FINGERPRINTING JUNIPERUS COMMUNIS L. CULTIVARS USING RAPD MARKERS

8 Pages·1999·4.7 MB·English

by  Vanessa E T M Ashworth

Madrono, Vol. 46, No. 3, pp. 134-141, 1999
FINGERPRINTING JUNIPERUS COMMUNIS CULTIVARS
L.
USING RAPD MARKERS
Vanessa E. T. M. Ashworth1, Bart C. O'Brien, and Elizabeth A. Friar
Rancho Santa Ana Botanic Garden, 1500 North College Avenue, Claremont,
CA 91711-3157
Abstract
Eight of eleven cultivars of Juniperus communis L. growing at Rancho Santa Ana Botanic Garden
exhibit a morphology atypical of the wild populations from which they were reportedly derived. The
"exotic" morphology consists ofbranchlets arranged at almost 90° to the branch axis and more spreading
leaves that are of a bluer color than those ofthe wild plants. One ofthe "exotic" cultivars additionally
shows a chimaeric distribution of acicular and scale-like leaves along its branches. Scale-like leaves are
characteristics ofJuniperus section Sabina and not section Juniperus, to which J. communis belongs. A
RAPD marker study was initiated to compare RAPD fingerprints of the cultivars with those of their
putative wild ancestors and representatives ofotherJuniperus species in both sections. Results suggested
that the eight cultivars having an "exotic" morphology were either hybrids between J. communis and J.
chinensis, or pedomorphic forms of J. chinensis. The three remaining cultivars that have a "native"
morphology clustered with J. communis progenitors.
Rancho Santa Ana Botanic Garden (RSABG) is semble J. communis found in the wild. CV2 has
home to eleven plants of Juniperus communis L. longer, incurved, leaves and a less prostrate habit
(common juniper). All were established from cut- with a moderately erect stem. A mat-like habit and
tings reportedly taken from wild populations native incurved leaves characterize cultivars CV5 and
to the California Floristic Province (Raven and Ax- CV11 (Table 1). The former is of greener and the
elrod 1978). However, not all the plants in question latter of bluer coloring.
exhibit the morphology generally seen in the wild.
Instead of having a prostrate habit and somewhat Juniperus communis Varieties in the Western
incurved leaves, plants produce branches with fair- United States. Juniperus communis is a circumbo-
ly upright branchlets and spreading leaves. Given real species ofjuniper (Franco 1962) characterized
that RSABG specializes in the cultivation ofplants by acicular leaves. Two varieties of J. communis
native to California, a RAPD (Random Amplified (Cronquist et al. 1972; Flora of North America
Polymorphic DNA) marker study was initiatedwith Committee 1993) are encountered in the western
the aim oftracing the origins ofthe putatively "ex- United States. Juniperus communis var. depressa
otic" specimens and to match up the remainingju- Pursh is native to the Great Basin Floristic Prov-
nipers with their wild progenitors. ince. It ranges farther north into Alaska and east-
ward across much of Canada and the Great Lakes
Cultivars at Rancho Santa Ana Botanic Garden. region, arching south along the east coast to North
Table 1 summarizes the salient characteristics and Carolina. Juniperus communis var. montana Aiton
collection information of the junipers included in occurs from British Columbia southward into Cal-
this study. Five of the eleven plants are of known ifornia in the Cascade Ranges, North CoastRanges,
geographic origin, but documentation for the re- and SierraNevada. The two varietiesdifferinhabit,
mainder is either questionable or missing. Fourdis- leaf size and shape and width of the glaucous sto-
tinctive morphologies are represented. The "exot- matal band on the adaxial leaf surface. Although
ic" form consists of long branches from which
both are low-growing, variety depressa develops a
short branchlets emerge at an upward angle of al- somewhat erect main stem whereas variety mon-
most 90°. Leaves are short and spreading and the
tana is entirely prostrate. Leaf dimensions are ca.
