Next Article in Journal
Uric Acid and Oxidative Stress—Relationship with Cardiovascular, Metabolic, and Renal Impairment
Next Article in Special Issue
ASTool: An Easy-to-Use Tool to Accurately Identify Alternative Splicing Events from Plant RNA-Seq Data
Previous Article in Journal
The Hematopoietic TALE-Code Shows Normal Activity of IRX1 in Myeloid Progenitors and Reveals Ectopic Expression of IRX3 and IRX5 in Acute Myeloid Leukemia
Previous Article in Special Issue
Deciphering the Host–Pathogen Interactome of the Wheat–Common Bunt System: A Step towards Enhanced Resilience in Next Generation Wheat
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 6
10.3390/ijms23063191
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Draft Sequencing Crested Wheatgrass Chromosomes Identified Evolutionary Structural Changes and Genes and Facilitated the Development of SSR Markers

by
Jana Zwyrtková
,
Nicolas Blavet
,
Alžběta Doležalová
,
Petr Cápal
,
Mahmoud Said
,
István Molnár
,
Jan Vrána
,
Jaroslav Doležel
and
Eva Hřibová
*
Institute of Experimental Botany of the Czech Academy of Sciences, Centre of the Region Haná for Biotechnological and Agricultural Research, CZ-77900 Olomouc, Czech Republic
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2022, 23(6), 3191; https://doi.org/10.3390/ijms23063191
Submission received: 15 February 2022 / Revised: 13 March 2022 / Accepted: 14 March 2022 / Published: 16 March 2022
(This article belongs to the Special Issue Plant Biology and Biotechnology: Focus on Genomics and Bioinformatics)
Browse Figures
Review Reports Versions Notes

Abstract

:
Crested wheatgrass (Agropyron cristatum), a wild relative of wheat, is an attractive source of genes and alleles for their improvement. Its wider use is hampered by limited knowledge of its complex genome. In this work, individual chromosomes were purified by flow sorting, and DNA shotgun sequencing was performed. The annotation of chromosome-specific sequences characterized the DNA-repeat content and led to the identification of genic sequences. Among them, genic sequences homologous to genes conferring plant disease resistance and involved in plant tolerance to biotic and abiotic stress were identified. Genes belonging to the important groups for breeders involved in different functional categories were found. The analysis of the DNA-repeat content identified a new LTR element, Agrocen, which is enriched in centromeric regions. The colocalization of the element with the centromeric histone H3 variant CENH3 suggested its functional role in the grass centromere. Finally, 159 polymorphic simple-sequence-repeat (SSR) markers were identified, with 72 of them being chromosome- or chromosome-arm-specific, 16 mapping to more than one chromosome, and 71 mapping to all the Agropyron chromosomes. The markers were used to characterize orthologous relationships between A. cristatum and common wheat that will facilitate the introgression breeding of wheat using A. cristatum.

1. Introduction

Crested wheatgrass (Agropyron cristatum (L.) Gaertn.) belongs to the family Poaceae and the tribe Triticeae. It is a perennial, low-maintenance grass mainly used as forage and is grown worldwide [1,2]. Wheatgrasses are resistant to drought and cold [3,4,5,6], tolerant to environmental stress [7,8,9] and cadmium [10], and moderately tolerant to salinity. Wheatgrasses exhibit large genetic variation, which, together with their resistance to drought and biotic and abiotic stress, makes them a potential source of new genes for crop improvement [7,11,12]. Most Agropyron species are autotetraploids (2n = 4x = 28, PPPP) and can be found across Europe and Asia. Diploids (2n = 2x = 14, PP) are located on the same continents, excluding Turkey, Iran, and Georgia. Hexaploid species (2n = 6x = 42, PPPPPP) are less common and are predominantly located in Turkey, Iran, and Georgia [8]. Several artificial hybrids between common wheat (Triticum aestivum) and A. cristatum have been developed [13,14], and the introgression lines [15,16,17,18] have been used to improve wheat, for example, to increase the number of florets and kernels and enhance the grain weight and spike length [16,19].
The development of alien introgression lines, which involves hybridization and backcrossing with parental lines, is an important step in transferring useful genes and alleles from wild relatives to wheat [20]. A number of wheat–Agropyron chromosome addition and translocation lines have been developed and are used in wheat pre-breeding programs [17,21,22,23,24,25]. Wheat–Agropyron translocation lines bearing leaf-rust resistance, chromosomal loci in A. cristatum, conferring resistance to powdery mildew, and Agropyron chromosomes responsible for increased numbers of florets and kernels were recently identified [17,19,26]. The introgressions of genes of interest from wild relatives to important crops are hampered by linkage drag and extra chromatin parts that are transferred with the main trait loci [27]. Thus, the availability of molecular tools specific for Agropyron that can be used for the rapid characterization of breeding progeny is essential.
Several types of molecular markers originally developed for wheat, such as EST, STS, and COS markers, have also been applied for the characterization of A. cristatum [28,29,30]. Simple-sequence repeats (SSRs), or microsatellites, are a common and informative type of molecular marker, with high reproducibility and transferability and polymorphic patterns. They are one of the most informative genetic markers [31] used for the integration of genetic and physical maps [32] and for the ordering of genome scaffolds to larger genome-scale assemblies [33]. SSR markers have been widely used in association studies [34] and to analyze the genetic variability of natural populations of different plant species [34,35,36,37,38], as well as for fingerprinting and determining the genetic similarity of breeding lines [39]. One of the greatest advantages of SSR markers is their co-dominant character and their simple use as PCR-based markers.
Given the large genome size of A. cristatum (1C~6.3 Gbp) [40], its dissection into individual chromosomes by flow sorting could simplify genomic analyses [41]. This approach has facilitated the draft genome sequencing of barley, rye, wheat, chickpeas, and peas [42,43,44,45,46]. Sequencing data obtained from individual chromosomes or chromosomal arms can be used to identify genes of interest and to analyze genome structure through the creation of draft assemblies [43,44,47]. Chromosome-specific sequencing data were also used to develop different types of molecular markers and to map agronomic traits of interest [43,48,49,50]. However, obtaining chromosome-specific sequences may be hampered by difficulties in discriminating and sorting the chromosomes of interest. To overcome this obstacle, wheat-alien chromosome-addition lines have been used to separate individual chromosomes or chromosomal arms [19,25]. Alternatively, single chromosomes have been sorted from lines with standard karyotypes, followed by single-chromosome amplification [51].
In this work, we report on developing new genomic sources for A. cristatum. These include molecular markers to provide insights into the genome organization of A. cristatum and facilitate the introgression breeding of wheat.

2. Results

2.1. Chromosome Sorting and the Creation of Partial Assemblies

A. cristatum chromosomes 2P, 3P, 5P, and 6P were flow-sorted from bread wheat–A. cristatum addition lines at high purities, ranging from 81% (6P) to 98% (3P, 5P) (Table 1, Figure S1B,D) and then amplified by whole-genome amplification [52]. Chromosomes 1P and 4P, which could not be discriminated from bivariate flow karyotypes (Figure S1A,C), were sorted from cytogenetic stocks in a single-chromosome mode and used for single-chromosome DNA amplification [51]. Chromosome 7P was flow-sorted in a single-chromosome mode from a composite peak representing chromosomes 7P and 2P of the diploid genotype of A. cristatum cv. Parkway. Single-chromosome amplification [51] was followed by the identification of fractions of chromosomes 1P, 4P, and 7P. The first two chromosomes were identified using PCR markers targeting Agropyron-specific retroelements and chromosome 7P by PCR markers developed from cDNA sequences [40]. Each chromosome was amplified in at least four independent reactions to avoid DNA-amplification bias. Illumina sequencing resulted in 23.5×–42.8× coverage by the sequencing data, which were assembled separately for individual chromosomes. As expected, the quality of the partial chromosome assemblies differed between individual chromosomes (Table 1). Chromosomes 1P, 4P, and 7P, the sequencing libraries of which were prepared from DNA amplified in single-chromosome mode [51], resulted in assemblies of lower quality and represented only 4.3% to 15.4% of whole chromosome lengths according to Said et al. [40] (Table 1). The assemblies specific to chromosomes 2P, 3P, 5P, and 6P, which were sorted and amplified according to Šimková et al. [52], represented a higher percentage of their chromosome lengths (Table 1).

