Diversification of the P genome among Agropyron Gaertn. (Poaceae) species detected by FISH

Abstract The genomes of five Agropyron Gaertner, 1770 species were characterized using all potential di- or trinucleotide simple sequence repeat (SSR) motifs and four satellite DNA repeats as fluorescence in situ hybridization (FISH) probes. The sites of 5S and 45S rDNA were relatively conserved among the diploid and tetraploid species. A number of sites for the dinucleotide SSRs AC, AG, and pSc119.2 was detected in all investigated species except A. mongolicum Keng, 1938. Several different trinucleotide SSRs were identified in different tetraploid species. All Agropyron species were suggested to include the basic P genome, although genome differentiation was still observed. The P genome of A. mongolicum was distinct from that of the diploid A. cristatum (Linnaeus, 1753) Gaertner, 1770. and other tetraploid species, with no hybridizations for AC, AG, or pSc119.2 observed. This finding supports designation of the P genomes of A. cristatum and A. mongolicum as Pc and Pm, respectively. An exceptional 5S rDNA site revealed in one set of homoeologous chromosomes strongly supports the allopolyploid origin of A. desertorum (Fischer ex Link, 1821) Schultes, 1824. However, the diploid donors to A. desertorum need further investigation. Similarly, the unique FISH pattern of a pair of 5S rDNA-carrying chromosomes was indicative of a potential allopolyploid origin for A. fragile (Roth, 1800) Candargy, 1984. The conserved distribution of 5S and 45S rDNA suggests A. cristatum (4x) and A. michnoi Roshevitz, 1929 are closely related. Two forms of B chromosomes were identified among individuals A. mongolicum and A. desertorum plants.


Introduction
The genus Agropyron Gaertner, 1770, also referred to as the crested wheatgrass complex, includes 10-15 species (Asay and Jensen 1996). The genus is distributed in Eurasia and comprises a series of diploid (2n = 14), tetraploid (2n = 28), and hexaploid (2n = 42) species (Löve 1982, 1984, Dewey 1984. The diploid species are less common and distributed from Europe to Mongolia, whereas tetraploids commonly occur in central Europe, the Middle East, and central Asia. Hexaploids are rare in Turkey and Iran (Dewey and Asay 1982). The spike morphology is important for identification for the species, and varies in a continuous fashion from broad, pubescent, pectinate spikes to narrow, linear, glabrous spikes (Dewey and Asay 1982). The species taxonomy is confounded by overlapping geographic distributions and high cross-compatibility among species. The taxonomy of Agropyron spp. has been characterized as "a multitude of taxonomic binomials" (Dewey and Pendese 1967). Although morphologically diverse, all crested wheat grasses are considered to share the same basic genome; this was previously termed the C genome and is currently referred to as the P genome (Dewey and Pendese 1967).
Inter-specific differentiation in Agropyron has been extensively studied using morphology, cytology and molecular markers. Agropyron mongolicum Keng, 1938 is a diploid species indigenous to northern China. It is distinguished from the diploid species A. cristatum (Linnaeus, 1753) Gaertner, 1770 based on its narrow, linear spikes (Dewey 1981). Hsiao et al. (1986Hsiao et al. ( , 1989 revealed A. cristatum and A. mongolicum share the same basic P genome but differ as a result of structural rearrangement of some chromosomes. These findings were based on a karyotype analysis and meiotic analysis of interspecific hybrids. Data from amplified fragment length polymorphism (AFLP) markers showed that A. mongolicum clusters separately from other Agropyron species, including A. cristatum and A. fragile (Roth, 1800) Candargy, 1984 (Mellish et al. 2002). These findings suggest that the P genomes of A. cristatum and A. mongolicum should be designated "P c " and "P m ", respectively (Mellish et al. 2002). The origin of A. desertorum (Fischer ex Link, 1821) Schultes, 1824 has been extensively discussed. Taylor and McCoy (1973) concluded that A. desertorum originated from amphidiploids between A. imbricatum Roemer & Schultes, 1817 and A. pectiniforme Roemer & Schultes, 1817 on the basis of chromatographic and karyotypic analysis. Both A. imbricatum and A. pectiniforme have been reclassified subsequently as subspecies of A. cristatum (Tzvelev 1976). On the basis of morphology and meiotic relationship in hybrids between A. mogolicum × A. cristatum amphiploid and A. desertorum, Asay et al. (1992) suggested that the ancestral germplasm of A. desertorum was derived from hybridization between A. mongolicum and A. cristatum, followed by chromosome doubling. In the same study, the authors further proposed that A. fragile is an autotetraploid derivative of A. mongolicum, and that variants of A. desertorum could be generated from hybrids between A. fragile and tetraploid A. cristatum (Asay et al. 1992). Molecular data from AFLP markers suggest that A. desertorum is an alloploid incorporating the P genomes of A. mongolicum and A. cristatum, and thus the genome of A. desertorum could be designated "P c P c P m P m " (Mellish et al. 2002). However, Vogel et al. (1999) concluded that the genome length of A. desertorum was more consistent with that of an autoploid of A. cristatum than that of an allotetraploid between A. cristatum and A. mongolicum. Moreover, Che et al. (2015) distinguished populations of A. desertorum, A. mongolicum and A. michnoi Roshev. from populations of A. cristatum by means of principal coordinate analysis. The results suggest that A. desertorum, A. mongolicum and A. michnoi Roshevitz, 1929 are derivative species of A. cristatum that share the same basic genome as the ancestral species.
Fluorescence in situ hybridization (FISH) is a powerful tool for characterization of genome composition based on a few repetitive sequences. Species-level phylogenies across species can be derived from repeat-based comparative FISH karyotyping (Jiang andGill 2006, Heslop-Harrison andSchwarzacher 2011). Comparative FISH may allow to detect genome differentiation between closely related species , Dou et al. 2016.
The genomes of Triticeae species are large, of which 75% of the genome comprises repetitive sequences (Flavell et al. 1974(Flavell et al. , 1977. The genome composition in Triticeae species can be characterized using different microsatellite or satellite repetitive sequences via FISH Dou et al. 2016). In the present study, comparative FISH was conducted using all potential dinucleotide and trinucleotide simple sequence repeats (SSRs), 5S and 45S ribosomal DNA (rDNA), pSc119.2, and pAs1 repeats on mitotic chromosomes of five Agropyron species. The objective was to examine the genome differentiation among species within the genus Agropyron and infer genetic relationships among the investigated Agropyron species.

