Previous molecular investigations into Rubus species diversity have yielded significant knowledge about species relatedness. However, little work has been focused at the cultivar level. Random amplified polymorphic DNA(RAPD)-PCR studies have successfully differentiated closely related cultivars. The ability to definitively distinguish blackberry and red raspberry cultivars based on other molecular methods could prove useful in many aspects of breeding and proprietary protection. In this study, the nuclear ribosomal DNA internal transcribed spacer regions (ITS) of six Rubus cultivars were sequenced. DNA sequencing revealed little genetic variation among blackberry cultivars, but revealed distinctions between blackberry and red raspberry cultivars. Analysis by maximum-parsimony and maximum-likelihood confirmed the small variation among blackberry cultivars, although cultivars Apache, Brazos, and APF-12 did organize a weak sub-cluster within the blackberry genotypes. However, ITS region sequences do not appear to differentiate among closely related blackberry genotypes for purposes of cultivar discrimination or plant patent protection

Clark, John R.

Stafne, Eric T.

Szalanski, Allen L.

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 57 Article 242003Nuclear Ribosomal ITS Region Sequences forDifferentiation of Rubus GenotypesEric T. StafneUniversity of Arkansas, FayettevilleJohn R. ClarkUniversity of Arkansas, FayettevilleAllen L. SzalanskiUniversity of Arkansas, FayettevilleFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Botany Commons, and the Entomology CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academyof Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu.Recommended CitationStafne, Eric T.; Clark, John R.; and Szalanski, Allen L. (2003) "Nuclear Ribosomal ITS Region Sequences for Differentiation of RubusGenotypes," Journal of the Arkansas Academy of Science: Vol. 57 , Article 24.Available at: http://scholarworks.uark.edu/jaas/vol57/iss1/24176Nuclear Ribosomal ITS Region Sequences forDifferentiation of Rubus GenotypesEric T.Stafhe* and John R. ClarkDepartment of HorticultureAllen L.SzalanskiDepartment of EntomologyUniversity of ArkansasFayetteville, AR 72701University of ArkansasFayetteville, AR 72701Corresponding AuthorAbstractPrevious molecular investigations into Rubus species diversity have yielded significant knowledge about speciesrelatedness. However, little work has been focused at the cultivar level. Random amplified polymorphic DNA (RAPD)-PCRstudies have successfully differentiated closely related cultivars. The ability to definitively distinguish blackberry and redraspberry cultivars based on other molecular methods could prove useful in many aspects of breeding and proprietaryprotection. In this study, the nuclear ribosomal DNAinternal transcribed spacer regions (ITS) of six Rubus cultivars weresequenced. DNA sequencing revealed little genetic variation among blackberry cultivars, but revealed distinctions betweenblackberry and red raspberry cultivars. Analysis by maximum-parsimony and maximum-likelihood confirmed the smallvariation among blackberry cultivars, although cultivars Apache, Brazos, and APF-12 did organize a weak sub-cluster withinthe blackberry genotypes. However, ITS region sequences do not appear to differentiate among closely related blackberrygenotypes for purposes of cultivar discrimination or plant patent protection.IntroductionBlackberries (Rubus subgenus Rubus Watson; subgenusEubatus) and red raspberries (R. idaeus L.; subgenusIdaeobatus) are economically and ecologically importantfruiting plants which belong to the diverse genus Rubus.Most modern Eastern American blackberry cultivars werederived from diploid blackberry species R. allegheniensisPorter and R. argutus L. (Hall, 1990). Morphologicaldifferentiation between blackberries and raspberries is fairlywelldelineated withdistinct fruiting characteristics being theprimary recognition, but these identifiers are not presentthroughout the year (Parent and Page, 1992). Also,raspberry species have been used in blackberry breeding,thus donating