain the infor mation nec essary for pr ocess ing ( Harris et al, 2000). Further more, this arr angement is flanked at each end by an external tr anscribed spacer (ETS) (Coté et al, 2001). 6.

(7) Fig. 1 Gener al str ucture of IGS/ETS region in eukaryotes It is know n that r DNA is prone to c oncerted ev olution, w hich ov er time r esults in a homogenization of all multigene families pres ent in the genome ( Harris et al, 2000). How ever, the IGS/ETS region has been reported to vary signific antly within indiv iduals and populations , w ith poly mor phisms that v ary in the order of kilobas e pairs ( Coté et al, 2001) due to a very low concer ted ev olution rate (Coté et al, 2001, Chen et al, 2000, Harr is et al, 2000).. Pr evious studies have repor ted IGS/ETS r egions of 1.8 kb for the octocor al Junceella fr agilis ( Chen et al 2000) and 2.5 kb for the Pavona cac tus cor al (Smith et al, 1997), w here the r egions of both organis ms present a pattern consistent w ith a tandem arrangement as pr eviously mentioned. It w as the aim of this study to deter mine the s ize and repetit ive natur e of the IGS/ETS of sever al oc toc or als and blac k c orals, as w ell as deter mining the effectiv eness of this region as a molec ular mar ker for inters pecific variation us ing a RFLP tec hnique. Also, an alternativ e protocol for sequencing large fragments (such as IGS/ETS) w as ex plor ed so as to omit clonin g methods .. Multiple c opies of this r egion betw een 600 and 1700 bp w ere obs erved in both groups and a gr eat deal of interspecific v ariation w as assessed, res ulting in what appears to be a v ery pr omising molecular mar ker for further s tudies regarding cor als . How ever, a muc h lar ger s ample number must be inc lu ded in studies s imilar to this so as to elabor ate a mor e exhaustive model for the IGS/ETS regions in cor als and although interspec ific var iation w as deter mined, various s amples of a same species must be included in or der to assess intraspecific v ariation of this r egion. Finally, it is imperativ e that standar ization of the alternativ e s equenc ing pr otocol be c ontinued so that its effectiveness c an be incr eas ed and it can be employed in the futur e w ith a much higher efficiency.. 7.

(8) MATERIALS AND METHODS Sample collection Black cor al s amples w ere collec ted on September of 2005 at The Flow er Gar den Banks National Mar ine Sanctuary in the Gulf of Mexico by NURC phantom S2, a remotely oper ated vehic le. Octocoral samples w ere collec ted on September of 2005 at different loc ations of Cartagena, Colombia and on September of 2007 at the island of Gorgona, Colombia by members of the BIO MMAR lab from the Universidad de los Andes by scuba div ing (Annex 1). A total of 40 samples w ere collected and pr eserv ed in 95% ethanol.. DNA Extra ction DNA from all blac k cor al s amples w as extracted using the extraction kit DNeasy by Qiagen® under c onditions r ec ommended by the manufac tur er.. DNA from all the octocoral samples w as extr acted using a modified v ersion of the protoc ol by Coffroth et al, 1992. Approx imately 2 mm cube fragments of eac h s ample w ere used for DNA extr action. Each fragment w as ground using ster ile pestles in 300 µL of CTA B 2X ( Cetyltr imethyl ammonium br omide) at 65 C, and 2 µL of pr oteinase K w ere added in order to lys e any pr oteins pres ent, including DNAs es. This lys ate w as inc ubated at 65 C over night. After this , 300 µL of PCIA ( Phenol/chloroform – isoamy lalc ohol 25:24:1) w ere added to the lys ate, agitated and s pun in a centrifuge dur ing 10 minutes at 12000 rpm. After centr ifugation, the aqueous phase w as recuperated and 300 µL of CIA ( Chloroform is oamylalcohol 24:1) were added to this phase and centrifuged again for 10 minutes at 12000 rpm.. 8.

