c Indian Academy of Sciences
RESEARCH NOTE
Molecular cytogenetic identification of a wheat–Thinopyrum ponticum
translocation line resistant to powdery mildew
FANG HE1 , YINGUANG BAO1 , XIAOLEI QI2 , YINGXUE MA1 , XINGFENG LI1 and HONGGANG WANG1 ∗
1
State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, College of Agronomy, Shandong
Agricultural University, Taian 271018, People’s Republic of China
2
Taian Academy of Agricultural Sciences, Taian 271018, People’s Republic of China
Abstract
Thinopyrum ponticum (2n = 70) serves as a valuable gene pool for wheat improvement. Line SN0224, derived from crosses
between Th. ponticum and the common wheat cultivar Yannong15, was identified in the present study. Cytogenetic observations showed that SN0224 contains 42 chromosomes in the root-tip cells and 21 bivalents in the pollen mother cells, thereby
demonstrating its cytogenetic stability. Genomic in situ hybridization, probed with the total genomic DNA of Th. ponticum,
produced hybridization signals in the distal region of two wheat chromosome arms. After inoculation with the Blumeria
graminis f. sp. tritici (Bgt) isolates, SN0224 exhibited immunity. Segregation in F1 s and F2 s from the cross SN0224/cv. Huixianhong indicated that SN0224 carries a single dominant gene for powdery mildew (Pm) resistance, which was temporarily
designated PmSn0224. Three markers Barc212, Xwmc522 and Xbarc1138 were detected to be linked with PmSn0224. Based
on the locations of the markers, PmSn0224 was located on the chromosome 2A. None of the three markers above is linked
with the previously reported PM resistance genes on chromosome 2A, and none of the previously reported PM resistance genes on chromosome 2A is related to Th. ponticum. Therefore, PmSn0224 is likely a novel gene putatively from
Th. ponticum.
[He F., Bao Y., Qi X., Ma Y., Li X. and Wang H. 2017 Molecular cytogenetic identification of a wheat–Thinopyrum ponticum translocation
line resistant to powdery mildew. J. Genet. 96, 165–169]
Introduction
Powdery mildew (Pm), which is caused by Blumeria graminis f. sp. tritici (Bgt), is a serious global wheat disease (Alam
et al. 2011). Breeding resistant cultivars has been recognized as the most economical, effective, and environmentally
appropriate strategy to control Pm. To date, in wheat and
its wild relative, more than 73 alleles at 50 loci that confer
resistance to Pm have been identified (Chhuneja et al. 2015).
Unfortunately, genetic resistance is frequently overcome by
new Bgt isolates due to frequent changes in the pathogen
population, especially when a single resistance gene is
deployed over a wide area (Hsam and Zeller 2002). Therefore it is necessary to continuously identify novel genes for
Pm resistance in wheat-breeding programmes.
∗ For correspondence. E-mail: [email protected].
Fang He and Yinguang Bao contributed equally to this work.
Thinopyrum ponticum (Podp.) Barkworth and D. R.
Dewey (2n = 10x = 70), has long been known to exhibit
strong resistance to numerous diseases. Since Th. ponticum
was initially hybridized with wheat ∼70 years ago, some
value of resistance genes, such as Sr25, Lr19 and Cmc2
have been transferred through wheat–Th. ponticum chromosome translocations. These translocations have supported the
development of several wheat germplasms that are in use
by wheat improvement programmes throughout the world
(Friebe et al. 1996; Li and Wang 2009). Recently, a putative Th. ponticum-derived gene, Pm51, was identified in an
introgression line and mapped to the chromosome 2BL (Zhan
et al. 2014).
Line SN0224 derived from the BC1 F5 line of a cross
between Th. ponticum and common wheat cultivar Yannong15 display excellent resistance to Pm as well as other
advantageous agronomic characteristics, including the production of numerous tillers and a vigorous growth habit.
Here, we report the cytogenetic verification and molecular
analysis of Pm resistance in SN0224.
Keywords. translocation; powdery mildew; genomic in situ hybridization; Thinopyrum ponticum.
