Dosage Effect of the Ph1 Locus on Homologous Crossovers in a Segment of Chromosome 1B of Bread Wheat (Triticum aestivum L.).

INTRODUCTION: The Ph1 locus in polyploid wheat enforces strictly bivalent behaviour in meiotic metaphase I, by preventing homoeologues from crossing over. It has always been considered as completely dominant as no homoeologous metaphase I pairing has ever been detected with its single dose present. However, Ph1 also affects pairing and crossing over of homologous chromosomes. METHODS: Homologous crossover frequencies with Ph1 in two, one, and null doses were scored cytologically, as exchanges within a ca. 9.5-9.9 Mbp terminal wheat segment of a wheat-rye translocation T-9 and corresponding segments in chromosome arms 1BS originating from four wheat cultivars. RESULTS: In all cases, the crossover rates in the tested homologous segment of wheat genome, with a single dose of Ph1 present, were intermediate between those at two and null Ph1 doses. Averaging across all four chromosomes, the crossover rate with a single dose of Ph1 present was 37% higher from that with two doses and 46.4% lower of that with a zero dosage. CONCLUSION: The Ph1 locus in wheat affects homologues and appears to operate in a dosage-dependent manner.

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.

Direct evidence for crossover and chromatid interference in meiosis of two plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei).

Crossing over, in addition to its strictly genetic role, also performs a critical mechanical function, by bonding homologues in meiosis. Hence, it is responsible for an orderly reduction of the chromosome number. As such, it is strictly controlled in frequency and distribution. The well-known crossover control is positive crossover interference which reduces the probability of a crossover in the vicinity of an already formed crossover. A poorly studied aspect of the control is chromatid interference. Such analyses are possible in very few organisms as they require observation of all four products of a single meiosis. Here, we provide direct evidence of chromatid interference. Using in situ probing in two interspecific plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei) during anaphase I, we demonstrate that the involvement of four chromatids in double crossovers is significantly more frequent than expected (64% versus 25%). We also provide a physical measure of the crossover interference distance, covering ~30-40% of the relative chromosome arm length, and show that the centromere acts as a barrier for crossover interference. The two arms of a chromosome appear to act as independent units in the process of crossing over. Chromatid interference has to be seriously addressed in genetic mapping approaches and further studies.

Out-of-position telomeres in meiotic leptotene appear responsible for chiasmate pairing in an inversion heterozygote in wheat (Triticum aestivum L.).

Chromosome pairing in meiosis usually starts in the vicinity of the telomere attachment to the nuclear membrane and congregation of telomeres in the leptotene bouquet is believed responsible for bringing homologue pairs together. In a heterozygote for an inversion of a rye (Secale cereale L.) chromosome arm in wheat, a distal segment of the normal homologue is capable of chiasmate pairing with its counterpart in the inverted arm, located near the centromere. Using 3D imaging confocal microscopy, we observed that some telomeres failed to be incorporated into the bouquet and occupied various positions throughout the entire volume of the nucleus, including the centromere pole. Rye telomeres appeared ca. 21 times more likely to fail to be included in the telomere bouquet than wheat telomeres. The frequency of the out-of-bouquet rye telomere position in leptotene was virtually identical to the frequency of telomeres deviating from Rabl's orientation in the nuclei of somatic cells, and was similar to the frequency of synapsis of the normal and inverted chromosome arms, but lower than the MI pairing frequency of segments of these two arms normally positioned across the volume of the nucleus. Out-of-position placement of the rye telomeres may be responsible for reduced MI pairing of rye chromosomes in hybrids with wheat and their disproportionate contribution to aneuploidy, but appears responsible for initiating chiasmate pairing of distantly positioned segments of homology in an inversion heterozygote.

Misdivision of Telocentrics and Isochromosomes in Wheat.