significant traits which were not previouslyknown in blackberry germplasm (e.g., fall-fruiting habit).Breeding of new Rubus cultivars has resulted in a narrowingof the genetic diversity, thus making definitive cultivaridentification difficult (Jennings, 1988). However, little isknown about the actual relationships among cultivars totheir progenitors. Therefore, improvement in cultivaridentification could be ofsignificant benefit to verifyidentityand assist inconfirming proprietary rights.Since the internal transcribed spacer (ITS) region isrepeated within plant nuclear genomes, amplification andsequencing of the nuclear ribosomal DNA is easily achieved(Baldwin et al., 1995). Previous studies on Rubus involvingnuclear ribosomal DNAITS region sequences have focusedentirely at the species level (Alice, 2002; Alice andCampbell, 1999; Alice et al., 2001). Alice and Campbell(1999) reported that Rubus ITS sequences are mainlyinformative among subgenera, but variability is lowbetween closely related species. Since the lineages of mostblackberry cultivars include several distinct species, itmaybe possible to discriminate among the cultivars based onITS sequences.The objectives of this study were to distinguishblackberry and raspberry genotypes with the use of ITSsequences and also to ascertain ifthe results obtained wouldbe useful for incorporation into a Rubus breeding programand plant patent protection.Materials and MethodsActively growing leaf material was sampled from sixRubus cultivars (Table 1) growing in the fieldduring summer2001 at the Univ. of Arkansas Fruit Research Substation,Clarksville or at the Agricultural Research and ExtensionCenter in Fayetteville. Allplant material was stored in afreezer (-20° C) until the DNA extraction procedure wasinitiated, which was generally within 24 hours. Activelygrowing leaf tissue (100 mg) was ground to a powder with amortar and pestle in the presence of liquid nitrogen. DNAextraction followed manufacturer's protocols (QiagenDneasy Plant Mini Kit, Valencia, CA). DNA was re-precipitated with ammonium acetate (one-half of theoriginal extraction volume) and 100% ethanol (three timesthe extraction volume plus the ammonium acetate). TheJournal of the Arkansas Academy of Science, Vol. 57, 2003*176Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24Published by Arkansas Academy of Science, 2003177Nuclear Ribosomal ITS Region Sequences for Differentiation of Rubus GenotypesTable 1. Parentage and program of origination of six Rubus genotypes studied.Genotype Parentage Program locationApache (SIUS 68-6-15 x Comanche) xNavaho Fayetteville, ArkansasAPF-12 2 Arapaho x A-830 FayettevilleArapaho (A-550 x Cherokee) xA-883 FayettevilleBrazos Lawton x Nessberry College Station, TexasHeritage v (Milton x Cuthbert) x Durham Geneva, New YorkIlliniHardy NY95 x Chester Thornless Carbondale, IllinoiszUnreleased breeding selection, not an item of commerce.vRaspberry cultivar.Table 2. Pairwise genetic distances among nine Rubus genotypes and two outgroup taxa.No. Genotype 1 2 3 I 5 6 7 8 <) 10 II1 APF-122 Apache 0.0093 Brazos 0.009 0.0034 Heritage 0.039 0.036 0.0385 R. idaeus 0.040 0.035 0.036 0.0036 IlliniHardy 0.014 0.007 0.009 0.028 0.030IR. allegheniensis 0.012 0.004 0.006 0.027R. argutus 0.012 0.006 0.008 0.028 0.000. . 0.030 0.032 0.001 0.0009 Arapaho 0.014 0.004 0.006 0.031 0.033 0.003 0.000 0.00110 A. argute? 0.702 0.697 0.699 0.696 0.699 0.699 0.699 0.696 0.69711 V. corymbosum 2 0.705 0.703 0.703 0.705 0.704 0.2870.705 0.708 0.706 0.703zOutgroup taxa.Iesulting DNApellet was dried and then washed with 70%thanol and re-suspended in 50 /uh of TE buffer (pH 7.4).tThe PCR reaction mixtures for each sample weremprised of 5 [xLof 10X Taq buffer withMgCl2 (Promega,adison, WI), 4 /uL dNTP (Pharmacia Biotechnology,scataway, NJ), 1 /uL primer ITS 4 (5'TCCTCCGCTTATTGATATGC- 3'), 1 /uL ITS 5 (5' -GGAAGTAAAAGTCGTAACAAGG- 3') (White et al.,1990), 0.4 Taq polymerase (Promega), and 40 /xL doubledistilled water all of which were master mixed to ensureuniformity among reagents. An aliquot of 48 was addedto a 0.5 ml thin- walled microcentrifuge tube (Sarstedt, NC),Journal of the Arkansas Academy of Science, Vol. 57, 2003177Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24http://scholarworks.uark.edu/jaas/vol57/iss1/24I178Eric T. Stafne, AllenL. Szalanski, and John R. ClarkFig. 