Possible use of RFLP in Repeated Sequence - assbt

January.June 1994
Possible Use of RFLP in Repeated Sequence Families
43
Possible Use of RFLP in Repeated Sequence Families in Sugar Beet Breeding and For Management of Sugar Beet Genetic Resources Jean Fram;ois Bonavent, Anne Sophie Bournay,
Sylvain Santonf and Andre BerviUe2
INRA-Dijon, Station d'Amelioration des Plantes, BV 1540, F- 21034 Dijon Cedex, France. I Present address: INRA, Ferme du Mouton, F-91190 Gifsur Yvette, France. 2Present address: INRA-ENSAM, Station d'Ameiioration des Plantes, 2 Place Pierre Via/a, F-34060 Montpellier Cedex, France. ABSTRACT
Because they usually correspond to unique sequences,
RFLP markers are used in genomic mapping, variety
distinction, variety homogeneity and in reconstruction of
phylogenies. Nevertheless, they are not the most suitable
markers for all these applications. Their use in taxonomy
is not always straightforward, since these sequences may be
highly conserved as in the genus Beta L. The analysis of
genetic variability allows calculation of genetic distances
and enables the construction of phenograms, but not
phylogenetic trees. Phylogenetic trees are constructed using
other methods based on parsimony analysis and, hence,
they do not always fit within taxonomic groups. Therefore,
we have turned to other molecular markers which have
already been found in sections Beta, Corollinae and
Procumbentes, and, therefore, fit well into the main
taxonomic divisions. RFLPs with repeated sequences are
generally not used, although they offer a wide range of
potential applications. Some have already been used and
others appear suitable for use as markers. Here we present
results with moderately and highly repeated sequences
which display RFLP or are genome specific when used as
probes. The main advantages and disadvantages of both
RFLP types are discussed.
Additional Key Words: moderately and highly repeated sequences,
satellite DNA, molecular taxonomy, genetic variability, variety
identification.
44
Journal of Sugar Beet Research
Vol 31 No 1 and 2
The polymorphisms that exist at the DNA level can now be
revealed through a wide range of methods for use as genetic markers
in plant breeding (Landry et aI., 1987). The specific advantages of
each kind of marker have already been analyzed. In the case of RFLP,
several classes of DNA are involved. It appears that each class displays
differential utility for various applications. Usually the polymor­
phisms are sought among unique sequences, a class that corresponds
to truly unique sequences or sequences present in few copies.
Isozymes, RFLP and some RAPD correspond to the same class of
unique sequences. The polymorphisms that might exist in the
moderately or highly repeated sequences are generally not useful,
primarily because of the difficulties in visualizing the markers.
Recently, several studies have been carried out on sugar beet involv­
ing RFLP types from non-repetitive DNA (Pillen et aI., 1992), and
in moderately (Santoni and Berville, 1992a) and highly repetitive DNA
sequences (Schmidt and Metzlaff, 1991; Santoni and Berville, 1992b)
which enabled us to choose the most suitable field of applications
for both types of RFLP.
Some reviews of the classical RFLP methods are necessary. The
DNA fragments coming either from cDNA libraries or from genomic
libraries have to be checked for the degree of repetition in the genome.
To avoid selection of repetitive fragments, which leads to redundan­
cy among clones, techniques are recommended which take advan­
tage of the high degree of methylation in repeated sequences.
Genomic clones are obtained using a methylation sensitive restric­
tion enzyme (usually PstI) in order to enrich the library for unique
sequences. However, in the library obtained, it is still necessary to
eliminate those clones corresponding to repeated sequences. The col­
ony dot hybridization method, with genomic DNA as a probe, allows
us to differentiate unique fragments, which give no or a faint signal,
compared to cloned repeated fragments, which give a strong
hybridization signal. The technique has been improved using
minipreparations of plasmid DNA probed with genomic DNA. In
fine probes corresponding to unique sequences are evaluated during
autoradiography. The time of autoradiogram exposure must be two
to three days prior to hybridization signal appearance to provide for
identification of unique sequences within a genomic size of 1.3 pg.
All the sequences that correspond to moderately and highly repetitive
genomic DNA are thus eliminated in the RFLP probe screening steps.
