Download molecular marker-based characterization of barley powdery mildew

______AGRONOMIJAS VĒSTIS (Latvian Journal of Agronomy), No.11, LLU, 2008____
exhibited the highest direct effect on seed yield both in dry and rainy growing seasons. Therefore,
both these traits seem to be good selection criteria to improve sunflower seed yield.
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SAULESPUĖU (Helianthus annuus L) RAŽAS PAZĪMJU KORELĀCIJU ANALĪZE
Kaya Y., Evci G., Pekcan V., Gucer T., Durak S., Yilmaz M.I.
Saulespuėu selekcionāriem jāzina, ka pazīmes, kuras galvenokārt nosaka sēklu ražu, ir
kvantitatīvas un tās ietekmē vide, kā arī vides un pazīmes mijiedarbība. Korelācijas koeficientu
analīze palīdz selekcionāram izskaidrot tiešās un netiešās ietekmes, tādejādi šī metode plaši tiek
izmantota selekcijas darbā. Pētījums veikts, izmantojot ražas datus un korelāciju analīzes Nacionālā
Saulespuėu pētījumu projekta izmēăinājumā iekĜautajiem hibrīdiem. Projekts notiek Edirne
provincē, kurā ražo 20 % no Turcijas saulespuėu produkcijas. Kopumā 2932 saulespuėu hibrīdi
tika pētīti 118 izmēăinājumos šī pētījuma gaitā. Augstražīgu hibrīdu selekcijā pazīmei - 1000
graudu svars - bija būtiskākā nozīme. Ziedkopas diametrs un auga garums gan sausos, gan lietainos
augšanas apstākĜos bija nākamie nozīmīgākie, balstoties uz vienkāršām korelācijas analīzēm.
MOLECULAR MARKER-BASED CHARACTERIZATION OF BARLEY POWDERY
MILDEW MLO RESISTANCE LOCUS IN EUROPEAN VARIETIES AND BREEDING
LINES
1
Kokina A.1, LegzdiĦa L.2, BērziĦa I.2, Bleidere M.3, Rashal I.4 and Rostoks N. 1
Faculty of Biology, University of Latvia, 4 Kronvalda Blvd., Rīga, LV-1586, Latvia , phone: +371
6703 4867, e-mail: [email protected]
2
State Priekuli Plant Breeding Institute, Zinatnes str. 1a, Priekuli, Latvia
3
State Stende Cereal Breeding Institute, Dižstende, Talsu raj., Latvia
4
Institute of Biology, University of Latvia, Miera str. 3, Salaspils, LV-2169, Latvia
Abstract
Powdery mildew is an economically important barley disease, caused by a fungal pathogen
Blumeria (Erysiphe) graminis f.sp. hordei. While the pathogen is relatively easily controlled by
fungicides, it may represent a serious threat for barley produced in low input and organic
agriculture. Alternatively, the disease can be controlled using resistance genes that are either
specific for certain fungal pathotypes or confer resistance to a broad range of pathotypes. Naturally
occurring and induced recessive mutations in the Mlo gene provide race nonspecific resistance that
has been effective against almost all powdery mildew pathotypes. The Mlo gene has been cloned
and several resistance allele have been characterized at the DNA sequence level. Here we report the
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______AGRONOMIJAS VĒSTIS (Latvian Journal of Agronomy), No.11, LLU, 2008____
identification of a novel mlo allele in a mutant in the cultivar Maja, which represents an amino acid
Gly318 to Arg exchange. Available CAPS (Cleaved Amplified Polymorphic Sequence) and indel
markers for mlo-5, mlo-9 and mlo-11 alleles were used to screen a selection of European barley
varieties, as well as Latvian and foreign breeding lines for the presence of mlo resistance. The
results of our study demonstrate the advantages of using molecular markers for the selection of
mildew resistant hybrids.
Key words: barley, molecular marker, Mlo gene, Cleaved Amplified Polymorphic Sequences,
CAPS, indel
Introduction
The barley crop was the fourth largest in the world by production in 2005 (FAO,
http://www.fao.org ) with the end uses being mostly food, feed and malt production. The most
important factors affecting barley production are abiotic stresses, such as drought, cold and soil
salinity (Stanca, 2003), as well as biotic stresses, such as fungal pathogens (Weibull et al., 2003).
