Page 9 - PGTv3no6

Plant Gene and Trait 2012, Vol.3, No.6, 28-33
http://pgt.sophiapublisher.com
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Tp069 with genetic distance 0.01 cM; the QTL
qECP-9- 1 located on linkage group 9, which closely
linked to the Sca901 with genetic distance 0.1 cM.
Two QTLs related to easy curing potential should be
further validated to be used for markerassisted
selection breeding, while to further mine and utilize
the marker with big contribution rate would be
benefit to improvement of the target traits in marker-
assisted breeding.
3 Materials and Methods
3.1 Materials
The experiamental materials are the flue-cured
tobacco cultivar, Yunyan85, with good easy curing
potential and Dabaijing599, with bad easy curing
potential, which came from the National Tobacco
Interim Germplasm Bank. The F2 mapping population
consisting of 176 individuals was derived from the
cross of above mentioned parents. SSR primers used
in this study employed from the self-developed
primers by Tobacco Research Institute of Chinese
Academy of Agricultural Sciences and the Tobacco
Institute of Agricultural Sciences and the primers
published by Bindler (2007).
3.2 Field experimental design
The yellowing index was determined in the seedling
stage to represent the indicator of easy curing potential
in flue curing tobacco. Experiments in 2011 were
carried ut at the Jimo farm of Tobacco Research
Institute of Chinese Academy of Agricultural Sciences;
P1 (Yunyan85), P2 (Dabaijing599), F1, F2, were sown
at greenhouse in the Jimo farm. The tobacco seedlings
were transplanted into small pots with the 13~14 cm
diameter when the seedlings grew 4 to 5 true leaves;
the top spout was trimmed when the 8th leaf has
appeared. Three leaves from top to bottom were
picked up after 14 day later, and wrapped book-like
shape by using newspaper, and placed in a chamber
with constant temperature and humidity (temperature
36 ℃ , relative humidity 90%). The proportion of
yellowing was measured seven times once every 24 h,
and calculated the yellowing index (Takashi and
Tajima, 1984, Chinese tobacco, (3): 45-48).
3.3 DNA extraction and SSR analysis
DNA was extracted followed the modified CTAB
method (Yang et al., 2005), and its quality detected
by agarose gel electrophoresis and stored at - 20℃
refrigerator. The PCR reaction system: PCR reaction
system of total 15 μL including 1 × PCR buffer
solution, MgCl 2 1 mmol/L, dNTPs 1.2 mmol/L for
each, primer 0.4 μmol/L, 1 U Taq enzyme, 20 ng
genomic DNA. The PCR program: pre-denaturation
for 5 min at 94 ℃, and then 35 cycles for denaturation
45 s at 94℃, annealing for 45 s at the temperature
55℃, or 58℃ or 60℃ based on primer annealing
temperature), extension for 45 s , finally, extension for
10 min at 72℃.
3.4 Genetic linkage map construction
The linkage map was conducted by using the soft-
ware Mapmaker 3.0. The steps were as follows: First,
all markers grouped (LOD=3.0) by the “group”
command. Each linkage group was sorted by using
the "compare" and "order" command; the remaining
markers were added to the sorted sequence by using
“ry” command, and then using the “Kosambi”
function transformed the recombinant value into map
units (cM), finally a genetic linkage map was built by
“map” command.
3.5 QTLAnalysis of and naming
QTL analysis software Windows QTL Cartographer
2.5 was employed to map QTL related to easy curing
potential by using composite interval mapping (CIM)
and to detect QTL effect. The parameters for CIM
analysis were set as the follows, 2.0 cM of step, the 1
000 times of regression calculation, significant level at
0.01, and 2.0 of the LOD value.
QTL was named following as QTL + trait + chromo-
some + the number of QTL, of which the QTL with a
lowercase “q”, the trait shown as abbreviation, if there
is one more QTL on the same chromosome, a number
of different sites of the QTL on same chromosome
would be assigned to the number “1”, “2”, “3” in
order to distinguish the different locus (McCouch et
al., 1997).
Authors ' Contributions
XLT designed and carried out the experiment; ZFZ and XHX
conceived this program, directed the experiment, analyzed the
data, as well as wrote and modified the manuscript; XWZ
helped to analyze the data; NCW and JLX was responsible for