For personal use only!
49
Euphytica 93: 49–54, 1997.
c 1997 Kluwer Academic Publishers. Printed in the Netherlands.
Transfer of powdery mildew resistance from Aegilops variabilis into bread
wheat
Penko Spetsov1, Dominique Mingeot2, Jean Marie Jacquemin2, Krassiana Samardjieva3 &
Elena Marinova3
1
Institute of Wheat and Sunflower 9520, General Toshevo, Bulgaria; 2 Station D’Amelioration des Plantes, 5030
Gembloux, Belgium; 3 Institute of Genetic Engineering 2232, Kostinbrod-2, Bulgaria
Received 15 December 1994; accepted 25 June 1996
Key words: Triticum aestivum, Aegilops variabilis, powdery mildew resistance, addition and substitution lines
Summary
Winter hexaploid wheat (Triticum aestivum L.) was crossed with Aegilops variabilis to transfer resistance to
powdery mildew into wheat. Following two backcrosses to wheat and from 5 to 9 generations of selfing, several
disomic addition and substitution lines of hexaploid wheat resistant to the mildew pathogen were isolated.
A pair of short satellited chromosomes was always observed in the resistant lines. Further evidence utilizing
as markers for homoeologous group 1 HMW glutenin subunits and DNA hybridization with probe pGBX 3076
showed that an alien substitution involved this homoeologous group.
Introduction
Materials and methods
Aegilops variabilis Eig (syn. Triticum peregrinum
Hackel) is an allotetraploid species (2n = 4 = 28,
genomically UUSS). It has been recognized as a source
of genes for resistance to fungi, nematodes and high
protein content in the grain (Spetsov, 1989).
Information is available showing that the ‘Chinese
Spring’-Ae. variabilis addition lines G and O carry
genes conferring resistance to stem rust and cereal cyst
nematode, respectively (Driscoll, 1975). Recently, Yu
et al. (1990) succeeded in transferring root knot nematode resistance from Ae. variabilis into bread wheat,
while Bang and Hulsbergen (1992) obtained stable
lines with 2n = 42, having eyespot resistance, being
introgressed from Ae. kotschyi (UUSS).
A programme, aimed at exploiting genes for powdery mildew resistance present in Aegilops species for
wheat improvement, is being currently worked on by
our group. The most advanced lines involve Ae. variabilis, from which several 44- and 42-chromosome
lines have been selected and studied (Spetsov et al.,
1993). The genetics and breeding values of ten out of
21 lines produced, are reported in this paper.
Pedigree and generation of lines
Lines with 44- and 42-chromosomes have been produced by crossing the winter bread wheat variety, T.
aestivum cv. ‘Rusalka’, to an accession of Ae. variabilis from the IWS – General Toshevo collection. The
subsequent crosses, as well as the selected lines and
their generations in the year of isolation, are shown in
Table 1. F1 plants deriving from the ‘Rusalka’ Ae.
variabilis cross were divided into four groups (1–4).
The first group was backcrossed to ‘Rusalka’ wheat,
in the second group the pollen probably came from
an unidentified wheat variety resembling ‘Rusalka’,
called ‘Rusalka*’. Irradiation of F1 seeds was applied
to the third group and the corresponding plants were
backcrossed to ‘Rusalka*’. Pollen from ‘Rusalka’ and
from the wheat cultivar ‘Pliska’ was then used to produce the BC1 seeds of the fourth group.
Powdery mildew resistance
The resistance to the powdery mildew pathogen,
Erysiphe graminis tritici, was estimated mainly at the
For personal use only!