1. Consensus parsimony tree with nine Rubus genotypes and two outgroups based on ITS region DNA sequences.Bootstrap values greater than 50% are indicated at nodes.then 2 /uh of template DNA was added to comprise a totalreaction volume of50 /uh. A Techne Touchgene 40 x 0.5 ml(Techne Corp., Princeton, NJ) was used toperform the PCR.The thermocycling program used for this study consisted ofan initial denaturation of 94 °C for 2min., then 40 cycles of94° C for 45 s, 46° C for 1min, and 72°C for 1min, followedby a final extension for 5 min at 72°C.A 1% agarose solution (Fisher Scientific, St. Louis, MO),was placed into the gel box (Owl Scientific, Cambridge,MA) filled with 0.5X TBEbuffer solution. The PCR productand dye buffer totaled 10 fA.Ten fA of 100 bp DNA ladder(Promega) was added in the first well. Ethidium Bromidewas added to the buffer solution and the voltage was set at~165 V. The electrophoresis ran for 50 min. The gel wasremoved and photographed in the BioDoc-It imagingsystem (UVP, Inc., Upland, CA).Amplified DNA of six sample genotypes (fiveblackberry and one raspberry) was purified as described inJournal of the Arkansas Academy of Science, Vol. 57, 2003178Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24Published by Arkansas Academy of Science, 2003:>I•.179Nuclear Ribosomal ITS Region Sequences for Differentiation of Rubus GenotypesSzalanski et al. (2000). The purified DNA samples were sentto the DNA core sequencing lab at the Univ. of Arkansas(Fayetteville) to be sequenced in both directions with anABIPrism 377 DNA sequencer. An amplified fragment ofDNA between 700 and 800 bp in size was sequenced for allsamples. However, to understand genetic relationships withexisting Genbank DNA sequences, the sequences werereduced to 643 bp.Sequences derived from ribosomal DNA were alignedwith the Genetics Computer Group (GCG, Madison, WI)PILEUP program (gap weight = 5.0, gap length weight =1.0) and used Vaccinium corymbosum L. and Actinidia arguta(Sieb. & Zucc.) Planch, ex Miq. as outgroups. Maximumlikelihood and maximum parsimony analyses wereperformed with PAUP* 4.0b 10 (Swofford, 2002). Bootstraptesting (100 re-sampled datasets) was used to determine treerobustness (Felsenstein, 1985).Results and DiscussionBases were revealed at the frequency of 0.20967,0.28372, 0.27825, and 0.22836 for A, C, G, and T,respectively. Existing Genbank sequences for A. arguta(AF323836; Liet al., 2002) and V. corymbosum (AF419778;Powell and Kron, unpublished data) were used as outgroups.R. idaeus (AF0557552), R. allegheniensis (AF055772), and R.argutus (AF0557742) were existing Genbank DNAsequencesthat were included to compare species with the moderncultivars included in the study (Alice and Campbell, 1999).Parsimony analysis resulted in 643 total charactersconsisting of 147 constant characters (22.8%), 176 variablecharacters that were parsimony-uninformative (27.3%), and320 parsimony-informative characters (49.7%). Pairwisegenetic distances (Tajima and Nei, 1984) ranged from 0.00('IlliniHardy 7/2. allegheniensis and R. allegheniensis/ R. argutus)to 0.04 (APF-12//?. idaeus) within the Rubus cultivars (Table2). Alice and Campbell (1999) reported that ITS sequencedivergence among species of the blackberry subgenus averagedonly 1.2%, and this appears also be a close representation ofthe data obtained in this study.Only one parsimonious tree was derived from the dataset (Fig. 1) with a length of 603 and a consistency index (CI)of 0.982. Bootstrap analysis indicated little divergenceamong blackberry cultivars and species, but suggested aweak sub-cluster of APF-12, 'Brazos', and Apache'. Thevalue of 51% at the APF-12/'Brazos' node implies a veryweak support for that separation. The maximum-likelihoodanalysis starting with the parsimonious tree found 3 trees,Dut all were similar and none of the changes resulted in asignificant change in topology. The