ࡱ> ]_^q`$+bjbjqPqP$4b::"%84,%"::::R ^`%b%b%b%b%b%b%$&hA)%!::%::%"""L::`%"`%""":  PԽ "<%$%0%"*.!x*""&*$#f&"(fff%%"Xfff%$  QTL MAPPING OF ALPHA ACID CONTENT IN HOP Cerenak Andreja1, Satovic Zlatko2, Jakae Jernej3, Luthar Zlata3, Carovic-Stanko Klaudija2 and Javornik Branka1,3 1Slovenian Institute of Hop Research and Brewing, Cesta }alskega tabora 2, SI-3310 }alec, Slovenia 2Faculty of Agriculture, University of Zagreb, Svetosimunska 25, 10000 Zagreb, Croatia, 3Centre for Plant Biotechnology and Breeding, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, Ljubljana, Slovenia; E-mail: branka.javornik@bf.uni-lj.si Abstract Effects of quantitative trait loci (QTLs) and the map locations were determined for alpha-acid content in hop (Humulus lupulus L.) using amplified fragment length polymorphism (AFLP) and microsatellite markers (SSRs). Genetic linkage maps were constructed from mapping population consisted of 111 progeny from a double pseudo-testcross. A total of 194 markers (150 AFLPs, 43 SSRs, 1 hypothetical sex marker) were located on the 20 linkage groups (LGs) of the maternal and paternal maps, covering total map lengths of 706 and 616 cM, respectively, due to the presence of 16 common biparental SSR markers the homology of seven LGs between parental maps could be inferred. The progeny segregated quantitatively for alpha-acid content determined in the years from 2002 - 2006. Thirteen putative QTLs were identified on both maps. Possible homologies between QTLs for alpha-acid content detected in different years as well as in both maps could be established in at least three chromosomal regions. Previously reported genetic map was successfully saturated with additional SSR markers providing a practical tool for the development of marker-assisted selection programme in hop. Key words: Humulus lupulus L., alpha acid content, genetic mapping, quantitative trait loci, quality Introduction Hop production is important agricultural branch in Slovenia, intensive for more than 100 years. At the moment there are 1467 ha hop fields in production (out of 1784 ha of hop fields together), more than 95 % are planted with Slovenian varieties. The most established is Aurora, presenting 62 % of hop acreage, following Savinjski golding with 13,2 %, Bobek with 9,7 %, Celeia with 7,5 %, Magnum with 4,5 %, Taurus with 0,2 % and on 4,9 % of hop fields other varieties are grown and hybrids. Hop breeding programme at the Slovenian Institute of Hop Research and Brewing (SIHRB) has over 50 years long tradition. The main purpose is to develop hop cultivars with improved quantity and quality (bitterness, aroma) of the yield, resistance against fungal diseases and pests, combining classical and molecular approaches. Regarding to all aims involved in breeding process different selection criteria and methods are included during the season. A general problem in developing new hop varieties is the long time required (at least ten years) especially because of the characteristics of hop plant. The aim of the research was to saturate existed genetic map and identify new QTLs involved in expressing alpha acid content in hop. Methods The F1 full-sib family was obtained by crossing the German variety Magnum with the Slovene breeding line 2/1. The analysis included 111 full-sib genotypes, 97 female and 14 male plants and the two parents (female/male ratio ( 6,9 : 1). Male hops were assigned as bearing the hypothetical male-determining locus M (Seefelder et al., 2000). Total genomic DNA was extracted from young leaves using a modified CTAB method according to Kump and Javornik (1996). The AFLP protocol was carried out according to Vos et al. (1995). The progenies of the family were selectively amplified with 18 EcoRI/MseI and 18 PstI/MseI primer pair combinations. Forty-six microsatellites developed by Jake and Javornik (2001), Jake et al. (2001), tajner et al. (2004) and new not-published SSRs were added to the analysis. The content of alpha-acids was measured in years 2002, 2003, 2004, 2005 and 2006 with the lead conductance value (LCV) method (Analytica EBC 1998). Linkage analysis was carried out using the same procedure as reported previous (Cerenak et al., 2006) with JoinMap Ver. 3.0 programme (Van Ooijen and Voorrips, 2001). The map was constructed using the LOD value of 5.0 for the grouping of markers. MapQTL version 4.0 (Van Ooijen et al., 2002) was used to identify and locate QTLs associated with alpha-acid