Introduction. Genome 53: (2010) doi: /g Published by NRC Research Press. 1 Corresponding author (

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1 Identification of new winter wheat winter barley addition lines (6HS and 7H) using fluorescence in situ hybridization and the stability of the whole Martonvásári 9 kr1 Igri addition set 35 É. Szakács and M. Molnár-Láng Abstract: A previous paper reported the development of disomic addition lines (2H, 3H, 4H, and 1HS isochromosomic) from hybrids between the winter wheat Martonvásári 9 kr1 and the two-rowed winter barley cultivar Igri. The present paper describes the isolation of two new additions, the 7H disomic and 6HS ditelosomic additions, using fluorescence in situ hybridization with the repetitive DNA probes Afa-family and HvT01. The identification of the barley chromosomes in the wheat genome was confirmed with simple sequence repeat markers. The morphological characterization of the new addition lines is also discussed. Studies of the genetic stability of the whole set (2H, 3H, 4H, 7H, 1HS iso, 6HS) of Martonvásári 9 kr1 Igri additions revealed that the most stable disomic additions are 2H and 3H and the most unstable line is the 1HS isochromosomic addition. Key words: Triticum aestivum, Hordeum vulgare, winter wheat winter barley addition lines, in situ hybridization, SSR markers, genetic stability. Résumé : Dans un article antérieur, les auteurs ont rapporté le développement de lignées d addition disomiques (2H, 3H, 4H et un isochromosome 1HS) à partir d hybrides entre le blé d automne Martonvásári 9 kr1 et l orge d automne à deux rangs Igri. Le présent article décrit l isolement de deux nouvelles additions, une lignée disomique 7H et une lignée ditélosomique 6HS, identifiées à l aide de l hybridation in situ en fluorescence au moyen des sondes d ADN répété de la famille Afa et HvT01. L identification des chromosomes de l orge au sein du génome du blé aété confirmée àl aide de marqueurs microsatellites. La caractérisation morphologique des nouvelles lignées d addition est également discutée. Des études sur la stabilité du jeu complet d additions (2H, 3H, 4H, 7H, 1HS iso, 6HS) Martonvásári 9 kr1 Igri révèle que les additions disomiques les plus stables sont 2H et 3H et que la lignée la plus instable est l addition isochromosomique 1HS. Mots-clés : Triticum aestivum, Hordeum vulgare, lignées d addition blé d automne orge d automne, hybridation in situ, marqueurs SSR, stabilité génétique. [Traduit par la Rédaction] Introduction Bread wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) are two of the most important cereal crops worldwide. Hybridization between these species makes it possible to expand the gene pool available to breeders to increase genetic variation in bread wheat. The production of intergeneric hybrids between wheat and barley by sexual cross and a first backcross with wheat is difficult and in some hybrid combinations it appears to be impossible (Wojciechowska and Pudelska 1993; Jauhar 1995; Taketa et al. 1998). The first wheat barley hybrid was produced by Kruse (1973) and not much later a set of Chinese Spring Betzes spring wheat spring barley addition lines was developed by Islam et al. (1978). In 1997 Koba et al. reported the isolation of 5H and 6H addition lines from a hybrid between the wheat cultivar Shinchunaga and the barley cultivar New Golden. The development of winter wheat winter barley ( Martonvásári 9 kr1 Igri ) disomic addition lines (disomic 2H, 3H, and 4H and isochromosomic 1HS) was first reported by Szakács and Molnár-Láng (2007). Addition lines form the starting point for producing translocations from selected chromosomes and thus are promising tools for transferring desirable traits (e.g., earliness, tolerance to drought and soil salinity, and various nutritional quality parameters) from barley into wheat and are suitable genetic materials for physical mapping of genes localized Received 1 April Accepted 30 October Published on the NRC Research Press Web site at genome.nrc.ca on 18 December Corresponding Editor: M. Puertas. É. Szakács and M. Molnár-Láng. 1 Agricultural Research Institute of the Hungarian Academy of Sciences, H-2462 Martonvásár, P.O. Box 19, Hungary. 1 Corresponding author ( molnarm@mail.mgki.hu). Genome 53: (2010) doi: /g09-085

