S. M. Reader

Molecular characterization of Ph1 as a major chromosome pairing locus in polyploid wheat

The foundation of western civilization owes much to the high fertility of bread wheat, which results from the stability of its polyploid genome. Despite possessing multiple sets of related chromosomes, hexaploid (bread) and tetraploid (pasta) wheat both behave as diploids at meiosis. Correct pairing of homologous chromosomes is controlled by the Ph1 locus. In wheat hybrids, Ph1 prevents pairing between related chromosomes. Lack of Ph1 activity in diploid relatives of wheat suggests that Ph1 arose on polyploidization. Absence of phenotypic variation, apart from dosage effects, and the failure of ethylmethane sulphonate treatment to yield mutants, indicates that Ph1 has a complex structure. Here we have localized Ph1 to a 2.5-megabase interstitial region of wheat chromosome 5B containing a structure consisting of a segment of subtelomeric heterochromatin that inserted into a cluster of cdc2-related genes after polyploidization. The correlation of the presence of this structure with Ph1 activity in related species, and the involvement of heterochromatin with Ph1 (ref. 6) and cdc2 genes with meiosis, makes the structure a good candidate for the Ph1 locus.

Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

SummaryGenomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.

A cereal centromeric sequence

We report the identification of a family of sequences located by in situ hybridisation to the centromeres of all theTriticeae chromosomes studied, including the supernumerary and midget chromosomes, the centromeres ofall maize chromosomes and the heterochromatic regions of rice chromosomes. This family of sequences, (CCS1), together with the cereal genome alignments, will allow the evolution of the cereal centromeres and their sites to be studied. The family of sequences also shows homology to the CENP-B box. The centromeres of the cereal species and the proteins that interact with them can now be characterised.

RFLP-based maps of the homoeologous group-6 chromosomes of wheat and their application in the tagging of Pm12, a powdery mildew resistance gene transferred from Aegilops speltoides to wheat

Genetic maps of the homoeologous group-6 chromosomes of bread wheat, Triticum aestivum, have been constructed spanning 103 cM on 6A, 90 cM on 6B and 124 cM on 6D. These maps were transferred to a Chinese Spring (CS) x line #31 cross to locate a dominant powdery mildew resistance gene, Pm12, introgressed into line #31 from Aegilops speltoides. Pm12 was shown to lie on the short arm of translocation chromosome 6BS-6SS.6SL in line #31, but could not be mapped more precisely due to the lack of recombination between the 6S Ae. speltoides segment and chromosome 6B. Possible strategies to reduce the size of the alien segment, which probably encompasses the complete long arm and more than 82% of the short arm of chromosome 6B, are discussed.

Association of homologous chromosomes during floral development

Reduction in chromosome number and genetic recombination during meiosis require the prior association of homologous chromosomes, and this has been assumed to be a central event in meiosis. Various studies have suggested, however, that while the reduction division of meiosis is a universally conserved process, the pre-meiotic association of homologues differs among organisms. In the fruit fly Drosophila melanogaster, some somatic tissues also show association of homologues [1,2]. In the budding yeast Saccharomyces cerevisiae, there is some evidence for homologue association during the interphase before meiotic division [3,4], and it has been argued that such associations lead directly to meiotic homologue pairing during prophase I [5]. The available evidence for mammals suggests that homologous chromosomes do not associate in germ cells prior to meiotic prophase [6]. To study the occurrence of homologue pairing in wheat, we have used vibratome tissue sections of wheat florets to determine the location of homologous chromosomes, centromeres and telomeres in different cell types of developing anthers. Fluorescence in situ hybridization followed by confocal microscopy demonstrated that homologous chromosomes associate pre-meiotically in meiocytes (germ-line cells). Surprisingly, association of homologues was observed simultaneously in all the surrounding somatic tapetum cells. Homologues failed to associate at equivalent stages in a homologue recognition mutant. These results demonstrate that the factors responsible for the recognition and association of homologues in wheat act before the onset of meiotic prophase. The observation of homologue association in somatic tapetum cells demonstrates that this process and meiotic division are separable.

