prime objective of plant breeding is to produce new varieties
(cultivars) of crops of agricultural and horticultural importance
that represent a genuine improvement in yield and/or quality over
existing varieties. An increasingly important trend is towards
the production of hybrid varieties. Hybrids offer farmers increased
yields, wider adaptability and reliability of both performance
and quality by the exploitation of heterosis (`hybrid vigour').
There are also benefits to the plant breeding and seed industries
in the commercialisation of hybrids, both in terms of better protection
of intellectual property rights and of guaranteed annual seed
sales to farmers.
there is also a need to be able to assess the hybrid (genetic)
purity of seed lots, as part of the seed certification process
and as a means of general quality control. Only then can the genetic
gains introduced by the breeding process be fully available to
purity testing can be carried out in the field by careful observation
of morphological characters to recognise non-hybrid plants. However,
these procedures are slow, labour-intensive and require large
areas of land. A rapid laboratory-based test is required, which
allows decisions to be made on seed lots prior to sowing.
Despite some success with biochemical methods, they are unlikely
to offer a long-term solution in most crops, especially those
being developed as hybrid varieties. Methods also need to be rapid,
highly automated and analyse multiple gene loci simultaneously.
Second generation DNA profiling methods that are being developed
and evaluated offer the greatest potential for such a system,
particularly the use of sequence-tagged site microsatellites and
AFLP (amplified fragment length polymorphism).
bred varieties of crop plants must undergo statutory testing before
they can be marketed within the EU to determine their eligibility
for inclusion on a National List of Varieties. This testing requires
that varieties must be distinct (D) from others known to exist,
as well as uniform (U) and stable (S) in the characteristics used
to distinguish and describe them. Plant breeders may protect their
intellectual property in new varieties of crops through the statutory
Plant Breeders Rights' (PBR) schemes, based on the same DUS principles.
Current DUS testing relies mainly on morphological comparison
of new (candidate) and existing varieties in the same trial. Guidelines
for the testing of different crops are laid down by UPOV, consisting
of lists of such characters and protocols. Many of the morphological
characters can be affected by environmental interactions, being
multigenic and continuously expressed, and this necessitates replication
of observation. Some species also have insufficient descriptors
to allow identification or discrimination of all varieties. There
are thus compelling reasons to find more rapid and cost-effective
procedures that could augment the morphologically-based approach.
To develop where necessary, optimise and evaluate the use of DNA
profiling methods (microsatellites and AFLP) for plant variety
identification and registration and for hybrid purity assessment
in crops of interest to European agriculture (maize, sunflower,
and Brassica crops, primarily oilseed rape and the vegetable white
To use maize as a model species for examining hybrids of various
types and for comparison with the recognised and widely used biochemical
methods for hybrid purity assessments
To use oilseed rape and kale as models for other Brassica crops
where hybrid varieties of different types are commonly produced
(e.g. Brussels sprouts, cabbage, cauliflower), to compare the
results obtained with those from existing biochemical/molecular
methods and to examine the transportability of techniques between
species within the genus.
To develop not only the necessary profiling technology, but also
the overall methodology required for the use of the techniques
in the seed testing and certification context and to devise an
operational system. This would involve consideration of both technical
and statistical issues.
To organise 'ring tests' to compare data obtained from the use
of the optimised method(s) with a particular seed lot with those
obtained from (any) existing biochemical analysis, and observed
in field plots of the same lot. Whilst this comparison would apply
primarily to the overall level of hybrid purity determined by
the different approaches, it would also include the examination
of any 'off-types'. This would form part of the assessment of
an optimised operational system for seed certification.