The genus Beta has recently been divided into 15 species includ­
ed within 4 sections. The section Beta includes the cultivated beets,
sugar beet, table beet, Swiss chard, forage beet and B. maritima. The
section Corollinae includes Eastern European species. The section
January.June 1994
Possible Use of RFLP in Repeated Sequence Families
4S
Nanae, from Anatolia, includes one species. The Procumbentes are
from the coast of Morocco and the Canary Islands.
Repeated gene families, such as ribosomal DNA and 5S DNA, have
been widely used in plant analyses because homologies between species
allow the use of heterologous probes to probe similarities. In sugar beet
there are about 1,500 rDNA and 50,000 sDNA copies. For example, the
phylogenetic study by Santoni and Berville (1992a) was developed for
Beta based upon rDNA polymorphisms, which correspond to the
chloroplastic DNA studies (Fritzche et aI., 1987) and agrees with the
taxonomy proposed by Barocka (1966). RFLP in rDNA were used to
examine gene flux between cultivated and wild beets (Santoni and
Berville, 1992c). Bolting plants in sugar beet fields displayed both
rDNA unit types found in cultivated and wild beet such as B. maritima.
It is therefore likely that they have hybridized with the B. maritima that
surround hybrid seed production fields.
Cloning of highly repeated fragments has been carried out in three
ways in Beta. The first way was to pick clones which yield intense
hybridization signals when genomic DNA was used as a probe, as
described above (Santoni and Berville, 1992b). However, here the library
has to be prepared with a restriction enzyme which cuts methylated
DNA. The second way was to cut genomic DNA with one of a series of
restriction enzymes in order to detect discrete bands of the same size in
the smear. These bands are selected , cloned and verified for the degree
of repetition (Pillen et aI., 1992; Santoni and Berville, 1992b). The third
method involves pulse field gel electrophoresis (PFGE) to detect large
size fragments which may correspond to tandemly repeated sequences.
In this case the restriction enzyme chosen does not restrict within the
repeated sequences (lung et al. 1990; Schmidt and Metzlaff, 1991).
For sugar beet and wild beet species all these methods have been
used to obtain RFLP probes in unique and highly repeated DNA
sequences, also called satellite DNAs.
MATERIALS AND METHODS
The list of plant materials is given in Table I. The plants were
cultivated in a glasshouse at 25 °C for 16h (day) and 15°C (night). Leaves
were harvested on at least 3-month-old plants. Three to five
preparations were made using 5g fresh weight. Seeds from accessions
unable to germinate were used to prepare DNAs.
The methods used to obtain total DNA from dry seeds (Santoni et
aI., 1991) or leaves, and for DNA preparation, DNA restriction,
membrane transfer, hybridization and autoradiography were described
by Santoni and Berville (l992a). Total DNA was restricted with ten units
Table 1. List, origin and code numbers of wild and cultivated beet accessions. WB refers to the IVT stocks. Other
codes are from INRA.
Section
Procumbentes
Species
Subspecies or
common name
B. procumbens
B. patel/aris
B. webbiana
Corollinae
Beta
B. corol/iflora
B. trigyna
B. lomatogona
B. macrorhyza
B. intermedia
B. maritima
B. maritima
B. maritima
B. maritima
B. maritima
B. maritima
B. maritima
B. maritima
var. maritima
Origin of variety
Rosenhof, Germany
Rosenhof, Germany
Rosenhof, Germany
East Germany
Turkish
Beltsville, MD
Turkish
Turkish
INRA Colmar, France
Universite LilIe, France
INRA Colmar, France
INRA Colmar, France
INRA Colmar, France
Collection Deleplanque
INRA Colmar, France
INRA Dijon, France
Author
Reference
Chr. & Sm.
Moq.
Moq.
Zoss.
Waldst.
Fisch.
Stev.
Bunge
Arcang.
Arcang.
Arcang.
Arcang.
Arcang.
Arcang.
Arcang.
Arcang.
WB 21, H 2148
WB 20, F 3977
WB 22, F 3981
WB 12
WB 8, G 2377
WB 5, G 2362
G 2365, A 1326
F 3971
H 2151
I 9001
F 3494
F 3954
F 4001
F 3903
H 2151
F 3997
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48
Journal of Sugar Beet Research
Vol 31 No 1 and 2
of enzyme per g DNA at 37°C for 2.5 h. Submarine agarose gel elec­
trophoresis or vertical 6070 polyacrylamide gels were used in TAE buf­
fer (Maniatis et al., 1982). Cloning of DNA fragments and data
analysis of distances are given in Santoni and Berville (1992b). Every
probe corresponding to a unique fragment (25ng) or to total DNA
(200ng) was labelled using the Random Primed DNA or the nick
translation DNA labelling kit of Boehringer, using 20ttCi of 32p_
aCTP (3,000Cilmmol).
