Detailed analysis of rye B-located high copy sequences revealed that Bs contain a similar proportion of repeats as A chromosomes, but differ substantially in their repeat composition (Martis et al. 2012; Klemme et al. 2013; Marques et al. 2013). More specifically, B-specific accumulation of Gypsy retrotransposons and other repeated sequences have been reported for rye (Sandery et al. 1990). The accumulation of active retrotransposons on Bs might be rooted in a relaxed selection pressure. Reduced crossing-over in Bs might further facilitate retroelement accumulation.

As mentioned above, B-located genic sequences originated from A chromosomes or from organellar DNA. Because Bs are dispensable, it is expected that they are prone to mutation accumulation as they undergo pseudogenization (Banaei-Moghaddam et al. 2015). Pseudogenes can encode endo-siRNAs and regulate the expression of their parental A chromosome genes (Johnsson et al. 2014). Thus, as B-specific transcripts would might be aberrant due to less selective pressure, they can serve as a substrate for RNA-directed RNA polymerase, which would make double strand RNA (dsRNA) (Banaei-Moghaddam et al. 2015). The dsRNA is then processed to small regulatory RNA. This model is consistent with the cumulative effects of an increasing number of Bs (Banaei-Moghaddam et al. 2015). Alternatively, pseudogene transcripts can function as indirect post-transcriptional regulators. For example, they might act as miRNA sponges. Due to high similarity between parental and pseudogene transcripts, both could compete for miRNA, leading to degradation of parental gene transcripts (Muro et al. 2011). Further, it has been shown that some pseudogene transcripts translate and produce short peptides or truncated proteins of functional importance (Johnsson et al. 2014). Pseudogene transcripts could also act as a source of siRNAs via pathways involving RNA-directed RNA polymerases (Banaei-Moghaddam et al. 2015). It is not yet known whether B-derived siRNAs acting as regulators exist.

Little information is available on the chromatin composition of Bs. Preliminary classical cytological observations (e.g., Giemsa-banding) suggested that the Bs in about half of the plant species carrying them are heterochromatic (Jones 1975). In rye, the sub-terminal heterochromatic domain of the B is characterized by a unique combination of histone methylation marks (Carchilan et al. 2007; Marques et al. 2013). Contrary to the heterochromatic regions of the A chromosomes, this domain is simultaneously marked by tri-methylated histone H3K4 and tri-methylated H3K27. In addition, this domain shows a dark Giemsa band at mitosis, but undergoes decondensation during interphase and transcription of B-specific high copy repeat families (Banaei-Moghaddam et al. 2015).

### 4.3 Preferential Transmission (Accumulation Mechanism) of B Chromosomes

B chromosomes dispensability, i.e., non-essentiality for normal development of their host, remains undetermined. In addition, while they may have negative effects on nuclear physiology and phenotype, it remains unclear if Bs have any selective advantages and how they manage to persist in specific populations. Certainly, their maintenance requires means of survival against selection. To counteract elimination, Bs of many species have evolved a 'drive' mechanisms, which ensures their transmission to the next generation at frequencies that are higher than expected according to Mendelian rules (Jones 1991; Jones et al. 2008a). Such transmission enables the maintenance of Bs in natural populations. The variety of mechanisms, including segregation failure, by which B chromosomes gain heritable advantage in transmission, are known as accumulation or drive mechanisms. Depending on the species, B chromosome drive can be pre-meiotic, meiotic, or post-meiotic, but the underlying molecular process remains unclear (Jones 1991; Burt and Trivers 2006). Post-meiotic drive is frequent in flowering plants during gametophyte maturation.

Survival in populations is achieved by various mechanisms of mitotic and meiotic drive (Jones 1991, 1995). The more common drive process for Bs in plants, especially in the family Poaceae, is based on directed nondisjunction in the gametophyte phase of the life cycle. In rye, it takes place both at first pollen mitosis and at first egg cell mitosis, in maize, it occurs at the second pollen mitosis, and at other time points in other species (Jones and Rees 1982).

Matthews and Jones (1982, 1983) assumed that the differential transmission of Bs in rye is strong enough to overcome their negative effects on plant vigor and fertility. Moreover, they suggested that the main factor enabling variation in population equilibrium for B-frequency is the level of bivalent or multivalent pairing of Bs with themselves at meiosis. Confirmation of the predicted variation in pairing levels was achieved when selection for high and low transmission genotypes was found to correlate with the level of B pairing at first meiotic metaphase in lines of Korean rye (Jiménez et al. 1997). It was found that plants with 2B of the low line formed bivalents in only 20% of first meiotic metaphase cells, whereas, in the high line, there were more than 90% of bivalents. Puertas et al. (1998, 2000) later proposed that what Jiménez et al. (1997) assumed were 'genes' dictating transmission rate, were actually the sites of chiasma formation, or the binding sites, in the Bs themselves.

In most species carrying Bs, the mitotic transmission of Bs during growth and development is normal and hence, all cells within the individual carry the same number of Bs (Houben et al. 2014). However, there are some exceptions in which the Bs show mitotic instability and are therefore present in variable numbers, sometimes characterizing specific tissues and/or organs. For example, in the grasses Aegilops speltoides and Amblyopyrum muticum, Bs exist in aerial organs but not in roots (Mendelson and Zohary 1972; Ohta 1996).

The behavior of rye Bs during pollen mitosis was first studied by Hasegawa (1934), who described how the two chromatids of the B chromosome do not separate at anaphase of the first pollen grain mitosis and, in most cases, are included in the generative nucleus. In the second pollen grain mitosis, the generative nucleus divides to produce two sperm nuclei, each with an unreduced number of Bs. A similar nondisjunction process may occur in the female gametophytes of rye as well (Håkansson 1948).

Banaei-Moghaddam et al. (2012) proposed a model describing the B chromosome accumulation mechanism in rye. The model is based on analysis of the cellular mechanism of B chromosome drive in the male gametophyte of rye. At all mitotic stages of microgametogenesis, the As and Bs centromeres are active. However, at first pollen mitosis, sister chromatid cohesion differs between As and Bs. The B-specific pericentromeric repeats are involved in the formation of pericentric heterochromatin, which plays a critical role in the cohesion of sister chromatids and in their non-disjunction at the first pollen mitosis. Failure to resolve the pericentromeric cohesion is under the control of the B-specific nondisjunction control element. The asymmetry of this division plays a critical role in the determination and subsequent fate of the two unequal mitotic products: the vegetative and the generative cells. Due to unequal spindle formation, joined B chromatids preferentially migrate towards the generative pole. Thus, at first pollen mitosis of rye, the mitotic spindle asymmetry seems to play a central role in B accumulation. In the second pollen mitosis, the generative nucleus divides to produce two sperm nuclei, each with an unreduced number of Bs. Hence, a combination of nondisjunction and of unequal spindle formation at first pollen mitosis, results in the directed accumulation of Bs to the generative nucleus, which consequently ensures their transmission at a higher than Mendelian rate to the next generation.

The B centromeres demonstrate standard behavior at anaphase of the first pollen mitosis, and can be seen separated and pulling to opposite poles. In contrast, the B chromatids appear to be transiently held together at sensitive sticking sites on either side of the centromere (the receptors), and since the spindle is asymmetric, the equator is closer to the pole which will include the B chromatids in the generative nucleus. The question to be answered is how the B-specific region signals the receptors to remain conjoined just long enough to facilitate directed nondisjunction? This is a fundamental question in terms of genome evolution, since the mechanism had to arise de novo, and then become rapidly established in a highly conserved way to allow the rye Bs to survive following their origin. Furthermore, nondisjunction works equally well when the rye B is introduced as an additional chromosome into hexaploid wheat (Lindström 1965; Müntzing 1970; Niwa et al. 1997; Endo et al. 2008), into hypo-pentaploid Triticale (Kishikawa and Suzuki 1982), or into Secale vavilovii (Puertas et al. 1985). Thus, the B autonomously controls the process of nondisjunction (Matthews and Jones 1983; Romera et al. 1991).

Müntzing (1946) demonstrated in Secale cereale that in the standard B chromosome, as well as in isochromosomes consisting of the large and small arms of the standard B chromosome, the centromeres divided normally at anaphase of the pollen mitosis. However, in the standard and large isochromosomes, there were sticking sites on either side of the centromere which prevented normal anaphase separation of the chromatids, thereby causing nondisjunction of these two types. The chromosomal region that carries the element controlling nondisjunction is comprised of a concentration of B-specific sequences from two families, E3900 and D1100, assembled from a variety of repetitive elements, some of which are also represented in the A genome (Sandery et al. 1990; Blunden et al. 1993; Houben et al. 1996; Langdon et al. 2000). No genes have been found in the region, which raises questions regarding the genetic process controlling nondisjunction (Matthews and Jones 1983; Ortiz et al. 1996).

Deficient Bs lacking the heterochromatic terminal region of the long arm, undergo normal disjunction at first pollen anaphase. Therefore, it seems that the accumulation mechanism of the B by nondisjunction requires factors located at the end of the long arm (Müntzing 1948; Håkansson 1959; Endo et al. 2008). This factor can act in trans because, if a standard B (Lima-de-Faria 1962) or the terminal region of the long arm of the B (Endo et al. 2008) is present in the same cell containing a deficient B, nondisjunction occurs for both the standard and the deficient B. The nondisjunction control region is enriched in B-specific repeats, which are highly transcriptionally active in anthers (Banaei-Moghaddam et al. (2012). In addition, the distal heterochromatin of the long arm is marked with the euchromatin-specific histone modification mark H3K4me3 (Carchilan et al. 2007).

Lima-de-Faria (1962) initially proposed that the rye B drive mechanism is controlled by the sub-telomeric domain of the B long arm, where two sequence families, D1100 (Sandery et al. 1990) and E3900 (Blunden et al. 1993), accumulate (Langdon et al. 2000). Later, analysis of chromosome behavior of wheat lines with introgressed fragments of rye Bs, established that non-disjunction is in fact dependent on the D1100 and E3900 sequence families (Endo et al. 2008). More recently, a B-specific chromatid adhesion site involving the pericentromeric repeat ScCI11, was implicated in the delay of sister chromatid separation (Banaei-Moghaddam et al. 2012). Langdon et al. (2000) suggested that E3900 and D1100 repeats evolved via amplification of ancestral A-located sequences within the dynamic nondisjunction control region on rye B. The B-enriched tandem repeats could have been amplified via unequal crossover (Smith 1976).

It has been previously suggested that Bs of the two subspecies of rye have a monophyletic origin (Niwa and Sakamoto 1995; Marques et al. 2013), and that the organization of D1100 and E3900 is highly conserved (Klemme et al. 2013). The complex organization of the E3900 and D1100 sequence families as high copy repetitive DNA is specific to Bs (Klemme et al. 2013). FISH showed that D1100 accumulates in two zones in the sub-telomeric region of the long B arm, physically separated by an interstitial less labeled space, while E3900 has a more homogeneous and distal signal that overlaps with the D1100 domain closer to the telomere (Wilkes et al. 1995). Besides the 3.9-kb form of E3900, a shorter 2.7-kb E3900-related sequence, which also accumulates on the B-specific domain, has been identified (Pereira et al. 2009). Estimates of copy number have shown that E3900 sequences are highly conserved and are present in 100–150 copies on Bs and in single or low copy numbers in A chromosome (Pereira et al. 2009). Importantly, E3900 sequences are differentially expressed in a tissue- and developmental stage-specific manner in plants with and without B chromosomes (Pereira et al. 2009). While the expression levels of E3900 do not vary in leaves from plants with and without Bs, they are significantly upregulated during meiosis exclusively in plants with Bs, maintaining a high level of transcription in the gametophyte (Pereira et al. 2009).

Transcripts of the recently identified D1100 and E3900 tandem repeats were only observed in + B plants, mostly restricted to anthers, where post-meiotic nondisjunction of rye Bs takes place (Carchilan et al. 2007). Although it remains to be directly demonstrated, anther-specific transcripts of sequences residing within the terminal nondisjunction control region might be related to the non-Mendelian accumulation of Bs, for example, by mediating stickiness of sister pericentromeric regions.

The discovery that some of the nondisjunction control region-specific repeats produce noncoding RNA, predominantly in anthers of rye (Prestel et al. 2010), suggests an intriguing possibility that the nondisjunction of Bs occurs because the control region somehow maintains cohesion in key regions of B-sister chromatids. Failure to resolve the pericentromeric heterochromatin during first pollen mitosis leads to the question: in which aspect does the first pollen mitosis differ from other mitotic events in other cell types? It is argued that either a haploid tissue type-specific expression of nondisjunction controlling transcripts (Carchilan et al. 2007) and/or the formation of a contrasting chromatin composition during first pollen mitosis (Houben et al. 2011) ensures this non-disjunction that results in specific accumulation of B chromosomes.

### 4.4 Effect on Morphology, Fitness and Meiotic Chromosomal Pairing in Species and Hybrids

### 4.4.1 Effect on Morphology and Fitness

The presence of Bs is associated with mild or no obvious change in phenotype in many species. This feature led to the conclusion that Bs are depleted of functional genes. However, an excessive number of Bs can cause phenotypic effects and may reduce host fertility (Jones and Rees 1982). The maximum number of Bs tolerated by individuals varies among different species. The harmful effects of Bs on fitness were charged to the energy cost of their maintenance and their potential interference with the proper assortment of A chroosomes during meiosis (Jones and Rees 1982). Besides reduction of fitness, further phenotypic effects have been associated with the presence of Bs in several plant species (Jones and Rees 1982).

The phenotypic effects of the presence of B chromosomes are usually cumulative, depending upon the number of Bs, with a positive correlation between severity of effects and Bs counts (Jones and Rees 1982; Jones 1995; Bougourd and Jones 1997; Carlson 2009; Houben et al. 2013a).

A significant amount of information is available regarding the effects of rye Bs upon sporophyte and gametophyte fitness and viability, from seed germination to seed set (Jones and Rees 1982). As the number of Bs increases, negative effects on fertility (Müntzing 1943), seed germination timing (Moss 1966) and vigor (Müntzing, 1963) have been described. Bs also induce nuclear physiological effects in proportion to their number, such as increased cell cycle length (Evans et al. 1972) and decreased nuclear protein and RNA levels (Kirk and Jones 1970). These extra chromosomes have various effects on mitotic and meiotic A chromosome behavior. For example, Bs alter rDNA condensation patterns in mitotic cells (Morais-Cecílio et al. 1997; Delgado et al. 2004) and induce alterations in the frequency and distribution of A chromosome chiasmata at meiosis (Jones and Rees 1982). A dosage-dependent increase in the frequency of anomalous adherences between sister chromatids at anaphase and metaphase cells in the first mitosis of pollen grains provided more direct evidence that Bs affect A chromosome behavior (Pereira et al. 2009). Pereira et al. (2017) provided detailed cytogenetic and molecular insight into the effects of heat stress during reproductive development on meiosis in rye plants with 0 and 2B chromosomes. Their findings are the first indication that rye B chromosomes have implications in heat tolerance and protection against heat stress-induced damage at early stages of meiosis.