etCmrnoVatir1itre0se.issppClrsaVeenae7tdniinaisglnsboChlaVubd1eiit-,f,gferCareVsne3dnb,yainCciVhtcs4io,mlgoarreCe.rVein6Tce,hridsiafsnotdslruiiiaCbtguVeet,8ioo-anf sl1ao.)n0,g-1a.{n6mdomn(t1m.a2n)bar)1o.a(5dC-r1o.X8nq(mu6i)mst1b0er-to1aa8ld.mX1m9752l-)o1.n0gT(h{1ed2e)pgrlmeasum--
of leaf shapes along its branches and branchlets. ceoaucsh gsrtoemeantmalarbgainnd{idseapsrebsrsoaa)doras2,-o3rtniamreroswaesr,brtohaadn
Zones of acicular, spreading leaves alternate with as each green margin {montana). Two other varie-
appressed, scale-like leaves reminiscent of species
in section Sabina. The three remaining cultivars re- ties are occasionally distinguished in California.Ju-
niperus communis var.jackii Rehder (Rehder 1940)
differs from var. montana by having longer, more
1 Present address: Department ofBotany and Plant Sci- sparsely branched lateral branches. It is a form
ences, University ofCalifornia, Riverside, CA 92521. common to serpentinite substrates in inland coastal
•
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MADRONO
136 [Vol. 46
areas of northern California and Oregon. Juniperus transilluminator (Fotodyne). Product size was de-
communis var. sibirica Rydb. describes avery pros- terminedusing aDNA standard (1 kb ladder; Gibco
trate, almost mat-like, form found on coastal bluffs BRL, Inc.). Bands were scored as present or absent
in the extreme northwest of California and south- by the first and last author. The scores were ana-
UPGMA
western Oregon, and at Ebbett's Pass in the Sierra lyzed using the clustering algorithm (Un-
Nevada. According to Roof (1973), this variety is weighted pair group method with arithmetic aver-
characterized by leaves that are more incurved, ages; average link) and Neighbor-Joining (NJ)
making it less prickly to the touch thanJ. communis available on PAUP* version 4.0 (31 (Swofford
vars.jackii or montana. 1998).
RAPD analysis was chosen as a quick and rela-
tively inexpensive means ofgetting a fingerprint of Results
the genome ofeach plant which could then be com- Ofa total of65 primers screened forRAPDanal-
pared against similar fingerprints generated from ysis, six showed scorable and reproducible banding
the native populations. This technique has been ap- patterns and were entered into the final analysis.
plied successfully to Juniperus in other studies Scorable bands per primer ranged from one (Op-
(Adams and Demeke 1993) addressing affinitiesbe- eron Al) to nine (UBC-244). A total of 34 bands
tween species ofJuniperus. were scored.
The preliminary analysis, which included all
Methods RSABG cultivars (CV), J. communis var. depressa
PlantMaterial. Plant material was gathered from (de) and all but the Ebbett's Pass population of J.
all communis cultivars growing at RSABG and communis var. montana (mo), revealed a strikingly
from seven wild populations growing at localities different banding pattern of cultivars CV1, CV3,
from which the original cultivar cuttings had re- CV4, and CV6 through CV10. All had numerous
portedly been collected. In some cases, plants had bands that were missing from the three remaining
been acquired from a nursery that had reportedly cultivars and all wild populations. Clearly, the an-
established its plants from wild-collected stock. cestry of these cultivars included an as yet unsam-
Where the source populations were no longer alive pled genotype. The three cultivars having a set of
or accessible (CV11 and CV5, respectively) adja- bands more consistent with that of the wild popu-
cent populations were collected instead. Details of lations were CV2, CV5 and CV11.