Journal of Genetics, DOI 10.1007/s12041-017-0754-2, Vol. 96, No. 1, March 2017
165
Fang He et al.
Materials and methods
Plant materials
The plant materials included Th. ponticum, SN0224,
Yannong15 and Huixianhong wheat varieties. A F2 segregation population was developed from a cross between SN0224
and the susceptible parent Huixianhong. SN0224 was developed from the progeny of Yannong15 and Th. Ponticum at
the Agronomy College of Shandong Agricultural University,
Taian, China. Th. Ponticum was provided by Prof. Zhensheng
Li, formerly of the Northwest Institute of Botany, Chinese
Academy of Sciences, Yangling, China. All plant materials
were maintained through selfing at the Tai’an Subcenter of
the National Wheat Improvement Center, Shandong, China.
Cytogenetic analysis
Seeds were germinated in Petri dishes maintained at 4◦ C
for ∼24 h and then cultivated at 25◦ C. Roots 1–2 cm were
cut and treated in ice water for ∼24 h before fixing in
Carnoy’s solution. After fixation, the root tips were stained
and squashed in carbol fuchsin and mitotic chromosomes
were observed under a microscope. When plants reached the
flag leaf stage, spikes were sampled and anthers at metaphase
I (MI) of meiosis were fixed in Carnoy’s solution, dissociated in 1M HCl at 60◦ C for 6–8 min and squashed in 1%
acetocarmine.
Genomic in situ hybridization (GISH)
Total genomic DNA from Th. ponticum was labelled with
Texas-red-5-dCTP using the nick translation method and
is available as a probe. Sheared genomic DNA from Yannong15 (AABBDD) was invoked as blocking DNA. The
detailed procedures of the chromosome maintenance and
hybridization mixture have been described originally by Han
et al. (2009). Photographs were captured using the Olympus
BX-60 equipped with a CCD camera.
Evaluation of Pm responses
Two Bgt isolates, E09 and E20, were utilized to test the
response of seedlings of Th. ponticum, SN0224, Yannong15
and Huixianhong in the greenhouse. Isolate E09, a dominant pathotype in the Beijing region, i.e. contagious to the
majority of Pm genes, including Pm1, Pm3a, Pm3c, Pm5,
Pm7, Pm8, Pm17 and Pm19 (Zhou et al. 2005) was used to
test the F2 segregating population of SN0224/Huixianhong.
The infection types (ITs) of the seedlings of each line were
rated using a scale from 0 to 4: 0, no noticeable symptoms; 0n , necrotic flecks; 1, strongly resistant; 2, resistant; 3,
susceptible; 4, strongly susceptible (Liu et al. 2005).
polymorphisms. EST-SSR primers were supplied by Prof.
Sishen Li, Agronomy College of Shandong Agricultural University, Tai’an, China and the SSR primers were synthesized according to sequences published in the GrainGenes
database (http://wheat.pw.usda.gov).
Each 15 μL polymerase chain reaction (PCR) solution contained 1.5 μL of 10× PCR buffer, 1.2 μL of
Mg2+ (2.5 mM), 0.9 μL of deoxynucleotide triphosphates
(2.5 nM), 0.12 U of Taq polymerase, 3.0 μL of the primer
pairs (5 μM), 5.28 μL of double distilled H2 O, and 3.0 μL
of DNA (30 ng/μL). Amplification was accomplished by
touchdown PCR and the amplification products were kept
at 10◦ C using a Bio-Rad 9600 Thermal Cycler (Hercules,
USA). The PCR products were segregated on 8% nondenaturing polyacrylamide gel and subsequently stained with
silver nitrate.
Data analysis
Chi-square tests were conducted to examine the goodness
of fit of the observed and expected ratios for Pm reactions
and molecular markers. The linkage relationship between the
resistance gene and the markers was analysed using JoinMap
software, ver. 4.0.
Results
Cytological assessment of SN0224
Chromosome counts were determined in nine plants of
SN0224. Each plant contained 42 chromosomes in the roottip cells (figure 1a) and 21 bivalents in pollen-mother cells
(figure 1b), indicating the cytogenetic stability of SN0224.