Plant material
Six accessions belonging to A. mongolicum, A. cristatum, A. desertorum, A. fragile, and A. michnoi were used in this study ( Table 1). Seeds of each accession were randomly selected for cytogenetic investigation.

Slide preparation
The seeds were germinated on moist filter paper in Petri dishes at room temperature. Root tips of of 1-2 cm length were excised and pretreated with nitrous oxide at a pressure of 8 atm for 2 h at room temperature following the method of Kato (1999). Subsequently, the root tips were fixed in ethanol-glacial acetic acid (3:1, v/v) for at least 30 min at room temperature. Each root tip was squashed in a drop of 45% acetic acid.

DNA probes and labelling
All potential dinucleotide and trinucleotide SSRs and four satellite DNA sequences, namely pAs1 (Rayburn and Gill 1986), pSc119.2 (Bedbrook et al. 1980), 5S and 45S rDNA, were used to generate DNA probes. The procedure for labeling the above sequences was performed in accordance with the method of Dou et al. (2016).

FISH and microphotometry
The FISH experiments were conducted following the method of Dou et al. (2009;2016). The images were captured with a Photometrics CoolSNAP CCD camera (Roper Scientific, Trenton, NJ, USA) using a fluorescence microscope (model DM R HC, Leica Microsystems, Wetzlar, Germany) and processed with the Meta imaging system (Universal Imaging Corporation, West Chester, PA, USA). Finally, the images were adjusted with Adobe Photoshop 6.0 for contrast and background optimization.

