C, Q, and restriction enzyme banding of the chromosomes in brook trout (Salvelinus fontinalis) and Arctic charr (Salvelinus

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1 Hereditas 114: (1991) C, Q, and restriction enzyme banding of the chromosomes in brook trout (Salvelinus fontinalis) and Arctic charr (Salvelinus S. E. HARTLEY Department of Biological and Molecular Sciences, School of Natural Sciences, University of Stirling, Scotland HARTLEY, S. E C. Q, and restriction enzyme banding of the chromosomes in brook trout (Sali,elinus fontinalis) and Arctic charr (Salvelinus alpinus). - Hereditas 114: , Lund, Sweden. ISSN Received January 7, Accepted April 16, 1991 The brook trout (Salvelinus fontinalis) and the Arctic charr (Salvelinus alpinus) represent different phylogenetic lines of the salmonid fish genus Salvelinus. Chromosome banding studies utilizing C, Q, and restriction enzymes reveal differences between the two species for the amount and sequence composition of their heterochromatic DNA. Arctic charr, the more recently evolved of the two species, possesses more C and Q bands than the brook trout. With the exception of Alu I, the banding patterns produced by restriction enzymes bear a close resemblance to the C band pattern. Alu I eliminates staining from large pericentromeric regions, including the centromeric heterochromatin, in the chromosomes of both species and from some telomeric regions in Arctic charr. These findings show that the evolutionary divergence of the Arctic cham from the brook trout has been accompanied by accumulation of heterochromatic DNA and subsequent sequence divergence. S. E. Hartley, Department of Biological and Molecular Sciences, School of Natural Sciences, Uniivrsiry of Stirling, Stirling FK9 401, Scotland The brook trout and the Arctic cham are both members of the salmonid fish genus Salvelinus but represent divergent phyletic lines, with the brook trout being of more ancient origin (BEHNKE 1972, 1980; SAVVAITOVA 1980). The brook trout, Salvelinus fonrincrlis, is indigenous to North America but has been widely introducd throughout the world and is the most generalised of the charr species (BEHNKE 1980). A diploid chromosome number (2n) of 84 with a chromosome arm number (NF) of 100 has been widely reported (SVARDSON 1945; UYENO chromomycin A3 (CMA3) and silver staining for 1972; DAVISSON et al. 1973; L ~~and WRIGHT 1981; nucleolar organizer regions (NORs) (AMEMIYA and CAVENDER 1984; DISNEY and WRIGHT 1987). The GOLD 1986, 1988; GOLD 1984; GOLD et al. 1986; Arctic charr, Salvelinus alpinus, has a circumpolar KLIGERMAN and BLOOM 1977; PHILLIPS and HARTdistribution in the northern hemisphere and shows LEY 1988). a plasticity of form and behaviour which has led to The use of these techniques in salmonid fishes complex systematic relationships being postulated has revealed polymorphisms for both heterochro- for the species (BEHNKE 1980; SAVVAITOVA 1980). A karyotype of 2n = 78, NF = 98 has been reported for fish from Canada, Norway, and Scotland (CAVENDER 1984; HARTLEY 1989; PLEYTE et al. 1989) while fish from Sweden, Kamchatka and Maine, USA have been reported with 2n = 80, NF = 100 karyotypes (SVARDSON 1945; NYGREN et al. 1971; VASIL YEV 1975; DISNEY and WRIGHT 1987). Since detailed linear banding patterns similar to the G and R banding patterns of higher vertebrates have not been obtained in fish chromosomes (HART- LEY and HORNE 1985; SCHMID and GUTTENBACH 1988) chromosome banding in fishes has largely been confined to those techniques which stain regions containing repeated sequences: C and quinacrine (Q) banding for constitutive heterochromatin, matin bands and NORs within species (PHILLIPS and ZAJICEK 1982; PHILLIPS et al. 1985, 1988; PLEYTE et al. 1989), variation between closely related species (HARTLEY and HORNE 1984; PHILLIPS et al. 1986, 1989) and has suggested that subsets of heterochromatin exist within species (PHILLIPS and HARTLEY 1988; HARTLEY 1989).

