Chromosomal localization of 45S and 5S rDNA in 14

ORIGINAL ARTICLEChromosomal localization of 45S and 5S rDNA in 14species and the implications for genome evolution of genus EpimediumMao-Yin Sheng •Ling-Jiao WangReceived:17April 2010/Accepted:13August 2010/Published online:17September 2010ÓSpringer-Verlag 2010Abstract Studying the genome structure of Epimedium has been hindered by the large genomes and uniform kar-yotypes.Consequently our understanding of the genome organization and evolutionary changes of Epimedium is extremely limited.In the present study,the 45S and 5S rDNA loci of 14Epimedium species were physically mapped by double-probe FISH for the first time.Results showed the following:(1)Chromosomes I and II of all 14species examined,except for E.shuichengense ,hosted one pair of 45S rDNA sites,respectively.Most of the 45S rDNA sites gave clear signals and were positioned in the distal regions of the short arms.(2)All species studied of section Diphyllon were found to have one pair of 5S rDNA sites localized in the interstitial regions of the long arm of chromosome IV,and the two species of section Epimedi-um ,E.alpinum and E.pubigerum ,had two pairs of 5S rDNA sites localized in the interstitial regions of the long arm of chromosomes IV and V,respectively.(3)In section Diphyllon ,all species of small flower taxa,except E.shuichengense ,had three pairs of 45S rDNA sites,clearly more than species of big flower taxa,except E.davidii ,with two pairs of 45S rDNA sites.Based on the45S and 5S rDNA distribution patterns and other chro-mosomal morphological characteristics,six pairs of chro-mosomes can be unambiguously identified in all 14Epimedium species.The stable differentiation in 45S and 5S rDNA FISH patterns between the two sections suggests that chromosomal rearrangements and transpositional events played a role in the splitting of the two sections,and section Diphyllon may be more primitive than section Epimedium .In the same way,big flower taxa may be more primitive than small flower taxa in section Diphyllon .Keywords 45S and 5S rDNA ÁFISH ÁEpimedium ÁPhylogenesisIntroductionBarrenwort (Epimedium ,Berberidaceae)is a traditional Chinese medicine known as Yinyanghuo,Xianlinpi,and Yangheye in China (Zhang et al.2002;Xie and Sun 2006).The plant is effective in strengthening the kidneys and ameliorating rheumatism,and is widely used in the treat-ment of osteoporosis,hypertension,coronary heart disease (Guo and Xiao 1999),senility,cancer,etc.(Xie and Sun 2006;Zhang et al.2002).Many flavonol glycosides have been isolated from a number of Epimedium plants,although the relation between these flavonol glycosides and the pharmacological activities of the drug is still obscure (Guo and Xiao 1999).In Japan,Europe,and North America,barrenwort species are also very popular as graceful garden plants.Epimedium is a genus of the Old World.Members are dispersed from Japan to Algeria and mainly occur in eastern Asia and the Mediterranean countries (Stearn 2002).Approximately 80%of the species are found inM.-Y.Sheng (&)Postgraduate Administration Office,Guizhou Normal University,Guiyang,Guizhou 550001,China e-mail:shmoy@M.-Y.ShengSchool of Life Science,Nanjing University,Nanjing,Jiangsu 210093,China L.-J.WangSchool of Vocation and Technology,Guizhou Normal University,Guiyang,Guizhou 550001,ChinaPlant Syst Evol (2010)290:65–73DOI 10.1007/s00606-010-0349-3central-southeastern China(Ying2002).Linnaeus recorded this genus and its type species E.alpinum in1753.After that,Morren and Decaisne(1834),Franchet(1886), Komarov(1908),and Stearn(1938,2002)made mono-graphic and systematic studies of Epimedium(Stearn 2002).Thus far,more than60species of Epimedium are recognized.Stearn(2002)established the most compre-hensive classification system of this genus.In his mono-graph,he arranged the genus into two subgenera, Epimedium L.and Rhizophyllum(Fischer&Meyer)Stearn, mainly based on whether theflowering stem has leaves or not,and he divided the subgenus,Epimedium,into four sections mainly according to their geographical distribution and infrageneric relationship.Among the four sections, members of section Diphyllon(Kom.)Stearn were all endemic to China and subdivided into four series:Cam-panulatae Stearn,Davidianae Stearn,Dolichocerae Stearn, and Brachycerae Stearn based on corolla characteristics such as petal type,the form and