Chromosome pairing in meiosis of partially fertile wheat/rye hybrids

The normal course of meiosis depends on regular pairing of homologous chromosomes. In intergeneric hybrids, including those of wheat, there is no chromosome pairing because there are no homologs. In F₁ wheat/rye hybrids, pairing is largely prevented by the pairing homoeologous1 (Ph1) gene. In its presence, there are only rare instances of pairing; most chromosomes are univalent, and their orientation at metaphase I initiates different pathways of the meiotic cycle. The meiotic-like pathway includes a combination of the reductional and the equational + reductional steps at AI followed by the second division. The resulting gametes are mostly non-functional. The mitotic-like pathway involves equational division of univalents at AI and the absence of the second division. Any fertility of wheat/rye hybrids depends on the production of unreduced gametes arising from meiotic restitution (mitotic-like division). We examined the meiotic pairing in wheat/rye hybrids created from wheat lines with single rye chromosome substitutions and Ph1 present. This guaranteed F₁ meiosis with one pair of rye homologs. All hybrids formed bivalents, but proportions of meiocytes with bivalents varied. In the meiocytes where bivalents were present, there was a higher tendency for the meiotic-like pathway, while in meiocytes where bivalent pairing failed, the tendency was stronger for the mitotic-like pathway. Among the equationally dividing cells, we observed more than 90 % of meiocytes without bivalents, where rye homologs did not form bivalents, too. The data indicate a potential application of wheat/rye lines in producing genetic stocks of amphidiploids with designated genomic constitutions.     

Centromere association is an unlikely mechanism by which the wheat Ph1 locus regulates metaphase I chromosome pairing between homoeologous chromosomes

Chiasmate pairing between homoeologous chromosomes at metaphase I (MI) of meiosis in wheat is prevented by the activity of the Ph1 locus on chromosome 5B. Several hypotheses have been proposed sharing the assumption that Ph1 regulates MI chromosome pairing by regulating centromere-mediated chromosome alignment before the onset of meiosis. To test the relevance of the putative predetermination of chromosome pairing at MI by the centromere-mediated chromosome association prior to meiosis, a 2BL.2RL homoeoisochromosome was constructed and its MI pairing was assessed in the presence and absence of the Ph1 locus. Although the 2BL and 2RL arms of the homoeoisochromosome paired with each other at MI in the absence of Ph1, they never paired with each other at MI in the presence of Ph1. Since the two arms were permanently associated in the homoeoisochromosome via a common centromere, it is unlikely that Ph1 predetermines MI pairing between homoeologous chromosomes solely by controlling premeiotic association of centromeres. These findings are consistent with the idea that Ph1 determines the chromosome pairing pattern at MI by scrutinizing homology across the entire chromosome.     

Cytogenetics of chromosome pairing in wheat

Meiotic chromosome pairing in Triticum aestivum is controlled by genetic systems promoting and reducing pairing. The pairing of homoeologous chromosomes is prevented principally by the activity of a single locus (Ph) distally located on the long arm of chromosome 5B. In certain hybrids, supernumerary chromosomes (B chromosomes) from Aegilops species can compensate for the absence of chromosome 5B preventing or reducing homoeologous pairing. Temperature-dependent variants and colchicine sensitivity have been used to show that there are at least two stages in the G1 of meiosis at which the occurrence of meiotic pairing is determined. Wheat may differ from lily in the detailed organization of meiosis.     

Homoeologous recombination in the presence of <Emphasis Type="Italic">Ph1</Emphasis> gene in wheat

