Identification and comparison of individual chromosomes of three Hordeum chilense accessions, Hordeum vulgare and Triticum aestivum by FISH

Karyotypes of three accessions of Hordeum chilense (H1, H16 and H7), Hordeum vulgare and Triticum aestivum were characterized by physical mapping of several repetitive sequences. A total of fourteen repetitive sequences were used as probes for fluorescence in situ hybridization (FISH) with the aim of identifying inter‐ and intra-species polymorphisms. The (AG)12 and 4P6 probes only produced hybridization signals in wheat, the BAC7 probe only hybridized to the centromeric region of H. vulgare, and the pSc119.2 probe hybridized to both wheat and H. chilense, but not to H. vulgare. The remaining repetitive sequences used in this study produced a hybridization signal in all the genotypes. Probes pAs1, pTa535, pTa71, CCS1 and CRW were much conserved, showing no significant polymorphism among the genotypes studied. Probes GAA, (AAC)5, (CTA)5, HvT01 and pTa794 produced the most different hybridization pattern. We identified large polymorphisms in the three accessions of H. chilense studied, supporting the proposal of the existence of different groups inside H. chilense species. The set of probes described in this work allowed the identification of every single chromosome in all three species, providing a complete cytogenetic karyotype of H. chilense, H. vulgare and T. aestivum chromosomes, useful in wheat and tritordeum breeding programs.

), resistance to pests and diseases (Martín 53 et al. 1996) and high seed carotenoid content (Atienza et al. 2004). Several substitution and addition lines 54 have been developed to transfer these traits into wheat (Miller et al. 1982   Seeds of each genotype used in this study were germinated on wet filter paper in the dark for 5 days at 97 4°C, followed by a period of 24h-48h at 25°C under constant light. Root tips were obtained from these 98 seedlings and occasionally, from adult plants grown in

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10% dextran sulfate, 2x saline sodium citrate (SSC), 0.125% sodium dodecyl sulfate (SDS) and 0.1 mg of 127 salmon sperm DNA. The concentration of each probe in the hybridization mixture is described in Table 1.

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The hybridization mixture was denatured for 8 min at 80 °C and cooled on ice for 5 min. A 40 μl-aliquot 129 of the hybridization mixture was added to the cross-linked samples and a cover-slip applied. Slides were  (Table 1). Slides were then washed in TNT for 5 min and dehydrated 138 in 70% and 100% ethanol for 1 min. After counter-staining with 4', 6-diamidino-2-phenylindol (DAPI) for 139 5 min, slides were washed in water for 5 min, dehydrated again and mounted in Vectashield (Vector

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Repetitive sequences hybridized to mostly terminal and interstitial regions: pAs1 and pTa-535

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The pAs1 probe is a repetitive DNA sequence isolated from Aegilops tauschii Coss. (formerly known 155 as Ae. squarrosa L.) (Rayburn and Gill 1986). The pTa-535 is a 342-bp tandem repeat isolated from T. 7 and 4H ch exhibited weaker signals than the rest of the chromosomes; while in H. vulgare, chromosomes 164 3H v , 4H v and 5H v exhibited the weakest signals (Fig. 1).

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In wheat, both probes mainly hybridized to both chromosome arms in all D genome chromosomes as 166 previously described (Rayburn and Gill 1986;Komuro et al. 2013). Signals were also predominantly 167 telomeric and subtelomeric, as was observed in the Hordeum analysed. Some chromosomes from the A and 168 B-genomes occasionally hybridized to both probes, but these signals were weak and unsteady, so only D-169 genome chromosomes were identified in this work.

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In H. chilense H1, six pairs and seven pairs of chromosomes were hybridized to pSc119.2 and HvT01, 177 respectively ( Fig. 2). Probe pSc119.2 did not hybridize on chromosome 3H ch , and the signals produced on 178 chromosome 7H ch were weak (Fig. 2). The pSc119.2 signals were detected on the short arm of 1H ch , 2H ch , 179 5H ch and 7H ch (1H ch S, 2H ch S, 5H ch S and 7H ch S), on the long arm of 6H ch (6H ch L) and on both arms of 4H ch 180 ( Fig. 2). Probe HvT01 was detected on 2H ch S, 4H ch S, 5H ch S, 6H ch L and 7H ch S, and on both arms of 1H ch 181 and 3H ch (Fig. 2). In H. chilense H16, four pairs of chromosomes were labelled with pSc119.2 and HvT01 182 (Fig. 2). The pSc119.2 signals were detected on 1H ch S, 5H ch S, 6H ch L and on both arms of 4H ch (Fig. 2).

