Application of random amplified polymorphic DNA

RESEARCH ARTICLEApplication of random amplified polymorphic DNA andinter-simple sequence repeat markers in the genus Crataegus H.Dai1,X.Guo1,Y.Zhang2,Y.Li1,L.Chang1&Z.Zhang1,21College of Horticulture,Shenyang Agricultural University,Shenyang,Liaoning,China2Key Laboratory of Agriculture Biotechnology of Liaoning Province,Shenyang,ChinaKeywordsCrataegus;genetic diversity;ISSR;RAPD.CorrespondenceZ.Zhang,College of Horticulture,Shenyang Agricultural University,Dongling Road120, Shenyang,Liaoning110161,China.Email:zhangz@Received:23September2007;revised version accepted:20June2008.doi:10.1111/j.1744-7348.2008.00290.x AbstractHawthorn(Crataegus spp.)has a long history as an ornamental and a source of medicine.We report the use of random amplified polymorphic DNA(RAPD) and inter-simple sequence repeat(ISSR)markers to determine genetic rela-tionships in the genus Crataegus.Twenty-eight accessions,including eight species(Crataegus pinnatifida,Crataegus bretschneideri,Crataegus maximowiczii, Crataegus kansuensis,Crataegus altaica,Crataegus songarica,Crataegus dahurica and Crataegus sanguinea)and two botanical varieties(C.pinnatifida var.major and C.maximowiczii var.ninganensis)were analysed.Twelve RAPD primers repro-ducibly and strongly amplified128fragments of which116were polymorphic; similarly,13ISSR primers generated127products of which119were poly-morphic.Dendrograms based on unweighted pair group method with arith-metic average analysis were constructed from both the RAPD and the ISSR data.Similarity coefficient based on RAPD and ISSR markers ranged from0.22 to0.98and0.23to0.98,respectively.The range in similarity coefficient indi-cated that the genus has a high level of genetic diversity.The Mantel test on the similarity matrices produced by RAPD and ISSR markers gave r=0.86, showing high correlation between RAPD and ISSR markers in their ability to detect genetic relationships between Crataegus accessions.RAPD and ISSR appear to be reliable methods for the analysis of genetic relationships among hawthorns.IntroductionHawthorn(Crataegus spp.),a genus of the Rosaceae fam-ily,grows as small shrubs or spreading trees with thorny branches,three-tofive-lobed deciduous leaves,white flowers and red,orange,yellow-green or yellow fruits (Dai et al.,2007).Including numerous hybrids,the Cra-taegus genus contains anywhere between140and200 species(Phipps et al.,2003).Besides its worldwide use as an ornamental,hawthorn ranks as one of the most ancient of pharmaceutical plants and is described in vari-ous pharmacopoeias(Rigelsky&Sweet,2002;Kao et al., 2005).The hawthorn fruit,which may be consumed fresh or in the form of sauce,preserved slices or juice, has been used in China as an aid to digestion for at least 2500years.As many as18species and6botanical varie-ties of Crataegus originated in China(Zhao&Feng,1996),among which the large fruited(6–17g)Chinese hawthorn(Crataegus pinnatifida Bge.var.major N.E.Br.) is represented by>100cultivars in northern China.A characterisation of diversity present is a prerequisite for the scientific exploitation of genetic resources.To date,in hawthorn,this has been based on variation for morpholog-ical traits,a method that suffers from low numbers of inde-pendent characters and often poor heritability(Archak et al., 2003).In contrast,DNA-based assays are generally highly heritable and available in unlimited numbers.Random amplified polymorphic DNA(RAPD)and inter-simple sequence repeat(ISSR)are molecular marker techniques based on PCR.The former employs a single short primer of arbitrary nucleotide sequence(Williams et al.,1990),while the latter targets the genomic DNA lying between the fre-quently occurring microsatellites(or simple sequence re-peats)by anchoring the primers to either end of theseAnnals of Applied Biology ISSN0003-4746Ann Appl Biol154(2009)175–181ª2008The Authors175features(Zietkiewicz et al.,1994).Both RAPD and ISSR techniques have several advantages such as simplicity of use,the short time required to obtain results,highly informative nature,lower cost and the use of a small amount of plant material(Qian et al.,2001).And there are many different RAPD primers that can be available commercially.RAPD and ISSR have been widely applied for DNAfingerprinting(Blair et al.,1999;Raina et al., 2001),for gene tagging(Molnar et al.,2000),for pedigree verification(Harada et al.,1992),for cultivar identification (Martins et al.,2003)and for assessing the level of genetic diversity in collections of germplasm(Qian et al.,2001; Souframanien&Gopalakrishna,2004).Molecular marker technology has yet to be applied to genus Crataegus.In the present paper,we describe a diver-sity study of hawthorn,based on RAPD and ISSR mark-ers,and compare the utility of these two techniques.Materials and methodsPlant materialsThe sample set of accessions consisted of28of the col-lection of130accessions of hawthorn,including eight species and two botanical varieties originating from China, maintained in the National Hawthorn Germplasm Re-pository at Shenyang.The sample included one represen-tative each of the wild species[Crataegus maximowiczii Schneid.,Crataegus kansuensis Wils.,Crataegus altaica (Loud.)Lange,Crataegus songarica C.,Crataegus dahurica Koehne,Crataegus sanguinea Pall.]and the botanical vari-ety C.maximowiczii Schneid.var.ninganensis S.Q.Nie& B.J.Jen,four accessions each of Crataegus bretschneideri Schneid.and C.pinnatifida Bge.,and a set of13ran-domly chosen cultivars of C.pinnatifida Bge.var.major N.E.Br.(Table1).Table1Informativeness of RAPD and ISSR primers used to genotype accessions of Crataegus spp.Number Name ofAccession SpeciesRAPD ISSRNo.ofScoredBandsNo.ofPolymorphicBandsPolymorphism(%)No.ofScoredBandsNo.ofPolymorphicBandsPolymorphism(%)1Maoshanzha Crataegus maximowiczii494183.7322475.02Ninganshanzha C.maximowicziivar.ninganensis514384.3443681.83Liaoningshanzha Crataegus sanguinea463882.6443681.84Ganshushanzha Crataegus kansuensis534584.9453782.25Aertaishanzha Crataegus altaica453782.2282071.46Zhungeershanzha Crataegus songarica393179.5393179.57Guangyeshanzha Crataegus dahurica524484.6393179.58Zuofu2Crataegus bretschneideri524484.6605286.79Jifu2 C.bretschneideri534584.9655787.710Fulihong C.bretschneideri544685.2645687.511Jifu1 C.bretschneideri534584.9625487.112Dashanlihong Crataegus pinnatifida544685.2484083.313Xinbinruanzi C.pinnatifida574986.0514384.314Fenshanlihong C.pinnatifida544685.2453782.215Chuizhishanlihong C.pinnatifida534584.9423481.016Yidu C.pinnatifida var.major554785.5524484.617Jilindawang C.pinnatifida var.major605286.7574986.018Xuzhoudahuo C.pinnatifida var.major554785.5534584.919Qiujingxing C.pinnatifida var.major635587.3605286.720Mopan C.pinnatifida var.major585086.2524484.621Liaohong C.pinnatifida var.major564885.7554785.522Qiuhong C.pinnatifida var.major675988.1564885.723Jianzirou C.pinnatifida var.major605286.7574986.024Tongliaohong C.pinnatifida var.major564885.7554785.525Wulinghong C.pinnatifida var.major564885.7524484.626Luanhong C.pinnatifida var.major635587.3564885.727Tugu1 C.pinnatifida var.major564885.7595186.428Yubeihong C.pinnatifida var.major524484.6554785.5ISSR,inter-simple sequence repeat;RAPD,random amplified polymorphic DNA.RAPD and ISSR markers in Crataegus H.Dai et al. 176Ann Appl Biol154(2009)175–181ª2008The AuthorsTotal DNA extractionGenomic DNA was extracted from leaves using the CTAB (cetyltrimethylammonium bromide)method described by Doyle&Doyle(1990),with minor modifications.The con-centration of CTAB in the extraction buffer was3%w/v. The concentration of dissolved DNA was estimated using a DU800spectrophotometer(Beckman Coulter,Fullerton, CA,USA).Random amplified polymorphic DNA and inter-simple sequence repeat amplificationsA set of148RAPD primers was tested,including133 10-mer primers purchased from Sangon Biological Engi-neering and Technology and Service Co.,Ltd.