A crossover (CO) and its cytological signature, the chiasma, are major features of eukaryotic meiosis. The formation of at least one CO/chiasma between homologous chromosome pairs is essential for accurate chromosome segregation at the first meiotic division and genetic recombination. Polyploid organisms with multiple sets of homoeologous chromosomes have evolved additional mechanisms for the regulation of CO/chiasma. In hexaploid wheat (2n = 6× = 42), this is accomplished by pairing homoeologous (Ph) genes, with Ph1 having the strongest effect on suppressing homoeologous recombination and homoeologous COs. In this study, we observed homoeologous COs between chromosome 5M^g of Aegilops geniculata and 5D of wheat in plants where Ph1 was fully active, indicating that chromosome 5M^g harbors a homoeologous recombination promoter factor(s). Further cytogenetic analysis, with different 5M^g/5D recombinants, showed that the homoeologous recombination promoting factor(s) may be located in proximal regions of 5M^g. In addition, we observed a higher frequency of homoeologous COs in the pericentromeric region between chromosome combination of rec5M^g#2S·5M^g#2L and 5D compared to 5M^g#1/5D, which may be caused by a small terminal region of 5DL homology present in chromosome rec5M^g#2. The genetic stocks reported here will be useful for analyzing the mechanism of Ph1 action and the nature of homoeologous COs.     

Effect of the pairing gene Ph1 on centromere misdivision in common wheat.

The cytologically diploid-like meiotic behavior of hexaploid wheat (i.e., exclusive bivalent pairing of homologues) is largely controlled by the pairing homoeologous gene Ph1. This gene suppresses pairing between homoeologous (partially homologous) chromosomes of the three closely related genomes that compose the hexaploid wheat complement. It has been previously proposed that Ph1 regulates meiotic pairing by determining the pattern of premeiotic arrangement of homologous and homoeologous chromosomes. We therefore assume that Ph1 action may be targeted at the interaction of centromeres with spindle microtubules--an interaction that is critical for movement of chromosomes to their specific interphase positions. Using monosomic lines of common wheat, we studied the effect of this gene on types and rates of centromere division of univalents at meiosis. In the presence of the normal two doses of Ph1, the frequency of transverse breakage (misdivision) of the centromere of univalent chromosomes was high in both first and second meiotic divisions; whereas with zero dose of the gene, this frequency was drastically reduced. The results suggest that Ph1 is a trans-acting gene affecting centromere-microtubules interaction. The findings are discussed in the context of the effect of Ph1 on interphase chromosome arrangement.     

Identification of a chromosome-specific probe that maps within the Ph1 deletions in common and durum wheat

 The Ph1 (pairing homoeologous) gene is the major factor that determines the diploid-like chromosome behavior of polyploid wheat. This gene, which is located on the long arm of chromosome 5B (5BL), suppresses homoeologous pairing at meiosis while allowing exclusive homologous pairing. In an effort to tag the specific chromosomal region where this gene is located, we have previously microdissected chromosome arm 5BL from bread wheat and produced a plasmid library by random PCR amplification and cloning. In this work we isolated from this library a 5BL-specific probe, WPG90, and mapped it within the interstitial deleted chromosome fragments carrying Ph1 in common and durum wheat. A PCR assay of Ph1 based on WPG90 was developed that allows an easy identification of homozygous genotypes deficient for this gene. Received: 19 June 1996 / Accepted: 18 October 1996     

Suppression of Homoeologous Pairing by <Emphasis Type="Italic">B</Emphasis> Chromosomes in a <Emphasis Type="Italic">Lolium</Emphasis> Species Hybrid

INTERSPECIFIC hybridization together with polyploidy has been an important force in the evolution of many of our graminaceous crop plants. Both wheat (Triticum aestivum) and oats (Avena sativa), for example, are natural allohexaploids derived in each case from the hybridization of three separate but related diploid species. The efforts of plant breeders to synthesize stable and fertile polyploids of this kind have, on the whole, been unsuccessful. The main reason for this is that whereas meiosis in natural allopolyploids such as wheat is extremely regular this is not the case with “synthetic” polyploids. In wheat precise control over pairing at meiosis is achieved by a gene or a cluster of genes on chromosome SB. The gene acts by restricting the pairing to homologous chromosomes with the result that only bivalents are formed, disjunction is regular and inheritance is completely disomic^1,2. In the artificial polyploids at pachytene there is pairing between both homologous chromosomes (from the same species) and “corresponding” homoeologous chromosomes (from different species). The result is an extremely irregular metaphase 1 comprising multivalents and univalents as well as bivalents. Segregation is irregular and a certain degree of infertility is inevitable.     