2 254 5 E HAKI'LEY Hereditas 114 (1991) Recently, evidence for the existence of subclas- 5es of highly repeated DNA in constitutive heterochromatin of insects, mammals and amphibians has come from the digestion of fixed metaphase chromosomes with restriction enzymes (FI<RRL.CCI et al. 1987: LoPF.~-F~~K~A\~~I-/. et al. 1989: SC-HMID and DE AI.W:IDA 1988). In the four fish species studied to date. modified C banding patterns have been found following treatment of chromosomes with rehction enzymes. In the anguilliform fish, MI~~CICIIN hc~knu, the existence of different classer of highly repetitive DNA in centromeres and telomeres was demonstrated (C.41. et al. 1988). In the rainbow trout. Oric.or.Ji~,i~~/1zts n~$i.s.s. (LI-OYD and Ttma&wi) 19x8) and the brown trout. Su/nio triitr~. (H,AKTLL,Y I991 ) more distinct C banding patterns with more telomeric bands were obtained with several restriction enzymes uhile in the Atlantic salmon. Sulmo.ctrlur.. differences in the centromeric hetcrochromatin between metacentric and acrocentric chromosomes are suggested following digestion with Ddc I (HmrL.r;Y 19')l ). The salmonid fishes are ancient tetraploid species that have practically completed diploidization ( ALt.r:xL)oK!. and TtioRc; WRI) 1983). During this process extensive chromosome rearrangements involving both centric fusions and pericentric inversions have taken place during the evolution of modern \ahonid karyotypes (H,xrI.F.Y 19x7). It has been wgge\ted that during the evolutionary divergcnce of brook trout and Arctic charr difrerences in type well as number of fusions occurred (DISYEJ. and WRI(;H.T 1987). In this paper C. Q. and restriction enzyme banding of chromosomes have been used to investigate the relationships between these two species. Materials and methods Chromosome preparations u cre made from short term blood lymphocyte culture4 of 6 brook trout and 15 Arctic charr by previouslj described mehods (11 WI'I i Y and HORYE: 19x5). The fish Lvere a gift from the Freihivater Fisheries Laboratory. Pitlochr>. Thc restriction enzymes used were Alir I. IIiic, I. //w 111. Hi/i fi. Mho I. P\.it I1 and Rstr I i Gihco-HRi. or Phatmacia). For chromosome iiigestion I5 unit> of enzyme in 100 ~1 of the appropriate buffer nme applied to the chromosome preparation. covered with a coverslip and incubated ovcmight in a moist chamber at 37 C. The reaction \\a\ itopped ty washing the slides with distilled water and the chromosomes were stained in 4 % Giemsa in ph 6.8 buffer for min. C banding was performed according to SUMNER (1972). and Q banding according to PHILLIPS and HARTLEY (1988). Between 10 and 20 metaphases were examined for each treattnent from each individual. Slides were viewed with a Zeiss Universal microscope and photographed with Kodak Technical Pan film. Results In this study brook trout were found to have 2n = 84, NF = 100 ( 16 metacentrics/68 acrocentrics) and Arctic charr 2n = 78. NF = 98 (20 metacentrics/58 acrocentrics): karyotypes identical to those observed in previous studies. Distinct banding patterns were produced in both species with C banding, Q banding and following digestion with A h I, Dde I and Hue 111 (Fig. 1, 2, 3, 4-c and 5). Mho 1 (Fig. 4d) and Rsa 1 had effects on brook trout chromosomes that were not observed in Arctic charr. C banding In the brook trout. C banding reveals prominent C bands at the centromeres of all chromosomes, in the short arms of the largest acrocentric chromosome pair and at one telomere of a metacentric chromosome pair. while many other telomeres appear to be faintly banded (Fig. 1). The Arctic charr possesses prominent C bands at all centromeres and at some telomeres