Budapest, Hungary, 2011 AGRISAFE
ASSESSMENT OF BARLEY BREEDING GERMPLASM BY SSR
MARKERS ASSOCIATED WITH CERTAIN QTL’S REGARDING
ABIOTIC STRESS TOLERANCE AND QUALITY
Ivan ABIČIĆ1 – Alojzije LALIĆ1 – Silvio ŠIMON2 – Ivan PEJIĆ
Agricultural Institute Osijek, Južno predgrađe 17, HR-31000 Osijek, Croatia
University of Zagreb, Faculty of agriculture, Svetošimunska cesta 25, 10000 Zagreb, Croatia
Breeders around the globe consider breeding for abiotic stress tolerance as something that preoccupies their
attention more than ever before. Also, the malting industry has some specific demands towards barley quality
improvement. A total of 126 barley cultivars, 78 winter and 48 spring type from different background were
screened by SSR markers in order to assess genetic variability among them. Croatian barley cultivars are
represented with 66 winter and 27 spring type (total 93). Preliminary data presented in this paper is derived for
markers Bmac181 (chr. 4H), Bmag211 (chr. 1H), EBmac755 (chr. 7H) and HVM54 (chr. 2H). All of them are
linked to certain QTLs, either for abiotic stress tolerance or quality, published in a number of studies done on
different populations. Specified QTLs are for drought tolerance, (pinpointed by markers Bmac181 and
EBmac755), viscosity (Bmag211) and hull content (HVM54). The number of alleles per SSR locus averaged
5.5 among elite breeding lines. A number of cultivars for which specific allele amplification has been
successful, were selected and their distribution displayed. This type of assay will be useful for determination of
the informativeness level of SSR markers used when generating a meaningful classification of elite germplasm.
Also, some pointers considering carriers of QTLs of interest can be highlighted for future selection of parental
lines in hybridization process.
Key words: barley, abiotic stress, quality, SSR, QTL
It is well known that barley (Hordeum vulgare L.) is one of the most important crop
species in the world, and also one of the most representative cereals grown nowadays.
Barley also has different roles (dual-role) dependant on what will be its purpose, whether
we use it for animal and/or human consumption or as a raw material in brewing and
pharmaceutical industry. It is thought that it originated from H. vulgare L. ssp.
spontaneum which resides even today in the area of the Fertile Crescent (Nevo, 1992).
Because of its origin and vast diversity, barley is considered to be relatively resistant to
water deficit in general, even though it has the least developed root system in
comparison to other small cereals. More knowledge regarding the genetic structure of
breeding materials could help to maintain genetic diversity, which would sustain long-
term selection responses and reduce vulnerability of breeding pograms in that matter
(Troyer et al. 1998; Liu et al. 2000). Results of this work will provide some better width
and understanding of germplasm compiled mostly of Croatian cultivars and comparing
and detecting the ones which are potential carriers of QTLs for drought tolerance and
Material and methods
A total of 126 barley (Hordeum vulgare L.) cultivars (78 winter and 48 spring type) from
different background were screened by SSR markers in order to assess genetic variability
among them. Group of Croatian barley cultivars consists of 66 winter and 27 spring
types (total 93). DNA was isolated by CTAB method (Doyle and Doyle, 1990) from leaf
tissue grown from three seeds (for every sample/cultivar) sown into plastic container
filled with substrate. Before DNA isolation, the tissue needed to be lyophilized and
grounded with steel beads in order to get powdery texture of the sample. After isolation
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the concentration of genome DNA was determined by spectrophotometer. Amplification
process (PCR reaction in Veriti Thermal Cycler, Applied Biosystems) of microsatellite
markers was carried according to Liu et al. (1996) and Li et al. (2003). PCR products
were then being analyzed with genetic analyzer (ABI 3130 - size standard used:
GeneScan 500LIZ, Applied Biosystems) and data was retrieved afterwards via
GeneMapper 4.0 (Applied Biosystems) software.
