Molecular Plant Breeding 2015, Vol.6, No.21, 1
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criteria for uniformity assessment of wheat varieties
using SSR markers were established as follows.
(1) The variety shows uniformity if seed purity and
SSR-HLR are both > 95%;
(2) A variety with an SSR-HLR < 91% is not uniform,
even if the seed purity is > 95%;
(3) A variety with seed purity ≤ 95% is not uniform,
even if the SSR-HLR is > 95%;
(4) A variety with an SSR-HLR ranging from 91% to
95% requires additional uniformity assessments in the
field, even if the seed purity is >95%.
Technical procedures for wheat variety uniformity
assessment using SSR markers
Our studies demonstrate that 30 and 80 SSR markers
(Table 4) are reasonable for detection of seed purity
and SSR-HLR, respectively (Wang et al., 2009b,
2014a, 2014b; Liu et al., 2013), and the appropriate
sample sizes are 100 and 20 individuals, respectively.
Of 80 SSR markers, each marker with an average PIC
of 0.68 produced a single amplification product. The
total number of alleles observed in 633 varieties was
666, with an average of 8.3 alleles on each locus.
Considering the experimental cost and cycle of
detection of SSR-HLRs, we proposed to divide the 80
SSR markers into 21 first-grades, 29 second-grade, and
30 third-grade markers (Table 4) based on their DNA
banding clarity and polymorphism information
content (PIC). Uniformity assessment of most of
varieties relies on the 21 first-grade markers, and that
of minority of varieties are completed by 50 markers
(21 first-grade and 29 second-grade markers) or 80
markers.
The 21 first-grade markers have sharper banding
patterns and higher PIC (0.55-0.82). SSR-HLRs of the
varieties with 21 homozygous loci are generally >
95% (Wang et al., 2014a). The 21 first-grade markers
can be used to complete about 60% of uniformity
assessment because the 21 loci of ~60% of the varieties
tested are homozygous. The 29 second-grade markers
are used in combination with the first-grade markers
to complete assessment of a further 10% of varieties
based on SSR-HLR of 50 loci. Only about 30% of
varieties need to be tested with 80 SSR markers. The
three-grade marker test system significantly reduces the
time and cost involved in wheat variety uniformity
assessment. The methods for the discrimination
of contaminant individuals from non-homozygous
SSR loci and for the detection of SSR-HLRs and the
seed purity using SSR markers are described in the
Materials and Methods. Here, the procedure for wheat
variety uniformity assessment on the basis of seed
purity and SSR-HLRs is outlined in the following
flow chart.
2 Discussion
Innovations in this study
Non-homozygous loci are one cause of inter-plant
genotypic and phenotypic differences among individuals
of a variety. Nevertheless, it is not possible to result in
phenotypic differences in a variety if the ratio of
non-homozygous SSR loci is < 5% because a variety
is classified as uniform if the SSR-HLR is > 95% on
the basis of the 3-year study described aforementioned.
Confusion of inter-plant genotypic differences caused
by non-homozygous loci and contaminant plants can
result in a false determination of non-uniformity for a
variety, even if the seed purity and SSR-HLR of the
variety are both > 95%.
The method for uniformity assessment of wheat
varieties proposed here allows researchers to correctly
distinguish inter-plant genotypic differences caused by
contaminant plants from those caused by non-homo-
zygous SSR loci. It enables more accurate and rapid
uniformity assessment of wheat varieties. On average,
assessment of one variety takes only seven days in our
laboratory. The assessment report informs breeders of
the seed purity and SSR-HLRs of their varieties. If the
tested variety is not uniform, the report will help
breeders identify the problem with the variety, and
direct breeders to improve the seed purity or
SSR-HLR, or both, of the variety. The concepts and
methods proposed here will also be of benefit for the
assessment of other crops and cultivated plants.
The same technical points on assessment of seed
purity and DUS using SSR markers
To eliminate the interference of non-homozygous loci
and accurately identify contaminant individuals when
the seed purity of a wheat variety is assessed, it is
necessary to discriminate the genotypes of contaminants
from non-homozygous loci (Wang et al., 2014b). It is
also necessary to discriminate the genotypes of
contaminants from non-homozygous loci when
distinctness of a wheat variety is assessed. To avoid
errors in distinctness assessments of wheat varieties,
we propose elimination of contaminant plants from
