sequenced region, a candidate gene approach can
be used with sequencing in the mutant of the
candidate gene. Alternatively, one could do a
whole genome sequence of the mutant and look
in the mapped region for a mutated gene(s) (Liao
et al. 2012 ). This approach also led to the iden-
tification of a candidate gene forsnf4(Sandal and
Andersen, unpublished). To prove that you found
the right gene you need to sequence several
alleles and/or complement the mutant with
transformation with a wild-type gene construct in
Agrobacterium rhizogenes or Agrobacterium
tumefaciens. For most symbiotic mutants, the
phenotype is determined by the root genotype,
and therefore, hairy root transformation withA.
rhizogenescan be used. For more information
about the map-based cloning procedure, see
Sandal et al. ( 2005 ).
In Fig.3.1, the map positions of 49 symbiotic
(Sym) genes from L. japonicus MG-20 are
shown. In Fig.3.2, the same map is shown for
Gifu. Notice that the map position in Gifu is
different on chromosome I and the bottom of
chromosome II because of a translocation of the
region corresponding to the top 10 cm of Gifu
chromosome I to the bottom of MG-20 chro-
mosome II. Most of these genes have now been
isolated with map-based cloning. Information on
some of the map positions can be found in
Sandal et al. ( 2006 ). ForSym105see Hossain
et al. ( 2006 ). Information about the gene isola-
tion/map-based cloning projects can be found for
many genes (Table3.2).Nin was isolated by
transposon tagging andNapby a combination of
retrotransposon tagging and mapping.
3.3 Physical Mapping
Fluorescent in situ hybridisation (FISH) analysis
with BAC clones with known genetic map posi-
tions gave the link to the physical chromosomes
(Pedrosa et al. 2002 ; Hayashi et al. 2001 ; Ohmido
et al. 2010 ). Therefore, the chromosomes and
linkage groups could be numbered with chro-
mosome I as the largest Gifu chromosome cor-
responding to linkage group I and chromosome II
as the second largest chromosome corresponding
to linkage group II and so on. For the Gifu x MG-
20 map, chromosomes I and II presented a
problem. This was solved by a comparison to the
genetic map developed from L. filicaulis x
L. japonicusGifu and FISH analysis. It turned out
that there is a translocation between the lower part
of Gifu chromosome II to the upper part of MG-
20 chromosome I. On the other hand,L.filicaulis
xL. japonicusGifu has problems with low seed
set, distorted segregation and several regions with
suppressed recombination. Therefore, the align-
ment of the two genetic maps was of great help in
ordering the markers and sequences from the
BAC and TAC sequencing made by Kazusa DNA
Research Institute.3.4 AdditionalLotusSpeciesL. burttii(Sz.-Borsos et al. 1972 ; Kawaguchi
et al. 2005 ) originates from Pakistan. It is a good
crossing partner forL. japonicusas it provides a
better seed set, shorter generation times and has
fewer chromosome regions with distorted segre-
gation thanL.filicaulisxL. japonicusGifu. For
some regions with suppression of recombination
in Gifu x MG-20, such as the top of MG-20
chromosome I, the cross toL. burttiicould be
advantageous (Kawaguchi et al. 2005 ).L. burttii
has a number of phenotypic differences to
L. japonicus,and therefore, the developed RILs
are of use in QTL detection for a number of
traits. One example is a QTL forSinorhizobium
frediinodulation (Sandal et al. 2012 ).
Evolutionarily, L. kryloviiappears to be as
close toL. japonicusasL. burttiiand closer than
L.filicaulis(Degtjareva et al. 2008 ).L. krylovii
has been found in Russia and China, and an F2
population ofL. japonicusGifu x L. krylovii
(Russian accession) has been obtained (Sandal,
unpublished). This population could be used for
mapping various phenotypic traits and be further
developed to RILs.
The evolutionary relationship between the
different Lotus species based on nrITS phylog-
eny was determined by Degtjareva et al. ( 2008 ).
As part of the Lotassa collaboration, we have
further investigated the level of polymorphism3 Genetic Linkage Maps, Synteny and Map-based Cloning 23