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The pSc119.2 signals were observed on 5H ch S and on both arms of 1H ch and 4H ch (Fig. 2). The HvT01 186 signals were detected on 3H ch S, 4H ch S and 5H ch S (Fig. 2). The HvT01 signals obtained in H1 and H7 agreed

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In H. vulgare, no hybridization signal was detected using the pSc119.2 probe (Fig. 2), which agreed 189 with previous studies (Gupta et al. 1989). However, all pairs of chromosomes were hybridized to HvT01  208 chilense H1, several pericentromeric signals were detected on chromosomes 3H ch and 7H ch , and several 209 interstitial signals on 4H ch S and 2H ch S. Chromosome 1H ch L was the only chromosome showing a strong 210 terminal signal. Chromosome 5H ch S and 7H ch S showed occasionally a weak interstitial and telomeric signal, 211 respectively. In H. chilense H16, the hybridization pattern was slightly different to H1. Chromosomes 6H ch 212 and 7H ch showed several pericentromeric and centromeric signals, chromosome 4H ch S and 5H ch L showed 213 several interstitial signals and chromosome 2H ch L showed a strong interstitial signal. Chromosomes 1H ch L 214 and 3H ch L showed a strong terminal signal. Chromosomes 1H ch S and 4H ch S showed occasionally a weak 215 telomeric signal, and 2H ch L and 7H ch L a weak interstitial one (Fig. 3). Hordeum chilense H7 showed less 216 signals than H1 and H16. Chromosomes 2H ch , 3H ch , 4H ch , 5H ch and 6H ch showed several pericentromeric 217 signals, and chromosomes 4H ch S and 7H ch S showed several interstitial signals. Chromosome 2H ch L showed 218 a strong interstitial signal. Chromosomes 1H ch L, 3H ch L and 7H ch S showed a terminal signal (Fig. 3). The 219 hybridization pattern detected with the GAA probe on H. vulgare was quite different to the pattern observed 220 9 in H. chilense. All chromosomes showed centromeric or pericentromeric signals, with only chromosome 221 3H v L showing a strong distal signal (Fig. 3). As mentioned above, in wheat, the GAA probe hybridized to 222 all B-genome chromosomes, with strong signals distributed along the whole chromosomes (Fig. 5). All 223 chromosomes from the A-genome plus 1DS, 7DS and both arms of 2D, also showed some GAA signal, but 224 the number and intensity were much lower than the ones observed in the B-genome (Fig. 5). The GAA 225 signals pattern agreed with the one described by Pedersen and Langridge (1997).

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The (AAC)5 probe produced an intense signal at the centromeric region of all H1, H16 and H7 227 chromosomes. An exception was chromosomes 5H ch , which did not show any signal, and 7H ch , which only 228 showed an interstitial signal on the long arm (Fig. 3). This probe was quite conserved in the three H. chilense 229 accessions used in this work. The only difference was on chromosome 4H ch L, which showed some 230 interstitial signals in H1 and H16 accessions, but not in H7. In H. vulgare, the (AAC)5 probe hybridized to 231 the centromeric regions of all chromosomes (Fig. 3). In wheat, this probe hybridized to all B-genome 232 chromosomes, and it is mainly distributed around the centromeric region, although some interstitial signals 233 were also observed in some of the chromosomes (Fig. 5). Chromosomes 2AS, 4AL and 7AL also showed 234 (AAC)5 signals close to the centromere (Fig. 5).

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The hybridization pattern of (CTA)5 was similar in H1 and H16, hybridizing both to the centromeric 236 regions of chromosomes 2H ch and 3H ch , and to 4H ch S in the case of H16 (Fig. 3). However, H7 showed a 237 higher number of signals, with all chromosomes except for 5H ch showing signal (Fig. 3). In H7, signals 238 were pericentromeric on chromosomes 1H ch L, 2H ch L, 3H ch L and 7H ch L and on both arms of 4H ch . On 239 chromosome 6H ch , the signal was located at the NOR region (Fig. 3). Hordeum vulgare showed (CTA)5 240 signals on all chromosomes, except for chromosomes 1H v and 3H v . It hybridized to the pericentromeric and 241 subtelomeric regions on chromosomes 5H v L and 6H v S and only to the subtelomeric region on chromosomes

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The 4P6 probe did not produce any signal in any of the Hordeum used in this study (Fig. S1). This probe 245 only hybridized to the wheat D-genome (Fig. 5). In wheat, signals were detected on five D-genome 246 chromosome pairs: on chromosomes 2DL, 4DL, 5DS, 6DS, and on both arms of chromosome 1D (Fig. 5).