2 36 Genome Vol. 53, 2010 on barley chromosomes. They can also be used to produce chromosome dissection lines suitable for the creation of deletion-based physical maps of the alien chromosomes (Serizawa et al. 2001; Ashida et al. 2007) and microdissected (Schondelmaier et al. 1993) or flow-sorted (Suchánková et al. 2006) alien chromosome samples for isolation of chromosome-specific sequences. Sophisticated molecular cytogenetic and molecular genetic methods help to analyse hybrids and their progenies in plants. Monosomic and disomic addition lines can have specific morphological characteristics, but more often they need additional confirmation by molecular marker analyses and assessment by fluorescence in situ hybridization with genomic and chromosome-specific DNA as probes. Genomic in situ hybridization (GISH) provides a direct, visual method of distinguishing entire parental genomes in both intergeneric (Schwarzacher et al. 1989; Le et al. 1989; Anamthawat- Jónsson et al. 1990; Mukai and Gill 1991) and interspecific (Gale and Miller 1987) hybrids, while fluorescence in situ hybridization (FISH) is a powerful technique for detecting specific nucleic acid sequences and localizing highly repetitive DNA sequences in specific regions of individual chromosomes, thus allowing their identification (Rayburn and Gill 1985; Leitch and Heslop-Harrison 1992; Mukai et al. 1993). Afa-family, a D-genome-specific pas1-like (Rayburn and Gill 1986) tandem repetitive DNA sequence of 340 bp, is present in numerous Triticeae species (Nagaki et al. 1995). In contrast to the probe pas1, Afa-family shows strong and typical hybridization patterns not only on wheat but also on barley chromosomes. HvT01 is a subtelomeric tandem repeat amplified from barley (Schubert et al. 1998) that gives subtelomeric signals on one or both barley chromosome arms. One very advantageous feature of this probe is that, with the exception of the 4BL chromosome arm, where a very weak signal is detectable, it does not hybridize to the wheat genome. Combination of these two probes makes it possible to detect and identify all the barley chromosomes in a wheat background directly after FISH without performing GISH. PCR-based markers for barley chromosomes (Murai et al. 2000; Sherman et al. 2001), in particular those based on simple sequence repeats (SSR) (Ramsay et al. 2000), are available to detect or confirm the presence of barley genetic material in a host genome. The main objective of the present study was to identify wheat barley addition lines different from the 2H, 3H, 4H, and 1HS isochromosomic addition lines described earlier (Szakács and Molnár-Láng 2007) in the progenies of the Martonvásári 9 kr1 Igri hybrid using in situ hybridization and SSR markers, and to determine the genetic stability of the Martonvásári 9 kr1 Igri addition set. Materials and methods Plant materials Wheat barley hybrids were produced in Martonvásár using the Hungarian winter wheat (Triticum aestivum L., 2n =6x = 42) line Martonvásári 9 kr1 (Mv9kr1) as maternal parent and the two-rowed winter barley (Hordeum vulgare L., 2n =2x = 14) cultivar Igri as the male parent. The kr1 gene responsible for crossability was transferred from the spring wheat cultivar Chinese Spring into the cultivar Martonvásári 9 by Molnár-Láng et al. (1996a). The Mv9kr1 Igri hybrids were multiplied in tissue culture as described earlier (Molnár-Láng et al. 2000a). The backcross pollination was carried out using Mv9kr1. The BC 1 plants were backcrossed again with Mv9kr1 and 24 BC 2 seeds were obtained, 14 of which grew into fertile plants. Selfed progenies of these 14 BC 2 plants grown in phytotron climatic chambers were screened using in situ hybridization to find new addition lines. Differences in morphological characteristics (plant height, spikes per plant, spike length, spikelets per spike, grains per spike, and fertility) among the disomic addition lines (7H and 6HS) and the control Mv9kr1 were determined by means of single-factor analysis of variance (ANOVA) at the P < 0.01 or P < 0.05 level. The results are the means of 10 measurements on the disomic addition lines and the parental wheat genotype, Mv9kr1. Fertility was quantified as the percentage of fertile florets (with grain) among the total number of competent florets of a spike. To study genetic stability (that is, what percentage