The control of pathogens is usually achieved by the application of fungicides during the growing
season, but such practice has associated cost penalties as well as often unpredictable effects on
wildlife biodiversity. Moreover, the use of pesticides is unacceptable in organic agriculture. The
combination of these factores requires the development of barley varieties that combine high yield
and defined quality parameters with natural disease resistance.
In barley, specific powdery mildew resistance is conferred by several loci of which the Mla locus is
probably the best studied and the most important. The complex Mla locus is located on the short
arm of the barley chromosome 1H and consists of several NBS-LRR type disease resistance genes
providing resistance to different fungal pathotypes (Wei et al., 1999). The locus has been
completely sequenced from the susceptible cultivar Morex and the resistance alleles Mla1 (Zhou et
al., 2001), Mla6 (Halterman et al., 2001), Mla12 (Shen et al., 2003) and Mla13 (Halterman et al.,
2003) have been characterized (Mejlhede et al., 2006). However, gene-for-gene disease resistance
provided by Mla genes is relatively rapidly overcome by the evolution of pathogen avirulence
factors (Weibull et al., 2003), thus, race nonspecific resistance genes are desirable.
Naturally occurring and induced recessive mutations at the Mlo locus on the barley chromosome
4H confer race nonspecific resistance against most of the powdery mildew pathotypes (Jørgensen,
1992). The Mlo gene has been cloned, multiple resistance alleles have been characterized at the
DNA sequence level and molecular markers are available for marker-assisted selection (Buschges
et al., 1997; Piffanelli et al., 2004). mlo-mediated resistance comes with a yield penalty because of
the necrotic lesions on plant leaves (Wolter et al., 1993), therefore mlo-11 allele is often used in
plant breeding as it exhibits a residual production of a functional MLO protein (Piffanelli et al.,
2004). However, many varieties also possess the mlo-5 and mlo-9 alleles resulting from single
nucleotide mutations in the Mlo gene (Buschges et al., 1997).
CAPS (Cleaved Amplified Polymorphic Sequences) markers are PCR-amplified DNA regions that
exhibit polymorphic DNA fragments after digestion with restriction endonucleases (Konieczny and
Ausubel, 1993). In case of cloned genes, the CAPS markers can be designed based on the DNA
sequence targeting specific DNA polymorphisms that alter restriction sites. CAPS markers are
available for mlo-5 and mlo-9 alleles since mutations destroy the EcoRV and HhaI sites,
respectively (Schulze-Lefert et al., 1998). In addition to the CAPS marker, a SNaPShot assay has
been reported for the mlo-9 allele (Ovesna et al., 2003). The naturally occurring mlo-11 allele is
caused by a complex sequence duplication of the Mlo locus, therefore detection of the mlo-11 allele
is based on the genotyping of closely linked loci (Piffanelli et al., 2004; Tacconi et al., 2006).
In this study we used molecular markers to characterize resistance alleles at the Mlo locus in a
selection of European barley varieties and breeding lines, and we also sequenced a novel mlo allele.
Molecular markers flanking the Mlo gene and targeting the induced mutations could be used to
determine the presence and segregation of mlo-5, mlo-9 and mlo-11 alleles in hybrids.
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______AGRONOMIJAS VĒSTIS (Latvian Journal of Agronomy), No.11, LLU, 2008____
Materials and Methods
DNA from 44 European barley varieties and 15 Latvians breeding lines (Table 1) was extracted
from a single barley plant using a modified procedure by (Edwards et al., 1991). Plant tissue was
collected either from a field-grown plant or a seedling germinated on Petri dish in the laboratory.
Seeds were obtained from the State Priekuli Plant Breeding institute and State Stende Cereal
Breeding institute. DNA extractions from barley hybrid lines were performed from a combined
tissue sample originating from five plants. The Mildew resistant barley mutant line 792-99
previously characterized as allelic to mlo (I. Rashal, unpublished), was obtained from the Institute
of Biology, University of Latvia. A single mutant plant was used for DNA extractions and
sequence analysis.
