Genome Analysis of Thinopyrum intermedium and Its Potential Progenitor Species Using Oligo-FISH

The genome composition of intermediate wheatgrass (IWG) is complex and continues to be a subject of investigation. In this study, molecular cytogenetics were used to investigate the karyotype composition of Th. intermedium and its relative diploid species. St2-80 developed from Pseudowroegneria strigose and pDb12H developed from Dasypyrum breviaristatum were used as probes in fluorescence in situ hybridization (FISH) to classify the chromosomes of Th. intermedium into three groups, expressed as JvsJvsJrJrStSt. A combined multiplex oligonucleotide probe, including pSc119.2-1, (GAA)10, AFA-3, AFA-4, pAs1-1, Pas1-3, pAs1-4, and pAs1-6, was used to establish the FISH karyotype of ten accessions of Th. intermedium. Variability among and within the studied accessions of intermediate wheatgrass was observed in their FISH patterns. Results of this study led to the conclusions that Jvs had largely been contributed from Da. breviaristatum, but not the present-day Da. villosum; IWG had only one J genome, Jr, which was related to either Th. elongatum or Th. bessarabicum; and St was contributed from the genus Pseudoroegneria by hybridization with Th. junceiforme or Th. sartorii.

The genome constitution of IWG had been proposed as E 1 E 2 X [12], J e J e S [12] (while the symbol S was later changed to St [13]).The presence of the St genome of Pseudoroegneria in IWG was substantiated by all subsequent studies [14][15][16][17].Using St genomic DNA as a probe, there were eight to ten chromosomes showing the signals at centromeres [14][15][16]18,19], which was given the symbol J s [15] even though it was not a complete set of 14 chromosomes.Later, St 2 -80 was developed as a new FISH marker for the St genome, useful in genome analysis of Triticeae species and hybrids [20].When genomic DNA of Dasypyrum villosum or an oligo DNA from D. breviaristatum (H.Lindb.)Fred.(syn.Dasypyrum hordeaceum (Hack.)P. Candargy) pDb12H [21,22] was used as a probe, fourteen chromosomes, including those eight to ten J s type chromosomes, showed hybridization signals.On the other hand, the 14 J chromosomes were distinguished from J s and St chromosomes by a long terminal repeat (LTR) sequence pMD232-500, from Secale [18].Therefore, J s was changed to J vs and J was changed to J r , and J r J r J vs J vs StSt was proposed to be the genome symbol for Th.intermedium [23].Hence, this genome constitution had been adopted by other researchers [24,25].It should be noted that J r was proposed as an ancestral genome to the present-day J b and J e genome in the diploid species Th. bessarabicum (Savul.and Rayss) A. Löve and Th.elongatum (Host) D. Dewey, respectively [23], based on the relationships between R and the J genomes revealed by restriction site differences [26].
Genomic in situ hybridization (GISH) had been a valuable cytogenetic technique widely used to determine genome constitutions of plant species.However, the oligonucleotide fluorescence in situ hybridization (oFISH) could pinpoint the chromosomal locations of known genes whose DNA sequences were used as probes.
Karyotypes of oligonucleotide fluorescence in situ hybridization, coupled with molecular markers, would be useful for aiding the precise identification of individual chromosomes in IWG.Using dual and multiplex oligonucleotide FISH, we studied ten accessions of IWG having widely different origins along with IWG-related or progenitor species of the ploidy ranging from diploid to hexaploid.The objectives are (1) to test if pDb12H and St 2 -80 are sufficient to distinguish J r , J vs , and St in Thinopyrum species, (2) to determine if variability in IWG can be visualized with oFISH, and (3) to refine the relationships between IWG and its progenitor diploid species.Results will provide a clear picture of the relationships among studied species in the tribe Triticeae.

