How to date the correct partner? - Role of the Ph1 locus

Graham Moore

Crop Genetics, John Innes Centre, Colney, Norwich, UK
E-mail address: graham.moore@bbsrc.ac.uk

Summary

There is a difference in the behaviour of heterochromatin regions at the centromeres, telomeres and interstitial sites with Ph1. In the presence of Ph1, this may reduce the ability of homologous repeats to be involved in the pairing process. Although homoeologues have a similar gene order along their chromosome length, the expression pattern of the genes on the homoeologues will be different. This may make the genic regions on the homoeologues distinct in terms of the chromatin structure. Thus the ability to reduce the involvement of repetitive DNA in the pairing process and the uniqueness of genic regions (as result of differential expression between the homoeologues) may be sufficient to stabilise correct pairing at meiosis.

Key words: Ph1 locus, telomeres, centromeres, meiosis, chromosome pairing

It can be argued that Western Civilisation owes much of its foundation to wheat. In turn, hexaploid and tetraploid wheat only exists because the Ph1 locus stabilises the pairing of its multiple related genomes at meiosis. It provides a high level of fertility and seed set. The following article reviews a current knowledge of this important locus.

Some of the world's most important crops are allopolyploids, including wheat, canola, oats, cotton, tobacco, cultivated sugarcane and cooking banana; a few are autopolyploid (e.g. potato). Others, such as maize are cryptic allopolyploids. Following polyploidisation, extensive genome arrangements have now partially concealed their polyploidy origin. In fact more than 70% of flowering plants are polyploids possessing two or more sets of related chromosomes. This occurs as a result of either the doubling of chromosomes following sexual hybridisation within the same species (autopolyploidy), or between closely related species containing related but not completely homologous (homoeologous) genomes (allopolyploidy). For allopolyploids to produce viable gametes and be fertile, they must behave as diploids during meiosis with only identical chromosomes (homologous) pairing instead of homoeologues.

Breeders make use of the interspecific crosses to introduce desirable genes from exotic germplasms. These hybrids are produced by sexual hybridisation between a polyploid and a wild relative generally and contain a haploid set of polyploid and wild relative chromosomes. In some cases, there is a low level of pairing and recombination between the wheat and wild relative chromosomes which is a problem for breeding. Understanding the barriers during meiosis that prevent pairing and recombination of the chromosomes from the two parental species is therefore important to crop improvement strategies.

During meiosis, the number of chromosomes needs to be halved before sexual reproduction, thereby ensuring that chromosome number does not double with each generation (reviewed by Zickler and Kleckner, 1999). To this end, each of the two homologues is replicated before meiosis, forming two sister chromatids that remain linked together (four chromatids in total). Homologues are correctly segregated so that each gamete carries only a single copy of each chromosome. To achieve this, each chromosome must recognise its homologue from among all the chromosomes present in the nucleus. The homologues must then become intimately aligned along their entire lengths and a proteinaceous structure known as the synaptonemal complex (SC) must be assembled between them, a process called synapsis. In this way, meiotic recombination (the exchange of DNA strands between the homologues) is completed, resulting in the formation of chiasmata, physical links that hold the chromosomes together after disassembly of the SC. After the resolution of the physical links, the homologues separate during the first meiotic division. Many components of meiotic recombination and synapsis machinery are known, especially from studies of yeast. However little is understood about how homologues first recognise each other and how this is controlled. An unlikely model system, namely hexaploid wheat is starting to provide an insight into this process.

In hexaploid (bread) wheat (an allopolyploid), chromosome lA has a similar gene order to 1B and 1D (karyotype reviewed Gill et al. 1991; RFLP maps reviewed in Gale et al.1997). Tetraploid (pasta) wheat possesses just the A and B homoeologous genomes, and lacks the D genome. Although there is a similar gene order on the homoeologues, the expression pattern between the homoeologues will be different (Mochida et al. 2004). However the major difference will be in the repetitive DNA between the genes. Hexaploid and tetraploid wheat both behave as diploids at meiosis, with regular pairing at metaphase I. Thus, at meiosis, wheat chromosome 1A pairs with 1A, and not with either 1B or 1D.