Karyotype features of the P genome among species
A chromosome number of 2n = 14 was detected in A. mongolicum ( Fig. 1a-c) and A. cristatum ( Fig. 1d-h), whereas a chromosome number of 2n = 28 was detected in most individuals of the other Agropyron species (Figs 1i-r, 2a-r). Both diploid and tetraploid species were detected. However, monosomic chromosomes were identified in many cases in tetraploid species (Figs 1o, r 2k, r). The repetitive sequence pAs1 showed multiple subtelomeric, intercalary, or pericentromeric hybridization sites on all chromosomes. The hybridization pattern of pAs1 was much more useful for distinguishing each chromosome of the P genome in all species compared with the other repetitive sequences. The tentative karyotypes of all individuals analyzed were determined using pAs1 as a land marker and the chromosome arm ratio and relative length as references (Fig. 3). Four pairs of metacentric chromosomes and three pairs of submetacentric chromosomes were identified in both A. cristatum and A. mongolicum. In addition,  eight pairs of metacentric chromosomes and six pairs of submetacentric chromosomes were identified in the other tetraploid species. Although a high level of intra-and inter-species chromosomal polymorphisms were detected, homoeologous chromosomes among species were tentatively identified and designated 1-7 (1^-7^) (Fig. 3). In this designation, the numerals do not correspond with those of the homoeologous groups used in the nomenclature system of the chromosomes of wheat and barley. Seven homoeologous chromosomes were identified: • Chromosome 1 is a metacentric chromosome with pAs1 hybridization signals in pericentromeric regions in most cases. • Chromosome 2 is a metacentric chromosome lacking pAs1 hybridization signals in the subtelomeric region of the short arm in most cases. • Chromosome 3 is a metacentric chromosome with pAs1 hybridization signals distributed from the intercalary to subtelomeric regions of both arms. • Chromosome 4 is the smallest metacentric chromosome.
• Chromosome 5 is the largest submetacentric chromosome.
• Chromosome 6 is a submetacentric chromosome with the smallest arm ratio (short arm to long arm). • Chromosome 7 is a submetacentric chromosome lacking pAs1 hybridization signals in the subtelomeric region of the short arm.

Chromosomal distribution of 5S rDNA, 45S rDNA and pSc119.2 among species
The hybridization sites of 5S rDNA and 45S rDNA were stably detected in all analyzed species. The 5S rDNA hybridization signals were detected in one pair of chromosomes in the diploid species A. mongolicum and A. cristatum (Fig. 1a, d) and in two pairs of chromosomes in the other tetraploid species (Figs 1i, 2a, f, m). The physical position of the 5S rDNA sites were the most conserved, which were stably detected in the intercalary regions of the short arms of homoeologous chromosome 6 (6 and 6^ chromosomes). Notably, additional 5S rDNA sites in the pericentromeric regions of the long arms of chromosome 6^ and additional 5S rDNA sites in the intercalary regions of the short arms of chromosome 6^ were detected in A. desertorum and A. fragile, respectively (Fig. 3). Four highly conserved 45S rDNA sites were stably revealed in subtelomeric regions in both diploid species (Fig. 1b and e). However, variable numbers of 45S rDNA sites (from four to six) were identified in tetraploid species: six for A. cristatum (Fig. 1j), five for A. desertorum (Fig. 2b), four for A. fragile (Fig. 2j) and five for A. michnoi (Fig. 2n). Most of the 45S rDNA sites were physically mapped to the subtelomeric regions of homoeologous chromosomes 2, 5, 6, and 7 among the tetraploid species. Occasionally, 45S rDNA sites were detected on chromosome 1 and in the subtelomeric regions of the long arms of chromosomes 4 and 6 in A. desertorum (Fig. 3). The 45S rDNA sites on homoeologous chromosome 6 were the most conserved, which were detected on Figure 3. Molecular karyotypes of six Agropyron species probed using pAs1 (red) combined with 5S rDNA (green), 45S rDNA (green) or pSc119.2 (green) sequences. The numbers 1 to 7 designate seven different homologous chromosomes, whereas the same number followed by "^" indicates the respective homoeologous chromosomes in each genome of tetraploid species.
Although the (AC) 15 hybridization signals were frequently detected in homoeologous 1 chromosomes in most species, the conserved distribution of the (AC) 15 signal among all investigated species was not observed. Agropyron fragile (Fig. 2k) and A. michnoi (Fig. 2r) carried a high number of (AG) 15 hybridization signals (6-8), whereas the others carried low number of (AG) 15 hybridization signals (3-4; Table 2). Numbers followed by " or ' are the number of hybridization sites of the respective sequence. " Indicates a pair of homologous chromosomes; ' indicates one of the homologous chromosomes. Numbers in parentheses indicate the chromosome designation shown in Fig. 3 All ten potential trinucleotide SSRs probes, (AAG) 10 , (AAC) 10 , (AAT) 10 , (ACG) 10 , (ACT) 10 , (AGG) 10 , (CAC) 10 , (CAG) 10 , (CAT) 10 , and (GGC) 10 , were used to characterize the chromosomes of all species. No hybridization signals for any trinucleotide SSR probes were detected in the diploid species A. mongolicum and A. cristatum 'Fairway' or in the tetraploid species A. desertorum. A small number of SSR hybridization signals were identified in A. cristatum (4x), A. fragile, and A. michnoi, although these signals were not commonly shared among the species. Agropyron cristatum (4x) harbored the greatest number of trinucleotide SSR hybridization sites. One hybridization site of (AAG) 10 , (ACG) 10 , (AGG) 10 , (CAG) 10 and (CAT) 10 was well identified in the individuals of A. cristatum (4x) (Fig. 1l-p). All (AAG) 10 , (ACG) 10 , (CAG) 10 and (CAT) 10 sites were physically mapped to the pericentromeric regions of the long arms of chromosome 2. This finding implies the co-localized distribution of (AAG) 10 , (ACG) 10 , (CAG) 10 , and (CAT) 10 in A. cristatum (4x). However, (AGG) 10 was physically mapped to the pericentromeric region of the short arm of chromosome 5 (Table  2). Two trinucleotide SSRs (ACT) 10 and (AGG) 10 , gave rise to hybridization signals in A. fragile ( Fig. 2i and j). The SSR (ACT) 10 , which was not detected in the other species analyzed, was localized in the intercalary regions of the short arms of chromosome 4 in A. fragile (Fig. 2j).The (AGG) 10 was hybridization site was physically mapped to subtelomeric regions in A. fragile (Fig. 2i) rather than to pericentromeric regions as in A. cristatum (4x) (Fig. 1n). Only one trinucleotide SSR, (CAT) 10 , was detected in A. michnoi. The (CAT) 10 was hybridizatin site was localized to intercalary regions in A. michnoi (Fig. 2p) rather than to pericentromeric regions as in A. cristatum (4x) (Fig. 1p).