OligoFISH of Ten Accessions of IWG
In our study, chromosomes in ten accessions of IWG (Table 1) were probed with two oligonucleotides, pDb12H and St 2 -80, to distinguish the J r , J vs , and St genomes.Then, the same chromosome spreads were probed with bulked oligonucleotides consisting of pSc119.2-1,(GAA) 10 , AFA-3, AFA-4, pAs1-1, Pas1-3, pAs1-4, and pAs1-6 after the slides were cleaned of the pDb12H and St 2 -80 probes.The probe pDb12H labeled 14 chromosomes of the J vs genome with green color on both arms, whereas the probe St 2 -80 gave red-colored signals at distal ends of 14 chromosomes, each of the J vs and J r genomes, and in the interstitial regions of 14 short chromosomes of the St genome (Figure 1).The probe pDb12H labeled 14 chromosomes of the J vs genome with green color on both arms, whereas the probe St2-80 gave red-colored signals at distal ends of 14 chromosomes, each of the J vs and J r genomes, and in the interstitial regions of 14 short chromosomes of the St genome (Figure 1).All ten accessions of IWG had the same FISH pattern from the two probes pDb12H and St2-80, showing their common genome constitution J r J r J vs J vs StSt (left side of Figure 2).However, the multiplex oligonucleotide FISH patterns revealed variable signals on some chromosomes of the J vs and J r genomes among accessions, or even within an accession (right side of Figures 1 and 2).All ten accessions of IWG had the same FISH pattern from the two probes pDb12H and St 2 -80, showing their common genome constitution J r J r J vs J vs StSt (left side of Figure 2).However, the multiplex oligonucleotide FISH patterns revealed variable signals on some chromosomes of the J vs and J r genomes among accessions, or even within an accession (right side of Figures 1 and 2).

OligoFISH of Three Tetraploid Species of Thinopyrum Genus
Both Th. junceiforme and Th.sartorii had the J vs J vs J r J r genome constitution, as shown in Figures 3 and 4, respectively.Variation in FISH signals between homologous chromosomes could be detected.On the other hand, Th. scirpeum was found to be an autotetraploid having the J r J r J r J r genome composition (Figure 5).The former two species differ from Th. intermedium by lacking the St genome.

OligoFISH of Four Diploid Species That Were Implicated as Progenitors of IWG
Three diploid species, Th. elongatum, Th. bessarabicum, and Ps.sspicata, were studied using the two probes pDb12H and St 2 -80 and multiplex oligos pSc119.2-1,(GAA) 10 (green), AFA-3, AFA-4, pAs1-1, Pas1-3, pAs1-4, and pAs1-6 (red) (Figure 6).None of them showed strong green signals from pDb12H, indicating these species could not be the donor of the J vs genome in IWG.Some chromosomes of Th. elongatum had very faint green signals in the interstitial regions, whereas no such signals were detected in Th. bessarabicum (Figure 6A-D).Weak green signals of the pDb12H probe were detected at the centromere region of eight chromosomes in Da.Villosum probed by pDb12H and St 2 -80 (Supplementary Figure S1).

OligoFISH of Three Tetraploid Species of Thinopyrum Genus
Both Th. junceiforme and Th.sartorii had the J vs J vs J r J r genome constitution, as show Figures 3 and 4, respectively.Variation in FISH signals between homologous chro

OligoFISH of Four Diploid Species That Were Implicated as Progenitors of IWG
Three diploid species, Th. elongatum, Th. bessarabicum, and Ps.sspicata, were studied using the two probes pDb12H and St2-80 and multiplex oligos pSc119.2-1,(GAA)10 (green), AFA-3, AFA-4, pAs1-1, Pas1-3, pAs1-4, and pAs1-6 (red) (Figure 6).None of them showed strong green signals from pDb12H, indicating these species could not be the donor of the J vs genome in IWG.Some chromosomes of Th. elongatum had very faint green signals in the interstitial regions, whereas no such signals were detected in Th. bessarabicum (Figure 6A-D Interestingly, the red signal of St 2 -80 occurring in Th. elongatum, Th. bessarabicum, and Ps.spicata differed in its distribution on chromosomes.This red color was intense and located on distal ends of chromosomes in the two diploid Thinopyrum species.It was mostly spread out in the interstitial regions of Pseudorogneria chromosomes.There were more chromosomes with the red hybridization signal in Th. bessarabicum (Figure 7) than Th.elongatum (Figure 6).