Chromosome painting in meiosis reveals pairing of specific chromosomes in polyploid <Emphasis Type="Italic">Solanum</Emphasis> species

Analysis of chromosome pairing has been an important tool to assess the genetic similarity of homologous and homoeologous chromosomes in polyploids. However, it is technically challenging to monitor the pairing of specific chromosomes in polyploid species, especially for plant species with a large number of small chromosomes. We developed oligonucleotide-based painting probes for four different potato chromosomes. We demonstrate that these probes are robust enough to monitor a single chromosome throughout the prophase I of meiosis in polyploid Solanum species. Cultivated potato (Solanum tuberosum, 2n?=?4x?=?48) is an autotetraploid. We demonstrate that the four copies of each potato chromosome pair as a quadrivalent in 66–78% of the meiotic cells at the pachytene stage. Solanum demissum (2n?=?6x?=?72) is a hexaploid and has been controversial regarding its nature as an autopolyploid or allopolyploid. Interestingly, no hexavalent pairing was observed in meiosis. Instead, we observed three independent bivalents in 83–98% of the meiotic cells at late diakinesis and early metaphase I for the four chromosomes. These results suggest that S. demissum has evolved into a cytologically stable state with predominantly bivalent pairing in meiosis.     

Effect of a rye B chromosome and its segments on homoeologous pairing in hybrids between common wheat and Aegilops variabilis

Rye B chromosomes, which are supernumerary chromosomes dispensable for the host but increase in number by non-disjunction after meiosis, have been reported to affect meiotic homoeologous pairing in wheat-rye hybrids. The effect of a rye B chromosome (B) and its segments (B-9 and B-10) on homoeologous pairing was studied in hybrids between common wheat (2n=42) and Aegilops variabilis (2n=28), with reference to the Ph1 gene located on wheat chromosome 5B. The B-9 and B-10 chromosomes are derived from reciprocal translocations between a wheat and the B chromosomes, and the former had the B pericentromeric segment and the latter had the B distal segment. Both the B and B-9 chromosomes suppressed homoeologous pairing when chromosome 5B was absent. On the other hand, the B-9 and B-10 chromosomes promoted homoeologous pairing when 5B was present. On pairing suppression, B-9 had a greater effect in one dose than in two doses, and B-9 had a greater effect than B-10 had in one dose. These results suggested that the effect of the B chromosomes on homoeologous pairing was not confined to a specific region and that the intensity of the effect varied depending on the presence or absence of 5B and also on the segment and dose of the B chromosome. The mean chiasma frequency (10.23) in a hybrid (2n=36) possessing 5B and one B-9 was considerably higher than that (2.78) of a hybrid (2n=35) possessing 5B alone, and was comparable with that (14.09) of a hybrid (2n=34) lacking 5B. This fact suggested that the B chromosome or its segment can be used in introducing alien genes into wheat by inducing homoeologous pairing between wheat and alien chromosome.     

Dual effect of the wheat <Emphasis Type="BoldItalic">Ph1</Emphasis> locus on chromosome synapsis and crossover

Allopolyploids must possess a mechanism for facilitating synapsis and crossover (CO) between homologues, in preference to homoeologues (related chromosomes), to ensure successful meiosis. In hexaploid wheat, the Ph1 locus has a major effect on the control of these processes. Studying a wheat mutant lacking Ph1 provides an opportunity to explore the underlying mechanisms. Recently, it was proposed that Ph1 stabilises wheat during meiosis, both by promoting homologue synapsis during early meiosis and preventing MLH1 sites on synapsed homoeologues from becoming COs later in meiosis. Here, we explore these two effects and demonstrate firstly that whether or not Ph1 is present, synapsis between homoeologues does not take place during the telomere bouquet stage, with only homologous synapsis taking place during this stage. Furthermore, in wheat lacking Ph1, overall synapsis is delayed with respect to the telomere bouquet, with more synapsis occurring after the bouquet stage, when homoeologous synapsis is also possible. Secondly, we show that in the absence of Ph1, we can increase the number of MLH1 sites progressing to COs by altering environmental growing conditions; we show that higher nutrient levels in the soil or lower temperatures increase the level of both homologue and homoeologue COs. These observations suggest opportunities to improve the exploitation of the Ph1 wheat mutant in breeding programmes.     

