Effect of the pairing gene Ph1 and premeiotic colchicine treatment on intra- and interchromosome pairing of isochromosomes in common wheat.

The analysis of the pattern of isochromosome pairing allows one to distinguish factors affecting presynaptic alignment of homologous chromosomes from those affecting synapsis and crossing-over. Because the two homologous arms in an isochromosome are invariably associated by a common centromere, the suppression of pairing between these arms (intrachromosome pairing) would indicate that synaptic or postsynaptic events were impaired. In contrast, the suppression of pairing between an isochromosome and its homologous chromosome (interchromosome pairing), without affecting intrachromosome pairing, would suggest that homologous presynaptic alignment was impaired. We used such an isochromosome system to determine which of the processes associated with chromosome pairing was affected by the Ph1 gene of common wheat-the main gene that restricts pairing to homologues. Ph1 reduced the frequency of interchromosome pairing without affecting intrachromosome pairing. In contrast, intrachromosome pairing was strongly reduced in the absence of the synaptic gene Syn-B1. Premeiotic colchicine treatment, which drastically decreased pairing of conventional chromosomes, reduced interchromosome but not intrachromosome pairing. The results support the hypothesis that premeiotic alignment is a necessary stage for the regularity of meiotic pairing and that Ph1 relaxes this alignment. We suggest that Ph1 acts on premeiotic alignment of homologues and homeologues as a means of ensuring diploid-like meiotic behavior in polyploid wheat.     

Cytological and molecular analysis of centromere misdivision in maize.

B chromosome derivatives suffering from breaks within their centromere were examined cytologically and molecularly. We showed by high resolution FISH that misdivision of the centromere of a univalent chromosome can occur during meiosis. The breaks divide the centromere repeat sequence cluster. A telocentric chromosome formed by misdivision was found to have the addition of telomeric repeats to the broken centromere. A ring chromosome formed after misdivision occurred by fusion of the broken centromere to the telomere. Pulsed-field electrophoresis analyses were performed on the telocentric and ring chromosomes to identify fragments that hybridize to both the telomeric repeat and the B-specific centromeric repeat. We conclude that healing of broken maize centromeres can be achieved through the mechanisms of addition or fusion of telomeric repeat sequences to the broken centromere.     

Transfer of Ph I genes promoting homoeologous pairing from Triticum speltoides to common wheat

Diploid-like chromosome pairing in polyploid wheat is controlled by several Ph (pairing homoeologous) genes with major and minor effects. Homoeologous pairing occurs in either the absence of these genes or their inhibition by genes from other species (Ph ^I genes). We transferred Ph ^I genes from Triticum speltoides (syn Aegilops speltoides) to T. aestivum, and on the basis of further analysis it appears that two duplicate and independent Ph ^I genes were transferred. Since Ph ^I genes are epistatic to the Ph genes of wheat, homoeologous pairing between the wheat and alien chromosomes occurs in the F₁ hybrids. Using the Ph ^I gene stock, we could demonstrate homoeologous pairing between the wheat and Haynaldia villosa chromosomes. Since homoeologous pairing occurs in F₁ hybrids and no cytogenetic manipulation is needed, the Ph ^I gene stock may be a versatile tool for effecting rapid and efficient alien genetic transfers to wheat.Contribution no. 93-435-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506-5502, USA     

The effect of the wheat Ph1 locus on chromatin organisation and meiotic chromosome pairing analysed by genome painting