In order to make results valid in this paper and comparable to some point, another
method of data collection and analysis has been used in a way of choosing a subset of
winter barley cultivars (14) where we collated their yield stability parameter with
seasonal vegetation periods of accentuated drought stress. In this way a simple pattern
mechanism is devised which helps determine varieties which have proven themselves as
drought tolerant or susceptible through long term research in situ, combining them with
data collected by SSR marker analysis. Subset of cultivars consists from number of
varieties which originate from Agricultural Institute Osijek – Croatia (Sladoran, Rex,
Zlatko, Titan, Prometej, Barun, Spartak, Bingo, Lord and Princ), BC Institute from
Zagreb – Croatia (Favorit), Germany (Tiffany and Vanessa) and France (Plaisant).
Software used for data analysis: IRRISTAT, IRRI of Manila, for AMMI1 and AMMI2
bi-plot analysis. These trials were set on the locality Osijek and seasonal periods
included in data comparison last from the season of 2002/2003 till 2009/2010. Also, to
determine which samples have high grain quality and suspected QTLs for the trait, we
included spring barley cultivars well known for their high potential in this matter and
grouped samples which posses the same set of alleles. Briefly said, the focus on
determining drought stress is on barley winter types and quality on spring types. The
reason for this approach is in the fact that winter barley has prolonged vegetation and
therefore it is much likely to be longer under negative influence of water deficiency
which, at the end, greatly impacts yield. On the other hand when we speak about quality,
the spring barley cultivars have the edge over winter types, according to brewing
industry data and demands.
Results and discussion
Barun Spartak 2006/2007
0.44 IPCA1 Plaisant 2008/2009
2003/2004 Bingo Spartak Vanessa
2002/2003 -0.12 Princ 2007/2008
IPCA2 Sladoran Rex Bingo Barun
Titan Sladoran Zlatko
Favorit -0.66 2004/2005
2009/2010 Lo 2003/2004 Prometej Titan
2004/2005 rd 2009/2010
2008/2009 5.9 6.82 7.74 8.66 9.58 10.5
Plaisant MEANS, t/ha
-1.2 -0.66 -0.12 0.42 0.96
Figure 1. AMMI2 and AMMI1 biplot models of adaptability and stability of cultivars (subset of 14 winter
types) for grain yield; variety – year (blue – years with over the average rainfall and below average
temperature; red – years with below average rainfall and over the average temperature; black – years with
average rainfall and temperature)
Budapest, Hungary, 2011 AGRISAFE
Results (Figure 1.) clearly display when drought took place by seasonal overview.
Seasons 2002/2003, 2006/2007 and 2008/2009 were overall dry seasons with water
deficit and sometimes extremely high temperatures throughout the whole vegetation
period, or through some certain key vegetation growth stages (tillering, flowering, etc.).
