Development of wheat—Hordeum chilense Chromosome 2Hch Introgression Lines Potentially Useful for Improving Grain Quality Traits

The chromosome 2Hch of Hordeum chilense. has the potential to improve seed carotenoid content in wheat as it carries a set of endosperm carotenoid-related genes. We have obtained structural changes in chromosome 2Hch in a common wheat (Triticum aestivum L. “Chinese Spring”) background by crossing a wheat double disomic substitution 2Hch(2D) and 7Hch(7D) line with a disomic addition line carrying chromosome 2Cc from Aegilops cylindrica Host.. Seven introgressions of chromosome 2Hch into wheat were characterized by fluorescence in situ hybridization (FISH) and DNA markers. Chromosome-specific simple sequence repeats (SSRs) were used for identifying wheat chromosomes. In addition, we tested 82 conserved orthologous set (COS) markers for homoeologous group 2, of which 65 amplified targets in H. chilense and 26 showed polymorphism between H. chilense and wheat. A total of 24 markers were assigned to chromosome 2Hch with eight allocated to 2HchS and 16 to 2HchL. Among the seven introgressions there was a disomic substitution line 2Hch(2D), a ditelosomic addition line for the 2HchL arm, an isochromosome for the 2HchL arm, a homozygous centromeric 2HchS·2DL translocation, a double monosomic 2HchS·2DL plus 7HchS·D translocation, a homozygous centromeric 7HchS·2HchL translocation and, finally, a 2HchL·7HchL translocation. Wheat—H. chilense macrosyntenic comparisons using COS markers revealed that H. chilense chromosome 2Hch exhibits synteny to wheat homoeologous group 2 chromosomes, and the COS markers assigned to this chromosome will facilitate alien gene introgression into wheat. The genetic stocks developed here include new wheat—H. chilense recombinations which are useful for studying the effect of chromosome 2Hch on grain quality traits.


Introduction
Narrow genetic diversity often limits the improvement of many traits in wheat. The introgression of genes from wild relatives to wheat has become a widely recognized genetic approach for increasing genetic diversity and, hence, the need to explore primary, secondary and tertiary gene pools of wheat has grown [1,2]. Hordeum chilense Roem. et Schultz. is a diploid wild barley that exhibits advantageous agronomic and quality characteristics [3][4][5][6]. Furthermore, its high crossability with other species of the tribe Triticeae, such as both durum and common wheat, [3][4][5] make it useful in cereal breeding.
Addition and substitution lines of alien chromosomes in a common wheat background, are useful for introgressing alien chromosomal segments carrying genes of agronomical interest into wheat. Chromosome addition lines of H. chilense in the Triticum aestivum L. cultivar "Chinese Spring" have been obtained including five for chromosomes 1H ch , 4H ch , 5H ch , 6H ch and 7H ch and the ditelosomic addition line for the short arm of chromosome 2H ch [7]. Fertile wheat lines carrying deletions and translocations involving chromosome 3H ch from H. chilense have also been obtained [8]. However, no addition or substitution lines in a common wheat background have been developed for chromosome 2H ch .
Chromosome 2H ch has the potential to improve seed carotenoid content in wheat. Genetic studies of yellow pigment content (YPC) in H. chilense revealed that chromosome 2H ch showed a significant association with YPC [19] and four endosperm carotenoid-related genes have been genetically mapped to chromosome 2H ch , such as geranyl geranyl pyrophosphate synthase (Ggpps1) for geranylgeranyl diphosphate synthesis, zeta-carotene desaturase (Zds), beta-carotene hydroxylase 3 (Hyd3) from the carotenoid biosynthetic pathway and polyphenol oxidase 1 gene (Ppo1) implicated in plant tissue enzymatic browning [20].
Molecular markers that are able to distinguish H. chilense chromosome 2H ch in wheat background provide a useful tool for selection. The conserved orthologous set (COS) [21] represents an important reservoir of markers that allow comparative studies with wheat and barley and their transference to H. chilense is a main goal.
The aims of this work were the following: (a) to obtain wheat-H. chilense chromosome 2H ch introgression lines; (b) to characterize the lines obtained by fluorescence in situ hybridization (FISH) and chromosome-specific simple sequence repeat (SSR) markers; (c) to transfer COS markers to H. chilense and to determine their arm location within 2H ch and (d) to compare the arm location with wheat and barley homoeologous group 2.

