Download Rye SCAR markers for male fertility restoration in the P cytoplasm

J Appl Genet 46(4), 2005, pp. 371-373
Short communication
Rye SCAR markers for male fertility restoration
in the P cytoplasm are also applicable
to marker-assisted selection in the C cytoplasm
Stefan Stoja³owski1, Mi³os³awa Jaciubek2, Piotr Masojæ1
1
2
Department of Genetics and Plant Breeding, Agricultural University, Szczecin, Poland
Polish Academy of Sciences, The F. Górski Institute of Plant Physiology, Kraków, Poland
Abstract. The study aimed at testing the usefulness of recently developed SCAR markers on rye (Secale
cereale L.) chromosome 4R in hybrid breeding based on the C source of male sterility-inducing cytoplasm.
Of 10 markers studied, 4 revealed polymorphisms between 2 inbred lines (544cms-C and Ot0-20) crossed to develop F2 and BC1 mapping populations. Analyses performed on 94 F2 and 93 BC1 plants allowed to extend a formerly constructed genetic map of chromosome arm 4RL. Three SCAR markers (SCP14M55, SCP15M55
and SCP16M58) were mapped in the vicinity of gene Rfc1, which restores male fertility in the C cytoplasm.
The 3 tested SCAR markers proved to be effective in marker-assisted selection (MAS) for male fertility/sterility.
Key words: cytoplasmic male sterility, SCAR markers, Secale cereale.
The first hybrid cultivars of rye were developed
20 years ago and their productivity soon exceeded
that of population varieties (Miedaner 2004). Generally, hybrid rye breeding makes the use of
the Pampa (P) male sterility-inducing cytoplasm
(Geiger and Schnell 1970). Other sterilizing
cytoplasms (C, G, R) were also studied extensively (£apiñski 1990; Melz and Adolf 1991;
Kobyljanskij et al. 1994; Börner et al. 1998) but,
with the exception of the G cytoplasm, they were
not employed in commercial breeding
programmes. Applying the alternative sources
of cytoplasmic male sterility (CMS) in rye is recommended to reduce risks arising from
plasmotype uniformity.
The C cytoplasm was detected by £apiñski
(1972) in the Polish rye variety Smolickie. In contrast to the P cytoplasm, the C cytoplasm is difficult to maintain, i.e. there is a low frequency of
non-restorer genotypes in common rye populations. Identification of new non-restorer lines for
the C cytoplasm may be facilitated by using molecular markers.
Male sterility/fertility in rye with cytoplasm C
is controlled by at least 3 loci, of which the restorer
gene Rfc1, located on chromosome 4RL, plays
a major role (Stoja³owski et al. 2004b). Interestingly, the same chromosomal region contains
the strong restorer genes Rfp1 and Rfp2 for
the P cytoplasm (Miedaner et al. 2000). A set of
SCAR markers tightly linked with those restorer
genes has been recently developed by
Stracke et al. (2003). Additionally, 3 other SCAR
markers from chromosome 4R were described by
Gonzales et al. (2002). The possibility of application of these markers for selection in the CMS-C
system has not yet been investigated, so in this
study we analysed the linkage relationship of
Received: March 10, 2005. Accepted: September 6, 2005.
Correspondence: S. Stoja³owski, Department of Genetics and Plant Breeding, Agricultural University, S³owackiego 17,
71-437 Szczecin, Poland, e-mail: [email protected]
372
S. Stoja³owski et al.
the recently developed SCAR markers for the fertility gene Rfc1 on chromosome 4R.
Two inbred lines 544cms-C (male sterile)
and Ot0-20 (restorer) were crossed in order to develop F2 and BC1 [544cms-C × (544cms-C ×
Ot0-20)] mapping populations. The 94 F2
and 93 BC1 plants were grown in a field near
Szczecin in the 1999/2000 season. Male fertility of
each plant was visually assessed on a scale of 1–9
according to Geiger and Morgenstern (1975).
On the basis of the results, the plants were divided
into 3 groups: male sterile (score 1–3), partly male
sterile (score 4–6), and male-fertile (score 7–9).