The most extreme was the season of 2002/2003, and it was used as a reference point for
determining the adaptable and stable cultivars. One can see (Figure 1.) that foreign
cultivars Tiffany and especially Vanessa showed very good adaptability towards
stressful conditions under the season of 2002/2003. This is in accordance to their SSR
profiles shown by Bmac181 and EBmac755 which are the same (Vanessa and Tiffany:
Bmac181 – 180 bp allele; EBmac755 – 136 bp allele). This type of SSR signature can
also be found on Croatian cultivar Lord where by pedigree one of the parents is variety
Plaisant with almost the same SSR profile (Bmac181 – 180 bp allele; EBmac755 – 138
bp allele). Both of these varieties show similar response to yield stability (AMMI1 biplot
model – low IPCA1 values!) and little less to adaptability (Lord is more adaptable)
which is logically sound considering Plaisant being foreign cultivar. Interestingly,
Croatian cultivar Rex is, according to results, one of the most adaptable cultivars in this
research even though its SSR profiling is somewhat different then the ones mentioned
above (Bmac181 – 178 bp allele; EBmac755 – 136 bp allele). But pedigree inquiry
shows that Rex also has some foreign germplasm within, to be precise a genotype also
included in SSR profiling, by the name Alpha (Bmac181 – 180 bp allele; EBmac755 –
136 bp allele) of French origin (Rex: DORAT//ALPHA/MURSA/3/OSK.5-59-6-7); in
fact, most of Croatian cultivars show differences in allele length on locus Bmac181
(Croatian cultivars – 178 and 176 bp allele length; foreign cultivars (mostly German and
French) – 180 bp allele length). We may conclude that cultivars mentioned above do
possess excellent adaptability towards weather imposed drought stress, especially
varieties Rex and Vanessa. Also, this conclusion is concurrent with SSR data acquired
where common signature for the two loci can be found (Bmac181 – 180 bp and/or
EBmac755 – 136 bp). Ivandic et al. (2003.) stated detection of highly significant
associations at the loci Bmac181 on chromosome 4H for water stress tolerance in barley,
explaining that this locus expresses additional effects for grain yield under well watered
conditions and in an adaptive response to water stress. Chen et al. (2010) used, among
others, molecular marker EBmac755 placed on chromosome 7H for barley QTL
mapping of traits controlling drought resistance, where it was found that this specific
marker is in close proximity to wilting time trait closely related to osmolarity. Chen et al.
(2010) further pointed out the importance of mechanism where high osmolarity means a
low osmotic potential that results in postponing of plant wilting. I the midst of all this we
can conclude that it is possible to determine carriers for drought tolerance QTLs among
cultivars which partake about 21.8% (17) of the whole number of 78 winter barley
cultivars screened in this paper.
Spring barley cultivars were screened with SSR markers (Bmag211 – chr. 1H and
HVM54 – chr. 2H) to determine the potential carriers of desirable traits among them.
Regions on chromosome 1H are well known for their associations with malt extract and
other malting attributes (Marquez-Cedillo et al. 2000; Collins et al. 2003; Coventry et al.
2003; Panozzo et al. 2003). First of all, distinct foreign high quality (malting) barley
genotypes were determined in order to establish a possible pattern within SSR data.
“Pattern group” consists from four high quality cultivars: Triumph (Bmag211 – 184 bp
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allele; HVM54 – 161 bp allele), Scarlett (Bmag211 – 182 bp allele; HVM54 – 161 bp
allele), Prestige (Bmag211 – 182 bp allele; HVM54 – 145 bp allele) and Barke
(Bmag211 – 186 bp allele; HVM54 – 157 bp allele). Genomic region of Bmag211 has
been reported to be linked with viscosity trait (Raman et al., 2003) and we can also try to
determine which ones of 48 spring barley cultivars possess the same sized allele. An
interesting parallel occurred because only about 19% (9) from the whole number of
cultivars have 182 and 186 bp allele size, and seven of those have very high values of
viscosity documented. Screenings for hull content also showed congruent results, almost
65% (34) of cultivars examined showed 145 and 161 bp allele size. Von Korff et al.
(2008) state that as viscosity and friability are mainly affected by the breakdown of b-
glucan and other cell wall polysaccharides, genes affecting b-glucan or b-glucanase
activity may underlie the QTLs detected for viscosity and friability. It is necessary to
point out the need for further investigation of these samples (48) in order to collect and
analyze data for viscosity and hull content traits to confirm displayed presumptions.
The results hereby shown are very promising and can be used to give certain pointers for
SSR markers used. Nevertheless, further analysis of data is needed, and also data from
field trials and laboratory tests must be obtained in order to achieve clear and broad
picture of germplasm involved. In addition, it will be possible to determine and display
groups of elite barley lines who presumably carry QTL’s for traits of interest, from
which parental lines can be picked for development of future breeding populations.
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Budapest, Hungary, 2011 AGRISAFE