GISH was produced using total genomic DNA from Th. ponticum as a probe and ABD-genomic DNA from Yannong
15 wheat as a blocker to detect alien genetic materials in
SN0224. In mitotic metaphase cells, two chromosomes were
labelled red in the terminal regions of the short arms, four
were labelled bright red on the short arms, and others were
counterstained with blue (figure 1c). Four bright fluorescent
segments appeared consistently in common wheat when
GISH was performed using total genomic DNA from Th.
ponticum as a probe (data not shown). Therefore, SN0224
was shown to be a translocation line with a tiny segment from
Th. ponticum translocated to the apical region of a wheat
chromosome.
Reactions to Pm
After inoculation with isolates E09 and E20, both Yannong15 parent and Huixianhong control were observed to
be susceptible, whereas SN0224 and Th. ponticum were
immune (table 1). These results prove that SN0224 conferred
resistance to Pm similar to its wild donor Th. ponticum.
Microsatellite screening and electrophoretic analysis
Microsatellite primers, including expressed sequence tagderived SSRs (EST-SSRs) and simple sequence repeats
(SSRs), were used to scan the plant materials for
166
Inheritance of Pm resistance in SN0224
To assess the succession of resistance, SN0224 was crossed
with Huixianhong. When inoculated with isolate E09, F1
Journal of Genetics, Vol. 96, No. 1, March 2017
Cytogenetic identification of a translocation line
Figure 1. Cytological patterns of SN0224. (a) A root-tip cell at mitotic metaphase (2n = 42). (b) A
pollen mother cell at meiotic metaphase I (2n =21II). (c) GISH pattern of SN0224 probed with total
genomic DNA from Th. ponticum. Arrows indicated the translocation segments from Th. ponticum.
Asterisks indicate the segments detected in common wheat when GISH was conducted using total
genomic DNA from Th. ponticum as a probe.
Table 1. Reactions of Huixianhong, SN0224 and their parents to Pm.
Material
Material type
Infection type
Isolate
Th. ponticum
Yannong15
SN0224
Huixianhong
E09
E20
Donor
0
0
Receptor
4
4
Translocation
0–0n
0–0n
Control
4
4
Table 2. Inheritance of powdery-mildew resistance in SN0224.
No. of F2 plants investigated
F2 plants of SN0224/Huixianhong
R
S
Total
2
χ(3:1)
P value
272
83
355
0.50
0.25–0.50
R, resistant; S, susceptible. P = 0.05 and 1df = 3.84.
plants showed infection types similar to SN0224 and F2 s
showed apparent symptoms of reactions at a resistant: sus2
, P > 0.05) (table 2).
ceptible ratio of 3:1 (χ 2 = 0.50 < χ0.05
Thus, the observed Pm resistance in SN0224 indicated typical dominant inheritance controlled by a single gene or locus,
which was temporarily appointed as PmSn0224.
Molecular linkage and chromosome assignment for PmSn0224
The F2 population of SN0224/Huixianhong was responsible for mapping the PmSn0224 gene. Approximately 289 of
the 800 SSR and EST-SSR primer pairs chosen for the preliminary screening were polymorphic between SN0224 and
Huixianhong. These primers were rechecked among a preferred small groups (PSG) consisting of 10 typical resistant individuals and 10 typical susceptible individuals from
the F2 population of SN0224/Huixianhong. Three primer
pairs BARC212, Xwmc522 and Xbarc1138, were identified as
potential linkage markers for PmSn0224 (figure 2).
To obtain genomic data, we further analysed the genotypes
of the above three loci in 205 F2 individuals (table 3). Each
locus exhibited a 1AA:2Aa:1aa segregation ratio, which
upheld the involvement of a single gene in their succession of resistance. Utilizing JoinMap 4.0 software, we determined the linkage relationships of BARC212, Xwmc522 and
Xbarc1138 with PmSn0224 (figure 3). The locus order was
identified as PmSn0224–BARC212–Xwmc522–Xbarc1138
with genetic distances of 5.1, 5.3 and 7.0 cM, respectively.