The diploid chromosome pairing behaviour which restricts pairing to homologues rather than homoeologues in wheat is under genetic control. A single locus (termed Pairing homoeologous 1, Ph1) on the long arm of chromosome 5B has a major controlling effect (Riley and Chapman 1958; Sears and Okamoto 1958). Other loci that affect pairing in wheat have also been identified (e.g. on chromosomes 5AL, 5DL, 5AS, 3DS, 3AL, 3BL and 3DL,) (reviewed by Feldman 1993). However they do not compensate for the absence of the Ph1 locus. Moreover to date little research has been undertaken on these loci compared to that on Ph1. In the presence of this locus (i.e. two copies of Ph1), chromosome pairing in tetraploid and hexaploid wheat during meiosis is largely restricted to homologous chromosomes, but in deletion mutants lacking Ph1, increasing aberrant pairing can be observed at metaphase I with each generation of the mutant. These Ph1 mutants accumulate rearrangements with each propagation (Sanchez-Moran et al. 2001). To date, no chromosome substituted for 5B in wheat from any wild species has been shown to compensate fully for the Ph1 locus. However the 5G chromosome from wheat relative Triticum timopheevi, when substituted for 5B does provide a similar effect (Ozkan et al. 2001). It is likely that other species do carry loci equivalent to Ph1 on 5B, but that their effect is not so dramatic.

The Ph1 locus is specific to 5B and to a region defined by 70 megabase deletions in hexaploid wheat and tetraploid wheat (Sears 1977; Gill et al. 1993). The chromosome 5B carrying the Ph1 locus has meiotic drive compared to a 5B chromosome lacking the Ph1 locus (Sears 1977). It is not found in either the A or D genomes, nor in the diploid A, D or S genome progenitors. It is present on the 5G chromosome of T. timopheevi (AAGG) (Ozkan et al. 2001). EMS treatment has failed to generate Ph1 mutants but large deletions generated by either X-ray or fast-neutron treatments have yielded mutants (Wall et al. 1971; Sears 1977; Roberts et al. 1999). The failure of treatments which yield point mutations to produce Ph1 mutants suggests that the phenotype is neither due to a single gene nor several different genes contributing to a phenotype. Thus it seems likely to be several related genes contributing to the phenotype, in which knocking out any would not produce an altered phenotype. B chromosomes which are essentially lumps of heterochromatin can compensate for the Ph1 locus (Dover and Riley 1972). The ability of Ph1 to hinder chromosome pairing between homoeologues is dosage dependent. In tetraploid and hexaploid wheat carrying chromosomes from two related rye species- Secale montanum (Rm) and Secale cereale (Rc)- the homoeologous rye chromosomes pair freely at metaphase I with one Ph1 copy, but there are few pairs formed by the rye chromosomes in the presence of two Ph1 copies (Riley and Miller 1970; Miller and Riley 1972). A single copy of the Ph1 is sufficient to prevent wheat-rye chromosome pairing at metaphase I in tetraploid and hexaploid wheat-rye hybrids (but there is pairing in its absence). In contrast six doses of Ph1 prevents not only homoeologous pairing but also homologous pairing as it results in unpaired homologous chromosomes at metaphase I (Feldman 1966; Yacobi et al. 1982; Holm and Wang 1988). Thus Ph1 is encoding something which reduces overall chromosome pairing with its effect being both somatic and meiotic cells. Thus at zero doses of Ph1, homoeologous chromosomes regularly pair, with a single dose, some homoeologues still can pair, with two doses homoeologous chromosomes can not pair and at 6 doses some homologous chromosomes are unable to pair.

The 42 of hexaploid wheat chromosomes pair as 21 bivalents as scored at metaphase I in the presence of Ph1. In the absence of Ph1, close to 21 bivalents are observed in most meiocytes at metaphase I (Roberts et al. 1999). Thus Ph1 does not affect substantially overall pairing and recombination. If it is not affecting pairing and recombination, does it affect how chromosomes recognise each other? Given the homoeologues possess similar gene order how can a pair of homologues be marked to assess the level of homologue pairing in the presence and absence of Ph1. A wheat line carrying a rye segment covering 15% of the distal chromosome arm substituted for the equivalent region of the 1D pair of wheat chromosomes provides a solution. By visualising the rye segments using genomic in situ hybridisation, the homologues bearing these segments paired with each other at metaphase I in all the melocytes examined in the presence of Ph1. In contrast, these homologues are observed pairing in 66% of the meiocytes examined from the Ph1 mutant (Prieto et al. 2004). This suggests the Ph1 is changing the ability of chromosomes to recognise their homologue from the homoeologue and that this process is distinct from the mechanism of pairing and recombination which is basically unaffected in the presence and absence of the Ph1 locus.

