Molecular Plant Breeding 2015, Vol.6, No.21, 1
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high PIC, clear DNA banding patterns making
different genotypes easy to distinguish, and stable
PCR amplification. The SSR and EST-SSR primer
sequences presented in Table 4 were obtained from
and cited from papers
(Röder et al., 1998; Pestsova et al., 2000; Sourdille et
al., 2001; Guyomarc’h et al., 2002; Gupta et al., 2002;
Gao 2003; Yu et al., 2004; Chen et al., 2005; Song et
al., 2005).
Recording the genotypes at an SSR locus of
individuals in a variety
When recording the genotype at an SSR locus among
individuals in a variety, the most common homozygous
genotype was designated aa, the second-most common
was designated bb, and so on. The genotypes were
generally designated from aa to ff in order of most to
least common. Heterozygotes were designated ab, ac,
bc, and so on. The genotype at each SSR locus of each
individual in this study was recorded in a table, as
represented by Table 5.
Determination of non-homozygous SSR loci
The causes of non-homozygous SSR loci in wheat
varieties have been addressed in the Introduction.
Three genotypes at a non-homozygous locus among
individuals of a variety are possible, namely the
maternal and paternal homozygous genotypes, and the
parental heterozygous genotype (aa, bb, and ab). The
ratio of individuals with maternal and paternal
homozygous genotypes should theoretically be 1:1
(Wang et al., 2014a). However, because of genetic
segregation distortion of heterozygotes or for other
reasons, it is often the case that the number of
individuals with maternal or paternal homozygous
genotypes at a non-homozygous locus is overwhelmingly
large in a population, and the ratio of individuals with
two parental homozygous genotypes is almost 10:1 or
higher. We termed these as non-homozygous SSR loci
due to distortion of allele distribution, and treated
them as homozygous ones because that locus had little
influence on the uniformity of the variety (Wang et al.,
2009b, 2014a).
20 or 100 individual plants sampled from each variety
tested were genotyped by the SSR markers mentioned.
The 1:1 goodness-of-fit test was employed to
determine whether a non-homozygous SSR locus had
an allele frequency be apt to only one parent. When χ
2
≤ 6.63 (χ
2
0.01, 1
= 6.63), the ratio of individuals with
maternal and paternal genotypes at a non-homozygous
SSR locus in 20 individuals was considered to meet
1:1, so that the locus was a theoretical non-homoz-
ygous one. Otherwise, when χ
2
0.01, 1
> 6.63, the locus
was considered the non-homozygous locus due to
distortion of allele distribution.
Detection of the homozygous SSR loci ratio
Twenty individuals of each variety were detected by
50 or 80 SSR markers. A homozygous locus was one
with the same genotype in 20 individuals. A locus
with two homozygous genotypes among individuals
of a variety (if there were no contaminant individuals)
was regarded as a non-homozygous locus whether or
not heterozygotes were observed. The SSR-HLR is
defined as the number of the homozygous loci as a
percentage of the total number of loci tested.
The following formula was used to calculate the
SSR-HLR (Wang et al 2014a):
Where
n
is the number of loci detected, and
x
is the
number of non-homozygous SSR loci.
Identification of contaminant individuals
Thirty SSR markers (labeled by * in Table 4)
including 26 genomic-SSRs and four EST-SSRs used
to detect seed purity. Each individual plants sampled
from the tested varieties were genotyped by the 30
SSR markers. The genotypes of each SSR locus
among most plants of each variety were identical;
these plants served as the control (Ck) for identifying
contaminants. The locus with different genotype from
that of Ck was deemed to be an off-type locus. The
individual was deemed to be a contaminant for
assessment of seed purity if there were > 2 off-type
and/or heterozygous loci at the 30 SSR loci of the
individual (Wang et al., 2014b).
Calculation of seed purity
Seed purity is defined as the number of true
individuals as a percentage of the total number of
seeds tested (100 seeds), and was calculated as
follows:
100 −
n
Seed purity (%) = ×100%
n
