in a short time (Vos et al. 1995 ). The level of
polymorphisms is very high between Gifu andL.
filicaulis. In that way, a number of genetic
markers were found and they could be grouped
into six linkage groups corresponding to the six
chromosomes of L. japonicus (Sandal et al.
2002 ). Recombinant inbred lines (RILs), gener-
ation S8, have been developed fromL.filicaulis
xL. japonicusGifu (Sandal et al. 2006 ). The
advantage of RILs is that they are almost com-
pletely homozygous, and therefore, mapping
information can be shared between groups
working with these lines.
3.2 Ecotype Miyakojima MG-20
The ecotypeL. japonicusMiyakojima MG-20
was found on one of the southern islands of
Japan (Kawaguchi 2000 ). Because of the large
geographical distance to most of the other eco-
types, it has a high level of polymorphisms to the
Gifu ecotype.L. japonicusMG-20 also has the
advantage of growing very well in indoor sys-
tems (Kawaguchi et al. 2001 ) and was therefore
chosen for genome sequencing by Kazusa DNA
Research Institute in Japan with a BAC/TAC
sequencing strategy (Sato et al. 2008 and refer-
ences therein). With the sequences of BAC or
TAC clones, it became possible to look for
microsatellite sequences such as TATATATA
that are highly polymorphic in, for example, TA
repeat number and can therefore be used to
develop microsatellite size markers that are easier
to work with than AFLP markers. Microsatellite
markers can be tested in high percentage (2–4%)
agarose gels to see small size differences. The
microsatellite markers were developed to work
with the same PCR conditions, such as an
annealing temperature of 55°C. AFLP markers
were also found in the cross between Gifu and
MG-20. The AFLP and microsatellite markers
were used to develop an F2 genetic map for this
cross (Hayashi et al. 2001 ). Some of the micro-
satellite markers were included in both maps, and
therefore, it was possible to see the colinearity
between the two maps. With the substantial
increase in the number of microsatellite markers,
the high level of colinearity between the maps of
L.filicaulisx Gifu and Gifu x MG-20 was clearly
seen (Sandal et al. 2006 ). Only the genetic dis-
tances between markers varied in the two
crosses. So, with the combination of the mapping
information from the two crosses, most of the
BAC and TAC sequences from the genome
sequence efforts could be ordered on the six
linkage groups ofL. japonicus. The number of
AFLP markers in the Gifu x MG-20 population
has been substantially increased to 2,053 markers
by Wang et al. ( 2008 ). In that case, the mapping
was done on RILs of Gifu x MG-20 developed at
Kazusa DNA Research Institute. This made it
possible to look for codominant markers as the
RILs are almost 100 % homozygotic. These RILs
are available through LegumeBase (http://www.
legumebase.brc.miyazaki-u.ac.jp/lotus/
rilStrainListAction.do). RILs are developed by
selfing up to S8 from F2 populations from the
crosses. The genotypes of the different lines can
be downloaded from http://www.kazusa.or.jp/
lotus/RIline/index.html. These lines have been
used for the QTL analysis of 13 agronomic traits
by Gondo et al. ( 2007 ). There are so many dif-
ferences between Gifu and MG-20 that MG-20
has been suggested as a new species,Lotus mi-
yakojimae(Degtjareva et al. 2008 ).
As more and more BAC and TAC sequences
became available, more microsatellite markers
were developed and microsatellite markers
became the chosen method for the mapping of,
for example, symbiotic genes known from
mutants. A set of such markers distributed over
the six chromosomes (Table 3.1) can quickly
give an approximate map position of the gene.
Further, microsatellite markers from the region
can then be used to narrow down the region of
the gene. It should be noted that in the cross
between Gifu and MG-20, the markers on the top
of chromosome I and the bottom of chromosome
II appear linked because of the translocation
occurring between these ecotypes. Information
about primer sequences, map positions, and the
quality of the marker for hundreds of microsat-
ellite markers can be found athttp://www.kazusa.
or.jp/lotus/markerdb_index.html. When the
region is narrowed down to less than 200 kb in a22 N. Sandal and S. Sato