Both markers Xwmc522 and BARC212 were previously
mapped on wheat chromosome 2A (Somers et al. 2004) and
Xbarc1138 on 2A and 2D (http://wheat.pw.usda.gov/cgi-bin
/GG3/report.cgi?class=marker&name=BARC1138). Therefore, nine additional polymorphic markers between SN0224
and Huixianhong located on chromosome 2D were tested
in the PSG. However, none of these markers was linked to
PmSn0224. Thus, PmSn0224 was deduced to be located on
chromosome 2A.
Discussion
Th. ponticum is an influential perennial Triticeae species
with a considerable number of traits that can be potentially
used for wheat improvement. Chromosome translocation
Journal of Genetics, Vol. 96, No. 1, March 2017
167
Fang He et al.
Figure 2. Amplification products of BARC212, Xwmc522 and Xbarc1138 from the susceptible parent, H, Huixianhong; N, the resistant
parent SN0224; S, 10 typical susceptible individuals; R, 10 typical resistant individuals; M, DL2000 marker.
Table 3. F2 phenotypes and genotypes revealed by BARC212, Xwmc522 and Xbarc1138.
Genotype
Marker
BARC212
Xwmc522
Xbarc1138
Phenotype
AA
Aa
aa
Total
R
S
Total
R
S
Total
R
S
Total
50
3
53
46
3
49
49
3
52
98
6
104
100
4
104
96
6
102
48
48
2
50
52
3
48
51
148
57
205
148
57
205
148
57
205
2
χ(1:2:1)
P value
0.29
0.75–0.90
0.13
0.90–0.95
0.02
0.98–0.99
AA homozygous for the SN0224 allele; aa, homozygous for the Huixianhong allele; Aa, heterozygous.
Table value of χ 2 at P = 0.05 and 2df is 5.99.
is a convenient method for transferring alien genes from
Th. ponticum to common wheat. In the present study,
SN0224 was obtained from a single wheat × Th. ponticum
hybrid backcross and selffertilized for five generation. When
analysed using GISH, two chromosomes of SN0224 were
labelled red in terminal regions, and four were labelled bright
red on the short arms (figure 1c). The four bright fluorescent segments appeared consistently in common wheat when
GISH was performed using total genomic DNA from Th.
ponticum as a probe. These segments might be interlinking
regions of the satellite and the short arms. However, additional research is needed to substantiate this assumption. The
results indicate that SN0224 is a translocation line with a tiny
fragment from Th. ponticum translocated to the apical region
of a wheat chromosome.
To date, five PM resistance loci/genes have been located
on chromosome 2A; Pm4 (The et al. 1979), PmLK906
168
(Niu et al. 2008), PmPS5A (Zhu et al. 2005), PmHNK54 (Xu
et al. 2011) and Pm50 (Mohler et al. 2013). In the present
study, we identified a PM resistance gene, PmSn0224, in the
wheat–Th. ponticum translocation line SN0224 and localized
it to chromosome 2A using the markers BARC212, Xwmc522
and Xbarc1138. None of the three markers is linked to
the other five PM resistance loci/genes. In addition, the
four designated alleles of Pm4, Pm4a, Pm4b, Pm4c and
Pm4d originated from T. dicoccum, T. carthlicum, T. aestivum and T. monococcum, respectively, whereas PmLK906,
PmPS5A, PmHNK54 and Pm50 originated from T. aestivum,
T. carthlicum, T. aestivum and T. dicoccum, respectively.
None of these genes is related to Th. ponticum. Recently, a
new PM resistance gene Pm51, was identified in a putative
wheat–Th. ponticum introgression line (Zhan et al. 2014).
However, it was localized distally on chromosome 2BL.
Therefore, PmSn0224 may be a novel Pm gene. Compared
Journal of Genetics, Vol. 96, No. 1, March 2017
Cytogenetic identification of a translocation line
Figure 3. Linkage map of PmSn0224 on the wheat chromosome
2A.
with incorporation from other exotic sources, incorporating
the high-resistance gene PmSn0224 into various wheat cultivars should be relatively straightforward owing to the common wheat background and other positive agronomic traits
of SN0224.
Acknowledgements
This research was supported by The National Key Research and
Development Plan of China (2016YFD0102004) and the Shandong
Natural Science Foundation of China (no. ZR2016CB37).