(Shanghai, China)1212-mer primers obtained from Wako Inc. (Osaka,Japan)and three designed by the National Insti-tute of Vegetable and Tea Science at Tsu in Japan.RAPD amplifications were carried out in20l L reactions contain-ing1ÂPCR buffer[50mM KCl,10mM Tris–HCl(pH9.0), 0.1%v/v Triton X-100,2mM MgCl2],0.2mM dNTP, 0.3l M primer,0.5U Taq DNA polymerase(Tiangen, Shanghai,China)and50ng template DNA.Amplification was achieved via an initial denaturing step(94°C/30s), followed by45cycles of94°C/30s,40°C/120s,72°C/ 180s and ending with an incubation of72°C/7min.For ISSR,26primers were selected from the University of British Columbia series(UBC807–812,814,815,823,824, 834–836,840–844,852,853,868,881and888–891),eight [VBV(AC)7,HBH(CT)7,GCV(TC)7,VCG(TC)7,BDV(AG)7, HVH(TGT)5,BDB(CAC)5and BDV(CAG)5]were as reported by Arnau et al.(2002)and were nominated st-01to st-08, respectively,in our laboratory and two[(BDB(AC)7, (AG)8YG],nominated SAU01and SAU02,were designed in our laboratory.ISSR amplifications were carried out in 20l L reactions containing1ÂPCR buffer[50mM KCl; 10mM Tris–HCl(pH9.0);0.1%v/v Triton X-100,3mM MgCl2],0.2mM dNTP,0.3l M primer,0.5U Taq DNA polymerase(Tiangen)and50ng template DNA.Amplifica-tion was achieved via an initial denaturing step(94°C/ 3min),followed by38cycles of94°C/30s,annealing tem-perature for60s,72°C/120s and ending with an incubation of72°C/7min.The optimal annealing temperature of each primer wasfirst established by temperature gradient PCR. Amplicons were separated by agarose gel electrophore-sis in TBE buffer,and profiles were visualised under UV light after staining in ethidium bromide.The experimental reproducibility of the RAPD and ISSR markers was as-sessed by performing two independent amplifications. Data analysisRAPD and ISSR products were scored as present or absent, disregarding variation in band intensity.Data analyses were performed using NTSYS-pc v2.10e(Rohlf,1992). Jaccard’s coefficient was used to estimate genetic iden-tity,calculated as N AB/(N AB+N A+N B),where N AB represents the number of shared bands between two profiles,N A the number of fragments unique to sample A and N B the number unique to sample B.A cluster analysis was based on the resulting similarity matrices, and the relationships between accessions were displayed as dendrograms based on the unweighted pair group method with arithmetic average(UPGMA)algorithm. Correlation between the two matrices obtained with two marker types was estimated by means of the Mantel test (Mantel,1967)with1000random permutations. ResultsRAPD banding patternsOf the148RAPD primers,30amplified clear and repro-ducible profiles.Fig.1is the representative of the extent of polymorphism of four accessions of Crataegus revealed by primers of A15and A17.Of the30RAPD primers,the 12that proved the most highly informative(Table2) were used to genotype the full panel of accessions. RAPDfingerprinting was based on128clear,reproduc-ible fragments,equivalent to a mean of10.7fragments per primer.Of these,116(9.7per primer)were poly-morphic.The size of the amplified fragmentsrangedFigure1Random amplified polymorphic DNA profiles of hawthorn accessions generated by primers A15and A17.M,100bp DNA ladder. 