So what sites do chromosomes first use to recognise each other at the start of meiosis and does Ph1 affect them? Studies of diploids- yeast (Schizosaccharomyces pombe and Saccharomyces cerevisiae), mammals and rye, and polyploids- maize (a cryptic polyploid) and wheat show that telomeres of chromosomes aggregate on the nuclear envelope forming a telomere cluster or bouquet during meiotic prophase I (Dawe et al. 1994; Bass et al. 1997, 2000; Chikashige et al. 1997; Trelles-Sticken et al. 1999). The proposal is that this structure facilitates in some way the sorting of chromosomes into homologous pairs. Synaptonemal complex formation initiates near the telomeres in many plants (including maize and rye) (Gillies 1975,1985). A deletion of the telomere region of one of the homologues is generally sufficient to reduce or eliminate subsequent pairing between these chromosomes at metaphase I, presumably because of the synaptonemal complex initiation (Curtis et al. 1991; Lukaszewski 1977). Thus homologous chromosomes are tethered at a region close to the telomeres during early prophase I and then intimately aligned along their length as prophase I pairing proceeds. Two doses of Ph1 change the specificity of the telomere regions so that the telomere regions only pair homologously in the presence of homologues and homoeologues (Prieto et al. 2004). However in the absence of homologues, a single dose of Ph1 can not prevent homoeologues pairing via their telomere regions (Prieto et al. 2004).

Are other chromosome sites being used as initial recognition sites? Centromeres in meiosis are involved in segregating the homologues to opposite poles at metaphase I but are they involved in the process of homologue recognition? In early floral tissue, the centromeres associate as pairs in both hexaploid and tetraploid wheat (Aragon-Alcaide et al. 1997; Martinez-Perez et al. 2000, 2001). The centromeres do not pair premeiotically in the progenitor diploids (Martinez-Perez et al. 2000). A third of these associations are homologous (Aragon-Alcaide et al. 1997). At early meiosis, these pairs of centromere form into 7 groups corresponding to the seven homoeologous groups. After centromere assortment within these groups, the centrorneres resolve as homologous pairs (Martinez-Perez et al. 2001, 2003). Thus the centromeres do act as initial sites for homologue recognition. Moreover Ph1 affects the specificity of centromere interactions. In wheat-rye hybrids, their 28 centromeres form 14 centromere sites prior to meiosis and 7 groups at meiosis. However the 14 centromere sites in the presence of Ph1 are composed of 7 wheat-rye pairs and 7 wheat-wheat pairs, while in the absence of Ph1, they are 7 sites composed of 21 wheat centromeres and 7 single rye centromeres (Prieto et al. 2004).

Thus Ph1 affects the specificity of subtelomere and centromere regions. Does Ph1 affect the behaviour of other chromatin? Studies exploiting maize and Caenorhabditis elegans have indicated that the onset of pairing at meiosis is associated with conformational changes in the chromosomes (Dawe et al. 1994; MacQueen and Villeneuve 2001). In maize, it is observed that this is not a generalised conformational change of the whole chromosome, but is a localised effect. Thus these conformational changes are distinct from the generalised condensation of chromosomes which is initiated at the onset of meiosis and end with condensed chromosomes at metaphase I. As two homologues pair or "zip up", the chromatin immediately preceding the "pairing fork" undergoes a conformational change becoming elongated after which it intimately pairs. However as yet no mutants have been identified in either C. elegans or maize which affect this conformational change. Recently this "localised" conformational change has also been observed in hexaploid wheat chromosomes which are pairing at meiosis and interestingly the behaviour of the conformational change is affected by the Ph1 locus. In the presence of Ph1, euchromatin but not some telomeric heterochromatin can undergo the conformational change, while in its absence; both euchromatin and the telomeric heterochromatin undergo the change. Thus in hexaploid wheat, when two chromosomes recognise each other by their telomere (or centromere regions), the chromatin immediately adjacent to these regions undergoes a conformational change. The conformational change is confined to these interacting chromosomes. The signal to change conformation does not involve a signal diffusing throughout the nucleus. The changes in the conformation occur chromosome by chromosome. Moreover the fact that the comparable chromosome segments on the homologues so closely mirror each other in condensation state in the presence of Ph1, suggests that the signal to initiate the conformation change occurs at the same time on both homologues. Thus as the chromosome regions pair along the chromosome, the chromatin immediately adjacent to these regions then undergoes a conformational change followed by its intimate pairing (Dawe et al. 1994; Prieto et al. 2004).