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The (AG)12 probe, as 4P6, was absent in all Hordeum species used in this work (Fig. S1). In wheat, 248 some signals were detected on the pericentromeric region of chromosomes 3BS, 5BS and 6BL (Fig. 5).

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On the contrary, the pTa794 probe showed a different pattern among the Hordeum studied. In H. 259 chilense H1, H16 and H7, this probe was only detected on chromosome 5H ch S (Fig. 4). However, in H.

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The BAC7 probe is a centromere-specific large insert clone from H. vulgare L. (Hudakova et al. 2001).

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The CRW probe is a wheat centromeric retrotransposon from Ae. speltoides Tausch. and Ae. tauschii Coss.

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The centromeric probe CRW (Liu et al. 2008) was detected on all chromosomes of the three accessions 273 of H. chilense (H1, H16, H7) and H. vulgare (Fig. 6). However, non-specific signals were also frequently 274 observed along chromosomes (Fig. 6). In wheat, probe CRW also hybridized to the centromeric regions of 275 all chromosomes (Fig. 6). Unlike the genus Hordeum, signals on wheat were strong and clear, labelling 276 11 exclusively the centromeric region. Chromosomes from the D-genome showed weaker signals than A and 277 B-genome chromosomes, which is due to the fewer number of CRW copies as previously described (Liu et

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The CCS1 probe (Aragón-Alcaide et al. 1996) was also detected in H1, H16, H7, H. vulgare and T. 280 aestivum at the centromeric region (Fig. 6). However, as happened with the CRW probe, non-specific 281 signals were frequently observed along chromosomes (Fig. 6). In wheat, the CCS1 pattern was the same as 282 CRW: signals were strong, labelled the centromeric region and chromosomes from the D-genome showed 283 weaker signals than A and B-genome chromosomes (Fig. 6).

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Up until now, no H. chilense specific centromere probe has been described. Therefore, in an attempt to

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Here, fourteen repetitive probes were used, which accurately identified all individual chromosomes 309 from three accessions of H. chilense (H1, H7 and H16), H. vulgare and T. aestivum. Briefly, the 4P6 and 310 the (AG12) sequences were specific to wheat, and the BAC7 sequence was specific to H. vulgare. None of 311 the probes described here was specific to H. chilense. The pSc119.2 probe hybridized to both wheat and H.  wheat. In the case of both CCS1 and CRW, they are conserved in the three species, labelling the centromeres 323 of all chromosomes (Fig. 6). However, the hybridization signal on wheat was much stronger and clearer 324 than in barley species (H1, H16, H7 and H. vulgare), which frequently showed some background signals.

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Moreover, a confusing result is that neither the CCS1 nor the CRW probes, showed any signal on H. 326 chilense chromosomes when present in the background of tritordeum (Fig. S2). In tritordeum line HT27,

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wheat chromosomes always showed a centromeric signal, however, this signal was absent in H. chilense 328 chromosomes. A possible explanation for this result is that barley carries fewer copies of both CCS1 and 329 CRW and when placed in the background of wheat, the signal is too weak to be detected. This is observed 330 in the case of the wheat D-genome, which carries less copies of CCS1 and CRW sequences compared to 331 the A and B-genome, and accordingly, the hybridization signal in weaker (Liu et al. 2008). None of the 332 centromeric sequences described, can therefore be used to identify the centromeric region of H. chilense 333 chromosomes in tritordeum lines. However, an alternative is to use the (AAC)5 probe. This repetitive 334 sequence, hybridizes to the centromeric and pericentromeric region of all chromosomes of H. chilense, 335 13 except chromosome 5H ch and 7H ch , and to all chromosomes of H. vulgare. We tested the (AAC)5 sequence 336 in tritordeum line HT27, and H. chilense chromosomes were perfectly labelled (Fig. S2). Therefore, the 337 (AAC)5 sequence can be used to identify the centromeric region of most H. chilense chromosomes in 338 tritordeum or any other wheat background.

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It has been suggested that H. chilense consists of at least three morphologically and genetically distinct                        Table 1