2.2. Genic Sequences Identified in the Assemblies

Partial chromosome-specific contigs and scaffolds enabled the identification of sequences homologous to important genes. Using the MAKER-P pipeline [53], we identified 45,126 coding and noncoding sequences and 44,814 coding/genic sequences (Table 1). The highest representation of genic sequences was found for chromosome 4P (~12%), and the lowest for chromosome 7P (~3%) (Table S1), whereas the number of genic sequences per chromosome ranged from 1470 in chromosome 4P to 11,694 in chromosome 5P. The functional annotation provided a putative function for about 15% of the total number of these sequences. Of 20,616 gene families, most (15,877) were connected to proteins of unknown function. From the remaining 4,739 gene families with known function, two families were the most frequent: a family of kinase proteins, which included the rust-resistance kinases Lr10, and a cluster of germin-like proteins were found to be most abundant, especially in the genomic data for chromosome 3P (Figure 1). The most abundant genic sequences with a predicted function, which were annotated for all chromosomes, are summarized in Table S2. Genes connected to forms of biotic resistance, such as the rust-resistance kinase Lr10 (14 copies) and disease-resistance protein Piks-2 (three copies), were the most frequent on chromosome 3P, and the multidrug-resistance protein (four copies) was the most frequent on 4P. On the other hand, only a single copy of genes for tolerance, such as metal-tolerance protein C4 and 1, were found on chromosome 2P, and metal tolerance protein 7 and the chloroplast enhancing stress tolerance protein were found on chromosome 3P. Finally, genes increasing the fitness of the plant, such as abscisic stress-ripening protein, were found on chromosomes 2P, 7P (each with a single copy), and 3P (eight copies); heat-stress transcription factors were found on 2P, 3P, 6P (each with two copies), and 5P (seven copies); the zinc-finger-domain-containing stress-associated protein was found on 2P (single copy) and 5P (two copies); the salt stress root protein was found on 3P (single copy); transcription factors of the WRKY family were found on chromosome 3P (four copies) and 5P (three copies); and between two and ten copies of the transcription factors of the MYB family were found on each chromosome except for 4P (Table S3).
Despite the low quality of the chromosome assemblies, sequences homologous to genes encoding agronomically important traits that can be utilized for wheat improvement were identified. Almost a complete sequence of the β-glucan-biosynthesis genes HvCslF10 and HvCslF3 was identified in A. cristatum data specific to chromosomes 2P and 3P; the arabinoxylan biosynthesis gene TaGT47-13 was identified on 3P; and the bread-making-quality genes puroindoline A and B were identified on chromosome 5P. Genic sequences homologous to the leaf-rust-resistance gene Lr1 were identified in the data for chromosome 5P, and the salt-tolerance genes BP2A, SOS1, and HKT1 were found on chromosomes 3P and 7P. Sequences homologous to the vernalization locus VRN2 were identified on chromosome 2P.
On chromosomes 2P and 5P, 5S rRNA genic sequences were found, whereas 45S rRNA genic sequences were found on chromosomes 1P, 4P, and 5P. The average representation of genic sequences on individual chromosomes was about ~5% of the assembly length.

2.3. Repetitive DNA Sequences Identified in the Assemblies

Repetitive DNA accounted for ~80% of the assemblies, excluding specific sequences, of 1P, which contained ~65% of DNA repeats, and 4P, with ~40% of repeats. To annotate repeats in the assemblies, public repeat databases (GenBank and Repbase), as well as the database of repeats identified and characterized in A. cristatum in our previous study [40], were used. As expected, DNA repeats were represented mainly by different types of transposable elements, and out of them, the Athila element of the Ty3/gypsy family was the most abundant (Table S1, Figure S2A). The proportion of individual types of transposable elements, as well as tandem organized repeats in chromosome assemblies, was similar to that in our previous study on the global analysis of repetitive DNA sequences identified in partial genomic Illumina sequence reads of A. cristatum cv. Parkway [40].
Our previous study revealed the presence of a relatively high number of tandem organized repeats, which were used as probes for FISH to identify wheatgrass chromosomes in situ [41]. Given that none of the tandem repeats were specifically localized to the centromeric regions of wheatgrass chromosomes, we speculated that centromeres of wheatgrass were enriched for specific LTR elements, similar to other related species in the Poaceae family [54]. Thus, we performed homology searches and phylogenetic analysis of Cereba-like elements, the most probable candidate of centromere-specific retroelements in Gramineae [54,55,56,57]. Complete sequences homologous to Cereba-like elements were identified in many scaffolds of all the chromosomes of Agropyron and were called Agrocen LTR elements. A phylogenetic analysis of the RT domain of Agrocen LTR elements confirmed their close relationship to centromeric retrotransposons (Figure S2B; [57]). To confirm their location in the A. cristatum genome, FISH with probes specific to the RT domain of the most abundant Agrocen element was carried out. This work confirmed the presence of the Agrocen element in the centromeric regions of all the Agropyron chromosomes. Moreover, a combination of FISH and immunostaining with the grass centromere-specific histone H3 (CENH3; [58]) resulted in strong, overlapping signals (Figure 2).

2.4. Development of SSR Markers

The MISA program identified more than 134,000 SSRs with at least a 12-bp-long repetitive region containing 2- to 6-nt repetitive units. A majority of the SSRs were identified in the assemblies of chromosomes 2P, 3P, 5P, and 6P. The lowest number of SSRs (1136) was identified in the assembly of chromosome 4P, which represents only ~4% of its chromosome length (Table 1).
To increase the probability of identifying unique chromosome-specific markers, Agropyron scaffolds containing SSRs were aligned to the reference genome sequence of T. aestivum [46]. Scaffolds of Agropyron that mapped to a unique position in the wheat D-subgenome were then used to identify chromosome-specific SSRs. These SSRs were selected based on their positions in different locations along collinear chromosomes of the wheat D-subgenome and further verified by PCR on a set of wheat–Agropyron addition lines. Using this approach, we selected sets of 29 to 50 SSRs specific for each Agropyron chromosome (Table S4).

2.5. Experimental Verification of Newly Designed SSR Markers

In total, 250 SSR primer pairs were selected for experimental verification. Out of them, 159 were found to be specific to Agropyron, including 16 that showed a polymorphic pattern (different lengths of the amplified PCR products) between Agropyron and bread wheat. A total of 72 SSR markers were specific for only one Agropyron chromosome, and 53 were specific for the chromosomal arm. There were 16 SSR markers that mapped to more than one chromosome or chromosomal arm and 71 SSRs that mapped to all the Agropyron chromosomes. Newly developed SSR markers were marked as “olomouc crested wheatgrass markers” (ocwgm).
The use of wheat chromosome-addition lines containing Agropyron chromosomal arms enabled us to assign some of the new SSR markers more precisely (Table 2, Figure 3). We identified six new SSR markers specific for chromosome 1P, with one of them unambiguously mapping on TH4 (1PS + 1BL) addition lines containing the short arm of chromosome 1P. In total, 13 SSRs mapped to chromosome 2P; out of them, 6 SSRs also mapped to the 2PS addition line and 6 other SSRs mapped to the 2PL addition line. The marker ocwgm019, which was found to be specific to chromosome 2P, also gave an amplification product on both the 2PS and 2PL cytogenetic lines. Out of the 14 SSRs specific to chromosome 3P, 9 were mapped to the 3PS addition line. Eleven SSR markers were found to be specific for chromosome 4P, with five of them also localizing to the 4PS addition line. Only four new SSR markers were found to be specific to chromosome 5P, with three of them mapping to the 5PL addition line. Out of the 15 SSRs specific to chromosome 6P, 8 were found on 6PS and 3 others on 6PL addition lines. Four markers that were unambiguously mapped to the addition line of chromosome 6P were not amplified on cytogenetic lines of 6PS or on 6PL. Finally, nine new SSR markers were found to be specific to chromosome 7P (the results are listed in Table S4). As the addition line of chromosome 7P was not available to us, 7P-specific SSR markers were considered those that gave a specific product on Agropyron genomic DNA and simultaneously did not provide any positive PCR product on wheat–Agropyron addition lines. Given the fact that the wheat–Agropyron addition lines were created from different Agropyron cultivars, which could be genetically variable, we cannot exclude the possibility that some of the newly developed SSR markers could be absent on chromosome 7P.
Fourteen SSR markers were mapped to the addition line 3P + 3PS (the addition line including chromosome 3P and the extra short chromosomal arm 3PS), and out of them, five SSRs were missing in the addition line specific to 3PS; therefore, these SSR markers are most probably localized on the long chromosomal arm of 3P, the addition line of which was unavailable. A similar situation occurred during the analyses of chromosomes 4P and 5P. Six SSRs provided amplification products only in the addition line for chromosome 4P and simultaneously did not provide any amplification products in the addition line of 4PS; therefore, they were assigned to 4PL. One marker that only gave an amplification product in the addition line for chromosome 5P and not for 5PL was assigned to 5PS (Table S4).

2.6. Orthologous Relationships between A. cristatum and Bread Wheat Chromosomes

Out of 72 (98.6%) single loci SSR markers, 71 showed synteny between bread wheat and the diploid A. cristatum genome (Figure 3, Table S4). Only one marker (ocwgm059) identified in the scaffold of A. cristatum, which shared homology with the genomic sequence of chromosome group 1 of wheat, was mapped onto the nonhomologous chromosome 6PL in crested wheatgrass (Figure 3). Three SSR markers specific to chromosome 2P (ocwgm007, ocwgm011, and ocwgm013) were located in opposite chromosomal arms compared to wheat subgenomes, which indicated intrachromosomal rearrangement in Agropyron compared to wheat (Figure 4A). A similar situation was observed for 11 SSRs specific to chromosome 4P (ocwgm034–ocwgm044). Their position on short and long arms indicated the same structural rearrangement as observed for wheat chromosome 4A, which has an inverted structure in comparison to wheat chromosomes 4B and 4D (Figure 4B).
Besides the SSRs pointing to intrachromosomal changes, 16 additional SSR markers found at single loci in the wheat genome were detected as duplications in the genome of the diploid wheatgrass cultivar Parkway (Table S4). For example, three markers specific to chromosome group 6 in wheat showed the duplication of 2P/6P, two markers showed the duplication of 3P/4P, and two SSRs indicated the duplication of 1P/5P/6P. Other duplicated regions in the Agropyron genome were indicated by the multiple localization of nine other SSRs, which are summarized in Table S4.
Finally, 25 out of 159 (15.7%) SSR markers were localized to scaffolds that unambiguously carried genic sequences. Ten of them localized to the specific chromosomes (2P, 3P, 5P, and 6P), and three other markers localized to chromosome 7P. Only 11 markers shared their location on the scaffold with the respective genic sequence on chromosomes 1P, 3P, 4P, 5P, 6P, and 7P (Table S5).