For normal transition through meiosis, chromosomes rely on pairing with their homologues. Chromosomes which fail to pair, univalents, behave irregularly and may undergo various types of breakage across their centromeres. Here, we analyzed the meiotic behavior of misdivision products themselves: isochromosomes and telocentrics in wheat. Both types of chromosomes behaved in the same fashion as standard 2-armed chromosomes. The 2 most frequent scenarios were separation of sister chromatids in anaphase I or monopolar/bipolar attachment of the univalent to the spindle apparatus with unseparated chromatids. Misdivision was rare, and its frequency appeared directly related to the size of the centromere. The previously deduced relationship between misdivision frequency and chromosome size was likely erroneous and can be explained by a general relationship between chromosome length and the size of its centromere. Pairing of identical arms in isochromosomes did not protect them from misdivision. It is not chiasmate pairing that protects from misdivision but mechanistic issues that arise through that pairing.

Instability of Alien Chromosome Introgressions in Wheat Associated with Improper Positioning in the Nucleus.

Alien introgressions introduce beneficial alleles into existing crops and hence, are widely used in plant breeding. Generally, introgressed alien chromosomes show reduced meiotic pairing relative to the host genome, and may be eliminated over generations. Reduced pairing appears to result from a failure of some telomeres of alien chromosomes to incorporate into the leptotene bouquet at the onset of meiosis, thereby preventing chiasmate pairing. In this study, we analysed somatic nuclei of rye introgressions in wheat using 3D-FISH and found that while introgressed rye chromosomes or chromosome arms occupied discrete positions in the Rabl's orientation similar to chromosomes of the wheat host, their telomeres frequently occupied positions away from the nuclear periphery. The frequencies of such abnormal telomere positioning were similar to the frequencies of out-of-bouquet telomere positioning at leptotene, and of pairing failure at metaphase I. This study indicates that improper positioning of alien chromosomes that leads to reduced pairing is not a strictly meiotic event but rather a consequence of a more systemic problem. Improper positioning in the nuclei probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at the onset of meiosis, preventing synapsis and chiasma establishment, and leading to their gradual elimination over generations.

Correction to: Development and characterization of a spring hexaploid wheat line with no functional VRN2 genes.

In the original version of this article, PCR fragments and digestion product sizes for the VRN-B2 and VRN-D2 markers were not accurate. The corrected sizes are detailed below.

Chromosomes 1BS and 1RS for control of male fertility in wheats and triticales with cytoplasms of Aegilops kotschyi, Ae. mutica and Ae. uniaristata.

KEY MESSAGE: Engineered chromosomes 1BS and 1RS offer a new alternative in the development of hybrid systems in bread wheat and triticale. In the cytoplasmic male sterility system for hybrid wheat based on the cytoplasm of Triticum timopheevi fertility restoration is difficult, with few good restorer genes available. In the system based on the cytoplasms of Aegilops kotschyi, Ae. uniaristata and Ae. mutica, essentially all chromosomes 1B carry locus Rf multi that restores male fertility; male sterility manifests itself in wheats with the 1RS.1BL translocation where 1BS chromosome arm is missing. To generate male sterile wheats without the 1RS.1BL translocation, the 1BS arm was cytogenetically engineered to replace the segment with Rf multi with two short inserts of rye chromatin. Conversely, to enhance fertility restoration by doubling the number of restorers present for eventual use in wheat and triticale, a region of 1BS with Rf multi was inserted into 1RS. Alloplasmic wheats with Rf multi removed were completely male sterile; alloplasmic wheats with engineered 1RS carrying Rf multi and without normal 1B were male fertile. An exception to the ubiquitous presence of Rf multi is T. spelta var. duhamelianum; four accessions tested in this study gave inconsistent results but some did not restore male fertility. Engineered chromosomes 1BS and 1RS and chromosomes 1B of T. spelta offer a new alternative for practical application of a cytoplasmic male sterility system in the development of hybrid wheat and hexaploid triticale.

Engineering the 1BS chromosome arm in wheat to remove the Rf (multi) locus restoring male fertility in cytoplasms of Aegilops kotschyi, Ae. uniaristata and Ae. mutica.