Inducing chromosome pairing through premature condensation: analysis of wheat interspecific hybrids

At the onset of meiosis, chromosomes first decondense and then condense as the process of recognition and intimate pairing occurs between homologous chromosomes. We show here that okadaic acid, a drug known to induce chromosome condensation, can be introduced into wheat interspecific hybrids prior to meiosis to induce chromosome pairing. This pairing occurs in the presence of the Ph1 locus, which usually suppresses pairing of related chromosomes and which we show here delays condensation. Thus the timing of chromosome condensation during the onset of meiosis is an important factor in controlling chromosome pairing.     

Genetic Control of Chromosome Pairing in Hexaploid Oats

THE common oat, Arena sativa, is a hexaploid (2n=6x=42) whose chromosomes form exclusively bivalents during meiosis. The bivalents are formed by the pairing of exact homologues resulting in disomic inheritance. Gene duplications and triplications were revealed by early inheritance studies¹ and homoeologous relationships of the three genomes were later supported by the good tolerance of deficiency for single and for pairs of chromosomes².     

Isolation and cytogenetic characterization of male meiotic mutants of Drosophila melanogaster

Proper segregation of homologous chromosomes in meiosis I is ensured by pairing of homologs and maintenance of sister chromatid cohesion. In male Drosophila melanogaster, meiosis is achiasmatic and homologs pair at limited chromosome regions called pairing sites. We screened for male meiotic mutants to identify genes required for normal pairing and disjunction of homologs. Nondisjunction of the sex and the fourth chromosomes in male meiosis was scored as a mutant phenotype. We screened 2306 mutagenized and 226 natural population-derived second and third chromosomes and obtained seven mutants representing different loci on the second chromosome and one on the third. Five mutants showed relatively mild effects (<10% nondisjunction). mei(2)yh149 and mei(2)yoh7134 affected both the sex and the fourth chromosomes, mei(2)yh217 produced possible sex chromosome-specific nondisjunction, and mei(2)yh15 and mei(2)yh137 produced fourth chromosome-specific nondisjunction. mei(2)yh137 was allelic to the teflon gene required for autosomal pairing. Three mutants exhibited severe defects, producing >10% nondisjunction of the sex and/or the fourth chromosomes. mei(2)ys91 (a new allele of the orientation disruptor gene) and mei(3)M20 induced precocious separation of sister chromatids as early as prometa-phase I. mei(2)yh92 predominantly induced nondisjunction at meiosis I that appeared to be the consequence of failure of the separation of paired homologous chromosomes.     

Genesis of bivalent pairing in hexaploid clump Verbena

Both 6x Verbena aubletia (n=15) and 2x V. tenuisecta (n=5) form bivalents during meiosis, however, their 4x F₁ hybrid (V. aubletia × V. tenuisecta) shows almost complete homoeologous pairing involving on average 19.74 out of its 20 chromosomes. In 10% cells there are 4IV+2II indicating that essentially there may be 4 homoeologous sets of 5 chromosomes each in the F₁ hybrid. Evidently, V. aubletia is segmental allo-hexaploid involving 3 homoeologous genomes (A₁A₁ A₂A₂ A₃A₃). Whether its cytologically diploid behaviour is the result of a multivalent suppressor system or due to an acute property of preferential pairing, cannot be answered with certainty. In either case intergenomal homoeologies are totally suppressed resulting in bivalent pairing, meiotic isolation of the 3 genomes and institution of normal fertility.     