3. Discussion

The present study increases the range of genomic resources for A. cristatum, a potential source of important genes for wheat improvement. Chromosome-specific sequence data obtained after the flow sorting of individual chromosomes made it possible to create draft chromosome assemblies and develop new SSR markers. Chromosome-centric genomics has been instrumental for creating and/or improving the draft assemblies of species with large genomes, characterizing genic sequences, creating gene orders, and identifying specific molecular markers (as reviewed by Zwyrtková et al. [58]).
In our study, two chromosome-sorting strategies developed by Šimková et al. [52] and Cápal et al. [51] were used to sequence DNA from individual flow-sorted chromosomes of Agropyron. The strategy of Šimková et al. [52] was used to obtain DNA from many copies of chromosomes that were sorted at high purity from specific cytogenetic lines. This approach resulted in draft assemblies that represented a higher proportion of whole chromosome lengths. In comparison, draft assemblies obtained from DNA prepared from single copies of chromosomes, as described by Cápal et al. [51], were less complete, most probably due to higher bias during DNA amplification [51].
Despite the short cumulative length of the draft chromosome assemblies, we were able to identify wild alleles of agronomically important genes highlighting the importance of these genomic resources. The Agropyron variants of HvCslF10 and HvCslF3 genes on the chromosomes 2P and 3P, respectively, or the gene TaGT47-13 on 3P could be suitable targets for gene introgression programs aimed at modifying the dietary fiber composition of wheat [59]. The upregulation of transporter genes SOS1 (a plasma membrane Na+/H+ transporter) and HKT1, which mediates Na distribution between roots and shoots, is a typical response for salt stress in tolerant wheat genotypes [60]. The fact that these genes were found on chromosomes 3P and 7P, respectively, may indicate the role of these chromosomes in the salt tolerance of A. cristatum. A careful stress–physiological investigation of wheat–A. cristatum addition lines will be needed to confirm this hypothesis. Several gene homologs connected with rust resistance or genes for multidrug resistance were also identified on the chromosome assemblies of A. cristatum. In previous studies, SSR and STS molecular markers helped to localize leaf-rust resistance on the 1PS chromosome arm. The 1BL translocation line [17] and other genes of resistance to stripe rust and leaf rust were mapped to chromosome 6PS [61,62], in contrast to in our study, in which the genes homologous to rust resistance were found in draft assemblies of chromosomes 1P, 3P, and 5P. The known positions of genes on individual chromosomes of Agropyron or another alien species are necessary to simplify introgression breeding, especially for the creation of better-targeted addition lines in a breeding strategy.
The representation of repetitive DNA sequences identified in draft chromosome assemblies was in concordance with our previous study describing the characterization of the repetitive landscape of the wheatgrass genome from partial genome sequencing [40]. As in other Poaceae species, a large part of the complex genome of A. cristatum consists of different types of retrotransposons, with Ty3/gypsy elements being more abundant than Ty1/copia. Variable lineages of Ty3/gypsy elements were also found to be most abundant in closely related species, such as barley [42], rice [63], maize [64], wheat [65], oat [66], rye [67], Aegilops [68], fescues, and ryegrasses [68]. However, the genome of wheatgrass contains a relatively large number of different satellite repeats, which enabled the identification of individual chromosomes in situ [40]. Interestingly, none of the satellite repeats were found in centromeric regions. In this study, we identified and further confirmed the presence of Agrocen’s LTR element in the centromeric regions of all the wheatgrass chromosomes. Its close phylogenetic relationship to Cereba-like elements in Gramineae [54,55,56,57], together with its colocalization with histone CENH3 [69], indicated the role of Agrocen retrotransposon in centromere function.
Simple-sequence repeats or microsatellites (SSRs) are highly abundant in plant genomes and widely used in association studies and genetic-diversity studies for the fingerprinting of breeding lines or for the construction of genetic linkage maps [34,37]. In our study, we have identified and developed new Agropyron-specific SSR markers that will facilitate the rapid and easy characterization of wheat–A. cristatum addition lines. Our chromosome draft assemblies enabled the characterization of 72 chromosome-specific and/or chromosome-arm-specific SSRs, including those for chromosome 7P, expanding our previous study, in which co-dominant COS markers, originally developed for wheat [70,71], were mapped onto six out of seven chromosomes of Agropyon [30]. Moreover, we have developed Agropyron-specific markers, which have been found distributed in multiple loci on all the Agropyron chromosomes but are missing in wheat and, thus, can be used for initial screening for the presence of fragments of Agropyron genomes in newly developed wheat–A. cristatum introgression lines. In the future, the new SSR markers can be integrated into a marker-assisted selection system to follow the introgressed Agropyron chromatin during the transfer into elite cultivars. Our dataset contains many more SSRs identified in silico, which can be used for the development of additional PCR-based chromosome-specific markers or together with other types of markers, e.g., SNPs, for the creation of genetic linkage maps and to enable direct map-based cloning, QTL analysis, or anchoring physical and genetic maps.
In our study, we used newly developed chromosome-specific SSR markers to compare the organization of homologous chromosomes of Agropyron and bread wheat. This knowledge is essential for breeders to minimize and/or avoid the possibility of synapsis and chiasma formation in addition lines, which can lead to the elimination of alien (Agropyron) chromosomes [72,73]. However, the chromosomes of the tribe Triticeae are highly collinear [74], and a number of chromosomal rearrangements, including inversions and translocations, occurred during their evolution [75]. Newly developed markers confirmed the presence of paracentric inversions on chromosome 4P, which correlated with the previous study of Said et al. [40], who discovered paracentric inversion on 4P using FISH with single-copy cDNA probes. Our findings showed that the structure of chromosome 4P of wheatgrass is collinear with wheat chromosome 4A, which has an inverted structure with respect to chromosomes 4B and 4D [74]. Our results also indicate a short shift in the centromeric region of chromosome 2P and the presence of the non-reciprocal translocation of the pericentromeric region of 1PL to 6PL (Figure 3, Table S4).
Newly developed SSR markers provided important information about the structure of the chromosomal arms present in wheat–Agropyron telosomic addition lines. Our results show that the additional line of chromosomes 6PS and 6PL did not contain whole chromosomal arms and that a part of the centromeric region was missing. This can be explained by the unequal breakage of chromosome 6P and the transfer of incomplete parts into the wheat background or the use of different, structurally heterozygous genotypes for transferring the short and long arms of chromosome 6P into bread wheat. A similar situation was detected for chromosome 2P. Similarly, the amplification of several SSR markers on cytogenetic lines containing chromosome arms 2PS and 2PL most probably indicates that these additional lines consist of overlapping centromeric regions. Our results point to the fact that specific wheat–Agropyron addition lines were created from different tetraploid cultivar(s) of wheatgrass and illustrate their genomic variability [76]. Due to the cross-pollinating nature of Agropyron, structural variability is also typical for its diploid species [40].
The application of other genomic tools and approaches will be needed to analyze the chromosome structural variation in Agropyron more precisely [77]. Although Hi-C and Bionano optical mapping technologies are suitable for the comparison of organisms with highly similar DNA sequences, e.g., different plant cultivars [78,79], the creation of high-density genetic maps or the use of third-generation sequencing technologies, such as Oxford Nanopore, could enable the comparison of the structure of chromosomes between different related species [80,81,82,83,84].

4. Materials and Methods

4.1. Plant Material and the Isolation of Genomic DNA

Seeds of crested wheatgrass (Agropyron cristatum) cv. Parkway (2n = 2x = 14, PP genome) were provided by Dr. Joseph Robins (ARS Forage and Range Research Laboratory, USDA, Logan, UT, USA). Seeds of common wheat (Triticum aestivum) cv. Chinese Spring were provided by Dr. Pierre Sourdille (INRA, Clermont-Ferrand, France). Seeds of wheat (cv. Chinese Spring)–A. cristatum disomic chromosome addition lines 1P, 2P, 3P, 4P, 5P, and 6P and wheat (cv. Chinese Spring)–A. cristatum telosome addition lines 2PS, 2PL, 4PS, 5PL, 6PS, and 6PL were produced by Chen et al. [21,22] and provided to us by Dr. Adoración Cabrera (University of Córdoba, Córdoba, Spain). Seeds of wheat–A. cristatum disomic chromosome 3P addition lines were renamed the 3P + 3PS line in this work, following the results of Said et al. [30]. The wheat (cv. Chinese Spring)–A. cristatum 3PS telosome addition line was developed by Said et al. [30]. Seeds of wheat (cv. Chinese Spring)–A. cristatum with the Robertsonian translocation line TH4, comprising the long arm of wheat chromosome 1B and the short arm of chromosome 1PS of tetraploid A. cristatum, were developed by Ochoa et al. [17] and provided to us by Dr. Adoración Cabrera (University of Córdoba, Córdoba, Spain). Genomic DNA was isolated from the young leaf tissues of plants grown under controlled temperature, light, and humidity regimes using the NucleoSpin® Plant II kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), following the manufacturer’s instructions.

4.2. Chromosome Sorting and the Amplification of DNA

A flow cytometric analysis of suspensions of intact mitotic metaphase chromosomes and chromosome sorting was performed as described by Said et al. [85]. Chromosomes 1P, 2P, 3P, 4P, 5P, and 6P were flow-sorted from wheat–Agropyron addition lines, whereas the chromosome 7P, for which the chromosome addition line was not available, was sorted from A. cristatum cv. Parkway in a single-chromosome mode [51]. Three independent batches of 100,000 copies of chromosomes 2P, 3P, 5P, and 6P were flow-sorted and checked for identity and contamination using genomic in situ hybridization (GISH). Their DNA was amplified using an Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Chicago, IL, USA) following Šimková et al. [52]. The amplification products from each chromosome were combined to reduce the potential amplification bias. Chromosomes 1P and 4P were sorted as single copies from respective addition lines, and chromosome 7P was sorted from diploid cultivar Parkway. The DNA of chromosomes 1P, 4P, and 7P was amplified using the single-chromosome amplification procedure according to Cápal et al. [51]. Their identity was assessed using a PCR marker targeting Agropyron-specific retroelements [40] prior to sequencing. To identify chromosome 7P, DNA samples were checked using a set of PCR-based markers developed from cDNA sequences, which were previously mapped to chromosome 7P [40]. Five independent and PCR-confirmed amplified products of chromosomes 1P and 7P and six products of chromosome 4P were pooled and used for Illumina sequencing.