AFLP-Based mRNA Fingerprinting

AFLP, a robust and rapid technique for displaying large numbers of DNA polymorphisms, is being used extensively for genetic mapping and fingerprinting in plants (1). Use of this technique avoids problems which may be encountered with reproducibility and optimisation of reaction conditions when using arbitrarily primed PCR. Since the primer sets are readily available (Life Technologies and Perkin Elmer), we have explored whether AFLP could be applied to generating mRNA fingerprints in polyploid crop plants. In polyploid species, mutant stocks can be created by the deletion of short chromosome segments on one of the constituent homoeologous genomes. Messenger RNA fingerprints would be useful for identifying genes located within these deleted segments. However, the expression of monomorphic mRNAs from homoeologous genes in polyploids would make fingerprints indistinguishable when comparing normal and deletion stocks. If the homoeologous gene sequences were polymorphic, e.g. for a restriction enzyme site, then their AFLP fingerprints would be easily distinguishable and the expressed sequences could be genetically mapped. To test the sensitivity of this approach we used hexaploid wheat (2n = 6x arranged in seven groups of three homoeologous chromosome pairs) and one of its deletion mutants. Messenger RNA was extracted from immature spikes taken from wheat cv Chinese Spring and a mutant of this variety with deletions on chromosomes 3A and 5B (2,3). We used an mRNA Quickprep kit (Pharmacia) following the manufacturer’s instructions, but including treatment with DNase I before final precipitation. The mRNA was dissolved in 100 μl dH2O and purified using an RNeasy kit (Qiagen). Synthesis of double-stranded cDNA was performed according to instructions supplied with Superscript reverse transcriptase (Life Technologies). An equimolar mixture of three oligonucleotides with the sequence 5′-AGTCTGCAGT12V-3′ (where V denotes A, C or G) was used to prime first strand cDNA synthesis. As this primer contains a recognition sequence for PstI, the doublestranded cDNA can be cut with this restriction enzyme. This dual-purpose primer was designed to enable us to make use of available AFLP adapter and primer stocks without having to modify the PCR conditions used in the technique. The variable 3′ nucleotide adjacent to the T12 tract ensures that synthesis begins at the junction between the poly A tail and the sequence at the 3′ end of the mRNA (4,5). First strand cDNA synthesis was performed at 42 C using 0.5 μg of the oligonucleotide mix, 1 μg mRNA and 200 U reverse transcriptase, omitting [α-32P]dCTP but including RNAguard RNase inhibitor (Pharmacia). The cDNA was purified using a QiaQuick column (Qiagen). After second strand synthesis, cDNA was digested with 5.0 U each of PstI (NBL) and MseI (New England Biolabs). Digested cDNA was ligated with an MseI-adapter and a biotinylated PstI-adapter [patent application, Zabeau and Vos (1993), EP 0534858] and affinity-purified using streptavidin-linked paramagnetic beads (Dynal). Preamplification was carried out using non-selective primers to conserve purified cDNA stocks. All subsequent steps were performed as previously reported for genomic DNA AFLP (1). Labelled selective amplification products were run on standard 6% acrylamide sequencing gels and visualised by exposure to Kodak BioMax-MR film for ∼50 h. RNA fingerprints were generated from Chinese Spring and the deletion mutant cDNA templates using 49 MseI-primers with two or three selective bases. Amplification products ranged in size from 600 bp. Examples of the fingerprints obtained are shown in Figure 1a. Sixteen amplification products, which differed between Chinese Spring and the deletion mutant, were excised and reamplified. Purified reamplification products were labelled and hybridised with various digests of genomic DNA from Chinese Spring and the deletion mutant. Five of the products gave similar hybridisation patterns and these five probes were subsequently found to cross-hybridise with the 18S–5.8S–26S ribosomal RNA genes of wheat. The remaining 11 probes hybridised to single or low copy sequences. Two of these (M12B and M48A) detected DNA fragments that are present in Chinese Spring but absent in the deletion mutant (Fig. 1b). Further analysis revealed that the fragments are located on chromosome 3A (data not shown). The reproducibility of the technique was demonstrated by synthesising fresh cDNA from Chinese Spring and deletion mutant mRNA and processing this (including non-selective preamplification) as described. In the selective amplification, 10 different MseI primers were used for direct comparison of the banding patterns obtained using the duplicate cDNA templates. There was little or no variation between the two batches of cDNA and differences between Chinese Spring and the mutant were consistent (Fig. 1a). We have shown that the AFLP technique can be modified to allow display of mRNAs and used to isolate sequences mapping to deleted chromosome segments in hexaploid wheat. Since our protocol used existing MseI-adapters and primers, cDNA sequences forming an MseI recognition site (5′-TTAA-3′) at the junction of