of both metacentric and acrocentric chromosomes, while other telomeres appear to be faintly banded. There is inter-individual variation for both the number and size of the prominent C bands (Fig. 3). (2 banding In brook trout (Fig. 3a) the centromeres of all the metacentric and one pair of acrocentric chromosomes are stained. while in Arctic charr (Fig. 3 b) Q bands are found primarily at telomeres. with fainter fluorescence of metacentric centromeres. In the Arctic charr there are always fewer Q banded telomeres than C banded telomeres (HARILEY 1989; PI.E\.TL: et al. 1989). Restriction enzyme banding The banding patterns obtained with restriction enzymes are shown in Fig. 4 and 5 and Table I, where

3 Herrditas 114 (1991) CHROMOSOME BANDING IN BROOK TROUT AND ARCTIC CHARR 255 Fig. 1. C banded brook trout metaphase with the prominently banded acrocentric short arms indicated by arrows. Bar = 10wm they are compared with those found in other salmonid fishes. The banding pattern produced by Alu I is similar in both brook trout and Arctic cham (Fig. 4a and 5a). All centromeric staining is abolished and dark bands become visible at the ends of all chromosomes. In some chromosomes in the Arctic charr these bands are not at the telomeres as a pale region distal to the dark band is discernible (Fig. 5). In brook trout Dde I (Fig. 4b), Hue I11 (Fig. 4c), and Mbo I (Fig. 4d) produce C-like banding patterns with many more telomeric bands in the Dde I and Hue I11 digested chromosomes than in conventionally C banded chromosomes. In all cases the C band positive short arms of the large acrocentric chromosomes are no longer visible. Treatment with Rsa I digests the chromosomes so that only a small amount of centromeric heterochromatin remains stained. In the Arctic charr Dde I (Fig. 5b) and Hue I11 produce patterns very similar to C banding although the Hue I11 pattern is indistinct. After Mhn 1 and Rsu I treatment no banding patterns are observed and the chromosomes are uniformly stained. Hin fi, Pvu I1 and buffer alone had no effect on either species chromosomes. Discussion Based on a number of morphological and meristic characteristics the brook trout is well separated from and of more ancient lineage than the Arctic

4 256 s t IIARILLY Her-editas 114 (1991) Fig. 2. C' banded karqotype\ ot Arctic charr to show interindividual differences in \ire and number ot prominent bands. The large acrocentric chromosonie pair with intenritial C hands are xro%ned. Bar. = I0 pm. charr tc \\I-.\DEK 1980; S-\L.\ uro\..\ 1980). This is \upported by the possession of a higher chromoiorne arid chromosome a n number although all the inember\ of %ih.rlinirs posses this primitive type ot Lar! otype dominated by acrocentric chromo- \onics th XKI I t.\ I?X7j. Centric fusions have played ;I primar) role in the evolution of salmonid karyotype5 Oi i.! rim 1?87) and the change in chromomne number from 84 with 16 metacentrics in brook trout to 78 with 20 metacentrics in Arctic charr may be accomplished by four centric fusions involving eight acrocentric chromosomes. The re- duction in chromosome arm number from 100 to 98 may have been accomplished in three ways: loss of two acrocentric chromosomes, tandem fusion of four acrocentrics, or centric fusion of four acrocentrics to produce two metacentric chromosomes followed by pericentric inversions in these nietacentrics. The latter two methods will result in a pair of large acrocentric chromosomes. Such a pair of chromosomes with a small interstitial C band exists in the Arctic charr (Fig. 2). A similar pair of chromosomes is present in the Dolly Varden, Salvefinus mulmu, (UEDA and OJIMA 1983), a sister species to