### 4.4.2 Effect on Meiotic Chromosome Pairing in Species and Hybrids

B chromosomes have been reported to have an effect on meiotic chromosome pairing in specie. Using the C-banding technique, Alvarez et al. (1991) studied the effect of Bs on homologous pairing, by examining first metaphase association of S. cereale chromosomes both in normal plants (2n = 14) and in plants with B chromosomes (2n = 14 + Bs). They noted a promoting effect on homologous meiotic chromosome pairing by B chromosome, particularly by its short arm.

Müntzing et al. (1969) studied meiosis in common wheat plants bearing one to several S. cereale B chromosomes that were transferred to common wheat from Secale cereale by Lindström in 1965. The effect of these B chromosomes on meiotic pairing of common wheat chromosomes was insignificant, whereas the meiotic pairing of the Bs was poor and the frequency of B chromosome univalents was much higher than in the corresponding strain of rye. The reason for this difference must involve an influence of the wheat chromosomes or the wheat cytoplasm on the rye chromosomes. Likewise, the influence of B-chromosomes of Secale cereale on homologous chromosome pairing at first meiotic metaphase of the inter-varietal common wheat hybrid, Chinese Spring x Lindström (carrying B chromosomes of S. cereale) was studied by Viegas (1979) in the presence and absence of chromosome 5D of common wheat, that carries a promoter of homologous pairing (Feldman 1966). The presence of these B chromosomes did not change the normal pattern of chromosome pairing in disomic plants, although it slightly increased chiasma frequency in monosomic 5D plants at 20 °C. When chromosome 5D was absent, e.g., nullisomic 5D, the increase in chiasma frequency was more pronounced, especially the number of ring bivalents. In nullisomic 5D plants, at 10 °C, where a high degree of asynapsis was observed, the addition of B chromosomes increased chromosome pairing, but no increase in the frequency of monosomics was observed, which at 10 °C showed a slight reduction in pairing.

The effect of B chromosomes on homoeologous pairing in hybrids with common wheat is ambiguous. B chromosomes of Amblyopyrum muticum and Aegilops speltoides suppress homoeologous meiotic pairing of A chromosomes in intergeneric hybrids with common wheat lacking the ph1 gene, which suppresses homoeologous pairing (Mochizuki 1964; Vardi and Dover 1972; Dover and Riley 1972; Ohta and Tanaka 1982). Thus, the effect of Bs is similar to that of Ph1 of wheat on homoeologous pairing in wheat hybrids with alien species. The B chromosomes of A. muticum do not affect homologous pairing but suppress homoeologous pairing in interspecific hybrids (Mochizuki 1964; Dover and Riley 1972; Vardi and Dover 1972). Similarly, studies on meiotic chromosomal pairing in F1 hybrids between A. muticum and most of the diploid species of Aegilops and Triticum containing B-chromosomes of A. muticum, showed notably reduced chromosomal pairing (Vardi and Dover 1972; Ohta and Tanaka 1983; Ohta 1990, 1991).

Vardi and Dover (1972) assumed that B chromosomes of Ae. speltoides and A. muticum interact with specific gene loci of the A chromosome complement that inactivate the suppression of Ph1. Furthermore, they suggested that Bs affect chromosomal pairing by causing disturbances in the mitotic and meiotic spindle. The similarity between the effects of Ph1 and B chromosomes on chromosomal pairing suggests that the ph1 gene of polyploid wheat was transferred from B chromosomes of A. muticum or Ae. speltoides to chromosome 5B of common wheat, via an ancestral translocation between a B chromosome and an A chromosome, presumably 5B (Vardi and Dover 1972). However, the absence of meiotic pairing between the B chromosome and chromosome 5B of common wheat refutes this idea. Alternatively, since B chromosomes derived from one of the A chromosomes in these two diploid species and contain many DNA sequences of other A chromosomes, one of the most essential prerequisites for the establishment of the B chromosome as an independent entity was to prevent pairing and recombination between B and A chromosomes. Thus, the development of a genetic system suppressing homoeologous pairing was a necessary event for the independent existence and evolution of B chromosomes.

On the other hand, conflicting results were obtained concerning the effect of Secale cereale B chromosomes on homoeologous pairing in hybrids with common wheat. Viegas (1980) reported that B chromosomes of S. cereale suppressed homoeologous pairing in hybrids with common wheat, irrespective of the presence or absence of chromosome Ph1, while Cuadrado et al. (1988) found that cereale B chromosomes only suppressed homoeologous pairing in hybrids with common wheat in the absence of Ph1. Romero and Lacadena (1980) found that cereale B chromosomes also suppress homoeologous pairing in hybrids with common wheat lacking the pairing suppressors on wheat chromosomes 3A and 3D, although they increased the level of pairing when a chromosome with a promoter effect (3B, 5A or 5D) was absent. Roothaan and Sybenga (1976) found that B chromosomes of S. cereale do not compensate for the absence of Ph1 in hybrids with common wheat lacking Ph1. Estepa et al. (1993) studied the effect of different numbers of B chromosomes on homoeologous pairing in common wheat x S. cereale hybrids and found no significant quantity-related effect, but the variance of distribution of means of bivalent and paired chromosome complements was significantly increased when odd numbers (3 or 5 B-chromosomes) were present. Jenkins and Jones (2004) concluded that B-chromosomes of S. cereale carry genes that act together with the pairing control genes of common wheat.

Kousaka and Endo (2012) studied the effect of a rye B chromosome and its segments (B-9 and B-10) on homoeologous pairing in hybrids between common wheat and Aegilops peregrina. The B-9 and B-10 chromosome segments are derived from reciprocal translocations between a wheat and B chromosome; B-9 had the B pericentromeric segment, whereas B-10 had the B distal segment. Kousaka and Endo (2012) found that both the complete B chromosome and the B-9 segment suppressed homoeologous pairing when chromosome 5B was absent. On the other hand, the B-9 and B-10 segments promoted homoeologous pairing when 5B was present. The mean chiasma frequency (10.23/cel) in the hybrid of common wheat x Ae. Peregrina possessing 5B and one B-9, was considerably higher than that of a hybrid possessing 5B alone (2.78/cell), and was comparable to that of a hybrid lacking 5B (14.09/cell). The results suggested that the effect of the B chromosomes on homoeologous pairing was not confined to a specific region, i.e., it resulted from a polygenic system, and that the intensity of the effect varied and depended on the presence or absence of Ph1 and on the dose of the B chromosome and the B segments.

### 4.5 Transcriptional Activity of B Chromosomes

Considering the intra- or interspecific origin of Bs and the above-listed B-associated effects, many have sought to determine whether Bs carry genes. Studies have led to different conclusions regarding the transcriptional activity of Bs. In contrast to single- or low-copy genes that were rarely found on Bs in early studies, rRNA genes have been frequently identified on Bs of many species (Green 1990; Bougourd and Jones 1997). This is most likely due to the fact that their detection is rather easy by cytogenetic techniques in contrast to unique genes. In contrast to the prevalent view that Bs do not harbor genes, recent analysis revealed that Bs of sequenced species are rich in gene-derived sequences (Houben et al. 2014). Recent application of next generation sequencing-based approaches revealed that rye Bs contain more than 4000 putative genic sequences (Martis et al. 2012), many of which are partly transcriptionally active (Banaei-Moghaddam et al. 2013). Some of the rye B gene sequences had lower similarity to their A-located counterparts, reflecting their faster degeneration or earlier insertion in Bs (Banaei-Moghaddam et al. 2013). These studies suggest that B chromosomes carry transcriptionally active genic sequences that could affect the transcriptome profile of their host genome (Banaei-Moghaddam et al. 2015). Thus, the view that B chromosomes are genetically inert, selfish elements without any functional genes, is gradually changing. This can partly explain the deleterious effects associated with their presence as well as the possible advantages that Bs confer on their host.

A comparative cDNA-AFLP analysis indicated that rye Bs can modulate the transcription of corresponding gene copies on A chromosomes (Carchilan et al. 2009) and, from these studies, regulatory interactions between A- and B-located coding sequences have been proposed (Banaei-Moghaddam et al. 2015). It is likely that Bs influence A-localized sequences through epigenetic mechanisms, such as homology-dependent RNA interference pathways (Slotkin and Martienssen 2007), as has been proposed for the modulation of gene-activity in newly formed hybrids and allopolyploids (Comai 2005; Kenan-Eichler et al. 2011). Bs may also exert control over A chromosomes via the spatial organization of As in interphase nuclei, and it has been suggested that spatial positioning of genes and chromosomes can influence gene expression (Misteli 2007).

As Bs are dispensable, it is expected that they are prone to accumulate mutations as they undergo pseudogenization (Banaei-Moghaddam et al. 2013). Indeed, next-generation sequencing technologies have shown that B chromosomes of S. cereale harbor many pseudogenes originating from the A chromosomes and organellar DNA sequences (Houben et al. 2013b). It has been further demonstrated that some of the A-derived sequences are transcribed in a genotype-specific manner (Banaei-Moghaddam et al. 2013). This could explain the apparently contradictory findings and complex interactions associated with the effect of B chromosomes on homoeologous pairing in hybrids with common wheat.

It is possible that only part of the B-located genes is inactive (Houben et al. 2014). If Bs share many, almost identical, genic sequences with As, why is the presence of Bs not associated with more severe phenotypes, particularly assuming that some sequence variants may still have a biological effect? Bearing in mind that the relative dosage of a chromosome is critical for normal development, it is striking that organisms with an additional B are little affected. It is probable that during early evolution of a proto B, A-derived genes are likely to be downregulated by dosage compensation.

Transcripts coming from a B chromosome in combination with their related A-located genes, provide additional complexity to the transcriptome of their host and this may partially explain the phenotypes and effects associated with the presence of Bs. The transcriptional activity of Bs could form regulatory transcripts such as siRNAs which have the potential to modulate the level of A-derived transcripts or to change the chromatin status of a target region by DNA or chromatin modification (Dalakouras and Wassenegger 2013; Filipowicz et al. 2005). In addition, transcripts from Bs similar to pseudogenes may lead to indirect effects by competing with A-derived transcripts for regulatory factors such as miRNAs (Pink et al. 2011). If truncated proteins or overexpression of functional proteins are produced in the presence of Bs, they can cause overloading of the proteasome machinery, required to process these unfolded, misfolded, aggregated, and/or uncomplexed proteins, thereby imparting an energetic burden in the host (Gordon et al. 2012). But Bs may also produce functional proteins that may have some role in maintenance of B chromosomes.

Banaei-Moghaddam et al. (2015) postulated the potential activity modulation of A chromosome-located genes by homologous sequences on B chromosomes. When the B chromosome is absent, each gene on A has a defined level and pattern of expression. In the presence of a B chromosome, when the B-located gene is transcribed, transcripts can serve as a substrate for RNA-directed RNA polymerase, which would generate dsRNA, which is then processed to small RNAs. These small RNAs could cause mRNA degradation or epigenetic changes of A-located sequences. Alternatively, the B-transcripts may compete for regulatory factors responsible for regulation of A-located genes, yielding up- or down-regulation of the counterpart A gene. In the presence of B, if the corresponding B-located gene is inactive, the expression level and pattern of an A-located gene will not change. Likewise, when both A- and B-located genes express from the same strand, the expression level of that gene may remain unchanged compared to that of plants lacking B, due to dosage compensation.

Transcription of B-enriched repetitive sequences have also been demonstrated in rye. A comparative analysis of RNA-seq reads obtained from rye plants with and without Bs, was recently performed to assess the transcriptional activity of repetitive sequences. In this species, several B-repeats are active in a tissue-dependent manner (Klemme et al. 2013; Carchilan et al. 2007). Transcripts derived from vegetative (root and leaf) and generative (anthers) tissues, represented by 26–151 million RNA-seq reads, were screened for their sequence similarity to a complete set of previously identified rye repetitive elements (Martis et al. 2012). Low levels of transcription were found for most repetitive elements, which represented a combined total of 3.1–4.9% of transcripts in analyzed tissues (excluding rRNAs). Repeat expression profiles were similar for plants with and without B chromosomes, with the exception of highly expressed B-specific satellite E3900, which was found in both vegetative tissues and anthers. This finding confirms previously reported data based on Northern blot experiments (Carchilan et al. 2007). On the other hand, none of the other satellites enriched on or specific to B chromosomes (Martis et al. 2012; Klemme et al. 2013) were expressed at comparable levels.

Dosage-sensitive genes are less likely a part of an evolving B, as duplication of a B chromosome-donor fragment containing such genes could result in a detrimental phenotype. In contrast, dosage-insensitive genes, such as structural genes bearing no regulatory roles in transcription or translation, could be B-hosted and eventually undergo loss or pseudogenization. Duplicated genes are often associated with detrimental effects and are removed by natural selection. As most of the mutations are degenerative, it is more likely that duplicated gene underwent inactivation rather than acquiring a new function. Nevertheless, evidence of the beneficial role of duplicated genes, especially in stressful conditions, exists. In some circumstances, selective retention of duplicated genes could occur, e.g., when their redundancy protects corresponding parental genes from immediate detrimental mutations, or when overdominance exists between their products. In this case, duplicated genes could convert to new genes by achieving a beneficial mutation. Therefore, B-located duplicated genes may accelerate the evolution of their carriers.

Could a B-located genic sequence with the ability to modulate the activity of a corresponding A-located gene cause long-term evolutionary effects? If the product of the B-located gene would become beneficial, positive selection would act to preserve the respective B present in the population. This could release the pressure on a B-specific differential transmission, namely, a former selfish B chromosome would transform into a beneficial genome component. It would be intriguing to test whether Bs affect the epigenetic status of As, by comparing epigenetic modifications and expression levels of parental A genes in the presence and absence of Bs, over several generations.

### References

Jones RN (2012) B chromosomes in plants. Plant Biosyst 146:727–737

Jones RN, Houben A (2003) B chromosomes in plants: escapeees from

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# 5.1 General Description of the Subtribe

Simmonds

Whereas the economically important cereals, Triticum L., Secale L., Hordeum L., and Aegilops L., have been subjected to intensive taxonomic, cytogenetic, molecular, and evolutionary studies, several other Triticeae genera received less attention. These "orphan" genera are Agropyron Gaertner, Eremopyrum (Ledeb.) Jaub. & Spach, Henrardia C. E. Hubbard, Dasypyrum (Coss. & Dur.) Dur., Heteranthelium Jaub. & Spach., Taeniatherum Nevski, and Crithopsis Jaub. & Spach. Several diploid Elymus species, having the St and E genomes, are also included in this group. They are small genera containing few species, most of which diploids that are characterized by a distinct morphology, and grow in different regions of the tribe distribution area, some in more arid environments. Their genetic relationships to the other well-studied genera of the tribe are vaguely known. Studies of these small genera may provide additional knowhow on the range of genetic diversity in the tribe, on processes that have led to diverge evolutionary developments as well as on the phylogenetic relationships among members of the tribe. As relatives of the crops, species of these orphan genera may contain valuable genes that, may be transferred to the crops and enhance greater tolerance to biotic and abiotic stresses, improve quality and performance. As such, these orphan genera deserve greater attention.

