Mapping microsatellites in hexaploid wheat.

G.J. Bryan, P. Stephenson, A.J. Collins, C.S. Busso, and M.D. Gale.

Microsatellites or simple sequence repeats consist of tandem arrays of 2, 3, or 4 base-pair repeats, for example (GA)n or (CA)n. These markers have been shown to provide the basis for extremely polymorphic genetic marker systems in hexaploid bread wheat, by virtue of their inherent length variability. This variability can be accessed by the use of PCR primers designed for sequences flanking the microsatellite array.

We have isolated a relatively large number of microsatellites from the wheat genome by screening small insert genomic libraries with different types of simple-sequence repeat probes. More than 200 clones containing microsatellites were sequenced and it was possible to make 153 primer pairs for sequences flanking microsatellite repeats. These microsatellites were characterized using wheat germplasm and aneuploid lines. At the present time, 49 primer pairs amplify a PCR fragment of the predicted size. The remaining primer sets either amplify no products or a large number of fragments that are impossible to analyze. On average, the 49 microsatellite primers are detecting 3.3 alleles per locus and an average PIC value of 0.47, using a set of 10 adapted wheat cultivars. Higher levels of polymorphism are seen using large samples of wheat germplasm. In general, these microsatellites show low levels of transferability to other cereal genomes or species.

To date, 69 of 72 loci have been assigned to particular chromosomes using the nullisomic-tetrasomic lines of wheat. An average of 1.5 loci per primer pair are detected. A significant correlation occurrs between the length of the microsatellite repeat and the degree of polymorphism observed (r = 0.61). Microsatellites showing polymorphism between the parents of our main mapping cross now are being mapped using the `Chinese Spring x Synthetic' F2 mapping population.

The physical organization of cereal nuclei during meiosis.

T. Schwarzacher, S. Wu, and C.B. Gillies.

Some of the critically important, unanswered questions about meiosis relate to timing and mechanisms of homologous chromosome recognition and alignment and pairing during meiotic prophase and the preceding interphase. Molecular cytogenetic methods now enable us to look at the earliest stages, before individual chromosomes become visible in pachytene, when their organization is vastly different from that at mitosis. Wheat genotypes carrying a single pair of rye chromosome arms (such as the 5AS·5RL translocation) grow normally, and their chromosomes all pair as bivalents at meiosis. The rye chromosome arms can be painted by in situ hybridization with genomic DNA as a probe. In somatic interphase nuclei, two labelled rye chromosome arms are visible, normally in separate domains. In cereals with large amounts of DNA, the meiotic interphase nucleus starts with a Rabl configuration with centromeric and telomeric Polfeldern. The model postulates three stages of chromosome pairing; cognition, alignment, and then synapsis. Cognition can happen at the centromere, or wherever chromosome-specific DNA sequences are present, whereas synapsis can initiate at one, probably near the telomere, or more independent locations.

Comparison of genetic and physical maps of large cereal genomes have revealed substantial discrepancies. Many genes and markers are clustered genetically near the centromere, but they are located physically in the distal region of the chromosome. Hence, the distal segments include most of the genetic length and recombination of the chromosomes, which agrees with the distal location of chiasmata near the subtelomeric heterochromatin of rye bivalents at metaphase I of meiosis. In situ hybridization with the rDNA probe identifies the bivalent of chromosome 1 where 84 % of chiasmata were distal to the rDNA locus and 16 % were proximal, corresponding with the location on the genetic map of Devos et al. 1993.

The large-scale genomic organization of repetitive DNA families at the telomere of rye chromosomes.

A.A. Vershinin, T. Schwarzacher, E. Alkhimova, and J.S. Heslop-Harrison.