relative size of the inner sepals and petals,andflower dimension.Up to now,many studies have been dedicated to deducing the system and evolution of this genus,including studies of pollen morphology(Zhang and Wang1983;Liang and Yan1991),isozymes(Koga et al.1991),molecular biology (Nakai et al.1996;Sun2004;Sun et al.2005),biogeography (Ying2002;Zhang et al.2007),and chemical classification (Guo and Xiao1999;Koga et al.1991;Guo et al.2008). Results showed that the infrageneric classification was gen-erally consistent with its phytogeographic distribution,and the phylogenetic relationship among sections was basically clear in Epimedium.However,the system and evolution of Chinese Epimedium species still is not revealed well for many Epimedium species with multifarious petals are nested together in China and the petal evolution is unintelligible (Guo et al.2008).Recently,much additional research has been performed to study the system and phylogeny of Epi-medium(Kim et al.2004;Zhang et al.2007;Sheng et al. 2008).As an intractable genus in taxonomy,however,several problems remain regarding the classification and phyloge-netic relationship of Epimedium species,especially for the Chinese Epimedium species.Many works on karyotype and chromosome C-banding of the genus Epimedium have also been undertaken to deduce the phylogenesis of Epimedium(Tanaka and Takahashi1981;Takahashi1989;Sheng and Chen 2007a,b,2008;Zhang et al.2008).The results of all of this work showed that karyotypes of all species studied were symmetrical(1A,2A,or3A of Stebbins)and similar to each other.Thus,it is very difficult to identify the chro-mosomes and study the genome organization and evolution of this genus based on the common karyotype analyses.Ribosomal DNAs(45S and5S rDNA)are valuable genetic units in plant genome research.The45S rDNA,encoding18S–5.8S–25S ribosomal RNA,is present in many hundreds of repeated units generally arranged in tandem arrays corresponding to the nucleolus organizer region(NOR)(Lapitan1992;Sastri et al.1992).Although the5S rDNA,encoding5S ribosomal RNA,has a similar organization to45S rDNA,its site along the chromosome is not revealed by any morphological characteristics.Ribo-somal DNAs have been physically mapped in many plants byfluorescence in situ hybridization(FISH),and the dis-tribution and numbers of rDNA in the genome differ among different plants(Abbo et al.1994;Hanson et al. 1996;Adachi et al.1997;Taketa et al.1999;Martina et al. 2010).They are excellent cytological markers for karyo-type analysis especially in species with many small and equal chromosomes.The use of FISH for mapping and detecting ribosomal DNAs directly on the chromosomes provides a useful approach for chromosome identification and studies of genome organization and evolution.Thus far,however,the physical location of the rDNA genes of barrenwort has not been reported yet.Thus,there is a clear need tofind out more about the localization of rDNA loci in Epimedium to generalize the patterns of rDNA loci distribution in this genus and the differentiation among species.In the present study,the45S and5S rDNA loci localization patterns in14species of Epimedium are investigated using DNA probes for the45S rDNA and5S rDNA to address the following questions:(1)Do the number and location of rDNA loci differ among species of Epimedium?(2)Does the difference in rDNA loci number correlate with a difference in the genome of Epimedium?(3)Can the variations in FISH patterns be explained with a phylogenetic framework of Epimedium?Materials and methodsPlant materialsFourteen species of Epimedium were examined,including 12species of section Diphyllon endemic to China and2 species of section Epimedium collected from the Munich Arboretum,Germany(Table1).They were cultivated in pots at a greenhouse of the Institute of Plant Genetics and Breeding,Guizhou Normal University,Guiyang,Guizhou, China.Voucher samples were stored at the Institute.Preparation of chromosome spreadsChromosome slides were prepared according to Sheng and Chen(2007a).Briefly,the root tips werefirst treated with 0.3%8-hydroxyquinoline for15–20°C for3h,fixed in Carnoy Isolution overnight,treated in4%(v/v)cellulose-pectinase at37°C for4h,then washed and soaked in66M.Sheng,L.WangT a b l e 1T h e E p i m e d i u m m a t e r i a l s t u d i e d a n d t h e 45S a n d 5S r D N A l o c iT a x aP o p u l