PCR was performed in a 20 1 reaction consisting of ca. 50 ng DNA, 10 M primers, 1 x reaction
buffer (Fermentas, Vilnius, Lithuania), 2.5 mM MgCl2, 0.2 mM dNTPs and 1 u of Hot Start Taq
polymerase (Fermentas, Vilnius, Lithuania). PCR primers and conditions for mlo-11 allele are
described (Piffanelli et al., 2004). Barley CAPS markers for mlo-5 and mlo-9 alleles are described
(Schulze-Lefert
et
al.,
1998),
except
that
the
primers
Y14573_F02
(5’CGCCAGCAAACCAGACACAC-3’) and Y14573_R01 (5’-TTCCATGAGGACGGACACGA-3’)
were used to amplify a 321 bp fragment. Digestion with enzymes HhaI and EcoR32I (Fermentas,
Vilnius, Lithuania) was done according to manufacturer’s recommendations. Restriction digests
were analyzed on 1.5% w/v agarose gels (Fermentas, Vilnius, Lithuania).
Primers for PCR amplification and the sequencing of the Mlo coding region were from (Mejlhede
et al., 2006). Sequencing was done using BigDye 3.1. terminator mix (Applied Biosystems, Foster
City, CA, USA) on an ABI3730 sequencer according to the manufacturer’s recommendations. Base
calling and the sequence assembly was done using the Staden software package (Staden, 1996).
The sequence alignment of mutant and wild type Mlo genes was done with Clustal 1.83 software
(Thompson et al., 1997).
Results and Discussion
CAPS markers for mlo-5 and mlo-9 alleles (Figure 1A for their location in Mlo gene) and an indel
marker for the mlo-11 allele (Piffanelli et al., 2004) were used to screen a selection of European
barley varieties and breeding lines (Table 1) for the presence of mlo resistance alleles. We
identified one variety with the mlo-5 allele, two varieties with mlo-9 alleles and five varieties with
the mlo-11 allele (Table 1) based on DNA fragment sizes in agarose gels. In addition, two Latvian
breeding lines appeared to carry mlo-11. The same markers were used to screen several barley
hybrid lines (Figure 1B). Homozygous mlo-9 and mlo-11 alleles were detected in several hybrid
lines, however, some hybrids showed a mlo-9/Mlo and mlo-11/Mlo restriction fragment pattern
(Figure 1B, lanes 2 and 9, respectively). Because each hybrid line was represented by a pool of five
plants, we could not distinguish between heterozygous plants and heterogeneous pools (Figure 1B),
but it was a clear indication that the line was still segregating for mlo resistance. Registered Latvian
barley varieties are known to be Mlo wild type, therefore introgression of mlo alleles in locally
adapted germplasm could be beneficial for traditional and organic barley growers in Latvia.
Application of the mlo molecular markers (Buschges et al., 1997; Piffanelli et al., 2004; SchulzeLefert et al., 1998) for barley breeding have the potential to facilitate the identification of mlo
resistant progeny in crosses, in particular in the presence of other powdery mildew resistance
genes, e.g., Mla.
Table 1. Molecular marker-based assessment of mlo alleles in a set of barley accessions
Accession
Alamo
Alexis
Ametist
Annabell
Anni
Ballistu
Country of
origin
CA
DE
CZ
DE
EE
FR
Length of a PCR product or restriction fragment in base
pairs for each marker 1
mlo-5 Eco32I
mlo-9 HhaI
mlo-11 (indel 3)
321 bp
195, 77, 49 bp
470 bp
321 bp
272, 49 bp
470 bp
321 bp
195, 77, 49 bp
470 bp
321 bp
195, 77, 49 bp
470 bp
321 bp
195, 77, 49 bp
470 bp
321 bp
195, 77, 49 bp
470 bp
79
Detected
allele 2
wt Mlo
mlo-9
wt Mlo
wt Mlo
wt Mlo
wt Mlo
______AGRONOMIJAS VĒSTIS (Latvian Journal of Agronomy), No.11, LLU, 2008____
Barke
DE
321 bp
272, 49 bp
470 bp
mlo-9
Beatrice
FR
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Dziugiai
LT
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Effendi
NL
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Emir
NL
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Fager
NO
218, 103 bp
195, 77, 49 bp
470 bp
mlo-5
Fibar
CA
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Fink
DE
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Fontana
FR
321 bp
195, 77, 49 bp
530 bp
mlo-11
Forace
IT
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Fredrikson
US
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Freedom
CA
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Golf
UK
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Hellana
AT
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Heris
CZ
321 bp
195, 77, 49 bp
530 bp
mlo-11
Hily
SE
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Hiproly
DK
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Hockey
UK
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Ivana
FI
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Jaspis
CZ
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Jet
ET
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Justina
DE
321 bp
195, 77, 49 bp
530 bp
mlo-11
Karat
CZ
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Keti
DK
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Klaxon
UK
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Kompakt
SK
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Lawina
DE
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Lysimax
DK
321 bp
195, 77, 49 bp
530 bp
mlo-11
Magda
NL
321 bp
195, 77, 49 bp
530 bp
mlo-11
Merlin
US
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Peggy
DE
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Prosa
AT
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Rattan
CA
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Rupal
SE
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Samson
CA
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Steffi
DE
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Wanubet
US
321 bp
195, 77, 49 bp
470 bp
wt Mlo
Washonubet
US
321 bp
195, 77, 49 bp
470 bp
wt Mlo
L-3118
LV
321 bp
195, 77, 49 bp
530 bp
mlo-11
PR-2797
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3005
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3282
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3297
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3335
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3345
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3348
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3351
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3353
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3455
LV
321 bp
195, 77, 49 bp
530 bp
mlo-11
PR-3487
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3520
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3522
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
PR-3524
LV
321 bp
195, 77, 49 bp
470 bp
wt Mlo
1
Length of PCR amplicons and restriction fragments is predicted from the known sequence of the Mlo locus.
