=Paper=
{{Paper
|id=Vol-2030/HAICTA_2017_paper60
|storemode=property
|title=Molecular Detection of the Resistance to Biotic Stress Conditions in Hellenic Bread Wheat Commercial Cultivars
|pdfUrl=https://ceur-ws.org/Vol-2030/HAICTA_2017_paper60.pdf
|volume=Vol-2030
|authors=Natalia Kozub,Anatolii Karelov,Igor Sozinov,Oksana Sozinova,Ioannis Xynias
|dblpUrl=https://dblp.org/rec/conf/haicta/KozubKSSX17
}}
==Molecular Detection of the Resistance to Biotic Stress Conditions in Hellenic Bread Wheat Commercial Cultivars==
https://ceur-ws.org/Vol-2030/HAICTA_2017_paper60.pdf
Molecular Detection of the Resistance to Biotic Stress
Conditions in Hellenic Bread Wheat Commercial
Cultivars
A.V. Karelov1,2, N. I. Kozub1,2, I.A Sozinov2, O. Sozinova2,3 and I. N. Xynias 4*
1
Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine,
Osipovskogo St. 2a, 04123, Kyiv, Ukraine
2
Institute of Plant Protection, Ukrainian Academy of Agrarian Sciences, Vasilkovska St., 33,
03022, Kyiv, Ukraine
3
Taras Shevchenko National University of Kyiv, Ukraine
4
School of Agricultural Technology & Food Technology and Nutrition,
Western Macedonia University of Applied Sciences, Terma Kontopoulou, 53 100 Florina,
Greece.
*
corresponding author: ixynias@teiwm.gr; ioannis_xynias@hotmail.com
Abstract: Biotic stress conditions are the most serious obstacle in bread wheat
cultivation resulting in yield reduction and consumption safety problems
(poisonous toxic production). For this, the identification of resistant cultivars
and their respective genes is the main prerequisite in most breeding programs.
In order to exploit the benefits of molecular technology in studying their
genetic background, eight Hellenic bread wheat cultivars were analyzed to
determine their gene constitution at some important disease resistance loci. It
was revealed that cultivar Elissavet carries genes conferring resistance to tan
spot (insensitivity to toxins A and B), rusts, powdery mildew, and barley
yellow dwarf virus (Lr34/Yr18/Pm38/Sr57/Bdv1 in combination with the genes
on the wheat-rye 1BL/1RS translocation). Cultivar Strymonas has three genes
for resistance to necrotrophic diseases. Cultivar Yecora E carries the genes
conferring resistance to tan spot and rusts (Lr34/Yr18/Pm38/Sr57/Bdv1) but
lacks the translocation. The third cultivar, i. e. (Acheron) which carries the
1BL.1RS wheat-rye chromosome translocation, also has genes for resistance to
tan spot (due to insensitivity to toxin B) and Fusarium head blight but lacks the
resistance allele of the Lr34 gene. It is concluded from the results that cultivar
Elissavet constitutes a remarkable combination of favorable genes and must be
more extensively used as a parental line in breeding programs to developing
novel wheat germplasm.
Key words: resistance, fungal diseases, bread wheat, resistance genes
1 Introduction
Biotic stressing factors and more precisely foliar diseases represent the most serious
obstacle in bread wheat cultivation (Faris et al. 2010). In addition to decreased yields
(e. g. caused by rusts), biotic stressing factors could cause safety problems to
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consumers (e. g. poisonous toxins produced by Fusarium), or, additionally carry
other pathogens in various crops (e. g. viruses transferred by aphids) (Moreno et al.
2012). For all these reasons, identifying resistant varieties and their corresponding
genes is a key goal in many breeding programs (Diethelm et al. 2014). Modern
molecular breeding with very detailed technology available can greatly contribute to
this objective (Abeysekara et al. 2010). It is well established that the
Lr34/Yr18/Pm38/Sr57/Bdv1 gene conferring moderate resistance to biotrophic
pathogens is located on chromosome 7D (Krattinger et al. 2009; Lagudah et al. 2009;
Dakouri et al. 2010), the Tsn1 gene, responsible for sensitivity to the toxins A of the
necrotrophic fungi Pyrenophora tritici-repentis (Died.) Drechesта Stagonos
poranodorum (Berk.) E. Castell. & Germano is located on chromosome 5A (Faris et
al. 2010), and the TDF_076_2D gene conferring moderate resistance to Fusarium
head blight is located on chromosome 2D (Diethelm et al. 2014). The Tsc2 gene
responsible for sensitivity to the toxin B of P. tritici-repentis was mapped on
chromosome 3B (Abeysekara et al. 2010). Biochemical screening of the existing
germplasm could also facilitate this identification (Xynias et al. 2007). In a previous
article we reported the presence of the 1BL.1RS wheat rye chromosome translocation
in Hellenic bread wheat cultivars after applying biochemical analysis (Xynias et al.
