Molecular Ecology Notes 2002, 2 (2), . DOI: 10.1046/j.1471-8278 .2001.00161.x
PRIMER NOTE
Polymorphic microsatellite repeats
are not conserved between
Leishmania donovani and
Leishmania major
M. B Jamjoom,* R. W Ashford,* P. A Bates,* S. J
Kemp† and H.A Noyes†
*Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK,
†School of Biological Sciences, University of Liverpool, L69 7ZD,
UK
To cite this article:
Jamjoom, M. B, Ashford, R.
W, Bates, P. A, Kemp, S. J
& Noyes, H.A. Polymorphic
microsatellite repeats are not
conserved between
Leishmania donovani and
Leishmania major. Molecular
Ecology Notes 2002, 2 (2), . Available
from: http://dx.doi.org/10.1046/j.14718278 .2001.00161.x
Medline Author Search
Jamjoom, M
Ashford, R
Bates, P
Kemp, S
Noyes, H
Medline Keyword Search
Leishmania tropica
Leishmania mexicana
mapping.
Leishmania infantum
Correspondence: H.A. Noyes. Fax:
0151 794 3655; E-mail:
harry@liv.ac.uk
Abstract
Thirteen sets of polymerase chain reaction (PCR) primers
were designed to amplify microsatellite loci identified in the
genome sequence of Leishmania major. Polymorphisms
were detected in L. major at all loci. In Leishmania
donovani only two of these loci were informative for
classification purposes with this data setQuery 1. The PCR
products of all loci from one L. donovani strain were
sequenced and it was found that the number of repeats in the
microsatellite loci were either substantially reduced with
respect to L. major or absent altogether. Consequently it is
unlikely to be possible to use the genome sequence of L.
major to identify polymorphic microsatellite loci in other
Leishmania species.
Received 12 August 2001; revision received 15 October
2001; accepted 7 November 2001
Introduction
Leishmania is a protozoan parasite that infects macrophages
causing a broad spectrum of diseases in humans. There are
approximately 30 species of Leishmania that infect
mammals which are found in large areas of the tropics and
subtropics . There are approximately 360 million people at
risk and 400 000 cases of disease per yearQuery 3 (Ashford
et al. 1992). High-resolution markers are required to track
parasite strains through human populations and identify
animal reservoirs of the strains circulating in humans.
Microsatellite markers have been found in L. infantum and
others have been shown to discriminate between some
Leishmania (Viannia) populations (Rossi et al. 1994;
Rodriguez et al. 1997; Russell et al. 1999). The present
study was undertaken to test whether it would be possible to
use the L. major genome project sequences in public
databases to identify microsatellites that would be
informative in L. donovani and other L. (Leishmania)
species.
Twenty-seven independent microsatellite loci were
identified by BLASTQuery 4 search of L. major nucleotide
sequence information held at the European Bioinformatics
Institute (http://www.ebi.ac.uk/blast2/parasites.html. PCR
primers were designed using PRIMER3 software
(http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi)
(Table 1). The lengths of PCR products of microsatellite loci
were determined on an ABI 377 genetic analyser using the
GENOTYPER software. The sequence of the microsatellite loci in
L. donovani was determined by cloning the PCR product
into pCR 2.1 TOPO (Invitrogen) and cycle sequencing and
analysis on the ABI 377.
Table 1 L. major Accession Number indicates the GenBank
accession number of the L. major clone against which the primers
were designed. Chromosome; Indicates the number of the
chromosome in strain MHOM/IL/80/Friedlin; FV1 from which
the sequence was derived. Repeat; indicates repeat sequence and
repeat number in L. major. Nmaj indicates the number of alleles
observed in the 5 L major strains tested. HO and HE indicate the
proportion of heterozygotes that were observed and expected in L.
major. Ndon indicates the number of alleles observed in the 10 L
donovani strains tested. Rdon indicates the repeat number in L.