KEY MESSAGE: By removing the Rf (multi) locus from chromosome 1BS of wheat via chromosome engineering we were able to generate a resource for the production of male sterile wheats in three new cytoplasms. Cytoplasmic male sterility is an essential component in the development of many hybrid crops. In wheat (Triticum aestivum L.) only the cytoplasm of T. timopheevi cytoplasm has been extensively tested even though many other cytoplasms are also known to produce male sterility. Among them are the cytoplasms of Ae. kotschyi, Ae. uniaristata and Ae. mutica but here male sterility manifests itself only when the 1RS.1BL rye-wheat translocation is present in the nuclear genome. The location of the male fertility restoring gene on the chromosome arm 1BS (Rf (multi) ) has recently been determined using a set of primary recombinants of chromosome arms 1RS with 1BS. Using this knowledge the same recombinants were used to create chromosome arm 1BS in wheat with a small insert from rye that removes the restorer locus. The disomic engineered chromosome 1B1:6 assures male sterility in all three cytoplasms and any standard chromosome 1B in wheat is capable of restoring it. This newly engineered chromosome in combination with the three cytoplasms of Aegilops sp extends the range of possibilities in attempts to create a viable system for hybrid wheat production.

Development and characterization of a spring hexaploid wheat line with no functional VRN2 genes.

KEY MESSAGE: The combination of three non-functional alleles of the flowering repressor VRN2 results in a spring growth habit in wheat. In temperate cereals with a winter growth habit, a prolonged exposure to low temperatures (vernalization) accelerates flowering. Before vernalization, the VRN2 locus plays a central role in maintaining flowering repression. Non-functional VRN2 alleles result in spring growth habit and are frequent in diploid wheat and barley. However, in hexaploid wheat, the effect of these non-functional VRN2 alleles is masked by gene redundancy. In this study, we developed a triple VRN2 mutant (synthetic vrn2-null) in hexaploid wheat by combining the non-functional VRN-A2 allele present in most polyploid wheats with a VRN-B2 deletion from tetraploid wheat, and a non-functional VRN-D2 allele from Aegilops tauschii (Ae. tauschii) (the donor of hexaploid wheat D genome). Non-vernalized vrn2-null plants flowered 118 days (P < 2.8E-07) earlier than the winter control, and showed a limited vernalization response. The functional VRN-B2 allele is expressed at higher levels than the functional VRN-D2 allele and showed a stronger repressive effect under partial vernalization (4 °C for 4 weeks), and also in non-vernalized plants carrying only a functional VRN-B2 or VRN-D2 in heterozygous state. These results suggest that different combinations of VRN-B2 and VRN-D2 alleles can be a used to modulate the vernalization response in regions with mild winters. Spring vrn2-null mutants have been selected repeatedly in diploid wheat and barley, suggesting that they may have an adaptative value and that may be useful in hexaploid wheat. Spring wheat breeders can use these new alleles to improve wheat adaptation to different or changing environments.

The origin of unusual chromosome constitutions among newly formed allopolyploids.

PREMISE OF THE STUDY: Wide hybridization followed by spontaneous chromosome doubling of the resulting hybrids plays an important role in plant speciation. Such chromosome doubling is usually accomplished via unreduced gametes produced by altered meiosis, the so-called 'meiotic restitution'. Unreduced gametes are expected to carry somatic chromosome numbers and constitutions. However, it has been shown recently that new allopolyploids often carry unusual chromosome constitutions which include compensating and noncompensating nulli-tetrasomies and monotrisomies, and translocations of homoeologues. METHODS: We have reanalyzed meiotic divisions in a wheat-rye hybrid by in situ probing with labeled DNA focusing on deviations from the standard pattern of meiotic restitution. KEY RESULTS: In a typical first division restitution in a wide hybrid, there is no chromosome pairing, univalents separate sister chromatids in anaphase I, and there is no meiosis II. Here we illustrate that occasional pairing of homoeologous chromosomes in metaphase I, combined with separation of sister chromatids of univalents, generates diads with compensating nulli-disomies and associated translocations of homoeologues. Similarly, precocious metaphase I migration to the poles of some undivided univalents generates a wide range of noncompensating simple and complex nulli-disomies in the gametes. CONCLUSIONS: Both alterations to the standard pattern of meiotic restitution tend to maintain the somatic chromosome numbers in the gametes; chromosome constitutions are variable but mostly genetically balanced. This source of variation among progeny may be an important factor contributing to greater success of natural allopolyploids.

