Molecular cytogenetic and morphological characterization of two wheat-barley translocation lines

Abstract Barley chromosome 5H, carrying important QTLs for plant adaptation and tolerance to abiotic stresses, is extremely instable in the wheat genetic background and is eliminated in the early generations of wheat-barley crosses. A spontaneous wheat-barley 5HS-7DS.7DL translocation was previously obtained among the progenies of the Mv9kr1 x Igri hybrid. The present work reports on the transfer of the 5HS-7DS.7DL translocation into a modern wheat cultivar, Mv Bodri, in order to use it in the wheat breeding program. The comparison of the hybridization bands of DNA repeats HvT01, pTa71, (GAA)n and the barley centromere-specific (AGGGAG)n in Igri barley and the 5HS-7DS.7DL translocation, together with the visualization of the barley chromatin made it possible to determine the size of the introgressed barley segment, which was approximately 74% of the whole 5HS. Of the 29 newly developed PCR markers, whose source ESTs were selected from the Genome Zipper of barley chromosome 5H, 23 were mapped in the introgressed 1–0.26 FL 5HS bin, three were located in the missing C-0.26 FL region, while three markers were specific for 5HL. The translocation breakpoint was flanked by markers Hv7502 and Hv3949. A comparison of the parental wheat cultivars and the wheat-barley introgression lines indicated that the presence of the translocation improved tillering ability in the Mv9kr1 and Mv Bodri genetic background. The similar or better yield components under high- or low-input cultivation environments, respectively, indicated that the 5HS-7DS.7DL translocation had little or no negative effect on yield components, making it a promising genotype to improve wheat genetic diversity. These results promise to accelerate functional genomic studies on barley chromosome 5H and to support pre-breeding and breeding research on wheat.


Introduction
The genetic diversity of common wheat (Triticum aestivum L.) can be extended via interspecific hybridization. One of the promising crossing partners is barley (Hordeum vulgare L.), a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the translocation in disomic form was selfed three times to obtain a sufficient number of uniform progenies for agronomic investigation. These genotypes, containing a mixed wheat genetic backround originating from Mv9kr1 and Mv Bodri, were designated as 5HS-7DS.7DL/ Mv9kr1/Mv Bodri.
The barley cultivar Betzes (Hordeum vulgare L., 2n = 2x = 14) and the Chinese Spring-Betzes wheat-barley 5H disomic addition line [13] were used as positive controls and the wheat genotype Chinese Spring as negative control for the PCR validation of EST-derived markers.

Field trials and agronomic investigation of plants
Agronomic investigation of the genotypes was carried out in a low-input field (Tükrös Nursery, Martonvásár, Hungary; geographic coordinates: 47˚18'40"N 18˚46'56"E) in the 2015-2016 season and in a high-input location (Breeders nursery, Martonvásár, Hungary; 47˚19'58"N 184 7'08"E) in 2016-2017. As both areas are owned by the Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, no specific permission was required for the experiments. It is also confirmed that the field studies did not involve endangered or protected species.
The genotypes 5HS-7DS.7DL/Mv9kr1, 5HS-7DS.7DL/Mv9kr1/Mv Bodri, Mv9kr1 and Mv Bodri were grown in chernozem soil in both locations. In the low-input field, which was treated with herbicides [27], each genotype was sown in 5 × 1 m rows with 10 seeds per row and a row distance of 15 cm. In the high input field, each genotype was raised in a 4 m 2 plot with 6 x 3 m rows, 50 seeds per row, and a row distance of 20 cm. Ten typical plants per genotype were randomly selected and analyzed for agronomic traits. Plant height and tillering were recorded directly before harvest, while seeds/spikelet, length of the main spike, number of seeds/main spike, number of spikelets/main spike and number of seeds/plant were assessed after harvest.
After documentation of the GISH sites, the slides were washed and rehybridized using the repetitive DNA probes HvT01, pTa71 and (GAA) n and the barley centromere-specific sequence (AGGGAG) n . The FISH experiments was carried out according to the methods of Linc et al. [36] with modifications as described by Szakács and Molnár-Láng [14].
Pretreatments and stringency washings before the FISH experiments on the barley chromosome preparations were the same as those used for GISH and the same hybridization solutions containing repetitive DNA probes HvT01, pTa71, (GAA) n and (AGGGAG) n were used as for the 5HS-7DS.7DL translocation lines.
Mitotic cells were examined with a Zeiss Axio Imager M2 fluorescence microscope equipped with the appropriate filter sets (Carl Zeiss Mikroskopie, Jena, Germany). Images were captured with a Zeiss AxioCam MRm CCD camera (Diagnostic Instruments, Sterling Heights, Mich., USA) and processed with Zeiss Axiovision 4.8.2. software.
HvT01 is a barley-specific repetitive sequence located on the subtelomeric region of 5HS, pTa71 is an rDNA probe specific for the NOR region and able to detect the position of the secondary constriction on the 5HS arm, the (GAA) n oligonucleotide is located on the pericentric region of 5HS, while the (AGGGAG) n repeat is specific for the barley centromere. The length of the chromosome segments and the distances between the FISH signals were measured using Image Pro Plus 5.1 software. The positions of the translocation breakpoint and each of the FISH signals on the barley chromatin were expressed as fraction length (FL) values from the centromere relative to the length of the complete 5HS arm.