50
Table 1. Summary of the origin, actions performed and generation in which selections
were isolated following the original wheat Ae. variabilis cross (see text)
1
2
Groups
Selected lines
Generation of
selection
1. Backcrossing with ‘Rusalka’
2. Backcrossing with ‘Rusalka*’1
3. Irradiated F1 backcrossed
with ‘Rusalka*’
4. Irradiated F1 backcrossed
with ‘Rusalka-Pliska’2
Line 3
Lines 2; 8, 28-7
Lines 4; 5; 6; 7;
10; 6-9
Lines 9; 11; 12; 11-8;
11-9; 5-2; 18-1; 121
37-3; 52-1; 104-5
BC1 F6
BC1 F6 ; BC2 F8
BC1 F6 -F7
BC1 F5 -F9 ; BC2 F7 -F9
‘Rusalka*’ was an unidentified cultivar, resembling ‘Rusalka’
Both ‘Rusalka’ and ‘Pliska’ varieties were used for obtaining BC1 seeds.
adult plant stage, as described earlier (Spetsov and
Iliev, 1991). Three major classes of host reactions were
distinguished: resistant (r), intermediate (i) and susceptible (s). The wheat parent showed susceptibility (s),
while the Aegilops variabilis and lines selected from
the cross exhibited resistance (r). Mildew resistance at
the adult plant stage was estimated in the greenhouse
and by infection field, using a mixture of races of the
pathogen that commonly occur in Bulgaria.
gel. The DNA was transferred onto nylon membranes
(Hybond N+). Filters were hybridized at 65 C for
16 h with the dCTP labeled cDNA probe pGBX 3076,
obtained from Dr. B.S. Gill, Kansas State University.
They were given a final stringency wash of 0.5X SSC,
0.1% SDS at 65 C, and exposed for autoradiography.
Cytology and protein analysis
Genetics of the addition lines
Somatic chromosomes were counted using root-tip
cells and univalent number was assessed in PMCs at
meiotic metaphase I, using the acetocarmine squashing
method. Genetic stability of lines was determined by
somatic chromosome counts of 10–15 plants, taking
samples from two subsequent self-generations.
Satellite number was counted according the procedure described by Waines and Kimber (1973), but
differently from that investigation, the material was
not coded and the analyses were preferentially carried
out by one person.
The high molecular weight glutenin subunit electrophoretic profiles were determined from a ground
single seed of each line, using the method of Payne et
al. (1987), after separation in 12% PAGE with SDS.
At least 10 disomic addition lines have been produced
which showed similar powdery mildew resistance at
the adult plant stage as the alien species parent.
Three lines (2, 3 and 8), originating from nonirradiated hybrid seeds, turned out to be stable disomic
addition lines and morphologically well differentiated
(Spetsov et al., 1993). Line 3 was phenotypically characterized by having a red coleoptile, brown coloured
spike, reduced ear fertility, and lower grain weight.
This line was the only one selected from the backcross of the ‘Rusalka’ Ae. variabilis F1 with normal
‘Rusalka’ (Table 1). Lines 2 and 8 showed a similar
phenotype, but originated from the second crossing
group (2).
Meiotic configurations at metaphase I of F1 plants
from crosses between the three addition lines, have
been investigated (Table 2). It was evident that lines
2 and 8 showed a greater degree of genetic similarity in the F1 (about 0.65 univalents per PMC), while
line 3 had apparently a different cytogenetic structure.
Cytological observations revealed the presence of a
pair of short satellited chromosomes in the additions,
which were visually different from the wheat satel-
DNA hybridization
DNA was extracted from leaves of the different lines,
including common wheat cv. ‘Chinese Spring’ and
its nullisomic 1A – tetrasomic 1B line (= N1AT1B).
Genomic DNA (10 g per line) was digested with
Hind III and DraI and run overnight on a 0.8% agarose
Results and discussion
For personal use only!