A number of interspecific and intergeneric hybrids involving those genera were produced during the years (e.g., Cauderon 1966; Sakamoto 1967, 1968, 1969, 1972, 1973, 1974, 1979; Sakamoto and Muramatsu 1963; Dewey 1969, 1970, 1984; Frederiksen 1991a, b, 1993; Frederiksen and von Bothmer 1986, 1989, 1995). Successful production of the hybrids suggests fairly good genetic or cytoplasmic compatibility among those species. However, there is very little chromosome pairing in F1 hybrids between them as well as between wheat and these species, indicating limited homology between their genomes. Hence, these genera seem to be highly differentiated from the taxonomic and the genetic viewpoints.

# 5.2 Elymus Species with St or E Genome

### 5.2.1 Group Description

The delimitation of the genera Elymus L. and Agropyron Gaertner has been the subject of controversy over the years (Assadi and Runemark 1995), primarily due to the absence of clear-cut generic characters and from the presence of numerous intergeneric hybrids that gave rise to conflicting results as discussed below (Melderis 1978). Their delimitation using different taxonomic treatments was changed several times over the last eight decades. Nevski (1933), Bor (1968), Tzvelev (1976), and Sakamoto (1974) kept these two genera separated, while Gould (1947) and Runemark and Heneen (1968), assuming that the traditional subdivision into Elymus s. l. and Agropyron s. l. (including Pseudoroegneria, Elytrigia, and Thinopyrum) is artificial, united them into a single genus, Elymus.

The species within the Elymus-Agropyron group have traditionally been referred to as Agropyron, if the spikelets are solitary, and as Elymus, if they are arranged in pairs or larger numbers at each rachis node. Yet, this division is not very distinct and Runemark and Heneen (1968) and Melderis (1978) pointed out that the number of spikelets at each node has a limited taxonomic value since several Agropyron species contain pairs of spikelets at several rachis nodes, especially in the lower or in the middle part of the spike, while several species of Elymus contain only one spikelet on each rachis node. Also, with regard to leaf anatomy, no difference was found between Elymus and Elytrigia that was included in Agropyron (Runemark and Heneen 1968). Dewey (1969, 1970) found homology between the genomes of several Elymus and Elytrigia species and Runemark and Heneen (1968) noted similar chromosome morphology in Agropyron elongatum (now Elymus elongatus) and Elymus

caninus. In reality, the two genera only represent different levels in the reduction of a paniculate inflorescence (Runemark and Heneen 1968). Cytogenetic studies (e.g., Cauderon 1966; Sakamoto 1973; Dewey 1984, and reference therein) contributed to a better understanding of the genomic relationships among species of Elymus and Agropyron and, as a result, to modification of the delimitation of the species in these two genera. Based on the above information, as well as on the absence of morphological discontinuities between the taxa Pseudoroegneria, Elytrigia, Thinopyrum, and Elymus, Melderis (1978, 1980) included these taxa in the genus Elymus s. l., while retaining Agropyron s. str. as a separate genus for the crested wheatgrasses, that contains only species with a solitary spikelet at each rachis node. The restricted Agropyron genus contains diploid and polyploid species that are based on the P genome (Table 2.4) and are morphologically distinct from other genera in Triticeae. The genus Elymus is treated by Melderis (1978, 1980, 1985a, b) in a broad sense, as comprising the genera Elytrigia Desv., Pseudoroegneria (Nevski) A. Löve, Thinopyrum A. Löve, Lophopyrum A. Löve, and Trichopyrum A. Löve (Table 2.2 ). Wang (1989) supported Melderis' classification which viewed separation of Pseudoroegneria from Elymus as unjustified for both evolutionary and morphological reasons, since several Elymus species include St genomes from different Pseudoroegneria diploids, and Pseudoroegneria and Elymus can hardly be distinguished from each other. This taxonomic classification makes the discrimination between these two genera more straightforward and has been accepted by various taxonomists, e.g., Clayton and Renvoize (1986), Assadi and Runemark (1995), Watson and Dallwitz (1992), Watson et al. (1985). Therefore, this book follows

A large number of hybrids within and between the Elymus s. l. and Agropyron s. str. genera have spontaneously emerged in nature. Many hybrids are sterile, but a considerable number are more or less fertile, at least upon backcross to one of the parents. Apparently, introgressive hybridization has played an important role in the evolution of these two genera.

the Melderis classification.

Melderis (1978, 1980) transferred the following two sections from Agropyron s. l. to Elymus s. l.: Caespitosae (Rouy) Melderis, comb. nov. (Syn.: Agropyron sect. Caespitosa Rouy; Elytrigia sect. Caespitosae (Rouy) Tzvelev) and Junceae (Prat) Melderis, comb. nov. [Syn.: Agropyron sect. Junceae (Prat) Tzvelev]. The subdivision of these two sections was mainly based on caespitose or rhizomatous habit. The constituent species contain the St, Ee (=E) and the Eb (=J) genomes (Table 5.1), three genomes that occur in species that were included by Nevski's classification (1933), in the genus Elytrigia, but, later on, Nevski himself (1934a, b) included Elytrigia as a section in Agropyron. Tzvelev (1973) maintained the generic status of Elytrigia, but pointed out that species of this genus are close to species of Elymus.

The Elymus L. section Caespitosae is characterized by caespitose plant, lax and erect spikes, tough rachis, solitary or sometimes two spikelets on the node at the lower part of the spike, usually with 6–13 florets, and glumes 5–8 mm long, unkeeled with 5–9 veins. In some species, the rachilla is fragile and disarticulates above the glumes and beneath each floret (floret-type disarticulation). This section contains about 13 species (Table 5.1) comprising a polyploid series (2n = 14, 28, 56, and 70), and most of them are allogamous, and have long anthers.

The Elymus L. section Junceae is characterized by rhizomatous or caespitose plants, lax, erect, and sometimes curved spikes, a fragile rachis, disarticulating at maturity into spikelets with the rachis segment below them (Fig. 2.3; wedge type dispersal unit), solitary spikelets on the rachis, with 2–9 florets and keeled glumes, 5–18 mm long. This section comprises only three species, E. farctus (Viv.) Runemark ex Melderis, occurring in Europe and the Middle East, E. curvifolius(Lange) Melderis, occurring in south and central Spain, and E. distichus (Thumb.) Melderis, native to South Africa. Elymus farctus and E. distichus grow on maritime coasts.

The species of sections Caespitosa and Junceae presumably originate in Europe or west Asia. Genome St is found in several diploid and polyploid species, Ee exists in the diploid taxon E. elongatus (Host) Runemark subsp. elongatus [formerly Agropyron elongatum (Host) Beauv.] and in several auto- and allo- polyploids, whereas genome Eb occurs in the diploid taxon E. farctus (Viv.) Runemark ex Melderis subsp. bessarabicus (Savul. & Rayss) Melderis [formerly Agropyron junceum subsp. bessarabicum Savul. & Rayss; Thinopyrum bessarabicum (Savul. & Rayyss) A. Löve], in the allopolyploids of this species and of E. distichus (Thunb.) Melderis and in the autopolyploid E. curvifolius (Lange) Melderis (Table 5.1).

Löve (1984) used the genome symbols J for the genome of Elymus farctus and E for that of Elymus elongatus. Endo and Gill (1984) questioned the equivalence of J and E and based on differences in C-banding patterns, justified the separation of these two genomes. However, Dewey (1984) and Dvorak et al. (1984b), based on evidence from karyotype and genome analyses, considered the J and E genomes as the same basic genome. Previous studies of chromosome pairing in hybrids carrying these two genomes had already shown that they are closely related (Cauderon and Saigne 1961; Heneen and Runemark 1972; Dvorak 1981a; McGuire 1984) and more recent studies supported Dewey's consideration (Wang 1985b; Wang and Hsiao 1989). However, Jauhar (1988) reached a different conclusion by studying chromosome pairing in the hybrids analyzed by Wang (1985b) and Wang and Hsiao (1989). Since a recent

<sup>a</sup> These species were transferred from Agropyron to Elymus by Melderis (1978, 1980) <sup>b</sup> Genome symbols according to Wang et al. (1995) <sup>c</sup> Genome symbol according to Zhang et al. (1996) and Li and Zhang (2002)

literature review indicated that most studies regarded J and E genomes as members of the same cluster (see Table 1 in Wang and Lu 2014), it is now generally accepted to regard them as very closely related genomes, supporting the use of a common basic genome symbol, E (Seberg and Frederiksen 2001; Yen et al. 2005; Fan et al. 2007; Liu et al. 2008; Sha et al. 2010; Yan et al. 2011; Wang and Lu 2014). Thus, these genomes were designated Ee for Elymus elongatus and Eb for Elymus farctus, respectively, as proposed by Dvorak (1981a) and McGuire (1984).

While the use of one basic genome symbol for these two species was rejected by some researchers (Jauhar 1988, 1990a, b; Jarvie and Barkworth 1992; Jauhar et al. 2004), several studies using different methodologies, have further confirmed the close relationship between genomes Ee and Eb. The studies included chromosome pairing (de V Pienaar et al. 1988; Forster and Miller 1989; Wang and Hsiao 1989), random amplified polymorphic DNA (RAPD) and sequence-tagged site (STS) markers (Wei and Wang 1995; Li et al. 2007), genomic in situ hybridization (GISH) (Kosina and Heslop-Harrison 1996; Chen et al. 1998a, b, 2003), chloroplast DNA sequences (Mason-Gamer et al. 2002; Liu et al. 2008), sequences of a gene encoding plastid acetyl-CoA carboxylase (Fan et al. 2007, 2009), and nuclear rDNA internal transcribed spacer (ITS) sequences (Hsiao et al. 1995; Liu et al. 2008; Yu et al. 2008). At the second International Triticeae Symposium, the Genome Designation Committee (Wang et al. 1995) adopted a system for the application of nuclear genome symbols in the tribe Triticeae. This system is based mainly on prevailing symbols, but since the number of basic nuclear genomes in the Triticeae exceeds the number of single letters in the Roman alphabet, some basic genomes are designated with an uppercase letter followed by a lowercase letter, e.g., Ee or Eb, for the genome in Elymus elongatus and E. farctus, respectively. An uppercase letter followed by a superscript in small letters are used when modified versions of a basic genome is referred to, e.g., A<sup>m</sup> for the genome found in Triticum monococcum.

Melderis transferred the Asiatic diploid species of Elymus, namely, libanoticus, reflexiaristatus (subsp. reflexiaristatus and subsp. strigosus), and the diploid cytotypes of stipifolius and tauri from Agropyron (=Elytrigia) to Elymus (1978, 1980). These species carry the St genome (Wang et al. 1995) that also exists in the Elymus polyploid species containing the Ee genome, i.e., elongatus, nodosum, bungeanus, and hispidus (Table 5.1). The St genome is related to the Ee and Eb genomes (Wang 1989). Bieniek et al. (2015) found that nucleotide sequences of the diploid Ee, Eb and St taxa are almost identical, with only one substitution within the matK gene, differentiating genome Eb from the Ee and St genomes. Petersen and Seberg (1997) and Wang and Lu (2014) confirmed this very close relationship between the three genomes. The St genome almost always has a dominant influence on the morphology of the taxa of which it is a component (Assadi and Runemark 1995) and since exists in more primitive Elymus species, it is reasonable to assume that Ee and Eb evolved from St.

### 5.2.2 Elymus Species with St Genome

<IP_ADDRESS> Species Description The St-genome species of Elymus were previously recognized as a biological unit and placed as a separate section, Elytrigia, in the traditional Agropyron s. l. (Nevski 1934a). Due to the fact that all these species contain one genome, Love (1980), treated them as a separate genus, Pseudoroegneria. However, due to the absence of morphological discontinuities between Pseudoroegneria and Elymus, Melderis (1978, 1980) included Pseudoroegneria in the genus Elymus s. l.

The Elymus species bearing the St genome include approximately 15 different taxa that consists of about equal numbers of diploids and tetraploids. The type species of this group is E. reflexiaristatus (Nevski) Melderis subsp. strigosus (M. Bieb.) Melderis [formerly Pseudoroegneria strigosa (M. Bieb.) Á. Löve] (Dewey 1984; Lӧve 1984; Watson and Dallwitz 1992; Yan and Sun 2011). Interspecific hybrids between the St diploid species exhibit almost complete chromosome pairing at fist meiotic metaphase, but with high or complete sterility, indicating divergence of the same basic genome in each diploid (Stebbins and Pun 1953; Dewey 1975). Some of the species, e.g., stipifolius, and spicatus, have diploid and tetraploid cytotypes and the tetraploids behave cytologically as autotetraploids or near autoploids (Dewey 1975). The tetraploid taxa of two other species, tauri and panormitanus, are allopolyploids containing the St and P subgenomes.

A large amount of the allopolyploid species of Elymus s.l. share a common St genome with diploid Elymus species in different combinations with H, Y, P, and W subgenomes (Table 2.4). The maximum likelihood tree constructed, using nuclear ribosomal internal transcribed spacer region (nrITS) data, showed that diploid Elymus, Hordeum and Agropyron species served as the St, H and P subgenomes donors, respectively, for the Elymus allopolyploids (Dong et al. 2015). The maximum likelihood tree for the chloroplast genes (matK and the intergenic region of trnH-psbA) suggests that the Elymus diploid donors of the St genome to Elymus allopolyploids served, in most cases, as the maternal donor. Moreover, the chloroplast genes data suggest that diploid St Elymus species from Central Asia and Europe are more ancient than those in North America (Dong et al. 2015). Thus, it was hypothesized that the Elymus s. l. species originated in Central Asia and Europe, and then spread to North America.

The St genome species are perennials, caespitose, and cross-pollinating, with culms between 30 and 90 cm tall, narrow, linear spikes with single, distantly spaced spikelets, 5–8 mm long glumes of equal length in E. reflexiaristatus, or unequal in E. spicatus, tauri and libanoticus, 8–30 mm long glume awns, absent in E. tauri and libanoticus, and long anthers. These species grow in the northern Hemisphere, from southwestern and southeastern Europe, the Middle East, Transcaucasia across Central Asia and Northern China to Western North America (Dewey 1984). They occur on open rocky hillsides, are exceptionally drought and salt tolerant and have excellent quality forage that is palatable to animals (Dewey 1984).

The Elymus libanoticus-related species, that exemplify all St genome diploid species, are described below.