Plant Material
A "Chinese Spring" (CS) wheat-H. chilense double 2H ch (2D)-7H ch (7D) disomic substitution line, previously obtained at the University of Córdoba (results not shown), was used for inducing structural changes in chromosome 2H ch using gametocidal chromosome 2C c from Aegilops cylindrica host. The double 2H ch (2D) and 7H ch (7D) disomic substitution line was obtained by pollinating tritordeum (the fertile amphiploid between H. chilense and T. turgidum L., AABBH ch H ch , 2n = 6x = 42) with a wheat disomic addition line for gametocidal chromosome 2C c from Ae. cylindrica Host. following the breeding procedure described in [8]. The double substitution 2H ch (2D) and 7H ch (7D) line was pollinated with the wheat disomic addition line for the gametocidal chromosome 2C c from Ae. cylindrica. The F1 plants monosomic for 2H ch , 7H ch and 2C c were selfed for four generations.

Fluorescence In Situ Hybridization (FISH)
The excised root tips were pretreated with ice water for 24 h and then fixed in acetic ethanol: acetic acid (3:1, v/v), as described previously [8]. The FISH protocol was carried out as described by [22]. The pAs1 sequence (1 kb) isolated from Aegilops tauschii Coss. [23] and H. chilense genomic DNA were used as probes. The pAs1 probe hybridizes to D-genome chromosomes of wheat [24] and H ch -genome chromosomes from H. chilense [25]. The pAs1 probe and H. chilense DNA were labeled with biotin-16-dUTP (Roche Diagnostics, Switzerland) and with digoxigenin-11-dUTP (Roche Diagnostics, Switzerland), respectively, by nick translation. Three plants per each introgression line were analyzed.

Molecular Marker Analysis
A total of 82 COS markers from wheat homoeologous group 2 [21] were studied for their utility in H. chilense (File S1). H. chilense (line H7) and common wheat CS were used as controls. The CTAB method [26] was used for DNA extraction of young leaf tissue. The concentration of each sample was estimated using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Amplifications were made using a TGradient thermocycler (Biometra, Göttingen, Germany) with 60 ng of template DNA in a 25 µl volume reaction containing 5 µl of 10× PCR Buffer, 0. In addition, four chromosome-specific SSR markers for the wheat D-genome were used for molecular characterization of the introgression lines [27,28]. Xgwm261 and Xgwm157 markers were used to detect 2DS and 2DL chromosome arms, respectively. Xcfd66 and Xbarc111 were used to detect 7DS and 7DL chromosome arms, respectively. Amplifications were carried out as described at GrainGenes [29] One plant from each introgression line was used for the molecular characterization. "Chinese Spring", H. chilense, a ditelosomic 2H ch S line, a ditelosomic 7H ch S line, a ditelosomic 7H ch L line and disomic substitution line CS 7H ch (7D) were used as controls. Ditelosomic 2H ch S and ditelosomic 7H ch S lines were provided by the John Innes Centre (UK). The ditelosomic 7H ch L line and disomic CS 7H ch (7D) substitution line were obtained previously [17].
The amplified products were resolved using 2% agarose gels (SSRs) or polyacrylamide gels (10%, w/v; C: 2.67%) (COS) and stained with ethidium bromide or SafeView Nucleic Acid Stain (NBS Biologicals, Huntingdon, UK) incorporated in the gel. A 100 bp DNA ladder (Solis BioDyne, Tartu, Estonia) was used as a standard molecular weight marker. Kodak Digital Science 1D software (version 2.0) was used to determine the amplicon lengths.

Comparative Mapping
The orthologous relationship between the 2A, 2B, and 2D genome chromosomes of bread wheat and the 2H ch chromosome from H. chilense has been studied from the genomic perspective of wheat as described previously [30]. For the construction of the physical map, the expressed sequence tag (EST) source sequences (File S2) were used as queries in BLASTn searches against the wheat reference pseudomolecules [31] to identify the start positions (bp) of the ESTs. In this study, BLAST hits with E values smaller than 1e −10 , identity % > 58. 44 and alignment length > 100 bp were considered significant. The genomic start positions in bp of the best hits in wheat pseudomolecules (File S3) were used to construct a physical map of the polymorphic COS markers. The wheat reference genome sequence [31] was used to determine the centromere positions for 2A, 2B and 2D wheat chromosomes. Both the length in bp of wheat pseudomolecules, as well as the start genomic positions of the ESTs, were converted to pixels. Then, the data from the BLASTn searches were used to construct a physical map for 2A, 2B, and 2D wheat chromosomes showing the position of the source EST of the COS markers assigned to H. chilense chromosome 2H ch .
The rice locus (RAP) [21] was used to locate the COS markers in the barley genome zipper [32]. The RAP locus identifier was retrieved using the ID Converter tool [33]. The full-length barley cDNA corresponding to each rice locus was used for determination of the barley Unigene corresponding to each COS marker. The Unigene sequences were aligned in Barleymap [34] to obtain their positions in the International Barley Sequencing Consortium map [32,35].