of the plants retained the disomic state in the next generation), 50 seeds from each line (containing 44 chromosomes) of the addition set were watered to germinate. Chromosome composition was determined in root cells using GISH and FISH. GISH and FISH Mitotic metaphase chromosome preparations from the roots of germinating seeds of the BC 2 plants and their selfed progenies were used for in situ hybridization. Preparation followed the method described by Jiang et al. (1994). GISH was carried out according to Reader et al. (1994) with minor modifications (Molnár-Láng et al. 2000b). Total barley genomic DNA was labelled with Fluorored (rhodamine-5- dutp, Roche Diagnostics GmbH, Mannheim, Germany) by nick translation and used as a probe. Unlabelled wheat genomic DNA was sheared by autoclaving and used as blocking DNA at 30 times the quantity of the probe. FISH was carried out as reported by Linc et al. (1999) with the DNA probes Afa-family (Nagaki et al. 1995) and HvT01 (Schubert et al. 1998). The probes were amplified from the genomic DNA of barley (H. vulgare L.) and labelled by PCR with Fluorored or Fluorogreen (fluorescein-12-dutp, Roche Diagnostics GmbH, Mannheim, Germany) according to Vrána et al. (2000). The composition of the GISH and FISH hybridization mixtures and the procedures for hybridization and stringency washing were detailed earlier (Szakács and Molnár-Láng 2007). The slides were counterstained with 1 mg/ml DAPI (4,6- diamidino-2-phenylindole, Amersham). A Zeiss Axioskop 2 epifluorescence microscope equipped with Filter 10 for FITC, Filter 15 for Texas Red, and Filter 01 for DAPI and fitted with a Spot CCD camera (Diagnostic Instruments, Sterling Heights, Michigan, USA) was used to document the hybridization signals. The images were compiled with Image-Pro Plus 4.0 software (Media Cybernetics, Silver Spring, Maryland, USA). SSR marker analysis Genomic DNA from the Mv9kr1 Igri 7H and 6HS disomic addition lines and from Mv9kr1 and Igri (as controls) was isolated according to Csanádi et al. (2001). SSR

3 Szakács and Molnár-Láng 37 Table 1. Occurrence of different barley chromosomes in the selfed progenies of the 14 BC 2 plants. BC 2 plant No. Chromosome number of BC 2 plant Fertility (seeds/plant) of BC 2 plant Barley chromosome(s) in the progeny 3 NC 44 4H 4 43/ telo 16 6HS H, 3H H t 77 4H HS iso, 3H 14 NC H H H, 3H, 4H Note: NC, not countable. markers Bmac0163 (5HS), Bmac0316 (6HS), Bmag0021 (7HS), and Bmac0156 (7HL) were selected from a highly saturated genetic map of barley (Ramsay et al. 2000). The PCRs were performed as described by Nagy et al. (2002). The 15 ml reaction mixture contained 30 ng genomic DNA, 5 PCR buffer without MgCl 2, 0.3 mmol/l MgCl 2, 0.45 U Taq DNA polymerase (all three from Invitrogen Ltd., Paisley, UK), 0.3 mmol/l forward and reverse primers, and 200 mmol/l deoxynucleoside triphosphates. The PCRs were run on an Eppendorf MasterCycler (Eppendorf, Germany). The amplification profiles were chosen from Appendix S2, PCR Profiles used for the Barley SSRs, reported by Ramsay et al. (2000). The PCR products were separated on 1.5% agarose gels. A 100-bp DNA ladder (Invitrogen) was used as a size marker. The bands were visualized by ethidium bromide staining applied to the agarose gel at a final concentration of 1%. Results Identification of 6HS and 7H barley chromosomes Selfed generations derived from the 14 fertile BC 2 plants were screened to find the full set of barley additions in the wheat background. The 2H, 3H, and 4H additions reported earlier (Szakács and Molnár-Láng 2007) were detected again during the present research, beside which the disomic 7H and ditelosomic 6HS addition lines could be identified. The occurrence of different barley chromosomes in the selfed progenies of the BC 2 plants is summarized in Table 1. 4H addition lines were derived from 4 of the BC 2 plants, while 2H and 3H addition lines were derived from 3 of the BC 2 plants and the 6HS, 1HS isochromosomic, and 7H addition lines were each derived from a single BC 2 plant. 