To develop databases of varietal DNA profiles and potential applications
of such techniques and databases to varietal registration.
research thus promotes collaborative research and the development
of harmonised and efficient systems for plant variety testing
throughout the EU, as well as the availability of high quality
varieties. It will provide tools for production of higher quality
raw materials and increase the efficiency of the agri-industries
within Europe. The DNA profiling systems can be used readily to
trace varieties (and the products derived from them) throughout
'total quality management' systems, from propagative material
to the end product, and contribute to the improvement of the quality
work will develop, optimise and evaluate the use of two DNA profiling
methods. Microsatellite and AFLP `Variety Test Sets' will be constructed,
comprising a number of pairs of oligonucleotide primers revealing
polymorphisms within a collection of reference varieties of maize,
sunflower, oilseed rape and white cabbage. These microsatellite
and AFLP Variety Test Sets will then be screened against a wide
range of varieties, inbred lines and hybrids of different types
for each of the four species. Comparative ring tests of two DNA
profiling methods (sequence tagged site microsatellites and AFLP)
for determining levels of hybrid purity in maize, sunflower, oilseed
rape and white cabbage will then be organised.
purity results obtained from the use of microsatellites and AFLP
will be compared with those obtained from the use of existing
biochemical (maize) and molecular (oilseed rape) methods of hybrid
purity assessment and morphological assessment of field trials
(maize, sunflower, oilseed rape).
of microsatellite and AFLP primers developed will be investigated
for oilseed rape and white cabbage to other Brassica species.
Consideration will be given to the development of operational
systems for the use of microsatellites and AFLP for hybrid purity
assessment in the context of seed testing and certification, in
a range of different types of hybrid of each of the species. Databases
of DNA profiling information will be compiled for varieties and
hybrids for each of the species being considered.
oilseed rape, NIAB has identified some microsatellites using
a LiCor automated sequencer. Others have been obtained from the
literature, database searches and enriched library cloning. These
have been evaluated for polymorphism and reliability. A total
of 64 primer pairs were evaluated in total. From these, 11 were
found to amplify successfully and were polymorphic in oilseed
Sunflower, 25 putative microsatellite clones have been sequenced
by NIAB; 14 of which show microsatellites or SSRs, but only four
were suitable for primer design. Of these, two have been tested
with a small number of varieties but no polymorphism was found.
A further 2 microsatellites were obtained from the literature
(Brunel, 1994) but found to be non-polymorphic. Of 14 microsatellites
published on the WWW (Huestis, 1997) and tested, 12 amplify satisfactorily
and 5 reveal some polymorphism.
White cabbage, PRI (formerly CPRO) has tested 19 primer
sets from the literature, the 10 sets designed from EMBL database
sequences and the 36 sets designed on sequences obtained from
PRI enriched. The 12 most informative primer pairs, which gave
good quality products with clear and readily scored profiles,
were taken forward for further testing and evaluation.
for Maize, UHOH has developed maize microsatellites by
thorough examination of the publicly available primer sequences.
(PCR, MetaPhor gel electrophoresis) for analysing 19 selected
microsatellites were also established; 10 fluorescence-labelled
maize primer pairs have been employed with the ALF.
rape: PGS tested a large set of AFLP primer combinations on
two Brassica napus lines. 79 combinations yielding high-quality
patterns were then evaluated on the set of ten representative
lines from different countries. Two additional primer combinations
(E32-M48 and E32-M62) were tested on the ten lines. The combination
of MseI primers with EcoRI primer E32 appear to
yield the highest average level of polymorphisms. PGS decided
to use this primer as one common EcoRI primer in combination
with six MseI primers for further development. The six
combinations can be multiplexed cost-effectively in two sets of
three combinations in the ABI377-assisted non-radioactive assay.
the AFLP methods have been adapted by NIAB to utilise the LiCor
for analysis. Evaluation of potential primer combinations was
initially by assessment of band patterns produced using DNA pooled
from 10 plants each of two parent lines and their hybrid variety.
Other criteria for selecting primer combinations for further analysis
were applied at this stage, including uniform intensity and distribution
of bands across the gel. Sixty four primer combinations have been
tested on this basis - Eco RI primers with the following selective
bases; AAC, AAG, ACA, ACC, ACG, ACT, AGC & AGG, in all combinations
with the following Mse I primers; CAA, CAC, CAG, CAT, CTA, CTC,
CTG & CTT. From this initial investigation, 32 combinations
were selected for polymorphism analysis.
cabbage: two primer combinations were extensively tested by
PRI and a third primer combination chosen from the previously
selected fourteen optimal ones, based on band quality, degree
of polymorphism and reproducibility in a test set of OPVs as well
as hybrid varieties with their parent lines. The three AFLP primer
combinations chosen are E39 + M36, E39 + M38 and E37 + M33.