Studies by Dvorak and. colleagues found that in the absence of Ph1, recombination occurs between a pair of wheat chromosomes composed of combinations of homoeologous and homologous segments, but in the presence of Ph1, recombination is restricted to homologous segments (Dubcovsky et al. 1995; Luo et al. 1996,2000). The ability to propagate the conformational signal along two chromosomes pairing intimately may explain Ph1's ability to restrict recombination between homoeologous segments embedded within homologous chromosomes. In the presence of two copies of Ph1, the signal to change conformation does not propagate through the homoeologous segment. Without this change in conformation, those regions will not intimately pair correctly and therefore recombine. While in the absence of Ph1, the two segments will undergo the conformational change and intimately pair and recombine. At present it is unclear in the presence of Ph1 whether the failure of the rye telomeric heterochromatin to undergo conformational changes stops subsequent changes in conformation in regions proximal to the heterochromatin. The effect on the telomeric heterocbromatin knobs could help to explain the basis for homoeologous pairing-in the absence of Ph1 . In the presence of Ph1, regions which are highly homologous (such as heterochromatin) do not undergo the conformational change and may therefore be excluded from the pairing process. In contrast in the absence of Ph1, the chromatin changes do occur in these highly homologous regions and they can engage in multiple associations between homologous, homoeologous and non-homologous chromosomes. Moreover the whole chromosome may be slightly more condensed in the presence of Ph1 when pairing than in its absence which might further exclude highly homologous repeats from the pairing process. It has also been observed in the absence of Ph1 that the elongation of chromatin associated with pairing can occur in meiocytes which have not fully formed the telomere bouquet (Prieto et al. 2004). In contrast, the conformational changes associated with pairing are only observed at the telomere bouquet formation in the presence of Ph1 (Prieto et al. 2004). This implies that pairing is being initiated earlier in the absence of the Ph1 as the telomeres are beginning to duster. One explanation for this is that the stringency at which interactions between telomere regions can trigger the pairing process is reduced in the absence of Ph1. Thus there will be a higher chance of an interaction occurring between telomere regions as they cluster in the absence of Ph1 which triggers the pairing process. An earlier initiation of pairing in the absence of Ph1 implies that the chromosomes will be pair in slightly different overall condensation states in the presence and absence of Ph1. Thus in the absence of Ph1, the chromosomes will be less condensed than in the presence of Ph1. This is consistent with the proposal by Maestra and colleagues that although there is no apparent difference in the overall structure of chromosomes in the presence and absence of Ph1 prior to meiosis, the chromosomes may be less condensed when pairing during early meiosis in the absence of Ph1 than its presence (Maestra et al. 2002). The more "open chromatin" (less condensed) of the entire chromosome at the time of pairing combined with the ability of highly homologous heterochromatin to extensively elongate as the chromosomes pair may explain the basis of marked increase in homoeologous and non-homologous interactions in the absence of Ph1 . However it is difficult to provide clear-cut data for this proposal at the telomere bouquet as visualising the behaviour of whole chromosome additions is difficult to interpret at this stage while reducing the complexity by visualising single arm additions (telosomes) has additional complications. The two telomeres of the telosome join the telomere bouquet bringing centromere of the telosome into the bouquet. Thus the telosome is looped back at this stage and in some cases can stretch right around the nucleus (Martinez-Perez et al. 1999; Maestra et al. 2002; Canton and Cande 2002).