7H addition lines were first found as monosomic additions using a combination of the Afa-family and HvT01 repetitive DNA probes. As the karyogram made with Afafamily shows (Fig. 1A), 7H can be unequivocally differentiated from other barley chromosomes and wheat D-genome chromosomes, having intense telomeric and subtelomeric signals on both arms and a very specific hybridization site near the centromere. Only the 6H chromosome has similar (though weaker) patterns, but being a satellite chromosome it is easily distinguishable from 7H. As Figs. 1B and 1C show, with the help of the barley-specific DNA probe HvT01 (labelled with Fluorogreen), the 7H chromosome could easily be detected in the wheat background, being the only chromosome that showed subtelomeric hybridization patterns. The first disomic addition line was found after checking 45 selfed progeny plants of the monosomic lines using the HvT01 probe (Fig. 1D). The SSR markers Bmag0021 (7HS) and Bmac0156 (7HL) confirmed the presence of the 7H chromosome in these lines (Fig. 2A). The 6HS ditelosomic addition line was detected by GISH (Fig. 1E). 6HS is a satellite chromosome arm that is indistinguishable from the short arm of the 5H chromosome with the DNA probes GAA (Pedersen et al. 1996), HvT01, and Afa-family, which are generally used to identify barley chromosomes. A study using the SSR markers Bmac0163 (5HS) and Bmac0316 (6HS) confirmed the presence of the 6H short arm in this addition line (Fig. 2B). Morphological characterization of the 6HS and 7H Mv9kr1 Igri addition lines Results obtained by ANOVA can be found in Table 2. There were significant differences in morphological characters between the addition lines and the control genotype Mv9kr1. The 6HS addition line was significantly shorter (42.9 cm) and the 7H addition line was significantly taller (63.4 cm) than the control genotype (54.4 cm). Both addition lines had longer spikes but lower grain yield per spike than Mv9kr1. Addition of the barley 6HS chromosome pair to the wheat genome significantly reduced the spikelet number of the main spikes, while addition of the 7H chromosome increased the number of tillers per plant. A significant decrease in fertility (from 69.5% to 42.3%) was found only for the 7H addition line. The spike morphology of the new addition lines is shown in Fig. 3. The spikes of the 6HS ditelosomic addition line had awn stubs and were loose at the bottom but denser at the top. The spikes of the 7H disomic addition line were straighter and more compact and had short awns, especially at the upper part of the spike.

4 38 Genome Vol. 53, 2010 Fig. 1. (A) FISH karyogram of barley (Hordeum vulgare L. Igri ) chromosomes with the DNA probe Afa-family labelled with Fluorored. The strong 7H-specific hybridization pattern near the centromere is indicated with an arrow. (B) Detection of the 7H chromosome (indicated with an arrow) in a partial metaphase cell of a monosomic addition line with the barley-specific HvT01 probe labelled with Fluorogreen. FISH signals cannot be detected on wheat chromosomes except for the 4BL chromosome arm, indicated with an arrowhead (photo taken through a green filter). (C) Identification of the 7H chromosome (arrows) in the same cell with the repetitive DNA probes Afa-family (red) and HvT01 (green) (photo taken through a double bandpass filter). The magnified 7H chromosome is presented in the bottom right corner. The red arrow points to the 7H-specific Afa-family signal and yellow arrows indicate the HvT01 (yellow = green + red) pattern. (D) A pair of 7H chromosomes (arrows) in a disomic addition line selected from the progeny of a monosomic line. Hybridization was carried out with the probe HvT01 (labelled with Fluorored). (E) Detection of a 6HS ditelosomic addition line with GISH. Chromosomes were hybridized with total barley genomic DNA labelled with Fluorored. Scale bars = 5 mm. Stability of the Mv9kr1 Igri addition lines The genetic stability of the Mv9kr1 Igri disomic addition lines was analysed in 42, 50, 47, 43, 44, and 40 roottip preparations made from the 1HS isochromosomic and 2H, 3H, 4H, 6HS, and 7H disomic addition lines, respectively. The results are summarized in Table 3. The most stable addition lines were the 2H and 3H disomic additions, where all the progeny plants contained 44 chromosomes. A