Sequences of the chosen cloned monomers were obtained by the
enzymatic chain-termination procedure with 35S-')IATP and the
Multiwell Sequencing kit from Amersham (Sanger et al., 1977). Se­
quence recovery from GenBank and EMBL databases was perform­
ed using the GCG program (Devereux et al., 1984). Sequence analysis
was performed according to Wilburg and Lipman (1983).
All other methods, cited, but not used by ourselves, are describ­
ed in the referenced articles.
RESULTS AND DISCUSSION
Moderately repeated, tandemly arranged sequences were cloned
first from B. corolliflora; they correspond to satellite DNAs. Two novel
sequences were found. The pBCE cloned fragment is about 258 bp
long and is about 46% AT rich (Fig. 1). The degree of repetition of
the BCESAT family is about 2,000 copies per haploid genome. pBCE
contains several direct or inverted repeats (Fig. lA). The AluI and
EcoRI patterns are shown for two species in Fig. 2. The EcoRI
polymorphism in various species is displayed in Fig. 3. The copy
number determination in a wide range of species is displayed in Fig.
4. Incomplete restriction, due to variable DNA methylation within
species, is likely.
The pBCA sequence is shown in Fig. lB. The pBCA fragment
used as a probe for AluI restricted DNA from B. trigyna and B.
lomatogona displays several signals at 160, 320 and 480bp, thus sug­
gesting a tandem array of fragments (Fig. 5). The BCASAT family
contains about 15,000 motifs of 150bp and represents about 0.02%
of the haploid genome. The EcoRI polymorphism from various
species is displayed Fig. 6. The Beta, Corollinae and Procumbentes
species are differentiated.
The pBVE and pBVA DNA fragments reveal two highly repeated
sequence families. BVESAT and BVBSAT families have been found
in the section Beta. The PTS family (BPESAT) has been found in
the section Procumbentes (Schmidt et al., 1991). BVESAT and
BPESAT share homologies at medium stringency. However, at high
January.June 1994
Possible Use of RFLP in Repeated Sequence Families
49
1
~
CCTTCGAAGG CCAAAATCGG ACCAAAATGG CCTTCATTTA CCCAAAATGG
51
100
GTTTCAAAGC ATATGAGTGA ACTTTAATTG ACTCTTATAG TTATATATGT
101
150
ACCTATTATA ACTATATATG ACCTAACATG TGGCTAAATG CGCGAAACTA
151
161
AGTCAAA TGA G
A
1
~
CTAGCTCTTC CAGAGTGGTA TCTCACTGAT GGCTCGGGCC CCCCGGAAGC
51
100
CTTCTTCGCC TTCCACCTAA GCTGCGCAGG AAAAGCCCAA AGCCAATCCC
101
150
AGGGAACAGT AAAGCTTCAT AGGGTCTTTC TGTCCAGGTG CAGGTAGTCC
151
200
GCATCTTCAC AGACATGTCT ATTTCACCGA GTTTCTCTCC GAGACAGTGC
201
250
CCAGATCGTT ACGCCTTTCG TGCGGGTCGG AACTTACCCG ACAAGGAATT
251
258
TCGCTACC
B
Figure 1. A = Sequences of the pBCE fragment. B = Sequences of the
pBCA fragment.
stringency, BVESAT and BPESAT are perfectly distinguished. It is
worth noting that Jung et al. (1990) and Schmidt et al. (1991) have found
highly repeated sequences when looking for B. procumbens specific
DNA probes. Surprisingly we were not able to clone a highly repeated
DNA family in the section Corollinae. The pBCASAT1, pBCESATl,
and pBVESATl-4 sequences are registered in the EMBL data bank
under numbers X69916, X69917, and X69918, respectively.
Dispersed repeated sequences were searched in the three Beta
sections without success. There is no specific strategy for cloning such
sequences, which might explain why we failed to clone them. However,
the number of clones checked in this way is not large enough to draw
a definitive conclusion.