Development of EST-derived markers and PCR analysis
Genomic DNA was extracted from fresh young leaves (plants in the 2-leaf stage) from wheat genotypes Mv9kr1, Mv Bodri and Chinese Spring, barley cultivars Igri and Betzes, the Chinese Spring-Betzes 5H addition and the 5HS-7DS.7DL/Mv9kr1 and 5HS-7DS.7DL/Mv9kr1/Mv Bodri translocation lines using Quick Gene-Mini80 (FujiFilm, Japan) with a QuickGene DNA tissue kit (FujiFilm, Japan) according to the manufacturer's instructions [37].
The barley ESTs specific for chromosome 5H were selected from the publicly available barley Genome Zipper (http://pgsb.helmholtz-muenchen.de/plant/barley/gz/tablejsp/index.jsp) [26,38] and then aligned to the genomic sequences of the corresponding wheat homeologous chromosomes using BLASTn (https://urgi.versailles.inra.fr/blast/blast.php) to find ESTs showing polymorphism relative to wheat. BLASTn hits that met certain criteria (at least 7-8 bp InDel polymorphism and at least 80% homology between barley and wheat sequences) were considered as significant. The pairwise alignment of the selected ESTs and wheat contigs was carried out with UGENE software (v.1.23.0) to identify InDel regions between wheat and barley and to design barley-specific primers. The markers specific for barley chromosome 5H were validated by PCR on the genotypes Chinese Spring and Betzes and on the Chinese Spring-Betzes 5H addition lines. Two or three primer pairs were designed for each barley EST (Integrated DNA Technologies, Coralville, Iowa, USA) and the primer pair producing the most typical barley signal was chosen for subsequent analysis. The translocation genotypes were analyzed using primer pairs selected after the validation test.
The PCR reactions were performed in a final volume of 15 μL containing 20 ng of template DNA, 1.5 μL of 10× key reaction buffer (MgCl 2 final concentration of 1.5 mmol/L), 200 μmol/ L of each dNTP, 0.2 μmol/L of forward and reverse primers, and 0.375 U of TEMPase Hot Start DNA Polymerase (VWR International, Belgium). The reactions were carried out in an Eppendorf Mastercycler (Eppendorf, Germany) using the following PCR profile: 15 min at 95˚C, 35 cycles of 20 s at 95˚C, 20 s at 58˚C, 30 s at 72˚C, and a final extension at 72˚C for 5 min, following the manufacturer's instructions (VWR International). All the molecular markers were constructed to have the same melting point (58˚C). The PCR products were separated using a Fragment Analyzer™ Automated CE System equipped with a 96-Capillary Array Cartridge (Advanced Analytical Technologies, USA). The separated nucleic acid fragments were visualized as digital capillary electrophoretic gel images, provided by the PROsize v2.0 software (Advanced Analytical Technologies, USA), in order to compare the size and concentration data of all the genotypes investigated.
The amount of introgressed barley chromatin was compared in the two introgression lines 5HS-7DS.7DL/Mv9kr1/Mv Bodri and 5HS-7DS.7DL/Mv9kr1 using Student's t-tests for paired data at the P = 0.05 significance level.
The agronomic traits of lines 5HS-7DS.7DL/Mv9kr1/Mv Bodri and 5HS-7DS.7DL/Mv9kr1 and of parental wheat cultivars Mv9kr1 and Mv Bodri were compared pair-wise with each other. Differences in agronomic traits between the genotypes were evaluated by means of Tukey's post hoc test (SPSS 16.0) at the P = 0.05 significance level.

