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of a specific parental band. Genetic linkage maps were
constructed using MAPL 98 mapping software (Ukai.
1998). Genetic map distances in centimorgans (cM)
were calculated using the Kosambi mapping function
(Kosambi. 1944).
QTL analyses were carried out using all markers mapped
onto genetic linkage maps of ‘Fuji’ and ‘Maypole’.
Windows QTL cartographer version 2.5 (Wang et al.,
2010) was used to perform composite interval mapping
with a step size of 1.0 cM. The logarism of odds (LOD)
threshold for declaring the presence of a QTL was
defined by 1,000 permutation tests at a 5% level of
significance. The position at which the LOD score curve
reached its maximum was used as the estimate of the
QTL location. The percentage of the phenotypic variance
explained by a QTL was estimated as the coefficient of
determination (
R
2
).
3.5 Marker development for physical mapping of
QTLs
Sequences of two SSR markers, CH02a03 and
CH05c06, which flanked the QTLs on linkage group
16 of ‘Fuji’, were obtained from the High-Quality
Disease Resistant Apples for a Sustainable Agriculture
website (http://www.hidras.unimi.it/). The markers
were BLAST-searched against the genomic sequence
of the cultivar ‘Golden Delicious’ using the GDR
BLAST server (http://www.rosaceae.org). DNA
sequences of the QTL region
delimited by the two
flanking markers were screened for the presence of
SSR motifs using Tandem Repeats Finder (Benson et
al., 1999). Primer pairs flanking SSR motifs were
designed with Primer 3 Plus (Untergasser et al., 2007).
PCR amplification and SSR analysis were conducted
as described above.
3.6 Candidate gene searching
We searched for predicted gene transcripts within the
estimated QTL
region on the ‘Golden Delicious’
genomic sequence using GDR GBrowse. Their
locations and putative functions were based on
Malus ×
domestica
whole genome v1.0 assembly & annotation,
GDR GBrowse.
Author contributions
TM and KB designed the experiment. TM and KY performed
experiments. TM, KY and HO analyzed data. TM and KB
drafted the manuscript. All authors read and approved the final
manuscript.
Acknowledgments
The research was financially supported by the Sasagawa
Scintific Reserch Grant from The Japan Science Society.
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