5 Ilrredrtus 114 (19911 CHROMOSOME BANDING IN BROOK TROUT AND ARCTIC CHARR 257 Fig. 3a and b. Q banded metaphases of a brook trout, and b Arctic charr. Bar = 10 pm

6 Hereditus 114 (1991) Fig..la-d. Hi-cioh irotii nieraphacec following treatmeni uiih a.a//( 1. h Dclc 1. c Htrc 111 and d Mho 1. Bar = 10 pm. the 4riiic ch'iri CC'\\ I \m ii 1980) and i \ considered chioino\ome\ of the Atlantic salmon, Solnio wlai h> C.i\ I K ( 19x4) to h,i\e anwn b\ the third a \pecies with a much reduced arm number ielative altcnidi\c outlined hove Thi\ mcchaiiism i\ also to the other \almond\, have been derived (~IAKTLF Y thought to be the \\J> in mhich the large acrocentric 1987) The suggestion by D I ~ Y I and WRKJIT

7 Hereditas 114 (1991) CHROMOSOME BANDING IN BROOK TROUT AND ARCTIC CHARR 259 Fig. 5a and b. Arctic charr metaphases following treatment with a Alu I and b Dde I. Arrows in a indicate telomeric regions that have been digested. Bar = 10 pm. ( 1987) that different types of fusion have occurred during the evolutionary divergence of brook trout and Arctic cham is based on their study of silver 4tained nucleolar organizer regions (NORs) where they found no NORs on metacentric chromosomes. Silver stains only those NORs that were active during the previous interphase. However, when brook trout chromosomes are stained with chromomycin A3 (CMA3), which stains NORs regardless of activity (AMEMIYA and GOLD 1986), a NOR site is found at a metacentric telomere (PHILLIPS et al. 1989). Thus, it seems unlikely that there have been different types of fusions in the two lines. Chromosome banding reveals that the chromosome changes that have taken place have been accompanied by accumulation of heterochromatic DNA and subsequent sequence divergence to form subsets within the heterochromatin. C and Q banding reveal substantial differences between the two species for possession of prominent chromosome bands, with many more bands present in the Arctic charr. No telomeric Q bands were observed in brook trout and there were always fewer telomeric Q bands than telomeric C bands in the Arctic charr. This suggests differences in sequence composition within the heterochromatin since quinacrine is known to stain AT rich DNA (COMINGS 1978). However, another AT specific fluorochrome, Hoechst 33258, produces fluorescent bands only at the centromeres of both brook trout and Arctic charr chromosomes (HARTLEY, unpublished), while only metacentric centromeres are stained in brook trout with DAPI, another AT binding dye (MAYR et al. 1988). This suggests that the telomeric Q bands of Arctic charr are not composed entirely of AT rich DNA sequences and that differences may exist between the centromeric heterochromatin of acrocentric and metacentric chromosomes of both species. The heterochromatin associated with the NORs in brook trout and Arctic charr is GC rich as revealed by its fluorescence with CMA3 (AMEMIYA and GOLD 1986). In other fish species it has been found to be digested by Alu I (AG!CT) and Ddc I (C!TNAG) as well as by Hue 111 (GG!CC) but not Mho I (!GATC) (CAU et al. 1988; HARTLEY 1991). The multichromosomal location of NORs and the interindividual variability for number in both brook trout and Arctic charr (PHILLIPS et al. 1989) make it difficult to positively identify the effects of restriction enzymes on their NOR associated heterochromatin. However, in brook trout, where a NOR is situated on the short arm of the longest acrocentric chromosome pair (PHILLIPS et al. 1989). all staining was eliminated from the short arms of the large acrocentric pair with those enzymes that produced banding patterns. The majority of banding patterns generated by digestion of chromosomes with restriction enzymes in many organisms are very similar to the C band pattern for that organism. Fish are no exception to this, with faint telomeric C bands often becoming more prominent following restriction enzyme treat- ment (CAU et al. 1988; LLOYD and THORGAARD 1988; HARTLEY 1991). The same effects are seen in both brook trout and Arctic charr with the exception of Ah I. In these two species, but not the other