2.4.
In Situ Hybridization of Th. intermedium Using Genomic DNA of Th. elongatum and Th.bessarabicum, and Oligo Probes pDb12H and St 2 -80 When Th. intermedium was probed with genomic DNA of Th. elongatum and Th.bessarabicum, and the two oligos pDb12H and St 2 -80, it was found that the genomic DNA of both diploid Thinopyrum species hybridized the same 14 chromosomes (Figure 7A,B).This result indicated that IWG had only one J genome.On the other hand, the oligo probes pDb12H and St 2 -80 hybridized the other 28 chromosomes, 14 each of the J vs and St genome, respectively (Figure 7C).Interestingly, the red signal of St2-80 occurring in Th. elongatum, Th. bessarabicum, and Ps.spicata differed in its distribution on chromosomes.This red color was intense and located on distal ends of chromosomes in the two diploid Thinopyrum species.It was mostly spread out in the interstitial regions of Pseudorogneria chromosomes.There were more chromosomes with the red hybridization signal in Th. bessarabicum (Figure 7) than Th.elongatum (Figure 6).When Th. intermedium was probed with genomic DNA of Th. elongatum and Th.bessarabicum, and the two oligos pDb12H and St2-80, it was found that the genomic DNA of both diploid Thinopyrum species hybridized the same 14 chromosomes (Figure 7A,B).This bridize the same chromosomes of IWG in a root-tip cell (Figure 8).Both V genomic DNA (Figure 8A) and pDb12H (Figure 8B) hybridized the same 14 chromosomes in Th. intermedium.The St2-80 probe hybridized all 42 chromosomes, but at different sites and at a different intensity.Chromosomes showing green signals of pDb12H had the red signals from St2-80 at telomeric ends.Intense red signals were on 14 short chromosomes, and another 14 chromosomes had red signals at the ends of chromosome arms that embraced bluish interstitial segments.

Prior Studies on Thinopyrum intermedium and Related Species
The genome constitution of IWG had been investigated by many research groups and its genome symbol changed over the years, as described by Wang and Lu (2014) [34].The genome constitution of IWG had been designated as E 1 E 2 X [12].Using the methods of chromosome karyotyping, Giemsa C-banding, and meiotic pairing in hybrids, Liu and Wang [35] concluded that the X genome in Th. intermedium is the S genome from an unspecified Pseudoroegneri pecies.Three tetraploid species, Elytrigia caespitosa, Lophopyrum nodosum, Pseudoroegneria geniculata ssp.scythica, were determined to have the J e S genome constitution in the same study.Then, the symbol S was changed to St in 1995 [13].The karyotype of Th. intermedium revealed two sets of seven chromosomes that were longer than the third set of seven chromosomes, which was the St genome.When Th. intermedium × Th. bessarabium hybrid was analyzed, seven long chromosomes were attributed to Th. bessarabium (J b genome); 14 intermediate (J e ) and seven short chromosomes (St) were from Th. Intermedium.It was shown in this study that when J b was present with St, the ratio between the longest and shortest chromosome was about 2.3.The ratio was around 1.8 when J e and St were present in combination.This ratio was 1,3 within the J b genome.
In 1992, Liu and Wang [36] had given the genome symbol J b J e to both Th.junceiforme and Th.sartorri, but noted variations in the satellite number and size as well as C-banding.
A few years earlier, Pienaar et al. [37] studied the genome relationships in Thinopyrum species.Genome constitution was determined in Th. scirpeum as J e J e , Th. distichum J d J d , Th. junceiforme J 1 J 2 .
Using in situ hybridization and molecular markers, Wang and Zhang [14] first used the St genomic DNA in a GISH study of two translocation lines involving Th. intermedium that conferred the resistance to either wheat streak mosaic virus or barley yellow dwarf virus.The presence of St chromatin in these two translocation lines CI17766 and TC14 was substantiated also by St-and E-specific RAPD cloned marker OPB08 525 and OPC03 340 , respectively.
In Canada, Chen et al. (1998) [15] analyzed both Th.intermedium and Th.ponticum using the genomic DNA from Th. elongatum (Host) D.R. Dewey (genome E, 2n = 14), Th. bessarabicum (Savul.& Rayss) Á. Löve (genome J, 2n = 14), and Ps.strigosa (M.Bieb.)Á. Löve (genome St, 2n = 14).They gave Th. intermedium the genome designation JJ s S, where J was related to the E genome of Th. elongatum and the J genome of Th. bessarabicum, the S genome was homologous to the S genome of Ps. strigosa, while the J s genome referred to modified J-or E-type chromosomes distinguished by the presence of S genome-specific sequences close to the centromere.However, the J s genome was not composed of fourteen, but only nine to eleven chromosomes.
Later, Kishii et al. (2005) [16] showed that Th. intermedium contains three kinds of genomes: St, E/J, and the third genome might be close to the V genome.However, PCR analysis disconfirmed the presence of the present-day V genome in Th. intermedium, but showed some similarity in the R genome.Thus, the genomic formula of Th. intermedium was tentatively re-designated as StJ s (V-J-R) s .Then in 2011, the study of Mahelka et al. [17] further complicated the genome constitution of Th. intermedium by GISH and nuclear GBSSI sequences suggesting that present-day genera Pseudoroegneria, Dasypyrum, Taeniatherum, Aegilops, and Thinopyrum were involved in the evolution of IWG.The DNA of the former two genera consistently hybridized to two genomes of Th. intermedium, but the third genome could be hybridized by those of the other three genera.