2
wt Mlo – wild type Mlo allele.
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______AGRONOMIJAS VĒSTIS (Latvian Journal of Agronomy), No.11, LLU, 2008____
3
indel – PCR fragment length polymorphism caused by an insertion of transposable element (Piffanelly et
al., 2004).
While mlo-5, mlo-9 and mlo-11 alleles are mostly used in plant breeding
(http://www.crpmb.org/mlo/), there is still a need for the characterization of additional mutant
alleles both for the understanding of the Mlo function in plants and as a material for breeding. The
complete coding sequence of the Mlo gene from the 792-99 mutant and the variety Maja was
obtained and used to identify the cause of mutation. A single base exchange at position 2004
relative to the ATG codon was identified as the only difference between the mutant 792-99 and the
wild type variety Maja (Figure 1A).
Figure 1. Molecular markers for the mlo alleles
A Structure of the Mlo gene with the mlo-5 and mlo-9 mutations indicated. A G to A transition in the 10th
exon is a novel mutation identified in the variety Maja. Boxes represent exons; filled boxes represent coding
regions, open boxes represent 5’- and 3’-untranslated regions. B Molecular marker-based detection of mlo-9
and mlo-11 alleles in barley hybrids. Lanes 1 – 4 – four hybrid lines analyzed with the mlo-9 CAPS marker
(Schulze-Lefert et al., 1998); lane 5 – MW marker; lanes 6 – 9 – the same four hybrid lines analyzed with the
mlo-11 indel marker (Piffanelli et al., 2004).
The mutation caused the amino acid Gly to Arg exchange at position 318 of the MLO protein, a
position that has also been mutated in the mlo-27 allele (Gly to Glu) (Piffanelli et al., 2002). Thus,
the predicted Gly to Arg exchange was likely to be responsible for the observed resistance
phenotype and further confirmed the functional significance of this amino acid residue.
Powdery mildew is still one of the most damaging barley diseases in Europe regnising extensive
use of pesticides. The exploitation of natural plant disease resistance provides a sustainable solution
for pest control, however, breeding for disease resistance is often complicated because of difficulty
of carrying out field phenotyping tests and the linkage drag of undesired traits with resistance
genes. Molecular markers have the potential to facilitate plant breeding by the enabling one to carry
out selection at seedling stage based on molecular marker genotypes and thus avoiding time- and
labor-intensive field tests (Varshney et al., 2005). Molecular markers also provide the opportunity
to screen a large number of progeny to select for the rare recombinants that uncouple the resistance
from closely linked deleterious alleles (Thomas et al., 1998).
Acknowledgements
The study was funded by the Latvian National programme in agrobiotechnology, the Latvian
Council of Science project 07.2055 and University of Latvia grant ZP-59.
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MOLEKULĀRO MARĖIERU PIELIETOJUMS MILTRASAS IZTURĪBAS LOKUSA
MLO RAKSTUROJUMAM EIROPAS MIEŽU ŠĖIRNĒS UN SELEKCIJAS LĪNIJĀS
Kokina A, LegzdiĦa L, BērziĦa I, Bleidere M, Rashal I un Rostoks N.
