Plant Molecular Biology 35: 943–948, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
simple sequence repeat DNA in the alga Chlamydomonas
Tae-Jin Kang and Marvin W. Fawley
Department of Botany, North Dakota State University, P.O. Box 5517 Fargo, ND 58105-5517, USA (
Received 13 November 1996; accepted in revised form 28 May 1997
Key words: Chlamydomonas, Chlorophyta, microsatellite, polymorphism, simple sequence repeat DNA
A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)
probe for the presence
simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic
library, we estimate that (CA/GT)
repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome.
Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets ﬂanking (CA/GT)
sequences were constructed for four loci. The PCR was used to speciﬁcally amplify these regions from multiple
isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian
inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross
between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful
for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.
Loci with short tandemly repeated motifs such as
with n varying from 10 to 60 have been
termed short tandem repeat , microsatellites , or
simple sequence repeats (SSRs) . SSRs are com-
mon in the genomes of eukaryotes [10, 19], highly
polymorphic [20, 24], and easily typed using the PCR
Compared to restriction fragment length poly-
morphisms (RFLP), SSRs typically have more alleles
and the procedures do not require Southern blotting.
Another type of molecular marker, randomly ampli-
ﬁed polymorphic DNA (RAPDs), is also PCR-based
[3, 13]. However, RAPD markers in general behave
as dominant genetic markers and do not discriminate
between heterozygous individuals and one homozy-
gote. This contrasts with SSR markers which are gen-
erallycodominantandthereforecan distinguish among
heterozygotes and homozygotes .
SSRs have been reported in several land plants, but
have not yet been fully characterized from any alga.
Because the genetics of Chlamydomonas reinhardtii
have been studied more than for any other alga, it
provides an attractivesystem in which to investigate the
presence and features of SSR DNA sequences. Morris
et al.  assayed (CA/GT)
SSRs for C. reinhardtii
as well as several other prokaryotic and eukaryotic
organismsanddeterminedthatC. reinhardtii possessed
a large number of these repeats.
The present study addresses (1) the frequency of
SSRs in Chlamydomonas, (2) construc-
tion of PCR primers to amplify the SSR loci, (3)
polymorphism of these loci in different C. reinhardtii
strains, and (4) the mode of inheritance of the SSR loci
C. reinhardtii Dangeard (strains UTEX 89, CC-
125, CC-1418, CC-2342, CC-2344, and CC-2931),
C. asymmetrica Korshikov (SAG 70.72), C. frankii
Pascher (UTEX 1057), C. humicola Lucksch (UTEX
225), C. mexicana Lewin (UTEX 729), C. moewusii
Gerloff (UTEX 97), C. noctigama Korshikov (SAG
6.73), and C. segnis Ettl (UTEX 1919) were obtained
from the Culture Collection of Algae at the University
of Texas at Austin, the Chlamydomonas Genetics Cen-
ter at Duke University, or Sammlung von Algenkul-
ottingen. Vegetative cells were grown in WH