Molecular Plant Breeding 2015, Vol.6, No.21, 1
-
17
15
Where 100 is the number of individuals tested and
n
is
the number of contaminant individuals.
Discriminance of contaminant individual genotypes
from non-homozygous SSR loci
As the discussion aforementioned, it is necessary to
discriminate contaminant individual genotypes from
non-homozygous SSR loci in assessments of seed
purity and DUS of wheat variety using SSR marker.
For clarity, the discriminance have to be reproduced
here. To illustrate the distinction of the two phenomena,
the 20 SSR loci genotypes of 30 individuals from the
wheat variety WJ02 are presented in Table 5, which
was given as an example in an article about the
assessment of wheat seed purity (Wang et al., 2014b).
At the 20 SSR loci, the genotypes of only six loci
(
Xgdm72
,
Xcfd76
,
Xksum62
,
Xgwm304
,
Xbarc198
,
and
Xcfa2028
) were uniform among individuals of
WJ02. The genotypes at 14 loci differed among
individuals. These results showed the presence of
contaminants and non-homozygous SSR loci in WJ02.
The genotypes at > 3 out of 20 loci of WJ02 individuals
2, 3, 5, 8, 10, and 27 differed from those of other
individuals. These six individuals were considered to be
contaminants according to the criteria aforementioned.
After omitting the data for these six contaminant
individuals, whether SSR loci are non-homozygous is
readily estimated from the remaining data. The
genotypes at 18 loci of 24 individuals were aa, with
the exception of
Xgwm294
and
Xcfd72
. Three
genotypes (aa, bb, and ab) were detected at
gwm294
.
The numbers of individuals with the three genotypes
were 19, 3, and 2, respectively. This locus was
deemed to be a non-homozygous SSR locus due to
distortion of allele distribution because χ
2
= 9.38 on
the basis of the 1:1 goodness-of-fit test aforementioned,
and thus
gwm294
was treated as a homozygous locus.
Two genotypes (aa and bb) were detected at
Xcfd72
.
The number of individuals with the two genotypes
were 17 and 7 (χ
2
= 3.38). This locus was deemed to
be a theoretical non-homozygous locus.
Observation of morphological traits
To determine the relationship between the SSR-HLR
and the uniformity of the wheat varieties, the grains of
individual plants of nearly 60 varieties with an
SSR-HLR ≤ 95% from the national winter wheat
regional trials in 2008, 2009 and 2010 were harvested,
and were then grown by the plant-to-row method in
the following years (2008-2009, 2009-2010 and
2010-2011). The phenotypic and agronomic traits
were compared among different rows of a variety. If
the rows of a variety were distinct from each other,
according to the criteria in Table 6, it indicated that
the variety was still segregating and was not uniform.
Concurrently, 10-20 varieties with an SSR-HLR >
95% were grown in blocks. Phenotypic and
agronomic traits were then compared among different
individuals of a variety. 30 to 50 individuals were
sampled from each row or block to investigate the
phenotypic and agronomic traits listed in Table 6.
Calculation of genetic similarity coefficient
In this paper, the GS was used to evaluate the
similarity between two plants. The GS between two
plants is close to 1.0 if they share the same genotype.
When two plants were scored as having the same
genotype at a locus, the locus was increased by 1 as
the numerator of the formula for calculating GS. For a
heterozygous locus in a plant, 0.5 was added as the
numerator if its genotype for one allele was the same
as the homozygous genotype at the same locus of
another plant. For example, the SSR locus
Xgwm294
of plant 18 and 19 of variety WJ02 is homozygous and
heterozygous, respectively (Table 5), therefore 0.5
was added as the numerator of the formula for
calculating GS because the genotype of one allele of
plant 19 is the same as that of plant 18. Moreover, if a
locus for two plants was heterozygous, and the genotypes
for one allele were the same but those for another
allele differed between the two plants, 0.5 was added
also as the numerator because one allelic genotype
differed between the two plants. The numerator was
divided by the total number of loci compared between
two plants.
The formula for calculating GS was:
Where
x
is the number of loci with the same genotype
between two plants,
n
is the number of loci compared
between the two plants, and 1 is a correction value
owing to the limited number of markers tested.