a t i o n sV o u c h e r s N o .o f c h r o m o s o m e s N o .o f r D N A l o c i45S 5SS e c t i o n D i p h y l l o n (K o m .)S t e a r n (n a t i v e t o C h i n a )B i g flo w e r t a x aE .a c u m i n a t u mF r a n c h .K a i y a n g ,G u i z h o u ,C h i n a2004050101K Y 2n =124(I S S A T ,I I S )2(I V L )E .l e p t o r r h i z u m S t e a r n W u c h u a n ,G u i z h o u ,C h i n a 2009020101W C 2n =124(I S S A T,I I S )2(I V L )E .w u s h a n e n s e T .S .Y i n gK a i l i ,G u i z h o u ,C h i n a2007030201K L 2n =124(I S S A T ,I I S )2(I V L )E .l i s h i h c h e n i i S t e a r nY i s h a n ,T o n g r e n ,G u i z h o u ,C h i n a2008030201Y S 2n =124(I S S A T ,I I S )2(I V L )E .p a u c i flo r u m K .C .Y e nM a o x i a n ,S i c h u a n ,C h i n a2008060210M X 2n =124(I S S A T ,I I S )2(I V L )E .d a v i d i iF r a n c h .K u n m i n g ,Y u n n a n ,C h i n a2007070301K M 2n =126(I SS A T,I I S ,I V S )2(I V L )E .b a o j i n g e n s e Q .L .C h e n &B .M .Y a n g B a o x i n g ,S i c h u a n ,C h i n a2008060210B X 2n =124(I SS A T,I I S )2(I V L )S m a l l flo w e r t a x aE .p u b e s c e n s M a x i m .G u i y a n g A r b o r e t u m ,G u i z h o u ,C h i n a2007052001G Y2n =126(I S S A T ,I I S ,I V S )2(I V L )E .e c a l c a r a t u m G .Y .Z h o n gQ i n g c h e n g s h a n ,S i c h u a n ,C h i n a2008030201Q C2n =126(I S S A T ,I I S ,I V S )2(I V L )E .s a g i t t a t u m (S i e b .&Z u c c .)M a x i m .K a i y a n g ,G u i z h o u ,C h i n a2007110405K Y2n =126(I SS A T,I I S ,I V S )2(I V L )E .d o l i c h o s t e m o n S t e a r n Q i n g c h e n g s h a n ,G u i z h o u ,C h i n a2008110502Q C2n =126(I SS A T,I I S ,I V S )2(I V L )E .s h u i c h e n g e n s e S .Z .H e L i u p a n s h u i ,G u i z h o u ,C h i n a2009032001L P S2n =124(I SS A T,I V S )2(I V L )S e c t i o n E p i m e d i u m (n a t i v e t o E u r o p e a n d C a u c a s u s )E .a l p i n u m L .M u n i c h ,G e r m a n y2008121401M U2n =126(I S S A T ,I I S ,I V S )4(I V L ,V L )E .p u b i g e r u m M o r r .&D e c n e .M u n i c h ,G e r m a n y2008121405M U2n =128(I SS A T,I I S ,I V S ,V S )4(I V L ,V L )I S S h o r t a r m o f c h r o m o s o m e I ,I I S s h o r t a r m o f c h r o m o s o m e I I ,I V S s h o r t a r m o f c h r o m o s o m e I V ,V S s h o r t a r m o f c h r o m o s o m e V ,I V L l o n g a r m o f c h r o m o s o m e I V ,V L l o n g a r m o f c h r o m o s o m e V ,S A T s a t e l l i t e c h r o m o s o m eChromosomal localization of 45S and 5S rDNA in 14species and their implications67distilled water for90min,followed by smear preparation. The prepared slides were stored at-20°C or immediately used for FISH.Probe preparation and FISHThe probes of45S and5S rDNA cloned in the vector were kindly provided by Prof.Adelaide,Heidelberg University, and were labelled by the random priming method using Klenow fragment(Promega).Biotin-16-dUTP(Roche)was used for labeling5S rDNA and digoxigenin(DIG)-11-dUTP(Roche)for45S rDNA.FISH was carried out according to the manufacturer’s instructions.The chromosomal slides were treated with 100l L RNase(100l g mL-1in29SSC)at37°C for1h and then soaked twice for5min in29SSC.The chro-mosomes were then digested with proteinase K (1l g mL-1,Promega)at37°C for10min.The slides were dehydrated in a series of70,95,and100%ethanol and then air-dried.A20l L hybridization solution containing10% dextran sulfate,50%formamide,10ng l L-1of sheared salmon sperm DNA,2ng l L-1of denatured probes,and 29SSC was added to each slide.The slides were placed in a moisture chamber,denatured at85°C for13min,then immediately placed in a hybridization oven and kept at 37°C overnight for probe hybridization.The slides were then washed with20%formamide in0.19SSC at42°C for 10min,followed by two washes with29SSC at37°C for 5min,and29SSC at room temperature for5min.In situ hybridization was repeated on three tofive chromosome spreads for each species.DetectionBefore detection,the slides were soaked in detection buffer (5%BSA,100mM Tris-HCl pH7.5,150mM NaCl,0.1% Triton X-100)at37°C for30min.The45S rDNA signals were detected using anti-DIG-rhodamine(2ng mL-1; Roche),and5S rDNA signals were detected using avidin-FITC(2l g mL-1;Sigma,USA).The slides were coun-terstained with100l L DAPI(2l g mL-1)and observed under afluorescence microscope(BX51,Olympus)at 100910magnification.The FISH images