### <IP_ADDRESS> Elymus libanoticus (Hack.) Melderis—A Representative Example

### Morphological and Geographical Notes

Elymus libanoticus (Hackel) Melderis [Synonym: Agropyron libanoticum Hackel; Pseudoroegneria libanotica (Hackel) D.R. Dewey; Elytrigia libanotica (Hackel) Holub); Pseudoroegneria tauri ssp. libanotica (Hackel) Á. Löve; Agropyron sosnovskyi Hackel; Elytrigia sosnovskyi (Hackel) Nevski; Elymus sosnovskyi (Hack.) Melderis; Pseudoroegneria sosnovskyi (Hackel) A. Love; Agropyron gracillimum Nevski; Elytrigia gracillima (Nevski) Nevski; Pseudoroegneria gracillima (Nevski) Á. Löve], is perennial, caespitose, with short rhizomes, 45–85 cm high culms, 5–15 cm long linear spikes, with 4–7 spikelets, each 10–15 mm long, one per node, tough rachis, 3–6 florets, unequal, lanceolate, 3– 5-veined glumes, the lower ones 6–8 mm long, typically 3/4 or nearly as long as lower floret, and upper ones 7–9 mm long, 8–9 mm long, lanceolate, 3-veined, unawned, lemma, palea shorter than lemma, sparsely ciliate on keels, 4–5 mm long anthers, and caryopsis adherent to palea and lemma. Chromosome number 2n = 2x = 14 (Dewey 1972) (Fig. 5.1a).

Unlike E. libanoticus, E. sosnovskyi (Hack.) Melderis [=Agropyron sosnovskyi Hack.: Elytrigia sosnovskyi (Hack.) Nevski] bears acuminate glumes with 3 veins. Agropyron gracillimum Nevski differs from E. libanoticus by their smaller leaf thickness. These differences, however, fall within the variation of the Iranian E. libanoticus material (Assadi 1996). Moreover, hybrids between E. sosnovskyi or A. gracillimum with E. libanoticus were highly fertile, with regular meiotic metaphase. Therefore, the three names are considered synonymous (Assadi 1996). E. libanoticus is closely related to E. tauri subsp. libanoticus, differing only in several morphological traits (Assadi 1996).

Elymus libanoticus grows in Lebanon, Syria, northern Israel, south and southeastern Anatolia, northern Iraq, Iran, and Caucasus. It thrives on dry mountain slopes and limestone ravines, usually on more xeric habitats, 1000–3050 m a.s.l. It is an Irano-Turanian element.

### Cytology, Cytogenetics and Evolution

Hsiao et al. (1986) analyzed the karyotype of diploid St genome species, including Elymus spicatus, E. Reflexiaristatus subsp. Strigosus, E. libanoticus, and E. stipifolius. All four species possess similar karyotypes and chromosomal

Fig. 5.1 Mediterranean Elymus species; <sup>a</sup> A plant and spikes of E. libanoticus (Hack.) Melderis (2n = 2x = 14), (Photographed by the late Prof. Avinoam Danin); b A plant and spike of E. elongatus (Host)

Runemark ssp. elongatus (2n = 2x = 14); c A plant and spike of E. farctus (Viv.) Runemark ex Melderis ssp. farctus Runemark ex Melderis (2n = 6x = 42)

lengths. The karyotypes of all species have one pair of small and one pair of large satellites on the short arms of chromosomes 2 and 5, respectively (Hsiao et al. 1986). The karyotypes are symmetric; most chromosomes are metacentric and a few are sub-metacentric (Wang et al. 1985; Hsiao et al. 1986; Deng et al. 2004). The St genome consists of smaller chromosomes than those of the R, P, and Eb genomes. Despite their wide geographical distribution, the karyotype patterns of the St genome species have not been dramatically altered. The karyotype of E. spicatus has been reported previously (Schulz-Schaeffer and Jurasits 1962; Dvorak et al. 1984a, b).

Endo and Gill (1984), using the acetocarmine-Giemsa C-banding technique, studied heterochromatin distribution in somatic chromosomes of diploid Elymus and Agropyron species. With the exception of E. elongatus, which is moderately self-fertile, all other species are cross-pollinating and self-sterile. The cross-pollinating species showed large terminal C-bands and a high level of C-band polymorphism, whereas E. elongatus showed small terminal and interstitial bands and a minimal C-band polymorphism. C-banding patterns show that the Eb genome of diploid E. farctus appears to be distinct from the Ee genome of diploid E. elongatus and may constitute an intermediate link between the Ee and St genomes (Endo and Gill 1984).

E. spicatus, E. libanoticus, and E. stipifolius have similar C-band patterns, although C-bands were less prominent in E. stipifolius than in the others. Thus, the C-banding patterns and morphology of satellite chromosomes supported previous evidence that E. spicatus, E. libanoticus, and E. stipifolius share a common St genome. Variation in the intensity of terminal C-bands was observed in E. stipifolius, which is to be expected in a basic genome of species with worldwide distribution (Dewey 1981).

Wang (1989) produced the tetraploid hybrid (genome StStStH) from crossing the hexaploid Elymus transhyrcanus (genome StStStStHH) with E. libanoticus (genome StSt). This F1 hybrid exhibited at first meiotic metaphase 13.94 univalents, 0.16 rod and 6.78 ring bivalents (6.94 total bivalents) and 0.06 trivalents. The reciprocal hybrid showed an average of 10.22 univalents, 2.34 rod and 5.24 ring bivalents (7.58 total bivalents), 0.74 trivalents and 0.10 quadrivalents. The amount of pairing in the hybrid and particularly that of trivalent cnfiguraton was much less than expected in the case of three fully homologous St genomes. Hence, either the two St sugenomes of the hexaploid had diverged from one another or both had diverged from the St genome of the diploid.

In the F1 tetraploid hybrid (genome StStEeEe) between Elymus libanoticus (genome StSt) and E. hispidus (=Thinopyrum intermedium; genome StStEeEeEeEe) Wang (1989) observed at first meiotic metaphase an average of 6.68 univalents, 4.96 rod and 3.66 ring bivalents (8.62 total bivalents), 1.06 trivalents, 0.20 quadrivalents and 0.03 pentavalents. These data show that, in addition to the autosyndetic pairing in the form of bivalents between the Ee subgenomes, the presence of multivalents indicates some allosyndetic pairing between St and Ee chromosomes, indicating that the two genomes are related.

### Crosses with Other Triticineae Species

Studies of meiotic chromosome pairing in F1 hybrids between diploid Agropyron cristatum (genome PP) and several different diploids species of Elymus with St genome (genome of all hybrids was PSt), showed that the two genomes, P and St, are related (Wang 1985a, 1986, 1987a, b, 1988, 1989, 1990, 1992; Wang et al. 1985). Size differences between Agropyron (large) and St genome Elymus (small) chromosomes facilitated interpretation of chromosome pairing in the F1 hybrids. The average chromosome pairing at first meiotic metaphase of the diploid hybrid A. cristatum x E. libanoticus included 7.71 univalents, 2.77 bivalents, 0.22 trivalents, 0.01 quadrivalents and 0.01 pentavalents (Wang 1986), while that between A. cristatum and E. stipifolius displayed a similar amount and pattern of pairing (Wang 1985a). These pairing data indicate allosyndetic pairing between the homoeologous chromosomes of the two genomes, demonstrating a close relation between the St and the P genomes.

Meiotic chromosome pairing in the F1 hybrid Elymus spicatus (genome StSt) x Secale strictum (genome RR) exhibited an average of 12.97 univalents, 0.49 bivalents and 0.01 trivalent (Wang 1987b). The F1 hybrid Agropyron. mongolicum x S. strictum, which had the PR genome, showed an average of 12.86 univalents, 0.51 bivalents, 0.03 trivalents and 0.004 quadrivalents. The hybrid between Elymus spicatus and A. mongolicum (genome StP) had a mean configuration of 8.05 univalents, 2.86 bivalents, 0.07 trivalents and 0.01 quadrivalents. All hybrids were sterile. The meiotic pairings of these hybrids indicated that chromosome homology between the St and P genomes is higher than between St and R and between P and R. The degree of meiotic pairing in the E. spicatus x A. mongoicum hybrid was similar to those in other diploid hybrids bearing the same genome constitution, i.e., A. cristatum x E. stipifolius and A. cristatum x E. libanoticus (Wang et al. 1985; Wang 1986).

Following hybridization of the diploid Elymus Stipifolius (genome StSt) with tetraploid Elymus elongatus (genome EeEeEeEe), Dvorak (1981a) obtained a triploid hybrid (genome StEeEe), that exhibited 7.8 univalents, 5.9 bivalents and 0.41 trivalents at first meiotic metaphase. This pattern of pairing was attributed primarily to autosyndesis between homologous chromosomes of the Ee genomes. Stebbins and Pun (1953) had speculated that the Ee and St genomes might be variations of the same basic genome, yet the hypothesis was contradicted by Dvorak's (1981a) data, which showed that the Ee and St genomes are distinctly different.

Wang (1989) crossed Elymus libanoticus (genome StSt) with the tetraploid cytotype of Agropyron cristatum (genome PPPP) and observed an average meiotic pairing profile of 11.30 univalents, 3.40 rod and 1.50 ring bivalents (4.90 total bivalents) in the resulting triploid hybrid. Most pairing in this triploid hybrid was autosyndetic, indicating a difficulty in learning about the relationship between two genomes when one of the genomes exists in two doses.

Interpretation of chromosome pairing in St-Elymus and Agropyron hybrids is aided by size differences between the Agropyron (large) and St-Elymus (small) chromosomes. Chromosomes of autotetraploid Elymus spicatus (genome StStStSt) paired only rarely with chromosomes of diploid Agropyron cristatum (genome PP) in their triploid hybrids (StStP) (Dewey 1964). In the tetraploid hybrids (PPStSt) of A. desertorum (genome PPPP) and tetraploid E. spicattus (genome StStStSt), all chromosome pairing was attributed to autosyndesis between the PP and StSt genomes (Dewey 1967). Wang et al. (1985) crossed Agropyron desertorum (2n = 4x = 28; genome PPPP) with the autotetraploid cytotype of Elymus stipifolius (2n = 4x = 28; genome StStStSt). The tetraploid hybrid averaged 3.09 bivalents, most of which resulted from autosyndetic pairing between the P or the St genomes (Wang et al. 1985). In this tetraploid hybrid, because of the presence of homologous chromosomes, the P genome chromosomes rarely paired with the St genome chromosomes.

Wang (1989) used the mean C-values (the ratio between the number of chiasmata and the number of chromosome arms) to assess the relationships between genomes in diploid hybrids of the perennial Triticeae. He found that a C-value of 0.55 in diploid hybrids can serve as a critical value (in conjunction with other evidence, e.g., karyotype characteristics) to separate intergenomic from intragenomic divergence. Using this rule, he found that the Secale R genome, the Hordeum H genome and the Psathyrostachys N genome are distinct from each other and from other Triticeae genomes (C-values 0.03–0.17), while the St, Ee, Eb, and P genomes show considerable homoeology (C-values 0.24– 0.36). Thus, the Eb and St genomes, despite considerable differences in total genome size (Hsiao et al. 1986), show considerable homoeology, with a mean C-value of 0.35 in the diploid hybrid between them (Wang 1989). Similar homoeology was recorded by Liu and Wang (1993) in the triploid hybrids (genomes StStEb and StEeEe).

### 5.2.3 E. elongatus (Host) Runemark (Based on Ee Genome)

<IP_ADDRESS> Species Description Elymus elongatus [syn. Triticum elongatum Host; Agropyron elongatum (Host) Beauv.; Agropyron elongatum subsp. scirpeum; Elytrigia elongata (Host) Nevski; Lophopyrum elongatum (Host) Á. Löve; Thinopyrum elongatum (Host) D. R. Dewey] is a perennial, caespitose, 30– 100 cm high, with robust, glabrous culms, 10–25 cm long lax and erect spikes, tough rachis, 10–25 mm long solitary spikelets on each rachis node, sometimes two spikelets on one node, with 6–13 (9–25) awnless florets, glumes shorter than spikelet, 6–8 mm, 5–9 veined, without keels, 7–10 mm long, keeled lemma, keeled palea, 4–4.5 mm long anthers, caryopsis with adherent pericarp. The rachilla is fragile and disarticulates above the glumes and beneath each floret (floret-type disarticulation). This type of seed dispersal is characteristic of the Arctic-Temperate group and especially of species of Elymus.

The cytotaxonomy of E. elongatus was studied by several researchers, e.g., Peto (1930), Simonet (1935), Cauderon (1958, 1966), Schulz-Schaeffer and Jurasits (1962), Schulz-Schaeffer et al. (1971), Evans (1962), Runemark and Heneen (1968) Heneen (1972), Heneen and Runemark (1972, 1977), and Luria (1983), who showed that E. elongatus comprises a polyploid complex of diploid, tetraploid, octoploid, and decaploid taxa (Table 5.1). The diploid and decaploid taxa are well documented in the literature and evidence desmonstrates that the autotetraploid taxon also belongs to this group (Heneen and Runemark 1972). Hexaploid chromosome number was also found in material collected from Istria (Heneen and Runemark unpubl.). Schulz-Schaeffer and Jura (1967) reported the existence of hexaploid types in plants collected from Turkey. However, this hexaploid was not recognized as a valid subspecies. In addition to the diploid and tetraploid subspecies, an octoploid subsp. of Elongatus, subsp. turcicus (P. E. McGuire) Melderis, from Turkey, was described (McGuire 1984). Thus, it appears that the Elymus elongatus complex is represented in nature by types that form a complete polyploid series, ranging from diploids to decaploids.

Several authors described variants of this species as separate species or as subordinate taxa. Since the morphological differences between these taxa are not clear, Melderis (1980) recognized only two taxa that merit the subspecies status, namely, subsp. elongatus, a diploid, and subsp. ponticus, a decaploid. Later, Melderis (1985a) recognized an additional subspecies, subsp. turcicus (P. E. McGuire) Melderis, an octoploid taxon. Heneen and Runemark (1972) included an autotetraploid taxon from Cyprus and the Aegean islands in E. elongatus as ssp. flaccidifolius. Breton-Sintes and Cauderon (1978) classified an accession from Sicily of Heneen and Runemark (1972) autotetraploid subspecies as Agropyron elongatum (Host) ssp. scirpeum (C. Presl.) Cifferi et Giacom. The taxon ssp. flaccidifolius was elevated by Melderis (1978) to the specific rank Elymus flaccidifolius (Boiss. & Heldr.) Melderis. However, since there are only minor morphological differences (mainly quantitative) between this species and other autotetraploids of E. elongatus, it is more appropriate to classify it, along with all the other autoetraploids of E. elongatus, as a subspecies of elongatus. Hence, in this book, all the autotetraploid taxa of E. elongatus (=Agropyron elongatum var. flaccidifolium Boiss. & Heldr.; Agropyron flaccidifolium (Boiss. & Heldr.) Candargy; Elymus flaccidifolius (Boiss. & Heldr.) Melderis; Agropyron elongatum Host subsp. scirpeum (C. presl.) Ciferri & Giacom.; Lophopyrum scirpeum (C. Presl) Á. Löve; Thinopyrum scirpeum (Presl) D. R. Dewey; Agropyron scirpeum C. Presl; Elytrigia scirpea (C. Presl) Holub) are referred to as Elymus elongatus (Host) Runemark ssp. flaccidiffolius (Boiss. & Heldr.) Runemark, and were grouped together as one subspecies.

