ITEMS FROM THE UNITED KINGDOM

JOHN INNES CENTRE
Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.

A new hexaploid wheat mutant exhibiting high homoeologous chromosome pairing.

J.W. Snape, T.E. Miller, M. Roberts, S.M. Reader, L.J. Fish, T.N. Foote, and G. Moore.

As part of the strategy to positionally clone the genes controlling homoeologous chromosome pairing in wheat, lines need to be identified that carry deletions of these genes. Such lines then can be used to precisely delimit their chromosomal location. Therefore, approximately 3,000 seed from a Highbury DH line containing a Chinese Spring chromosome 5B substitution were irradiated with fast neutrons, and M2 lines were selected that exhibited reduced fertility. A number of RFLP markers located on the long arm of wheat homoeologous group 5 were turned into PCR-based markers so that individual loci on chromosome 5B could be identified by a simple + or - PCR assay. The lines exhibiting reduced fertility were screened using the PCR-based approach to identify those carrying deletions on chromosome 5BL. One line was identified that was assessed for its level of homoeologous pairing and shown to have a higher level than that observed in the Sears' mutant (ph1b) but equivalent to the homoeologous pairing observed in nullisomic 5B lines. The data suggest that more than one gene (or locus) may controll homoeologous pairing on wheat chromosome 5BL.

Association of homologous chromosomes during floral development in wheat.

L. Aragón-Alcaide, S.M. Reader, A.F. Beven, P.J. Shaw, T.E. Miller, and G. Moore.

To study the occurrence of homologous chromosome pairing in wheat, we used vibratome sections of wheat florets to determine the location of homologous chromosomes, centromeres, and telomeres in different cell types of developing anthers. Fluorescent in situ hybridization followed by confocal microscopy demonstrated that during floral development, when the meiocytes and tapetal cells are not distinguishable, homologous chromosomes are not associated. However, at the stage when meiocytes and tapetal cells are visually distinguishable, homologous chromosomes (homologues) associate at their centromeres. Then, during premeiotic interphase, the homologous chromosomes align and associate. The association of homologues surprisingly was observed simultaneously in all the surrounding somatic tapetum cells. In a line lacking 5B, the homologues fail to come together at their centromeres and, hence, fail to align and associate premeiotically. Therefore, they align and associate during meiotic prophase. In a previous study using the centromere as a reporter, we had shown that the loci Ph1 and Ph2 and loci present in Ae. speltoides affect chromatin organization at the centromere, which is consistent with the confocal observations on chromosome pairing.

Resistance of Dasypyrum villosum to the eyespot pathogenes, Tapesia yallundea and T. acuformis.

E. Uslu, T.E. Miller, N.H. Rezanoor, and P. Nicholson.

Resistance to T. yallundae and T. acuformis was studied in addition lines of D. villosum chromosomes to Chinese Spring wheat and in five D. villosum accessions. In order to investigate the resistance of D. villosum chromosomes to the two eyespot pathogens, inoculated seedlings were scored by visual estimates of disease severity 10 weeks after inoculation. In addition, DNA was isolated from these plants, and the fungal DNAs of both species were quantified by competitive PCR. Visual assessment showed D. villosum to be resistant (to both T. yallundae and T. acuformis) compared to Chinese Spring. The level of pathogen DNA also was generally lower in the D. villosum accessions than in Chinese Spring. The resistance to T. yallundae was determined by factors on chromosomes 1V, 2V, 3V, and 4V. In contrast, resistance to T. acuformis was not found on 4V but on chromosomes 1V, 2V, 3V, and to a lesser extent 5V. The genetic basis of resistance to the two pathogens differs in D. villosum.

Allelic variants at the Rht dwarfing gene locus of wheat.

A.J. Worland and V. Korzun (IPK Gatersleben).

Many new molecular marker systems have been developed and used to establish a very precise genetic map of wheat in recent years. Where molecular markers can be associated with agronomic traits, they can be used to ease selection in segregating breeding populations and to increase the precision of studying the character in genetic experiments.