were captured by a cooled CCD color chromatic camera.The arm lengths of chromosomes in the CCD-captured images of ten cells from each of two or three chromosome spreads of each species were measured.The arm ratio was calculated,and the average values were used to construct an ideogram of the karyotype for each species.The chro-mosomes were assigned consecutively according to their lengths,with the longest being assigned as I and the shortest as VI.For chromosomes of equal length,the chromosome with the longest short arm was assignedfirst.In addition to the length and arm ratios of the chromo-somes,secondary constrictions and FISH patterns are also informative characters for karyotyping.The location of rDNA loci on each chromosome is shown on the ideo-grams.The mapped positions are not precise quantitative representations,although the relative distances of each rDNA site from the centromere and telomere were con-sidered when constructing the ideograms.ResultsThe FISH results showed that the metaphase chromosomes were blue,while the signals were green(5S rDNA)and red (45S rDNA)after counterstaining(Figs.1,2).Clear,repro-ducible,unambiguous FISH signals from three tofive FISH replicates of each species were registered.Weak signals in centromeric regions were not analyzed.Examples of the45S and5S rDNA FISH patterns obtained for each species are shown in Figs.1and2,and an ideogram of the karyotypes is presented in Fig.3.The karyotype features of the14Epi-medium species are similar to those reported by Tanaka and Takahashi(1981),Takahashi(1989),and Sheng and Chen (2007a,b,2008).Chromosomes I–VI were found in all cases to be metacentric or submetacentric and to have similar lengths,except that chromosomes VI of E.sagittatum and E.letorrhizum were subtelocentric.However,the chromo-somal distribution patterns of the45S and5S rDNA sites differed considerably among the14species.In E.acuminatum(Figs.1a,3a), E.leptorrhizum (Figs.1b,3b),E.wushanense(Figs.1c,3c),E.lishihchenii (Figs.1d,3d),E.pauciflorum(Figs.1e,3e),and E.bao-jingense(Figs.1f,3f),two pairs of intense45rDNA site signals were detected,one localized on the secondary constrictions of their SAT chromosomes(the short arm of chromosome I),another localized at the end of the short arm of chromosome II.One pair of5S rDNA sites was detected in the interstitial region close to the centromere in the long arm of chromosome VI.In E.davidii(Figs.2a,3g),E.pubescens(Figs.2b,3h), E.ecalcaratum(Figs.2c,3i),E.sagittatum(Figs.2d,3j), and E.dolichostemon(Figs.2e,3k),three pairs of45S rDNA and one pair of5S rDNA sites were detected.One pair of45S rDNA loci was localized in the secondary constrictions of their SAT chromosomes(the short arm of chromosome I),and the other two were localized at the end of the short arms of chromosomes II and VI,respectively. The5S rDNA sites were localized in the interstitial region of the long arm of chromosome VI.A notable feature was that the short and long arms of chromosome VI hosted one pair of45S and5S rDNA sites,respectively.In E.shuichengense(Figs.2f,3l),two pairs of45S rDNA and one pair of5S rDNA sites were detected.One68M.Sheng,L.Wangpair of 45S rDNA was found in the secondary constrictions of the SAT chromosomes (the short arm of chromosome I)and another in the distal region on the long arm of chro-mosome VI.One pair of 5S rDNA sites was found in the interstitial regions of the long arm of chromosome VI.In E.alpinum (Figs.2g,3m),three pairs of 45S rDNA and two pairs of 5S rDNA sites were detected.One pair of 45S rDNA loci was found in the secondary constric-tions of the SAT chromosomes (the short arm of chro-mosome I),and the other two were found on the ends of the short arms of chromosomes II and VI,respectively.In contrast to the above species studied,two pairs of 5S rDNA sites were detected and localized in the interstitial regions of the long arms of chromosomes VI and V,respectively.In E.pubigerum (Figs.2h,3n),four pairs of 45S rDNA and three pairs of 5S rDNA sites were detected.Three pairs of 45S rDNA sites were localized in the distal regions of the short arms of chromosomes II,VI,and V,respectively.The others were localized in the secondary constrictions of the SAT chromosomes (the short arm of chromosome I).Similar to E.alpinum ,two pairs of 5S rDNA sites were found in the interstitial regions of the long arms of chro-mosomes VI