5 Szakács and Molnár-Láng 39 Fig. 2. (A) Identification of 7H addition lines with SSR markers Bmag0021 (7HS) and Bmac0156 (7HL). 7H-specific products were detected in both the control sample ( Igri ) and two different 7H disomic addition lines (7H-1, 7H-2). (B) Confirmation of the presence of 6HS chromosome arms in two ditelosomic addition lines (6HS-1, 6HS-2) was carried out with SSR markers Bmac0163 (5HS) and Bmac0316 (6HS). The bands of 5HS- and 6HS-specific PCR products are indicated with arrows. A 5HS-specific (Bmac0163) product was detected only in the barley cultivar Igri. Bands under 100 bp are not chromosome-specific PCR products (e.g., primer dimers). high degree of stability was also observed in the 7H disomic addition line, where 96.4% of the plants retained the disomic state in the next generation, while 3.6% were monosomic. In the case of the 6HS ditelosomic addition line, the percentages of disomic and monosomic plants were 90.0% and 10.0%, respectively. In the 4H addition line, 68.4% of the progeny plants were disomic and 10.5% were monosomic, and the barley chromosomes were completely eliminated from 21.1% of the progeny plants. The addition line disomic for 1HS isochromosomes was the most unstable. The added chromosome pair was retained in 5.7% of the plants, while 22.9% of the progeny plants contained a single chromosome. The composition observed most frequently (28.6%) included one isochromosome and one telocentric 1HS chromosome. The percentage of telosomics carrying only one 1HS chromosome arm was 11.4%. The barley chromosomes were completely eliminated from 31.4% of the selfed progeny. Discussion The analysis of progenies in several selfed generations of the 14 BC 2 plants led to the identification of 4 disomic (2H, 3H, 4H, 7H), 1 ditelosomic (6HS), and 1 isochromosomic (1HS) Mv9kr1 Igri addition line. In comparison, Islam et al. (1981) identified 5 Chinese Spring Betzes disomic

6 40 Genome Vol. 53, 2010 Table 2. Comparison of the morphological characteristics of the 6HS and 7H Mv9kr1 Igri addition lines with those of the parent genotype Mv9kr1. Plant height (cm) Tillering (spikes/plant) Length of main spike (cm) Spikelets / main spike Grains / main spike Fertility (%) Mv9kr HS 42.9** 3.0 NS 10.7* 16.5* 28.7* 56.8 NS 7H 63.4** 4.2** 10.4* 20.1 NS 25.4** 42.3** Note: NS, not significant; *, significantly different from Mv9kr1 at the P < 0.05 level; **, significantly different from Mv9kr1 at the P < 0.01 level. Average of 10 plants/genotype (2009, Martonvásár, phytotron). Fig. 3. Spike morphology of the Mv9kr1 Igri disomic addition set compared with that of the parental Mv9kr1 (wheat) and Igri (barley) genotypes. addition lines when screening 789 progenies of selfed monosomics. The 5H Igri chromosome was apparently completely eliminated from the progenies of the hybrid plant, indicating the low tolerance of its presence in wheat. The elimination of alien chromosomes from intergeneric hybrids is a well-known phenomenon. In the BC 2 plants of another winter wheat ( Asakaze komugi ) winter barley ( Manas ) hybrid, the 5H chromosome was heavily underrepresented compared with the other barley chromosomes (Molnár-Láng et al. 2005). This chromosome was also eliminated with high frequency in the spring wheat spring barley hybrids examined by Koba et al. (1991) and in the Hordeum lechleri H. vulgare hybrid (Linde-Laursen and von Bothmer 1988), and was the least stable among the Chinese Spring Betzes addition lines (Islam et al. 1981). Morphological characteristics of the 6HS and 7H disomic addition lines The spikes of the 6HS and 7H disomic addition lines had specific morphological characteristics, differing both from each other and from the spike types of the addition lines reported earlier (Szakács and Molnár-Láng 2007). The greatest similarities were observed between the 6HS line and the disomic addition line containing 1HS isochromosomes. The spikes of both lines were narrow and had awn stubs, but spikes of the 6HS line were lax in the lower part and denser in the top. The long narrow spike of the 7H disomic addition line bore the greatest resemblance to that of the 2H addition line, but it was more compact. Similar alterations in wheat spike morphology due to the addition of barley chromosomes were described in the Chinese Spring Betzes (Islam et al. 1981) and Shinchunaga New Golden (Koba et al. 1997) addition lines (both containing H. vulgare chromosomes) and in the Shinchunaga H. spontaneum OUH602 (Taketa and Takeda 2001) addition lines containing chromosomes from the wild barley Hordeum vulgare subsp. spontaneum. Owing to the effect of the 6H (or 6HS) and 7H barley chromosomes, the wheat spikes became narrower. Surprising similarity was also observed between the spikes of the Mv9kr1 Igri 7H disomic addition line and those of the Mv9kr1 Ae. biuncialis 7M disomic addition line (Schneider et al. 2005). Similarly, the addition of the 2H chromosome from various Hordeum species and the 2S chromosome from Aegilops speltoides (Friebe et al. 2000) resulted in narrower