4.3. Illumina Sequencing

Illumina libraries were prepared from amplified DNA for each chromosome separately using a TruSeq® DNA PCR-Free High Throughput Library Prep Kit (Illumina, San Diego, CA, USA). Chromosomes 2P and 6P were paired-end sequenced on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA) with 300 bp PE reads in several runs to achieve a minimal output of ~20× sequencing depth for each chromosome. Chromosomes 1P, 3P, 4P, 5P, and 7P were sequenced on the HiSeq 2500 instrument in 250 bp PE Rapid Run mode to achieve a minimal output of ~20× sequencing depth for each chromosome. The sequence reads were deposited in the Sequence Read Archive (BioProject ID: PRJNA801633; accessions SAMN25358569, SAMN25358571, SAMN25358572, SAMN25358575, SAMN25358577, SAMN25358579, SAMN25358581, SAMN25358582, and SAMN25358585). Assembled contigs and scaffolds specific for individual wheatgrass chromosomes are publicly available in the Dryad Digital Repository: https://doi.org/10.5061/dryad.gqnk98spv (accessed on 12 February 2022).

4.4. De Novo Assembly, Identification, and Verification of Chromosome-Specific SSR Markers

Raw datasets obtained after the Illumina sequencing of the seven A. cristatum chromosomes were trimmed to the appropriate length and quality (-q 20 -p 90) using Trimmomatic [86] and assembled using a Ray de novo assembler [87] with the following settings: a k-mer length of 75, a minimum contig length of 100 bp.
The identification of SSR markers was carried out using the microsatellite identification tool (MISA) [88], and the primers for the PCR amplification of the identified SSR markers were designed using Primer3 [89]. With the aim of increasing the probability of identifying chromosome-specific markers, Agropyron chromosomal scaffolds containing unique SSRs were mapped onto the wheat genome sequence assembly RefSeq v1.0 [46] using Gmap [90]. Based on the collinearity with bread wheat, a set of SSR markers that mapped to unique sites on the wheat D-subgenome were selected and further verified using PCR.
The PCR contained 20 ng of template DNA, a 1.25 µM mix of forward and reverse primers, 200 µM dNTPs, 1 mM MgCl2, and 2U/100 µL of the final reaction’s volume Taq polymerase with 1× Taq buffer (New England Biolabs, Ipswich, MA, USA) and was performed under the following conditions: 94 °C for 5 min, 35× (94 °C for 50 s, an appropriate annealing temperature for each primer pair for 50 s, and 72 °C for 50 s), and 72 °C for 5 min. Genomic DNA was isolated from wheat–Agropyron telosome addition and translocation lines, diploid A. cristatum cv. Parkway, and T. aestivum cv. Chinese Spring for use as the template DNA. The best annealing conditions were chosen after gradient PCR. The PCR products were analyzed by electrophoresis on a 2% agarose gel.

4.5. Annotation of Chromosome-Specific Sequences

The annotation of A. cristatum scaffolds was performed using the MAKER-P pipeline [53]. The use of the pipeline comprised two steps. The first step involved masking the genome using RepeatMasker [91] and the de novo reconstruction of DNA repeats of A. cristatum (https://olomouc.ueb.cas.cz/en/content/dna-repeats (accessed on 12 February 2022)); [40]. The resulting annotation of repeated elements was then added as an input GFF file for the second step. Genic sequences were identified using the gene-prediction tools AUGUSTUS [92] and FGENESH (http://www.softberry.com (accessed on 12 February 2022)) with the wheat and monocot models, respectively. The genic sequence prediction was supported by protein homologies with Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa, Triticum aestivum, Sorghum bicolor, Zea mays, and Aegilops tauschii, the sequences of which were retrieved from Ensembl Plants release 45 [93], and Hordeum vulgare proteins from the second assembly version [94]. The resulting potentially expressed sequences and proteins were then filtered to keep only the longest splicing variant for each genic sequence.
Following the MAKER-P pipeline, the functional annotation of the genic sequences was performed using InterProScan version 5.36-75.0 [95] and BLAST [96] results with the UniProt/SwissProt version from 2019/08 [97]. The clustering of genic sequences into functional families was conducted using SiLiX [98].

4.6. Immunostaining of Interphase Nuclei

BLASTN homology searches and further phylogenetic analysis of reverse transcriptase domains of representatives of Ty3/gypsy elements [57] and repetitive DNA sequences characterized in our previous study [40] were performed according to Novák et al. [99]. Multiple sequence alignment of reverse transcriptase domains was done with MAFFT v7.029 (–globalpair –maxiterate 1000) [100]. Phylogenetic trees were constructed with PhyML 3.0 [101] implemented in SeaView v5.0.2 [102]. The approximate likelihood ratio test [103] was performed to assess branch support. The phylogram was depicted in FigTree (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 12 February 2022)). The Agropyron-specific DNA element, which was identified as the closest relative to the Cereba element, was selected and used for the creation of the cytogenetic probe, which was co-localized with the anti-CENH3 antibody.
The cell nuclei of A. cristatum cv. Parkway were flow-sorted into PRINS buffer supplemented with 2.5% sucrose [104]. Microscopic slides with the sorted nuclei were stored at room temperature overnight. Fluorescence in situ hybridization (FISH) and immunostaining were carried out according to [68], with minor modifications: probes derived from the reverse transcriptase (RT) domain of the centromeric element were labeled with biotin-16-dUTP (Roche Applied Science) using PCR with specific primers (forward: 5′-GATGGTACGTCGCGTATGTG-3′; reverse: 5′-CGTCCAATGAAAGCACGTAA-3′); the primary antibody anti-CENH3 [105] was diluted 1:125, and the secondary antibodies (anti-rabbit antibodies STAR 635P, ST635-1002-500UG, Abberior, Göttingen, Germany) were diluted 1:200. The slides were examined with a confocal microscope (Leica Microsystems, Wetzlar, Germany) with appropriate laser lines, and images were prepared with Leica Application Suite X (LAS-X) software version 3.5.5 with the Leica Lightning module (Leica, Buffalo Grove, IL, USA). The pictures were deconvoluted with the Leica Lightning module based on the refractive index of the mounting medium. The final images were arranged in Adobe Photoshop 12.0 (Adobe Systems Corporation, San Jose, CA, USA).

5. Conclusions

Our work has created new genomic resources and increased the knowledge about the genome/chromosome structure of A. cristatum. Chromosome draft assemblies were created, and regions homologous to agronomically important genes were identified. The classification of repetitive DNA sequences revealed the centromere-specific LTR element Agrocen, whose colocalization with histone CENH3 indicates its role in centromere function. Simple-sequence repeats were identified in chromosome draft assemblies, and 72 new SSR markers specific to all seven chromosomes or chromosome arms of Agropyron were developed and used to compare the homologous chromosome structures between Agropyron and bread wheat. We demonstrate the potential of newly developed SSR markers for the rapid characterization of wheat–A. cristatum introgression lines to accelerate introgression breeding programs.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms23063191/s1.

Author Contributions

E.H. and J.D. conceived the project. J.Z., N.B., A.D., P.C., M.S. and J.V. conducted the study and processed the data. J.Z., E.H. and J.D. wrote the manuscript. J.Z., E.H., N.B., A.D., I.M. and J.D. discussed the results and contributed to the writing of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the ERDF project “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827), the Ministry of Education, Youth, and Sports of the Czech Republic.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The sequence reads were deposited in the Sequence Read Archive (BioProject ID: PRJNA801633; accessions SAMN25358569, SAMN25358571, SAMN25358572, SAMN25358575, SAMN25358577, SAMN25358579, SAMN25358581, SAMN25358582, and SAMN25358585). Assembled contigs and scaffolds specific for individual wheatgrass chromosomes are publicly available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.gqnk98spv (accessed on 12 February 2022).