and V,respectively.Fig.1FISH localization of 45S rDNA (red )and 5S rDNA (green )loci on somatic chromosomes in a E.acuminatum ,b E.letorrhizum ,c E.wushanense ,d E.lishihchenii ,e E.pauciflorum ,andf E.baojingense .Arrowheadsindicate the 5S rDNA signals.Scale bar 5l mChromosomal localization of 45S and 5S rDNA in 14species and their implications 69Fig.2FISH localization of 45S rDNA (red )and 5S rDNA (green )loci on somaticchromosomes in a E.davidii ,b E.pubescens ,c E.ecalcaratum ,d E.sagittatum ,e E.dolichostemon ,f E.shuichengense ,g E.alpinum ,andh E.pubigerum .Arrowheads indicate the 5S rDNA signals.Scale bar 5l m70M.Sheng,L.WangIn the Epimedium species examined,except for E.shuichengense ,chromosomes I and II all hosted one pair of 45S rDNA sites,respectively.Most of the 45S rDNA sites gave clear signals and were positioned in the distal regions of the short arms.No clear,unambiguous 45S signals were observed at the centromere in any case,although weak signals were detected in centromeric regions of one or two pairs of chromosomes in some species.All species studied were found with one pair of 5S rDNA sites localized in the interstitial regions of the long arm of chromosome VI,except two species,E.alpinum and E.pubigerum ,with two pairs of 5S rDNA sites localized in interstitial regions of the long arms of chromosomes VI and V,respectively.Based on the 45S and 5S rDNA distribution patterns and other chromo-somal morphological characteristics,six pairs of chro-mosomes can be unambiguously identified in all 14Epimedium species.In E.davidii , E.pubescens ,E.ecalcaratum ,E.sagittatum ,E.dolichostemon ,E.alp-inum ,and E.pubigerum ,chromosome VI hosted one pair of 45S and 5S rDNA sites on its short and long arms,respectively.DiscussionSince Kuroki did the studies of chromosome comparisons of Epimedium species in 1967(Kuroki 1967),the karyo-types of more than 40species of this genus have been observed (Kuroki 1970;Tanaka and Takahashi 1981;Takahashi 1989;Sheng and Chen 2007a ,b ,2008;Zhang et al.2008).All of these results indicate that all species studied are diploid (2n =12)with only two types of chromosomes,metacentric and submetacentric.Karyotypes of all species studied were symmetrical—1A,2A,or 3A of Stebbins—and similar to each other.Karyotype analyses of Epimedium species have revealed that the chromosomal morphology is highly conserved within the genus,and that most of the chromosomes have similar lengths and arm ratios,making precise karyotyping difficult (Sheng and Chen 2007a ).The chromosomes of Epimedium can not be identified precisely by the karyotype analyses.The correct ordering of chromosomes II–V has been ambiguous to date (Sheng and Chen 2007a ).The use of double-or multiple-probe FISH,in combination with other chromosomal characters,has facilitated the identification ofhomologousFig.3Ideograms of a E.acuminatum ,b E.letorrhizum ,c E.wushan-ense ,d E.lishihchenii ,e E.pauciflorum ,f E.baojingense ,g E.davidii ,h E.pubescens ,i E.ecalcaratum ,j E.sagittatum ,k E.dolichostemon ,l E.shuichengense ,m E.alpinum ,and n E.pubigerum karyotypes.The white dots represent signals of 45S rDNA and the white asterisks represent 45S rDNA signalsChromosomal localization of 45S and 5S rDNA in 14species and their implications 71chromosomes and the inference of genome evolution among plant species(Brown et al.1999;Heslop-Harrison 2000;Marcon et al.2005;Taketa et al.2005;Vaio et al. 2005;Cai et al.2006;Martina et al.2010).In the present study,among14species studied,chromosomes I,II,VI, and V can be detected with hybridization signals.In combination with other chromosomal characters,the alignment of all six homologous chromosomes of the barrenwort genome can be made precisely.This study reports,for thefirst time,the physical locations of45S and5S rDNA loci in species of genus Epimedium as determined by double FISH.The features of45S and5S rDNA loci distribution in the14Epimedium species can be discerned.The numbers of45S and5S rDNA loci ranged from two to four pairs,and from one to two pairs,respec-tively(Table1;Fig.3),similar to other plants in Berberid-aceae(Liu et al.2006),which suggests that the number of changes of45S and5S rDNA loci has been small within family Berberidaceae.According to the classification by Stearn,12species among the14species studied were from section Diphyllon,native to China,including7bigflower