collecting locality and morphology of the native To identify the unknown parent or parental com-
populations are summarized in Table 2. The seven ponent, fourteen creeping cultivars of J. chinensis
wild-collected populations represent J. communis (3), J. conferta (1), /. horizontalis (6) and J. sabina
v(adresD.-dmeoFn)t.anaUnd(emroA—tmheoC,altmeronGa)tiveandtadxeopnroemsisca a(4n)dw2ersehoawddtehde rtoesutlhteinsgtuUdyPG(TMaAblean3d).NFJigpuhreenso-1
scheme (Table 2), populations moA and moG cor- grams. In both figures, the "exotic" and "com-
respond to J. communis var. sibirica, and popula- mercial" cultivars were more similar to each other,
tfioounrstereanoBcualtnidvamrsoCadtdoedJ.tcootmhmeunaniaslyvsairs.jaafctkeiri.cTohme- fwiolrdminpgopaula"tnioonn-snat(i"vneatcilvuestecrl"u,stterh"a)n.toAmanoyngof tthhee
pletion of the preliminary screens are identified in "commercial" cultivars, allJ. horizontaliscultivars
except hor 6 ('Wiltonii') formed a well-defined
Table 3. They represent different cultivars of spe-
cies ofcreepingjunipercommonly sold in the nurs- ccluulsttievra,rsa,nadsawneoltlhears hclourst6e.rPceornhtaapisnehdorall6Jw.assabmiinsa-
ery trade and are henceforth called "commercial"
labeled at the nursery oforigin or has been mistak-
cultivars.
enly attributed toJ. horizontalis. Juniperusconferta
DNA Analysis. Leaf samples weighing 0.2-0.5 g clustered with J. sabina (UPGMA; Fig. 1) or at the
were ground in liquid nitrogen, followed by extrac- base ofaclusterincluding the "exotics" and "com-
DNA
tion of genomic using a modification of mercial" cultivars (NJ; Fig. 2). Juniperus chinensis
Doyle and Doyle (1987). Reaction mixtures (25 |xl) var. sargentii 'Viridis' (chi 3), did not cluster with
f|uol1rgaemnploimfiiccaDtiNonAof(1R0AnPg/Dulb),an1d8s.8co|nult1adiHne20d,0.21.-51.(j0lI tthhee doitshtearnctewoalg/.orcihtihnmenussiesd.cuIlntsitveaards,,irtecglaursdtleersesdoaft
sequencing buffer (Tris-HCl (pH 9.0), KC1, MgCl2, the base of a cluster including the exotic RSABG
glycerin), 1.5 jjlI dNTP's (2.5 mM), 1.0 jjlI primer cultivars, chi 1 and chi 2, and J. horizontalis (ex-
(10 pmol/(xl), and 0.05 (xl Taq polymerase. Details cluding hor 6).
of primer nucleotide sequences are given in Table Both clustering algorithms placed the "exotic"
4. Amplifications were performed on a PTC-100 RSABG cultivars in a cluster with J. chinensis var.
thermocycler (MJ Research, Inc.) programmed for procumbens 'Nana' (chi 1) and J. chinensis 'San
1 cycle at 94°C for 1 min, 44 cycles at 94°C for 1 Jose' (chi 2). CV7 associated more closely with chi
min, 42°C for 1 min and 72°C for 2 min, followed 1 and chi 2 than the other "exotics" in theNJphen-
by a final extension time of7 min at 72°C. Reaction ogram (Fig. 2). Even when chi 1 or chi 2 were
product was run out on a 1.5% agarose gel, stained excluded from the analysis the "exotics" still clus-
with ethidium bromide to visualize the bands, and tered with the "commercial" cultivars (not shown).
electrophoregrams were photographed on a UV Only three RSABG cultivars clustered with the
1999] ASHWORTH ET AL.: FINGERPRINTING JUN1PERUS COMMUNIS CULTIVARS 137
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MADRONO
138 [Vol. 46
Table 3. Designation, Species and Cultivar Name, and Sectional Placement of Fourteen "Commercial" Cul-
tivars of Juniper Species Added to the Study to Trace the Parentage of the "Exotic" RSABG Cultivars.