AFLP development has been completed by UHOH using P33-labelled
primers. In total, 10 maize inbred lines were evaluated with 10
AFLP primer combinations.
Variety Test Set Development, microsatellites.
oilseed rape, the most useful microsatellites identified above
by NIAB were taken and used with 50 varieties to assess polymorphism
levels. Varieties included winter, spring and forage types. 64
primer pairs were evaluated for size, quality and polymorphism
and 11 were found to be polymorphic and reliable, but four showed
only two alleles and a fifth was incompatible for multiplexing.
Bn9, 12, 26 and 59 can be multiplexed and amplified successfully
with a 600 anneal. Bn 9 and 26 overlap in size but
are labelled with IR 700 and 800 dyes respectively. Clone 33 and
MB5 can be multiplexed at 550 and overlap in size,
but the use of different dyes avoids detection problems. The latter
markers cannot be run on the same gel because they are similar
in size to Bn9 and 26.
sunflower, the available microsatellite primers that were
found by NIAB to show polymorphism between and within varieties
were tested using the LiCor.
white cabbage PRI took the twelve most informative primer
sets from Task 1, that is the sets that amplified unambiguously
scorable products with the highest degree of polymorphism. These
were combined into three multiplexes of four microsatellites each.
One primer set was redesigned to obtain a PCR product size that
fits into the multiplex C.
have developed a variety test set (VTS) for maize consisting
of 5 primer pairs. A major criterion for selecting the primer
pairs was the size range of alleles detected within 57 maize inbred
lines: phi064 (Chromosome 1) covers the range of 75 to 113 bp,
phi073 (Chromosome 3) 90 to 102 bp, phi077 (Chromosome 6) 123
to 149 bp, phi057 (Chromosome 7) 153 to 157 bp, and phi071 (Chromosome
10) 207 to 214 bp. Non-overlapping allele ranges are important
for the future establishment of multiplex reactions. In order
to cover most of the genome, microsatellites were selected from
different chromosomes and with high PIC values to increase the
chance of detecting impurities.
Variety Test Set Development, AFLP.
oilseed rape, the AFLP variety test set for oilseed rape
has been defined by PGS. Assessing genome coverage obtained with
the VTS was also considered. Both polymorphism and genome coverage
were considered as a criterion for VTS selection. To assess the
genome coverage provided by the informative markers generated
by the six primer combinations these markers were integrated in
a published linkage map of B.napus. It was developed using
a doubled-haploid mapping population derived from a cross between
the spring variety Stellar and the winter variety Major and contains
RFLP markers. This map has been aligned (unpublished information)
with two other maps. The maps show a high degree of co-linearity.
They are considered as reference maps for B.napus. AgrEvo-PGS
has accessed the Stellar x Major mapping population. The six AFLP
primer combinations were run on this population and segregating
AFLP markers scored, added to the existing marker data and included
in a new linkage analysis. Nineteen linkage groups were obtained.
The markers generated by the proposed VTS covered 82% of the B.napus
genetic map. The following selective AFLP primer combinations
have been selected as the variety test set for B. napus:
E32 in combination with M47, M48, M50, M51, M61, M62.
analysed 32 primer combinations for sunflower which
fulfilled the criteria for selection with a wide range of varieties
to determine polymorphism levels and reproducibility. 8 hybrid
cultivars were chosen, including varieties from within one breeding
programme and from a number of unrelated programmes plus specialist
material. The number of polymorphic bands detected was taken as
a measure of informativeness, and the most informative primer
combinations taken for further analysis. Among these eight varieties,
good levels of polymorphism were produced by 12 primer combinations.
There were a minimum of six polymorphic bands with up to 19 polymorphisms.