DNA of each plant was extracted from leaves
cut at tillering and preserved at –70ºC by using
DNeasy Plant Mini Kit (Qiagen) and Genomic
Mini Kit (A&A Biotechnology). Twelve ng of
template DNA was diluted in 15 mL of the PCR
mixture containing ammonium sulphate buffer
(750 mM tris-HCl, pH 8.8, 200 mM (NH4)2SO4,
0.1% Tween 20), 1.5 mM MgCl2, 0.75 U Taq
polymerase (MBI Fermentas), 10 pM of each
primer, and 0.1 mM of each dNTP. The amplification reactions were performed in a GeneAmp ®
PCR System 9700 (Applied Biosystems) programmed for the initial 3-min denaturation step at
94ºC, followed by 10 cycles of 30 s at 92ºC, 45 s
at 65ºC (decreased 1oC per cycle), 1 min at 72ºC,
and 25 cycles of 30 s at 92ºC, 30 s at 55ºC
and 1 min at 72ºC, followed by an elongation step
for 5 min at 72ºC. Ten SCAR markers, including
OPA16_795, OPA16_1084 and OPS4_756 developed by Gonzales et al. (2002) and SCY03,
SCY09d, SCP12M56, SCP14M55, SCP15M55,
SCP16M58 and SCP44M51 described by Stracke
et al. (2003), were used in this study.
Amplification products were separated at
6 V/cm in 1.5% agarose gels containing TBE buffer
(90 mM tris-borate, 2 mM EDTA) and 0.4 mg mL–1
ethidium bromide. Electrophoregrams were visual-
Figure 1. Map position of markers linked with the restorer
gene Rfc1 on chromosome 4RL (constructed with the use
of data from F2 and BC1 mapping populations). Numbers
given on the left: distances in centimorgans (cM).
ized and documented by a Fluor S-Multi Imager
(Bio-Rad).
The segregation of the SCAR markers was
used to extend linkage maps of chromosome 4R
developed by means of RAPD markers
(Stoja³owski et al. 2004a, b). Integration of both
maps obtained in generations F2 and BC1 was
achieved by means of the JoinMap 3.0 software
(Van Ooijen and Voorrips 2001). The potential effectiveness of SCAR markers in the selection
of non-restorer lines was evaluated by the standard
c2 test.
Table 1. Number of male-sterile, partially male-fertile and male-fertile plants in three groups selected for
male-sterility by means of SCAR markers closely linked to restorer gene Rfc1 on rye chromosome arm 4RL
Number of plants 1)
Generation
F2
SCAR marker
SCP14M55
SCP15M55
SCP16M58
Total population
BC1
Total population
1)
SCP14M55
SCP15M55
SCP16M58
male-sterile
6
5
5
6
17
17
17
17
partly
male-sterile
male-fertile
c2 (vs. total
mapping
population)
5
5
5
7
10
10
10
26
16
16
16
81
11
13
11
50
17.33**
13.40**
13.40**
–
18.94**
16.33**
18.94**
–
classified on the basis of fertility scores according to Geiger and Morgenstern (1975)
Significant at P = 0.05 and P = 0.01, respectively
*, **
SCAR markers for Rfc1 gene
Among the 10 markers studied, only
OPA16_1084, SCP14M55, SCP15M55 and SCP
16M58 revealed polymorphisms between
the 2 parent lines. All segregated according to
a 3:1 ratio in the F2, and a 1:1 segregation ratio in
BC1 was observed for the last three markers.
The combined linkage map comprising F2
and BC1 segregation data (Figure 1) contains 3
SCAR markers located in the interval between
RAPD markers pr23/500 bp and pr743/750 bp,
flanking the Rfc1 gene according to a previous
study (Stoja³owski et al. 2004b). A strong linkage
of the SCAR markers with the restorer gene was
also confirmed by a significantly higher proportion of male-sterile plants in the respective marker
classes, as compared to the total population (Table 1), indicating their applicability as tools for
marker-assisted selection. Marker OPA16_1084
was not linked to any of the remaining markers.
Markers SCP14M55 and SCP15M55 are
tightly linked with the restorer locus Rfp1, which
was found in the primitive rye population IRAN
IX from the Middle East. Another restorer gene
for the P cytoplasm, Rfp2, was found in variety
Pico Gentario from Argentine (Geiger
and Miedaner 1996). Stracke et al. (2003) mapped
this locus in the vicinity of marker SCP16M58.
Unfortunately, the authors found no common
marker for the linkage groups of genes Rfp1
and Rfp2. In spite of this, Stracke et al. (2003)
speculated that these 2 restorer genes might be
identical. Results presented here (Figure 1) seem
to confirm this suggestion because all 3 analysed
SCAR markers are tightly linked. The same region
of chromosome 4RL was found to harbour restorer
genes Rfc1 (Stoja³owski et al. 2004 b) and Rfg1
(Börner et al. 1998). These data suggest that all
these restorer genes on chromosome arm 4RL are
either tightly linked or represent different alleles at
the same locus.
Acknowledgements. This work was supported by
the Agricultural University of Szczecin, Grant
No. BW/HK/02/03.
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