7 Szakács and Molnár-Láng 41 Table 3. Chromosome composition of progeny plants in the selfed generation of Mv9kr1 Igri addition lines containing 44 chromosomes. Status of the barley chromosomes in the progeny plants (%) Addition line n Disomic Monosomic Monosomic + telosomic Telosomic Eliminated 2H disomic H disomic H disomic H disomic HS ditelosomic HS isochromosomic Note: n, number of plants per addition line analysed by FISH and GISH. and elongated spikes, while the 3H chromosome as well as the 3M and 3U chromosomes (Schneider et al. 2005) had the opposite effect, resulting in shorter spikes. These data further confirm that the morphology of individual addition lines, whether derived from Hordeum, Aegilops, Secale, or other genera within the Triticeae, resembles the morphology of their homoeologous wheat tetrasomic lines, particularly if the wheat background is the same (Miller 1984). Both addition lines had reduced fertility compared with the parent Mv9kr1, but a significantly lower fertility rate was recorded only for the 7H disomic addition line. As the 6HS and 7H addition lines were grown in climatic chambers, fertility and other agronomic characteristics could not be compared with those of the addition lines grown in the field. Stability and applicability of the Mv9kr1 Igri addition set From the point of view of wheat breeding, addition lines are potential sources for increasing genetic diversity of common wheat in various characters. The prerequisite for transferring agronomically important traits from alien species to wheat is to induce recombination between the added alien chromosomes and the recipient genome. Recombinants with host plant chromosomes may occasionally be produced from addition lines through spontaneous translocations. For instance, a high rate of such translocations was observed in the progenies of 7 monosomic wheat rye addition lines (Ren et al. 1990). In most cases translocations are the result of chromosome manipulation using, for example, Ph1 mutants, irradiation with ionizing radiation, or tissue culture. The high stability of addition lines is an important criterion for applying them in the methods mentioned above. It seems that 4 of the 6 Mv9kr1 Igri addition lines (2H, 3H, 7H, and 6HS) meet this requirement. In contrast to the observation of Islam et al. (1981) that none of the Chinese Spring Betzes addition lines were completely stable, the 2H and 3H Igri chromosomes were fully recovered in the selfed progenies of these addition lines, though the 3H addition line was reported by the authors above to be the most stable (97.5%). The rate of disomic 2H progenies was 91.6% in the Chinese Spring Betzes addition line and 88.9% in another 2H addition line (Linc and Molnár-Láng 2003). The 7H disomic and 6HS ditelosomic addition lines can also be regarded as stable, as 96.4% and 90.0% of their progenies remained in the disomic state, respectively. Similar rates of 94.6% for the 7H and 93.5% for the disomic 6H were found in Chinese Spring Betzes addition lines. The stability of the 6H disomics (96.1%) reported by Linc and Molnár-Láng (2003) was nearly the same, but the 6HS ditelosomic addition line described by Molnár-Láng et al. (1996b) showed a stability of only 50.0%. Studies of preferential elimination (Ramsay and Dyer 1983; Wojciechowska 1985; Linde-Laursen and von Bothmer 1988) suggest that satellite chromosomes (5H and 6H in the case of barley) may be more frequently involved in numerical chromosome aberrations than other chromosomes. The lack of the 5H addition from the Mv9kr1 Igri addition set and the fact that only the short arms of the 6H Igri chromosome were transmitted from the hybrid to the addition line seem to support this suggestion. The Mv9kr1 Igri 4H addition line exhibited intermediate stability (68.4%), but this was lower than that of the Chinese Spring Betzes 4H addition line (86.0%), and 4H was