References
Alam M.A., Xue F., Wang C. and Ji W. 2011 Powdery mildew resistance genes in wheat: identification and genetic analysis. J. Mol.
Biol. Res. 1, 20–39.
Chhuneja P., Yadav B., Stirnweis D., Hurni S., Kaur S., Elkot A.
F. et al. 2015 Fine mapping of powdery mildew resistance genes
PmTb7A.1 and PmTb7A.2 in Triticumboeoticum (Boiss.) using
the shotgun sequence assembly of chromosome 7AL. Theor.
Appl. Genet. 128, 2099–2111.
Friebe B., Jiang J., Raupp W., McIntosh R. and Gill B. 1996 Characterization of wheat-alien translocations conferring resistance to
diseases and pests: current status. Euphytica 91, 59–87.
Han F., Gao Z. and Birchler J.A. 2009 Reactivation of an inactive centromere reveals epigenetic and structural components for
centromere specification in maize. Plant Cell 21, 1929–1939.
Hsam S. L. K. and Zeller F. J. 2002 Breeding for powdery mildew
resistance in common wheat (Triticum aestivum L.). In: The powdery mildews, a Comprehensive treatise (ed. R. R. Belanger, W.
R. Bushnell, A. J. Dik and T. L. W. Carver),. pp. 219–238. St.
Paul, USA.
Li H. and Wang X. 2009 Thinopyrum ponticum and Th. intermedium: the promising source of resistance to fungal and viral
diseases of wheat. J. Genet. Genomics 36, 557–565.
Liu S., Wang H., Zhang X., Li X., Li D., Duan X. et al.
2005 Molecular cytogenetic identification of a wheat-Thinopyron
intermedium (Host) Barkworth & DR Dewey Partial amphiploid
resistant to powdery mildew. J. Integr. Plant Biol. 47, 726–733.
Mohler V., Bauer C., Schweizer G., Kempf H. and Hartl L. 2013
Pm50: a new powdery mildew resistance gene in common wheat
derived from cultivated emmer. J. Appl. Genet. 54, 259–263.
Niu J., Wang B., Wang Y., Cao A., Qi Z. and Shen T. 2008 Chromosome location and microsatellite markers linked to a powdery mildew resistance gene in wheat line ‘Lankao 90(6)’. Plant
Breed. 127, 346–349.
Somers D. J., Isaac P. and Edwards K. 2004 A high-density
microsatellite consensus map for bread wheat (Triticum aestivum
L.). Theor. Appl. Genet. 109, 1105–1114.
The T. T., McIntosh R. A. and Bennett F. A. G. 1979 Cytogenetical
studies in wheat. IX. Monosomic analyses, telocentric mapping
and linkage relationships of genes Sr21, Pm4 and Mle. Aust. J.
Biol. Sci. 32, 115–126.
Xu W., Li C., Hu L., Wang H., Dong H., Zhang J. et al. 2011 Identification and molecular mapping of PmHNK54: a novel powdery mildew resistance gene in common wheat. Plant Breed. 130,
603–607.
Zhan H., Li G., Zhang X., Li X., Guo H., Gong W. and et al.
2014 Chromosomal location and comparative genomics analysis
of powdery mildew resistance gene Pm51 in a putative wheatThinopyrum ponticum introgression line. PloS One 9, e113455.
Zhou R., Zhu Z., Kong X., Huo N., Tian Q., Li P. et al. 2005
Development of wheat near-isogenic lines for powdery mildew
resistance. Theor. Appl. Genet. 110, 640–648.
Zhu Z., Zhou R., Kong X., Dong Y. and Jia J. 2005 Microsatellite markers linked to 2 powdery mildew resistance genes introgressed from Triticum carthlicum accession PS5 into common
wheat. Genome 48, 585–590.
Received 21 September 2015, in revised form 1 July 2016; accepted 11 July 2016
Unedited version published online: 13 July 2016
Final published online: 13 March 2017
Corresponding editor: UMESH C. LAVANIA
Journal of Genetics, Vol. 96, No. 1, March 2017
169