Sample
designation Juniper species and cultivar name Sectional placement
rChIIiI 11 J. Crllrlcrlblb VCLl. yrULUrrlUctlb INdlld section Sabina
chi 2 LfllflLflblb Ottll JU5>C oCCUUIl kJClOlria
Ctil j ./. LIIIrIt?riblb Veil, bL4tgt?tlltl VlilUld section Sabina
con 1 ./. LOilJcllU E/lllCIalU oca section Juniperus
hor 1 7. horizontalis 'Blue Chip' section Sabina
hor 2 7. horizontalis 'Emerald Isle' ('Emerald Spreader'?) section Sabina
hor 3 7. horizontalis 'Hughes' section Sabina
hor4 y. horizontalis 'Yukon Belle' section Sabina
hor 5 y. horizontalis 'Prince ofWales' section Sabina
hor 6 y. horizontalis 'Wiltonii' section Sabina
sab 1 y. sabina 'Calgary Carpet' section Sabina
sab 2 y. sabina 'Arcadia' section Sabina
sab 3 y. sabina 'Moor-Dense' section Sabina
sab 4 y. sabina 'Tamariscifolia' section Sabina
native populations (Figs. 1 and 2). CV5 clustered cluster analysis, most "exotics" associate closest
with moAl-moA4 (Fig. 1) or moB4 (Fig. 2). CV1 with y. communis var. montana population moQ\
was placed in a cluster with moA5 (Fig. 1) or with (not shown). However, the large proportion of
moG\-moG3 (Fig. 2). The long-leaved CV2 was bands shared with chi 1 and especially chi 2 (73-
less likely to group with any cluster. In the NJ 85%) does not exclude the possibility of a pure J.
phenogram (Fig. 2), it clustered with a four taxon chinensis origin. Under this scenario, the plants
cluster containing moB3 and deD, deE and deF. may represent pedomorphic J. chinensis mutants
However, UPGMA positioned CV2 at the base of that retain acicular (juvenile) foliage instead ofde-
the "native cluster'1 (Fig. 1). Results pertaining to veloping scale leaves typical of (mature) J. chinen-
the wild populations are discussed elsewhere (Ash- sis. Mutants are of common occurrence in Junip-
worth et al., in prep.). erus (p. 413, Flora of North America Committee
1993; Hall 1952).
Discussion If hybridity is invoked, the "exotics" may rep-
Phenetic analysis suggests that nine of eleven resent J. communis X J. chinensis hybrids that
cultivars growing at RSABG are either similar to hneanvseis.unTdheergNoJnetrmeuelt(iFpigl.e 2b)acpklcarcoesssCinVg7tocloJ.secrhit-o
yt.wecehninye.nsciosmmorunairse athned rJ.esuclhtineonfsihsy.brGiidvizeantitohnatbael-l pchoissi1balnyd cshuigg2estthianngit addodeistitohnealothebrac"kecxrootsiscisn,"g
"exotics" showed banding patterns far more rem-
events to J. chinensis, but this is not true of the
pinuirspcoerntteodfJy.. cchoimnemnusnisis{chpiro1geonritcohri, 2r)atthhearn tthheainr hUyPbrGidMsAapnhdenmoogrrpahomlo(gFiigc.al1).datIan,thUePcGasMeAofhaFs,
showing additivity, could reflect multiple back-
been shown to give more predictable placement of
crossing to the former (Hawkins and Harris 1998; a hybrid with one or both parents than NJ (Mc-
Rieseberg and Ellstrand 1993). Variability within
Dade 1997), but relative performances are un-
tna"haemetxoiovntwegiiclasdnt"cheepsaotnJpo.dur.lccahWtoiihmome1nunsonriamlcslahkibveaa2snridaesritteiseedhsixafcrfaleisudcudtlbehtedettfwopreuoetsmnaetlittevhhceeet kTalhnoeonwepnlfaofrcoeRrmAemnPotrDeofdacCtoaVm7apnldeclxcoasbserestseeitdonivncoghlivhii1nsgtaonmruidetsac,nhtilse.2t
is, however, consistent with the observation that
CV7 exhibits several J. chinensis characteristics,
Table 4. Nucleotide Sequences ofthe RAPD Primers notably scale-like leaves and spreading branches,