Cytogenetic and Molecular Characterization of Wheat-H. chilense Introgression Lines Involving Chromosome 2H ch
The pAs1 and H. chilense genomic DNA used as probes in FISH analysis allowed the identification of a pair of 2H ch chromosomes and the absence of the wheat 2D chromosome pair in one line with 42 chromosomes. This result indicated that this line was disomic for the substitution 2H ch (2D) (Figure 1a). The absence of 2D was tested using Xgwm261-2DS (Figure 2a) and Xgwm157-2DL (Figure 2b) molecular markers. A pair of telocentric chromosomes was identified by FISH in one line with 42 + 2t chromosomes (Figure 1b). To determine the chromosome arm involved in each introgression line, we used the c749557 COS marker mapped on the 2H ch S arm and c731690 mapped on 2H ch L, respectively (see Section 3.2). The presence of the c731690 marker for 2H ch L and the absence of the c749557 marker for 2H ch S showed that this line was ditelosomic for the 2H ch L arm (Figure 3a,b).
FISH analysis revealed a line apparently carrying chromosome 2H ch (Figure 1c). Marker c731690 for 2H ch L was amplified in this line, but there was no amplification of the c749557 marker for 2H ch S (Figure 3a,b). These results suggested that a 2H ch L·2H ch L isochromosome was present in this line, and it was named Iso 2H ch L. Both the ditelosomic 2H ch L and Iso 2H ch L lines were nullisomic for chromosome 2D, as demonstrated by the absence of amplification of both Xgwm261-2DS ( Figure 2a) and Xgwm157-2DL (Figure 2b) molecular markers.
We identified two lines carrying centromeric translocations involving the 2H ch S chromosome arm and wheat chromosomes. One of these lines was homozygous for the 2H ch S·2DL translocation (Figure 1d). The other translocation line was a double monosomic for 2H ch S·2DL and 7H ch S·D translocations (Figure 1e). Chromosome-specific SSR markers confirmed the absence of 2DS (Figure 2a) and the presence of 2DL (Figure 2b) in both lines. Amplification of the c749557 marker ( Figure 3a) and the absence of amplification of the c731690 marker (Figure 3b) demonstrated the presence of 2H ch S and the absence of 2H ch L, respectively, in both lines. COS markers c779791 and c759439, previously assigned to 7H ch S and 7H ch L, respectively [18], were used to detect introgression from chromosome 7H ch (Figure 3c,d). Amplification of the c779791 marker specific for the 7H ch S arm (Figure 3c) indicated the presence of 7H ch S translocated to an unidentified wheat fragment. The presence of pAs1 signals on the wheat small fragment indicated that the chromosome 7H ch S arm was translocated to an unidentified D-genome chromosome (Figure 1e). The absence of amplification of chromosome-specific markers Xcfd66-7DS and Xbarc111-7DL demonstrated the absence of a 7D chromosome pair in this line (Figure 2c,d).
In the remaining two lines, two centromeric translocations involving 2H ch and 7H ch H. chilense chromosomes were detected. One line was homozygous for the 7H ch S·2H ch L translocation (Figure 1f) and the other one was monosomic for the 2H ch L·7H ch L translocation. Both translocation lines were nullisomic for chromosome 2D (Figure 2a,b). Chromosome-specific SSR marker patterns for 2D (Xgwm261-2DS and Xgwm157-2DL) and 7D (Xcfd66-7DS and Xbarc111-7DL) genome chromosomes are given in Figure 2a,b and Figure 2c,d, respectively. Chromosome-specific marker results for chromosome 7H ch S and 7H ch L are given in Figure 3c,d. Table 1 shows the chromosome constitutions of all the H. chilense introgression lines. All lines were vigorous and seed set.  chromosome 7H ch (Figure 3c, d). Amplification of the c779791 marker specific for the 7H ch S arm (Figure 3c) indicated the presence of 7H ch S translocated to an unidentified wheat fragment. The presence of pAs1 signals on the wheat small fragment indicated that the chromosome 7H ch S arm was translocated to an unidentified D-genome chromosome (Figure 1e). The absence of amplification of chromosome-specific markers Xcfd66-7DS and Xbarc111-7DL demonstrated the absence of a 7D chromosome pair in this line (Figure 2c, d). In the remaining two lines, two centromeric translocations involving 2H ch and 7H ch H. chilense chromosomes were detected. One line was homozygous for the 7H ch S·2H ch L translocation (Figure 1f) and the other one was monosomic for the 2H ch L·7H ch L translocation. Both translocation lines were nullisomic for chromosome 2D (Figure 2 a, b). Chromosome-specific SSR marker patterns for 2D (Xgwm261-2DS and Xgwm157-2DL) and 7D (Xcfd66-7DS and Xbarc111-7DL) genome chromosomes are given in Figure 2 (a, b) and Figure 2 (c, d), respectively. Chromosome-specific marker results for chromosome 7H ch S and 7H ch L are given in Figure 3 (c, d). Table 1 shows the chromosome constitutions of all the H. chilense introgression lines. All lines were vigorous and seed set.

