ࡱ> PRMNO'`>bjbjLULU B.?.?7dVdVdVdVhhhiإإإ8ti|(d(($h ` -hY^YY dVdV6Y dVl hYͽc,hkX `wQcإ*LL0|lբrlTk"flh$5  Gj|YYYYiii(ԑiiiԑiiidVdVdVdVdVdV Assessment of barley breeding germplasm by SSR markers associated with certain QTL s regarding abiotic stress tolerance and quality Ivan ABI I1  Alojzije LALI1  Silvio `IMON2  Ivan PEJI 2 1Agricultural Institute Osijek, Ju~no predgrae 17, HR-31000 Osijek, Croatia 2University of Zagreb, Faculty of agriculture, Svetoimunska cesta 25, 10000 Zagreb, Croatia Abstract 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 Introduction 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 quality. 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 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  SHAPE \* MERGEFORMAT  SHAPE \* MERGEFORMAT  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) 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 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. Conclusions 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 QTLs for traits of interest, from which parental lines can be picked for development of future breeding populations. References Nevo, E. (1992): Origin, evolution, population genetics and resources for breeding of wild barley, Horedum spontaneum, in the Fertile Crescent. In Barley: Genetics, biochemistry, molecular biology and biotechnology. Edited by Peter Shewry CAB International Press, London. pp. 19-43. Troyer, A.F., Openshaw, S.J., and Knittle, K.H. (1998): Measurement of genetic diversity among popular commercial corn hybrids. Crop Sci. 28: 481485. Doyle, J.J. and J.L. Doyle. (1990): Isolation of plant DNA from fresh tissue. Focus 12:13-15. Liu Z-W, Biyashev RM, Saghai Maroof MA (1996): Development of simple sequence repeat DNA markers and their integration into a barley linkage map. Theor Appl Genet 93: 869876 J. Z. Li, T. G. Sjakste, M. S. Roder, M. W. (2003): Ganal Development and genetic mapping of 127 new microsatellite markers in barley. Theor Appl Genet 107: 10211027 V. Ivandic, W. T. B. Thomas, E. Nevo, Z. Zhang and B. P. Forster (2003): Associations of simple sequence repeats with quantitative trait variation including biotic and abiotic stress tolerance in Hordeum spontaneum. Plant Breeding 122, 300-304 G. Chen, T. Krugman, T. Fahima, K. Chen, Y. Hu, M. Roder, E. Nevo, A. Korol (2010): Chromosomal regions controlling seedling drought resistance in Israeli wild barley, Hordeum spontaneum C. Koch. Genet Resour Crop Evol 57:8599 Panozzo, J.F., Lim, O., Eckermann, P., Moody, D., Barr, A., Karakousis, A., Chalmers, K. and Cullis, B.R. (2003): Australian Journal of Agricultural Research 54 Collins, H.M., Panozzo, J., Logue, S.J., Jefferies, S.P. and Barr, A.R. (2003): Australian Journal of Agricultural Research 54 Marquez-Cedillo, L.A., Hayes, P.M., Kleinhofs, A., Legge, W.G., Rossnagel, B.G., Sato, K.,Ullrich, S.E., Wesenberg, D.M. and the NABGMP (2000): Theoretical and Applied Genetics 101:173-184. H. Raman, S. Venkatanagappa, A. Rehman, B. O'Bree, and B. Read (2003): Graphical genotyping of barley using molecular markers linked with malting quality, disease resistance and Al tolerance. Barley Technical/Cereal Chemistry 7 10 September 2003 M. von Korff, H. Wang, J. Leon, K. Pillen (2008): AB-QTL analysis in spring barley: III. Identification of exotic alleles for the improvement of malting quality in spring barley (H. vulgare ssp. spontaneum). 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