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Triticeae Genomics and Genetics 2011, Vol.2, No.1, 1
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6
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5
3.2 Phenotypic identification
TTSW
-
5 was mated with Jian 3 and Chuanmai 55 to
generate hybrid F
1
plants of 47 and 11, respectively.
All of F
1
plants self-crossing were to generate F
2
plants of 1107 and 176, respectively. The panicle
phenotypes of F
1
and F
2
were identified in ear
maturity. The phenotypic methods were as following:
harvesting the mature individual, measuring the
number of triple spikelet per ear and total number of
spikelet of whole ear to calculate the percentage by
the number of triple spikelet to total of spikelet, the
average percentage of each plant was assigned to be a
phenotypic data of each ear.
3.3 DNA extraction and SSR analysis
Genomic DNA was extracted from the parents and F
2
individuals derived from TTSW
-
5/Jian 3 by using
CTAB method. 944 SSR markers encoded with Barc,
Xgwm, Xgdm, Xwmc, Xcfd etc. (Röder et al., 1998;
Pestsova et al., 2000; Somers et al., 2004; Song et al.,
2005) applied to scan the polymorphism between
TTSW
-
5 and Jian 3. DNA pool of triple spikelet and
DNA pool of common spikelet were established by
using eight triple spikelet individuals and the common
according to the results of phenotypic identification,
respectively, find the polymorphism locus between the
traits of triple spikelet and common spikelet by using
the bulked segregant analysis (BSA) method.
PCR reaction was performed in the total volume of
20 μL containing 1×buffer (100 mmol/L Tris-HCl,
pH 8.3, 1.5 mmol/LMgCl
2
), 0.2 mmol/L dNTPs, 50 ng
random primers, 1 U
Taq
DNA polymer enzyme,
50~100 ng template DNA. PCR amplification procedures
were following as 94
℃
pre-denaturing for 5 min and
then following 35 cycles as 94
℃
denaturing for 1 min,
50
℃
/55
℃
/60
℃
annealing for 1 min, 72
℃
extending
for 1 min and finally 72
℃
extending for 10 min. PCR
amplification was carried out in a PTC
-
200, SSR
primers were synthesized by the TaKaRa Company.
PCR products was scored by 6% polyacrylamide gel
electrophoresis with 1×TBE of electrophoresis buffer
and 400 V voltages for 40 min, stained with nitrate
silver and digitalized by digital camera.
3.4 Genetic linkage map and QTL analysis
Linkage map was constructed by the Software of Map
Manager QTXb20, recombination rate was converted
to genetic distance (centiMorgan, cM) with Kosambi
function. QTL were detected by using composite
interval mapping of QTL Cartographer 2.5 software.
Author’s contribution
JL completed the experimental design, result analysis and
manuscript preparation and revision; JW, HTW and XRH are
the main executor of the experimental work, completed data
analysis and involved in writing. WYY conceived the project
and designed the experiments as well as wrote and revised
manuscript. All authors had read and agreed the final text.
Acknowledgement
This study was jointly supported by the National Foundation
of Natural Sciences (30871532), National Key Basic Research
Projects (2011CB100100), Specific Funds of Modern Agricultural
Technology System (CARS
-
3
-
2
-
41), Sichuan Provincial
Breeding Project, Sichuan International Cooperation Project
(2010HH0052, 2011HH0026) and Youth Fund and Excellent
Breeding Articles of Sichuan Province Finance.
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