The Ph1 locus in wheat influences homo(eo)logous chromosome pairing. We have analysed its effect on the behaviour and morphology of two 5RL rye telosomes in a wheat background, by genomic in situ hybridisation (GISH), using rye genomic DNA as a probe. Our main objective was to study the effect of different alleles of the Ph1 locus on the morphology and behaviour of the rye telosomes in interphase nuclei of tapetal cells and in pollen mother cells at early stages of meiosis. The telosomes, easily detectable at all stages, showed a brightly fluorescing chromomere in the distal region and a constriction in the proximal part. These diagnostic markers enabled us to define the centromere and telomere regions of the rye telosomes. In the presence of functional copies of Ph1, the rye telosomes associated at pre-leptotene, disjoined and reorganised their shape at leptotene, and became fully homologously paired at zygotene – pachytene. In plants without functional alleles (ph1bph1b), the rye telosomes displayed an aberrant morphology, their premeiotic associations were clearly disturbed and their pairing during zygotene and pachytene was reduced and irregular. The Ph1 locus also influenced the behaviour of rye telosomes in the interphase nuclei of tapetal cells: in Ph1Ph1 plants, the rye telosomes occupied distinct, parallel-oriented domains, whereas in tapetal nuclei of ph1bph1b plants they were intermingled with wheat chromosomes and showed a heavily distorted morphology. The results shed new light on the effect of Ph1, and suggest that this locus is involved in chromosome condensation and/or scaffold organisation. Our explanation might account for various apparently contradictory and pleiotropic effects of this locus on both premeiotic associations of homologues, the regulation of meiotic homo(eo)logous chromosome pairing and synapsis, the resolution of bivalent interlockings and centromere behaviour. Received: 27 April 1998; in revised form: 5 August 1998 / Accepted: 11 August 1998     

Effect of 5-azacytidine and trichostatin A on somatic centromere association in wheat.

Both homologous and non-homologous chromosomes in wheat associate via their centromeric hetero chromatin in the developing xylem vessel cells of the root. The antimetabolite 5-azacytidine (which reduces DNA methylation) decreases the overall level of centromere association. Treatment with 5-azacytidine caused a more marked reduction in the level of homologous chromosome association observed in a wheat line carrying a pair of marked chromosomes. On the other hand, treatment of wheat seedlings with trichostatin A (which increases histone acetylation) raises the overall level of centromere association. The Ph1 locus controls the specificity of both somatic and meiotic pairing of homologous centromeres in wheat. The level of non-homologously associated centromeres is, however, reduced in the presence of Ph1 compared with its absence, even after treatment with either drug. Thus these two drugs, which have been shown to affect chromatin structure, do affect chromosome association, but Ph1 must act at least in part by a different mechanism.     

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     

Ph1 gene derived from Aegilops speltoides induces homoeologous chromosome pairing in wide crosses of Triticum aestivum

The present study was conducted to investigate the effectiveness of the PhI gene transferred from Aegilops speltoides into bread wheat cultivar Chinese Spring (CS) in inducing homoeologous chromosome pairing in interspecific crosses using the Chinese Spring line, CS(PhI), carrying the gene. Chinese Spring, as well as CS(PhI), were crossed as female parents with three accessions of Ae. kotschyi (UUSS), one accession of Secale cereale (RR), two amphiploids of Triticum durum-Ae. caudata (AABBCC), and one amphiploid of Triticum durum-Ae. umbellulata (AABBUU). Meiotic metaphase I chromosome pairing was studied in all the interspecific crosses with CS as well as CS(PhI). There was significant increase in chiasma frequency in all the crosses with CS(PhI) over those with CS. The extent of induced homoeologous chromosome pairing by PhI in crosses of CS(PhI) with S. cereale was higher than with those of Ae. kotschyi, as indicated by higher chiasma frequency per pollen mother cell. Significant reduction in frequency of univalents and increase in bivalents (>14), multivalents, and chiasma frequency in crosses of amphiploids with CS(PhI) as compared to those of CS indicated induced homoeologous pairing between C and D, D and U, and C, D, and U genomes with AB genomes in the presence of PhI. The results of the present study unequivocally demonstrate the effectiveness of PhI gene transferred from Ae. speltoides in hexaploid wheat in inducing homoeologous chromosome pairing and suggest that the line CS(PhI) can be effectively used for precise transfer of useful alien genetic variations with least linkage drag.     

Dynamic nature of a wheat centromere with a functional gene

Centromeric regions of higher eukaryotes are comprised mainly of tandem and non-tandem repeat sequences with variable copy number, spacing, order and orientation; are heterochromatic in nature, and are believed to be devoid of actively transcribing genes. Here, we report an actively transcribing wheat homolog of HSP70 gene that maps in the functional wheat centromere, and copy number of which seems to change in response to centromeric breaks. The HSP70 gene physically maps on the short arm of chromosomes 1A and 1D of Chinese Spring (CS) and 1R of rye. Whereas, on chromosome 1B in both ‘CS’ and Pavon background, the gene maps in the functional centromere as evident from its presence in both cytologically confirmed true ditelosomic lines Dt1BS and Dt1BL. Sequence comparison of 11 ESTs showed three sequence patterns suggesting that all three homoeologous copies of the gene are expressing. The cDNA-single stranded conformation polymorphism analysis confirmed expression of the ‘CS’ 1B copy of the gene. Observed in two independently developed Dt1BL lines, the 1B copy number of the gene showed three to fivefold increase in response to chromosomal breaks around the centromere. Putative gene duplications seem to involve large chromosomal segments as only one of the ten restriction enzymes used for DNA gel-blot analysis showed unique extra fragment band in the Dt1BL line. Further investigations are warranted to uncover the nature and mechanism of these duplications.     