taxa and5smallflower taxa.The other two species,E.alp-inum and E.pubigerum,were from section Epimedium, collected from Germany(Table1).The results of the present study show the distribution pattern of5S rDNA loci of E.alpinum and E.pubigerum with two pairs of signals(4L and5L),obviously different from the other12species.The number of45S rDNA loci of E.alpnium and E.pubigerum—three and four pairs of signals,respectively—is also more than in the other12species.All of this supports the division of the sections Diphyllon and Epimedium within the genus Epimedium by Stearn(2002).The changes in rDNA loci may be attributed to the chromosomal rearrangement,exchange of unequal values, and amplification of latent rDNA copies by transposons, and during the course of the evolutionary process,the number of rDNA loci will have increased from the ancestral genome(Cai et al.2006).We therefore concluded that section Diphyllon should be a more ancestral taxon than section Epimedium in genus Epimedium.Results of previous karyotype analyses showed that the genome sizes of species E.alpinum and E.pubigerum were smaller than those of species of section Diphyllon(Sheng and Chen 2007a,b,2008).So there is a tendency for the genome to have decreased from the ancestral taxa in genus Epimedium over evolutionary time.For a long time,China has been considered as the origin centre of Epimedium,but this has not been confirmed owing to insufficient evidence.In the present study,results support China as the origin centre of Epimedium.In China,species of Epimedium can be divided into two groups based on theflower dimension:the smallflower and the bigflower groups(Guo and Xiao1999;Sheng and Chen2008).According to the present study,the5S rDNA loci were very conservative within section Diphyllon,and the12species studied all have one pair(4L)of sites. However,all species of smallflower taxa,except E.shui-chengense,have three pairs of45S rDNA sites,obviously more than the species of bigflower taxa,except E.davidii, with two pairs of45S rDNA loci,supporting the division based on theflower dimension and suggesting that the big flower taxa are more primitive than the smallflower taxa in Chinese Epimedium species.45S rDNA loci participate the structure of nucleolus organizer region(NOR),generally mapped on the sec-ondary constriction in morphology.Lima-de-Faria ana-lyzed the NOR of700species and found that the NOR site of87%species was on the short arm of the chromosome (Lima-de-Faria1973).In most species studied,the site of 45S rDNA was on the NOR or the short arm end of the chromosomes(Adachi et al.1997;Han et al.2002;Li and Arumuganathan2001;Liu et al.2006;Singh et al.2009). In the present study,the45S rDNA sites were located as expected on the NOR and the ends of the chromosomes. In the present study,the detection rate(about100%)of 45S and5S rDNA hybridization signals at interphase was higher than that at metaphase,suggesting that the target sequence with a lower degree of condensation was hybridized more easily at interphase.Results of the present study also showed that the signals of45S rDNA were stronger than those of5S rDNA,suggesting that the copies of45S rDNA should be more numerous than5S rDNA in the Epimedium genome.Until now,the physical location of45S or5S rDNA loci in family Berberidaceae had only be studied by Liu et al. (2006).In Liu et al.(2006),the45S rDNA of Berberis thunbergii DC(Berberidaceae)was single and physically located on the secondary constriction of the short arm of chromosome IX.Based on the data of chromosome num-bers,and the number and site of45S rDNA,the evolution of the genome in family Berberidaceae is likely to have experienced aneuploidy,chromosome break,rearrange-ment,cross,and so pared with45S rDNA,the change in the site and number of5S rDNA is smaller(Xu et al.2007).Mantovani et al.(2005)reported that there were only one or two pair sites of5S rDNA in most species studied,but did not state the pattern of distribution in chromosomes.However,the site,number,and distribution pattern of5S rDNA are observably conservative (Mantovani et al.2005;Liu et al.2003).In the present study,the5S rDNA was physically located on the long arm of the chromosome in the14Epimedium species for the first time,providing important data for genome evolution research in the genus 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