Current Studies on Thinopyrum intermedium and Related Species
The ten accessions of IWG studied here had the same genome constitution J r J r J vs J vs StSt, indicating that IWG populations collected from a wide range of regions (Table 1) had the same evolutionary end product even though it might have gone through different pathways, i.e., different diploid Pseudoroegneria species such as P. libanotica, P. stipieforlia, or P. strigosa could be hybridized by different tetraploid Thinopyrum species such as Th.junceiforme or Th.sartorii.The multiplex oligonucleotide FISH patterns of these IWG accessions, however, revealed variability among and within accessions.These variations could be attributed to the outcrossing nature of this species [38] and multiple hybridization from different sources [17].
Understanding the relationships among J, St, and ABD of wheat, Wang's laboratory [14,39,40] first used the genomic DNA of St as a probe for the GISH study of wheat hybrids with Th. ponticum and Th.intermedium.The use of St genomic DNA as a GISH probe for the detection of J chromosomes or chromosome fragments in hybrid derivatives of wheat × Thinopyrum intermedium was endorsed as a "landmark approach' for tracing the introgression of J chromatin into wheat [41].However, our findings of St 2 -80 hybridization signals at distal ends of chromosomes in Thinopyrum species (Figures 1-6) might set the limitation of this approach of GISH detection for useful alien genes.If the genes of interest were located in the interstitial region of J chromosomes, St genomic probe would likely fail to detect the introgression of alien chromatin.
Because IWG had been proposed to have a haplome formula of J e J e St [13], naturally occurring tetraploid Thinopyrum species having the genome composition J b J b , J b J e or J e J e would be a potential progenitor of IWG.Indeed, tetraploid Th. junceiforme and Th.sartorii previously shown to have the haplome composition J b J e [36] were found to have the J vs J r portion of J vs J r St in IWG (Figures 3 and 4).Thus, these two tetraploid Thinopyrum species are now the candidates for a progenitor of IWG.One the other hand, Th. scirpeum that is J r J r (Figure 5) could be excluded from the candidate progenitors of Th. intermedium.
One easy way to ascertain the presence of the St genome along with the J genome is to determine the chromosome length ratio between the longest and shortest chromosomes in the studied species.The ratio will be greater than 2.3 when J b and St are present; about 1.8 when J e and St are present; and about 1.3 when St is absent [35].The ratio around 2.3 was observed in karyotypes of Th. intermedium (Figures 1 and 2) where St was present.The ratio around 1.3 was found in Th. junceiforme, Th. sartorii, and Th.scirpeum (Figures 3-5), indicating the absence of St.The autotetraploidy of Th. scirpeum [37] is now confirmed by its J r J r J r J r genome composition.
Reporting the development of pDb12H, Yang et al. stated that this FISH probe detected all chromosomes of Da. breviaristatum (see Figure 6 of [21]), but the FISH signal was not detectable in Da. villosum chromosomes in diploid accessions [21].However, they did not provide the photographic evidence for the presence or absence of pDb12H in Da. villosum.
Our Figure S1 clearly shows that pDb12H could only weakly hybridize the pericentromeric regions of eight chromosomes of Da. villosum.This result supports the conclusion that Da. villoum and Da.breviaristatum are distinct, deserving the designation of genome symbols V v and V b , respectively [42,43].It also substantiates the conclusion that the present-day V genome of Da. villosum is not the J vs genome [23].
Most importantly, the observations that genomic DNA of Th. elongatum or Th.bessarabicum hybridized only to the same 14 chromosomes in IWG (Figure 7A,B) indicate that IWG had only one J genome.This observation is unique and different because the same root-tip cell was used in sequential GISH experiments.All other studies mentioned earlier had different root-tip cells used for GISH using different probes.Our finding in this study supports that reported for Th.junceiforme [44].Furthermore, the J vs genome could be hybridized by either genomic DNA of Da. villosum or oligo probe pDb12H (Figure 8), indicating that J vs was likely a progenitor of Da. breviaristatum and Da.villosum.Based on Figure S1, it can be inferred that the V v genome of the latter had a much lower copy number of the pDb12H sequence than V b of Da. breviaristatum.When more evidence becomes available, the genome symbol J vs might be changed to V b .