A DNA mismatch repair gene links to the Ph2 locus in wheat.

DNA mismatch repair is an essential system for maintaining genetic stability in bacteria and higher eukaryotes. Based on the conserved regions of the bacterial MutS gene and its homologues in yeast and human, a wheat cDNA homologue of MSH6, designated TaMSH7, was isolated by RT-PCR. The deduced amino acid sequence of TaMSH7 shows conserved domains characteristic of other MSH6 genes, with highest similarity to maize MSH7 and Arabidopsis MSH7. TaMSH7 is expressed in meristem tissue associated with a high level of mitotic and meiotic activity, with maximum expression in the reproductive organs of young flower spikes. TaMSH7 is located on the short arms of chromosomes 3A, 3B, and 3D and has been mapped within barley chromosome 3HS. The copy on 3DS is located within the region deleted in the wheat mutant ph2a, which shows altered recombination frequency in the interspecific hybrids. The relationship between the ph2a mutant and TaMSH7 gene function is discussed.     

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.     

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.     

Discovery and mapping of wheat Ph1 suppressors

Pairing between wheat (Triticum turgidum and T. aestivum) homeologous chromosomes is prevented by the expression of the Ph1 locus on the long arm of chromosome 5B. The genome of Aegilops speltoides suppresses Ph1 expression in wheat x Ae. speltoides hybrids. Suppressors with major effects were mapped as Mendelian loci on the long arms of Ae. speltoides chromosomes 3S and 7S. The chromosome 3S locus was designated Su1-Ph1 and the chromosome 7S locus was designated Su2-Ph1. A QTL with a minor effect was mapped on the short arm of chromosome 5S and was designated QPh.ucd-5S. The expression of Su1-Ph1 and Su2-Ph1 increased homeologous chromosome pairing in T. aestivum x Ae. speltoides hybrids by 8.4 and 5.8 chiasmata/cell, respectively. Su1-Ph1 was completely epistatic to Su2-Ph1, and the two genes acting together increased homeologous chromosome pairing in T. aestivum x Ae. speltoides hybrids to the same level as Su1-Ph1 acting alone. QPh.ucd-5S expression increased homeologous chromosome pairing by 1.6 chiasmata/cell in T. aestivum x Ae. speltoides hybrids and was additive to the expression of Su2-Ph1. It is hypothesized that the products of Su1-Ph1 and Su2-Ph1 affect pairing between homeologous chromosomes by regulating the expression of Ph1 but the product of QPh.ucd-5S may primarily regulate recombination between homologous chromosomes.     

Induction and characterization of Ph1 wheat mutants

The cloning of genes for complex traits in polyploid plants that possess large genomes, such as hexaploid wheat, requires an efficient strategy. We present here one such strategy focusing on the homologous pairing suppressor (Ph1) locus of wheat. This locus has been shown to affect both premeiotic and meiotic processes, possibly suggesting a complex control. The strategy combined the identification of lines carrying specific deletions using multiplex PCR screening of fast-neutron irradiated wheat populations with the approach of physically mapping the region in the rice genome equivalent to the deletion to reveal its gene content. As a result, we have located the Ph1 factor controlling the euploid-like level of homologous chromosome pairing to the region between two loci (Xrgc846 and Xpsr150A). These loci are located within 400 kb of each other in the rice genome. By sequencing this region of the rice genome, it should now be possible to define the nature of this factor.     

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.     

Physical location of a HSP70 gene homologue on the centromere of chromosome 1B of wheat ( Triticum aestivum L.)