New Observations of the Effects of the Cytoplasm of Aegilops kotschyi Boiss. in Bread Wheat Triticum aestivum L.

The cytoplasm of Aegilops kotschyi is known for the induction of male sterility and haploidy in wheat. Both systems originally appeared rather simple, but manipulation of the standard chromosome constitution of the nuclear genome revealed additional interactions. This study shows that while there is little or no allelic variation at the main fertility restorer locus Rfmulti on chromosome arm 1BS, additional genes may also be involved in the nuclear-mitochondrial genome interactions, affecting not only male fertility but also the growth rate, from pollen competition for fertilization and early endosperm divisions all the way to seed size and plant maturity. Some of these effects appear to be of a sporophytic nature; others are gametophytic. Induction of parthenogenesis by a rye inducer in conjunction with the Ae. kotschyi cytoplasm is well known. However, here we show that the cytoplasmic-nuclear interactions affect all aspects of double fertilization: producing maternal haploids from unfertilized eggs, diploids from fertilized eggs or synergids, embryo-less kernels, and fertilized eggs without fertilization of the double nucleus in the embryo sack. It is unclear how frequent the inducers of parthenogenesis are, as variation, if any, is obscured by suppressors present in the wheat genome. Genetic dissection of a single wheat accession revealed five distinct loci affecting the rate of maternal haploid production: four acting as suppressors and one as an enhancer. Only when the suppressing haplotypes are confirmed may it be possible to the identify genetic variation of haploidy inducers, map their position(s), and determine their nature and the mode of action.

Inversions of chromosome arms 4AL and 2BS in wheat invert the patterns of chiasma distribution.

In many species, including wheat, crossing over is distal, and the proximal regions of chromosome arms contribute little to genetic maps. This was thought to be a consequence of terminal initiation of synapsis favoring distal crossing over. However, in an inverted rye chromosome arm, the pattern of metaphase I chiasmata was also inverted, suggesting that crossover frequencies were specific to chromosome segments. Here, wheat chromosome arms 2BS and 4AL, with essentially entire arms inverted in reverse tandem duplications (rtd), were studied in the MI of meiosis. Inversion-duplication placed the recombining segments in the middle of the arms. While the overall pairing frequencies of the inverted-duplicated arms were considerably reduced relative to normal arms, chiasmata, if present, were always located in the same regions as in structurally normal arms, and relative chiasma frequencies remained the same. The frequencies of fragment or fragment + bridge configurations in AI and AII indicated that of the two tandemly arranged copies of segments in rtds, the more distal inverted segments were more likely to cross over than the segments in their original orientations. These observations show that also in wheat, relative crossover frequencies along chromosome arms are predetermined and independent of the segment location. The segments normally not licensed to cross over do not do so even when placed in seemingly most favorable positions for it.

C-Banding of Plant Chromosomes.

C-banding visualizes regions of chromosomes containing constitutive heterochromatin. It creates distinct patterns along the chromosome length and allows precise chromosome identification if C-bands are present in sufficient numbers. It is performed on chromosome spreads generated from fixed material, usually root tips or anthers. While there are numerous lab-specific modifications, all methods share the same steps: acidic hydrolysis, DNA denaturation in strong bases (usually saturated aqueous solution of barium hydroxide), washes in saline solution, and staining in Giemsa-type stain in a phosphate buffer. The method can be used for a wide range of cytogenetic tasks, from karyotyping, meiotic chromosome pairing analyses, to large-scale screening and selection of specific chromosome constructs.