the only disomic addition line where the total elimination of barley chromosomes occurred in the progenies. It is worth pointing out that chromosome 4H was eliminated with the lowest frequency from wheat barley hybrids (Islam et al. 1981; Koba et al. 1991; Szakács and Molnár- Láng 2007) but was one of the least stable chromosomes in selfed disomic addition lines. Similarly, when studying wheat rye and wheat barley addition lines, Riley (1960) and Islam et al. (1981) observed that there was no close relationship between stability and the meiotic regularity or the fertility of the lines. A fertile disomic addition line involving the entire barley chromosome 1H cannot be produced because of the Shw sterility gene present on the long arm. The addition line disomic for the 1HS isochromosome is selffertile, but very unstable, the occurrence of the full pair of 1HS isochromosomes being only 5.7% in the next generation after selfing. Because of the instability of the 4H disomic and 1HS isochromosomic additions, cytological monitoring is essential to maintain the integrity of these lines. In some rare cases, elimination of wheat chromosomes can also happen in wheat alien addition lines (Ren et al. 1990), but this phenomenon was not observed in the Mv9kr1 Igri addition set. Stable, self-maintaining wheat barley disomic addition lines can be used, among other purposes, for introducing abiotic stress resistance to the recipient species. It is predicted that both the quantity and the spatial and temporal distribution of the annual precipitation in Hungary will be unfavourable in the future (Várallyay 2008). Barley, being a

8 42 Genome Vol. 53, 2010 cereal relatively well adapted to water deficit (Ceccarelli 1987), is a potential gene source for improving the drought tolerance of cultivated wheat. Osmotic adjustment is a major trait for adaptation to drought and is associated with the whole-plant response to water stress. The quantitative trait loci (QTL) analyses carried out by Teulat et al. (1998) detected chromosomal regions on chromosomes 7H and 6H (6HS) that are considered to control osmotic adjustment. Recently, a field experiment carried out to test for drought tolerance included the parental genotypes of the Mv9kr1 Igri addition lines (Hoffmann et al. 2009). Data on plant height, root/shoot ratio, leaf water potential, ear length, thousandgrain weight, number of kernels, and grain yield revealed that Igri tolerated water deficit better than the wheat line Mv9kr1. On the basis of these data, the Mv9kr1 Igri 6HS and 7H addition lines can be expected to be appropriate genetic sources (after the production of translocations) for the transfer of drought tolerance from barley to wheat. The analysis of Na + and K + concentrations in the leaves of Chinese Spring Betzes (Islam et al. 1978) addition lines exposed to NaCl treatment (Gorham et al. 1990) revealed that there are genes on individual barley chromosomes, in particular on 7H and 6H, that could enhance the ability of bread wheat to maintain low Na + concentrations in the leaves. QTL analyses confirmed these results. QTLs for salt tolerance at the seedling stage were located on barley chromosome 6H (Mano and Takeda 1997) and clusters of loci were found on chromosome 7H (Ellis et al. 1997). The Mv9kr1 Igri 6HS and 7H addition lines will be tested for salt tolerance to ascertain whether Igri chromosomes 6HS and 7H have the same positive effect on Na + exclusion in wheat. Since all barley chromosome arms can be flow-sorted from ditelosomic addition lines (Suchánková et al. 2006), the Mv9kr1 Igri 6HS addition line is a suitable starting material for isolating the short arm of the 6H chromosome for purposes such as the physical mapping and (or) isolation of 6HS-specific barley genes and markers, the detection of Igri -specific transcripts, and their use for bacterial artificial chromosome library construction. Acknowledgements Thanks are due to Mrs. I. Bucsi and Mrs. J. Havasi for their technical assistance and to Mrs. B. Harasztos and Ms. A. Bacskovszky for revising the manuscript linguistically. This work was financed by the Generation Challenge Programme (CGIAR) GCP SP3 G and the Hungarian National Research Fund (K ) and supported by the AGRISAFE (No ) EU-FP7-REGPOT project. 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