Future Studies on Thinopyrum intermedium and Related Species Needed
The recent development of the precise identification of Th. intermedium chromosome compliment [45] and chromosome-specific bulked oligonucleotides for identifying E-genome chromosomes in both Th.bessarabicum and Th.elongatum [46] would be useful in future studies on all Thinopyrum species and wheat × Thinopyrum hybrid derivatives.
In addition, a polyhaploid of Th. intermedium (2n = 3x = 21, J vs J r St) and the hybrid between Th. intermedium and different Pseudoroegneria species (2n = 4x = 28, J vs J r StSt) should be made.Then, oFISH using pDb12H [21,22], St 2 -80 [20], and pMD232-500 [18] should be carried out on both root-tip cells and pollen mother cells of these plants.The results from oFISH of pollen mother cells would reveal whether the two genomes J vs and J r are capable of high pairing.If the two genomes can pair to form four or more bivalents, their genome symbols would stay.If the two genomes cannot form any bivalent, the J vs might have to be changed to V.

DNA Extraction and Probe Preparation
The CTAB method was used to extract total genomic DNA from Th. elongatum, Th. bessarabicum, Ps. strigose, Da. villosum.Two oligonucleotide probes used for the FISH studies were St 2 -80 [20] and pDb12H [21,22].pDbH12 could serve as a cytogenetic marker for tracing chromatin from the V b genome in wheat-alien introgression lines.St 2 -80 is a potential and useful FISH marker that can be used to distinguish St and other genomes in Triticeae.Fluorescent signals of Th. elongatum (E e = J e ), Th. bessarabicum (E b = J b ), and Ps.strigose (St) genomic DNA as well as St 2 -80 were labeled with Texas-red-5-dCTP, while pDb12H and Da.villosum (V) genomic DNA were labeled with fluorescein-12-dUTP using the nick translation method.Oligonucleotides (synthesized by Sangon Biotech, Shanghai, China) pSc119.2-1and (GAA) 10 were labeled with 5 -FAM (5-carboxyfluorescein),

2. 5 .
In Situ Hybridization of Th. intermedium Using Genomic DNA of Dasypyrum villosum and Oligo Probes pDb12H and St 2 -80Genomic DNA of Da. villosum and oligo probes pDb12H and St 2 -80 were used to hybridize the same chromosomes of IWG in a root-tip cell (Figure8).Both V genomic DNA (Figure8A) and pDb12H (Figure8B) hybridized the same 14 chromosomes in Th. intermedium.The St 2 -80 probe hybridized all 42 chromosomes, but at different sites and at a different intensity.Chromosomes showing green signals of pDb12H had the red signals from St 2 -80 at telomeric ends.Intense red signals were on 14 short chromosomes, and another 14 chromosomes had red signals at the ends of chromosome arms that embraced bluish interstitial segments.

Table 1 .
Plant materials used in this study.