51
Table 2. Percentage of cells with different number of univalents at metaphase I
of F1 hybrid plants between three common wheat Ae. variabilis addition lines
(2, 3 and 8)
F1 genotype
2
2
3
3
8
8
Cells
scored
% cells with univalents
0
1
2
3
4
Univalents/cell
(Mean SE)
238
271
239
6
47
10
0
0
7
1.92
0.65
1.76
24.4
42.4
24
63
9.6
52
6.5
0
7
0.051
0.042
0.063
Table 3. Somatic chromosome number and number of observable satellited chromosomes (%) in
common wheat cv. ‘Rusalka’, Ae. variabilis and substitution lines derived from them
Parents
Lines
2n
‘Rusalka’
Ae. variabilis
5-2
11-8
11-9
28-7
37-3
52-1
42
28
42
42
42
42
42
42, 43,
42 + t, 42 + 2t
Cells
scored
Cells with SAT-chromosomes (%)
0
1
2
3
4
5
6
100
160
80
240
125
180
165
110
8.2
17.2
10
9.3
23.1
15.2
30.4
20
0
3.2
4.8
0
0
2.6
0
0
0.02
0.04
0
0
0.01
0.03
0
0
lited chromosomes. They correspond to the shorter
SAT-chromosome pair of Ae. variabilis. From the data
presented in Table 2, it can be assumed that line 3 is
a disomic addition and substitution line, incorporating
two different alien chromosomes. Means of the two F1 s
involving line 3 showed a difference of 0.16 univalents,
that is significant at the 5% level, while the comparison
of these two with the third F1 (2 8) revealed a much
larger difference (significant at 0.1% level).
An attempt was made to discriminate the satellited
chromosomes of the three lines by conventional light
microscopy. On the basis of counts of 80–90 mitotic
cells per line, line 3 appeared to differ from the others
by having more cells (an average of 16.7%) showing
six satellites, which is an about a five times greater value than that exhibited by the two other lines (3.4% for
line 2 and 2.4% for line 8). In all cells showing satellites, the alien SAT-chromosome was always present
and easily distinguished due to its shorter length. The
added alien chromosome is readily recognized at mitotic metaphases and thus appears to be suppressing the
wheat 1B and 6B chromosomes in the addition lines.
Other examples indeed exist of a partial inactivation
of nucleolar activity of wheat SAT-chromosomes 1B
and 6B occurring in the presence of an added alien
0.9
6.6
1.5
0
2.8
0
0
0
38.8
22.3
21.6
28.2
51
18.8
26.1
31.4
37.8
17.2
20.6
26.3
7.7
34.1
8.7
25.2
14.3
33.4
41.5
36.2
15.4
29.3
34.8
23.4
chromosome (Martini et al. 1982; Friebe and Heun
1989).
Breeding and genetics of hexaploid lines
Some spontaneous hexaploid derivatives with complete powdery mildew resistance at the adult plant
stage, have been isolated. One of them, line 6–9,
has already been described as a disomic substitution
line, having a dark brown coloured spike (Spetsov et
al., 1993). Six additional lines have been cytologically
investigated (Table 3). The variety ‘Rusalka’ and Ae.
variabilis exhibited two pairs of SAT-chromosomes,
except for a few cells found with six satellites. In
‘Rusalka’, as in other cultivars of hexaploid wheat,
two chromosomes, 1B and 6B, have a secondary constriction, and can be visually differentiated in mitotic
metaphases. 1B and 6B of hexaploid wheat are similar in size, but differ in arm ratios. In common wheat,
sometimes NORs of 5D and rarely of 1A can also be
active (Lacadena and Cermeno, 1985). Ae. variabilis
possesses four satellited chromosomes, with one pair
shorter and easier to recognize than the other (see also
Cermeno et al., 1984). Investigations involving satellite number are difficult and risky, because secondary
For personal use only!
52
Table 4. Number of univalents at meiotic metaphase I of line 11-8 and its hybrid
derivatives
Genotype
Cells
scored
Number of univalents
1
2
3
Mean
Line 11-8
F1 (11-8 ‘Rusalka’)
F1 (11-8 ‘Chinese Spring’)
F1 (6-9 11-8)
86
183
50
278
8
66
26
73
0.19
1.60
1.63
1.40
4
101
15
133
0
8
8
11
SE
0.054
0.048
0.109
0.054
Figure 1. SDS PAGE patterns of the HMW glutenin subunits of the following wheat lines: 1-line 11-8; 2-line 6-9; 3-line 8; 4-line 3; 5-line 2;
6-‘Rusalka’; and 7-Ae. variabilis. Plants of lanes 1 to 5 are wheat Ae. variabilis selections (see text).
constrictions may appear at various places along the
chromosomes and be misclassified as NOR sites when
distally located.