The diploid and the tetraploid subspecies of E. elongatus exhibit wide morphological variation in the number of spikelets per spike, number of florets per spikelet, hairiness, and plant color. The decaploid subspecies, subsp. ponticus, exhibits wider variation than subsp. elongatus and flaccidifolius.

E. elongatus is found in all parts of the Mediterranean basin, in southwestern, southeastern, and eastern Europe, in North Africa, and the Middle East, Caucasus, western Asia, and Arabia. It was introduced or invaded Australasia, South America, and North America. It is a Mediterranean element (chorotype) and grows among Mediterranean plant communities. This species grows in salt marshes and near salty springs and is salt tolerant (Moxley et al. 1978; Dewey 1960; McGuire and Dvorak 1980).

### <IP_ADDRESS> Ssp. elongatus (2n = 2x = 14)

### Morphological and Geographical Notes

E. elongatus (Host) Runemark subsp. elongatus [Agropyron elongatum (Host) Beauv.; Lophopyrum elongatum (Host) Á. Löve; Thinopyrum elongatum (Host) D.R. Dewey] is a diploid subspecies with tall stems (50–80 cm high); 10– 25 cm long spikes, with 9–26 spikelets per spike, internodes at the base of the spike as long as the spikelets; 10–17 mm long spikelets with 7–8 florets, usually one spikelet at each rachis node, 7–10 mm long glumes with 5–9 veins, where the lower glume is shorter (about 2/3–3/4) than the lower floret, 9–10 mm long lemmas, 4–4.5 mm long anthers and 4 mm long caryopsis (Fig. 5.1b).

Subsp. elongatus grows in the Mediterranean basin. In Israel, the diploid taxon grows in the Coastal Plain from the Shfela (Einot Gibton) and northwards (Acre plain). It grows in salt marshes, near salty springs and on maritime sands, from sea level to 100 m a.s.l., throughout the range of the species. These saltmarsh habitats are characterized by high underground water that forms floods in the winter and salty soil with salty crust in the summer. The subspecies also grows on sandy soils near river mouths, on silt near river's banks or springs or on clay soil. When growing in wet soils, the amount of annual rainfall is not a limiting factor.

### Cytology, Cytogenetics and Evolution

Matsumura and Sakamoto (1956), Cauderon (1958), Evans (1962), Schulz-Schaeffer and Jurasits (1962), Runemark and Heneen (1968), Heneen (1972) and Luria (1983) described the karyotype of the diploid subspecies. The karyotype is symmetric, consisting of four metacentric pairs and three sub-metacentric pairs. The differences in length and arm ratio among the chromosomes of this subspecies are relatively small (Dvorak and Knott 1974). However, the homologous chromosomes can be visually identified (Evans 1962). Two chromosome pairs have satellites, with one metacentric pair carrying a large satellite and one sub-metacentric pair bearing a small satellite. The constrictions between the satellites and the chromosome arms carrying them are the nucleolar organizing regions (NORs). The NORs contain a set of argyrophilic proteins which are selectively stained by silver. After silver staining, the NORs can be easily identified as black dots that are called Ag-NORs. Thus, in agreement with the number of satellite (SAT)-chromosomes mentioned above, four Ag-NORs are regularly observed in somatic cells of diploid E. elongatus (Lacadena et al. 1984). Giemsa C-banding analysis of the chromosomes of several accessions of this subspecies revealed small terminal and interstitial bands and a minimal C-band polymorphism (Endo and Gill 1984).

Heneen and Runemark (1972) observed karyotype differences between plants of ssp. elongatus collected from different locations, the major difference lying in the appearance of the SAT-chromosomes. Runemark and Heneen (1968), comparing the karyotype of subsp. elongatus with that of diploid E. farctu (genome EbEb), found that the two karyotypes resemble one another, but the chromosomes of E. elongatus are somewhat smaller than those of E. farctus. In addition, differences in morphology of the SAT-chromosomes exist between the two taxa (Heneen 1962); the pair with large satellites in E. elongatus has more median centromeres than the equivalent pair in E. farctus. The constriction in this SAT-chromosome divides the short arm in E. elongatus into two unequal parts, with the part proximal to the centromere being longer than the satellite, which is not the case in E. farctus.

Chromosomal pairing at meiosis in the diploid subspecies is regular (0.08–0.15 univalents and 6.92–6.95 bivalents per meiocyte; 12.04–12.86 chiasmata/cell (Cauderon 1958; Luria 1983). However, in accordance with the karyological observations, meiotic analysis of several inter-varietal hybrids showed the existence of structural heterozygosity among several accessions of this subspecies (Heneen and Runemark 1972). Similarly, in one inter-varietal cross, Luria (1983) observed a quadrivalent, suggesting the existence of a reciprocal translocation between the two accessions. These findings may indicate the occurrence of initial steps of karyotype divergence among and within accessions of the diploid subspecies of E. elongatus.

### Crosses with Other Triticineae Species

### Crosses with Diploids Species

Cross of Elymus farctus ssp. bessarabicus (genome EbEb), as female, with E. elongatus ssp. elongatus (genome EeEe) was successful, while the reciprocal cross failed (Wang 1985a). Karyotypes of mitotic chromosomes in the parental species revealed that three of the seven chromosomes in the Eb and Ee genomes were similar in length and arm ratio. Meiosis in the F1 hybrids substantiated this observation, but four chromosomes had undergone some structural rearrangements such as reciprocal translocations (Wang 1985b). Chromosomal pairing at meiotic first metaphase of the F1 hybrid averaged 2.68 univalents, 4.68 bivalents, 0.27 trivalents, 0.27 quadrivalents, and 0.01 pentavalents (Wang 1985b). The F1 hybrids were completely sterile upon self-pollination. From the relatively high pairing, Wang (1985a) concluded that the Eb and Ee genomes are closely related, supported the transfer of Lophopyrum elongatum to the genus Thinopyrum, as was suggested by Dewey (1984), as opposed to keeping them as two separate genera, as suggested by Löve (1984). GISH studies substantiated this conclusion by showing that genomes Ee and Eb are closely similar in their repetitive DNA (Kosina and Heslop-Harrison 1996).

Considering the suppression of pairing by the Ph1 gene that inhibits homoeologous pairing between the chromosomes of ssp. bessarabicus and ssp. elongatus in the tri-generic hybrid with durum wheat (genome ABEbEe), Jauhar (1988, 1990a, b) argued that the genomes Eb and Ee are homoeologues rather than homologues and should be assigned distinct genome symbols (J and E, respectively).

Jauhar et al. (2004) later analyzed chromosomal pairing in meiosis of the tri-generic hybrids between durum wheat, with and without the Ph1 gene, and the amphidiploid E. farctus ssp. bessarabicus-E. elongatus ssp. elongatus. Meiotic chromosome pairing was studied using both conventional staining and fluorescent genomic in situ hybridization (fl-GISH). As expected, the Ph1-intergeneric hybrids (genome ABEbEe) showed low chromosome pairing (23.86% of the total chromosome complement paired), whereas 49.49% of the trigeneric hybrids without Ph1 showed pairing. Fl-GISH analysis provided insight to the study of the specificity of chromosome pairing: wheat with Elymus (AB with Ee and/or Eb), wheat with wheat (A with B), or E. elongatus with E. farctus (Ee with Eb). The analysis revealed that without the Ph1 gene in the tri-generic hybrid, there were 3.97 chiasmata/cell between chromosomes of the Eb and Ee genomes, 2.29 chiasmata/cell between wheat chromosomes, and 2.6 chiasmata/cell between wheat–Elymus chromosomes. Thus, the two E genomes are more closely related to each other than A and B to one another.

Similarly, Forster and Miller (1989) reported that the chromosomes of ssp. bessarabicus and subsp. elongatus rarely paired in the presence of the Ph1 gene, i.e., in the hybrid between the two amphiploids Triticum aestivumdiploid E. farctus x T. aestivum–diploid E. elongatus. However, they concluded that, because of the relative high frequency of pairing between chromosomes of these two species at the diploid level, their genomes warrant a common genome symbol. Yet, since the two genomes do not pair in a wheat genetic background, their differentiation should also be indicated. Therefore, Forster and Miller (1989) proposed that the genome symbol of E. elongatus be E and that of ssp. besarabicus Eb, as suggested by Dvorak (1981a) and McGuire (1984).

Crossess with other Triticeae diploids revealed very little homology. Dvorak (1981b) succeeded in crossing Aegilops tauschii (=Ae. squarrosa) with ssp. elongatus, while crosses between ssp. elongatus and Ae. speltoides or Triticum monococcum ssp. aegilopoides (=T. boeoticum), were not successful. Mean chromosome pairing at first meiotic metaphase of the diploid hybrid yielded 10.7 univalents, 1.5 (0–5) bivalents, 0.027 trivalents per cell, indicating a certain degree of homoeology between the genomes of the two species (Dvorak 1981b). The F1 hybrid plants were sterile, with very low pollen fertility.

### Crosses with Tetraploid Species

Cauderon (1958) and Cauderon and Saigne (1961) crossed the allotetraploid Elymus farctus ssp. boreo-atlanticus (=Agropyrum junceum boreo-atlanticum) (genome EbEbEeEe) with Elymus elongatus ssp. elongatus (genome EeEe) and studied chromosome pairing at first meiotic metaphase of the triploid F1 hybrid (genome EbEeEe). Meiotic pairing showed 3.40 univalents, 4.50 bivalents, 2.76 trivalents, and 0.08 quadrivalents. From the relatively high frequency of trivalents at the hybrid meiosis, they concluded that the Eb and Ee genomes are closely related. A similar conclusion was drawn following karyotype analysis (Cauderon 1958).

When diploid E. elongatus was crossed with tetraploid (durum) wheat (genome BBAA) (Jenkins and Mochizuki 1957; Mujeeb-Kazi and Rodriguez 1981), the F1 hybrids showed very little pairing (0.3–2.6 bivalents per cell), suggesting that in the presence of one dose of Phl, the Ee genome chromosomes of elongatus showed little, if any, pairing with those of the subgenomes A and B of durum wheat. However, the level of chromosomal pairing reported by Jenkins and Mochizuki (1957), i.e., 2.6 bivalents/cell in the hybrid durum wheat x diploid E. elongatus was significantly higher than expected on the basis of pairing in haploid durum wheat, i.e., 0.37 bivalents/cell, as reported by Kihara (1936) and Lacadena and Ramos (1968).

In a later study, Mochizuki (1960, 1962) studied chromosomal pairing between individual E. elongatus chromosomes and tetraploid wheat chromosomes in monosomic addition lines, where single elongatus chromosomes were added to the durum complement. No chromosome associations were observed between wheat and elongatus chromosomes in three lines, while a high frequency of trivalent associations was noted in the remaining four lines. From these results, he concluded that four elongatus chromosomes are partially homologous to durum chromosomes. However, Dvorak and Knott (1974) assumed that the trivalents resulted from translocations between the durum and elongatus chromosomes that occurred during the production of the monosomic addition lines and actually, in the presence of two doses of Ph1 of durum wheat, there was no pairing between the elongatus and the durum chromosomes. Following this controversy, Ono et al. (1983) re-examined Mochizuki (1962) durum-elongatus addition lines and found that apart from 5Ee, no elongatus chromosomes paired with wheat chromosomes. Evans (1962) found that the nucleolar of Elymus were suppressed in the amphiploid Triticum durum-diploid Elymus elongatus by the durum NORs.

### Crosses with Hexaploid Species

The F1 hybrid between hexaploid wheat (BBAADD) and diploid E. elongatus (EeEe) exhibited very little chromosomal pairing at meiosis (Jenkins 1957). A low level of pairing between Ee genome chromosomes of diploid E. elongatus and those of common wheat was also observed in elongatus addition lines to common wheat (Dvorak and Knott 1974). Study of pairing of single diploid E. elongatus chromosomes with common wheat chromosomes in monosomic addition lines, in the presence of two doses of the homoeologouspairing suppressor Ph1, showed that elongatus chromosomes do not pair with wheat chromosomes, with the exception of chromosome IV [assigned later to homoeologous group 3, and designated 3Ee by Dvorak (1980)], that very rarely paired with a wheat chromosome (Dvorak and Knott 1974). The researchers thus concluded that elongatus genomes did not play any role in the evolution of the polyploid series of Aegilops and Triticum.

When ten ditelosomic addition lines, comprising of diploid E. elongatus telosomes added to the common wheat complement, were crossed to Aegilops speltoides, that suppresses the activity of the Ph1 gene, all ten elongatus telosomes paired with common wheat chromosomes (Dvorak 1979). But, because this pairing only occurred when Ph1 was not active, Dvorak concluded that none of the ten elongatus-chromosome arms has a homologous partner among the three common wheat subgenomes A, B, and D, and the involved speltoides genome.

Likewise, in crosses between elongatus substitution lines, where the activity of Ph1 was suppressed, elongatus chromosome 6Ee paired, to some extent (4.6%), with wheat chromosomes of homoeologous group 6 (Dvorak 1979). Similarly, pairing between elongatus and wheat chromosomes was also observed by Johnson and Kimber (1967), Dvorak (1979, 1981b), and Sears (1973) in hybrids between elongatus and common wheat, when the Ph1 gene of wheat was suppressed or absent.

If the interpretation of these data, as well as that of Jenkins and Mochizuki (1957) on pairing in the hybrid T. durum x diploid E. elongatus, is correct, then there must be considerable homology between Elymus elongatus and common wheat chromosomes. However, Dvorak and Knott (1974) assumed that this degree of pairing does not result from chromosomal homology but, rather, from the presence of E. elongatus genes that promote homoeologous pairing. Indeed, Dvorak and Knott (1974) found that chromosome IV (designated later 3Ee by Dvorak 1980) and chromosome I (1Ee; Dvorak 1980) increased significantly the pairing of wheat chromosomes, i.e., they carry genes that promote pairing of homoeologous chromosomes. If this is the case, then, the pairing reported by Jenkins and Mochizuki (1957) between elongatus and durum wheat chromosomes, resulted presumably from homoeologous pairing between chromosomes of the A and B subgenomes of durum in addition to that between elongatus and durum.

Dvorak (1987) assumed that genes promoting or suppressing pairing of homoeologous chromosomes are ubiquitous among Triticeae diploid species. To identify such genes in diploid E. elongatus, he crossed common wheat lines with added or substituted E. elongatus chromosomes with Hordeum bulbosum to obtain haploids, and with Triticum urartu to obtain interspecific hybrids. Studies of chromosome pairing at first meiotic metaphase in the resulting haploids and hybrids and in the parental addition and substitution lines revealed genes affecting homologous or homoeologous chromosome pairing. Genes promoting pairing were found on the short and long arms of chromosome 3Ee, on the short arms of 4Ee and 5Ee, and on chromosome 6Ee of E. elongatus. Genes suppressing pairing of homoeologous chromosomes were found on the long arms of chromosomes 4Ee and 7Ee (Dvorak 1987). That may explain why different results were found when using lines that may contain different alleles of these pairing genes.