8 C c" C Uonr c C C - &mmids studied to date. Lvhere C band patterns '41 ~ii/ji~ll,[/,q[,r~if,iir\. - I am grateful to R.B. Greer and A.F. itre generated (Table 1 ) (Li.om and THORG.A.ARD Walher. DAFS. Freshwater Fisheries Laboratory. Pitlochry, Perth- \hire. Scotland who provided fish for the study, and to Professor 1988: HARTLEY 1991). a distinct pattern (here called C.M. Gosden. MRC Human Genetics Unit. Western General Hos- \Iir banding). in which large pericentromeric re- pital. Edinburgh. Scotland tor useful discus\ions. Thi\ work was pions including the centroineric heterochrornatin \upported b)?rant no. GR3/69X5 from the Natural Environment itre digested away. is produced. In the Arctic charr Research Ciiiiiicil of Great Britain. \ome telomeric C bands also appeal- to be renioved..nir I produces banding patterns by extraction of chromosomal DNA leading to a reduction of staining in those areas uith a high frequenq of the recognition sequence (Bi.\\<wi et a\. 1985: M wrt-t, et al. 1985: LLOYD and THORGAARU I The banding pattern obtained has been found. in a Lariety of species. to reflect the distribution of their highly repeated or sate e DNA sequences (DF S.irtf;\o et al. 1986: %IF. \\(mi: et al. 1986: FIX- KI (TI et al. 1987) and has revealed qualit:itive differences in the lieterochroiiiatiii anti highly repetitive DN.4 of the great apes and miin ( F ~KKI (XI et x7,. The dif'ferences obtained in banding pattern beiueen the two.sii/iv//tiu.\ >\pecks and other salmoriith when their chromosomes are digested with.4/u 1 are reflected in the handing patterns obtained in qarose gels following electrophoregs ot' A/ir I ilige\ted DNA. Families of repeated sequences iire ire\ealecl in brook trout and.4rctic charr. but not in hroun trout. Atlantic salmon. and rainbow trout 1 H \f<ii.f > and D,\\,iosou, unpublished). Preliminary results suggest that these two families are not L.tosely related and further experiments to characterire and locali7e them are under way.

9 Hereditac I14 (1991) CHROMOSOME BANDING IN BROOK TROUT AND ARCTIC CHARR 26 1 B. L. BURNS), Unir,. Manitoba Press, Winnipeg, p COMINGS, D. E Mechanisms of chromosome banding and implications for chromosome structure. - Annu. Rev. Genet. 12: 2546 DAVISSON, M. T., WRIGHT, J. E. and ATHERTON, L. M Cytogenetic analysis of pseudolinkage of LDH loci in the teleos1 genus Sulvelrnus. - Genetics 73: Dr STEFANO, G. F., ROMANO, E. and FERRUCI, L The Alu I-induced bands in metaphase chromosomes of orangutan (Pongo pygmaeus). Implications for the distribution pattern of highly repetitive DNA sequences. -Hum. Gener. 72: DIWEY. J. E. and WRIGHT, J. E. JR Cytogenetic analyses of a Sulwlrnus hybrid reveal an evolutionary relationship between the parental species. - Cytogenet. Cell Genet. 45: FERRLCCI, L., ROMANO, E. and DE STtbANO, G. F The Alu I-induced bands in great apes and man: implication for heterochromatin characterization and satellite DNA distribution. - CJro,qenet. Cell Genet. 44: GOI I), J. R Silver-staining and heteromorphism of chromosomal nucleolar organizer regions in North American cyprinid fithe\. - Copeia I : GOLD, J. R., AMEMIYA, C. T. and ELLISON, J. R Chromowmal heterochromatin differentiation in North American cyprinid fishes. - Cyrologiu 51: HAKTI.EY, S. E The chromosomes of salmonid fishes. - Biol Re\,. 