resulting tree was similarn topology to that of the consensus parsimony tree (data notshown). Blackberry and red raspberry genotypes wereseparated as distinct clusters by both the maximum-jarsimony and maximum-likelihood tests. The raspberrygenotypes displayed informative deletions at sites 75 and442, whereas the blackberry genotypes had a deletion at 515(data not shown). These are likely strong indicators fordifferentiating the two groups.The phylogenetic maximum likelihood tree derivedfrom PAUP* differentiated the subgenera Idaeobatus andEubatus (red raspberry and blackberry, respectively), but didnot recognize any notable differences within the blackberrycultivars. Thus, Rubus cultivar differentiation, which hasbeen done with other molecular techniques (Stafne et al.,2003), does not appear to be viable with ITS regionsequences. Blackberry cultivars containing more divergentspecies have yet to be tested and may show distinction fromthe other blackberry cultivars tested in this study.GonclusionsThe main benefit for Rubus appears to be ifmorphological ambiguity exists. ITS sequence analysis maydistinguish between red raspberry and blackberrygenotypes, but probably not within cultivars of those groupsunless they contain highly divergent progenitor species.Since ITS sequences cannot distinguish between similarcultivars, its utility for plant patent protection is limited.Literature CitedAlice, L. A. 2002. Evolutionary relationships in Rubus(Rosaceae) based on molecular data. Acta Hort. 585:79-83.Alice, L. A., and C. S. Campbell. 1999. Phylogeny ofRubus (Rosaceae) based on nuclear ribosomal DNAinternal transcribed spacer region sequences. Amer. J.Bot. 86:81-97.Alice, L. A., T. Eriksson, B. Eriksen, and C. S.Campbell. 2001. Hybridization and gene flowbetweendistantly related species of Rubus (Rosaceae): evidencefrom nuclear ribosomal DNA internal transcribedspacer region sequences. Syst. Bot. 26:769-778.Baldwin, B. G., M.J. Sanderson, J. M. Porter, M. F.Wojciechowski, C. S. Campbell, and M. J.Donoghue. 1995. The ITS region of nuclear ribosomalDNA: a valuable source of evidence on angiospermphylogeny. Ann. Missouri Bot. Garden 82:247-277.Felsenstein, J. S. 1985. Confidence limits on phylogenies:an approach using bootstrap. Evolution 39:783-791.Hall, H. K. 1990. Blackberry breeding. Pp. 249-312, InPlant Breeding Reviews (J. Janick ed.) Vol. 8. TimberPress, Portland, Oregon.Jennings, D. L. 1988. Raspberries and blackberries: theirbreeding, diseases and growth. Academic Press,London.Li,J., H.Huang, and T. Sang. 2002. Molecular phylogenyand infrageneric classification of ActinidiaJournal of the Arkansas Academy of Science, Vol. 57, 2003179Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24http://scholarworks.uark.edu/jaas/vol57/iss1/24180Eric T. Stafhe, AllenL.Szalanski, and John R. Clark(Actinidiaceae). Syst. Bot. 27:408-415.Parent, J. G., and D. Page. 1992. Identification ofraspberry cultivars by random amplified polymorphicDNA (RAPD) analysis. HortScience 27:1108-1110.Stafhe, E. T., J. R. Clark, M. C. Pelto, and J. T.Lindstrom. 2003. Discrimination of Rubus cultivarsusing RAPD markers and pedigree analysis. Acta Hort.2003. 626:119-124.Swofford, D.L.2002. PAUP*: phylogenetic analysis usingparsimony (*and other methods), version 4 beta 10.Sinauer, Sunderland, Massachusetts.Szalanski, A.L.,D. S. Sikes, R. Bischof, and M. Fritz.2000. Population genetics and phylogenetics of theendangered American burying beetle, Nicrophorusamericanus (Coleoptera: Silphidae). Ann. Ento. Soc.Amer. 93:589-594.Tajima, E, and M.Nei. 1984. Estimation of evolutionarydistance between nucleotide sequences. Mol.Biol.Evol.1:269-285.White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990.Amplification and direct sequencing of fungalribosomal RNA genes for phylogenetics, p. 315-322. InPCR protocols: a guide to methods and applications.(D.G.M. Innis, J. Sninsky, and T. White, eds.).Academic Press, San Diego, California.Journal of the Arkansas Academy of Science, Vol. 57, 2003180Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24Published by Arkansas Academy of Science, 2003