However, there were some discrepancies between these results and
the earlier screening, suggesting that further development work
cabbage, PRI selected the following AFLP primer combinations
from Task 2: E39/M36, E39/M38 and E37/M33. Finally, in maize,
UHOH investigated 51 maize inbreds with EcoRI/MseI and PstI/MseI
AFLP primer combinations. Generally the number of informative
bands was higher with the EcoRI/MseI primer combinations, so exclusively
EcoRI/MseI AFLP primer combinations were employed. Statistical
analyses of this data will facilitate selection of the most suitable
primer combinations as a variety test set.
Evaluation of Variety Test Sets.
has evaluated six primer pairs in oilseed rape, comprising
the microsatellite VTS using 50 varieties of various types. For
each primer pair, the separation rate (defined as s = number
of pairs of varieties separated / total number of pairs of varieties,
as a percentage) was determined, both alone and in combination
with others. Those primer pairs, which amplified multiple polymorphic
bands (alleles), tended to give much higher levels of separation
than those which have only a limited number of allelic states.
example, the primer pair Bn12, which amplifies 3-6 differently
sized bands per variety, gave a separation rate of 83.8%, whilst
SLA2G showed only two alleles with a ratio of 47:3, giving a separation
rate of 11.5%. Combining the data from more than one microsatellite
increased the separation rates. When used in combination the two
most polymorphic primers pairs, Bn12 and clone 33, provided a
separation rate of 95.1%. This could be increased further still
by the addition of data from other primer pairs, such that for
instance Bn12, clone 33, Bn26 and Bn27 in combination achieved
a 97.8% separation.
has started the AFLP variety test set screening for oilseed
rape on 33 varieties representing a wide range of germplasm.
A final evaluation of the variety test set will be done by calculating
the marker index for each primer combination based on the results
of the screening of 33 lines. The marker index not only considers
both the level of polymorphism and the frequency of the marker
alleles in germplasm.
has tested the five most useful and polymorphic microsatellite
primer pairs in sunflower in a range of varieties. These
are now being evaluated with a series of parent and hybrid lines.
In white cabbage the multiplexes of microsatellite primer
sets from Task 3 have been tested by PRI on a series of 70 hybrid
varieties of diverse types using bulks of four plants from each
variety. The multiplexes could distinguish all 70 varieties from
each other. This level of discrimination was accomplished using
only two of the multiplexes, and in several of the variety bulks
more than two alleles for some of the primer sets were found.
B. oleracea being essentially a diploid species, this implies
variability at these loci between the individuals of the bulk
sample, in turn suggesting variability of the parent lines at
these particular markers.
AFLP variety test set evaluation in white cabbage was
performed by PRI on the same 70 hybrid varieties as have been
used in the microsatellite analysis above, using the three primer
combinations from Task 3. All varieties could be distinguished
from each other. In this case, this could already be achieved
using only either primer combination E39/M36, which produces 53
polymorphic bands, or E39/M38, which produces 47 polymorphic bands.
Primer combination E37/M33, which produces the lowest number of
polymorphic bands (40) was by itself not able to distinguish all
has completed variety test set evaluation for maize microsatellites
using the five primer pairs selected above, plus an additional
14, to investigate 55 maize inbred lines. This information has
been used to confirm selection of the VTS. AFLP data obtained
in Task 2 using 10 AFLP primer combinations for 10 maize inbreds,
as well as those obtained using 51 maize inbreds and 8 AFLP primer
combinations, will be analysed within the next months to select,
confirm and evaluate the most appropriate AFLP VTS. Microsatellites
appear to be the preferable marker type compared to AFLPs for
the second half of this project in maize. Especially for highly
polymorphic species like maize, where many markers exist and multiplexing
is possible, microsatellites should detect impurities or heterogeneities
in seed lots quicker and with lower costs than AFLPs.
Hybrid Purity Assessment - Ring Tests.
oilseed rape, two hybrid varieties were analysed by NIAB
and their parental lines with four of the VTS microsatellites.