Repetitive DNA sequences in the terminal heterochromatin of rye, S. cereale, chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandem-repeat subtelomeric DNA families with complex but contrasting higher-order structural organizations, pSC119.2, pSC200, and pSc250. In situ hybridization showed that all three repeats are located in the major subtelomeric heterochromatic regions with pSc200 being most terminal and pSC119.2 most proximal. Clone pSC119.2 also has many intercalary sites and a clearly different organization after pulse field gel electrophoresis. Polymerase chain reaction analysis with a single primer showed that, apart from the commonly found head-to-tail orientation, a fraction of the repeat units of one family, pSc200, is organized in a head-to-head orientation. Such structures suggest evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. After Xbal digestion and pulse field gel electrophoresis, the telomeric and subtelomeric clones showed strong hybridization signals from 400ñ100 kb, with a maximum at 50 to 60 kb. We suggest that their fragments define a basic higher-order structure and DNA loop domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.

Investigations currently are underway to analyze the distribution and organization of these repetitive sequences in Secale and related species.

Wheat/Aegilops umbellulata recombinant chromosomes.

A. Castilho, T.E. Miller, and J.S. Heslop-Harrison.

Fluorescent genomic in situ hybridization was used to evaluate the extent of recombination and to physically map the translocation breakpoints of 11 wheatñAe umbellulata chromosome 1U recombinant lines, previously produced by N.M. Islam-Faridi. In situ hybridization was able to identify the alien material in the wheat background and showed breakpoints not only near the centromere, but also along chromosome arms. To characterize and identify the chromosomes, further simultaneous multiple target in situ hybridization was used to localize a tandemly repeated DNA sequence (pSc119.2) and the 18S-25S and 5S rRNA genes. From six independent original crosses, eight of the translocation lines included only two types of intercalary wheatñAe. umbellulata recombination events. Five occurred at the 5S rRNA locus on the short arm of the Ae. umbellulata chromosome with a distal wheat segment, and three breakpoints were proximal to the centromere in the long arm, so that most of the long arm was from Ae. umbellulata. The remaining three lines proved not to contain wheat/Ae.umbellulata recombinant chromosomes. One carried a telecentric 1U chromosome and two contained 1U chromosomes with terminal deletions.

Publications.

Abbo S, Dunford RP, Foote TN, Reader SM, Flavell RB, and Moore G. 1995. Organization of retro-element and stem-look repeat families in the genomes and nuclei of cereals. Chromosome Res 3:5-15.

Amoah BK, Rezanoor HN, Nicholson P, and Macdonald MV. 1995. Variation in the Fusarium section Liseola: pathogenicity and genetic studies of isolates of Fusarium moniliforme sheldon from different hosts in Ghana. Plant Path 44:563-572.

Anamthawat-Jónsson K and Reader SM. 1995. Pre-annealing of total genomic DNA probes for simultaneous genomic institu hybridization. Genome 38:814-816.

Ben Amer IM, Worland AJ, and Börner A. 1995. Chromosomal location of genes affecting tissue-culture response in wheat. Plant Breed 114:84-85.

Bonhomme A, Gale MD, Koebner RMD, Nicholas P, Jahier J, and Bernard M. 1995. RFLP analysis of an Aegilops ventricosa chromosome that carries a gene conferring resistance to leaf rust (Puccinia recondita) when transferred to hexaploid wheat. Theor Appl Genet 90:1042-1048.

Brown JKM. 1995. Pathogens' responses to the management of disease resistance genes. In: Advances in Plant Pathology (Andrews JJ and Tommerup I eds) London, Academic Press. Pp. 75-102.

Brown JKM. 1995. Recombination and selection in populations of plant pathogens. Plant Path 44:279-293.

Castilho A and Heslop-Harrison JS. 1995. Physical mapping of 5S and 18S-25S RDNA and repetitive DNA sequences in Aegilops umbellulata. Genome 38:91-96.

Cuadrado A, Jouve N, and Heslop-Harrison JS. 1995. Physical mapping of the 5S rRNA multigene family in 6x triticale and rye: identification of a new rye locus. Genome 38:623-626.

Devos KM, Bryan GJ, Collins AJ, Stephenson P, and Gale MD. 1995. Application of two microsatellite sequences in wheat storage proteins as molecular markers. Theor Appl Genet 90:247-252.

Devos KM, Dubcovsky J, Dvorak J, Chinoy CN, and Gale M D. 1995. Structura evolution of wheat chromosomes 4A, 5A and 7B and its impact on recombination. Theor Appl Genet 91:282-288.