Transferability and Chromosome Location of COS Markers in H. chilense
The transferability to H. chilense of 83 COS markers from wheat homoeologous group 2 was studied (File S1). First, all 83 markers were screened for polymorphisms (size polymorphisms or presence and absence) between H. chilense and common wheat. Of the 83 markers, 65 (78.3%) consistently amplified H. chilense products and 26 (40.0% of the total) were polymorphic between H.

Transferability and Chromosome Location of COS Markers in H. chilense
The transferability to H. chilense of 83 COS markers from wheat homoeologous group 2 was studied (File S1). First, all 83 markers were screened for polymorphisms (size polymorphisms or presence and absence) between H. chilense and common wheat. Of the 83 markers, 65 (78.3%) consistently amplified H. chilense products and 26 (40.0% of the total) were polymorphic between H. chilense and wheat (Table 2). Twenty-four of these 26 polymorphic markers were mapped to chromosome 2H ch , as demonstrated by their presence in the wheat-H. chilense 2H ch (2D) substitution line. We were unable to map the remaining two markers because they did not amplify products in any of the available wheat-H. chilense addition lines. Of the 24 COS markers mapped on chromosome 2H ch , eight were located on 2H ch S and 16 were located on 2H ch L, as demonstrated by their presence and absence in 2H ch S or 2H ch L ditelosomic lines, respectively. Table 2 summarize the characterization and chromosome arm location of wheat COS markers on H. chilense chromosome 2H ch . Figure 3a,b shows examples of amplification of homoeologous group 2 COS markers.

Wheat-H. chilense Group 2 Homoeology
To investigate wheat-H. chilense group 2 macrosyntenic relationships, the source ESTs of the 24 polymorphic COS markers mapped on chromosome 2H ch were BLASTed to the sequences of the wheat chromosomes [31]. All EST markers showed hits on wheat pseudomolecules (File S3). To produce a physical map (Figure 4), the start positions of the alignments of the best hits on the A, B, and D genomes were extracted. All markers assigned to 2H ch S and 2H ch L were located in the same arm of wheat homoeologous group 2 chromosomes (Table 2, Figure 4).
To determine the positions of COS markers in Barley-maps ( Table 2) the align tool implemented in Barleymap [34] was used to determine the positions of COS markers in barley maps ( Table 2). Four of the COS markers were not found in the barley genome zipper [32] so their position in barley could not be determined. A good correspondence between H. chilense and barley was found for the arm locations of COS markers ( Table 2).

Discussion
A set of introgression lines involving chromosome 2H ch from H. chilense in common wheat background was produced in this work using the gametocidal chromosome 2C c from Ae. cylindrica. Gametocidal genes have been used to produce structural chromosome aberrations in wheat [36,37], barley [38,39] and rye [40]. In H. chilense, structural changes have been previously obtained for