DNA fragments used to reveal polymorphisms in unique se­
quences enabled Pillen et al. (1992) to construct a linkage map of
sugar beet with 108 RFLP loci, six isozymes and a morphological trait.
The loci cover 789 cM with an average spacing of 6.9 cM. This
Journal of Sugar Beet Research
50
Vol 31 No 1 and 2
map is to be enlarged by public institutes and by companies.
We have previously reported results on ribosomal DNA probes
which reveal polymorphisms in moderately repeated sequences
(Santoni and Berville, 1992a). The polymorphisms in the rDNA genes
are larger in B. maritima L. than in sugar beet. Several unit types
exist in wild forms differing either in size (lOA, 10.7, 11, and 11.3
kb
4.2
2
3
4
4.2
1.618
1.618
0.516
0.516
0.258
0.258
Figure 2. Autoradiogram of the Southern transfer of restricted beet
total DNA, electrophoresed in 0.8 070 agarose and hybridized with
the pBCEl insert. MW markers: lkb and 123bp ladders from
Bethesda Research Laboratories. A: B. trigyna, lane 1: AluI; lane 2:
EcoRI; B: B. maritima F3903, lane 3: AluI; lane 4: EcoRI.
January.June 1994
51
Possible Use of RFLP in Repeated Sequence Families
or in sequences for the intergenic spacer (IGS) carrying 1,2 or 3 EcoRI
sites 0.3 kb apart. Several length variable units of each type have been
detected. They all represent less than 15070 of the units. In sugar beet we
have found the 11 kb unit carrying 3 EcoRI sites in the IGS. Although
sugar beet varieties have been introgressed by wild beet such as B.
maritima, the corresponding expected rDNA alleles were not found in
sugar beet or other cultivated beet varieties. We here suggest that the
rDNA locus is linked to a major characteristic found in cultivated beets,
and, therefore, wild alleles from B. maritima are eliminated because the
plants carrying wild alleles do not display the correct characteristic.
I,b
1
2
3 4
5
6 7
8
9 JO 11 12 13 14
15
I{b
11.6
11.6
10.5
8.37
10.5
8.37
6.0
6.0
1.75
1.75
1.63
1.63
516
394
Figure 3. Characterization of BCESAT moderately repeated family.
Autoradiogram of EcoRI restricted beet nuclear DNA electrophoresed in 0.80/0
agarose gel and stained with ethidium bromide, transferred to nylon membrane
and then probed with the pBCE fragnc :lt. For the following lanes, 12g DNA
per lane was hydrolyzed with 60U of rc iction enzyme. Lane I: sugar beet 059;
lane 2: table beet albin a FD 020; lane 3: B. maritima FD 010; lane 4: sugar beet
59; lane 5: B. maritima 19001; lane 6: table beet Crapaudine H2099; lane 7: B.
webbiana; lane 8: B. maritima F3494; lane 9: B. trigyna G2377; lane 10: B.
lomatogona G2362; lane 11: Petunia hybridaTlvl; lane 12: B. maritima F3903;
lane 13: B. corollijlora WBI2; lane 14: B. macrocarpa; lane IS: PCR pBCE
amplification.
52
Journal of Sugar Beet Research
____ pg ___
0.11
A
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Vol 31 No 1 and 2
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I
I
I
I
2
3
4
5
)
I
6 7
pBCE
5~g
0.5~g
I
8
9 10 11
Figure 4. Determination of copy numbers for the BCESAT families. Dots were hybridiz­
ed with the pBCEI insert. A: dots of pBVEI insert (ng or pg). B: dots of total DNA
(g); lane I: Petunia; lanes 2 and 3: sugar beet 59 and 059; lane 4: B. maritima F3903;
lane 5: table beet Albina; lane 6: B. corol/iflora; lane 7: B. lomatogona; lane 8: B.
maritima 19001; lane 9: B. maritima F3954; lane 10: table beet Crapaudine H2099;
lane 11: B. webbiana.
~
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0.483
0.322
0.483
0.322
0.161
0.161
Figure S. Autoradiogram of the Southern transfer of Alul restricted beet total DNA,
electrophoresed in 0.8070 agarose, transferred to nylon membranes, and hybridized with
the pBCAI insert. Lane I: Ikb ladder from BRL; lane 2: B. trigyna; lane 3: B.
lomatogona.