B chromosome in A. mongolicum and A. desertorum
B chromosomes were identified in a few individuals of A. mongolicum (Fig. 1a) and A. desertorum (Fig. 2b) but not in the other Agropyron species analyzed. The B chromosomes in A. mongolicum and A. desertorum were intensely stained by the pAs1 probe. A distinct primary constriction was identified in the B chromosomes of both species. On the basis of the position of the primary constriction, the B chromosome in A. mongolicum was submetacentric, whereas that of A. desertorum was of metacentric type (Fig. 4).

Genomic characterization of the P genome in Agropyron using repetitive sequences
In this study, all potential dinucleotide and trinucleotide SSRs and four satellite DNA repeats were used to characterize the P genome of five Agropyron species. Unlike other genomes such as the H, I, A, B, and D genomes in Triticeae (Cuadrado et al. 2007;2008;Dou et al. 2016), which include a number of SSR hybridization signals based on FISH, the P genome in Agropyron harbored only the dinucleotide SSRs AG and AC in all species, except A. mongolicum, and fewer trinucleotide SSRs in the different species. The distribution of AAG corresponded to the distribution of N bands, which are composed of heterochromatin in Triticeae (Pedersen et al. 1996). In the P genome, only one AAG site was detected in the intercalary regions in one accession, A. cristatum (4x). The pAs1 in the P genome was detected more frequently in subtelomeric or pericentromeric regions than in intercalary regions, and the repeatedly detected 45S rDNA, pSc119.2, AC, and AG repeats were localized to subtelomeric regions. Forty-eight P-genome-specific sequences have been identified from the DOP-PCR product of the 6P chromosome of A. cristatum, and 14 of 48 sequences have been physically mapped to chromosomes, centromeres, pericentromeric regions, or subtelomeric regions throughout the P genome (Han et al. 2017). Taken together, the results of the present study suggest that the tandem-repetitive sequences in the P genome are much more intensively localized to the pericentromeric and subtelomeric regions rather than the intercalary regions.
The lack of tandem-repetitive sequences in intercalary regions makes the accurate discrimination of each chromosome of the P genome more difficult. In this study, 5S rDNA, 45S rDNA, and pAs1 repeats were more stable and informative. Four to five pairs or homoeologous chromosomes could be accurately identified in each diploid and tetraploid species based on the above-mentioned chromosomal markers, the chromosome length and the arm ratio.