(9) This new aqueous phas e w as recov er ed and the DNA w as precipitated us ing 800 µL 95% ethanol. The DNA w as left to precipitate ov ernight, after w hich it was centr ifuged at 12000 r pm during 30 minutes. The s uper natant w as disc ar ded and 500 µL of 70% ethanol w ere added to the remaining pellet and centrifuged during 10 minutes at 12000 rpm. The ethanol w as discar ded and any remaining ethanol w as evaporated at room temper ature. Onc e the pellet w as dr ied completely, 50 µL of TE buffer w ere added to the pellet and stor ed at - 20 C.. Confirmati on of DNA extracti on Pr esence of DNA w as confir med by visualiz ing the extr actions in a 0.8% agarose gel w ith 0.5X TBE buffer after a 30 minute r un at 110 V using the Chemidoc. TM. XRS BioRad gel documentator and Quantity One 4.0 s oftw are.. IGS/ETS PCR standardization. Standardization of DNA concentration. In order to optimize DNA conc entration for PCR protocol, a ser ies of dilutions were ass ayed for each sample, accor ding to the band intens ity obtained in the prev ious s tep of confir mation of DNA ex trac tion. Extr actions w ith a higher band intens ity w ere diluted to 1/50 and 1/20, w hile samples w ith a low er band intens ity w ere diluted to 1/10 and 1/5.. 9.

(10) Standardization of PCR conditions The IGS/ETS r egion of the c orals analyzed w as amplified by a PCR pr otoc ol using the univers al. pr imers. 28NF:. 5' - GA TTA TGACT GAACGCCTCT. AAGTCAGAA T CC- 3' and 18S- 10B: 5'-TTACCA TCGA CA GTTGA TA GGGCA3' ( Smith et al, 1997).. The PCR cycle w as perfor med on a My Cyc le. ther mocyc ler (Bio-Rad) based on pr evious w ork for the ETS/IGS r egion of the octocor al Junceella fragilis (Chen et al, 2000). Specific cyc le conditio ns for the octocor al samples and the black cor al samples w ere further standariz ed as s o, for octocor als the thermal cycle cons isted of 1 cycle at 95 °C (3 min); 35 cyc les at 94 °C ( 30 s), 57 °C ( 1 min) , and 72 °C ( 2 min); and 1 cycle at 72 °C ( 3 min), and for black cor als 1 cyc le at 95 °C (3 min); 4 cycles at 94 °C ( 30 s), 50 °C (1 min) , and 72 °C ( 2 min); 30 cycles at 94 °C (30 s), 52 °C (1 min), and 72 °C (2 min) and 1 final cycle at 72 °C ( 3 min) . The annealing temperatur e of the primers w as standar dized us ing a temper atur e gr adient (54 °C, 55.4 °C, 56.4 °C, 57.2 °C and 58 °C for octoc orals and 51 °C, 52.1 °C, 53.4 °C, 54 °C for blac k c oral s amples). The optimal Mg Cl 2 conc entration w as chosen based on a prev ious w ork done on PCR c onditions for black cor als ( Umaña, unpublished) at the BIO MMAR la b.. The amplific atio n reac tion us ed 2.25 µL of the dilu ted template, 0.3 units of Pr omega Taq polymerase, 3 µL of 5X buffer pr ovided w ith the enzyme, 0.3 µL of 10mM Promega dNTPs, 2.1 µL of 25mM MgCl 2 (fin al c onc entr ation of 3.5mM), 0.225 µL eac h of forw ard and rev erse primer ( 10µM) and 6.6 µL of ddH2O for a final volume of 15 µL. PCR pr oduc ts w ere ver if ied by electrophores is in a 1% agarose gel w ith the conditions pr eviously mentioned in DNA extr action confir mation.. 10.