Acknowledgments

We thank Romana Šperková, Zdeňka Dubská, and Helena Tvardíková for excellent technical support and Petr Navrátil for excellent IT support. We are grateful to Joseph Robins (ARS Forage and Range Research Laboratory, USDA, Logan, UT, USA), Pierre Sourdille (INRA, Clermont-Ferrand, France), and Adoración Cabrera (University of Córdoba, Córdoba, Spain) for providing the plant material. The computational resources were supplied by the project “e-Infrastruktura CZ” (e-INFRA CZ LM2018140), which is supported by the Ministry of Education, Youth, and Sports of the Czech Republic and by the ELIXIR CZ project (LM2018131), part of the international ELIXIR infrastructure.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Asay, K.H.; Jensen, K.B. Wheatgrasses. Cool.-Seas. Forage Grasses 1996, 34, 691–724. [Google Scholar] [CrossRef]
  2. Asay, K.H.; Chatterton, N.J.; Jensen, K.B.; Jones, T.A.; Waldron, B.L.; Horton, W.H. Breeding Improved Grasses for Semiarid Rangelands. Arid. Land Res. Manag. 2003, 17, 469–478. [Google Scholar] [CrossRef]
  3. Zhang, J.; Liu, W.; Han, H.; Song, L.; Bai, L.; Gao, Z.; Zhang, Y.; Yang, X.; Li, X.; Gao, A.; et al. De novo transcriptome sequencing of Agropyron cristatum to identify available gene resources for the enhancement of wheat. Genomics 2015, 106, 129–136. [Google Scholar] [CrossRef] [PubMed]
  4. Dewey, D.R. The Genomic System of Classification as a Guide to Intergeneric Hybridization with the Perennial Triticeae; Springer: Boston, MA, USA, 1984; pp. 209–279. [Google Scholar] [CrossRef]
  5. Limin, A.E.; Fowler, D.B. Cold hardiness of forage grasses grown on the Canadian prairies. Can. J. Plant Sci. 1987, 67, 1111–1115. [Google Scholar] [CrossRef]
  6. Asay, K.H.; Johnson, D.A. Genetic Variances for Forage Yield in Crested Wheatgrass at Six Levels of Irrigation. Crop Sci. 1990, 30, 79–82. [Google Scholar] [CrossRef]
  7. Dong, Y.S.; Zhou, R.H.; Xu, S.J.; Li, L.H.; Cauderon, Y.; Wang, R.R.-C. Desirable characteristics in perennial Triticeae collected in China for wheat improvement. Hereditas 1992, 116, 175–178. [Google Scholar] [CrossRef]
  8. Copete, A.; Moreno, R.; Cabrera, A. Characterization of a world collection of Agropyron cristatum accessions. Genet. Resour. Crop Evol. 2018, 65, 1455–1469. [Google Scholar] [CrossRef]
  9. Li, H.; Lv, M.; Song, L.; Zhang, J.; Gao, A.; Li, L.; Liu, W. Production and identification of wheat–Agropyron cristatum 2P translocation lines. PLoS ONE 2016, 11, e0145928. [Google Scholar] [CrossRef] [Green Version]
  10. Guo, Q.; Meng, L.; Mao, P.C.; Tian, X.X. An assessment of Agropyron cristatum tolerance to cadmium contaminated soil. Biol. Plant. 2014, 58, 174–178. [Google Scholar] [CrossRef]
  11. Asay, K.H. Breeding potentials in perennial Triticeae grasses. Hereditas 2008, 116, 167–173. [Google Scholar] [CrossRef]
  12. Han, H.; Liu, W.; Zhang, J.; Zhou, S.; Yang, X.; Li, X.; Li, L. Identification of P genome chromosomes in Agropyron cristatum and wheat–A. cristatum derivative lines by FISH. Sci. Rep. 2019, 9, 9712. [Google Scholar] [CrossRef] [Green Version]
  13. Chen, Q.; Jahier, J.; Cauderon, Y. Production and cytogenetic analysis of BC1, BC2, and BC3 progenies of an intergeneric hybrid between Triticum aestivum (L.) Thell. and tetraploid Agropyron cristatum (L.) Gaertn. Theor. Appl. Genet. 1992, 84, 698–703. [Google Scholar] [CrossRef]
  14. Limin, A.E.; Flower, D.B. An interspecific hybrid and amphiploid produced from Triticum aestivum crosses with Agropyron cristatum and Agropyron desertorum. Genome 1990, 33, 581–584. [Google Scholar] [CrossRef]
  15. Soliman, M.H.; Cabrera, A.; Sillero, J.C.; Rubiales, D. Genomic constitution and expression of disease resistance in Agropyron cristatum x durum wheat derivatives. Breed. Sci. 2007, 57, 17–21. [Google Scholar] [CrossRef] [Green Version]
  16. Zhang, J.; Zhang, J.; Liu, W.; Han, H.; Lu, Y.; Yang, X.; Li, X.; Li, L. Introgression of Agropyron cristatum 6P chromosome segment into common wheat for enhanced thousand-grain weight and spike length. Theor. Appl. Genet. 2015, 128, 1827–1837. [Google Scholar] [CrossRef]
  17. Ochoa, V.; Madrid, E.; Said, M.; Rubiales, D.; Cabrera, A. Molecular and cytogenetic characterization of a common wheat–Agropyron cristatum chromosome translocation conferring resistance to leaf rust. Euphytica 2015, 201, 89–95. [Google Scholar] [CrossRef]
  18. Zhang, Z.; Han, H.; Liu, W.; Song, L.; Zhang, J.; Zhou, S.; Yang, X.; Li, X.; Li, L. Deletion mapping and verification of an enhanced-grain number per spike locus from the 6PL chromosome arm of Agropyron cristatum in common wheat. Theor. Appl. Genet. 2019, 132, 2815–2827. [Google Scholar] [CrossRef]
  19. Wu, J.; Yang, X.; Wang, H.; Li, H.; Li, L.; Li, X.; Liu, W. The introgression of chromosome 6P specifying for increased numbers of florets and kernels from Agropyron cristatum into wheat. Theor. Appl. Genet. 2006, 114, 13–20. [Google Scholar] [CrossRef]
  20. Hao, M.; Zhang, L.; Ning, S.; Huang, L.; Yuan, Z.; Wu, B.; Yan, Z.; Dai, S.; Jiang, B.; Zheng, Y.; et al. The resurgence of introgression breeding, as exemplified in wheat improvement. Front. Plant Sci. 2020, 11, 252. [Google Scholar] [CrossRef] [Green Version]
  21. Chen, Q.; Jahier, J.; Cauderon, Y. Production and cytogenetical studies of hybrids between Triticum aestivum L. Thell and Agropyron cristatum (L.) Gaertn. Comptes. Rendus. l’Acad. Des. Sci. Série 3 Sci. La Vie 1989, 308, 425–430. [Google Scholar]
  22. Chen, Q.; Lu, Y.L.; Jahier, J.; Bernard, M. Identification of wheat–Agropyron cristatum monosomic addition lines by RFLP analysis using a set of assigned wheat DNA probes. Theor. Appl. Genet. 1994, 89, 70–75. [Google Scholar] [CrossRef]
  23. Luan, Y.; Wang, X.; Liu, W.; Li, C.; Zhang, J.; Gao, A.; Wang, Y.; Yang, X.; Li, L. Production and identification of wheat–Agropyron cristatum 6P translocation lines. Planta 2010, 232, 501–510. [Google Scholar] [CrossRef] [Green Version]
  24. Song, L.; Jiang, L.; Han, H.; Gao, A.; Yang, X.; Li, L.; Liu, W. Efficient induction of wheat–Agropyron cristatum 6P translocation lines and GISH detection. PLoS ONE 2013, 8, e69501. [Google Scholar] [CrossRef] [Green Version]
  25. Han, H.; Bai, L.; Su, J.; Zhang, J.; Song, L.; Gao, A.; Yang, X.; Li, X.; Liu, W.; Li, L. Genetic rearrangements of six wheat–Agropyron cristatum 6P addition lines revealed by molecular markers. PLoS ONE 2014, 9, e91066. [Google Scholar] [CrossRef] [Green Version]
  26. Copete, A.; Cabrera, A. Chromosomal location of genes for resistance to powdery mildew in Agropyron cristatum and mapping of conserved orthologous set molecular markers. Euphytica 2017, 213, 1–9. [Google Scholar] [CrossRef]
  27. Rey, E.; Molnár, I.; Doležel, J. Genomics of wild relatives and alien introgressions. In Alien Introgression in Wheat; Springer International Publishing: Berlin/Heidelberg, Germany, 2015; pp. 347–381. [Google Scholar]
  28. Dai, C.; Zhang, J.-P.; Wu, X.-Y.; Yang, X.-M.; Li, X.-Q.; Liu, W.-H.; Gao, A.-N.; Li, L.-H. Development of EST markers specific to Agropyron cristatum chromosome 6P in common wheat background. Acta Agron. Sin. 2013, 38, 1791–1801. [Google Scholar] [CrossRef]
  29. Lu, M.; Lu, Y.; Li, H.; Pan, C.; Guo, Y.; Zhang, J.; Yang, X.; Li, X.; Liu, W.; Li, L. Transferring desirable genes from Agropyron cristatum 7P chromosome into common wheat. PLoS ONE 2016, 11, e0159577. [Google Scholar] [CrossRef] [Green Version]
  30. Said, M.; Parada, A.C.; Gaál, E.; Molnár, I.; Cabrera, A.; Doležel, J.; Vrána, J. Uncovering homeologous relationships between tetraploid Agropyron cristatum and bread wheat genomes using COS markers. Theor. Appl. Genet. 2019, 132, 2881–2898. [Google Scholar] [CrossRef] [Green Version]
  31. Taheri, S.; Abdullah, T.L.; Yusop, M.R.; Hanafi, M.M.; Sahebi, M.; Azizi, P.; Shamshiri, R.R. Mining and development of novel SSR markers using Next Generation Sequencing (NGS) data in plants. Molecules 2018, 23, 399. [Google Scholar] [CrossRef] [Green Version]
  32. Córdoba, J.M.; Chavarro, C.; Rojas, F.; Muñoz, C.; Blair, M.W. Identification and mapping of simple sequence repeat markers from common bean (Phaseolus vulgaris L.) bacterial artificial chromosome end sequences for genome characterization and genetic-physical map integration. Plant Genome 2010, 3, 154–165. [Google Scholar] [CrossRef]
  33. Ren, Y.; Zhao, H.; Kou, Q.; Jiang, J.; Guo, S.; Zhang, H.; Hou, W.; Zou, X.; Sun, H.; Gong, G.; et al. A high resolution genetic map anchoring scaffolds of the sequenced watermelon genome. PLoS ONE 2012, 7, e29453. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Tabkhkar, N.; Rabiei, B.; Samizadeh Lahiji, H.; Hosseini Chaleshtori, M. Genetic variation and association analysis of the SSR markers linked to the major drought-yield QTLs of rice. Biochem. Genet. 2018, 56, 356–374. [Google Scholar] [CrossRef] [PubMed]
  35. Shehata, A.I.; Al-Ghethar, H.A.; Al-Homaidan, A.A. Application of simple sequence repeat (SSR) markers for molecular diversity and heterozygosity analysis in maize inbred lines. Saudi J. Biol. Sci. 2009, 16, 57–62. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Komínková, E.; Dreiseitl, A.; Maleèková, E.; Doležel, J.; Valárik, M. Genetic diversity of Blumeria graminis f. sp. hordei in central Europe and its comparison with australian population. PLoS ONE 2016, 11, e0167099. [Google Scholar] [CrossRef] [PubMed]
  37. Christelová, P.; De Langhe, E.; Hřibová, E.; Čížková, J.; Sardos, J.; Hušáková, M.; Van den houwe, I.; Sutanto, A.; Kepler, A.K.; Swennen, R.; et al. Molecular and cytological characterization of the global Musa germplasm collection provides insights into the treasure of banana diversity. Biodivers. Conserv. 2017, 26, 801–824. [Google Scholar] [CrossRef]
  38. Nyine, M.; Uwimana, B.; Swennen, R.; Batte, M.; Brown, A.; Christelová, P.; Hribová, E.; Lorenzen, J.; Doleziel, J. Trait variation and genetic diversity in a banana genomic selection training population. PLoS ONE 2017, 12, e0178734. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Lu, X.; Adedze, Y.M.N.; Chofong, G.N.; Gandeka, M.; Deng, Z.; Teng, L.; Zhang, X.; Sun, G.; Si, L.; Li, W. Identification of high-efficiency SSR markers for assessing watermelon genetic purity. J. Genet. 2018, 97, 1295–1306. [Google Scholar] [CrossRef]
  40. Said, M.; Hřibová, E.; Danilova, T.V.; Karafiátová, M.; Čížková, J.; Friebe, B.; Doležel, J.; Gill, B.S.; Vrána, J. The Agropyron cristatum karyotype, chromosome structure and cross-genome homoeology as revealed by fluorescence in situ hybridization with tandem repeats and wheat single-gene probes. Theor. Appl. Genet. 2018, 131, 2213–2227. [Google Scholar] [CrossRef] [Green Version]