Studying the agronomically important dwarfing gene Rht8 has been difficult until recently, because the gene is of relatively small effect and has proved difficult to detect and classify in segregating populations. V. Korzun has now detected a microsatellite marker, WMS 261, which is extremely tightly linked to Rht8 (0.6 cM) and, therefore, can be used as a marker for detecting the gene in genetic stocks. Three main allelic variants exist at the WMS 261 locus that can be distinguished as fragments with different numbers of base pairs. Lines with Rht8 have a 192-bp allele. Most western and central European wheats have a 174-bp allele.

Single chromosome substitution lines between 2D chromosomes of the cultivars Ciano 67 and Mara and the 2D homologue of Cappelle-Desprez were developed at the John Innes Centre . The recombinant lines were developed primarily for the study of the photoperiod response gene Ppd1 on chromosome 2D. The material also is ideal for studying pleiotropic effects of allelic variants at the Rht8 locus that also is on chromosome 2D and linked to Ppd1 by approximately 13 cM. By chance, Cappelle-Desprez, Ciano 67, and Mara all carry different allelic variants at the WMS 261 (Rht8) locus. Cappelle-Desprez produces a 174 bp allele, Ciano 67­165 bp, and Mara 192 bp. Following classification of the Cappelle-Desprez (Mara 2D) recombinant lines for the presence of the 174 or 192-bp alleles of WMS 261, agronomic data from previous harvests was reanalyzed to associate pleiotropic effects to the WMS alleles. Results show that the WMS 261­192 bp allele, diagnostic of Rht8, reduces height by approximately 8 cm compared to the WMS 261­174 bp allele. No pleiotropic effects were detected for yield, spikelet fertility, grain size, or flowering time. Similar classification of the Cappelle-Desprez (Ciano 67 2D) recombinant lines for the presence of WMS 261, 165, or 174-bp alleles and reanalysis of existing data show that, compared to the 174-bp allele, the 165-bp allele present in CIMMYT wheats is associated with a height increase of 3 or 4 cm. No other significant pleiotropic effects could be associated with the WMS 261 alleles.

Therefore, these results indicate that the Rht8 gene can be used to modify height without affecting other agronomic characters. Interestingly, although cultivars carrying Rht8 are common in the pedigree of CIMMYT wheats, cultivars selected at CIMMYT have the stronger of the height promoting alleles. Perhaps Rht8 compensates, in part, for the presence of other strong dwarfing genes in these cultivars.

The control of adult plant resistance to stripe rust by the translocated T5BS-7BS chromosome.

C.N. Law and A.J. Worland.

The reciprocal translocations T5BL-7BL and T5BS-7BS were widespread in west European wheats 30 years ago and are probably present in many of their descendants today. In cultivars with a history of durable adult-plant resistance to stripe rust (yellow rust) and carrying this translocation, removal of the T5BS-7BS chromosome makes adult plants more susceptible to stripe rust. Therefore, this chromosome may carry the gene(s) responsible for a major part of their resistance and possibly their durability. A series of lines was developed in which the T5BS-7BS chromosomes from both resistant and susceptible cultivars were substituted into a number of the durably resistant cultivars to test this hypothesis. In every case, the substituted T5BS-7BS chromosome, irrespective of origin, was found to produce a resistant phenotype, indicating that background chromosomes were responsible for the differences between the cultivars. Therefore, the resistance and durability of the resistant cultivars cannot be due solely to the translocated chromosome. In similar experiments, the 5BS and 7BS arms from cultivars not carrying the translocation were substituted into a cultivar with the translocation. In each instance, the lines with the substituted arms were much more susceptible than their recipient, confirming the major effect of the T5BS-7BS chromosome on resistance. The complete correlation between the translocation and resistance and between increased susceptibility and its absence suggests that the gene(s) for adult-plant resistance, located on the T5BS-7BS chromosome, may be linked closely to the break point. Alternatively, it may be a consequence of the close relationship of some of the cultivars. The presence of this gene(s) might be a factor explaining the prevalence of this translocation in some west European wheats.

The influence of the group-1 chromosomes of wheat on ear-emergence times and their involvement with vernalization and day length.

C.N. Law, E. Suarez (INTA, Castelar, Argentina), T.E. Miller, and A.J. Worland.

Use of aneuploid lines of wheat in the cultivar Chinese Spring showed that each of the homoeologous group 1 chromosomes delayed ear emergence. More than one gene per chromosome probably was involved. Because of the delays in ear emergence, at least one of the genes may be responsible for producing an inhibitor of flowering. The genes interacted with each other and with vernalization and day length. The genes on chromosome 1A were the most potent, and those of 1B the least potent. The genes on the group-1 chromosomes may be related to the genes for vernalization and day-length sensitivity found on the homoeologous chromosome 1H of barley. Reciprocal monosomic analyses of three cultivars detected allelic variation between homologues of group 1 for ear-emergence time.

Further genetic mapping of vernalization and frost-tolerance genes on chromosomes 5A and 5D.

J.W. Snape, R.N. Sarma, A. Semikhodskii, and L.J. Fish; G. Galiba and J. Sutka (Agricultural Research Institute of the Hungarian Academy of Sciences, Mártonvasár, Hungary); B.S. Gill (Kansas State University, Manhattan, Kansas, USA); T. Sasaki (National Institute of Agrobiological Resources, Tsukuba, Ibraki, Japan), and D.A. Laurie.

We are using a molecular-marker based approach to identify and understand the mode of action of genes determining key aspects of wheat biology on the homoeologous group-5 chromosomes. In particular, work has continued to further study the genes for frost tolerance and vernalization requirement.

Recent work with chromosome 5A has concentrated on physical mapping of the vernalization gene Vrn-A1. This gene is the predominant one determining the spring/winter habit difference in bread wheat. Using the Chinese Spring 5A deletion lines, Vrn-A1 was physically mapped to a region flanked by deletion break points 0.68 and 0.78 on the long arm by evaluating the flowering times of the different long and short arm deletion lines under controlled environment conditions and by mapping RFLP loci closely linked with Vrn-A1 in our previous studies. Analysis of the chromosome 5A deletion lines also showed evidence for a second more proximal effect on flowering time located between deletion break points 0.56 and 0.64.

Comparative mapping studies with rice also were made to look for possible orthologues of Vrn-A1 in rice. The 0.68 to 0.78 interval on the long arm of 5A of wheat was shown to be homoeologous to a region of rice chromosome 3 that contains the flowering time QTL Hd-6, previously mapped in a 'Nipponbare / Kasalath' cross and FLTQ1, a novel QTL identified by us from analysis of 78 F3 families derived from a cross of the rice cultivars 'IR20 / 63-83'. The proximal part of chromosome 5A was shown to be homoeologous to rice chromosome 9, on which, interestingly, two QTL were detected in the 'IR20 / 63-83' rice cross.

Work on chromosome 5D has concentrated on extending the studies of 5A to 5D where the presumed homoeologous genes Vrn-D1 and Fr2 have been shown to reside on the long arm from chromosome-assay studies. For mapping, recombinant substitution lines were developed from the cross between the substitution line 'Chinese Spring (Cheyenne 5D) / Chinese Spring'. However, the very low levels of RFLP for this chromosome have made previous mapping difficult and, hence, here we used the new molecular marker system, AFLP, to fill gaps between `anchor' RFLPs. Flowering time was measured under controlled environment conditions, without previous vernalization of the lines, and a bimodal distribution around the parental means indicated the segregation of a single gene for flowering time, undoubtedly indicative of the location of Vrn-D1. Response to cold temperatures was measured using the standard freezing tests in the Mártonvasár phytotron. However, variation for frost tolerance did not show a clear bimodal segregation and, thus, QTL analysis was used to locate the gene. Analysis confirmed that Fr2 is located on the long arm of chromosome 5D in a segment homoeologous to the location to Fr1. Fr2 also was closely linked to Vrn-D1 but mapped about 10cM away, indicating that it is a separate gene and not a pleiotropic effect from Vrn-D1, as had been found for Vrn-A1/Fr1 on 5A. By using RFLP mapping data from chromosome 5A and 5D of the cross 'Chinese Spring x SQ1', it was possible to link the 5D map to the 5A map of Galiba et al. (1995) and hence show that the loci are probably homoeoallelic. Future work will now concentrate on the fine mapping of Vrn-A1 and Vrn-D1 and developing a strategy for cloning them using a comparative mapping approach based on the use of rice 'tools'.

Genes and markers for resistance to preharvest sprouting.

J.E. Flintham, R.E. Adlam, P.C. Bailey, and M.D. Gale.

Wheat orthologues of the maize transcription factor Vpl have been mapped to a set of homoeoloci on the long arms of the group-3 chromosomes. The wheat taVp1 loci are some 30 cM proximal to the R loci controlling grain color. Grain color in wheat clearly is not controlled by Vp1 orthologues. However, these may yet prove to be involved in the control of sprouting via a mechanism independent of grain color. Opposite arms of chromosome 3A have opposite effects on dormancy of Chinese Spring wheat without affecting grain color. The 3AL effect possibly might be due to a defective taVp1 allele. We have assigned the provisional gene symbol Phs to another major-gene effect on dormancy (see AWN 43:252). Phs is linked to AFLP markers and appears to map on chromosome 1A. Phs has a dormancy effect of similar magnitude to the R genes but does not affect grain color. The search for genes with large effects on resistance to preharvest sprouting is continuing.

Temporal flux in the genetic diversity of U.K. cereal crops.

R.M.D. Koebner, J.R. Law, P. Donini, J.C. Reeves, and R.J. Cooke.

We have employed AFLPs and SSRs to produce a historical perspective of the genetic diversity of the U.K. wheat and barley crops. The survey covers the past 60-70 years, starting in the 1930s when cultivar registration was established. Since thas time, a record has been maintained of the identity and the acreages of cultivars that were grown commercially. Therefore, these cultivars give us a representative picture of the genetic variation within the crop. We identified 89 AFLPs among the winter wheat sample of 55 cultivars, and 137 among the 61 spring barleys. The wheat profiles were sufficiently variable to require four differences before the level of discrimination between any pair of entries fell below 100 %. Both the wheats and barleys had an average of about five alleles per SSR locus (14 loci in wheat and 22 in barley). The temporal trends in diversity for wheat and barley were very similar. Analysis of molecular variance showed that more than 90 % of the observed variation occurred within decades, with less than 10 % attributed between decades. Principle coördinate analysis of the AFLP data generated a series of convex hulls representing the extremes of variation within each decade. These overlapped significantly, with the data for the 1990s cultivars encompassing the majority of the diversity expressed in previous decades. The analysis of several statistical indices of diversity confirmed that no significant temporal changes in the levels of diversity had occurred. Taken together, these results indicate that over time, plant breeding has resulted in a qualitative rather than a quantitative shift in the diversity of these cereal crops in the U.K.

Meiotic homologous chromosomes start pairing in the interphase before meiotic prophase in wheat.

T. Schwarzacher, S. Wu, and T. Garkoucha.

Chromosome painting enabled the study of homologous chromosome behavior prior to and during meiosis. Total genomic DNA from rye, used as a probe for in situ hybridization, identified the rye chromosome arm in a wheat­rye translocation line (T5AS·5RL) at meiotic prophase and the preceding interphase. Accurate staging of the development of the meiocytes was attained by parallel studies of chromatin morphology, nucleolar behavior, and synaptonemal complex formation in electron microscope thin sections and silver-stained surface spreads. Three stages of pairing were identified for the large cereal genomes that are organized in a Rabl configuration at interphase: first, cognition occurs during the long interphase before leptotene, bringing the homologous chromosome domains into close proximity and possibly starting at the centromere; second, homologous chromosome segments align at leptotene; and third, zygotene synapsis initiates near the telomere, although it also was observed to occur near the centromere. These findings are in contrast to maize and mammals, where homologous chromosomes are not associated when entering meiosis.

Flow cytometric analysis of the chromosomes and stability of a wheat cell culture line.

T. Schwarzacher, M.L. Wang, A. R. Leitch, N. Miller, G. Moore, and J.S. Heslop-Harrison.

A rapidly growing, long-term suspension culture derived from T. aestivum was synchronized using hydroxyurea and colchicine, and a chromosome suspension with 2 to 3 x 10^6 chromosomes/ml was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin A3, univariate and bivariate flow cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was nearly similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over 1 year, whereas a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data show that flow cytometric analysis of plant karyotypes enables accurate, statistically precise, chromosome classification and karyotyping of cereals. Little overlap occurred between individual flow histogram peaks, so the method is useful for flow sorting and the construction of chromosome-specific recombinant DNA libraries. Bivariate analysis showed that the AT:GC ratio of all the chromosomes was remarkably similar, indicating strong homogenization events that are in contrast to mammalian genomes and are likely to affect sequence evolution and distribution of repeated sequences.

The chromosomal organization of simple sequence repeats (SSRs) in Triticum, Aegilops, and Secale genomes.

T. Schwarzacher, A. Cuadrado, D. Bardsley, G.E. Harrison, and J.S. Heslop-Harrison.

We are studying the physical distribution of 10 SSRs on chromosomes of bread wheat, wild wheats, rye, and hexaploid triticale. Oligomers with repeated di- tri- or tetra-nucleotide motifs were used as probes for fluorescent in situ hybridization on root-tip metaphase and anther pachytene chromosomes. All motifs showed dispersed hybridization signal of varying strengths on all chromosomes. In addition, particularly the motifs (AAG)5 and (GACA)4, hybridized to pericentromeric and multiple intercalary sites on the B-genome chromosomes and on chromosome 4A of wheat, and gave diagnostic patterns and resembled N-banding. In rye, all chromosomes showed hybridization of (GACA)4 and (AAG)5 at many intercalary sites that did not correspond to any other known banding pattern, but allowed identification of all R-genome chromosome arms. Overall, an SSR hybridization signal was found in related chromosome positions independent of the motif used and showed remarkably similar distribution patterns in wheat and rye, indicating their special role in chromosome organization as a possible ancient component of the Triticeae genome. We are currently studying SSR distribution in Ae. ventricosa and wheat lines that incorporate DNA from Ae. ventricosa and are investigating the use of SSR banding as a diagnostic tool in Triticeae genetics.

Publications.

Anamthawat-Jónsson K, Bodvarsdottir SK, Brangason BTH, Gudmundsson J, Martin PK, and Koebner RMD. 1997. Wide hybridzation between wheat (Triticum L) and lymegrass (Leymus Hochst). Euphytica 93:293-300.

Aragón-Alcaide L, Miller TE, Schwarzacher T, Reader, and Moore G. 1997. A cereal centromeric sequence. Proceedings of the 1997 Aberystwyth Cell Genetic Group, Experimental Biology Online. http://science.springer.de/ebo/ebo-main.htm.

Aragón-Alcaide L, Reader S, Bevan A, Shaw P, Miller T, and Moore G. 1997. Association of homologous chromosomes during floral development. Curr Biol 7:905-908.

Ben Amer IM, Korzun V, Worland AJ, and Börner A. 1997. Genetic mapping of QTL controlling tissue-culture response on chromosome 2B of wheat (Triticum aestivum L) in relation to major genes and RFLP markers. Theor Appl Genet 94:1047-1052.

Bozorgipour R and Snape JW. 1997. An assessment of somoclonal variation as a breeding tool for generating herbicide tolerant genotypes in wheat (Triticum aestivum L). Euphytica 94: 335-340.

Brown JKM, Foster EM, and O'Hara RB. 1997. Adaptation of powdery mildew populations to cereal varieties in relation to durable and non-durable resistance. In: The Gene for Gene Relationship in Plant-Parasite Interactions (Crute IR, Holub EB, and Burdon JJ eds). Wallingford, CAB International. pp. 119-138.

Bryan GJ, Collins AJ, Stephenson P, Orry A, Smith JB, and Gale MD. 1997. Isolation and characterisation of microsatellites from hexaploid bread wheat. Theor Appl Genet 94:557-563.

Castilho A, Miller TE, and Heslop-Harrison JS. 1997. Analysis of a set of homoeologous group 1 wheat-Aegilops umbellulata recombinant chromosome lines using genetic markers. Theor Appl Genet 94:293-297.
Devos KM and Gale MD. 1997. Comparative genetics in the grasses. Plant Mol Biol 35:3-15.

Devos KM and Gale MD. 1997. Comparative genome analysis in the grass family.