While eight Ag-NORs were observed in many cells of the amphiploid common wheat–diploid E. elongatus, four on the wheat chromosomes 1B and 6B and on four on two elongatus chromosomes, in some cells the Ag-NORs of elongatus were suppressed by the wheat chromosomes (Lacadena et al. 1984).

### <IP_ADDRESS> Ssp. flaccidifolius (Boiss. & Heldr.) Runemark (2n = 4x = 28)

### Morphological and Geographical Notes

Ssp. flaccidifolius [Syn.: Boiss. & Heldr.) Runemaks [Syn.: Agropyron scirpeum C. Presl; Agropyron scirpeum var. flaccidifolium Boiss. & Heldr.; Agropyron elongatum var. flaccidifolium (Boiss. & Heldr.) Boiss. & Heldr.; Agropyron flaccidifolium (Boiss. & Heldr.) Candargy; Agropyron elongatum Host ssp. scirpeum (C. Presl.) Ciferri & Giacom.; Elymus elongatus ssp. flaccidifolius (Boiss. a Heldr.) Runemark; Elytrigia scirpea (C. Presl) Holub; Lophopyrum scirpeum (C. Presl) Á. Löve; Thinopyrum scirpeum (C. Presl) D. R. Dewey} is a perennial caespitose, more or less glaucous grass with erect culms, 70–115 cm high, with 11– 23 cm long spikes, with 5–17 spikelets per spike, 10–22 mm long spikelets, with 5–10 florets and glumes with 5–6 veins, 4–6 mm long anther and 5 mm long caryopsis. In several accessions, two spikelets are located at each rachis node in the lower part or the center of the spike.

The diploid and tetraploid subspecies of E. elongatus cannot be morphologically distinguished with certainty, primarily because they differ mainly in quantitative traits (Breton-Sintes and Cauderon 1978; Luria 1983). Luria (1983) found several tetraploid accessions of E. elongatus ssp. flaccidifolius in Israel, in addition to diploid accessions of ssp. elongatus. The tetraploid accessions morphologically resemble the tetraploid subsp flaccidifolius (Breton-Sintes and Cauderon 1978; Luria 1983). The Israeli tetraploid differs from the diploid cytotype of ssp. elongatus only in its somewhat taller plants, shorter flag leaf, larger stomata, larger pollen grains and longer caryopses.

The tetraploid subspecies grows in the Mediterranean basin (Heneen and Runemark 1972; Luria 1983; Gabi and Dogan 2010). In Israel, the tetraploid subspecies grows from Einot Gibton, Shfela, southwards (was found in Nahal-Zin springs in the Negev). The distribution of this taxon is fragmentary and the populations are isolated from one another. In the salty spring of Einot Gibton, the only site in Israel where the diploid and the tetraploid subspecies occur together, the diploid grows near the spring and the tetraploid in the outer ring (Luria 1983). Hence, the diploid subspecies can tolerate higher concentrations of salt than the tetraploid subspecies.

### Cytology, Cytogenetics and Evolution

Based on karyomorphological data, Heneen and Runemark (1972) assumed that the tetraploid subspecies (2n = 4x = 28; genome Ee Ee Ee Ee ) is an autotetraploid, derived from the diploid subspecies by chromosome doubling, or rather, via inter-varietal hybridizations followed by chromosome doubling. An inter-varietal origin of natural autopolyploids in different groups of plants is a widespread phenomenon, as discussed by Stebbins (1950, 1971).

Heneen and Runemark (1972), Breton-sintes and Cauderon (1978), and Luria (1983) arranged the chromosomes of the tetraploid subspecies in seven groups of four. These groups morphologically correspond to the seven pairs of the diploid subspecies, supporting the likelihood of an autopolyploid origin of the tetraploid (Heneen and Runemark 1972; Breton-Sintes and Cauderon 1978). Yet, detailed karyomorphological studies showed the existence of small differences between pairs within groups of four (Heneen and Runemark 1972; Breton-Sintes and Cauderon 1978; Luria 1983). Consequently, it was proposed that this taxon originated from hybridization between diploid varieties that underwent some chromosomal divergence and therefore, possess two partially diverged genomes, namely, Ee1Ee1Ee2Ee2 (Breton-Sintes and Cauderon 1978).

Chromosome pairing in the F1 triploid hybrid between the tetraploid and the diploid subspecies, is only slightly lower than that expected for a hybrid between autotetraploid and its diploid progenitor (Dvorak 1981b; Charpentier et al. 1986). The assessment of the homology between the two genomes of the tetraploid showed that differentiation had occurred in all chromosome arms that could be tested. From the pairing frequencies of individual telosomes of the diploid subspecies of E. elongatus with chromosomes of the tetraploid subspecies, Dvorak (1981a) concluded that slight differentiation occurred in every chromosome of the two subspecies. Thus, both genomes of the tetraploid subspecies appear to be a slightly modified version of the genome of the diploid subspecies (Dvorak 1981b).

To account for this genomic divergence, Heneen and Runemark (1972) suggested that the autotetraploid originated from hybridization(s) between different diploid lines whose karyotype underwent some structural chromosomal differentiation. On the other hand, Dvorak (1981b), Dvorak and Scheltgen (1973) and Dvorak and McGuire (1981) Fig. 5.2 Nuclear DNA content in diploid species and their polyploid derivatives, natural (black) and synthetic (gray): a Diploid and autotetraploid cytotypes of Elymus elongatus (taken from Eilam et al. 2009), and b Diploids, allotetraploid and allo-auto-hexaploid of Elymus farctus (taken from the Angiosperm C-value database (http://www.rbgkew.org.uk.cval/) at Kew Botanic Gardens. \*The expected value, for each polyploid derivative, is the sum of the observed values of its parents

proposed that this differentiation resulted from changes in nucleotide sequences, rather than chromosomal aberrations such as inversions, translocations and other structural rearrangements. Alternatively, Eilam et al. (2009, 2010) suggested that the tetraploid subspecies underwent some cytological diploidization at the tetraploid level due to elimination of DNA sequences from two chromosomes in each group of four. Indeed, the tetraploid subspecies contained a significantly smaller amount of nuclear DNA (about 10% less) than the expected additive value of the diploid parent (Eilam et al. 2009, 2010). Also, a newly synthesized autotetraploid line of E. elongatus, produced by Charpentier et al. (1986), had significantly less DNA (8.57%) than the expected additive value (Eilam et al. 2009) (Fig. 5.2). The similarity in nuclear DNA content between the synthesized and the natural autotetraploids of E. elongatus indicates that the reduction in DNA content in the natural autotetraploid occurred immediately after its production, with only small changes in genome size over the history of the autotetraploid. Elimination of DNA sequences from two out of the four homologous chromosomes in each set of four, or elimination of sequences from one pair and other sequences from the second pair, augments the differentiation between the constituent subgenomes. Hence, the two subgenomes that became slightly divergent as a consequence of this pattern of elimination, underwent cytological diploidization. This reduction in nuclear DNA may lead to exclusive bivalent pairing between fully homologous chromosomes and consequently, disomic inheritance. The eliminated sequences are likely to include those that participate in homologous recognition and initiation of meiotic pairing.

The chromosomes in other tetraploid Elymus species, such as E. farctus ssp. boreo-atlanticus (Heneen 1962) and E. rechingeri (Heneen and Runemark 1962), could not be grouped into groups of four. These species most likely have an allopolyploid origin. An autopolyploid origin of the tetraploid subspecies of E. elongatus is also indicated by the occasional formation of quadrivalents at meiosis (Heneen and Runemark 1972). This conclusion was also supported by genome analysis; Dvorak (1981b) and Charpentier et al. (1986) observed extensive pairing at the first meiotic metaphase in the triploid hybrid of these two subspecies and concluded that the three genomes of the triploid (Ee, Ee1, and Ee2) are closely related.

Meiosis is generally regular in subsp. flaccidifolius (Heneen and Runemak 1972; Dvorak 1981b; Luria 1983; Charpentier et al. 1986). For instance, Charpentier et al. (1986) observed 0.12 univalents, 13.9 bivalents 0.025 quadrivalents; 26.55 chaismata/cell at first meiotic metaphase. The majority of the cells had all the chromosomes paired as ring bivalents, indicating a high degree of homology within chromosome pairs. Multivalents, represented mainly by quadrivalents, occurred very rarely. The preferential bivalent pairing and the rarity of multivalent pairing in the autotetraploid subspecies, indicate either that this taxon is an autotetraploid that underwent cytological diploidization (Eilam et al. 2009, 2010) or that the tetraploid subspecies is a segmental allopolyploid (Breton-sintes and Cauderon 1978).

An induced autotetraploid of E. elongatus, produced by colchicine treatment of a diploid plant, was found morphologically indistinguishable from the natural tetraploid (Charpentier et al. 1986). The F1 hybrid between the natural and the induced autotetraploid had almost complete chromosome pairing, with an average of 1.0 univalents, 7.9 bivalents, 2.8 quadrivalents and 23.8 chiasmata per cell, nearly similar to the chromosomal pairing observed in the induced autotetraploid parent (Charpentier et al. 1986). This pairing pattern further supports the autopolyploid nature of the natural tetraploid subspecies. Because of this slight genomic divergence, Dvorak (1981a, b) suggested classifying the tetraploid and the diploid taxa in two separate species. However, since autotetraploids and their diploid progenitors are usually included in the same species (Stebbins 1950), diploid and tetraploid elongatus are two cytotypes and were classified as members of a single biological species (Heneen and Runemark 1972; Breton-Sintes and Cauderon 1978).

### Crosses with Other Triticineae Species

Homology between genomes Eel and Ee2 was also inferred from the meiotic behavior of the F1 hybrid between tetraploid E. elongatus and common wheat. Despite the presence of one dose of Phl, the F1 hybrid (2n = 5x = 35; genome BADEe1Ee2) exhibited at the first meiotic metaphase five to seven bivalents, interpreted as autosyndetic pairing of elongatus Eel and Ee2 chromosomes (El Gawas and Khalil 1973; Dvorak 1981b; Sharma and Gill 1983; Charpentier et al. 1988a). This number of bivalents shows that most, if not all, chromosomes of the two Ee subgenomes were involved in pairing. Pairing of the elongatus Ee1 and Ee2 chromosomes in the presence of one dose of the Ph1 gene indicated that these two genomes still retained their homology, further supporting the autoploid nature of tetraploid E. elongatus.

Indications that genomes Ee1 and Ee2 are closely related were also reported by Han and Li (1993). In two crossing combinations, Triticum timopheevii ssp. timopheevii (2n = 4x = 28; genome GGAA) x tetraploid E. elongatus and T. turgidum ssp. durum (2n = 4x = 28; genome BBAA) x tetraploid E. elongatus, chromosome pairing at first meiotic metaphase included a mean 9.10 univalents, 9.11 bivalents, 0.20 trivalents and 13.78 univalents, 6.87 bivalents, and 0.15 trivalents, respectively (Han and Li 1993). Since pairing between A and B wheat subgenomes is very low in haploids of tetraploid wheat containing the Ph1 gene (Kihara 1936; Lacadena and Ramos 1968), pairing in the hybrid containing the BAEe1Ee2 genomes was likely due to autosyndesis between Ee1 and Ee2 chromosomes of tetraploid E. elongataus. Similar homologous relationships were observd between the two Eb genomes of hexaploid E. farctus (Charpentier 1992) and between the two Eb genomes of tetraploid E. farctus (de V Pienaar et al. 1988).

The hybrid formed between tetraploid E. elongatus and common wheat, with zero dose of Ph1, exhibited a relatively high degree of autosyndetic pairing between elongatus two-subgenome chromosomes and between wheat-subgenome chromosomes and allosyndetic pairing between wheat and elongatus chromosomes (range of chromosomal pairing was 9.6–11.2 bivalents and 1.2–1.9 trivalents per cell; quadrivalents, and some pentavalents were also observed) (Charpentier et al. 1988a). In contrast, a drastic reduction in pairing was observed in hybrids carrying one dose of Ph1. Altogether they showed a means 4.6–7.7 bivalents per cell, multivalents were rare. The number of chiasmata/cell dropped from 19–20 in Ph1-deficient hybrids to 6–10 in hybrids with one dose of this gene (Charpentier et al. 1988a).

Ph1-deficient haploid bread wheat was found to form at first meiotic metaphase 3.2–4.2 bivalents, 0.9–2.0 trivalents and very few quadrivalents (0.02–0.12) and pentavalents per cell (Riley 1960). Assuming a similar level of pairing between the wheat chromosomes in hybrids generated from the tetraploid subspecies of E. elongatus and Ph1-deficient bread wheat, then 6–7 bivalents of the observed 9.6–11.2 should be the results of pairing between Eel and Ee2 elongatus chromosomes. The number of quadrivalents and pentavalents that were observed in these hybrids indicate allosyndetic pairing. The relatively high level of allosyndetic pairing in F1 hybrids between tetraploid E. elongatus and Ph1-deficient bread wheat, and the low level of allosyndetic pairing in the presence of Ph1, indicates that the subgenomes of tetraploid elongatum do not have genes that suppresses or promote homoeologous pairing.

Interestingly, in contrast to the effect of Ph-suppressors or homoeologous pairing promoters on diploid E. elongatus (Dvorak 1987), such an effect was not observed in hybrids between common wheat and tetraploid E. elongatus (Dvorak 1981b).

To determine the chromosomal location of these and other genes that control pairing in diploid E. elongatus, disomic addition lines of chromosomes derived from the diploid subspecies of E. elongatus in the background of Chinese Spring, were crossed with the tetraploid subspecies of E. elongatus, and pairing was then compared to those observed in hybrids between Chinese Spring and tetraploid E. elongatus, whose Ee1 and Ee2 chromosomes were previously defined as homologues (Charpentier et al. 1986, 1988a). The resultant F1 hybrids (2n = 5x = 36), each carrying three doses of a given elongatus chromosome, enabled evaluation of the effect of each elongatus chromosome on pairing of homologues (Eel with Ee2) and homoeologues (A, B, D, and Ee). The study of chromosomal pairing in these hybrids enabled classification of the elongatus chromosomes into those that suppress (6Ee), promote (5Ee, 3Ee, and possibly also 1Ee), or have no effect on pairing (4Ee). The effect of chromosomes 2Ee and 7Ee was not studied. Chromosomes 5Ee and 3Ee differed in their effect on the degree and pattern of chromosome pairing, namely, the effect of 5Ee was stronger than that of 3Ee. Pairing analysis in such addition lines and in substitution lines, in their haploid derivatives and in hybrids between these lines and Triticum urartu, led Dvorak (1987) to allocate genes that promoted homologous or homoeologous pairing to chromosome arms 3EeS, 3EeL, 4EeS, 5Eep and to chromosome 6Ee of diploid E. elongatus. Genes suppressing homoeologous pairing were allocated to chromosome arms 4EeL and 7Eeq. In accord with Charpentier et al. (1988b), chromosomes 3Ee and 5Ee of diploid elongatus promoted homoeologous pairing.