UsedtoFingerprintJuniperusGenotypesinthisStudy. that are not found in the other "exotics". Overall,
All nucleotide sequences arecited in a 3' to5' orientation. the RAPD data are in good agreement with mor-
phology. All cultivars at RSABG suggested to be
Primer name Nucleotide sequence "exotic" by virtue of their less prostrate growth
OPERON Al CAG GCC CTT C and more prickly leaves, display banding patterns
OPERON B18 CCA CAG CAG T atypical ofthe wild-collected plants while the cul-
UBC-108 GTA TTG CCC T tivars of native appearance cluster with the wild
UBC-1 11 AGT AGA CGG G populations. CV5 and CV11 exhibit the prostrate
UBC-184 CAA ACG GCA C growth habit associated with native populations of
UUBBCC--322494 GCCAGG ACACCA ACCTCC GC yo.fcComVm1u1niasndvart.hemongtraeenna.fTolhieagbeluoef-gCreVe5n fmolaitacghe
1999] ASHWORTH ET AL.: FINGERPRINTING JUNIPERUS COMMUNIS CULTIVARS 139
moA\,
moA3,
"Exotics"
J. horizontalis
Fig. 1. Unrooted UPGMA phenogram, showing two distinct clusters that group plants with typical Juniperus com-
munis morphology (native cluster) and "exotic" morphology (non-native cluster). All RSABG cultivars (CV1-CV11)
are bolded. Shaded ovals highlight the four species ofjuniper other than J. communis. Members ofJ. communis var.
depressa are also indicated. All otherindividuals representJ. communis var. montana. Sample designations are identical
to those used in Tables 1-3.
Roof's (1973) description of the Point St. George Conclusions
and Gold Beach populations, respectively. CV2
resembles J. communis var. depressa in habit and This study ofdwarfjunipers illustrates that arel-
leaf size, an affinity receiving partial support from atively simple molecular technique can be used to
the RAPD data (NJ; Fig. 2). test a hypothesis based on observations of aberrant
MADRONO
140 [Vol. 46
A770B6
-2, A770A6
moA^ a<$>™t7 moC2
A770C1
moG^
CV2 CV11
J. sabina
hoft
communis
J.
var. depressa
J. conferta
horS
J. chinensis CV7 J- horizontalis
CV10
CV1
"Exotics"
Fig. 2. Unrooted NJ phenogram. Abbreviations and explanations as in Figure 1.
plant morphology. Although the precise parentage were themselves hybrids or J. chinensis mutants,
of the "exotic" cultivars is unknown, the RAPD even though many species of this wind-pollinated
fingerprints nonetheless point to a major contribu- genus are able to interbreed (e.g., Flora of North
tion from J. chinensis. Careful research into garden America Committee 1993) and /. chinensis has
records suggests that all "exotics" trace back to been in cultivation since the last century (Rehder
three plants acquired from Louis L. Edmunds, Dan- 1940).
ville, CA, in 1950. These had been purportedly col-
lected as cuttings from "just east of Tioga Pass Acknowledgments
summit" in 1938. The most likely explanation is a We thank Mary Debacon fortechnical assistance in the
nursery mix-up, mislabeling, or inadvertent hybrid- earlier stages ofthe laboratory work, J. Travis Columbus,
ization with J. chinensis (suggesting propagation J. Mark Porter and Eric H. Roalson for valuable discus-
from seed) in the intervening twelve years. It seems sions, and the reviewers for helpful comments on the
unlikely that the original plants from Tioga Pass manuscript.
1999] ASHWORTH ET AL.: FINGERPRINTING JUNIPERUS COMMUNIS CULTIVARS 141
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