Professional Documents
Culture Documents
189–198 (2014)
DOI: 10.1556/CRC.42.2014.2.2
1
Department of Biochemistry and Biotechnology, Slovak University of Agriculture in Nitra,
Tr. A. Hlinku 2, 949 76, Nitra, Slovak Republic
2
Crop Genetics Department, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK
3
Institute of Chemistry, Centre of Excellence for White-green Biotechnology,
Tr. Andreja Hlinku 2, 949 76 Nitra, Slovak Republic
4
Institute of Plant Genetics and Biotechnology, Akademická 2, 950 07 Nitra, Slovak Republic
In this study we evaluate the genetic diversity of a selection of wheat accessions character-
istically grown and adapted to mid-European environments, using various molecular marker
systems. Thirty-three simple sequence repeat (SSR) markers were used alongside genic mark-
ers for known dwarfing genes, flowering time genes, and grain hardness genes, namely
Rht-B1, Rht-D1, Ppd-D1, Vrn-A1, Vrn-B1, Vrn-D1 and Pinb-D1. In addition, variation was
scored for the high-molecular-weight glutenin storage proteins, responsible for dough techno-
logical quality. A dendrogram was constructed using the UPGMA algorithm, based on the mo-
lecular data and the country of origin, giving an overview of their genetic similarity and rela-
tionships. The potential for the use of some agronomic traits in breeding, by providing a basis
for multi-trait genetic selection in wheat breeding programs is discussed. Estimating the
breeding values of crops using multiple genetic markers might help in breeding for varieties
with good technological quality for growing under desired climatic conditions.
Keywords: durum wheat, functional markers, glutenin subunits, spelt wheat, SSR
Introduction
Molecular markers that accurately discriminate alleles of a targeted gene are ideal for
marker-assisted selection in wheat breeding (Liu et al. 2012). DNA-based markers for
storage proteins, particularly for the high-molecular-weight glutenin sub-units (Luo et al.
2001) have been used successfully in the characterization of large sets of wheat cultivars
for dough quality (Jin et al. 2011). Milling and end-use quality affected by kernel hardness
can be determined by evaluating variation at the Pina-D1 and Pinb-D1 genes (reviewed
by Bhave and Morris 2008), while the functional marker for Pinb-D1b, associated with
superior milling and processing qualities, is most useful in wheat breeding for quality
traits (Yang et al. 2009).
For important agronomic traits like plant height, photoperiod response, yield and dis-
ease resistance, 27 functional markers have been developed (Liu et al. 2012). In wheat
there are three markers commonly used for screening of short-straw varieties, namely for
the alleles Rht-B1b (Rht1) and Rht-D1b (Rht2), that reduce plant height by 15% and in-
crease yield by 24% (Gale and Youseffian 1985), as well as the Rht8 gene with a smaller
effect on plant height (Worland et al. 2001). For the latter gene it has been observed that
the 192 bp allele of microsatellite locus Xgmw261 can be diagnostic in many germplasm
collections (Korzun et al. 1998). The varieties carrying this allele show a height reduction
of 7–8 cm, while varieties carrying the 165 bp allele show a reduction of only 3 cm.
Flowering time affects adaptation of wheat to environmental conditions, and is greatly
influenced by the homoeoloci controlling photoperiod response: Ppd-A1, Ppd-B1 and
Ppd-D1 located on the short arms of chromosomes 2A, 2B and 2D, respectively (Mohler
et al. 2004). Corresponding functional markers are being used successfully to screen for
adaptation to latitude. Further, information on the distribution of vernalization genes and
their association with growth habit is crucial to understand the adaptability of wheat
cultivars to sowing time, either autumn or spring sowing. The three loci, Vrn-A1, Vrn-B1,
Vrn-D1 and Vrn-B3 have previously been used to select for the dominant spring growth
habit (Zhang et al. 2008) conferred by specific alleles. Gene perfect markers for disease
resistance loci have also been identified and used (e.g. Wang et al. 2008).
In this study we used a combined approach of storage protein sub-unit detection, simple
sequence repeats (SSRs), and a set of ‘perfect’ genic markers for characterization as well
as assessing the allelic diversity in germplasm accessions of wheat varieties/lines grown in
Middle European environments. Emphasis was given to Slovak wheat cultivars that have
not so far been described in this respect. The molecular markers used were identified as us-
able for identification and differentiation of the wheat accessions. Several well-described
wheat varieties from other countries were also included in analyses to serve as reference
genotypes. All the data obtained were analyzed to provide a comprehensive view on their
genetic relationship, breeding potential for selected agronomic traits as well as character-
istics that might favour wheat breeding with respect to climate in the geographic area of
Middle Europe.
Statistical analysis
The program DARwin5 (Perrier and Jacquemoud-Collet 2006) was used to analyze the di-
versity and phylogenetics of the wheat varieties from the allelic variation of the micro-
satellites. Dendrograms were generated based on hierarchical cluster analysis using the
UPGMA algorithm.
Figure 1. Dendrogram of the 65 wheat genotypes constructed on the basis of variability from 38 DNA
markers, perfect genic markers, and from the glutenin quality score
On the right, the corresponding information is given: first number = GQ score (Payne et al. 1987),
E/L = early/late flowering, second number = fragment length of the allele of locus Xgwm261 (if scored),
RhtB1b – if detected, the last is country of origin (SK – Slovakia)
Results
Analyses of proteins
The protein data on the HMW-GS profiles showed that the subunits 5+10 (an allele at
Glu-1D) with a known positive effect on technological quality were present in 30 Triticum
aestivum L. accessions. The maximum GQ score of 10 (with glutenin subunits 2*; 7+8;
5+10) was calculated for only 3 genotypes (Table 1, Fig. 1). The lowest score was detected
for the wheat genotype Slovenská 2 (subunits 0; 6+8; 2+12). Most of the spelt wheats con-
tained the subunit pair 2+12 and had the GQ score of 6 (Table 2). Among the durum
wheats, subunit 0 controlled by the locus Glu-A1 was detected in each cultivar/line, while
the subunits 7+8 and 7+9 from the locus Glu-B1 contributed to the GQ score of 4 for most
of the durum wheat genotypes studied (Table 2).
Perfect marker analysis
To more accurately characterize the adaptation of the wheat genotypes from an agronomic
perspective, perfect markers were used. Mutant alleles responsible for a reduction in plant
height were analyzed, and the presence of the Rht-B1b allele was found in nine wheat ge-
notypes, and all of the durum wheat genotypes. All varieties were wild-type (tall) for
Rht-D1. In contrast, each spelt wheat genotype carried the wild type allele, Rht-B1a. Anal-
yses of alleles associated with Rht8, an alternate dwarfing gene, detected that 10 out of 21
different allele sizes known at Xgwm261, including the predominant fragments of 192 bp,
165 bp and 174 bp (Chebotar et al. 2001) (Table 1) were present in the collection. In
T. aestivum L. lines, the 192 bp fragment occurred with the greatest frequency, in spelt
wheats the alleles of 165 bp (4 genotypes) and 175 bp (3 genotypes) were the only ones
present (Table 2).
The wild type allele of Pinb-D1a was detected in each accession (including the spelt
wheats) reflecting soft kernel texture in all lines (Tables 1 and 2). The homoeoloci
Ppd-A1, Ppd-B1 and Ppd-D1, responsible for photoperiod response, had recessive,
photoperiod insensitive, alleles associated with later flowering in 22 of the screened
hexaploid wheat genotypes, and in all of the spelt wheats. The distribution of vernalization
genes indicated that all of the bread wheat and spelt wheat varieties analyzed were winter
wheats (vrn-a1, vrn-b1, vrn-d1). Exceptions were the two genotypes Krajová Brestovec
and Krajová Chmel’nica, which are spring types with the genotype vrn-a1, Vrn-b1,
vrn-d1 (Tables 1 and 2).
Analysis of SSRs
In the 40 genotypes of common wheat (Triticum aestivum L.) there were 289 (x = 8.76) al-
leles detected at 33 loci (Table 3), with the range from 3 to 21 alleles per locus. In the spelt
wheat accessions there were 149 (x = 4.52) alleles detected (Table 3) ranging from 1 to 9
alleles per locus. In the 17 genotypes of durum wheat, a total 85 alleles were detected at 16
SSR loci with an average number of 5.31 (Table 3).
Table 2. Alelles of the gene-perfect markers identified in the spelt and durum wheat cultivars
Table 3. Overview on the DNA markers used in the analysis of the wheat accessions
Cluster analysis
Origin and pedigree (if available), data on the microsatellite and the gene (perfect) mark-
ers, and the storage protein data, were used to construct a dendrogram of the relationships
between the 65 wheat genotypes (Fig. 1). Two major clusters were generated. The first (I)
comprised two subgroups of the common (Ia) and spelt wheat (Ib) accessions. Surpris-
ingly, a spelt line (3/96) clustered with the class Ia accessions (Fig. 1). The cluster of com-
mon wheats was further divided into 7 sub-clusters (Ia1–Ia7) of various sizes, and 5
non-clustered genotypes. In the second cluster, II, the durum wheat genotypes clustered
together while forming two sub-clusters (Fig. 1).
Discussion
Various molecular markers were used to evaluate a set of wheat, spelt wheat and durum
wheat cultivars that have been bred as being adapted to the environments of Central
Europe. Our results showed that all of the hexaploid genotypes tested (including the spelt
genotypes) were soft textured wheats that are preferable for milling and bread baking
(Martin et al. 2001). At the same time, 75% of genotypes carried the subunits 5+10 for
high baking quality, while 45% of the common wheat, and all the durum wheat genotypes,
carried sub-units for poor baking quality.
Most of the wheat genotypes (including the spelt wheats) have alleles for winter growth
habit. Of the hexaploid wheats, 55% have a genotype for early flowering. Photoperiod in-
sensitive wheat varieties are more abundant in regions where grain maturing is necessary
before the onset of high temperatures (e.g. in eastern and southern Europe). With respect
to the alleles at plant height loci, the Xgwm261 192 bp allele, with a major effect on reduc-
ing plant height, was found in 40% of the genotypes. Shorter-strawed genotypes are being
introduced into breeding programmes to avoid lodging, since this has become a serious
problem as higher-yielding varieties are bred that respond to increased levels of mineral
fertilizer (Knopf et al. 2008). In addition, carrying one of the two dwarf alleles has been
shown to provide a yield advantage to wheat (Gale and Youssefian 1985). Unfortunately,
they also appear to correlate with susceptibility to Fusarium head blight (Mesterhazy
1995). These data provide knowledge on the potential (dis)advantage of individual (Slo-
vak) wheat accessions for breeding/growth under specific climate condition either within
Europe or in non-European areas.
The dendrogram generated from these data, in general, confirmed the basic separation
into common, spelt and durum wheats. Furthermore, it provides a comprehensive view re-
garding the technological quality and growth habits. For example, among the common
wheats, the subclasses Ia1 and Ia5 were identified as having a potential for good techno-
logical quality, with a soft textured kernel, shorter-straw, earlier flowering and a winter
habit. The other class of early flowering common wheats, Ia3, can be described as having
relatively good technological quality, soft textured kernels, but are tall-strawed and com-
prising both winter and spring type genotypes from Slovakia. The late flowering,
tall-strawed genotypes from Slovakia (except for the one from Germany) with a potential
of good technological quality, clustered together within the subgroup Ia2, or remained
un-clustered within the branch Ia.
The group of spelt wheats from Poland, Germany, Switzerland and USA can be de-
scribed as having potential for medium baking quality, with a soft textured kernel, and tall
straw. The group of durum wheats comprises 2 Slovak varieties (Soldur and Vendur) and
15 newly bred Slovak lines (with a missing pedigree) that have low baking quality, and
carry the dwarf allele Rht-B1b.
The dendrogram thus clusters the middle-European wheat varieties based not only a
microsatellite marker set, but also based on their genetic potential for agronomic perfor-
mance. Although a selection carried out solely on the basis of genetic markers (without
confirming the estimated effects by phenotypic evaluation) is considered to be risky, their
use can enhance genetic improvement programmes (Dekkers and Hospital 2002). Build-
ing a more complex genetic architecture with breeding potential can be used in breeding
programmes to select parental lines with several desired properties. For example, under
the current trends for climate change, the focus on breeding short strawed plants is even
more justified, while additional knowledge on technological quality, flowering time, yield
potential, susceptibility to pathogens, and genetic background, can be helpful and benefi-
cial. The dendrogram describing the relationship between the studied wheat acces-
sions will be helpful in future breeding programs, especially in case of genotypes with an
unknown pedigree, by providing a more comprehensive description of their breeding po-
tential.
Acknowledgements
The work has been supported by Crop Genetics Department of the John Innes Centre (JIC)
and a project Grant Agency from the Slovak Republic, No. 1/0471/09. The authors thank
the Gene Bank of the Plant Production Research Centre Piešt’any of the Slovak Republic
for supplying the wheat cultivars. This contribution is the result of the project implementa-
tion: Centre of excellence for white-green biotechnology, ITMS 26220120054, supported
by the research Development Operational Programme funded by the ERDF (25%), KEGA
project No. 034SPU-4/2012 (25%) and VEGA project No. 1/0513/13 (50%).
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