In the presence of an extra dose of chromosome 6Ee of elongatus, the number of bivalents per cell was reduced, indicating suppression of pairing between the Eel and Ee2 chromosomes (Charpentier et al. 1988b), bringing Charpentier et al. to conclude that chromosome 6Ee of diploid E. elongatus carries gene(s) that inhibit(s) pairing or chiasma formation. This is in contrast to the finding of Dvorak (1987), who assigned a pairing-promoting effect to this chromosome. Chromosome 4E had no effect on pairing in hybrids with wheat (Charpentier et al. 1988b). This is in accord with Dvorak (1987), who reported pairing suppression by the long arm of chromosome 4Ee, but assumed the presence of a pairing promoter on the short arm of 4Ee, thus accounting for the lack of pairing effect by the entire 4E chromosome. He also found a suppressive effect of chromosome arm 7Eq, a chromosome arm that was not studied by Charpentier et al. (1988b).

Charpentier et al. (1988b) also studied chromosome pairing at first meiotic metaphase in hybrids between the bread wheat cultivar Chinese Spring and a synthetic autotetraploid line derived from diploid E. elongatus. The hybrids exhibited a high level of homoeologous pairing. Apparently, the genome of the diploid, from which the autotetraploid was synthesized, promoted pairing even in the presence of Phl. A similar effect was reported for gene(s) derived from another diploid accession of E. elongatus (Dvorak 1981b). Promotion of homoeologous pairing by diploid E. elongatus was also observed in an amphiploid between allotetraploid E. farctus (subsp. boreali-atlanticus; genome EbEbEeEe) and diploid E. elongatus (genome EeEe) (Yvonne Cauderon, personal communication). The amphiploid had genome (EbEbEeEeEeEe). While tetraploid and hexaploid E. farctus exhibited mostly bivalents at meiosis, the amphiploid had several multivalents per cell, mostly quadrivalents but also some hexavalents. Evidently, in this amphiploid, genome Ee of dipoid E. elongatus promoted pairing between the homologues Ee genomes and between homoeologues Eb with Ee genoes. Three different accessions of diploid E. elongatus were found to promote homoeologous pairing: the accession used by Jenkins (1957) to produce the initial hybrid from which Dvorak and Knott (1974) derived their disomic addition lines, an accession from south France used by Cauderon in the cross with the tetraploid form of E. farctus and the Israeli accession, from which the induced autotetraploid was derived. Thus, the ability to promote homoeologous pairing may be a common feature of many accessions of diploid E. elongatus. Promotion of homoeologous pairing in the presence of Phl was described in several diploid Triticinae, viz. Aegilops speltoides (Riley 1960; Riley et al. 1961; Dvorak 1972), Amblyopyrum muticum (Riley 1966a, b; Dover and Riley 1972), Ae. longissima (Mello-Sampayo 1971b), Secale cereale (Riley et al. 1973; Lelley 1976; Dvorak 1977) and Dasypyrum villosum (Blanco et al. 1988b) (Table 5.2). However, chromosomal allocation of the promoters was only determined in rye (Lelley 1976) and in E. elongatus (Dvorak 1987; Charpentier et al. 1988b). In rye (Lelley 1976) chromosome 3R, and possibly also 5R, 4R, and 7R, were found to carry genes that promote homoeologous pairing in hybrids with wheat. This finding corresponds to the allocation of pairing promoters in diploid E. elongatus (Dvorak 1987; Charpentier et al. 1988b); the genes of 3Ee and 5Ee are presumably homoeoalleles to those of rye (Lelley 1976), as well as to those of homoeologous groups 3A, 3B and 3DL and 5A, 5D, and 5BS in bread wheat (Sears 1976).

### <IP_ADDRESS> Ssp. turcicus (P. E. McGuire) Melderis (2n = 8x = 56)

### Morphological and Geographical Notes

Ssp. turcicus [=Elytrigia turcica P. E. Maguire; Elytrigia elongata ssp. turcica (P. E. McGuire) Valdés & H. Scholz; Elytrigia pontica ssp. turcica (P. E. McGuire) Jarvie & Barkworth; Lophopyrum turcicum (P. E. McGuire) McGuire ex Löve; Thinopyrum turcicum (P. E. Maguire) Cabi & Dogan], is a perennial caespitose, more or less glaucous grass with erect culms, 70–115 cm high, with 10–20 cm long spikes, spikelets with 7–9 florets and glumes with 7–9 veins. Anthers are 2.5–3.5 mm long.

Within the polyploid complex of E. elongatus, ssp. turcicus most resembles the decaploid ssp. ponticus. It differs from ponticus by the laxer leaves, less prominent ligules, more rounded apex of glumes, lack of hairs inside the glumes at the apex, smaller anthers, and in chromosome number (McGuire 1983). Although morphologically similar, the octoploid and the decaploid taxa were treated as separate species by McGurie (1983), but Melderis (1978), Dewey (1984), and Moustakas (1989) considered the morphological differences between the two taxa insufficient for separation on the specific level and consequently, classified them as two subspecies.

This subspecies distributes in Thassos Island, Greece (Moustakas 1993), Turkey, Georgia, and northern Iran (Jarvie 1992). It grows on dry calcareous, saline land from sea level to dry and saline mountain habitats, 1800 m above sea level, in low rainfall areas.

### Cytology, Cytogenetics and Evolution

Chromosome counts in accessions of Elymus elongatus from eastern Turkey and northern Iran, showed the presence of octoploid plants with 2n = 8x = 56 (Lorenz and Schulz-Schaeffer 1964; Sculz-Schaeffer et al. 1971; McGuire 1983), implying that the octoploids are not just sporadic individuals, arising in populations with other ploidy levels, but represent established populations (McGuire 1983).

Moustakas (1993) performed computer-aided karyotype analysis and found that the karyotype of the octoploid taxon is asymmetric, namely, it is composed of 10 metacentric chromosome pairs, 15 sub-metacentric chromosome pairs and 3 sub-telocentric chromosome pairs. Only two chromosome pairs have secondary constrictions, implying that a number of NORs are inactive in this octoploid.

All the chromosomes pairs of the octoploid can be matched to the chromosome pairs of the decaploid. Only the sub-telocentric satellited chromosome pairs differ slightly (Moustakas 1993). Yet, the karyotype analysis (Moustakas 1989, 1991, 1993) indicated that the chromosomes of the present-day diploid elongatus and those of the octoploid and decaploid diverged from each other.

Analysis of seed protein polymorphism patterns (Moustakas 1989) revealed that the octoploid originated from a speciation event more recent than that associated with the decaploid from which it presumably evolved. Moustakas (1989) found that the patterns of seed-protein electrophoresis of ssp. turcicus were qualitatively similar to those of ssp. ponticus.

Taking into consideration the results of the karyotype analysis, Moustakas (1993) concluded that ssp. turcicus is a segmental allopolyploid, with genome designation JjJjJjJjJeJeJeJe. (Genome designations Jj and Je represent the same genome but with some structural nuances.) On the other hand, Jarvie (1992) thought that the genome of subsp. turcicus is EEEEJJJJ is an auto-allo polyploid. Yet, if subsp. turcicus derived from the decaploid subsp. ponticus, its genome designation should be EeEeEbEbStStStSt.

### <IP_ADDRESS> Ssp. ponticus (Podp) Melderis (2n = 10x = 70)

### Morphological and Geographical Notes

Ssp. ponticus (commonly known as tall wheatgrass and rush wheatgrass) [Syn.: Triticum ponticum Podp; Agropyron elongatum ssp. ponticum (Podp.) Senghas; Agropyron incrustatum Adamovic; Elymus ponticus (Podp.) N. Snow; Elytrigia pontica (Podp.) Holub; Elytrigia elongata ssp. pontica (Podp.) Gamisans; Elytrigia ruthenica (Griseb.) Prokudin; Lophopyrum ponticum (Podp.) Á. Löve; Thinopyrum ponticum (Podp.) Barkworth & D. R. Dewey] is a perennial, caespitose, tall plant with a 50–100 cm high stem. Its leaves are green or glaucous bluish with flat to curling blades that are often covered with short, stiff hairs, its lower sheaths usually ciliate, spikes are 10–35 cm long, lower internodes are usually much longer than the spikelets, rachis is not fragile, spikelets are 17–25 mm long, with 8–18 florets, glumes are thick and hardened, and 9–11 mm long, with 5–7 veins, lemmas are also thick and hardened, and 10– 13 mm long, palea with cilia are seen along the entire length of keels and anthers are 4–7 mm long. Under certain conditions, this perennial grass can grow up to 2 m tall, and spikelets up to 3 cm long, each containing up to 12 flowers.

Ssp. ponticus is native to southeastern Europe, Turkey near the Black Sea and southern Russia. It grows well in dry and saline habitats, especially alkaline soils, as well as in disturbed habitats, such as waste ground and roadsides. This subspecies is found generally 360–1740 m above sea level.

### Cytology, Cytogenetics and Evolution

Peto (1930), Simonet (1935), and Vakar (1935) reported that accessions of E. elongatus from Russia are decaploid, with 2n = 10x = 70 chromosome number. Heneen and Runemark (1972) superficially described the karyotype of the decaploid subspecies. The large number of chromosomes rendered it difficult for them to construct the karyotype and to identify all the SAT-chromosomes. Generally, chromosome morphology and SAT-chromosome type seemed similar to those of the diploid and tetraploid subspecies of E. elongatus, indicating that ssp. ponticus is interrelated to these subspecies (Heneen and Runemark 1972), supporting the view that different subspecies of E. elongatus are involved in the origin of the decaploid subspecies.

On the diploid and tetraploid levels, there is a correlation between the number of SAT-chromosomes and degree of ploidy. This correlation is not obvious at the decaploid level,

since the number of the barely detectable SAT-chromosomes in the decaploid subspecies was not proportional to the degree of ploidy (Heneen and Runemark 1972).

Up to ten nucleoli were recorded in pre-meiotic cells of the decaploid subspecies (Schulz-Schaeffer and Jura 1967). Brasileiro-Vidal et al. (2003), using silver nitrate staining to determine the number of nucleoli and NORs, revealed 17 AG-NOR sites on mitotic metaphase cells—a number similar to that of 45S rDNA detected via FISH. However, the mean number of nucleoli per interphase nucleus was much lower; in most cells, the number ranged from four to nine, indicating that at interphase, the active Ag-NOR sites tend to coalesce, as suggested by Lacadena et al. (1988).

Li and Zhang (2002) used FISH to study the distribution of the 18S-5.8S-26S rDNA in the decaploid subspecies and in its related diploid taxa, E. elongatus ssp. elongatus (genome EeEe), E. farctus subsp. bessarabicus (genome EbEb) and E. stipifolius (=Pseudoroegneria stipifolia) (genome StSt). The distribution of rDNA genes was similar in all three diploid taxa, i.e., two pairs of loci were observed in each somatic cell at metaphase and interphase. The first pair was located near the terminal end and the second in the interstitial regions of the short arms of a pair of chromosomes. The maximum number of major rRNA loci detected on metaphase spreads of the decaploid subspecies was 20, which corresponded to the additive sum of that of its progenitors. However, in the decaploid, all of the major loci were located on the terminal end of the short arms of the chromosomes. Apparently, the interstitial loci that exist in the possible diploid donors of genomes to the decaploid, changed their position during the formation and evolutionary history of the decaploid. These results suggest that there has been distinct differentiation between ponticus and its diploid relatives during the evolutionary process (Li and Zhang 2002). Positional changes of 18S-5.8S-26S rDNA loci between ssp. ponticus and its candidate genome donors, indicate that it is almost impossible to find a genome in the decaploid that is completely identical to that of its diploid donors (Li and Zhang 2002). The interstitial position is likely an ancestral trait, whereas the terminal position is probably a later-derived trait (Dubcovsky and Dvorak 1995). During polyploidization of ssp. ponticus, all of the interstitial loci have been either deleted and novel loci have been positioned on terminal regions of the chromosomes, or, alternatively, migrated to terminal positions. A similar phenomenon has been observed in other Triticeae species (Gill and Apples 1988; Dubcovsky and Dvorak 1995), although the exact underlying mechanism remains unknown.

Using FISH to determine the number and position of 45S and 5S rDNA sites in another accession of ssp. ponticus, Brasileiro-Vidal et al. (2003) detected both 45S and 5S rDNA sites on the short arms of 17 chromosomes, while on three other chromosomes, only the 5S rDNA site was observed. In ssp. ponticus, the 45S rDNA loci were always distally located in relation to the 5S rDNA loci. The occurrence of these sites in 17 instead of 20 chromosomes, as observed by Li and Zhang (2002), most likely indicates a reduction in the number of 45S rDNA sites in the accession used by Brasileiro-Vidal et al. (2003).

In ssp. elongatus, the 5S rDNA sites were associated with chromosomes 1Ee and, possibly, 5Ee (Scoles et al. 1988; Dvorák et al. 1989). Considering the distribution of these sites in diploid elongatus, the chromosomes carrying the 5S rDNA in the decaploid might also belong to homoeologous groups 1 and 5 (Brasileiro-Vidal et al. 2003).

Meiosis in the decaploid was less ordered. Cauderon (1958) observed 1.04 univalents, 19.9 bivalents, and a number of chain and ring multivalents (0.76 trivalents, 2.71 quadrivalents, 0.81 pentavalents, 0.52 hexavalents, 0.76 heptavalents, 0.19 octovalents, 0.05 ennevalents (=nine valents), and 0.04 decavalents). Similar patterns of chromosomal pairing were also observed by Zhang et al (1993, 1996) and Muramatsu (1990). The high frequency of multivalents in the decaploid subspecies may be the results of the activity of pairing promoters that exist in the ssp. ponticus genome (Zhang et al. 1993, 1995; Cai and Jones 1997).

Genomic relationships between the genomes of ssp. ponticus and its related taxa, have been the subject of several studies, and, due to chromosome pairing complexity, different genome formulae have been proposed for the decaploid. Peto (1936), assuming that the decaploid is an auto-allo-polyploid, tentatively assigned the genome formula AAXXXXYYYY, whereas Matsumura (1949) proposed the genome formula BBXXXXYYYY. Both researchers assumed that the A and B subgenomes of Triticum exist in ssp. ponticus. Muramatsu (1990) and Wang et al. (1991) concluded from the high frequency of multivalents in pollen mother cells (PMCs) of both the decaploid and the polyhaploid of ssp. ponticus, that this taxon contains several closely related or identical genomes, and therefore, is an autodecaploid with the genomic formula J1J1J2J2J3J3J4J4J5J5 and JJJJJJJJJJ, respectively. The J genome is from E. farctus ssp. bessarabicus (currently designated as Eb), is closely related to the Ee genome of ssp. elongatus and possesses modified versions of the same basic genome, namely, E (Forster and Miller 1989; Wang and Hsiao 1989; Wang 1990). Dvorak (1975) and Wang et al. (1991) regarded ssp. ponticus to be an autodecaploid that behaves as an allodecaploid, due to a bivalentization system.