content, by performing the non-parametric test of Kruskal-Wallis as well as both interval mapping (Lander and Botstein 1989) and multiple-QTL mapping (MQM; Jansen and Stam 1994). In the regions of the putative QTLs, the markers with the highest LOD values were taken as co-factors. The individual parental effects and interaction effect of the each putative QTL were calculated according to Knott et al. (1997). Results and Discussion Both types of polymorphic markers (AFLPs, SSRs) were tested for their inheritance pattern in mapping population. Segregation ratios were tested using the chi-square test and as suspected great proportion of markers did not fit the expected Mendelian ratios (p<0.01). Nevertheless, 43 out of 46 SSRs were successfully mapped. A total of 116 markers (86 AFLPs, 30 SSRs) were placed on the Magnum map forming 8 major linkage groups, 2 triplets and 4 doublets which are assumed to be part of the other groups, defining 706 cM of total map distance. A total of 94 markers (64 AFLPs, 30 SSRs) were placed on the 2/1 map, defining 616 cM of total map distance, with an average interval of 11 cM between adjacent markers. Nine major linkage groups, 3 triplets and 2 doublets were detected which are also assumed to be part of major linkage groups. Due to the presence of 16 common biparental SSR markers the homology of seven LGs between parental maps could be inferred. QTL analyses were carried out using data of alpha acid contents obtained in the years 2002, 2003, 2004, 2005 and 2006. None of the 5 distributions deviated significantly from the expected normal distribution. A LOD score of 3.0 was used to declare the presence of a QTL linked to alpha acid content. A total of 13 putative QTLs were determined in all 5 years, 7 were detected on female map and 6 on male map. Two putative QTLs were identified in 2002 (one on female and the other on male map) and one QTL was associated with alpha acid content in 2003. Four QTLs were determined in 2004 and also in 2006 while 2 QTLs were localised on the map associated with 2005. Possible homologies between QTLs for alpha-acid content detected in different years as well as in both maps could be established in at least three chromosomal regions. The regions containing temporally stable QTLs will be saturated with additional markers for further fine-mapping. The existing hop map (Cerenak et al., 2006) has been saturated further with additional SSRs and the homology of seven LGs between parental maps has been established providing a practical tool for the development of marker-assisted selection programme in hop. References Cerenak A., Satovic Z., Javornik B. 2006. Genetic mapping of hop (Humulus lupulus L.) applied to the detection of QTLs for alpha-acid content. Genome 49: 485-494. Jake J., Javornik B. 2001. High throughput isolation of microsatellites in hop (Humulus lupulus L.). Plant. mol. biol. report.19: 217-226. Jansen, R.C., and Stam, P. 1994. High resolution of quantitative traits into multiple loci via interval mapping. Genetics 136: 1447-1455. Knott, S.A., Neale, D.B., Sewell, M.H., and Haley, C.S. 1997. Multiple marker mapping of quantitative trait loci in an outbred pedigree of loblolly pine. Theor. Appl. Genet. 94: 810-820. Kump, B., and Javornik, B. 1996. Evaluation of genetic variability among common buckwheat (Fagopyrum esculentum Moench.) populations by RAPD markers. Plant Sci. 114: 149-158. Lander, E.S., and Botstein, D. 1989. Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 121: 185199. Seefelder, S., Ehrmaier, H., Schweizer, G., and Seigner, E. 2000. Male and female genetic linkage map of hops, Humulus lupulus. Plant Breed. 119: 249-255. tajner, N., Jake, J., Kozjak, P., and Javornik, B. 2004. The isolation and characterisation of microsatellites in hop (Humulus lupulus L.). Plant sci. 168: 213-221. Van Ooijen J.W., Boer M.P., Jansen R.C., Maliepard C. 2002: MapQTL Version 4.0, Software for the calculation of QTL positions on genetic maps. Plant Research International, Wageningen, the Netherlands Van Ooijen J.W., Voorrips R.E. 2001: JoinMap Version 3.0, Software for the calculation of genetic linkage maps. Plant Research International, Wageningen, the Netherlands Vos, P., Hogers, R., Bleeker, M., Reijans, M., Lee, T., Hornes, M., Frijters, A., Pot, F., Peleman, J., Kuiper, M., and Zabeau, M. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 21: 4407-4414.     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