P genome differentiation and genetic relationships of the Agropyron species
The P genome of A. mongolicum, which includes no AG, AC or pSc119.2 repeats in the subtelomeric regions of any chromosome, is distinct from the P genome of the other Agropyron species analyzed. Structural rearrangements of some chromosomes were revealed between the two diploid species A. cristatum and A. mongolicum (Hsiao et al. 1986(Hsiao et al. , 1989. In the present study, genome rearrangements were difficult to identify accurately, reflecting the low resolution of the chromosomal markers. However, discrepancies in the localization of the 45S rDNA sites were revealed on chromosomes 5 and 3 in A. mongolicum and A. cristatum, respectively. This observation strongly suggested that the chromosome structure arrangements involved these two chromosomes. The differentiation of both diploid species revealed in the present study suggests that the P genome of A. cristatum should be designated P c , and the P genome of A. mongolicum should be designated P m , consistent with the suggestions of Mellish et al. (2002). Agropyron mongolicum differs from other Agropyron species in producing a narrow, linear, glabrous spike (Dewey 1981). Data from AFLP markers discriminated A. mongolicum from the other Agropyron species (Mellish et al. 2002). Taken together, these results are strongly suggestive of a distant relationship between A. mongolicum and the other Agropyron species.
The tetraploid A. cristatum is considered to be an autopolyploid originating from the diploid species A. cristatum. However, the molecular karyotype of this tetraploid was not consistent with a doubled diploid karyotype. High variation in the number of hybridization sites and localization of 45S rDNA, AC, AG, and pSc119.2 repeats was revealed in the present study. Notably, a few trinucleotide SSRs were detected in tetraploid species rather than in diploid species. These observations indicate that high genomic variation, including DNA sequence deletion, amplification and genomic rearrangement, might have occurred in the transition from diploid to tetraploid species.
Agropyron desertorum is considered to be an allotetraploid between diploid A. mongolicum and A. cristatum (Asay et al. 1992, Mellish et al. 2002. The localization of a numer of 45S rDNA sites in A. desertorum was highly variable compared with the other species analyzed. Notably, one homoeologous set of chromosome 6 (6^) contained additional 5S rDNA sites in the pericentromeric regions of the long arm. The homoeologous chromosome 6 stably carried 5S rDNA sites in the short arms of all Agropyron species studied. The distinct differences in the FISH patterns between chromosomes 6 and 6^ strongly support the allopolyploid origin of A. desertorum. However, the FISH pattern of chromosome 6^ in A. desertorum was not similar to either A. mongolicum or A. cristatum.
Agropyron fragile has been suggested to be an autotetraploid derivative of A. mongolicum (Asay et al. 1992). The chromosomes of A. fragile include a few AG, AC, and pSc119.2 hybridization sites. The genomic characters of A. fragile are different from those of A. mongolicum. The present results did not support the suggestion that A. fragile is an autotetraploid derivative of A. mongolicum. Moreover, additional 5S rDNA sites were revealed in the intercalary regions of the short arms of one homoeologous set of chromosome 6 (6^). Similar to A. desertorum, the unique FISH pattern revealed in chromosome 6^ is supportive of a potential allopolyploid origin for A. fragile.
Agropyron michnoi exhibited a FISH pattern more similar to that of A. cristatum (4x). Specifically, the chromosomal co-linearity of the detected 45S rDNA sites was well retained between the two species, suggesting that A. michnoi might be more closely related to A. cristatum (4x) than to other Agropyron species studied.

Variability of the repetitive sequences in the P genome
The repeats, such as AC, AG, and pSc119.2, that are localized in subtelomeric regions, showed a highly varying number of hybridizations among species with the P genome and within populations, particularly in tetraploid species. The presence or absence of hybridization signals might reflect the deletion or duplication of the repetitive sequences. Unequal crossing over is a type of gene duplication or deletion event (Graur et al. 2000). Highly variable repeats of the above-mentioned sequences in tetraploid species, which include two sets of the basic P genome, suggests that variability of repeats may be strongly driven by unequal crossing over during the interfered meiosis process in tetraploid species with two similar genomes. The small number of trinucleotide SSRs hybridization sites and aneuploids frequently detected in tetraploid species might further confirm interference of meiosis in tetraploid species.
Specific FISH patterns of trinucleotide SSRs were detected in three different Agropyron species. Extensive intra-or inter-population genetic variation has been detected in Agropyron species (Mellish et al. 2002, Che et al. 2011, Chen et al. 2013). However, limited materials of each species were analyzed in the present study. Whether the FISH patterns of trinucleotide SSRs also exhibit high intraspecific variation needs further investigation.