Line 52-1 is unstable, giving progeny with four
types of somatic chromosome constitution (Table 3).
All other lines are stable in this respect and exhibit four
satellited chromosomes. The shorter SAT-chromosome
coming from Ae. variabilis is recognizable in all the
new genotypes, except lines 37-3 and 28-7. Lines 11-8,
11-9, 52-1 and 5-2 are similar in phenotype and might
carry the same alien chromosome, that is the shorter
SAT-chromosome from Ae. variabilis.
Out of them, line 11-8 has been more deeply studied in crosses with other hexaploid cultivars and lines
(Table 4). It might correspond to a disomic substitution line, since in crosses involving both ‘Rusalka’
and ‘Chinese Spring’ 1.60 and 1.63 univalents have
been detected, respectively. The chromosome differ-
ence between lines 11-8 and 6-9 is also evident, with an
average of 1.4 univalents in MI, that is statistically different from the mean of line 11-8 (0.19 univalents per
cell). A comparison between F1 s produced by crossing
line 11-8 with line 6-9 and cv. ‘Rusalka’, showed a
significant difference at the 1% level of 0.2 univalents.
This supports the above mentioned statement about the
genetically different structure of the two substitution
lines. Both lines have brown coloured spikes, with a
slight difference in the intensity, and contain the shorter
satellited chromosome from Ae. variabilis.
In the past only Ae. variabilis chromosomes for
homoeologous groups 1 and 2 have been substituted into wheat (Shepherd and Islam, 1988). Besides
this, both the above mentioned lines possessed a brown
spike colour and genes controlling glume pigmentation
which are located in homoeologous group 1. Thus, it
can be hypothesized that the alien substitution they
For personal use only!
53
cross ‘Rusalka’ Ae. variabilis, i.e. lines 2, 3, 8 (2n
= 44); 6-9 and 11-8 (2n = 42), was analyzed by polyacrylamide gel electrophoresis (PAGE). HMW subunits with molecular weights between 95-140 kD were
clearly resolved (Figure 1). In the variety ‘Rusalka’,
four subunits were present and designated, according
to decreasing molecular weights, as alleles 2, 7, 9 and
12. The Ae. variabilis glutenin composition exhibited
three HMW subunits (called x, y and z). The presence
of the alien genetic material in the disomic addition
lines is expressed by the presence of bands y and z.
The HMW glutenin profile of both substitution lines
also contained the y and z bands, but lacked bands 7
and 9 from ‘Rusalka’, that are characteristic of wheat
chromosome 1B. These results suggest that the alien
substitution in lines 11-8 and 6-9 involve the long arm
or the whole chromosome 1B.
DNA hybridization
Figure 2. Southern blot hybridization pattern of total DNA from
wheat varieties, Ae. variabilis and selected lines from the cross (see
text), digested with Hind III (part A) and DraI (part B) and hybridized
with pGBX 3076. DNA molecular weights are indicated in Kb.
carry involved homoeologous group 1, and specifically chromosome 1B.
Seed storage protein analysis
The electrophoretic pattern of HMW glutenin subunits
from the parents and some of the selected lines from the
Figure 2 shows the hybridization pattern of total DNA
digested by HindIII (A) and DraI (B) and hybridized
with probe pGBX 3076. From the HindIII digestion, it
was evident that a DNA fragment, which was present
in both ‘Chinese Spring’ and ‘Rusalka’, was missing in
the N1AT1B line, and also in the N1BT1A, N1DT1B
(results not shown), thus indicating that probe pGBX
3076 was specific for wheat homoeologous group 1.
Moreover, Ae. variabilis showed two hybridization
bands, one of which was present in all the addition
and substitution lines produced (Figure 2).
