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Molecular Plant Breeding 2010, Vol.1 No.4
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And the genetic distances between
Pm21
and
molecular markers of
Xcfe164
,
Xedm129
and
Xcinau1-
88
were all small, 3.75 cM, 1.26 cM and 0.98 cM,
respectively, so they had use value in marker-assisted
selection for
Pm21
, and can be used for marker-assist-
ed breeding. The laboratory which used the above
three markers to assisted selection of
Pm21
has proven
their reliability.
3 Materials and methods
3.1 Wheat materials
The wheat germplasm line CB033 (containing the
powdery mildew resistance gene
Pm21
), the common
wheat Huixianhong, CB033 and Huixianhong produc-
ed the hybrids F
1
, F
2
and F
2:3
families. The wheat
germplasm line CB033 were favored by researcher Chen
xiao who was from the Chinese Academy of Agricultural
Sciences Department, and saved in the Tai’an Subcenter
of National Wheat Improvement Center.
3.2 Primers
Selected 258 pairs of primers which all covered on
wheat homoeologous group VI, included three types,
genomic SSR markers, EST-SSR markers and STS
markers (YuanN Yuan-Yuan et al., 2010). All of the
above primers were synthesized by Shanghai
Biological Engineering Technology Co., Ltd.; 10×PCR
Buffer, dNTPs and Taq enzymes were Takara
Biotechnology Co., Ltd. products.
3.3 Evaluation of wheat powdery mildew resistance
the susceptible parent Huixianhong was seeded into
the infection line as susceptible CK , and the E15
strains were vaccinated in the infection line; when
Huixianhong fully infected, surveyed the powdery
mildew resistance of parents, F
1
, F
2
and F
2:3
families,
and analysed the resistant and susceptible segregation
of progenies.
Powdery mildew grading standards were grading
standards:
0 type: Immunization, plant no lesion;
0 type: necrotic reaction, leaves have dead spots;
1 type: high resistance, small lesions (usually less than
the diameter 1 mm), hyphae thin layer of green leaves
can be seen, occasionally larger lesions, but through
the green, sporulation little;
2 type: middle resistance, leaf lesion diameter less
than 1 mm, but the mycelium layer thick, opaque
green, can produce a certain amount of spores;
3 type: middle susceptible, leaf lesions more common
diameter greater than 1 mm, slice thickness mycelium,
sporulation capacity, but the lesion is not contiguous;
4 type: high susceptible, leaf lesion diameter greater
than 1 mm, slice thickness mycelium, sporulation
more contiguous lesion (see Sheng Baoqin, 1988,
Plant Protection, 1: 49),
Type 0, type0, type 1, type 2 for the resistance; type3,
type4 as susceptible. The E15 strains of powdery
mildew was provided by Dr. Li Hongjie who came
from the Chinese Academy of Agricultural Sciences.
3.4 Extraction of total DNA
According to SDS-phenol extraction to extract the
DNA of plant young leaves (Devos et al., 1993).
3.5 Detection of PCR amplification and electropho-
resis
Reaction system was 15 μL, and amplification proced-
ures used the landing PCR (touchdown PCR), then
amplification save under 10
℃
(YuanN Yuan-Yuan et
al., 2010). Amplification equipment used by the U.S.
company Bio-Rad's 9600 Thermal Cycler. PCR products
were separated by 8% non-denaturing polyacrylamide
gel, then stained by silver nitrate.
3.6 Markers analysis
Preferred small groups methods were used to analyze
(Hao et al., 2008), extracted total DNA from the 10
typical resistant individuals (the plants of 0 or 0; type)
and the 10 typical susceptible individuals (powdery
mildew-level 4
-
type plants), then used PCR amplific-
ation and electrophoresis to search for the markers
which were consistent in the phenotype and the
genotype (Zong Hao et al., 2009), further verified
them in F
2
segregating populations, and calculated the
the linkage genetic distance between the marker and
the gene (
Pm21
). Using chi-square test (χ2), determined
the observations of the separate groups and the fitness
of the expected degree. The genetic distances between
Microsatellite markers and
Pm21
were calculated by
JoinMap software, finally, the genetic linkage map
was drawn by MapDraw (Liu Renhu et al., 2003).