Transfer of the 5HS-7DS.7DL translocation into a modern wheat cultivar, Mv Bodri
In order to investigate the effect of the 5HS-7DS.7DL translocation on the agronomic performance of an elite wheat cultivar and to introduce this translocation into wheat breeding programs, a cross was made between 5HS-7DS.7DL/Mv9kr1 and Mv Bodri. The inheritance of the translocation was traced by GISH in 40 F 2 progenies of this cross. Among the F 2 genotypes the barley chromosome was eliminated from 27 plants, while 12 progeny carried the translocation in monosomic form and one in disomic form. As the 5HS-7DS.7DL/Mv9kr1 genotype was used as a crossing partner, the barley chromosome segment was regarded as originating from the 5HS-7DS.7DL translocation.

Physical characterization of the barley 5H chromatin using GISH and FISH
To study the structure of the introgressed 5HS chromatin and to obtain information about the missing part of the 5HS chromosome arm, the introgression lines were investigated with sequential FISH and GISH (Fig 1A-1D). The barley cultivar 'Igri' was also investigated with four repetitive DNA probes to obtain a reference karyotype for the 5H chromosome of barley (Fig 2). The hybridization signals of these probes served as cytogenetic landmarks for 5HS and their relative positions on the chromosome made it possible to calculate which part of the 5HS arm was missing in the translocation lines.
Measurements on 30 intact barley 5HS chromosomes showed that the region between the telomere and the satellite (pTA71 signal) represented 30.8% (FL: 0.692) of the entire 5HS arm and that between the centromere-specific and pericentric (GAA) n signals 14.7% (FL: 0.147) ( Fig 3C). Sequential GISH and FISH indicated that the pericentric (GAA) n and centromeric (AGGGAG) n signals were missing in the 5HS-7DS.7DL translocation (Fig 3A and 3B), while the distance between the telomere and the satellite (pTa71 signal), which accounted for 30.8% of the intact 5HS arm, was the same for the translocation lines as for the introgressed 5HS chromatin. It was possible to measure the total length of the introgressed barley chromatin visualized by GISH. As the relative position of the pTa71 signal on 5HS was the same in both the intact chromosome and the translocated segment, the relative position of the translocation breakpoints could be calculated from the total length of intact 5HS and the translocation segments. Based on measurements on 20 and 40 chromosomes respectively, the position of the translocation breakpoint of the introgressed 5HS segment relative to the whole 5HS chromosome arm was 0.27 FL ± 0.027 in 5HS-7DS.7DL/Mv9kr1 and 0.26 FL ± 0.025 in 5HS-7DS.7DL/Mv9kr1/Mv Bodri, indicating that the size of the introgressed barley segment (~74% of the whole 5HS arm) remained constant despite changes in the wheat genetic background, approximately 26% of the proximal part of the 5H short arm being absent from the translocated chromosome. The lack of the (GAA) n and centromere-specific signals, also indicating that at least 14.7% of the proximal part of 5HS is missing, further supports these results.

Molecular marker analysis
A total of about a thousand barley ESTs were analyzed using BLASTn (https://urgi.versailles. inra.fr/blast/blast.php). The query sequences (barley ESTs) were compared with the genomic sequences of wheat chromosomes. Ninety-five barley ESTs containing insertion/deletion regions (>7-8 bp) and showing at least 80% homology relative to wheat were chosen. Pairwise alignment was performed between the 95 selected ESTs and the corresponding wheat genomic sequences. Primer pairs were designed for the identified InDel regions in order to obtain markers amplifying the barley-specific fragments.
Primer sequences were developed for 35 of the 95 ESTs and these were validated by PCR on the control lines. The markers designed in the present study are listed in Table 1 together with Characterization of 5HS-7DS.7DL translocation their primer sequences and genetic positions. Six of the 35 EST-derived primer pairs either failed to produce a PCR amplicon in the barley genotypes or generated products non-polymorphic between the wheat and barley genotypes ( Table 1).
The remaining 29 markers were tested in the translocation lines and twenty-three were found to map within the 0-51.30 cM interval in both 5HS-7DS.7DL/Mv9kr1 and 5HS-7DS.7DL/Mv9kr1/Mv Bodri (Table 2). Three markers (Hv3949, Hv18070, Hv18916) located in the most proximal EST bin (51.30 cM) at the centromere were missing from the translocation lines and three (Hv26278, Hv26490, Hv18171) mapped to the proximal region of 5HL gave no PCR product in either of the translocation lines (Fig 4). These results indicate that the translocation breakpoint is located in the 51.3 cM region. The breakpoint was delimited between markers Hv7502 and Hv3949, located at loci 653 and 725, respectively, which means that this part of the 51.3 cM region is equivalent to 73-74% of the 5HS chromosome arm (Fig 5). Marker analysis also revealed that the barley chromatin segment did not change in the modified wheat genetic background ( Table 2).

