Molecular Ecology Notes (2003) doi: 10.1046/j.1471-8286 .2003.00382.x

© 2003 Blackwell Publishing Ltd

Blackwell Publishing Ltd.

PRIMER NOTE

Polymorphic microsatellite loci in

Plantago lanceolata

MARIE L . HALE and KIRSTEN WOLFF

School of Biology, University of Newcastle, NE1 7RU, U.K.

Abstract

The genus

Plantago

is particularly interesting for evolutionary studies because of its widerange of mating systems. We have developed primers for five highly polymorphic micro-satellite loci isolated from

P. lanceolata

. All five loci amplified and were polymorphic inthe two populations examined, Lowsteads Beach in the United Kingdom and Duke in theUnited States. These new markers will allow a comparison of population structure betweenthe outcrossing species

P. lanceolata

, and the highly selfing species

P. major

.

Keywords

:

microsatellite primers,

Plantago lanceolata

Received 9 July 2002; revision received 23 August 2002; accepted 20 November 2002

Plantago

species have shown to be very useful species fora wide range of population genetic and evolutionarystudies (e.g. Wolff 1987; Van Dijk

et al

. 1988; Wolff 1991;Van Delden

et al

. 1992). One of the reasons is the broad rangeof mating systems in the genus and the fact that many ofthe species are common and widespread. In this studywe developed microsatellite primers for the obligatoryoutcrossing

P. lanceolata

. Primers are already available for

P. major

c.s., a highly selfing taxon (Squirrell & Wolff 2001).The availability of microsatellite primers for a number ofspecies within a genus allows for interesting comparisonsof levels of variability and evolution of the microsatellites.In addition, the primers will be very useful for studieswithin the species

P. lanceolata

, for example for QTLmapping of life history traits (Wolff 1987).

A DNA library enriched for dinucleotide microsatelliteswas created for

P. lanceolata

using the double enrichmentmethod described in Hale

et al

. (2002). Seventeen nano-grams of the twice-enriched DNA was ligated into 100 ng

Bam

HI digested Ready-To-Go

TM

pUC18 vector (Amersham)following the manufacturer’s instructions. Two microlitresof this enriched library was transformed into 100

µ

L JM109competent cells (Promega), and plated onto LB:agar platescontaining 50

µ

g/mL ampicillin. Plates were incubated at37

°

C overnight, and then plasmids from individual col-onies prepared using a QIAprep® Spin Miniprep Kit(Qiagen). Plasmid inserts were sequenced using BigDyeTerminator Cycle Sequencing chemistry (Applied Bio-systems), and sequences detected on an ABI 310 Prism®

automated sequencer. A total of 54

P. lanceolata

clones weresequenced.

Thirty-two clones possessed a microsatellite with 10 ormore repeats; however, the large number of duplicate lociand loci close to a restriction site greatly reduced thenumber of useful loci. Five loci were chosen for primer test-ing and polymorphism assessment across two popula-tions: Duke in the United States (

n

= 17), and LowsteadsBeach in the United Kingdom (

n

= 23).Microsatellite loci were amplified in 15

µ

L reactions [1

×

Taq

buffer (16 m

m

(NH4)

2

SO

4

, 67 m

m

Tris-HCl, 0.01%Tween-20), 2.5 m

m

MgCl

2

, 0.2 m

m

each dNTP, 0.33

µ

m

each primer, 0.6 U

Taq

(Bioline) and 0.5

µ

L template DNA]under the following conditions: 95

°

C for 12 min, 10 cyclesof 94

°

C for 15 s, 52

°

C for 15 s, 72

°

C for 15 s, followed by30 cycles of 89

°

C for 15 s, 52

°

C for 15 s, 72

°

C for 15 s, then72

°

C for 10 min. For each locus the forward primer waslabelled with an ABI fluorescent dye (either JOE, FAM orTAMRA), obtained from MWG Biotech. All reactions wereperformed in a PTC-100

TM

thermocycler (MJ Research).Microsatellite fragments were detected on an ABI 310Prism® automated sequencer using labelled primers andan internal size standard (GeneScan-500 [ROX]), and wereanalysed and sized using

genescan

® software (AppliedBiosystems). Amplification of the microsatellite loci wastested across both UK and US populations.