2006). This presence was further confirmed by molecular markers (Peros et al.
2014). The 1BL/1RS translocation from the rye Petkus (2x) of the Kavkaz type, is
the most widespread introgression among common wheat varieties (Rabinovich
1998). The importance of this translocation is due to certain important genes located
on the small arm of 1R chromosome. The main advantages of the translocation is
high yield potential of the host cultivar (Kim et al. 2004), and resistance to both
biotic and abiotic stressing factors (e. g. disease, insect and drought resistance,
Anonymous 2013; Peng et al. 2007; Xynias et al. 2007). For this, screening Hellenic
germplasm, to identifying the above and/ or other resistant genes is also important,
because this germplasm could be involved in crosses to transfer all the important
traits to new varieties.
In the present work eight Hellenic bread wheat varieties were studied in order to find
which ones carry resistance genes for some of the most serious biotic stress factors,
such as rusts, Fusarium head blight, tan spot, powdery mildew.
2 Material and methods
2.1 Plant material
Seven commercial bread wheat cultivars produced at Cereal Institute of Thessaloniki
(i. e. cvs. Yecora E, Elissavet, Xenia, Acheron, Strymonas, Louros, and Lydia), one
non commercial cultivar (cv. Chios) developed at the University of Thessaloniki,
Greece and the Russian cultivar KVZ/Cgn were used for the purpose of the study.
The cultivar ‘Chinese Spring’ was used as the control for the “tr” allele of the marker
Xfcp623 (associated with the tsn1 ToxA insensitiveness allele of the gene) (Faris et
490
al. 2010), the “tsr” allele of the marker XBE444541 (associated with the tsc2
PtrToxB insensitivity allele of the gene) (Abeysekara et al. 2010) and the allele “+”
of the Lr34 gene (presence of resistance) (Lagudah et al. 2009). The cultivar
‘Katepwa’ was used as the control for the “Тs” allele of the marker Xfcp623
(associated with the Tsn1 toxin sensitive dominant allele of the gene), the “Tss”
allele of the marker XBE444541 (associated with the Tsc2 toxin sensitive dominant
allele of the gene) and the allele “-” of the Lr34 gene (absence of resistance)
(http://wheatpedigree.net/sort/show/31123). For the TDF_076_2D gene, the cultivar
Mironovskaya 808 was used as the control for the allele 2 (associated with moderate
resistance to Fusarium head blight) and the cultivar ‘Chinese Spring’ – as the control
for the allele 1 (Diethelm et al. 2014).The cultivars for the control were kindly
provided by the National Center for Plant Genetic Resources of Ukraine of NAAS
(Kharkiv). The marker Xfcp623 has 2 alleles: 379 bp (associated with sensitivity,
further – “Ts”) and null-allele (associated with insensitivity, further – “tr”) (Faris et
al. 2010).
2.2 Method
DNA was extracted from the sample of 25-30 mg. obtained from grinding 5-7 seeds
with further use of a DiatomTM DNA Prep100 DNA isolation kit (the sales
representative in Ukraine is Neogene® Company) following the standard protocol.
PCR was performed using GenPak® PCR Core Kits (the sales representative in
Ukraine is Neogene® Company) according to the manufacturer’s recommendations.
The PCR was performed in the amplifier 2720 GeneAMP System using GenPak®
PCR Core kits (the sales representative in Ukraine is the Neogene® Company)
according to the manufacturer’s recommendations.
The marker XBE444541-STS has 2 alleles: 340 bp (associated with sensitivity,
further – “Tss”) and 509 bp (associated with insensitivity, further – “tsr”, on the
agarose gel electrophoresis it is masked by nonspecific bands) (Abeysekara et al.
2010).
To determine the allelic state of the Lr34 gene a combination of the gene-localized
marker SNP12 and the closely linked marker ISBP1 were used (Dakouri et al. 2010).
The amplified fragments of 509 and 234 bp in length are associated with the Lr34+
allele and the fragments of 391 bp in length – with the Lr34- allele.
For the TDF_076_2D gene the intron-localized marker INDEL1 was used (Diethelm
et al. 2014). In case of the resistance-associated allele 2 the amplifies fragments of
212 and 221 bp in length were obtained and in case of susceptibility associated allele
– only fragments of 212 bp in length.
The annealing temperature was lowered to 42°С for the primer pair flanking the
marker XBE444541. For the combination of the markers SNP12 and ISBP1 the
condition following conditions: dissociation/activation of the hot-start polymerase at
95°C for 7 minutes then 32 cycles with dissociation phase at 94°C for 30 s, annealing
491
phase at 62.5°C for 40 s and elongation at 72°C for 40 s; final elongation – for 5 m.