On the Possible Trade-Off between Shoot and Root Biomass in Wheat.

Numerous studies have shown that under a limited water supply, a larger root biomass is associated with an increased above-ground biomass. Root biomass, while genetically controlled, is also greatly affected by the environment with varying plasticity levels. In this context, understanding the relationship between the biomass of shoots and roots appears prudent. In this study, we analyze this relationship in a large dataset collected from multiple experiments conducted up to different growth stages in bread wheat (Triticum aestivum L.) and its wild relatives. Four bread wheat mapping populations as well as wild and domesticated members of the Triticeae tribe were evaluated for the root and shoot biomass allocation patterns. In the analyzed dataset the root and shoot biomasses were directly related to each other, and to the heading date, and the correlation values increased in proportion to the length of an experiment. On average, 84.1% of the observed variation was explained by a positive correlation between shoot and root biomass. Scatter plots generated from 6353 data points from numerous experiments with different wheats suggest that at some point, further increases in root biomass negatively impact the shoot biomass. Based on these results, a preliminary study with different water availability scenarios and growth conditions was designed with two cultivars, Pavon 76 and Yecora Rojo. The duration of drought and water level significantly affected the root/shoot biomass allocation patterns. However, the responses of the two cultivars were quite different, suggesting that the point of diminishing returns in increasing root biomass may be different for different wheats, reinforcing the need to breed wheats for specific environmental challenges.

Behavior of Centromeres during Restitution of the First Meiotic Division in a Wheat-Rye Hybrid.

In first division restitution (FDR)-type meiosis, univalents congregate on the metaphase I plate and separate sister chromatids in an orderly fashion, producing dyads with somatic chromosome numbers. The second meiotic division is abandoned. The separation of sister chromatids requires separation of otherwise fused sister centromeres and a bipolar attachment to the karyokinetic spindle. This study analyzed packaging of sister centromeres in pollen mother cells (PMCs) in a wheat-rye F1 hybrid with a mixture of standard reductional meiosis and FDR. No indication of sister centromere separation before MI was observed; such separation was clearly only visible in univalents placed on the metaphase plate itself, and only in PMCs undergoing FDR. Even in the FDR, PMCs univalents off the plate retained fused centromeres. Both the orientation and configuration of univalents suggest that some mechanism other than standard interactions with the karyokinetic spindle may be responsible for placing univalents on the plate, at which point sister centromeres are separated and normal amphitelic interaction with the spindle is established. At this point it is not clear at all what univalent delivery mechanism may be at play in the FDR.

Dissection of QTL effects for root traits using a chromosome arm-specific mapping population in bread wheat.

A high-resolution chromosome arm-specific mapping population was used in an attempt to locate/detect gene(s)/QTL for different root traits on the short arm of rye chromosome 1 (1RS) in bread wheat. This population consisted of induced homoeologous recombinants of 1RS with 1BS, each originating from a different crossover event and distinct from all other recombinants in the proportions of rye and wheat chromatin present. It provides a simple and powerful approach to detect even small QTL effects using fewer progeny. A promising empirical Bayes method was applied to estimate additive and epistatic effects for all possible marker pairs simultaneously in a single model. This method has an advantage for QTL analysis in minimizing the error variance and detecting interaction effects between loci with no main effect. A total of 15 QTL effects, 6 additive and 9 epistatic, were detected for different traits of root length and root weight in 1RS wheat. Epistatic interactions were further partitioned into inter-genomic (wheat and rye alleles) and intra-genomic (rye-rye or wheat-wheat alleles) interactions affecting various root traits. Four common regions were identified involving all the QTL for root traits. Two regions carried QTL for almost all the root traits and were responsible for all the epistatic interactions. Evidence for inter-genomic interactions is provided. Comparison of mean values supported the QTL detection.

Aneuploidy among androgenic progeny of hexaploid triticale (XTriticosecale Wittmack).