January.June 1994
Possible Use of RFLP in Repealed Sequence Families
53
kb
kb
10.5 8.37 -
- 10.5
- 8.37
6.0 -
- 6.0
1.75 1.63 -
Figure 6. Characterizationof the BCASAT moderately repeated family.
Autoradiogram of EcoRl restricted beet nuclear DNA electrophoresed in 0.8070
agarose gel and stained with ethidium bromide, transferred to nylon membrane
and then probed with the pBCA fragment. For the following lanes, 12g DNA
per lane were hydrolyzed with 60U of restriction enzyme. Lane I: B. atriplicijolia;
lane 2: table beet Crapaudine Vilmorin; lane 3: B. maritima F3494; lane 4: sugar
beet 059; lane 5: B. maritima FDOIO; lane 6: sugar beet 59; lane 7: table beet
H2099; lane 8: B. trigyna G2377; lane 9: sugar beet 059; lane 10: B. maritima
F3995; lane 11: B. procumbens; lane 12: B. maritima F4001; lane 13: B. maritima
F3903; lane 14: B. maritima 19001; lane 15: table beet albina; lane 16: B. maritima
F3960; lane 17: B. webbiana; lane 18: MW marker Raoul.
CONCLUSIONS
Sugar beet linkage maps are necesssary to speed up efficient beet
breeding. Many morphological and physiological traits are being map­
ped to enable breeders to split and recombine allele blocks in order
to obtain better associations. Selection assisted by molecular markers
should be used to introduce disease resistance genes from wild rela­
tives. DNA fragments used to reveal polymorphisms in tandemly ar­
ranged moderately repeated sequences identify polymorphisms suitable
for use, as are RFLP in unique sequences. Examples are taken from our
preceding work on RFLP in ribosomal DNA. Polymor­
phisms in the rDNA will allow mapping this gene family in a cross
54
Journal of Sugar Beet Research
Vol 31 Noland 2
between the two B. vulgaris and B. maritima subspecies. However,
the rDNA is assigned to the chromosome carrying a satellite (Chr
I of sugar beet). Pillen et a!. (1992) clearly established that linkage
group I carries major physiological functions and, because of the
satellite, the rDNA locus also. The 5S DNA genes were not studied,
but this family is likely to provide a mark for another chromosome.
The efficacy of each sort of molecular marker is given for several
applications (Table 2).
DNA fragments used to reveal polymorphisms in unique se­
quences and cDNA have been found to be conserved in related plant
species and particularly in beets (Nagamine et a!., 1989). The com­
parison of genotypes involves the computing of molecular similarities
into genetic distances. There are several methods possible to calculate
genetic distances. Each method takes into account the common
presence of fragments, the common absence, the distances through
correspondence analysis, etc. Each method establishes a specific
distance between genotypes. Consequently, the aggregation of
distances or similarities, which leads to a tree called a phenogram,
using either the Joining or the UPGMA methods displays several
phenograms. A phenogram is not a phylogram since it is unroated
and there is no possibility of checking which is the best phenogram
corresponding to the true phylogeny. The use of RFLP in phylogeny
involves analysing data with methods based upon weighted markers,
such as parsimony, in order to screen for the best suitable tree, or
segmentation analysis to detect groups and subgroups which may
possibly correspond to taxa.
DNA fragments used to reveal polymorphisms in highly repeated
sequences have been shown to vary considerably both in size and in
sequence between distantly related species of Beta or within other
genera, but have been shown to be highly conserved in closely related
species. Moreover, they have resulted in species groupings consistent
with the major taxonomic divisions delineated by botanists. Those
repeated sequences were obtained from two different Beta sections.
The differentiation, recognition and identification of RFLP in
unique sequences if lines and varieties have been approached in several
species because of the importance of varietal protection and to iden­
tify homogeneity. RFLP in unique sequences do not appear as the
most convenient tool for such uses since numerous probes have to
be checked, and, moreover, the greater the number of genotypes
analyzed the greater the number of probes needed. Consequently the
cost is prohibitive. In contrast, the polymorphisms found in highly
and moderately repeated sequences might offer a simpler, less expen­
sive method. The variability of macrosatellite DNA has been found
Table 2. Classification of molecular markers as tools according to use for applications in plant breeding. 'Yes' means
possible; 'Poorly' means difficult or expensive; 'Potent' means fruitful; 'Partially' means some risk to use. PFGE
means pulse field gel electrophoresis. 'No' means no guarantee for results.