Cereal centromeres consist of a complex organization of repetitive DNA sequences. Several repetitive DNA sequences are common amongst members of the Triticeae family, and others are unique to particular species. The organization of these repetitive elements and the abundance of other types of DNA sequences in cereal centromeres are largely unknown. In this study, we have used wheat-rye translocation lines to physically map 1BL.1RS centromeric breakpoints and molecular probes to obtain further information on the nature of other types of centromeric DNA sequences. Our results, using the rye-specific centromeric sequence, pAWRC.1, indicate that 1BL.1RS contains a small portion of the centromere from 1R of rye. Further studies used molecular markers to identify centromeric segments on wheat group-1 chromosomes. Selected RFLP markers, clustered around the centromere of wheat homoeologous group-1S chromosomes, were chosen as probes during Southern hybridization. One marker, PSR161, identified a small 1BS segment in all 1BL.1RS lines. This segment maps proximal to pAWRC.1 in 1BL.1RS and on the centromere of 1B. Sequence analysis of PSR161 showed high homology to HSP70 genes and Northern hybridization showed that this gene is constitutively expressed in leaf tissue and induced by heat shock and light stimuli. The significance of this work with respect to centromere organization and the possible significance of this HSP70 gene homologue are discussed. Received: 12 March 2001 / Accepted: 14 June 2001     

Physical distribution of translocation breakpoints in homoeologous recombinants induced by the absence of the Ph1 gene in wheat and triticale

The physical distribution of translocation breakpoints was analyzed in homoeologous recombinants involving chromosomes 1A, 1B, 1D of wheat and 1R of rye, and the long arms of chromosome 7S of Aegilops speltoides and 7A of wheat. Recombination between homoeologues was induced by removal of the Ph1 gene. In all instances, translocation breakpoints were concentrated in the distal ends of the chromosome arms and were absent in the proximal halves of the arms. The relationship between the relative distance from the centromere and the relative homoeologous recombination frequency was best explained by the function f(x)=0.0091e^0.0592x. The pattern of recombination in homoeologous chromosomes was essentially the same as in homologues except that there were practically no double exchanges. Among 313 recombinant chromosomes, only one resulted from a double crossing-over. The distribution of translocation breakpoints in translocated arms indicated that positive chiasma interference operated in homoeologous recombination. This implies that the reduction of the length of alien chromosome segments present in translocations with wheat chromosomes may be more difficult than the production of the original recombinants.     

The Ph1 and Ph2 loci play different roles in the synaptic behaviour of hexaploid wheat Triticum aestivum

Triticum aestivum is an allohexaploid wheat (AABBDD) that shows diploid-like behaviour at metaphase-I. This behaviour is influenced by the action of several loci, Ph1 and Ph2 being the main loci involved. To study the effect of these two loci on chromosome pairing in T. aestivum we have analysed the synaptic pattern in fully traced spread nuclei at mid- and late-zygotene, and at pachytene, of three different genotypes of cv Chinese Spring: standard line, ph1b and ph2b mutants. The analysis of the synaptic progression showed that only a few nuclei accomplish synapsis in the ph2b genotype, whereas most nuclei completed synapsis in the standard and ph1b genotypes. This result indicates that the Ph2 locus affects synaptic progression. The number of synaptonemal complex (SC) bivalents and of the different SC multivalent associations were determined in each nucleus. The mean number of lateral elements involved in SC multivalent associations (LEm) at mid- zygotene was relatively high and showed similar values in the three genotypes. These values decreased progressively between mid-zygotene and pachytene in the genotypes with the Ph1 locus because of the transformation of multivalents into bivalents. In the ph1b genotype, this value only decreased between late-zygotene and pachytene. Therefore, multivalent correction was more efficient in the presence than in the absence of the Ph1 locus.It is concluded that the Ph1 and Ph2 loci bring about diploidization of allohexaploid wheat via a different mechanism. Received: 31 July 2000 / Accepted: 15 November 2000     

Recombination frequency in the locus Gli-D1 of common wheat T. aestivum L

The recombination frequency at the gliadin locus Gli-D1 of common wheat was determined by the maximum likelihood method. Recombination was observed between the gene encoding the fastest omega-component of the allele Gli-D1j, and the genes encoding the other omega-gliadins of this allele. The frequency of recombination was 0.65 +/- 0.18% for the cross between the near-isogenic lines of winter common wheat with respect to gliadin loci Gli-D1-4 and Gli-B1-3 and 0.78 +/- 0.45% for the cross between the varieties Yunnat and B-16.