Nuclear Ribosomal ITS Region Sequences for Differentiation of Rubus Genotypes

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 57 Article 24 2003 Nuclear Ribosomal ITS Region Sequences for Differentiation of Rubus Genotypes Eric T. Stafne University of Arkansas, Fayetteville John R. Clark University of Arkansas, Fayetteville Allen L. Szalanski University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Botany Commons, and the Entomology Commons Recommended Citation Stafne, Eric T.; Clark, John R.; and Szalanski, Allen L. (2003) "Nuclear Ribosomal ITS Region Sequences for Differentiation of Rubus Genotypes," Journal of the Arkansas Academy of Science: Vol. 57 , Article 24. Available at: https://scholarworks.uark.edu/jaas/vol57/iss1/24 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This Article is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. 176Nuclear Ribosomal ITS Region Sequences forDifferentiation of Rubus GenotypesEric T.Stafhe* and John R. ClarkDepartment of HorticultureAllen L.SzalanskiDepartment of EntomologyUniversity of ArkansasFayetteville, AR 72701University of ArkansasFayetteville, AR 72701Corresponding AuthorAbstractPrevious molecular investigations into Rubus species diversity have yielded significant knowledge about speciesrelatedness. However, little work has been focused at the cultivar level. Random amplified polymorphic DNA (RAPD)-PCRstudies have successfully differentiated closely related cultivars. The ability to definitively distinguish blackberry and redraspberry cultivars based on other molecular methods could prove useful in many aspects of breeding and proprietaryprotection. In this study, the nuclear ribosomal DNAinternal transcribed spacer regions (ITS) of six Rubus cultivars weresequenced. DNA sequencing revealed little genetic variation among blackberry cultivars, but revealed distinctions betweenblackberry and red raspberry cultivars. Analysis by maximum-parsimony and maximum-likelihood confirmed the smallvariation among blackberry cultivars, although cultivars Apache, Brazos, and APF-12 did organize a weak sub-cluster withinthe blackberry genotypes. However, ITS region sequences do not appear to differentiate among closely related blackberrygenotypes for purposes of cultivar discrimination or plant patent protection.IntroductionBlackberries (Rubus subgenus Rubus Watson; subgenusEubatus) and red raspberries (R. idaeus L.; subgenusIdaeobatus) are economically and ecologically importantfruiting plants which belong to the diverse genus Rubus.Most modern Eastern American blackberry cultivars werederived from diploid blackberry species R. allegheniensisPorter and R. argutus L. (Hall, 1990). Morphologicaldifferentiation between blackberries and raspberries is fairlywelldelineated withdistinct fruiting characteristics being theprimary recognition, but these identifiers are not presentthroughout the year (Parent and Page, 1992). Also,raspberry species have been used in blackberry breeding,thus donating significant traits which were not previouslyknown in blackberry germplasm (e.g., fall-fruiting habit).Breeding of new Rubus cultivars has resulted in a narrowingof the genetic diversity, thus making definitive cultivaridentification difficult (Jennings, 1988). However, little isknown about the actual relationships among cultivars totheir progenitors. Therefore, improvement in cultivaridentification could be ofsignificant benefit to verifyidentityand assist inconfirming proprietary rights.Since the internal transcribed spacer (ITS) region isrepeated within plant nuclear genomes, amplification andsequencing of the nuclear ribosomal DNA is easily achieved(Baldwin et al., 1995). Previous studies on Rubus involvingnuclear ribosomal DNAITS region sequences have focusedentirely at the species level (Alice, 2002; Alice andCampbell, 1999; Alice et al., 2001). Alice and Campbell(1999) reported that Rubus ITS sequences are mainlyinformative among subgenera, but variability is lowbetween closely related species. Since the lineages of mostblackberry cultivars include several distinct species, itmaybe possible to discriminate among the cultivars based onITS sequences.The objectives of this study were to distinguishblackberry and raspberry genotypes with the use of ITSsequences and also to ascertain ifthe results obtained wouldbe useful for incorporation into a Rubus breeding programand plant patent protection.Materials and MethodsActively growing leaf material was sampled from sixRubus cultivars (Table 1) growing in the fieldduring summer2001 at the Univ. of Arkansas Fruit Research Substation,Clarksville or at the Agricultural Research and ExtensionCenter in Fayetteville. Allplant material was stored in afreezer (-20° C) until the DNA extraction procedure wasinitiated, which was generally within 24 hours. Activelygrowing leaf tissue (100 mg) was ground to a powder with amortar and pestle in the presence of liquid nitrogen. DNAextraction followed manufacturer's protocols (QiagenDneasy Plant Mini Kit, Valencia, CA). DNA was re-precipitated with ammonium acetate (one-half of theoriginal extraction volume) and 100% ethanol (three timesthe extraction volume plus the ammonium acetate). TheJournal of the Arkansas Academy of Science, Vol. 57, 2003*176Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24Published by Arkansas Academy of Science, 2003177Nuclear Ribosomal ITS Region Sequences for Differentiation of Rubus GenotypesTable 1. Parentage and program of origination of six Rubus genotypes studied.Genotype Parentage Program locationApache (SIUS 68-6-15 x Comanche) xNavaho Fayetteville, ArkansasAPF-12 2 Arapaho x A-830 FayettevilleArapaho (A-550 x Cherokee) xA-883 FayettevilleBrazos Lawton x Nessberry College Station, TexasHeritage v (Milton x Cuthbert) x Durham Geneva, New YorkIlliniHardy NY95 x Chester Thornless Carbondale, IllinoiszUnreleased breeding selection, not an item of commerce.vRaspberry cultivar.Table 2. Pairwise genetic distances among nine Rubus genotypes and two outgroup taxa.No. Genotype 1 2 3 I 5 6 7 8 <) 10 II1 APF-122 Apache 0.0093 Brazos 0.009 0.0034 Heritage 0.039 0.036 0.0385 R. idaeus 0.040 0.035 0.036 0.0036 IlliniHardy 0.014 0.007 0.009 0.028 0.030IR.allegheniensis 0.012 0.004 0.006 0.027R. argutus 0.012 0.006 0.0080.028 0.000. . 0.030 0.032 0.001 0.0009 Arapaho 0.014 0.004 0.006 0.031 0.033 0.003 0.000 0.00110 A. argute? 0.702 0.697 0.699 0.696 0.699 0.699 0.699 0.696 0.69711 V. corymbosum 2 0.705 0.703 0.703 0.705 0.704 0.2870.705 0.708 0.706 0.703zOutgroup taxa.Iesulting DNApellet was dried and then washed with 70%thanol and re-suspended in 50 /uh of TE buffer (pH 7.4).tThePCR reaction mixtures for each sample weremprised of 5 [xLof 10X Taq buffer withMgCl2 (Promega,adison, WI), 4 /uL dNTP (Pharmacia Biotechnology,scataway, NJ), 1 /uL primer ITS 4 (5'TCCTCCGCTTATTGATATGC- 3'), 1 /uL ITS 5 (5' -GGAAGTAAAAGTCGTAACAAGG- 3') (White et al.,1990), 0.4 Taq polymerase (Promega), and 40 /xL doubledistilled water all of which were master mixed to ensureuniformity among reagents. An aliquot of 48 was addedto a 0.5 ml thin- walled microcentrifuge tube (Sarstedt, NC),Journal of the Arkansas Academy of Science, Vol. 57, 2003177Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24https://scholarworks.uark.edu/jaas/vol57/iss1/24I178Eric T. Stafne, AllenL. Szalanski, and John R. ClarkFig. 