Taxonomy
Cytogenetic studies
Chromosome
phylogeny
karyotype
organisation
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Tools
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Genetic maps
Variety
identification
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Isozymes
no
no
no
yes
yes poor
Polypeptides
yes
no
no
yes
yes poor
:e
"'l
Isozymes
no
no
no
yes
yes poor
:i'
RFLP unique cDNA and g DNA
poor
no
no
yes
yes poor
RFLP moderately rDNA, 5S DNA
yes partially
yes partially
yes partially
yes
yes poor
"""
RFLP highly macrosatellite
yes potent
yes potent
yes potent
in interspecific
prospect
crosses
in PFGE
r"
-=
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M inisatellite
no no yes
yes
yes
VNTR or microsatellite
no yes yes
yes
yes
RAPD unique
no no no
yes
yes poor
RAPD repeated
yes yes yes
yes
yes
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56
Journal of Sugar Beet Research
Vol 31 No 1 and 2
for humans and mice under long blocks of repeats revealed by PFGE,
and they represent only a few loci (Mar<;:ais et aI., 1991). The varia­
tion in size is quite large (420 kb to 2,650 kb). The segregation of
polymorphisms has been found to be Mendelian and no recombina­
tion event was detected after 150 cycles of meiosis. Therefore, these
sequences constitute highly polymorphic, centromeric markers, which
can be used in linkage analysis and to compare lines through PFGE
using few macrosatellite probes. In contrast, RFLP probes targeting
unique sequences usually require numerous probes.
ACKNOWLEDGEMENTS
Thanks are due to l. P. Denisot for enabling us to access the beet
wild form collection and to 1. Delbut for his expertise in photography.
LITERATURE CITED
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(Toumef) L. Z. Pflanzenzucht. 56: 379-388.
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Research 12: 387-395.
Fritzsche, K., M. Mezlaff, R. Melzer and R. Hagemann. 1987. Com­
parative restriction endonuclease analysis and molecular
cloning of plastid DNAs from wild and cultivated varieties
of the genus Beta (L.). Theor. Appl. Genet. 74: 589-594.
lung, c., M. Kleine, F. Fisher and G. Hermann. 1990. Analysis of
DNA from a Beta procumbens chromosome fragment in
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Appl. Genet. 79: 663-672.
Landry, B.S., R. Kesseli, Hei Leung and R. W. Michelmore. 1987.
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646-653.
Maniatis, T., E.F. Fritsch and l. Sambrook. 1982. Molecular cloning
: a laboratory manual. Cold Spring Harbor Laboratory. Cold
Spring Harbor, NY, 545 p.
Mar<;:ais B., M. Bellis, A. Gerard, Y. Boublik and G. Roizes. 1991.
Structural organization and polymorphism of the alpha
satellite DNA sequences of chromosomes 13 and 21 as reveal­
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January-June 1994
Possible Use of RFLP in Repeated Sequence Families
57
Nagamine, T., G. A. Todd, K. P. McKann, H. 1. Newbury and B. V.
Ford-Lloyd. 1989. Use of restriction fragment length polymor­
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Sanger, F., Nicklen S. and A. R. Coulson. 1977. DNA sequencing with
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5463-5467.
Santoni, S., P. Faivre-Rampant, E. Moreau and A. Berville. 1991. Rapid
control of purity for the cytoplasm of male-sterile seed stocks
by means of a dot hybridization assay. Mol. Cell. Probes 5: 1-9.
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ribosomal DNA units and phylogeny of Beta L. wild forms
and cultivated beets. Theor. Appl. Genet. 83: 533-542.
Santoni, S. and A. Berville. 1992b. Two different satellite DNAs in Beta
vulgaris L.: evolution, quantification, and distribution in the
genus. Theor. Appl. Genet. 84: 1009-1016.
Santoni, S. and A. Berville. 1992c. Evidences for gene exchanges bet­
ween sugar beet (B. vulgaris L.) and wild beets: consequence
for transgenic sugar beets. Plant Mol. BioI. 20: 578-580.
Schmidt, T and M . Metzlaff. 1991. Cloning and characterization of a
Beta vulgaris satellite DNA family. Gene 101: 247-250.
Schmidt, T, C. J ung and M. Metzlaff. 1991. Distribution and evolution
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Wilburg, W. 1. and D. 1. Lipman. 1983. Description of part of the
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