1–4,accessions:‘Yidu’(Crataegus pinnatifida var.major),‘Fulihong’(Crataegus bretschneideri),‘Zuofu2’(C.bretschneideri)and‘Ningan-shanzha’(Crataegus maximowiczii var.ninganensis).H.Dai et al.RAPD and ISSR markers in Crataegus Ann Appl Biol154(2009)175–181ª2008The Authors177from250to2100bp.The proportion of polymorphic fragments in the profile generated by each primer in turn ranged from66.7%(A20)to100%(A53,A56and A88)(mean90.7%).The most effective RAPD primer was A88(Table2).The number of fragments present in any particular profile varied from accession to accession from39(C.songarica‘Zhungeershanzha’)to67(C.pin-natifida var.major‘Qiuhong’)(Table1).Inter-simple sequence repeat band patternsExcept for UBC868and UBC890,all the ISSR primers amplified some PCR product.UBC891,BDB(AC)7and HBH(CT)7produced only weak and smeared bands. Thirteen of the primers generated clear and reproducible polymorphic banding profiles(Table3).Fig.2is the representative of the extent of polymorphism of28ac-cessions of Crataegus revealed by UBC835.Seven of these13primers were3#-anchored poly(GA)or poly (AG)based.The annealing temperature greatly affected the efficiency of amplification,and the difference between the theoretical melting temperature(T m)and the optimum annealing temperature ranged from24°C to5°C.The primers amplified a variable number of dis-tinct products,with the poly(AG)primers producing a greater number(12.7per primer)than poly(GA)(8.8 per primer).The13selected primers generated127clear, reproducible bands(mean of9.8per primer)(Table3). The size of the amplified fragments ranged from200to 2000bp.All but eight of the bands were polymorphic across the genotype set(mean of9.2polymorphic prod-ucts per primer).Individual primers generated66.7–100%polymorphism(mean93.8%),somewhat more than had been achieved by the RAPD primers.The most informative primer was SAU02,which amplified20 polymorphic products(Table3).The total number of fragments per accession varied from less than40to more than60(Table1).Dendrogram:interspecies and intraspecies relationshipAll the clear and reproducible bands generated by the12 chosen RAPD primers(Table2)were scored for presence or absence among the28accessions of Crataegus and used for the UPGMA cluster analysis.The dendrogram based on UPGMA analysis of the RAPD data is shown in Fig.3.The similarity coefficients ranged from0.22to0.98, indicating a good level of genetic diversity in theTable2Informativeness of a core set of12RAPD primers used to assess genetic diversity in a panel of28accessions of Crataegus spp.PrimerName Sequence No.ofScoredBandsNo.ofPolymorphicBandsPolymorphism(%)A15GGTGATGTCC161487.5A17GGGTTGCCGT121083.3A20AGCACTGTCA6466.7A28GGGATCGTGT151493.3A53GATAGCCGAC1111100A56TCGGCCTGCT1111100A70TGCAGCACCG5480.0A76CCACAGCAGT4375.0A88GAGCCCTCCA1616100A91GTGCCTAACC9888.9A95TGCCCGTCGT121191.7A96CTCTCCGCCA111090.9Total128116Average10.79.790.7Table3Informativeness of a core set of13ISSR primers used to assess genetic diversity in a panel of28accessions of Crataegus spp.Primer Name Sequence a T m(°C)Optimum AnnealingTemperature(°C)No.of ScoredBandsNo.ofPolymorphic BandsPolymorphism(%)UBC810(GA)8T50519888.9UBC811(GA)8C525377100UBC824(TC)8G525288100UBC834(AG)8YT535355100UBC835(AG)8YC55581313100UBC840(GA)8YT53581010100UBC841(GA)8YC55589666.7UBC853(TC)8RT53501212100UBC868(GAA)6485233100SAU02(AG)8YG55582020100st-06HVH(TGT)54951111090.9st-07BDB(CAC)559601111100st-08BDV(CAG)559559666.7Total127119Average9.89.293.8a R=(A,G);Y=(C,T);B=(C,G,T);D=(A,G,T);H=(A,C,T);V=(A,C,G).RAPD and ISSR markers in Crataegus H.Dai et al. 178Ann Appl Biol154(2009)175–181ª2008The AuthorsH.Dai et al.RAPD and ISSR markers in CrataegusFigure2Inter-simple sequence repeat profiles of hawthorn accessions generated by primer UBC835.M,100bp DNA ladder.1–28,accessions as 1.identified in TableFigure3Genetic relationships derived from an UPGMA-based analysis among a panel of Crataegus accessions(listed in Table1)based on random amplified polymorphic DNA(RAPD)or inter-simple sequence repeat(ISSR)genotyping.Ann Appl Biol154(2009)175–181ª2008The Authors179germplasm sample.The28accessions could be grouped into three clusters.Cluster I comprisedfive of the wild species(C.sanguinea, C.altaica, C.dahurica, C.kansuensis and C.maximowiczii)and one botanical variety(C.maximo-wiczii var.ninganensis).The C.bretschneideri,C.pinnatifida and C.pinnatifida var.major accessions grouped as cluster II,within which subclusters IIa and IIb contained,respec-tively,the wild C.pinnatifida and the C.bretschneideri ac-cessions.All the C.pinnatifida var.major cultivars fell into subcluster IIc.