Doubled haploids are an established tool in plant breeding and research. Of several methods for their production, androgenesis is technically simple and can efficiently produce substantial numbers of lines. It is well suited to such crops as hexaploid triticale. Owing to meiotic irregularities of triticale hybrids, aneuploidy may affect the efficiency of androgenesis more severely than in meiotically stable crops. This study addresses the issue of aneuploidy among androgenic regenerants of triticale. Plant morphology, seed set and seed quality were better predictors of aneuploidy, as determined cytologically, than flow cytometry. Most aneuploids were hypoploids and these included nullisomics, telosomics, and translocation lines; among 42 chromosome plants were nulli-tetrasomics. Rye chromosomes involved in aneuploidy greatly outnumbered wheat chromosomes; in C(0) rye chromosomes 2R and 5R were most frequently involved. While the frequency of nullisomy 2R was fairly constant in most cross combinations, nullisomy 5R was more frequent in the most recalcitrant combination, and its frequency increased with time spent in culture with up to 70% of green plants recovered late being nullisomic 5R. Given that 5R was not involved in meiotic aberrations with an above-average frequency, it is possible that its absence promotes androgenesis or green plant regeneration. Overall, aneuploidy among tested combinations reduced the average efficiency of double haploid production by 35% and by 69% in one recalcitrant combination, seriously reducing the yield of useful lines.

Dosage effect of the short arm of chromosome 1 of rye on root morphology and anatomy in bread wheat.

The spontaneous translocation of the short arm of chromosome 1 of rye (1RS) in bread wheat is associated with higher root biomass and grain yield. Recent studies have confirmed the presence of QTL for different root morphological traits on the 1RS arm in bread wheat. This study was conducted to address two questions in wheat root genetics. First, does the presence of the 1RS arm in bread wheat affect its root anatomy? Second, how does root morphology and anatomy of bread wheat respond to different dosages of 1RS? Near-isogenic plants with a different number (0 to 4 dosages) of 1RS translocations were studied for root morphology and anatomy. The F(1) hybrid, with single doses of the 1RS and 1AS arms, showed heterosis for root and shoot biomass. In other genotypes, with 0, 2, or 4 doses of 1RS, root biomass was incremental with the increase in the dosage of 1RS in bread wheat. This study also provided evidence of the presence of gene(s) influencing root xylem vessel number, size, and distribution in bread wheat. It was found that root vasculature follows a specific developmental pattern along the length of the tap root and 1RS dosage tends to affect the transitions differentially in different positions. This study indicated that the inherent differences in root morphology and anatomy of different 1RS lines may be advantageous compared to normal bread wheat to survive under stress conditions.

Development and mapping of DArT markers within the Festuca - Lolium complex.

BACKGROUND: Grasses are among the most important and widely cultivated plants on Earth. They provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Among cultivated grasses, species within the Festuca-Lolium complex predominate, especially in temperate regions. To facilitate high-throughput genome profiling and genetic mapping within the complex, we have developed a Diversity Arrays Technology (DArT) array for five grass species: F. pratensis, F. arundinacea, F. glaucescens, L. perenne and L. multiflorum. RESULTS: The DArTFest array contains 7680 probes derived from methyl-filtered genomic representations. In a first marker discovery experiment performed on 40 genotypes from each species (with the exception of F. glaucescens for which only 7 genotypes were used), we identified 3884 polymorphic markers. The number of DArT markers identified in every single genotype varied from 821 to 1852. To test the usefulness of DArTFest array for physical mapping, DArT markers were assigned to each of the seven chromosomes of F. pratensis using single chromosome substitution lines while recombinants of F. pratensis chromosome 3 were used to allocate the markers to seven chromosome bins. CONCLUSION: The resources developed in this project will facilitate the development of genetic maps in Festuca and Lolium, the analysis on genetic diversity, and the monitoring of the genomic constitution of the Festuca x Lolium hybrids. They will also enable marker-assisted selection for multiple traits or for specific genome regions.