In hexaploid wheat, most chromosomes intimately pair (synapse) with only one partner during meiotic prophase I. However occasionally chromosomes start intimately pairing/synapsing with more than partner (some 12 chromosomes out of 42 can be observed initially in multiple associations in both the presence and absence of Ph1 (HoIm 1986,1988; Martinez et al. 2001). Thus the specificity at which chromosomes initially synapse from the telomeres is unaffected by the presence or absence of two copies of Ph1 but is increased in the presence of four copies reducing the number of chromosomes that initially engage in multiple associations from some 12 to less than 4. In the presence of Ph1 (two copies), the incorrectly paired sites are resolved later during prophase I. By contrast in the absence of Ph1, many incorrectly paired sites remain unresolved in later meiotic prophase I involving some 8-12 chromosomes in both hexaploid and tetraploid wheat. If the ability of Ph1 to stop recombination occurs very early in this process, then in the presence of Ph1, recombination can not proceed between incorrectly associated chromosomes while in the absence of Ph1, recombination can and does proceed. Thus the incorrectly synapsed chromosomes will not resolve as no recombination will be initiated in the presence of Ph1 while in its absence, the recombination process will continue between the incorrectly paired chromosomes and therefore the chromosomes will remain synapsed and multivalent.

What is the Ph1 locus? No allelic variation has been reported for the Ph1 locus. The only variation reported has been associated with dosage. Increasing Ph1 dosage reduces both homologous as well as homoeologous pairing, while at a single dosage homoeologous rye chromosomes will pair and recombine which can not at two doses. Thus Ph1 can suppress all pairing but at different doses affects whether homologous or homoeologous pairing is affected. B chromosomes can compensate for the absence of Ph1, which assuming that B chromosomes are heterochromatin with little if any genes, means that the mechanisms which maintain and regulate heterochromatin also regulate pairing and recognition as Ph1 does. EMS mutagenesis has failed to generate any Ph1 mutants while X-ray and fast neutron treatments have generated mutants. This suggests that Ph1 is not simply a "gene" which can be knocked out by point mutation, but is more complex. For example if Ph1 is a multi-gene family, knocking out one member would have little effect. This would also explain the lack of variation in the Ph1 phenotype in hexaploid wheat as allelic variation in any one member would not have much affect.

Is Ph1 important for plant breeding? Interspecific hybrids made between wheat lacking Ph1 and its wild relatives often exhibit extensive pairing at metaphase I between the homoeologues but less in the presence of a single copy (Kimber et al. 1981). This observation is of practical importance for plant breeding as there is the potential to generate recombinant chromosomes composed of part wheat and part non wheat chromatin, allowing the introgression of exotic genetic material from wild relatives. Sears transferred disease resistance loci from wild relatives of wheat by recombining the chromosomes with those of wheat in lines deficient in the Ph1 locus (Sears 1972). More recently CIMMYT exploited Ph1 to introgress from closely related species so that now 25% of all new varieties released in their programme, will contain material derived from related species.

Unfortunately an important complication is that there is still a hierarchy in preferred pairing partners even in the absence of Ph1. Most pairing observed at metaphase I(80%) in hexaploid wheat interspecific hybrids lacking Ph1 locus between wheat chromosome from A and D although chromosomes of A and B genomes of wheat can pair at metaphase I in interspecific hybrids between tetraploid wheat carrying just the A and B genomes and a diploid wild relative (Blanco et al. 1988; Jauhar et al. 1991).

What can we expect in the future? The characterisation of the region containing the Ph1 locus should be completed and revealed in the coining months. From this, it should become clear how the Ph1 locus evolved on polyploidisation. However more importantly this information can be used to assess whether it is possible to generate allelic variation at the locus. This variation can then be assessed for any difference in phenotype. The factors which suppress the Ph1 effect are found in Aegilops speltoides and Ae. mutica. These factors should be elucidated in the future from programmes being developed by Dvorak and colleagues for Ae. speltoides and by ours for Ae. mutica. Thus it should be possible in the future to promote more easily pairing between homoeologous either by suppressing the Ph1 effect or by exploiting allelic variation in the Ph1 locus.

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