All five microsatellite loci were polymorphic withinboth populations (Table 1). The number of alleles foundranged from eight to 27 alleles per locus, and was substan-tially higher in the UK population than the US populationfor two loci. Significant deviations from Hardy–Weinbergequilibrium were detected for half the loci. In all cases

Correspondence: M. L. Hale. Fax: + 44 (0)191 2225229; E-mail:m.l.hale@ncl.ac.uk

P R I M E R N O T E

135

© 2003 Blackwell Publishing Ltd, 3, 134–135

the observed frequency of heterozygotes was lower thanthe expected frequency. Such a deficit of heterozygotes,albeit being smaller and insignificant, has been noted pre-viously in allozyme data for this species (Van Dijk

et al

.1988) and may be the result of sampling across neigh-bourhoods, or nonrandom mating within the populations.Null alleles may be a contributing factor to the deficit ofheterozygotes in some populations for the locus Pll13.However, we found no evidence for null alleles in the otherfour loci. All five loci were tested for amplification intwo other

Plantago

species:

P. major

and

P. maritima

. OnlyPll1 amplified in these two species but was monomorphicin both.

References

Hale ML, Squirrell J, Borland AM, Wolff K (2002) Isolation of

polymorphic microsatellite loci in the genus

Clusia

(Clusiaceae).

Molecular Ecology Notes

,

2

, 506–508.Squirrell J, Wolff K (2001) Isolation of polymorphic microsatellite

loci in

Plantago major

and

P. intermedia

.

Molecular Ecology Notes

,

1

, 179–181.Van Delden W, Van Dijk H, Wolff K (1992) The genetics of

Plantago

species. In:

Plantago: a Multidisciplinary Study

(eds Kuiper PJC,Bos M), pp. 295–310. Springer-Verlag, Berlin.

Van Dijk H, Wolff K, de Vries A (1988) Genetic variability in

Plan-tago

species in relation to their ecology. 3. Genetic structure ofpopulations of

P. major

,

P. lanceolata

and

P. coronopus

.

Theoreticaland Applied Genetics

,

75

, 518–528.Wolff K (1987) Genetic analysis of ecologically relevant morpho-

logical variability in

Plantago lanceolata

L. II. Localisation andorganisation of quantitative trait loci.

Theoretical and AppliedGenetics

,

73

, 903–914.Wolff K (1991) Analysis of allozyme variability in three

Plantago

species and a comparison to morphological variability.

Theoret-ical and Applied Genetics

,

81

, 119–126.

Table 1 Primer sequences and polymorphism information for five microsatellite loci isolated from P. lanceolata

Locus Repeat in clone Primer sequence (5′–3′)Product size (bp)

Duke (USA) Lowsteads (UK)

No. alleles n HE HO No. alleles n HE HO

Pll1 [TA]4A[GC]6[AC]10

F: GATCAGAATGAATAACCTTCG 158–180 8 17 0.848 0.706 8 23 0.681 0.601R: CAATCCAGAGGACCAAATGC

Pll8 [CT]17 F: TGATGCATGTGACCCTGACT 171–203 11 17 0.920 0.882 13 23 0.912 0.696R: GATGGGGCTGAGTTTGAGAG

Pll10 [AG]43 F: TGTGAGATGTGTTAGCTCGGTTA 93–189 8 17 0.864 0.588* 27 23 0.976 0.783*R: TCTGGGTTTGCTTTCTTTCC

Pll11 [TC]15 F: TGGATTCTGATGTCAGCTCAAC 182–270 10 17 0.923 0.588* 22 23 0.959 0.826R: TTCCCAACATTAATCGAGAGG

Pll13 [AC]8AG[AC]27

F: AAATTCTTTTATTTACACAGTGATTGG190–220 12 17 0.906 0.588* 8 23 0.871 0.435*R: ATGCCAATCTGCCATGACTC

n = Number of individuals, HE = heterozygosity expected under Hardy–Weinberg equilibrium, HO = observed heterozygosity. *Significant deficit of heterozygotes from expected under Hardy–Weinberg equilibrium (P < 0.05). GenBank Accession nos AY124325–AY124329.