Centromeric behaviour in wheat with high and low homoeologous chromosomal pairing

Abstract.Control of homoeologous chromosomal pairing in hexaploid wheat stems from a balance between a number of suppressor and promoter genes. This study used centromeric behaviour as a tool to investigate the mechanism. Fluorescent in situ hybridization employing centromeric and telomeric sequences as probes was applied to pollen mother cells of wheat and wheat/alien hybrids having different pairing gene combinations. It showed: association of centromeres during pre-meiotic interphase; decondensation of centromeric structure; sister chromatid disjunction of univalent chromosomes in homoeologous pairing situations at anaphase I; and centromeric stretching between univalent sister chromatids in wheat/rye hybrids deficient for pairing genes. The implications of these results are discussed.

Detection of homoeologous chiasma formation in Triticum durum × Thinopyrum bessarabicum hybrids using genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to study the nature of homoeologous chiasma formation in crosses between Triticum durum cv. Creso, homozygous for the ph1c mutation and Thinopyrum bessarabicum. The relative frequencies of wheat/wheat and wheat/Th. bessarabicum chiasma formation were determined. Pairing between apparently non-homologous Th. bessarabicum chromosomes was also observed. The potential of GISH as a tool for analysing homoeologous chiasma formation in wheat/alien hybrids is discussed.

Determination of the frequency of wheat-rye chromosome pairing in wheat x rye hybrids with and without chromosome 5B

Genomic in-situ hybridization (GISH) was used to determine the amount of wheat-rye chromosome pairing in wheat (Triticum aestivum) x rye (Secale cereale) hybrids having chromosome 5B present, absent, or replaced by an extra dose of chromosome 5D. The levels of overall chromosome pairing were similar to those reported earlier but the levels of wheat-rye pairing were higher than earlier determinations using C-banding. Significant differences in chromosome pairing were found between the three genotypes studied. Both of the chromosome-5B-deficient hybrid genotypes showed much higher pairing than the euploid wheat hybrid. However, the 5B-deficient hybrid carrying an extra chromosome 5D had significantly less wheat-rye pairing than the simple 5B-deficient genotype, indicating the presence of a suppressing factor on chromosome 5D. Non-homologous/non-homoeologous chromosome pairing was observed in all three hybrid genotypes. The value of GISH for assessing the level of wheat-alien chromosome pairing in wheat/alien hybrids and the effectiveness of wheat genotypes that affect homoeologous chromosome pairing is demonstrated.

A study of the effect of a homoeologous pairing promoter on chromosome pairing in wheat/rye hybrids using genomic in situ hybridization

Genomic in situ hybridization was used to study the relative frequency of wheat/wheat and wheat/rye homoeologous chiasma formation in Triticum aestivum cv. Chinese Spring tetrasomic for chromosome 3B × Secale cereale cv. Petkus Spring hybrids. In addition to wheat/wheat and wheat/rye homoeologous chiasma formation, pairing between apparently non-homologous rye chromosomes was also observed. The implications of the results obtained for the introgression of rye chromatin into wheat is discussed.