1. Consensus parsimony tree with nine Rubus genotypes and two outgroups based on ITS region DNA sequences.Bootstrap values greater than 50% are indicated at nodes.then 2 /uh of template DNA was added to comprise a totalreaction volume of50 /uh. A Techne Touchgene 40 x 0.5 ml(Techne Corp., Princeton, NJ) was used toperform the PCR.The thermocycling program used for this study consisted ofan initial denaturation of 94 °C for 2min., then 40 cycles of94° C for 45 s, 46° C for 1min, and 72°C for 1min, followedby a final extension for 5 min at 72°C.A 1% agarose solution (Fisher Scientific, St. Louis, MO),was placed into the gel box (Owl Scientific, Cambridge,MA) filled with 0.5X TBEbuffer solution. The PCR productand dye buffer totaled 10 fA.Ten fA of 100 bp DNA ladder(Promega) was added in the first well. Ethidium Bromidewas added to the buffer solution and the voltage was set at~165 V. The electrophoresis ran for 50 min. The gel wasremoved and photographed in the BioDoc-It imagingsystem (UVP, Inc., Upland, CA).Amplified DNA of six sample genotypes (fiveblackberry and one raspberry) was purified as described inJournal of the Arkansas Academy of Science, Vol. 57, 2003178Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24Published by Arkansas Academy of Science, 2003:>I•.179Nuclear Ribosomal ITS Region Sequences for Differentiation of Rubus GenotypesSzalanski et al. (2000). The purified DNA samples were sentto the DNA core sequencing lab at the Univ. of Arkansas(Fayetteville) to be sequenced in both directions with anABIPrism 377 DNA sequencer. An amplified fragment ofDNA between 700 and 800 bp in size was sequenced for allsamples. However, to understand genetic relationships withexisting Genbank DNA sequences, the sequences werereduced to 643 bp.Sequences derived from ribosomal DNA were alignedwith the Genetics Computer Group (GCG, Madison, WI)PILEUP program (gap weight = 5.0, gap length weight =1.0) and used Vaccinium corymbosum L. and Actinidia arguta(Sieb. & Zucc.) Planch, ex Miq. as outgroups. Maximumlikelihood and maximum parsimony analyses wereperformed with PAUP* 4.0b 10 (Swofford, 2002). Bootstraptesting (100 re-sampled datasets) was used to determine treerobustness (Felsenstein, 1985).Results and DiscussionBases were revealed at the frequency of 0.20967,0.28372, 0.27825, and 0.22836 for A, C, G, and T,respectively. Existing Genbank sequences for A. arguta(AF323836; Liet al., 2002) and V. corymbosum (AF419778;Powell and Kron, unpublished data) were used as outgroups.R. idaeus (AF0557552), R. allegheniensis (AF055772), and R.argutus (AF0557742) were existing Genbank DNAsequencesthat were included to compare species with the moderncultivars included in the study (Alice and Campbell, 1999).Parsimony analysis resulted in 643 total charactersconsisting of 147 constant characters (22.8%), 176 variablecharacters that were parsimony-uninformative (27.3%), and320 parsimony-informative characters (49.7%). Pairwisegenetic distances (Tajima and Nei, 1984) ranged from 0.00('IlliniHardy 7/2. allegheniensis and R. allegheniensis/ R. argutus)to 0.04 (APF-12//?. idaeus) within the Rubus cultivars (Table2). Alice and Campbell (1999) reported that ITS sequencedivergence among species of the blackberry subgenus averagedonly 1.2%, and this appears also be a close representation ofthe data obtained in this study.Only one parsimonious tree was derived from the dataset (Fig. 1) with a length of 603 and a consistency index (CI)of 0.982. Bootstrap analysis indicated little divergenceamong blackberry cultivars and species, but suggested aweak sub-cluster of APF-12, 'Brazos', and Apache'. Thevalue of 51% at the APF-12/'Brazos' node implies a veryweak support for that separation. The maximum-likelihoodanalysis starting with the parsimonious tree found 3 trees,Dut all were similar and none of the changes resulted in asignificant change in topology. The resulting tree was similarn topology to that of the consensus parsimony tree (data notshown). Blackberry and red raspberry genotypes wereseparated as distinct clusters by both the maximum-jarsimony and maximum-likelihood tests. The raspberrygenotypes displayed informative deletions at sites 75 and442, whereas the blackberry genotypes had a deletion at 515(data not shown). These are likely strong indicators fordifferentiating the two groups.The phylogenetic maximum likelihood tree derivedfrom PAUP* differentiated the subgenera Idaeobatus andEubatus (red raspberry and blackberry, respectively), but didnot recognize any notable differences within the blackberrycultivars. Thus, Rubus cultivar differentiation, which hasbeen done with other molecular techniques (Stafne et al.,2003), does not appear to be viable with ITS regionsequences. Blackberry cultivars containing more divergentspecies have yet to be tested and may show distinction fromthe other blackberry cultivars tested in this study.GonclusionsThe main benefit for Rubus appears to be ifmorphological ambiguity exists. ITS sequence analysis maydistinguish between red raspberry and blackberrygenotypes, but probably not within cultivars of those groupsunless they contain highly divergent progenitor species.Since ITS sequences cannot distinguish between similarcultivars, its utility for plant patent protection is limited.Literature CitedAlice, L. A. 2002. Evolutionary relationships in Rubus(Rosaceae) based on molecular data. Acta Hort. 585:79-83.Alice, L. A., and C. S. Campbell. 1999. Phylogeny ofRubus (Rosaceae) based on nuclear ribosomal DNAinternal transcribed spacer region sequences. Amer. J.Bot. 86:81-97.Alice, L. A., T. Eriksson, B. Eriksen, and C. S.Campbell. 2001. Hybridization and gene flowbetweendistantly related species of Rubus (Rosaceae): evidencefrom nuclear ribosomal DNA internal transcribedspacer region sequences. Syst. Bot. 26:769-778.Baldwin, B. G., M.J. Sanderson, J. M. Porter, M. F.Wojciechowski, C. S. Campbell, and M. J.Donoghue. 1995. The ITS region of nuclear ribosomalDNA: a valuable source of evidence on angiospermphylogeny. Ann. Missouri Bot. Garden 82:247-277.Felsenstein, J. S. 1985. Confidence limits on phylogenies:an approach using bootstrap. Evolution 39:783-791.Hall, H. K. 1990. Blackberry breeding. Pp. 249-312, InPlant Breeding Reviews (J. Janick ed.) Vol. 8. TimberPress, Portland, Oregon.Jennings, D. L. 1988. Raspberries and blackberries: theirbreeding, diseases and growth. Academic Press,London.Li,J., H.Huang, and T. Sang. 2002. Molecular phylogenyand infrageneric classification of ActinidiaJournal of the Arkansas Academy of Science, Vol. 57, 2003179Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24https://scholarworks.uark.edu/jaas/vol57/iss1/24180Eric T. Stafhe, AllenL.Szalanski, and John R. Clark(Actinidiaceae). Syst. Bot. 27:408-415.Parent, J. G., and D. Page. 1992. Identification ofraspberry cultivars by random amplified polymorphicDNA (RAPD) analysis. HortScience 27:1108-1110.Stafhe, E. T., J. R. Clark, M. C. Pelto, and J. T.Lindstrom. 2003. Discrimination of Rubus cultivarsusing RAPD markers and pedigree analysis. Acta Hort.2003. 626:119-124.Swofford, D.L.2002. PAUP*: phylogenetic analysis usingparsimony (*and other methods), version 4 beta 10.Sinauer, Sunderland, Massachusetts.Szalanski, A.L.,D. S. Sikes, R. Bischof, and M. Fritz.2000. Population genetics and phylogenetics of theendangered American burying beetle, Nicrophorusamericanus (Coleoptera: Silphidae). Ann. Ento. Soc.Amer. 93:589-594.Tajima, E, and M.Nei. 1984. Estimation of evolutionarydistance between nucleotide sequences. Mol.Biol.Evol.1:269-285.White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990.Amplification and direct sequencing of fungalribosomal RNA genes for phylogenetics, p. 315-322. InPCR protocols: a guide to methods and applications.(D.G.M. Innis, J. Sninsky, and T. White, eds.).Academic Press, San Diego, California.Journal of the Arkansas Academy of Science, Vol. 57, 2003180Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 24Published by Arkansas Academy of Science, 2003

English

https://scholarworks.uark.edu/cgi/viewcontent.cgi?article=1612&context=jaas

Nuclear Ribosomal ITS Region Sequences for Differentiation of Rubus Genotypes

Abstract

Similar works

Full text

Available Versions

ScholarWorks@UARK

UARK (University of Arkansas )