Both hybrids showed a range of off-types, the origin of which
appeared to be due to heterogeneity among the parental lines,
contamination with stray pollen or seed lot contamination. Maternal
selfing could be ruled out with the majority of cases. Parental
lines showed a range of off-type contamination, from uniform to
20% off-types. This is presumably a result of the breeding process,
which does not focus on homozygosity at non-morphological loci.
also tested sunflower varieties and their parental lines,
using the microsatellite VTS. These analyses are currently being
white cabbage PRI have chosen two varieties from the 70
analysed in Task 4 to screen more extensively using two of the
microsatellite multiplexes. From >100 individuals of each variety,
variety 1, nine selfings could be shown. In both varieties, a
few unexpected alleles were also found, indicating some variability
in the parent lines used for the production of these hybrids.
In one case for variety 1, a possible "outside" pollination
could be inferred. Thus it is already possible to discern all
relevant aspects of genetic impurities in hybrid varieties using
a relatively small set of variable microsatellite markers, without
having to refer to the parent lines. The selfings inferred by
the use of microsatellites were also completely corroborated by
the AFLP data. The possible "foreign" pollination inferred
by microsatellite analysis was also corroborated.
used maize supplied by UHOH comprising six hybrids and
parent. In parallel, the respective hybrids and parent lines have
been grown at UHOH to produce leaf material from 50 individuals
per hybrid and 20 individuals per parent line. The results will
be compared with those obtained using the microsatellite and AFLP
VTS by UHOH.
Comparison of Hybrid Testing Data - maize/oilseed rape.
samples supplied by UHOH are also being used to compare results
from biochemical (zein and isozyme analysis) and molecular analysis
of the method and its simplification enables a direct comparison
of different hybrids or inbred lines. The optimised method is
being applied to the samples supplied by UHOH. Method development
indicates the possibilities for computer-based identification
Comparison of Hybrid Testing Data - field trials.
oilseed rape field trial has been planted at NIAB, comprising
5 hybrid varieties and 5 parental lines (one line is the female
parent of three of the hybrids and not all parent lines of all
hybrids are represented). Additional plots were drilled consisting
of hybrid varieties deliberately contaminated with known percentages
of other varieties or parental lines. DNA sampling of the plots
is now complete and gel analysis is underway. Problems in purity
have been noted for by INSPV for sunflower in five commercial
hybrids and their inbred lines. Androsterile plants have been
observed in some commercial varieties. This will facilitate provision
of mixtures in the trial at INSPV next year. There are five commercial
hybrids of sunflower being tested, together with the male and
female lines. Maize samples (mixtures of six varieties
and varying proportions of their female lines) have been prepared
and supplied by UHOH and sown at two trial sites in Spain. The
results from the two sites will be compared.
Transportability of cabbage and oilseed rape microsatellite and
microsatellite multiplexes comprising the white cabbage VTS have
been tested by PRI against four Savoy cabbage, two red cabbage,
nine cauliflower and four Brussels sprouts hybrid varieties. The
microsatellites worked well in these crop types, with good amplification
and some new alleles being noted. All varieties could be distinguished
from each other, using only two of the multiplexes. The crop types
also formed groups that could clearly be distinguished from each
other. Transportability of the microsatellites was not limited
to within Brassica oleracea alone, for two of the multiplexes
tested have also worked in twelve oilseed rape varieties that
were obtained from partner 1.
Institute of Agricultural Botany (NIAB)
+44 1223 34 22 72
+44 1223 27 76 02
- Albrecht MELCHINGER
Universität Hohenheim (UHOH)
Tel: +49 711 459 23 34
Fax: +49 711 459 23 43
- Bart LAMBERT
Plant Genetic Systems nv (PGS)
Jozef Plateaustraat 22
Tel: +32 9 235 84 85
Fax: +32 9 224 06 94
- Lioba Friese
Staatliche Landwirtschaftliche Untersuchungs- und Forschungsanstalt
Tel: +49 721 946 81 00
Fax: +49 721 946 82 09
- Ben VOSMAN
PRI - Agrotechnological Research Institute
P.O. Box 16
NL-6700 AA Wageningen
Tel: +31 317 47 69 80
Fax: +31 317 41 59 83
- Jose I. ORTEGA MOLINA
Jose Abascal 4
Tel: +34 91 347 41 62
Fax: +34 91 347 41 68