Discussion
A set of introgression lines involving chromosome 2H ch from H. chilense in common wheat background was produced in this work using the gametocidal chromosome 2C c from Ae. cylindrica. Gametocidal genes have been used to produce structural chromosome aberrations in wheat [36,37], barley [38,39] and rye [40]. In H. chilense, structural changes have been previously obtained for chromosomes 1H ch , 3H ch , 4H ch and 7H ch [8,11,17,18,41] and have been useful in determining the locations of genes and markers in this species. Breaks at both centromeric and interstitial regions of chromosomes have been induced by gametocidal chromosome 2C c [36]. In the present study, telocentric and translocations between chromosome 2H ch from H. chilense and wheat chromosomes have been generated Alien addition and translocation lines are an ideal template for PCR-based mapping to assign molecular markers to chromosomes of the wild relatives of wheat [8,11,[41][42][43]. Using gene-based conserved orthologous set (COS) markers on wheat-H. chilense introgression lines obtained in this work, we assigned a total of 24 markers to H. chilense chromosome 2H ch . A 78.3% transference rate of COS markers to H. chilense chromosome 2H ch was found. Since COS markers were intended for comparative studies among grasses, the high rate of transferability obtained in this work was expected [21]. Similar rates of transference of COS markers to H. chilense chromosome 7H ch have been found previously [17]. COS markers have also been transferred successfully to other Triticeae species such as Agropyron cristatum [29,41] and Aegilops spp. [44].
The relevance of chromosome 2H ch for endosperm carotenoid content has been highlighted by previous work. Association studies for YPC allowed the identification of three main chromosome regions for YPC variation in H. chilense, with the largest one located on chromosome 2H ch and smaller regions detected on chromosomes 3H ch and 7H ch [20]. Four candidate genes associated with YPC were genetically mapped to chromosome 2H ch : both Ggpps1 and Zds were tightly linked and mapped near the centromere, while Hyd3 and Ppo1 were mapped to the long arm of chromosome 2H ch [20]. Furthermore, a significant QTL at the distal part of chromosome 2H ch has also been found where no carotenoid-related genes have been mapped [19,20]. The importance of chromosome 2H ch L in grain carotenoid content has also been revealed in the new cereal tritordeum (amphiploid derived from a cross between the wild barley H. chilense and durum wheat), which has higher carotenoid pigment content in its grain than durum or bread wheat [45,46]. Chromosome 7H ch from H. chilense confers the capacity to accumulate higher carotene concentration in seeds [15]. The Psy1 gene controlling the first step of the carotenoid biosynthetic pathway was mapped to 7H ch S [47]. Wheat-H. chilense chromosome 7H ch introgression lines have been developed [17,18,48], and all the genetic stocks carrying Psy1 from H. chilense show increased carotenoid content relative to common wheat [48,49]. The obtention in this work of translocation T7H ch S·2H ch L could be of interest for studying the effect of the Psy1 gene located on the 7H ch S arm and both the Hyd3 and Ppo1 genes mapped to the long arm of chromosome 2H ch [20]. Furthermore, the lines carrying T2H ch S·2DL and T2H ch S·2DL + T7H ch S·D translocations could be of interest for studying the effect of both the Ggpps1 and Zds genes mapped to the short arm of 2H ch , in the absence and presence of the Psy1 gene from H. chilense, respectively.
The transference of desirable genes from wild relatives to wheat can be restrict by linkage drag and the lack of compensation for the wheat chromatin substituted. The identification of alien chromosomal regions carrying the genes of interest and the analysis of their homoeologous relationships with wheat chromosomes can overcome that difficulty. It has been pointed out that only translocations produced by homoeologous recombination are beneficial for wheat improvement [50,51]. In this work, wheat-H. chilense macrosyntenic comparisons using COS markers revealed that H. chilense chromosome 2H ch exhibits good synteny with wheat homoeologous group 2 chromosomes. Comparative mapping of carotenoid-related genes mapped to chromosome 2H ch also showed good collinearity between H. chilense and Triticeae species [20,46]. The 24 COS markers assigned to chromosome 2H ch in this work will facilitate introgression of alien genes associated with this chromosome into wheat.

Conclusions
We used in situ hybridization and genotyping to characterize genetic stocks harboring chromosome 2H ch from H. chilense in a wheat background. As far as we know, no previous translocation lines involving chromosome 2H ch introgressed into wheat have been described. The cytogenetic stocks developed here may constitute an important resource for studying the effect of chromosome 2H ch on wheat grain color. In addition, these cytogenetic stocks allowed the localization of a set of conserved orthologous set (COS) markers to specific arms in chromosome 2H ch .The genomic position of orthologous unigene EST-contigs used for the COS marker design revealed a good macrosyntenic relationship between H. chilense chromosome 2H ch and wheat homoeologous group 2. The new wheat-H. chilense recombinations are useful for genetic studies and might also serve as a bridge for transferring genes associated with yellow pigment into a wheat background.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/9/9/493/s1, File S1: COS markers used in this work together with their primer sequences, File S2: Source of the COS markers assigned to the H. chilense chromosome 2H ch , File S3: Results of BLASTn search for COS markers assigned to H. chilense chromosome 2H ch in the reference sequences of hexaploid wheat group 2 chromosomes (www.wheatgenome.org/) and the start positions (bp) of the marker-specific ESTs.