  41. Doležel, J.; Číhalíková, J.; Lucretti, S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L. Planta 1992, 188, 93–98. [Google Scholar] [CrossRef]
  42. Mayer, K.F.X.; Waugh, R.; Langridge, P.; Close, T.J.; Wise, R.P.; Graner, A.; Matsumoto, T.; Sato, K.; Schulman, A.; Ariyadasa, R.; et al. A physical, genetic and functional sequence assembly of the barley genome. Nature 2012, 491, 711–716. [Google Scholar] [CrossRef]
  43. Bartoš, J.; Paux, E.; Kofler, R.; Havránková, M.; Kopecký, D.; Suchánková, P.; Šafář, J.; Šimková, H.; Town, C.D.; Lelley, T.; et al. A first survey of the rye (Secale cereale) genome composition through BAC end sequencing of the short arm of chromosome 1R. BMC Plant Biol. 2008, 8, 95. [Google Scholar] [CrossRef]
  44. Varshney, R.K.; Song, C.; Saxena, R.K.; Azam, S.; Yu, S.; Sharpe, A.G.; Cannon, S.; Baek, J.; Rosen, B.D.; Tar’an, B.; et al. Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement. Nat. Biotechnol. 2013, 31, 240–246. [Google Scholar] [CrossRef] [Green Version]
  45. Kreplak, J.; Madoui, M.A.; Cápal, P.; Novák, P.; Labadie, K.; Aubert, G.; Bayer, P.E.; Gali, K.K.; Syme, R.A.; Main, D.; et al. A reference genome for pea provides insight into legume genome evolution. Nat. Genet. 2019, 51, 1411–1422. [Google Scholar] [CrossRef]
  46. Appels, R.; Eversole, K.; Feuillet, C.; Keller, B.; Rogers, J.; Stein, N.; Pozniak, C.J.; Choulet, F.; Distelfeld, A.; Poland, J.; et al. Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science 2018, 361, aar7191. [Google Scholar] [CrossRef] [Green Version]
  47. Lukaszewski, A.J.; Alberti, A.; Sharpe, A.; Kilian, A.; Stanca, A.M.; Keller, B.; Clavijo, B.J.; Friebe, B.; Gill, B.; Wulff, B.; et al. A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science 2014, 345, 1251788. [Google Scholar] [CrossRef]
  48. Požárková, D.; Koblížková, A.; Román, B.; Torres, A.M.; Lucretti, S.; Lysák, M.; Doležel, J.; Macas, J. Development and characterization of microsatellite markers from chromosome 1-specific DNA libraries of Vicia faba. Biol. Plant. 2002, 45, 337–345. [Google Scholar] [CrossRef]
  49. Shatalina, M.; Wicker, T.; Buchmann, J.P.; Oberhaensli, S.; Šimková, H.; Doležel, J.; Keller, B. Genotype-specific SNP map based on whole chromosome 3B sequence information from wheat cultivars Arina and Forno. Plant Biotechnol. J. 2013, 11, 23–32. [Google Scholar] [CrossRef] [Green Version]
  50. Mazaheri, M.; Kianian, P.M.A.; Mergoum, M.; Valentini, G.L.; Seetan, R.; Pirseyedi, S.M.; Kumar, A.; Gu, Y.Q.; Stein, N.; Kubaláková, M.; et al. Transposable element junctions in marker development and genomic characterization of barley. Plant Genome 2014, 7, plantgenome2013.10.0036. [Google Scholar] [CrossRef]
  51. Cápal, P.; Blavet, N.; Vrána, J.; Kubaláková, M.; Doležel, J. Multiple displacement amplification of the DNA from single flow-sorted plant chromosome. Plant J. 2015, 84, 838–844. [Google Scholar] [CrossRef]
  52. Šimková, H.; Svensson, J.T.; Condamine, P.; Hřibová, E.; Suchánková, P.; Bhat, P.R.; Bartoš, J.; Šafář, J.; Close, T.J.; Doležel, J. Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley. BMC Genom. 2008, 9, 294. [Google Scholar] [CrossRef] [Green Version]
  53. Campbell, M.S.; Holt, C.; Moore, B.; Yandell, M. Genome annotation and curation using MAKER and MAKER-P. Curr. Protoc. Bioinform. 2014, 2014, 4.11.1–14.11.39. [Google Scholar] [CrossRef] [Green Version]
  54. Zwyrtková, J.; Němečková, A.; Čížková, J.; Holušová, K.; Kapustová, V.; Svačina, R.; Kopecký, D.; Till, B.J.; Doležel, J.; Hřibová, E. Comparative analyses of DNA repeats and identification of a novel Fesreba centromeric element in fescues and ryegrasses. BMC Plant Biol. 2020, 20, 280. [Google Scholar] [CrossRef]
  55. Presting, G.G.; Malysheva, L.; Fuchs, J.; Schubert, I. A TY3/GYPSY retrotransposon-like sequence localizes to the centromeric regions of cereal chromosomes. Plant J. 1998, 16, 721–728. [Google Scholar] [CrossRef]
  56. Hudakova, S.; Michalek, W.; Presting, G.G.; Hoopen, R.T.; Dos Santos, K.; Jasencakova, Z.; Schubert, I. Sequence organization of barley centromeres. Nucleic Acids Res. 2001, 29, 5029–5035. [Google Scholar] [CrossRef]
  57. Neumann, P.; Navrátilová, A.; Koblížková, A.; Kejnovsk, E.; Hřibová, E.; Hobza, R.; Widmer, A.; Doležel, J.; MacAs, J. Plant centromeric retrotransposons: A structural and cytogenetic perspective. Mob. DNA 2011, 2, 4. [Google Scholar] [CrossRef] [Green Version]
  58. Zwyrtková, J.; Šimková, H.; Doležel, J. Chromosome genomics uncovers plant genome organization and function. Biotechnol. Adv. 2021, 46, 107659. [Google Scholar] [CrossRef]
  59. Schreiber, M.; Wright, F.; MacKenzie, K.; Hedley, P.E.; Schwerdt, J.G.; Little, A.; Burton, R.A.; Fincher, G.B.; Marshall, D.; Waugh, R.; et al. The barley genome sequence assembly reveals three additional members of the CslF (1,3;1,4)-β-Glucan Synthase gene family. PLoS ONE 2014, 9, e90888. [Google Scholar] [CrossRef] [Green Version]
  60. Darko, E.; Khalil, R.; Dobi, Z.; Kovács, V.; Szalai, G.; Janda, T.; Molnár, I. Addition of Aegilops biuncialis chromosomes 2M or 3M improves the salt tolerance of wheat in different way. Sci. Rep. 2020, 10, 22327. [Google Scholar] [CrossRef]
  61. Zhang, Z.; Song, L.; Han, H.; Zhou, S.; Zhang, J.; Yang, X.; Li, X.; Liu, W.; Li, L. Physical localization of a locus from Agropyron cristatum conferring resistance to stripe rust in common wheat. Int. J. Mol. Sci. 2017, 18, 2403. [Google Scholar] [CrossRef] [Green Version]
  62. Song, L.; Lu, Y.; Zhang, J.; Pan, C.; Yang, X.; Li, X.; Liu, W.; Li, L. Physical mapping of Agropyron cristatum chromosome 6P using deletion lines in common wheat background. Theor. Appl. Genet. 2016, 129, 1023–1034. [Google Scholar] [CrossRef]
  63. Matsumoto, T.; Wu, J.; Kanamori, H.; Katayose, Y.; Fujisawa, M.; Namiki, N.; Mizuno, H.; Yamamoto, K.; Antonio, B.A.; Baba, T.; et al. The map-based sequence of the rice genome. Nature 2005, 436, 793–800. [Google Scholar] [CrossRef]
  64. Schnable, P.S.; Ware, D.; Fulton, R.S.; Stein, J.C.; Wei, F.; Pasternak, S.; Liang, C.; Zhang, J.; Fulton, L.; Graves, T.A.; et al. The B73 maize genome: Complexity, diversity, and dynamics. Science 2009, 326, 1112–1115. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Wicker, T.; Gundlach, H.; Spannagl, M.; Uauy, C.; Borrill, P.; Ramírez-González, R.H.; De Oliveira, R.; Mayer, K.F.X.; Paux, E.; Choulet, F. Impact of transposable elements on genome structure and evolution in bread wheat. Genome Biol. 2018, 19, 103. [Google Scholar] [CrossRef] [PubMed]
  66. Liu, Q.; Li, X.; Zhou, X.; Li, M.; Zhang, F.; Schwarzacher, T.; Heslop-Harrison, J.S. The repetitive DNA landscape in Avena (Poaceae): Chromosome and genome evolution defined by major repeat classes in whole-genome sequence reads. BMC Plant Biol. 2019, 19, 226. [Google Scholar] [CrossRef] [Green Version]
  67. Zhang, Y.; Fan, C.; Li, S.; Chen, Y.; Wang, R.R.C.; Zhang, X.; Han, F.; Hu, Z. The diversity of sequence and chromosomal distribution of new transposable element-related segments in the rye genome revealed by FISH and lineage annotation. Front. Plant Sci. 2017, 8, 1706. [Google Scholar] [CrossRef] [Green Version]
  68. Raskina, O. Transposable elements in the organization and diversification of the genome of Aegilops speltoides Tausch (Poaceae, Triticeae). Int. J. Genom. 2018, 2018, 4373089. [Google Scholar] [CrossRef] [Green Version]
  69. Sanei, M.; Pickering, R.; Kumke, K.; Nasuda, S.; Houben, A. Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids. Proc. Natl. Acad. Sci. USA 2011, 108, E498–E505. [Google Scholar] [CrossRef] [Green Version]
  70. Quraishi, U.M.; Abrouk, M.; Bolot, S.; Pont, C.; Throude, M.; Guilhot, N.; Confolent, C.; Bortolini, F.; Praud, S.; Murigneux, A.; et al. Genomics in cereals: From genome-wide conserved orthologous set (COS) sequences to candidate genes for trait dissection. Funct. Integr. Genom. 2009, 9, 473–484. [Google Scholar] [CrossRef]
  71. Howard, T.; Rejab, N.A.; Griffiths, S.; Leigh, F.; Leverington-Waite, M.; Simmonds, J.; Uauy, C.; Trafford, K. Identification of a major QTL controlling the content of B-type starch granules in Aegilops. J. Exp. Bot. 2011, 62, 2217–2228. [Google Scholar] [CrossRef] [Green Version]
  72. Naranjo, T. Variable patterning of chromatin remodeling, telomere positioning, synapsis, and chiasma formation of individual rye chromosomes in meiosis of wheat-rye additions. Front. Plant Sci. 2018, 9, 880. [Google Scholar] [CrossRef]
  73. Perničková, K.; Koláčková, V.; Lukaszewski, A.J.; Fan, C.; Vrána, J.; Duchoslav, M.; Jenkins, G.; Phillips, D.; Šamajová, O.; Sedlářová, M.; et al. Instability of alien chromosome introgressions in wheat associated with improper positioning in the nucleus. Int. J. Mol. Sci. 2019, 20, 1448. [Google Scholar] [CrossRef] [Green Version]
  74. Danilova, T.V.; Friebe, B.; Gill, B.S. Development of a wheat single gene FISH map for analyzing homoeologous relationship and chromosomal rearrangements within the Triticeae. Theor. Appl. Genet. 2014, 127, 715–730. [Google Scholar] [CrossRef] [Green Version]
  75. Luo, M.C.; Deal, K.R.; Akhunov, E.D.; Akhunova, A.R.; Anderson, O.D.; Anderson, J.A.; Blake, N.; Clegg, M.T.; Coleman-Derr, D.; Conley, E.J.; et al. Genome comparisons reveal a dominant mechanism of chromosome number reduction in grasses and accelerated genome evolution in Triticeae. Proc. Natl. Acad. Sci. USA 2009, 106, 15780–15785. [Google Scholar] [CrossRef] [Green Version]
  76. Mellish, A.; Coulman, B.; Ferdinandez, Y. Genetic relationships among selected crested wheatgrass cultivars and species determined on the basis of AFLP markers. Crop Sci. 2002, 42, 1662–1668. [Google Scholar] [CrossRef]
  77. Wang, W.W.; Tan, Z.Y.; Xu, Y.Q.; Zhu, A.A.; Li, Y.; Yao, J.; Tian, R.; Fang, X.M.; Liu, X.Y.; Tian, Y.M.; et al. Chromosome structural variation of two cultivated tetraploid cottons and their ancestral diploid species based on a new high-density genetic map. Sci. Rep. 2017, 7, 7640. [Google Scholar] [CrossRef]
  78. Tang, H.; Lyons, E.; Town, C.D. Optical mapping in plant comparative genomics. Gigascience 2015, 4, 3. [Google Scholar] [CrossRef] [Green Version]
  79. Wang, W.; Guan, R.; Liu, X.; Zhang, H.; Song, B.; Xu, Q.; Fan, G.; Chen, W.; Wu, X.; Liu, X.; et al. Chromosome level comparative analysis of Brassica genomes. Plant Mol. Biol. 2019, 99, 237–249. [Google Scholar] [CrossRef]
  80. Jiao, W.B.; Schneeberger, K. The impact of third generation genomic technologies on plant genome assembly. Curr. Opin. Plant Biol. 2017, 36, 64–70. [Google Scholar] [CrossRef]
  81. Li, S.; Yang, G.; Yang, S.; Just, J.; Yan, H.; Zhou, N.; Jian, H.; Wang, Q.; Chen, M.; Qiu, X.; et al. The development of a high-density genetic map significantly improves the quality of reference genome assemblies for rose. Sci. Rep. 2019, 9, 5985. [Google Scholar] [CrossRef]