Konarev (1979) assumed that the diploid subspecies of E. elongatus contributed to the karyotype of the decaploid taxon. Moustakas (1993), based on karyotype analysis, concluded that ssp. ponticus is a segmental allopolyploid, with genome formula JjJjJjJjJjJjJeJeJeJe, where genome designations Jj and Je represent the same genome but with structural differences. Dvorak (1975, 1981a) showed pairing of the chromosomes of diploid elongatus with some of the chromosomes of the decaploid, but the pairing was poor in every case, indicating that differentiation of the chromosomes had occurred. Consequently, Dvorak (1975) postulated that the decaploid evolved from an ancestral elongatuslike diploid taxa, by primary and secondary chromosome doubling of inter-ecotypic or inter-specific hybrids. In accordance, the karyotype analysis performed by Moustakas (1989, 1991), indicated that the chromosomes of the present-day diploid elongatus and those of the decaploid diverged from one another.

According to Dvorak (1981b), the decaploid appears to have one group of three closely related genomes and another group of two closely related genomes. Moreover, he suggested that the chromosomes of the diploid elongatus are more closely related to the doublet of the decaploid genome than to the triplet. Thus, Zhang and Dvorak (1990) suggested the genome designation ExExExExEyEyEyEyEyEy for subsp. ponticus. Jarvie (1992), assuming that ssp. ponticus is an auto-allo-polyploid, proposed the genome symbol EEEEEEJJJJ. Zhang et al. (1996), on the basis of GISH studies and genome specific markers, also suggested that this subspecies is an auto-allo-polyploid, but with the genome symbol StStStStEeEeEbEbExEx, where the St genome is homologous to the St genome of Elymus stipifolius. Moreover, GISH revealed that the centromeric region might be the critical area for discrimination between the St and E subgenomes (either Ee or Eb) in ssp. ponticus. Mitotic cells of several accessions of ssp. ponticus, when hybridized with the St probe and blocked by E genomic DNA, had 28 chromosomes strongly hybridized by the St probe at regions near the centromere (Zhang et al. 1996). When Ee or Eb was labeled as the probe and St was used as the blocker, all 70 chromosomes were labeled with FITC (fluorescein isothiocyanate). However, there were about 28 chromosomes lacking hybridization signals at the centromeric regions (Zhang et al. 1996). These consistent results were interpreted by Zhang et al. (1996) to mean that ssp. ponticus has 28 St genome chromosomes and 42 E genome chromosomes. The chromosome pairing data of Wang (1992) and the molecular studies of Hsiao et al. (1995) showed that the St, Ee, and Eb genomes are very closely related. The GISH results of Zhang et al. (1996) also revealed the close relationships between these three genomes and that GISH cannot distinguish between Eb and Ee. Taken together, the centromere and the region nearby may be the critical areas that discriminate the St from the E genomes in ssp. ponticus.

The 70 chromosomes of ssp. ponticus all fluoresced bright yellow when probed either with DNA from the Ee genome of E. elongatus ssp. elongatus or from the Eb genome of E. farctus ssp. bessarabicus (Chen et al. 1998a, b). This demonstrated that a substantial affinity exists between these probes and the subgenomes present in ponticus. Conversely, no obvious hybridization signal was detected in ponticus when probing either with DNA from the Ee genome and blocking with Eb genome DNA, or in the reverse analysis, using Eb genome DNA as probe and Ee genome DNA as blocker. Since this is expected when an effective DNA probe is used to block itself, the results suggest that the Ee and Eb genomes are closely related to one another and to the chromosomes of ssp. ponticus.

Chen et al. (1998a, b) also performed GISH using genomic DNA probes from E. elongatus ssp. elongatus (genome Ee), E. farctus ssp. bessarabicus (genome Eb), and E. strigisus (=Pseudoroegneria strigosa) (genome St), to investigate the genomic constitution of ssp. ponticus. Their findings indicated that the decaploid subspecies had only the two basic genomes Eb and Ebs (=Js). The Ebs genome of ponticus is homologous with E (Ee and Eb) genomes, but is quite distinct at the centromeric regions, which strongly hybridize with the St genomic DNA probe. This may indicate that the Ebs genome is a modified Eb (=J) genome whose chromosomes exchanged St segments via translocations between the two (Chen et al. 1998a, b). Support of this hypothesis also came from lack of centromeric hybridization signals upon hybridization of mitotic chromosomes of the diploid subspecies of elongatus and farctus with St genome DNA in the presence of Eb or Ee genome blocker (Chen et al. 1998a, b). Likewise, mitotic chromosomes of E. strigosus, probed with Eb genome DNA and blocked with St genome DNA, showed no hybridization signal. It appears that the chromosomes of ponticus, which show hybridization affinity with the centromeres of E. strigosus DNA, were not simply derived from any of these three diploid species, but rather, have a more complicated origin (Chen et al. 1998a, b).

Consequently, the group proposed that ssp. ponticus contains only segments of the St genome rather than any intact St genome or chromosomes. Based on the GISH results, namely, that all 70 chromosomes of ssp. ponticus hybridized extensively with Eb or Ee genome DNA probes, even in the presence of St genome blocker, Chen et al. (1998a, b) redesignated the genomic formula of ssp. ponticus as EEEEEEEbsEbsEbsEbs, where E refers to the Ee- or Eb-type chromosomes closely related to the genomes of ssp. elongatus and ssp. bessarabicus, respectively, while Ebs refers to a modified Ee- or Eb-type chromosomes distinguished by the presence of St genome-specific sequences close to the centromere.

The major disagreement between Zhang et al. (1996) and Chen et al. (1998a, b) centered around the explanation of the GISH results of ssp. ponticus probed by St genomic DNA and blocked by E genomic DNA. The St genomic probe hybridized all 70 chromosomes, but more strongly hybridized with 28 chromosomes at their centromeres and nearby regions. In the reverse GISH analysis, these 28 chromosomes were also hybridized by the E genomic probe, except for their centromeric and nearby regions, that were completely blocked by the St genomic DNA (Zhang et al. 1996). Zhang et al. opined that the unexpected signals appearing beyond the probe genome chromosomes were mainly caused by cross-hybridization between St and E genomes, arising from their close relationship in ssp. ponticus. Therefore, Zhang et al. (1996) proposed that the centromeres and nearby regions might be critical in the discrimination of St and E genomes.

Li and Zhang (2002) and Liu et al. (2007) accepted this interpretation, and used StStStStEeEeEbEbExEx as the genome formula for ssp. ponticus. Accordingly, the candidate donors of genomes to ssp. ponticus have been narrowed down to a few species, including the diploid species of the genus Elymus, namely, E. elongatus ssp. elongatus, E. farctus ssp. bessarabicus and E. stipifolius.

Since cytogenetic data indicate that the St, Ee, Eb (=J), and Ebs (=Js) genomes are very closely related (Wang 1992), the latest genomic designations are consistent with the earlier autopolyploid designation for these subspecies (Fedak et al. (2000). The study of Chen et al. (1998a, b) indicates that ssp. ponticus is not a characteristic autodecaploid and its five subgenomes are most likely modified versions of the Eb or Ee genomes. Since 28 chromosomes of ssp. ponticus containing centromeric region of St chromosomes are recombinant chromosomes, they concluded that ponticus can be regarded as a segmental autodecaploid with three sets of the E genome (Ee or Eb) genomes plus two sets of Ebs genome (Chen et al. 1998a, b). In contrast, Zhang et al. (1996) and Li and Zhang (2002) found that the decaploid is an auto-allo-polyploid containing six Ee genomes and four St genomes. The existence of multivalents in meiosis of the decaploid subspecies indicates that intergenomic recombination occurs quite frequently in this subspecies. Therefore, the genomes in the decaploid are recombinant genomes and differ from those of their donors.

### Crosses with Other Triticineae Species

Peto (1936) suggested that the relatively high number of paired chromosomes in F1 hybrids between Triticum turgidum ssp. dicoccon (2n = 4x = 28; genome BBAA) x Elymus elongatus ssp. ponticus (2n = 10x = 70; genome EeEeEbEbEbEbStStStSt) indicates homology between some wheat and E. elongatus chromosomes. Although B-subgenome chromosomes of wheat occasionally paired with other chromosomes at first meiotic metaphase of these F1 hybrids, aceto-carmine Giemsa N-banding analysis of chromosomes in root tip cells and PMCs of the F1 hybrid between Triticum aestivum cv. Fukuhoc and ssp. ponticus, showed that the latter does not contain the B subgenome of wheat (Zhang et al. 1993).

The average pairing in first meiotic metaphase of the F1 hybrid between tetraploid wheat, Triticum turgidum subsp. durum and E. Elongatus ssp. ponticus included 14.93 (9–25) univalents, 11.92 (5–18) bivalents, 2.14 (0–5) trivalents, 0.54 (0–3) quadrivalents, and 0.42 (0–20) pentavalents (Zhang et al. 1993). The average chromosome pairing in the F1 hybrid between hexaploid wheat, Triticum aestivum cv. Fukuhoc and ssp. ponticus displayed 10.87 (5–19) univalents, 16.40 (6–22) bivalents, 2.78 (0–6) trivalents, 0.55 (0– 3) quadrivalents, and 0.23 (0–3) pentavalents (Zhang et al. 1993). Likewise, the F1 hybrid T. aestivum cv. Chinese Spring x ssp. ponticus had 11.19 (4–17) univalents, 14.73 (7–20) bivalents, 3.12 (0–6) trivalents, 0.67 (0–2) quadrivalents, and 0.63 (0–2) pentavalents (Zhang et al. 1993). Cai and Jones (1997) used GISH to distinguish autosyndetic from allosyndetic pairing in the hybrid between ssp. ponticus and Triticum aestivum cv. Chinese Spring. Chromosome pairing in this hybrid occurred mainly among wheat chromosomes and among ssp. ponticus chromosomes, whereas allosyndetic pairing between wheat and ponticus chromosomes was very low. These results showed that the relationships among T. aestivum subgenomes and among ssp. ponticus subgenomes are much closer than the relationship between the subgenomes of the two species. The higher frequencies of autosyndetic pairing among the chromosomes of ssp. ponticus than among bread wheat chromosomes in the hybrid, indicated that the relationships between the five subgenomes of ssp. ponticus are closer than those between the three subgenomes of T. aestivum.

Comparing the observations in aestivum x ponticus to those in durum x ponticus, shows that adding the D subgenome leads to pairing of more than eleven chromosomes. This may indicate either that ponticus contains gene(s) that promote homoeologous pairing in the presence of Ph1, located in several different subgenomes of ponticus (Zhang et al. 1993, 1995), or that ponticus has a subgenome(s) related to the D subgenome of bread wheat (Zhang et al. 1993). Cai and Jones (1997) reported relatively high autosyndetic pairing frequencies among bread wheat chromosomes in the hybrid of subsp. ponticus x Triticum aestivum cv. Chinese Spring. The mean autosyndetic pairing frequency between wheat chromosomes in the hybrid was much higher than between those of euhaploids Chinese Spring with Ph1 (Jauhar et al. 1991). Since this hybrid carries the Ph1 gene, Cai and Jones (1997) suggested that ssp. ponticus carries gene(s) that can promote homoeologous chromosome pairing in the presence of Ph1.

Jauhar (1995) analyzed the F1 hybrid between ssp. ponticus and bread wheat, which proved perennial and morphologically resembled the Elymus parent. The hybrid (2n = 8x = 56; genome EeEbEbStStBAD) showed high chromosome pairing (average pairing included 9.24 univalents, 6.23 rod- and 11.68 ring-bivalents, 2.07 trivalents, 1.09 quadrivalents, 0.02 pentavalents, and 0.05 hexavalents). Like Cai and Jones (1997), also Jauhar (1995) suggested that this high pairing was due to the inactivation of Ph1 by ssp. ponticus promoters.

Addition lines of bread wheat bearing chromosomes of diploid E. elongatus, which are homoeologous with wheat chromosomes of groups 6 and 7, were crossed with addition lines of bread wheat carrying chromosomes of the decaploid subspecies of E. elongatus, which are also homoeologous with wheat chromosomes of groups 6 and 7 (Dvorak 1975). The chromosomes of the two elongatus subspecies paired with one another in the presence of the Ph1 gene of common wheat. Since pairing between diploid E. elongatus and decaploid E. elongatus chromosomes in the presence of the Ph1 gene, was generally low, it was suggested that the chromosomes assigned to the same group are not homologous, but rather, closely homoeologous (Dvorak 1975). In contrast to the low pairing between chromosomes of the two elongatus subspecies in the presence of Ph1, Dvorak (1975) assumed that these chromosomes could pair quite regularly in the absence of Ph1.

Johnson and Kimber (1967) produced complex hybrids bearing 29 chromosomes, including one telocentric chromosome (in different hybrids, different telocentric chromosomes represent a different chromosome arm) and twenty complete chromosomes of T. aestivum (2n = 6x = 42), seven complete chromosomes of Aegilops. speltoides (2n = 2x = 14) and one telocentric chromosome derived from E. elongatus ssp. ponticus, corresponding to homoeologous group 6. The presence of the Ae. speltoides genome induced pairing between homoeologous chromosomes at meiosis, even in the presence of Ph1. The elongatus telocentric chromosome paired with wheat chromosomes homoeologous to group 6. There was no evidence that it paired with chromosomes of any other group.

### 5.2.4 E. farctus (Viv.) Runemark Ex Melderis (Based on Eb Genome)

<IP_ADDRESS> Species Description E. farctus (Viv.) Runemark ex Melderis [Syn. Triticum farctum Viv.; Triticum junceum L.; Agropyron junceum (L.) Beauv.; Agropyron junceum ssp. mediterraneum Simonet; Elytrigia juncea (L.) Nevski; Agropyron farctum (Viv.) Rothm.; Elymus multinodus Gould; Elymus farctus (Viv.) Runemark; Elytrigia juncea ssp. mediterranea (simonet) Hyl.; Thinopyrum junceum (L.) Á. Löve; Thinopyrum farctum (Viv.) Cabi & Dogan comb. nov.] is rhizomatous, perennial, with rigid and glabrous 30–60 cm high stems, 2– 5 mm broad, glaucous-green, leaves, densely pubescent on ribs of upper surface, with no auricles; 15–25 cm long spikes, fragile rachis (wedge type disarticulation), 5–12 veined and keeled 10–18 mm long glumes, 10–18 mm long lemmas and 6–12 mm long anthers, Caryopsis with adherent pericarp, 9.0 mm long.