Agronomic investigation
In order to clarify whether the translocation or the modification of the wheat genetic background had a greater influence on agronomic traits, the agronomic performance of the two translocation genotypes 5HS-7DS.7DL/Mv9kr1 and 5HS-7DS.7DL/Mv9kr1/Mv Bodri was compared, together with that of the parental wheat genotypes Mv9kr1 and Mv Bodri, in two field trials representing high-input (Table 3) and low-input (Table 4) conditions.
In the two field experiments Tukey's post-hoc analysis classified the plant height of both translocation lines in the same category, which differed from that of the wheat cultivars. Mv9kr1 was the tallest in both field trials. Mv Bodri was the shortest genotype in the low-input organic nursery, but grew much taller in the high-input area, where Mv Bodri was also taller than the two translocation genotypes. The length of the main spike was smaller in the introgression lines than in the wheat cultivars. 5HS-7DS.7DL/Mv9kr1/Mv Bodri had the highest number of spikes per plant at both locations, showing a significant difference compared with the parental lines. Both translocation lines had fewer spikelets per main spike than the wheat Table 1 The spike of 5HS-7DS.7DL/Mv9kr1/Mv Bodri had a dense structure with awned spikes, like those of the Mv Bodri wheat genotype. However, 5HS-7DS.7DL/Mv9kr1 had a main spike with apical awn stubs, like that of Mv9kr1 wheat (Fig 6). Characterization of 5HS-7DS.7DL translocation  Characterization of 5HS-7DS.7DL translocation