(11) Re stricti on and liga tion assay - RFLPs Amplified DNA from Plumapathes pennacea, Tanacetipathes b arbadensis, Antipathes atlantica ( black cor als), Leptogor gia sp. (sample L3) , Pacifigorgi a s p. (samples G93 and G96), Muricea atlantica and Muricea pinnata (oc toc or als) was treated w ith the Ms eI res triction enzyme from Inv itrogen. For the r estr iction and ligation r eac tion 10 µL of PCR product w ere us ed, as w ell as 0.5 µL of Ms eI enzyme, 2.5 µL of 10X buffer provided w ith the enzy me, 0.33 µL of T4 ligase from Promega, 5 µL of 10X buffer prov ided w ith this enzyme, 1.25 µL of DTT (5mM), 0.42 µL of ddH2O and 2.5 µL eac h of MseI adaptor I ( 5’TA CTCAGGA CTCA T-3’) and Ms eI adaptor II (5’- GA CGATGA GTCCTGA G-3’, Zane et al, 2002) for a final volume reaction of 25 µL.. This r eac tion w as incubated at 37 C for 3 hours after w hich the r estr iction fragment length poly morphisms ( RFLPs) w ere checked by 7% polyacry lamide gel electr ophoresis w ith 1X TBE buffer at 110 V during 50 minutes. A 100bp benchtop ladder (Pr omega) w as used to v isualize the fragments, as w ell as a 50bp molecular mar ker from Invitrogen. Eac h band present w as cut from the gel and res us pended in 50 µL of ddH2 O. Each res uspended DNA fragment w as left in a shaker ov er night, after w hich the super natant w as passed to another ster ile eppendorf and 450 µL of absolute ethanol w ere added in order to pr ec ipit ate the DNA at - 20 C during the next 2 days. After centr ifugation at 12000 rpm dur ing 30 minutes, each pellet w as left to dry and r esuspended in 15 µL of TE buffer.. Amplification of re stricti on fragments. MseI-M13 PCR. 11.

(12) Reamplif ic ation of eac h resus pended restriction fragment and addit ion of the M13 tail (5’- TGTAAAA CGA CGGCCA GT-3’) w as obtained us ing the MseI-M13 primer pair, MseI-M13 forw ard: 5’-GTA AAA CGA CGG CCA GCA TGA GTC CT- 3’and Ms eI- M13 revers e: 5’- GAGAGMTCC TGA GTA A CA GGA AA C A C3’, w here an M13 tail w as added to the Ms eI primer pair . For the amplific ation reaction 5 µL of a 1/10 dilution of pr eviously resus pended DNA w ere us ed, as well as 0.1 unit of Promega Taq polymeras e, 4 µL of buffer prov ided w ith the enzyme, 2.8 µL of MgCl 2 ( 3.5 Mm), 0.4 µL of Pr omega dNTPs, 0.225 µL each of forw ard and r everse primer (10 uM) and 2.46 µL of ddH2 O for a final volume of 15 µL. The ther mal cycle c onsisted of 26 cycles at 94 °C (30 s) , 53 °C ( 1 min) and 72 °C (1 min) (Zane et al, 2002) .. Confir mation of tailed PCR pr oducts w as carried out using a 2% agaros e gel electr ophoresis w ith 0.5X buffer at 110V dur ing 30 minutes. .. M13 PCR A PCR w ith a M13 primer pair (M13 Forw ard: 5’-GTA AAA CGA CGG CCA G- 3’ and M13 Rev erse: 5’- CAG GAA ACA GCT ATG AC- 3’) complementary to the M13 tail inser ted in the Ms eI- M13 PCR w as carr ied out as a confir matio n step prior to sequenc ing to deter mine if the M13 tail w as indeed being inser ted in the amplified res triction fragments. The ther mal cycle employed w as as follow s: 30 cycles at 94 °C (30 s), 53 °C (1 min) and 72 °C ( 1 min). After this, PCR pr oducts were visualiz ed using a 2% agar ose gel w ith conditions pr ev ious ly mentio ned in DNA ex traction c onfir mation.. RESULTS AND DISCUSSION. 12.

(13) Standardization of DNA concentration. Fig. 2 Standardiz ation of DNA dilutions for Pacifigor gia s pp. and Leptogor gia sp. Lane 1: Negativ e PCR contr ol ( no DNA).. Standardiz ation of template c onc entr ation for PCR w as critical due to the fact that the DNA w as extr acted us ing tw o different methods (extraction kit and manual ex trac tion). For eac h s ample a s eries of dilu tions w ere ass ayed bas ed on the quality of the DNA ex tracted, the necess ary dilutions deter mined by visualiz ation of the extr action performed. For most of the s amples s tudied, a dilution of 1/20 w as used (blac k c orals and octocor als alike), but in some cas es a minor and major dilution w as nec essary (for example Plumapathes pennac ea and Antipathes atlantic a respectively). With s ome samples the PCR pr oduc t yield improved w ith increas ing dilutions (as suc h w as the case w ith Leptogor gia sp. sample L8). A poss ible ex planation. 13.

(14) for this phenomenon could be the pr esenc e of PCR inhibitors in the extr acted DNA.. Standardization of PCR conditions. Fig. 3 Temperature gradient for octocor al samples (A), and blac k c oral samples Pp: Pl umapathes pennac ea, Tb: Tanacetipathes barbadensis, Aa: Antipathes atlantic a, Af: Antipathes furcata ( B). All samples ar e diluted 1/20. The first lane in eac h gel is a negativ e c ontrol.. A separate standardization of PCR conditions w as necessary for the octocor al group of samples and the blac k c oral samples, the same thermal cycle w as not suitable for both gr oups. Even though the pr imers used in both PCR cyc les. 14.

(15) were the s ame ( 28NF and 18S-10B), the optimal annealing temper ature var ied signif icantly betw een the oc toc orals and black cor als (57 º C and 52 º C respectively) . This temperature w as chosen bas ed on the amplific ation behaviour of all the samples included in the standar dization assay and on the intens ity of all the bands pres ent in eac h PCR pr oduct. For this reas on 54 º C was the poorest annealing temper atur e for black cor als sinc e Antipathes atlantic a w as no longer amplified and amongst the remaining temper atures from the gr adient, 52.1 º C w as the one w here all the bands amplified from each sample pr esented the most intens ity . A similar analys is w as perfor med w ith the octocor al temper atur e gradient.. The amplific ation efficiency prior to standardization r anged betw een 9-11% (le ast and most efficient amplification ass ay respectiv ely) . After the optimal conditions w ere established, an efficiency of 88-91% w as attained for both sample gr oups. This efficiency w as estimated bas ed on the number of samples amplified in a giv en PCR, w ith equal number of samples in each assay (data not show n).. 15.

(16) IGS/ETS PCR Products. Fig. 4 PCR products of octoc orals and black cor als. A) Band patter n of Aa: Antipathes atlantica, L: 100bp mar ker. B) PCR produc ts of blac k c orals Pp: Plumapathes pennacea (dilution 1/5), Tb: Tanacetipathes barbadensis (dilution 1/50), Aa: Antipathes atl antica (dilution 1/20) and oc toc orals Ma: Muric ea atlantic a (dilution 1/20) and Mp: Muric ea pinnata (dilu tion 1/20) L: 50 bp marker. Negative contr ols (-). The PCR pr oducts obtained in this study pr es ented multiple bands , vary ing in size and intensity. As the annealing temper ature w as increased for any giv en sample, all the bands obtained at a low er temperature w ere still pres ent and even in a mor e intense fashion. This indicates that the multiple band patterns were not the artefact of an uns pecific annealing of the pr imers. A lso, each multiple band patter n w as maintained in follow ing r eplicates. It w ould appear that the ETS r egion of the cor als analyzed is not ev olv ing in a conc erted manner sinc e there doesn’t s eem to be occurr ing a homogeniz ation proc ess of. 16.