When DNA was restricted by DraI, varieties
‘Rusalka’ and ‘Chinese Spring’ exhibited only two
fragments of approximately 9.4 and 6.6 Kb, while
the alien species showed one, of intermediate molecular weight. The Ae. variabilis fragment was present
in all derivative lines tested. In the two substitution
lines, the 9.4 Kb fragment of ‘Rusalka’ was missing,
conferring the absence of the T. aestivum chromosome.
Further, addition line No. 2 showed a hybridization pattern different from that of ‘Rusalka’, suggesting some
additional DNA rearrangements. We suggest from this
hybridization analysis, that in the substitution lines
one chromosome of the group 1 was involved, and that
other chromosome modifications might have occurred.
Further evidence is needed to reach a definite conclusion.
For personal use only!
54
Acknowledgements
This work is supported in part by a Grant-in-Aid from
the Ministry of Education, Science and Technology
of Bulgaria. We wish to thank Drs. John Snape and
Perry Gustafson for reading the manuscript and making
valuable suggestions.
References
Bang, R. & H. Hulsbergen, 1992. Triticum aestivum-Aegilops
kotschyi introgression lines with eyespot resistance. EWAC
Newsletter 1992: 50.
Cermeno, M.C., J. Orellana, J.L. Santos & J.R. Lacadena, 1984.
Nucleolar activity and competition (amphiplasty) in the genus
Aegilops. Heredity 53: 603–611.
Driscoll, C.J., 1975. First compendium of wheat-alien chromosome
lines. Wheat Newsletter 21: 16–32.
Friebe, B. & M. Heun, 1989. C-banding pattern and powdery mildew
resistance of Triticum ovatum and four T. aestivum-T. ovatum
chromosome addition lines. Theor. Appl. Genet. 78: 417–424.
Lacadena, J.R. & M.C. Cermeno, 1985. Nucleolus organizer competition in Triticum aestivum-Aegilops umbellulata chromosome
addition lines. Theor. Appl. Genet. 71: 278–283.
Martini, G., M. O’Dell & R.B. Flavell, 1982. Partial inactivation of
wheat nucleolus organisers by the nucleolus organiser chromosomes from Aegilops umbellulata. Chromosoma 84: 687–700.
Payne, P.I., M.A. Nightingale, A.F. Krattiger & L.M. Holt, 1987.
The relationship between HMW glutenin subunit composition
and the breadmaking quality of British-grown wheat varieties. J.
Sci. Food Agric. 40: 51–65.
Shepherd, K.W. & A.K.M.R. Islam, 1988. Fourth compendium of
wheat-alien chromosome lines. In: T.E. Miller & R.M.D. Koebner (Eds). Proc. 7th Intern. Wheat Genet. Symp. p. 1373–1395,
Cambridge, England.
Spetsov, P., 1989. Studies of the species Aegilops variabilis Eig (2n =
4 = 28) and its hybrids with bread wheat. Genetics & Breeding
1: 76–83 (in Bulg.).
Spetsov, P. & I. Iliev, 1991. Characterization of a disomic wheatAe. variabilis addition line resistant to powdery mildew fungus.
Wheat Information Service 73: 1–4.
Spetsov, P., I. Iliev, K. Samardjieva & E. Marinova, 1993. Studies on
wheat-Aegilops variabilis addition and substitution lines resistant
to powdery mildew. In: Z.S. Li & Z.Y. Xin (Eds). Proc. 8th Intern
Wheat Genet. Symp. p. 327–332, China, Beijing.
Waines, J.G. & G. Kimber, 1973. Satellite number and size in
Triticum monococcum L. and the evolution of the polyploid
wheats. Can. J. Genet. Cytol. 15: 117–122.
Yu, M.Q., F. Person-Dedryver & J. Jahier, 1990. Resistance to root
knot nematode, Meloidogyne naasi (Franklin), transferred from
Aegilops variabilis Eig to bread wheat. Agronomie 6: 451–456.