Discussion
As the 5H chromosome is eliminated most frequently in wheat x barley crossing programs [13,19,20], the importance of the 5HS-7DS.7DL translocation is underlined by the fact that this translocation has exhibited stable inheritance through 15 generations. In the present study, the 5HS chromosome segment of Igri barley was transferred into an elite wheat genetic background to obtain a genotype suitable for direct use in wheat breeding programs. The structure of the introgressed barley chromatin was compared in the two different wheat genetic backgrounds, Mv9kr1 and Mv Bodri. Both molecular marker and molecular cytogenetic analysis confirmed that the size of the 5HS chromatin segment did not change when the wheat genetic background was modified. GISH and FISH determined the physical position of the translocation breakpoint at 0.26 FL of the whole 5HS arm. As the estimated length of the 5H short arm is 301 Mbp, compared with 5100 Mbp for the barley genome [39,40], it is estimated that the 74% of the whole 5HS arm transferred into wheat corresponds to 223 Mbp. The PCR analysis showed that 23 markers developed in the present study were mapped in the introgressed 1-0.26 FL (223 Mbp) bin, while three markers (Hv3949, Hv18070, Hv18916) were located in the missing C-0.26 FL region (78 Mbp) in both translocation genotypes. Interestingly, the translocation breakpoint was flanked by markers Hv7502 and Hv3949, and seven of the ten markers, whose source ESTs were previously mapped within the centromeric region of 5HS (51.3-52.02 cM) [26,41], were located in the 1-0.26 FL region. These results indicate that the chromosomal breakage must be more distal from the centromere than was suggested by the genetic position of the source ESTs on the high-resolution EST map. In an earlier study Nagy et al. [22] delimited the position of this translocation breakpoint between the microsatellite markers Bmag387 and Bmag337, so the present results also indicate that the Bmag387 marker was located distally and the Bmag337 marker proximally from the 0.26 FL position. It was also hypothesized earlier that the terminal region of 5HS had already been deleted in the barley genotype Igri due to a chromosomal rearrangement with a breakpoint near the satellite [22]. However, the telomere specific EST-derived marker (Hv19657) amplified a 5HS-specific fragment in the introgression lines and in Igri barley, indicating that the terminal segment of 5HS is present in the translocation. This study supports the previous opinion that the linearly ordered virtual gene map of barley (Genome Zipper) is extremely useful in both fundamental and applied barley research [42]. The new EST-derived markers developed in this study will be used in wheat prebreeding programs aimed at shortening the introgressed 5HS segment. This marker development approach can also be applied for the marker saturation of 5HS chromosome regions containing desirable agronomic traits for wheat improvement. In this context, several QTLs related to abiotic stress tolerance were mapped on the 5HS barley chromatin. On the distal half of 5HS, two QTLs related to the osmotic adjustment of plant cells under water-limited and well-watered conditions were located [17]. Some physiological traits connected to the salt tolerance in barley were also mapped near the centromere of 5HS [18], such as a QTL affecting proline content and another related to the water-soluble carbohydrate content, which may facilitate heat or salt tolerance in the grain-filling period. Further studies will be needed in the future to prove that the introgressed 5HS segment has a real effect on the abiotic stress tolerance of wheat.
The present study also investigated how the presence of barley 5HS chromatin affects agronomic parameters as compared with the changes caused by the modification of the wheat genetic background. The number of spikes per plant is one of the most important parameters determining the yield of cereals. Both translocation lines had a higher number of spikes per plant than the wheat cultivars in both field trials, indicating that this parameter was influenced predominantly by the introgressed barley chromatin. The results for the effect of 5HS on tillering ability are in agreement with previous data published by Naz et al. [43], who identified five QTLs associated with tiller number per plant on barley chromosomes 1H, 2H, 4H and 5H, among which the strongest locus (QTil.S42IL.5H) was localized on 5H. Other studies also reported QTLs affecting tillering ability on chromosome 5H [44], such as the locus spp-5H-1, mapped close to the 50 cM region [45].
As no significant difference was observed in other yield components (seeds/main spike, seeds/spikelet and thousand-kernel weight) between 5HS-7DS.7DL/Mv9kr1/MvBodri and Mv Bodri, it can be concluded that increased tillering ability is the main yield component responsible for the higher yield (expressed as seeds/plant) in the 5HS-7DS.7DL/Mv9kr1/MvBodri genotype relative to the corresponding wheat genotype Mv Bodri under high-input field conditions. On the other hand, the increased fertility indicated by higher values of seeds per main spike and spikelets per main spike might have contributed to the increased yield of 5HS-7DS.7DL/Mv9kr1/MvBodri in comparison with 5HS-7DS.7DL/Mv9kr1, especially under high-input conditions, indicating the positive effect of the advanced wheat (Mv Bodri) genetic background. Further field trials using a larger plot area/genotype will be needed for a more exact comparison of yield parameters in 5HS-7DS.7DL/Mv9kr1/MvBodri and Mv Bodri.
Wheat genotype Mv Bodri was found to have lower plant height than Mv9kr1, which could be related to the fact that Mv Bodri carries the mutant allele of the semi-dwarfing gene RhtD1b on the short arm of 4D [46][47][48]. However, the deletion of the terminal region of 7DS in the translocation genotypes may also result in shorter plants. Khlestkina et al. [49] mapped the Ent-kaurenoic acid oxidase-coding (KAO) genes determining the gibberellin biosynthesis pathway and identified the KAO-D1 gene on the terminal segment of 7DS. Later, Kruppa et al. [23] reported that the two microsatellite markers (Xgwm1258, Xgwm1250) flanking the KAO-D1 locus were missing in the 5HS-7DS.7DL/Mv9kr1 genotype. Thus, the absence of the terminal region of the 7DS chromosome arm and consequently the absence of the KAO-D1 gene may also contribute to the reduced plant height of 5HS-7DS.7DL/Mv9kr1/MvBodri.

Conclusions
The improved tillering ability, together with the similar or better yield components under high-or low-input cultivation environments, respectively, indicate that the 5HS-7DS.7DL translocation in the Mv Bodri genetic background has small or negligible disadvantageous effects relative to the parental wheat Mv Bodri, which makes it a promising genotype for use in crossing and selection programs aimed at wheat improvement.
The exact size of the translocation was determined using in situ hybridization and molecular markers. As the new 5HS-7DS.7DL/Mv9kr1/MvBodri translocation, representing 74% of the whole 5HS arm, has stable inheritance in the wheat genetic background, this work represents an important step forward in the integration of the extremely instable barley chromosome 5H, carrying potential alleles for abiotic stress tolerance, into wheat breeding programs. The new EST-derived markers will be useful in pre-breeding programs to select wheat genotypes with introgressed 5HS chromatin or to identify genotypes with a shortened 5HS segment.
Altogether, the agronomic performance of the translocation line was improved by crossing it with a high-yielding wheat cultivar and the 5HS segment had a positive effect on tillering ability, which will be further tested in field experiments in the future. The new translocation line promise to accelerate functional genomic studies on barley chromosome 5H and the new molecular markers will support pre-breeding and breeding research on wheat, which will be required to meet the future challenges of food security and sustainable agriculture.