Devos KM, Moore G, and Gale MD. 1995. Conservation of marker synteny during evolution. Euphytica 84:367­372.

Donini P, Koebner RMD, and Ceoloni C. 1995. Cytogenetic and molecular mapping of the wheat-Aegilops longissima chromatin breakpoints in powdery mildew-resistant introgression lines. Theor Appl Genet 91:738-743.

Dunford RP, Kurata N, Laurie DA, Money TA, Monobe Y, and Moore G. 1995. Conservation of fine-scale DNA marker order in the genomes of rice and the Triticeae. Nuc Acids Res 23:2724-2728.

Evers AD, Flintham J, and Kotecha K. 1995. FONT SIZE=2 FACE="Symbol"a-amylase and grain size in wheat. J Cereal Sci 21:1-3.

Feldman M, Lupton FGH, and Miller TE. 1995. Wheat. In: Evolution of Crop Plants (Smartt J and Simmonds NW eds). Harlow, Longman Scientific & Technical. Pp. 184-192.

Flintham JE and Gale MD. 1995. Plant height and yield components of inbred isogenic and F1 hybrid Rht dwarf wheats. In: Hybrids and Heterosis II (Maluszynski M ed). IAEA, Vienna.

Francis HA, Leitch AR, and Koebner RMD. 1995. Conversion of a RAPD-generated PCR product, containing a novel dispersed repetitive element, into a fast and robust assay for the presence of rye chromatin in wheat. Theor Appl Genet 90:636-642.

Galiba G, Quarrie SA, Sutka J, Morgounov A, and Snape JW. 1995. RFLP mapping of the vernalization (Vrn) and frost resistance (Fr1) genes on chromosome 5A of wheat. Theor Appl Genet 90:1174-1179.

Galasso I, Blanco A, Pignone D, and Heslop-Harrison JS. 1995. Phyletic relationships between Dasypyrum villosum and D. breviaristatum investigated by in situ hybridization. Chromosome Res 3 Suppl 1, 49.

King IP, Purdie KA, Law CN, Worland AJ, Orford SE, Reader SM, and Miller TE. 1995. An assessment of the potential of 4DS.4DL translocation lines as a means of eliminating tall off types in semi-dwarf wheat varieties. In: Proc 9th EWAC Conference (Börner A and Worland AJ eds). Gatersleben-Wernigerode 1994; Eur Wheat Aneuploid Co-operative Newslet. Pp. 63-64.

King IP, Purdie KA, Reader SM, and Miller TE. 1995. Detection of homoeologous chiasma formation in wheat/alien hybrids. In: Proc 8th Inter Wheat Genet Symp (Li ZS and Xin ZY eds). China Agricultural Scientec Press, Beijing. Pp. 203-205.

Koebner RMD. 1995. Generation of PCR-based markers for the detection of rye chromatin in a wheat background. Theoret Appl Genet 90:740-745.

Koebner RMD. 1995. Predigestion of DNA template improves the level of polymorphism of random amplified polymorphic DNAs in wheat. Genet Anal: Biomolec Engin 12:63-67.

Law CN. 1995. Genetic manipulation in plant breeding - prospects and limitations. Euphytica 85:1-12.

Law CN. 1995. Inter-varietal chromosome substitution lines - re-visited. In: Proc 9th EWAC Conference (Börner A and Worland AJ eds). Gatersleben, Wernigerode. Eur Wheat Aneuploid Co-operative Newslet Pp. 46-47.

Lee SJ, Penner GA, and Devos KM. 1995. Characterization of loci containing microsatellite sequences among Canadian wheat cultivars. Genome 28:1037-1040.

Lelley T, Kazman E, Devos KM, and Gale MD. 1995. Use of RFLPs to determine the chromosome composition of tetraploid triticale (A/B)(A/B)RR. Genome 38:250-254.

Martin PK and Koebner RMD. 1995. Sodium and chloride ions contribute synergistically to salt toxicity in wheat. Biol Plant 37:265-271.