donovani strain MHOM/ET/67/HU3; LV9, note the contrast with
L. major. The remaining columns indicate the PCR conditions and
the product size range in the 5 L major strains. PCR was carried
out in 15 µL volume of ReddyMix PCR Master MixTM. (1.5 or
2.5 mM MgCl2) (Abgene, Surrey, UK) containing 25 ng genomic
DNA and 4.5 pmol of each primer. Cycling conditions are as
follows (XX indicates primer specific annealing temperature
indicated in the table): 1 × 95 °C, 3 min; 5x (95 °C 30 s; XX °C
30 s; 72 °C 40 s); 25 × (94 °C 30 s; XX °C 30 s; 72 °C 55 s);
1 × 72 °C 15 min
Locus
no
L. major L. donovani
Accession Primer Sequence
Accession Number
F primer was labeled
Chr
LIST7001 AL391629 AF389867 F CGATGAAGTCAAGCGAAACC
R CGAGGAAGAGTCGAAGAGCA
13
LIST7002 AL390114 AF389868 F CAGTCGCCACCAAGAGATT
R CAAACGGGTTCCTGTGCT
12/24
LIST7003 AL358712 AF391547 F TTCTTTGTGTCGTTCGCTCTT
R CTTGTGGCGGTAGTCATCCT
19
LIST7004 AL035264 AF389869 F AGTCACGAAGGGTGAACTCG
R CCTGTAACGGACGCTGCT
4
LIST7005 AC079026 AF389870 F GTGTCGTGGTGACTCGTGAT
R CACATCGGCTGGAGAAGG
35
LIST7006 AC004145 AF389871 F GTTGGTTTCGTTGCATTTGA
R AAAGCGAATAACACCCGAGA
3
LIST7007 AL139794 AF389872 F TGAGGATGAGGAAGGGAGAA
R CTGCTTCTGGTTTGGTTGGT
4
LIST7008 AL391263 AF389873 F TGATCATGGTTTTTGTGCAG
R GTCAAAGTCCTCTGCGTGAG
21
LIST7009 AL133468 AF389874 F ACCCATCAGAATGCCTTGAA
R CATACACCCGCACCTCTACC
19
LIST7010 AL512293 AF389875 F CGGTGAATGCCTAAAGAGAGA
R AGGAACGCATACTTGGAAGG
14
LIST7011 AL359773 AF389876 F CGGCGACATGCACACATA
R CACACACATTGAAGATGGAGGA
14
LIST7012 AL133468 AF389877 F GTCTCCGTCCCGCATAAT
R GCGCTTCTCTCTTGCGTA
19
LIST7013 AL354532 AF389878 F GTACAGCGACGACAAAGCAC
R TCCCACCTCCCTCCTCTC
21
L. major strains were polymorphic at all loci. Thirteen out of
the 27 primer pairs designed against L. major genome
sequence amplified a single product of approximately the
expected size from L. donovani DNA. These 13 primer pairs
were used to screen a panel of 18 stocks comprising 5 L
major (MHOM/SN/XX/LV622;DK72,
MRHO/SU/59/LV39;Pstrain, MHOM/SU/60/LV356;LRCL38, IDUB/SN/XX/LV599;DK57, MHOM/IL/80/Friedlin;
FV1); 10 L donovani (MHOM/ET/67/HU3;LV9,
MHOM/BD/97/LDON/BG1, MHOM/SD/90/D83,
MHOM/SD/90/D92, MHOM/SD/90/2828,
MHOM/SD/90/D100, MHOM/SD/90/D75,
MHOM/SD/90/2655, MHOM/SD/90/D99,
MHOM/SD/91/D1783); 1 L infantum
(MCAN/GB/96/LV755); 1 L tropica (MHOM/IQ/66/LV556)
and 1 L mexicana (MNYC/BZ/62/M379). In L. major all 13
loci were polymorphic, and each of the five stocks examined
represented unique 'microdemes' (Russell et al. 1999).
Interestingly, nine of the 13 loci were heterozygous within
one or more of the five L. major stocks (Table 1). This is a
much higher level of heterozygozity than has been observed
using isoenzymes or any other method although still below
the heterozygosity expected for a population in Hardy–
Weinberg equilibrium. Furthermore since these stocks were
not cloned it is not clear whether these represent
heterozygous parasites or mixed stocks.Query 5
In contrast nine out of the ten L. donovani stocks were
monomorphic at all loci tested except LIST7010 and
LVST7011, despite including representatives of three
different zymodemes. The sequence of the PCR product of
MHOM/ET/67/HU3;LV9 (the World Health
OrganisationQuery 6 L. donovani reference strain) was
obtained for all loci. This showed that the copy number of
the SSR was greatly reduced in this species compared to L.
major, and in some cases the repeats had been eliminated
altogether (Table 1). Ten primer pairs also amplified L.
infantum loci. The L. infantum loci were also smaller than
the L. major but seven out of the 10 differed in size from the
Sudanese L. donovani isolates. Nine primer pairs amplified
L. tropica loci; eight out of nine of the PCR products were
also smaller than the L. major loci but were bigger than L.
donovani loci so these lociQuery 7may be polymorphicQuery 8.
Six primer pairs amplified L. mexicana. All of the PCR
products were also smaller than the L. major loci and
therefore may not be polymorphic.
A panel of microsatellites has now been isolated de novo that
is polymorphic in L. donovani and is reported separately
(Jamjoom et al. In Press)
Acknowledgements
We wish to thank Dr M. L. Chance (Liverpool) and Dr F.
Pratlong (Montpellier) for the supply of Leishmania stocks.
Dr P.C. Watts assisted with data analysis. MB JamjoomQuery
9 was funded by a Saudi Department of Higher Education
scholarship.
References
Ashford RW. et al. (1992) Estimation of population at risk of infection
and number of cases of leishmaniasis. Parasitology Today, 8, 104–105.
Jamjoom MB, et al.(In Press) Towards a standard battery of
microsatellite markers for the analysis of the Leishmania donovani
complex. Annals of Tropical Medicine and Parasitology
Rodriguez N. et al. (1997) Genomic DNA repeat from Leishmania
(Viannia) braziliensis (Venezuelan strain) containing simple repeats and
microsatellites. Parasitology, 115, 349–358.
Rossi V, Wincker P, Ravel C et al. (1994) Structural organisation of
microsatellite families in the Leishmania genome and polymorphisms at
two (CA) n loci. Molecular and Biochemical Parasitology, 65, 271–282.
Russell R, Iribar MP, Lambson B. et al. (1999) Intra and inter-specific
microsatellite variation in the Leishmania subgenus Viannia. Molecular
and Biochemical Parasitology, 103, 71–77.
Query 1
Query 2
Query 3
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Informative - in what sense, please
elaborate briefly.
Please insert up to 6 keywords
Cases of what, or at risk of what?
Infection, disease, death - please
specify.
BLAST - please define
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not inlcuded on the original hard
copy - are they to remain?
WHO -please define.
Query 7
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'these' - does this refer to primer
pairs?
Informative - in what sense, please
elaborate briefly.
JM - please expand.