‘Zhungeershanzha’(C.songarica)formed a separate operational taxonomic unit III.The dendrogram based on the ISSR data is also shown in Fig.3.The accessions formed two clusters,one of which comprised six of the wild species(C.songarica, C.san-guinea,C.altaica,C.dahurica,C.kansuensis and C.maximo-wiczii)and other botanical variety C.maximowiczii var. ninganensis.C.bretschneideri,C.pinnatifida and C.pinnatifida var.major grouped into cluster II,within which three sub-clusters could be identified.Subcluster IIa comprised the C.bretschneideri genotypes,IIb the three wild accessions of C.pinnatifida and IIc all13cultivars of C.pinnatifida var.major along with one wild C.pinnatifida accession (‘Dashanlihong’).A difference emerged between the RAPD-and the ISSR-based dendrograms:in the latter,all the C.pinnatifida accessions(including the wild types and the cultivars)formed a larger cluster,and then grouped with the C.bretschneideri accessions.However,in the for-mer,the wild C.pinnatifida accessionsfirst clustered with C.bretschneideri.An analysis performed by means of a Mantel test (Mantel,1967)indicated a high correlation between RAPD and ISSR markers in their ability to detect genetic relationships between Crataegus accessions(r=0.86; t=9.00;P=0.002).DiscussionDifferences between molecular marker types with respect to their informativeness have been well rehearsed in the literature(e.g.for strawberry–Degani et al.,2001;Kuras et al.,2004;for pea–Ellis et al.,1998and for hop–Pat-zak,2001).In the present study,the RAPD markers gen-erated a genetic data set,which was more consistent than was the ISSR-based set with the geographical origins and the morphological variation of hawthorn.Crataegus has been divided into22sections(Phipps,1983),and the eight species and two botanical varieties of hawthorn used here belong to section Pinnatifidae(C.pinnatifida, C.pinnatifida var.major and C.bretschneideri),section San-guineae(C.maximowiczii,C.maximowiczii var.ninganensis, C.kansuensis, C.altaica, C.dahurica and C.sanguinea)or section Orientales(C.songarica).The phylogenetic tree of hawthorn based on RAPD(Fig.3)is completely consis-tent with these taxonomic sections,as all the accessions in cluster I belong to the Sanguineae,all those in cluster II to the Pinnatifidae and C.songarica(Orientales)was an out-lier.However,in the ISSR-based tree,the Sanguineae and Orientales accessions clustered together.Random amplified polymorphic DNA markers have sometimes been associated with a lack of reproducibility (Penner et al.,1993).However,if the PCR conditions are well controlled,a high level of reproducibility is attain-able(Mattioni et al.,2002).We devoted considerable ef-forts to optimise the components of the PCR,including the concentrations of MgCl2,dNTP,primer and Taq DNA polymerase and the quality and concentration of tem-plate DNA(data not shown).Furthermore,a relatively high annealing temperature(40°C)and a long annealing time(120s)were applied(Zhang et al.,2003).Although Penner et al.(1993)have drawn attention to the depen-dence of RAPD profiles on the PCR machine used,in our hands,two different thermocyclers–the Mastercycler Gradient produced by Eppendorf company and the PTC-200DNA Engine produced by MJ research company–gave highly consistent results,with nearly all the strongly amplified fragments proving to be fully repro-ducible.We conclude that RAPD analysis is a reliable technique for genotyping in hawthorn. 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