  82. Wang, X.; Liu, H.; Pang, M.; Fu, B.; Yu, X.; He, S.; Tong, J. Construction of a high-density genetic linkage map and mapping of quantitative trait loci for growth-related traits in silver carp (Hypophthalmichthys molitrix). Sci. Rep. 2019, 9, 17506. [Google Scholar] [CrossRef]
  83. Amarasinghe, S.L.; Su, S.; Dong, X.; Zappia, L.; Ritchie, M.E.; Gouil, Q. Opportunities and challenges in long-read sequencing data analysis. Genome Biol. 2020, 21, 30. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Chawla, H.S.; Lee, H.; Gabur, I.; Tamilselvan-Nattar-Amutha, S.; Obermeier, C.; Schiessl, S.; Song, J.; Liu, K.; Guo, L.; Parkin, I.; et al. Long-read sequencing reveals widespread intragenic structural variants in a recent allopolyploid crop plant. Plant Biotechnol. J. 2020, 19, 240–250. [Google Scholar] [CrossRef] [PubMed]
  85. Said, M.; Kubaláková, M.; Karafiátová, M.; Molnár, I.; Doležel, J.; Vrána, J. Dissecting the complex genome of crested wheatgrass by chromosome flow sorting. Plant Genome 2019, 12, 180096. [Google Scholar] [CrossRef] [PubMed]
  86. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [Green Version]
  87. Boisvert, S.; Laviolette, F.; Corbeil, J. Ray: Simultaneous assembly of reads from a mix of high-throughput sequencing technologies. J. Comput. Biol. 2010, 17, 1401–1415. [Google Scholar] [CrossRef]
  88. Thiel, T.; Michalek, W.; Varshney, R.K.; Graner, A. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor. Appl. Genet. 2003, 106, 411–422. [Google Scholar] [CrossRef]
  89. Untergasser, A.; Cutcutache, I.; Koressaar, T.; Ye, J.; Faircloth, B.C.; Remm, M.; Rozen, S.G. Primer3—new capabilities and interfaces. Nucleic Acids Res. 2012, 40, e115. [Google Scholar] [CrossRef] [Green Version]
  90. Wu, T.D.; Watanabe, C.K. GMAP: A genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 2005, 21, 1859–1875. [Google Scholar] [CrossRef] [Green Version]
  91. Smit, A.; Hubley, R.; Green, P. RepeatMasker Open-4.0. 2013–2015. 2013. Available online: http://www.repeatmasker.org (accessed on 12 February 2022).
  92. Stanke, M.; Steinkamp, R.; Waack, S.; Morgenstern, B. AUGUSTUS: A web server for gene finding in eukaryotes. Nucleic Acids Res. 2004, 32, W309–W312. [Google Scholar] [CrossRef] [Green Version]
  93. Bolser, D.M.; Staines, D.M.; Perry, E.; Kersey, P.J. Ensembl plants: Integrating tools for visualizing, mining, and analyzing plant genomic data. In Methods in Molecular Biology; Humana Press Inc.: Totowa, NJ, USA, 2017; Volume 1533, pp. 1–31. [Google Scholar]
  94. Mascher, M. Pseudomolecules and Annotation of the Second Version of the Reference Genome Sequence Assembly of Barley cv. Morex [Morex V2]; e!DAL-Plant Genomics & Phenomics Research Data Repository (2019-05-09); IPK Gatersleben: Gatersleben, Germany, 2019. [Google Scholar] [CrossRef]
  95. Hunter, S.; Apweiler, R.; Attwood, T.K.; Bairoch, A.; Bateman, A.; Binns, D.; Bork, P.; Das, U.; Daugherty, L.; Duquenne, L.; et al. InterPro: The integrative protein signature database. Nucleic Acids Res. 2008, 37, D211–D215. [Google Scholar] [CrossRef] [Green Version]
  96. Altschul, S.F.; Madden, T.L.; Schäffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389–3402. [Google Scholar] [CrossRef] [Green Version]
  97. Consortium, T.U. UniProt: A worldwide hub of protein knowledge. Nucleic Acids Res. 2018, 47, D506–D515. [Google Scholar] [CrossRef] [Green Version]
  98. Miele, V.; Penel, S.; Duret, L. Ultra-fast sequence clustering from similarity networks with SiLiX. BMC Bioinform. 2011, 12, 116. [Google Scholar] [CrossRef] [Green Version]
  99. Novák, P.; Neumann, P.; Macas, J. Graph-based clustering and characterization of repetitive sequences in next-generation sequencing data. BMC Bioinform. 2010, 11, 378. [Google Scholar] [CrossRef] [Green Version]
  100. Katoh, K.; Toh, H. Recent developments in the MAFFT multiple sequence alignment program. Brief. Bioinf. 2008, 9, 286–298. [Google Scholar] [CrossRef] [Green Version]
  101. Guindon, S.; Dufayard, J.F.; Lefort, V.; Anisimova, M.; Hordijk, W.; Gascuel, O. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol. 2010, 59, 307–321. [Google Scholar] [CrossRef] [Green Version]
  102. Gouy, M.; Guindon, S.; Gascuel, O. SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol. 2010, 27, 221–224. [Google Scholar] [CrossRef] [Green Version]
  103. Anisimova, M.; Gascuel, O. Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. Syst. Biol. 2006, 55, 539–552. [Google Scholar] [CrossRef]
  104. Kubaláková, M.; Macas, J.; Doležel, J. Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS. Theor. Appl. Genet. 1997, 94, 758–763. [Google Scholar] [CrossRef]
  105. Nagaki, K.; Cheng, Z.; Ouyang, S.; Talbert, P.B.; Kim, M.; Jones, K.M.; Henikoff, S.; Buell, C.R.; Jiang, J. Sequencing of a rice centromere uncovers active genes. Nat. Genet. 2004, 36, 138–145. [Google Scholar] [CrossRef]
Ijms 23 03191 g001 550
Figure 1. Chromosomal distribution of the two most represented gene functional families observed in the draft sequencing of A. cristatum. X-axis: individual Agropyron chromosomes; Y-axis: chromosome proportion (%).
Figure 1. Chromosomal distribution of the two most represented gene functional families observed in the draft sequencing of A. cristatum. X-axis: individual Agropyron chromosomes; Y-axis: chromosome proportion (%).
Ijms 23 03191 g001
Ijms 23 03191 g002 550
Figure 2. Colocalization of CENH3 with the Agrocen element in A. cristatum cv. Parkway. A combination of the immunolocalization of the histone H3 variant CENH3 (red) and FISH on interphase nuclei with probes for the reverse transcriptase (RT) domain (green). Nuclei were counterstained with DAPI (blue). Bar corresponds to 10 µm.
Figure 2. Colocalization of CENH3 with the Agrocen element in A. cristatum cv. Parkway. A combination of the immunolocalization of the histone H3 variant CENH3 (red) and FISH on interphase nuclei with probes for the reverse transcriptase (RT) domain (green). Nuclei were counterstained with DAPI (blue). Bar corresponds to 10 µm.
Ijms 23 03191 g002
Ijms 23 03191 g003 550
Figure 3. Position of the new Agropyron-specific SSR markers on collinear chromosomes of T. aestivum. Seven homoeologous groups of wheat chromosomes are differentiated by colors (red, green, yellow, turquoise, purple, pink and light green). Short chromosomal arms (S) are dark in color, and long chromosomal arms (L) are light in color. Chromosome-specific SSR markers are marked by frames. Chromosome-arm-specific SSR markers are marked by frames filled up by colors of the corresponding chromosome arms. Any specific highlighting is not used for SSR markers localized in multiple positions in the genome of A. cristatum. Arrows depict positions of centromeres, as observed by Appels et al. [46].
Figure 3. Position of the new Agropyron-specific SSR markers on collinear chromosomes of T. aestivum. Seven homoeologous groups of wheat chromosomes are differentiated by colors (red, green, yellow, turquoise, purple, pink and light green). Short chromosomal arms (S) are dark in color, and long chromosomal arms (L) are light in color. Chromosome-specific SSR markers are marked by frames. Chromosome-arm-specific SSR markers are marked by frames filled up by colors of the corresponding chromosome arms. Any specific highlighting is not used for SSR markers localized in multiple positions in the genome of A. cristatum. Arrows depict positions of centromeres, as observed by Appels et al. [46].
Ijms 23 03191 g003
Ijms 23 03191 g004 550
Figure 4. Rearrangements specific for chromosome group 2 between A. cristatum and common wheat [46] (A), and rearrangements specific for chromosome group 4 between A. cristatum and common wheat [46] (B), as revealed by newly developed SSR markers.
Figure 4. Rearrangements specific for chromosome group 2 between A. cristatum and common wheat [46] (A), and rearrangements specific for chromosome group 4 between A. cristatum and common wheat [46] (B), as revealed by newly developed SSR markers.
Ijms 23 03191 g004
Table
Table 1. Sequencing of A. cristatum chromosomes.
Table 1. Sequencing of A. cristatum chromosomes.
Chromosome *
Purity (%)Length (Mb) **RayAssembly (bp)N50
(bp)		Coverage (%) ***Number of SSRsMaker &Maker &&Genes &&&
1P10087099,700,722572411.6594933223307758
2P901034342,722,238377433.219,604775977001093
3P98791443,871,941597356.132,62411,04510,9761388
4P10075732,166,64884344.3113614721470749
5P98969463,809,210817547.934,40111,76911,6941564
6P81974258,122,996221826.530,93268176756867
7P100958147,241,484488615.4962429422911499
* Single copies of chromosomes 1P, 4P, and 7P were flow-sorted. ** Based on Said et al. [40]. *** Chromosomal coverage of the assemblies. & Potentially expressed sequences. && Genic sequences. &&& Genic sequences with known function.
Table
Table 2. The most accurate locations of specific SSR markers established with Agropyron addition lines.
Table 2. The most accurate locations of specific SSR markers established with Agropyron addition lines.
Chromosome
Addition Line		Chromosome-Short-Arm
Addition Line		Chromosome-Long-Arm
Addition Line
1P1PS + 1BLNo line
ocwgm001ocwgm001
ocwgm002-
ocwgm003-
ocwgm004-
ocwgm005-
ocwgm006-
2P2PS2PL
ocwgm007ocwgm007-
ocwgm008ocwgm008-
ocwgm009ocwgm009-
ocwgm010ocwgm010-
ocwgm011ocwgm011-
ocwgm012ocwgm012-
ocwgm013-ocwgm013
ocwgm014-ocwgm014
ocwgm015-ocwgm015
ocwgm016-ocwgm016
ocwgm017-ocwgm017
ocwgm018-ocwgm018
ocwgm019ocwgm019ocwgm019
3P + 3PS3PSNo line
ocwgm020ocwgm020
ocwgm021ocwgm021
ocwgm022ocwgm022
ocwgm023ocwgm023
ocwgm024ocwgm024
ocwgm025ocwgm025
ocwgm026ocwgm026
ocwgm027ocwgm027
ocwgm028ocwgm028
ocwgm029-
ocwgm030-
ocwgm031-
ocwgm032-
ocwgm033-
4P4PSNo line
ocwgm034ocwgm034
ocwgm035ocwgm035
ocwgm036ocwgm036
ocwgm037ocwgm037
ocwgm038ocwgm038
ocwgm039-
ocwgm040-
ocwgm041-
ocwgm042-
ocwgm043-
ocwgm044-
5PNo line5PL
ocwgm045 -
ocwgm046 ocwgm046
ocwgm047 ocwgm047
ocwgm048 ocwgm048
6P6PS6PL
ocwgm049ocwgm049-
ocwgm050ocwgm050-
ocwgm051ocwgm051-
ocwgm052ocwgm052-
ocwgm053ocwgm053-
ocwgm054ocwgm054-
ocwgm055ocwgm055-
ocwgm056ocwgm056-
ocwgm057-ocwgm057
ocwgm058-ocwgm058
--ocwgm059
ocwgm060--
ocwgm061--
ocwgm062--
ocwgm063--
7P *No lineNo line
ocwgm064
ocwgm065
ocwgm066
ocwgm067
ocwgm068
ocwgm069
ocwgm070
ocwgm071
ocwgm072
* Absent on other addition lines; present on gDNA.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Zwyrtková, J.; Blavet, N.; Doležalová, A.; Cápal, P.; Said, M.; Molnár, I.; Vrána, J.; Doležel, J.; Hřibová, E. Draft Sequencing Crested Wheatgrass Chromosomes Identified Evolutionary Structural Changes and Genes and Facilitated the Development of SSR Markers. Int. J. Mol. Sci. 2022, 23, 3191. https://doi.org/10.3390/ijms23063191