(Karelov et al. 2014).Besides this PCR was performed according the literature
conditions (Diethelm et al. 2014; Abeysekara, et al. 2010; Faris et al. 2010).
PCR results were visualized by electrophoresis in 2–2.5% agarose gel in 0.5 x TBE
buffer with subsequent staining with ethidium bromide or (in case of the INDEL1
marker) – by 8% the PAAGE with subsequent staining with AgNO3 and use of the
gel- visualization system VISION Gel.
2.3 Genes detected
The resistance-associated allele of the Lr34/Yr18/Pm38/Sr57/Bdv1 gene (Dakouri et
al. 2010) was marked as Lr34+, the allele associated with absence of resistance as
Lr34-; for the Tsn1 gene (the marker Xfcp623), the allele for insensitivity to the toxin
A (Faris et al. 2010) was designated as tr, the allele for sensitivity as Ts; for the Tsc2
gene (the marker XBE444541-STS), the allele for insensitivity to the toxin B
(Abeysekara et al. 2010) was marked as tsr, the allele for sensitivity as Tss; for the
TDF_076_2D gene, the allele conferring Fusarium head blight resistance (Diethelm
e al.2014) was designated as TDF-1, the allele for the absence of such resistance as
TDF-2. The marker INDEL1 of the TDF_076_2D gene was analyzed by the
procedure described in (Diethelm et al. 2014; Karelov et al. 2015).
The presence of the wheat-rye 1BL/1RS translocation and respective resistance genes
was marked as +, and the absence as - (according to Xynias et al. 2006).
3 Results and discussion
The results of the molecular analysis regarding the allele constitution of genes
conferring resistance to biotic factors examined and are expressed in Hellenic bread
wheat cultivars are presented in Table 1.
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Table 1. Allele constitution of genes conferring resistance to biotic stressing factors in
Hellenic bread wheat cultivars.
Cultivar Tsn1 Tsc2 Lr34 TDF_076_2D 1BL/1RS (Pm8,
Sr31, Lr26,Yr9)
Yecora Ε Ts trr + 2 -
Elissavet tr trr + 2 +
Xenia Ts trr - 1 -
Acheron Ts trr - 1 +
Strymonas tr trr - 1 -
Louros tr - 2 -
Lydia Ts - 1 -
Chios tr - 1 +/-
KVZ/Cgn Ts + 1 +
Where for the Tsn1 gene tr is the allele for insensitivity and Ts is the allele for
sensitivity; for the Tsc2 gene tsr is the allele for insensitivity, and Tss the allele for
sensitivity; for the Lr34 gene with (+) is marked the resistant allele and with (–) the
non-resistant; for the TDF_076_2D gene 1 is the resistant and 2 is the non resistant
allele; the presence of the 1BL.1RS wheat rye translocation is marked with (+) and
the absence with (-).
In the majority of varieties, combinations of two or more resistance genes were
revealed at the loci analyzed (Table1). Cultivar Elissavet carries genes conferring
resistance to tan spot due to insensitivity to toxins A and B of P. tritici-repentis,
resistance to rusts, powdery mildew due to the presence of the wheat-rye 1BL/1RS
translocation and the gene Lr34/Yr18/Pm38/Sr57/Bdv1, which confers moderate
race-nonspecific resistance to a number of biotrophic pathogens, including yellow
dwarf virus. Cultivar Yecora E has the gene for resistance to tan spot (insensitivity
to toxin B) and the gene for moderate race-nonspecific resistance to rusts and other
pathogens (Lr34/Yr18/Pm38/Sr57/Bdv1). It should be noted that the important gene
Lr34/Yr18/Pm38/Sr57/Bdv1 is rare among European wheats (Kolmer et al. 2008).
Cultivar Acheron, which also carries the 1ΒL.1RS wheat rye translocation, has
respective resistance genes as well as the gene for insensitivity to P. tritici-repentis
(tanspot) toxin B and moderate resistance to Fusarium head blight. The cultivar
Strymonas is characterized by three genes conferring resistance to nectrotrophic
493
pathogenes (tan spot and Fusarium head blight). At least two important disease
resistance genes were detected in cultivars Xenia, Chyos, and KVZ.
4 Conclusion
It can be concluded from the above results that cultivar Elissavet, which carries the
1BL.1RS wheat rye translocation, represents a good combination of favorable genes
and for this it must be extensively used as parental line in breeding programs for
producing new wheat germplasm. Other varieties can also be used as sources of
important resistance genes in marker-assisted selection.
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