(17) the multiple copies pres ent ( Elder and Tur ner, 1995). How ever this as pec t c an only be c onfir med analyzing the sequence of eac h band obtained in the PCR products.. Unfor tunately it w as not poss ible to continue w orking w ith a 100bp molecular weight marker (only the PCR pr oduct of Anti pathes atlantic a w as view ed w ith this marker), ther efore the PCR pr oducts of the r est of the samples w ere v iew ed using a 50bp molecular mar ker. Since this marker didn’t cover the molecular weight range of the ETS amplification, an extrapolation from comparison of eac h sample w ith Anti pathes atlantica w as performed. The fragment lengths ranged betw een 600 bp and 1700 bp approx imately ( Figur e 4, A nnex 2) . Although fragment length v ar ied interspecifically, a pattern could be obs erv ed betw een the blac k cor al s amples, and another patter n betw een the octocor al samples. The tendency for the octocor al samples analyzed (Muric ea pi nnata, Muricea atl antica, Leptogorgi a sp. and Pacifi gor gia spp.) w as of four bands and a larger difference betw een band length, appr oximately 600-700 bp difference betw een the last tw o bands amplified in Muricea s pp. s amples ( Figur e 4 lanes Ma and Mp). The band pattern betw een these tw o spec ies is very similar (Figur e 4) , as is the band pattern betw een the s amples of Pacifigor gia spp. and the Leptogor gia sp. s ample (Figur e 2).. On the contr ary, blac k c oral s amples pr esented 3 bands in average and a 300400 bp difference betw een the last 2 bands amplified ( Figur e 4, lanes Tb and Aa).. The most intens e PCR pr oduc t bands throughout the s amples studied r anged betw een 1400 and 1700 bp ( Figure 4) w hich is similar to the fragment length of 1800 bp reported prev ious ly for the octoc oral Juniceella fr agilis ( Chen et al, 2000).. 17.

(18) A much lar ger s et of PCR pr oducts from a mor e extensiv e sampling are required in or der to c onstruct a bigger picture on the given char acteris tics of the ETS region amongst cor als.. RFLP s (Re striction Fragment Length Pol ymorphi sm s). Fig. 5 RFLPs of blac k cor als and octocorals A) Pp: Plumapathes pennacea, Tb: Tanac eti pathes barbadensis, Aa: Antipathes atl antica, L: 50 bp marker, C: Antipathes atlantica PCR Produc t, Ma: Muricea atl antica, Mp: Muricea pi nnata. B) L8: Leptogorgi a sp. (L8), G55: Pacifigorgi a sp. ( G55), L: 50 bp mar ker, G93: Pacifigorgi a s p. ( G93), G96: Pacifi gorgia sp. ( G96), C: Pacifigor gia s p. ( G55) PCR pr oduct.. All samples ass ayed w ere cut by the restr iction enzy me Ms eI y ielding a s er ies of dif ferent length fragments ( Figur e 5), this implies that thes e c orals have a different number of MseI r estriction sites at dif ferent positions . Again, these. 18.

(19) restriction sites ar e not nec essar ily cons erved betw een species, although similar band patter ns w ere obs erv ed betw een Tanac etipathes barbadensis and Antipathes atl antica ( Figure 5, lanes Tb and Aa) , betw een Muricea atlantic a and Muricea pinnata (Figure 5 lanes Ma and Mp) and betw een Pac ifigorgia s p. Samples (Figur e 5, lanes G93 and G96). No gener al band patter n w as obs erv ed amongst all the samples analyzed, sugges ting that thes e s equences are not conserved betw een gr oups or s pecies. How ever a much lar ger sample number includin g other cor al spec ies must be studied in or der to c onfir m these generalized affir mations .. Amplification of re stricti on fragments. Fig. 6 A mplification of restriction fragments using M13 tailed pr imers.. Although a high intensity band w as obtain ed w ith the res tric tion fragments cut from the gel, the efficiency for the amplification of each fragment w as dramatically r educed s eeing that only one band is being amplified ( Figur e 6 lane Tb3). How ever, the M13 tail is being inser ted as c an be appr eciated by the reamplified fragment in figure 6.. 19.

(20) CONCLUSIONS The IGS/ETS in octocor als and blac k cor als is a multicopy r egion w ith different number and s izes of copies for the octoc orals and blac k cor als studied. Also, all the samples analyz ed pres ented different number of MseI restr iction s it es and at different positions. This translates into a high interspecific v ariability for this region due to a low concer ted ev olution r ate w hich makes it an ideal molecular mar ker for future study. A much larger number of samples must be analyz ed in or der to obtain a vas ter pic tur e of the dis pos it ion of the IGS/ETS region in c orals, as w ell as various samples of the same s pecies for the ass ess ment of the intraspecific var ia tion of this region.. Finally, an efficiency of approx imately 90% w as achiev ed for the IGS/ETS PCR. How ever, in subsequent PCR cycles the efficiency of the pr oducts obtained decr eased s ignificantly . Further standariz ation w ork is required to optimize these protoc ols s o as to facilitate the study of this region in cor als and be able to c ontinue w ith the alt er native method for s equencing w ith the MseI-M13 PCR.. 20.

(21) REFERENCES Coffroth, M.A, Mulaw ka, J.M. 1992. Identific ation of mar ine inv ertebr ate larv ae by. means. of. PCR- RA PD. s pecies-s pec ific. markers .. Limnology. and. Oc eanography, 40( I): 181-189. Coté, C., Pec ulis , B. 2001. Role of the ITS2-pr oximal s tem and evidence for inderec t r ecognition of process ing sites in pr e-r RNA pr ocess ing in y east. Nuc leic Acids Research, 29: 2106- 2116. Chen, C., Miller , D., Wei, N., Chang- Feng, D., Hsiao- pei, Y. (2000) The ETS/IGS. region. in. a. low er. animal,. that. seaw hip,. Junc ella. fr agilis. ( Cnidar ia:Anthoz oa: Oc toc orallia) : Compactness, Low Variation and apparent conservation of prer RNA pr ocess ing signal w ith fungi. Zoologic al Studies 39( 2): 138-143.. Elder, J. F. and Turner, B. J. 1995. Addendum to Conc erted Ev olution of Repetitive DNA Sequences in Eukaryotes. The Quarterl y Review of Biology, 71( 1): 79. Harr is, J., Cr andall, K. 2000. In tragenomic var ia tion w ithin ITS1 and ITS2 of freshw ater cr ayfishes( Dec apoda:Cambaridae) : Implications for phy logenetic and microsatellite studies. Mol. Biol. Evol. 17(2): 284-291.. Smith C, Chen C.A ., Y ang H.P., Miller D.J. 1997. A PCR- bas ed method for assay ing molecular var iation in cor als based on RFLP analysis of the ribos omal inter genic spacer regio n. Mol. Ecol. 6: 683- 685. Sollner-Webb B,. Mougey E.B. 1991. New s from the nucleolus: rRNA gene. expr ession. Tr ends Biochem. Sci. 16: 58- 62.. 21.

(22) Umaña, C. 2007. Div ers idad de corales negros (Antipatharia) en las regio nes del es pac iador transcrito externo – Inter génico (ETS- IGS) , y el espaciador transcrito inter no 2 ( ITS2), del ADN ribosomal. Under graduate thes is. Science Fac ulty. Univ ers idad de los Andes. ZA NE, L., BA RG ELLONI, L., PATARNELLO T., 2002. Str ategies for micros atellite isolation: a r eview . Molecular Ecology (2002) 11: 1- 16. 22.

(23) ANN EX 1 – SAMPLE COLLECTION CODE. SPECI ES. GROU P. LOC ATI ON. D ATE. DFH11-1A. Plu mapathes. Black C oral. EF GBN MS, Gulf. Septem ber 2005. pennacea DFH11-2A. Plu mapathes. of Mexic o Black C oral. pennacea DFH11-3A. Elat opathes. Elat opathes. Black C oral. Tanacetipat hes. Black C oral. Tanacetipat hes. Black C oral. Elat opathes. Black C oral. Elat opathes. Black C oral. Tanacetipat hes. Black C oral. Aphanipathes. Black C oral. Antipat hes. Black C oral. Antipat hes. Black C oral. Antipat hes. Black C oral. DFH11-19C. Septem ber 2005. EF GBN MS, Gulf. Septem ber 2005. EF GBN MS, Gulf. Septem ber 2005. EF GBN MS, Gulf. Septem ber 2005. EF GBN MS, Gulf. Septem ber 2005. EF GBN MS, Gulf. Septem ber 2005. of Mexic o Black C oral. furcat a DFH11-13B. EF GBN MS, Gulf. of Mexic o. furcat a DFH11-12C. Septem ber 2005. of Mexic o. atlantica DFH11-11A. EF GBN MS, Gulf. of Mexic o. pedat a DFH11-10A. Septem ber 2005. of Mexic o. barbadensis DFH11-8A. EF GBN MS, Gulf. of Mexic o. abietina DFH11-7A. Septem ber 2005. of Mexic o. abietina DFH11-6B. EF GBN MS, Gulf. of Mexic o. barbadensis DFH11-6A. Septem ber 2005. of Mexic o. barbadensis DFH11-5A. EF GBN MS, Gulf of Mexic o. abietina DFH11-4B. Septem ber 2005. of Mexic o. abietina DFH11-3B. EF GBN MS, Gulf. Volcano Banks,. Septem ber 2005. Gulf of Mexic o. Acanthopathes thyoides. Black C oral. Sticopat hes s p.. Black C oral. WFGBN MS,. Septem ber 2005. Gulf of Mexic o WFGBN MS,. Septem ber 2005. Gulf of Mexic o DFH11-20A. Antipat hes. Black C oral. atlantica DFH11-20B. Tanacetipat hes. WFGBN MS,. Septem ber 2005. Gulf of Mexic o Black C oral. tanac etum. WFGBN MS, Gulf of Mexic o. 23. Septem ber 2005.

(24) C7. Antipat hes. Black C oral. caribbeana C12. Antipat hes. Plu mapathes. Black C oral. Muric ea atlantica. WFGBN MS,. Septem ber 2005. Gulf of Mexic o Black C oral. pennacea A363. Septem ber 2005. Gulf of Mexic o. caribbeana C13. WFGBN MS,. WFGBN MS,. Septem ber 2005. Gulf of Mexic o Octoc oral. Isla Tes oro,. Septem ber 2005. Cartagena A486. Muric ea pinnata. Octoc oral. Salmedina,. Septem ber 2005. Cartagena A526. Muriceopsis. Octoc oral. flavida A545. Eunic ea. Leptogorgia sp.. Septem ber 2005. Cartagena Octoc oral. flexuosa L3. Burbujas,. Burbujas,. Septem ber 2005. Cartagena Octoc oral. Juan Chinc ho,. Septem ber 2005. Gorgona L5. Leptogorgia sp.. Octoc oral. Juan Chinc ho,. Septem ber 2005. Gorgona L8. Leptogorgia sp.. Octoc oral. Juan Chinc ho,. Septem ber 2007. Gorgona G55. Pacifigorgia s p.. Octoc oral. Mont añitas,. Septem ber 2007. Gorgona G93. Pacifigorgia s p.. Octoc oral. Mont añitas,. Septem ber 2007. Gorgona G96. Pacifigorgia s p.. Octoc oral. Mont añitas, Gorgona. EF GBN MS: East Flower Garden Bank National Marine Sanctuary WFGBN MS: West Flower Garden Bank National Marine Sanctuary. 24. Septem ber 2007.

(25) ANN EX 2 – IGS/ETS LENG TH SPECIES. BAND LENGTH. Plu mapathes pennac ea. 1000 bp 1200 bp 1300 bp. Tanacetipathes barbadens is. 850 bp 1000 bp 1100 bp. Antipat hes atlantica. 900 bp 1500 bp 1600 bp 1800 bp. Muricea pinnata. 500 bp 1000 bp 1200 bp 1250 bp. Muric ea atlantic a. 600 bp 1100 bp 1250 bp. 25.

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