62: H~RTLLY, S. E Chromosomes and constitutive heterochromatin distribution in Arctic cham, Salvelinus alpinus (L.) (Pisces: Salmonidae). - Generica 79: HZRTL~Y. S. E. 1991, Restriction enzyme banding in Atlantic salmon (Sulmo sulur) and brown trout (Sulmo rrurra). - Gener. Kes. 57: HARTLtY, S. E. and HORNE. M. T Chromosome relation- \hips in the genus Sulmo. - Chromosoma YO: HAR.ILLY. S. E. and HORNT., M. T Cytogenetic techniques in fish genetics. - J. Fish Bid. 26: KLIGERM4N. A. D. and BLOOM, S. E Distribution Of F bodies, heterochromatin and nucleolar organizers in the genome of the central mudminnow, Umbra limi. - Cytugenet. Cell Gmet 18: Ltt. G. M. and WRIGHT, J. E Mitotic and meiotic analysis of brook trout Soli,elinusfontinali.~. - J. Hered. 72: LLOYD, M. A. and THORGAARD, G. H Restriction endonuclease banding of rainbow trout chromosomes. - Chromosoma 96: LOPTZ-FER~AVDEZ, C., GOSALVEZ, J. and MEZZANOTTE, R Heterochromatin heterogeneity in Oedipodu germunica (Orthoptera) detected by in situ digestion with restriction endonucleaies. -Heredity 62: MAYR, B., K~LAT, M. and RAB, P Heterochromatins and band karyotypes in three species of salmonids. - Theor. Appl. Gener. 76: MEZZANOTTE, R., FERRUCCI, L., VAYNI, R. and SUMNEK, A. T Some factors affecting the action of restriction endonucleases on human metaphase chromosomes. - E.yp. Cell Re.\ 16/: NYGREN, A,, NILSSON, B. and JAHNKE, M Cytological studies in Salmo trutta and Salmo ulpinus. - Her-edrtus 67: PHILLIPS, R. R. and HARTL~Y, S. E Fluorescent banding patterns of the chromosomes of the genus Salmo. - Ge~imc 30: PHILLIPS, R. B. and ZAJICEK, K. D Q band chromo\omal poiymorphisms in lake trout (Sulvelinits numu!( uth). - Genrrics 101: PHILLIPS, R. B., ZAJICEK, K. D. and UTTER, F. M Q band chromosomal polymorphisms in chinook salmon (0nwr.- hynchus tshau,y/schu). - Cop& 2: PHILLIPS, R. B., ZAJICEK. D. and UTTER, F. M Chromosome banding in salmonid fishes: nucleolar organizer region\ in Oncorhynchus. - Can. J. Genet. Cyrol. 28: PHILLIPS, R. B., PLEY.I.E, K. A. and HARrLEY, S. E Stochspecific differences in the number and chromosomal position\ of the nucleolar organizer regions in the Arctic char (Snlwlimc.\ alpinus). - Cytugmet. Cell Gener. 48: 9-12 PHILLIPS, R. B., PLEYTE, K. A. and IHSS~N, P. E Pattern\ of chromosomal nucleolar organizer region (NOR) variation in fishes of the genus Sulvelinus. - Copera I : PLEYTE, K. A., PHILLIPS, R. B. and HARTLEY, S. E Q-band chromosomal polymorphisms in Arctic char (Salwlrnrrs olpinus). - Genome 32: SAVVAITOVA, K. A Taxonomy and biogeography of charrs in the Palearctic. - In: Chorrs: Salmonid Fi.rhes ofrhr Genus Salvelinus (ed E. K. BALON), Publ. Dr W.Junk, The Hugitr. p SCHMID, M. and DE ALMEIDA, C. G Chromosome banding in amphibia XII. Restriction endonuclease banding. - Chromosoma 96: SCHMID, M. and GUTTENBACH, M Evolutionary diversity of reverse (R) fluorescent chromosome bands in VertebrdteS. - Chromosoma Y I 14 SUMNER, A. T A simple technique for demonstrating centromeric heterochromatin. - Exp Cell Res 7.5: SVARDSON, G Chromosome studies on Salmonidae. - Krport from the Swedish State Institute of Freshn~atei- Fislierj Research, Drottningholm. 23: UEDA, T. and OJIMA, Y. 1983, Karyotypes with C-banding patterns of two species in the genus Salvelinus of the family Salmonidae. - Pro(.. Jpn. Acad. 59B: UYENO, T Chromosomes of offspring resulting from crossing coho salmon and brook trout. - Jpn. J. Ichth!ol. 19: VASIL'YEV, V. P Karyotypes of some forms of Arctic ch;m, Salvelinus alpinus from Kamchatka. -J. I(.hrh:o/. IS: