Agricultura – Ştiinţă şi practică
no. 1- 2(89-90)/2014
Agriculture - Science and Practice
MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF
ROMANIAN WINTER WHEAT (TRITICUM AESTIVUM L.)
LINES IN CONTEXT OF PRE-HARVEST SPROUTING
Vas Eszter 1,*, C. Botez1, N. Lupu2, I. Haş 1,2, Marion S. Röder3
1-University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Mănăştur street 3-5,
400372, Cluj Napoca, Romania;
2-Agricultural Research and Development Station Turda; Agriculturii Street no.27, 401100,
Cluj, Romania
3-Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3,
D-06466, Gatersleben, Germany; *Corresponding author: [email protected]
Abstract. The main aims of our study are the genotypic and phenotypic analysis of four
Romanian wheat (Triticum aestivum) lines. The results will be used in further breeding studies for
increasing the pre-harvest sprouting (PHS) resistance in wheat. PHS occurs when the germination of
the seed on the spike starts in the field before harvest because of high humidity and weather
conditions. This damage causes economic and yield losses all over the world. The four investigated
parental lines, with different sensibility to PHS, were Turda 95 (sensible), Turda 18-94 (resistant),
Lovrin 32 (sensible), Fundulea 29 (resistant). The molecular study was performed with 640 SSR
markers. The results showed that between Turda 95 and Turda 18-94 line 90 primers were
polymorphic (14%), between Turda 95 and Fundulea 29 line 75 primers were polymorphic
(11.17%) and between Turda 18-94 and Fundulea 29 line 83 primers were polymorphic (12.97%).
The result from the phenotypic characterization of heading date and plant height showed that the F2
population of the cross Turda 95 and Turda 18-94 has normal distribution, and the PHS scoring
shows bimodal distribution and these data can be used for next analysis.
Keywords: microsatellite markers, polymorphism, pre-harvest sprouting, grain quality,
Triticum aestivum L.
INTRODUCTION
Wheat (Triticum aestivum) is one of the most important crop plants, the bread
made from wheat flour it represents a major food in more than half of the world
population. Wheat is used for leavened bread, flat and steamed breads, biscuits, cakes,
pasta, noodles, couscous and beer. The raised bread loaf is possible, because the wheat
kernel contains gluten, an elastic form of protein that traps minute bubbles of carbon
dioxide when fermentation occurs in leavened dough, causing the dough to rise (Hanson et
al., 1982). Unfortunately wheat it is sensitive to abiotic stresses and pathogens. An
important damage is the germination of the wheat seeds on the spike in the field before
harvest at high humidity conditions, which is called pre-harvest sprouting (PHS). Preharvest sprouting is a damage caused by environment and gene interaction.
In PHS damaged wheat the alpha-amylase activity is high and this causes the
degradation of the starches and the result is a low quality of the bakery products and bread.
Therefore PHS causes economical and yield losses in the quality of kernels, thus limiting
their end use. Damaged kernels are a popular source of animal feed, particularly in years
when harvests are adversely affected by rain and significant quantities of the grain are
made unsuitable for food use. Such low-grade grain is often used by industry to make
adhesives, paper additives, and several other products and even in the production of
alcohol.
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Agricultura – Ştiinţă şi practică
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Agriculture - Science and Practice
Genetically speaking, PHS heredity is treated as quantitative trait. Seed dormancy
is conditioned by the pleiotropic effects of gene R, which determines the red pericarp of
the seeds but also by genes that control the physiological process of germination and have
a marking effect on the embryo dormancy. Therefore, PHS is controlled both by embryo
physiology and also by seed coat regulation, each caused by different genetic systems
(Gross et al., 2002).
Breeding for resistance to pathogens and damages including pre-harvest resistance
is an important and attractive research topic worldwide to increase the quality of wheat
(Chen et al., 2007; Groos et al., 2002; Lohwasser et al., 2005; Liu et al., 2008).
A phenotypic characterization of some crosses and backcrosses of Romanian
wheat was performed by Lupu et al. (2010, 2011).
The molecular and phenotypic research is needed in the improvement of a good
quality winter wheat in Romania and to reduce the time of breeding.
The simplicity and easiness of the SSR (Simple Sequence Repeats or
microsatelittes) markers made this method useful in plant breeding. SSR markers are often
utilized in molecular studies of the wheat (Zeb et al., 2009) especially that there are many
SSR markers special designed for wheat (Röder et al., 1998; Pestsova et al., 2000; Somers
et al., 2004; Ganal and Röder, 2007)
The first aim of this study was to fulfill the lack of knowledge of the polymorphic
percentage in four Romanian wheat lines.
The second aim was to make the phenotypic characterization of the two parental
lines, with highest polymorphic percentage, and the F2 population of these if they have
normal distribution.
MATERIAL AND METHODS
The plant material, four parental lines with different sensibility to PHS (Turda 95sensible, Turda 18-94-resitant, Lovrin 32-sensible, Fundulea 29-resitant) and three crosses
between the parental lines (Turda 95 x Turda 18-94, Turda 95 x Fundulea 29, Turda 18-94
x Lovrin 32) was obtained from ARDS Turda Romania and the research was made in
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany.
Genotypic characterization. The DNA isolation from parental lines and later
from the F2 population was performed according to the protocol of Doyle and Doyle
(1990) from two week old leafs.
The characterization for polymorphism of the parental lines was conducted with
640 SSR (Simple Sequence Repeats or Microsatellite) markers (gwm, wmc and barc)
specific for wheat (Röder et al., 1998; Ganal and Röder, 2007) and a Viviparous-1 allelic
variant primer (Vp1-B) (Yang et al., 2007b). For the SSRs the left primer was fluorescence
labeled and the label was recognized by the laser from the Automated Laser Fluorescent
(ALF) fragment analyzer. Fragment analysis was carried out as described in Röder et al. in
1998. The Vp1-B marker was tested on agarose gels as described by Xia et al. in 2008.
Marker which showed polymorphism between the two parental lines was used for studying
the F2 population with the highest percent of polymorphism, which was the cross between
Turda 95 and Turda 18-94. This population was used for further phenotypic
characterization (Vas et. al, 2013)
Phenotypic characterization. The cross between Turda 95 and Turda 18-94 was
scored for the heading date and plant height.PHS was checked from freshly harvested
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Agricultura – Ştiinţă şi practică
no. 1- 2(89-90)/2014
Agriculture - Science and Practice
mature ears stage (Rehman et al., 2012) kept in humid conditions in wet sand for 14 days.
PHS was scored from 1 to 7 (where 1 was no sprouting and 7 indicated 100% sprouting)
(Figure 1).
Fig. 1. Steps from direct method
RESULTS AND DISCUSSION
Results from genotypic analysis. The molecular study was performed with 640
SSR markers. The polymorphic analysis is important, because we can see in the population
the differences between two parental lines. The results from the analysis showed that
between Turda 95 and Turda 18-94 parental lines 90 primers were polymorphic (14%),
between Turda 95 and Fundulea 29 parental lines 75 primers were polymorphic (11.17%)
and between Turda 18-94 and Lovrin 32 parental lines 83 primers were polymorphic
(12.97%).
The results of the analysis for polymorphism from parental lines suggest that the
highest percentage of polymorphism is between Turda 95 and Turda 18-94 (Table 1, Vas et.
al)
Table 1
Turda 95 x Turda 18-94
14 %
Turda 95 x Fundulea 29
11.17 %
Turda 18-94 x Lovrin 32
12.97%
Results from phenotypic characterization. Result from the phenotypic
characterization with statistical program SigmaPlot (Figure 2) showed that the distribution
is normal (Gaussian distribution) for heading date and plant height.
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Agricultura – Ştiinţă şi practică
no. 1- 2(89-90)/2014
Agriculture - Science and Practice
Fig. 2 SigmaPlot result from Heading date and Plant heigh
The data, from scoring the PHS with direct method, illustrate a not normal,
bimodal distribution (Figure 3.), which can indicate an action of a major gene.
Fig. 3. SigmaPlot results from scoring pre-harvest sprouting
CONCLUSION
The investigation of Turda 95, Turda 18-94, Fundulea 29 and Lovrin 32
autochtonous wheat lines is a big step for the winter wheat research in Romania.
The polymorphic data describe the genetic diversity between the four parental
lines and this can be further used for QTL mapping studies to establish markers for markerassisted selection
The cross between the two parental lines Turda 95 and Turda 18-94 is good for
further morphological and molecular analysis and a future QTL-analysis.
ACKNOWLEDGMENT
We would like to thank Deutsches Bundestiftung Umwelt (DBU) for funding
Eszter Vas stay in Germany at IPK Gatersleben.
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Agriculture - Science and Practice
REFERENCES
1. Chen C.X., C.A.I. Shi-Bin, B.A.I. Gui-Hua. (2007). A major QTL controlling seed
dormancy and pre-harvest sprouting resistance on chromosome 4A in a Chinese wheat landrace.
Mol. Breeding 21:351–358.
2. Doyle J.J., J.L. Doyle. (1990). Isolation of plant DNA from fresh tissue. Focus 12, 1315.
3. Ganal M.W., M.S. Röder. (2007). Microsatellite and SNP markers in wheat breeding,
Vol. 2. In: Varshney RK, Tuberosa R, editors. Genomic assisted crop improvement: genomics
applications in crops. The Netherlands: Springer; p. 1-24.
4. Groos C, G. Gay, M.R. Perretant, L. Gervais, M. Bernard, F. Dedryver, G. Charmet.
(2002). Study of the relationship between pre-harvest sprouting and grain color by quantitative trait
loci analysis in a white x red grain bread-wheat cross. Theor. Appl. Genet., Jan; 104(1):39-47.
5. Hanson H., N. E. Borlaug, R.G. Anderson. (1982). Wheat in the third world. Boulder,
CO, USA, Westview Press.
6. Liu S., S. Cai, R. Graybosch, C. Chen, G. Bai. (2008). Quantitative trait loci for
resistance to pre-harvest sprouting in US hard white winter wheat Rio Blanco. Theor. Appl. Genet.
117:691–699.
7. Lohwasser U., M.S. Röder, A. Börner. (2005). QTL mapping of the domestication traits
pre-harvest sprouting and dormancy in wheat (Triticum aestivum L.). Euphytica 143: 247–249.
8. Lupu N., R. Kadar, V. Moldovan, I. Haş. (2010). Rezultate privind fenomenul de
încolţire în spic la grâul de toamnă, la S.C.D.A. Turda. An. I.N.C.D.A. Fundulea, LXXVII, 1: 29-36.
9. Lupu N., R. Kadar, V. Moldovan, I. Haş (2011) Studiul hibrizilor F1 şi al formelor
parentale în privinţa rezistenţei la încolţirea în spic la grâul de toamnă An. I.N.C.D.A. Fundulea,
LXXIX, 2:181-191.
10. Mohan A., P. Kulwal, R. Singh, V. Kumar, R. R. Mir, J. Kumar, M. Prasad, H. S.
Balyan, P. K. Gupta. (2009). Genome-wide QTL analysis for pre-harvest sprouting tolerance in
bread wheat. Euphytica 168:319–329
11. Pestsova E., M.W. Ganal, M.S. Röder. (2000). Isolation and mapping of microsatellite
markers specific for the D genome of bread wheat. Genome 43:689–697
12. Rehman Arif M. A., K. Neumann, M. Nagel, B. Kobiljski, U. Lohwasser, A. Börner.
(2012). An association mapping analysis of dormancy and pre-harvest sprouting in wheat,
Euphytica.
13. Röder M.S., V. Korzun, K. Wendehake, J. Plaschke, M. H. Tixier, P. Leroy, M.W.
Ganal. (1998). A microsatellite map of wheat. Genetics 149:2007–2023.
14. Somers D.J., Isaac P., Edwards K. (2004). A high-density microsatellite consensus map
for bread wheat (Triticum aestivum L.). Theor Appl Genet 109:1105–1114.
15. Vas E., C. Botez, I. Haş, M. S. Röder. (2013). Identification of Molecular Markers for
Resistance to Pre-Harvest Sprouting in Triticum aestivum. Bulletin UASMV 70(1), 283-286.
16. Xia L.Q., M.W. Ganal, P.R. Shewry, Z.H. He, Y. Yang, M.S. Röder. (2008). Exploiting
the diversity of Viviparous-1 gene associated with pre-harvest sprouting tolerance in European
wheat varieties. Euphytica, 159, 411- 417.
17. Yang Y., X.L. Zhao, L.Q. Xia, X.M. Chen, Z.H. He, M.S. Röder. (2007b). Development
and validation of a Vp-1 STS marker for pre-harvest sprouting in Chinese wheats. Theor Appl Genet
115:971–980
18. Zeb B., A. K. Imtiaz, A. Shahid, B. Sardar, M. Saqib, A. S. Zahoor. (2009). Study on
genetic diversity in Pakistani wheat varieties using simple sequence repeat (SSR), markers. African
Journal of Biotechnology Vol. 8 (17), pp. 4016-4019
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