AMA Style

Zwyrtková J, Blavet N, Doležalová A, Cápal P, Said M, Molnár I, Vrána J, Doležel J, Hřibová E. Draft Sequencing Crested Wheatgrass Chromosomes Identified Evolutionary Structural Changes and Genes and Facilitated the Development of SSR Markers. International Journal of Molecular Sciences. 2022; 23(6):3191. https://doi.org/10.3390/ijms23063191

Chicago/Turabian Style

Zwyrtková, Jana, Nicolas Blavet, Alžběta Doležalová, Petr Cápal, Mahmoud Said, István Molnár, Jan Vrána, Jaroslav Doležel, and Eva Hřibová. 2022. "Draft Sequencing Crested Wheatgrass Chromosomes Identified Evolutionary Structural Changes and Genes and Facilitated the Development of SSR Markers" International Journal of Molecular Sciences 23, no. 6: 3191. https://doi.org/10.3390/ijms23063191

APA Style

Zwyrtková, J., Blavet, N., Doležalová, A., Cápal, P., Said, M., Molnár, I., Vrána, J., Doležel, J., & Hřibová, E. (2022). Draft Sequencing Crested Wheatgrass Chromosomes Identified Evolutionary Structural Changes and Genes and Facilitated the Development of SSR Markers. International Journal of Molecular Sciences, 23(6), 3191. https://doi.org/10.3390/ijms23063191

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop