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Tree Genetics and Molecular Breeding 2014, Vol.4, No.2, 1
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in apple fruit juice. LAR catalyzes the conversion of
leucocyanidins into the flavan-3-ols catechin and
epicatechin. Catechin and epicatechin are the building
blocks of procyanidins (Bogs et al., 2005) and are
substrates for PPO, resulting in fruit flesh/juice
browning. MdLAR co-localizes on chromosome 16
with QTLs for phenolic compounds including
catechin, epicatechin, and procyanidin (Chagné et al.,
2012; Khan et al., 2012a), and LAR expression is
significantly correlated with the content of these
metabolites (Khan et al., 2012b). LAR is thus another
possible candidate gene for the fruit juice browning
QTL. Consequently, an understanding of the effects of
LAR expression on the degree of fruit juice browning
is required for elucidation of the genetic mechanisms
controlling fruit juice browning in apple.
3 Materials and Methods
3.1 Plant materials
A cross combinations between apple cultivars ‘Fuji’
and ‘Maypole’ were carried out in 1999, resulting in
‘Fuji’ × ‘Maypole’ F
1
population of 79 individuals.
Progeny used in this study were grown on their own
roots and planted 0.5 m apart in rows separated by 2.0
m at the experimental farm of the Laboratory of
Pomology, Faculty of Agriculture, Shinshu University,
Nagano, Japan. Orchard management was performed
according to commercial practices used in the
southern part of Nagano Prefecture.
For DNA extraction, fresh young leaves were
collected and stored frozen at −85°C until use.
Genomic DNA was extracted from 0.5 g of leaves
(fresh weight) using a Plant Genomic DNA Maxi
extraction kit (Viogene, New Taipei, Taiwan). The
extracted DNA was quantified using a GeneQuant Pro
spectrophotometer (Amersham, UK), confirmed by
agarose gel electrophoresis, and then diluted to 20
ng/µL with 0.1× TE buffer.
3.2 Phenotypic assessment
Fruit samples were harvested over 3 successive years
from 2011 to 2013. Fruit from individual genotypes
were picked at maturity, when the ground color at the
calyx end had changed from green to yellowish green
or creamy, and immediately before commencement of
fruit drop in mid-August to late October. The fruits of
each individual were harvested several time as they
reached maturity.
For phenotypic assessment, five fruits per genotype
were randomly selected and assessed for both fruit
acidity and fruit juice browning. Crude juice was
extracted with a grater from each fruit. After obtaining
fruit juice by passing the crude extract through two
layers of cheesecloth, 5 mL of this sample was diluted
with approximately 30 mL of distilled water.
Titratable acidity was determined by titrating the
diluted sample with 0.1 N NaOH. Titration results
were calculated as malic acid (g) per 100 mL of
sample juice. The degree of fruit juice browning was
visually evaluated 6 h after juice extraction by two
independent investigators and categorized into five
classes as follows: 1 (none), 2 (low), 3 (medium), 4
(high), and 5 (extremely high). Phenotypic values of
each genotype represented the mean values obtained
from the analysis over 3 successive years.
3.3 SSR analysis
A total of 120 SSR markers were selected from
previously reported markers (Gianfranceschi et al.,,
1998; Guilford et al.,, 1997; Hokanson et al.,, 1998;
Liebhard et al.,, 2002; Silfverberg-Dilworth et al.,,
2006; Yamamoto et al.,, 2002), and each primer was
labeled with fluorescent dye (6-FAM, HEX, or NED).
PCR amplifications were performed under the
following conditions: initial denaturation at 94 °C for
2 min, followed by 35 cycles at 94 °C for 1 min,
60°C~55°C for 1 min, and 72 °C for 2 min, and a final
extension at 72 °C for 8 min. Allele sizes of the
parental amplicons were determined using an ABI
PRISM 310 Automated Fluorescent DNA sequencer
(Applied Biosystems, Foster City, CA, USA), and the
amplified products from the population were separated
on a 6% denaturing polyacrylamide gel containing 8
M urea in 0.5× TBE buffer. Fragments were visualized
by silver staining using a Silver Sequence DNA
Sequencing system (Promega, WI, USA).
3.4 Genetic linkage mapping and QTL analysis
Segregation analysis based on the pseudo-test cross
mapping strategy (Grattapaglia and Sederoff 1994)
was performed on 79 progeny of Fj × Mp. Segregation
of each marker was scored by the presence or absence