Maurin N, Rezanoor HN, Lamkadmi Z, Some A, and Nicholson P. 1995. A comparison of biological, molecular and enzymatic markers to investigate variability within Microdochium nivale (Fries) Samuels and Hasllett. Agronomie 15:39-47.

Miller TE, Reader SM, Mahmood A, Purdie, KA, and King IP. 1995. Chromosome 3N of Aegilops uniaristata a source of tolerance to high levels of aluminium for what. In: Proc 8th Inter Wheat Genet Symp (Li Z S and Xin Z Y eds). China Agricultural Scientech Press, Beijing. Pp. 1037-1042.

Miller TE, Reader SM, Purdie KA, and King IP. 1995. Fluorescent in situ hybridisation - a useful aid to the introduction of alien genetic variation into wheat. In: Proc 9th EWAC Conference (Börner A and Worland AJ eds). Gatersleben, Wernigerode. Eur Wheat Aneuploid Co-operative Newslet Pp. 58-60.

Moore G. 1995. Cereal genome evolution: pastoral pursuits with `Lego' genomes. Curr Opinion Genet Dev 5:727-724.

Moore G, Devos KM, Wang Z, and Gale MD. 1995. Grasses, line up and form a circle. Curr Biol 5:737-739.

Moore G, Foote, Helentjaris T, Devos KM, Kurata N, and Gale MD. 1995. Was there a single ancestral cereal chromosome? Trends in Genet 11:81-82.

Neves N, Heslop-Harrison JS, and Viegas W. 1995. rRNA gene activity and control of expression mediated by methylation and imprinting during embryo development in wheat x rye hybrids. Theor Appl Genet 91:529-533.

Plaschke J, Korzun V, Koebner RMD, and Börner A. 1995. Mapping the GA3-insensitive dwarfing gene ct1 on chromosome 7 in rye. Plant Breed 114:113-116.

Reader SM, Miller TE, and Purdie KA. 1995. Cytological analysis of plant chromosomes using rapid in situ hybridisation. In: Proc 9th EWAC Conference (Börner A and Worland AJ eds). Gatersleben, Wernigerode. Eur Wheat Aneuploid Co-operative Newslet Pp. 60-61.

Schwarzacher T, Mann S, and Heslop-Harrison JS. 1995. Fluorescent in situ hybridization (FISH) using Cy3. Sigma Immunotes 13:1-3.

Snape JW, Quarrie SA, and Laurie DA. 1995. Comparative mapping and its use for the genetic analysis of agronomic characters in wheat. In: Proc 9th EWAC Conference (Börner A and Worland AJ eds). Gatersleben, Wernigerode. Eur Wheat Aneuploid Co-operative Newslet Pp. 51-54.

Vershinin A, Schwarzacher T, and Heslop-Harrison JS. 1995. The large scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes. Plant Cell 7:1823-1833.

Wang G, Hyne V, Chao S, Henry Y, De Buyser J, Gale MD, and Snape JW. 1995. A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes. Theor Appl Genet 91:744-746.

Wang YB, Hu H, and Snape JW. 1995. Spontaneous wheat/rye translocations from female meiotic products of hybrids between octoploid triticale and wheat. Euphytica 81:265-270.

Worland AJ and Sayers EJ. 1995. Rht1 (B.dw), an alternative allelic variant for breeding semi-dwarf varieties. Plant Breed 114:397-400.

Worland AJ, Sayers EJ, Kirby J, and Howie J. 1995. Progress report of wheat genetics. In: Proc 9th EWAC Conference (Börner A and Worland AJ eds). Gatersleben, Wernigerode. Eur Wheat Aneuploid Co-operative Newslet Pp. 55-57.

PLANT BREEDING INTERNATIONAL

Maris Lane, Trumpington, Cambridge CB2 2LQ, United Kingdom.

In addition to the long-standing program based at Cambridge, we now have two additional programs based in France and Germany. Both of these programs have purpose-built breeding stations at Louville la Chenard, in the Beauce region south of Paris, and southwest of Magdaberg east of the Hartz mountains, in Germany.

Crossing and F2 production remains at Cambridge for the three programs but all selection and testing are done in the region projected for that particular cross. Quality testing and research backup is at Cambridge.