Miltrasa ir plaši izplatīta miežu slimība, ko izraisa mikroskopiskā sēne Blumeria (Erysiphe)
graminis f.sp. hordei. Slimību var kontrolēt izmantojot pesticīdus, taču to pielietošana paaugstina
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______AGRONOMIJAS VĒSTIS (Latvian Journal of Agronomy), No.11, LLU, 2008____
ražošanas izmaksas un nav pieĦemama bioloăiskajā lauksaimniecībā. Miežiem piemīt dabiskā
izturība pret miltrasu, ko nosaka vairāki genoma rajoni, no kuriem svarīgākie ir Mlo un Mla lokusi.
Dabiskas vai inducētas recesīvas mutācijas Mlo lokusā nodrošina plaša spektra izturību pret gandrīz
visiem zināmajiem miltrasas patotipiem. Mlo gēns ir klonēts un vairākas mutācijas gēna DNS
sekvencē, kas piešėir slimību izturību, ir zināmas. Mēs raksturojām jaunu mlo allēli mutantā, kas
iegūts no šėirnes Maja, kurā notikusi aminoskābes Gly nomaiĦa par Arg 318 pozīcijā. Lai
raksturotu mlo miltrasas izturību Eiropas miežu šėirnēs, kā arī Latvijas un ārzemju selekcijas
līnijās, tika izmantoti CAPS marėieri mlo-5, mlo-9 un mlo-11 allēlēm. Iegūtie rezultāti apstiprina
molekulāro marėieru pielietojuma perspektīvu pret miltrasu izturīgu miežu hibrīdu selekcijā.
A COMPARISION OF THE YIELD AND QUALITY TRAITS OF WINTER AND SPRING
WHEAT
Koppel, R., Ingver, A.
Jõgeva Plant Breeding Institute, Aamisepa 1, Jõgeva alevik, Estonia, 48 309, phone +372 77 66
901, e-mail: [email protected]
Abstract
Traditionally winter wheat is known by its higher yield potential and spring wheat by better baking
quality. In this investigation we studied how yield and quality traits of spring and winter wheat
differed at the Jõgeva PBI trials during 2004-2007. Yield and 1000 kernel weight of winter wheat
exceeded spring wheat every year. Spring wheat had higher protein and gluten content and volume
weight. There was no clear trend for the falling number and gluten index.
According to variance analyses, the value of yield and 1000 kernel weight were determined by the
wheat type (spring or winter) but other characteristics were more affected by the weather
conditions of a particular year. The effect of the weather conditions for the year was greater for
yield, 1000 kernel weight, protein and gluten content, bread loaf volume and dough stability for the
both types of wheat. For falling number the influence of the year was greater than that of the
variety of spring wheat and the influence was revesed for winter wheat. Volume weight depended
more on the weather for spring wheat and on the variety for winter wheat.
Key words: spring wheat, winter wheat, quality, yield
Introduction
The climatic conditions in Estonia are suitable for cultivation of the both wheat types – spring and
winter wheat. The acreage of wheat cultivation has enlarged from 78 to 102 thousand ha during the
last 4 years (2004-2007). The acreage share of winter wheat is 1/3 smaller than that of spring wheat
(but has a tendency to increase). Traditionally winter wheat is known for its higher yield potential
and spring wheat for its better baking quality (Swenson, 2006; Baker and Townley-Smith, 1986).
Yield and quality potential is largely determined by the variety, but the extent to which this
potential is achieved depends upon factors such as seasonal weather conditions. Higher grain yields
are usually associated with lower protein concentration (Terman et al., 1969, Blackman and Payne,
1987). The protein is a primary quality component of cereal grains. The protein concentration is
influenced by both environmental and genotypic factors that are difficult to separate (Fowler et al,
1990). The protein content of wheat grains can vary from 6% up to as much as 25%, depending
upon the growing conditions (Blackman and Payne, 1987). Terman et al. (1969) noted that protein
content varied more widely among locations than among varieties at the growing location.
Differences among cultivars tended to be greatest under optimum growth conditions (Terman,
1979). Protein content and protein quality have been also shown to be significant for baking quality
(Johanson and Svensson, 1998). Fredericson et al. (1997, 1998) found that protein content was
positively correlated with wet gluten content, farinogram dough stability and bread loaf volume.
The great majority of wheat products are adversely affected by alfa-amylase. The activity of alfaamylase can be described by the falling number test. High levels of alfa-amylase activity in the
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