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Rice Genomics and Genetics 2012, Vol.3, No.8, 50
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less polymorphism in breeding population and
couldn't be applied in rice improvement (Andersen
and Lübberstedt, 2003). The best method for resolving
this problem is to develop functional markers. They
are superior to random DNA markers such as RFLPs,
SSRs and AFLPs owing to complete linkage with trait
locus alleles. In other words, functional marker based
on the sequence variation of target gene is the
guarantee of molecular marker assisted selection
(Bradbury et al., 2005; Wang et al., 2009).
In this study, according to a 3.5 kb nucleotide
sequences deletion in low glutelin content rice, we
firstly developed marker In-Del-Lgc1-A, which
amplified 4 328 bp in normal plants and 828 bp in
mutant containing
Lgc1
. By using this marker, we
detected F
2
population of W3660/Nanjing 46.
Unfortunately, whatever plant appeared only one band,
namely 4 328 bp or 828 bp, were all lack of heterozygous.
The results were obviously inconsistent with theory.
According to the situation, we thought that allelic
fragment amplified in heterozygote differed so much,
resulting in small fragments obtained significant
advantage of amplification; In contrast, large fragment
amplification efficiency was low, and so it was
difficult to detect. Two types of plant DNA were
mixed together and amplified for further confirmation
of this idea, and the large fragments were still not
detected. As can be seen, our idea was correct. To
shorten the gap of the amplified fragment length,
making the heterozygous genotype to be reflected, we
further have designed a new primer, and with one of
primer from In-Del-Lgc1-A formed a new marker
In-Del-Lgc1-B, which amplified 1 573 bp. So far,
three primers consist of two markers in the same
reaction system to detect
Lgc1
. Using these two
markers, all individuals in F
2
were effective to
distinguish. Because of the competition effect, normal
genome could only amplify 1 573 bp but not 4 328 bp,
anyway, it was not affected by functional marker
detection. Doubtlessly, they could be used in
marker-assisted selection of low glutelin-content rice.
In addition, to guarantee the phenotype and genotype
of correspondence, F
2
population used half-grain
method, half-grain to be cultivated seedling and
another to be done for total protein SDS-PAGE. Our
previous reports and experiments revealed that
half-grain seedlings were weak and vulnerable to
disease, and even dead early. For the reason, it was
difficult to get enough leaves for the test. Here, put
half-grain was disinfected into culture medium
containing 0.5‰ Yi Peilong bactericide, and obtained
a high of 95% plant survival rate without microbial
contamination in the process of cultivating seedling
(three weeks). It also provided an important reference
for half-grain of cultivating seedling in the future.
3 Materials and Methods
3.1 Tested materials
With low glutelin gene
Lgc1
of rice variety W3660, it
was based on low glutelin variety LGC-1 as female
parent and Japanese main cultivar Koshihikari
hybridization, and the latter was as the recurrent
parent continuously backcrossing. The rice variety
planted in Jiangsu has a good yield and adaptability.
12 normal gluten-content rice cultivars or lines
including Kanto 194 (Japonica rice variety, Crop
Research Institute of Ibaraki, Kanto region of
Japan), Nipponbare (Japonica rice variety, Aichi
comprehensive agricultural test site, Japan), Aichi
106 (Japonica rice variety, Aichi comprehensive
agricultural test site, Japan), Nanjing 44 (Early-maturing
late-season japonica rice cultivar, Institute of Food
Crops, Jiangsu Academy of Agricultural Sciences),
Ning 5 055 (Early-maturing late-season japonica rice
line, Institute of Food Crops, Jiangsu Academy of
Agricultural Sciences), Nanjing 11 (Conventional
indica rice cultivar, Institute of Food Crops, Jiangsu
Academy of Agricultural Sciences), Wuyunjing 7
(Early-maturing late-season japonica rice cultivar,
Wujin rice and wheat breeding field, Changzhou of
Jiangsu), Ningjing 3 (Early-maturing late-season
japonica rice cultivar, College of Agriculture,
Nanjing Agricultural University), Huajing 3 (Late
maturity japonica rice varietie, Jiangsu Dahua Seed
Limited Company), Yandao 9 (Late maturity japonica
rice varietie, Yanhai Institute of Agricultural Science,
Jiangsu). F
2
segregating population, which was
from the cross Nanjing 46×W3660 (female parent).
3.2 Materials planting
To extract DNA, the materials were planted in our