Forward genetic screens have led to the isolation of several genes involved in secondary cell wall formation. A variety of evidence, however, suggests that the list of genes identified is not exhaustive. To address this problem, microarray data have been generated from tissue undergoing secondary cell wall formation and used to identify genes that exhibit a similar expression pattern to the secondary cell wall-specific cellulose synthase genes IRREGULAR XYLEM1 (IRX1) and IRX3. Cross-referencing this analysis with publicly available microarray data resulted in the selection of 16 genes for reverse genetic analysis. Lines containing an insertion in seven of these genes exhibited a clear irx phenotype characteristic of a secondary cell wall defect. Only one line, containing an insertion in a member of the COBRA gene family, exhibited a large decrease in cellulose content. Five of the genes identified as being essential for secondary cell wall biosynthesis have not been previously characterized. These genes are likely to define entirely novel processes in secondary cell wall formation and illustrate the success of combining expression data with reverse genetics to address gene function.

Identification of Novel Genes in Arabidopsis Involved in Secondary Cell Wall Formation Using Expression Profiling and Reverse Genetics

The recent advances in alien gene transfer from distantly-related species into wheat are reviewed in the present paper The main achievements during the last ten years include the great expansion of the range of wide hybridization and development of new techniques for production and characterization of wheat-alien chromosome translocations Updated results of wide hybridization since 1983 and comprehensive characterization of wheat-alien translocation lines in our laboratory are compiled The future outlook for alien gene transfer in wheat is also discussed

Recent advances in alien gene transfer in wheat

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for...

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years.

Genomic 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) Love, 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.

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

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques.

In this paper we describe a repetitive DNA sequence unique to the rye genome. The corresponding DNA probe (pAW173) was cloned as a 450-bp fragment and does not hybridize to wheat or barley DNA and is not related to five other repeated DNA families characterised in rye. This new family (designated the R173 family) consists of moderately repeated DNA (~ 15 000 units per genome) and is present on all seven rye chromosomes. The hybridization patterns in Southern blots indicate that it is not organised as blocks of tandem arrays and in situ hybridization shows that it is dispersed throughout the rye chromosomes. The probe has been successfully used as a molecular marker in identifying rye genetic material in a wheat background.Key words: repetitive DNA sequence, rye-specific DNA, molecular marker.

Weining Song

Papers

Cloning and characterisation of a new rye-specific repeated sequence