Terminal regions of wheat chromosomes select their pairing partners in meiosis

Many plant species, including important crops like wheat, are polyploids that carry more than two sets of genetically related chromosomes capable of meiotic pairing. To safeguard a diploid-like behavior at meiosis, many polyploids evolved genetic loci that suppress incorrect pairing and recombination of homeologues. The Ph1 locus in wheat was proposed to ensure homologous pairing by controlling the specificity of centromere associations that precede chromosome pairing. Using wheat chromosomes that carry rye centromeres, we show that the centromere associations in early meiosis are not based on homology and that the Ph1 locus has no effect on such associations. Although centromeres indeed undergo a switch from nonhomologous to homologous associations in meiosis, this process is driven by the terminally initiated synapsis. The centromere has no effect on metaphase I chiasmate chromosome associations: homologs with identical or different centromeres, in the presence and absence of Ph1, pair the same. A FISH analysis of the behavior of centromeres and distal chromomeres in telocentric and bi-armed chromosomes demonstrates that it is not the centromeric, but rather the subtelomeric, regions that are involved in the correct partner recognition and selection.     

The effect of B chromosomes on homoeologous pairing in species hybrids

The effect of B chromosomes on chromosome pairing at meiosis was investigated in the species hybrid Lolium temulentum x L. perenne at both the diploid and tetraploid level. The presence of B chromosomes drastically reduced association of homoeologous chromosomes in both the diploids and tetraploids. This was evident from the high frequency of univalents recorded in PMC's of diploid hybrids with B's and from the predominantly bivalent association of homologous chromosomes in tetraploids of this type. In the absence of B's homoeologous pairing was extensive giving a high frequency of bivalents in the diploids and multivalents as well as bivalents and univalents in the tetraploids.     

The molecular features of chromosome pairing at meiosis: the polyploid challenge using wheat as a reference

During meiosis, chromosome numbers are halved, leading to haploid gametes, a process that is crucial for the maintenance of a stable genome through successive generations. The process for the accurate segregation of the homologues starts in pre-meiosis as each homologue is replicated and the respective products are held together as two sister chromatids via specific cohesion proteins. At the start of meiosis, each chromosome must recognise its homologue from amongst all the chromosomes present in the nucleus and then associate or pair with that homologue. This process of homologue recognition in meiosis is more complicated in polyploids because of the greater number of related chromosomes. Despite the presence of these related chromosomes, for polyploids such as wheat to produce viable gametes, they must behave as diploids during meiosis with only true homologues pairing. In this review, the relationship between the Ph1 cyclin-dependent kinase (CDK)-like genes in wheat and the CDK2 genes in mammals and their involvement in controlling this process at meiosis is examined.     

Some characteristics of crossing over in induced recombination between chromosomes of wheat and rye

Allopolyploid wheat (Triticum aestivum L.) carries three pairs of homoeologous genomes but its meiotic pairing is diploid-like. This is the effect of the Ph (pairing homoeologous) system which restricts chromosome pairing to strictly homologous. Ph1 is the locus with the strongest effect. Disabling Ph1 permits pairing between homoeologues and is routinely used in chromosome engineering to introgress alien variation into breeding stocks. Whereas the efficiency of Ph1 and the general pattern of homoeologous crossovers in its absence are quite well known from numerous studies, other characteristics of such crossovers remain unknown. This study analyzed the crossover points in four sets of the ph1b-induced recombinants between wheat homologues as well as between three wheat and rye (Secale cereale) homoeologous chromosome arms, and compared them to crossovers between homologues in a reference wheat population. The results show the Ph1 locus also controls crossing over of homologues, and the general patterns of homologous (with Ph1) and homoeologous (with ph1b) crossing over are the same. In all intervals analyzed, homoeologous crossovers fell within the range of frequency distribution of homologous crossovers among individual families of the reference population. No specific DNA sequence characteristics were identified that could be recognized by the Ph1 locus; the only difference between homologous and homoeologous crossing over appears to be in frequency. It is concluded that the Ph1 locus likely recognizes DNA sequence similarity; crossing over is permitted between very similar sequences. In the absence of Ph1 dissimilarities are ignored, in proportion to the level of the sequence divergence.