Chromosomal assignment of characterized cDNA's and genes using RFLP analysis of ditelosomic addition lines

M. Cannell, M. Mulcahy, A. Karp, and P. R. Shewry
AFRC Institute of Arable Crops Research
Rothamsted Experimental Station
Harpenden, Herts AL5 2JQ
United Kingdom, "R"


As part of a project to produce a map of the barley genome based on DNA RFLP's, we have obtained cDNA's and genes for characterized proteins from various laboratories, and are determining their chromosomal locations using the ditelosomic addition lines of barley (cv. Betzes) into wheat (cv. Chinese Spring) produced by Islam and Shepherd (1983). The plants were grown in the glasshouse at Rothamsted and their karyotype confirmed by root tip analysis. Standard methods for DNA extraction and RFLP analysis were used which will be reported in detail elsewhere.

Some preliminary results using six clones are summarized in Table 1, and a typical result shown in Fig. 1.

Table 1.Chromosomal locations of genomic fragments hybridizing to cloned cDNA's and genes.

Fig. 1. RFLP mapping of the thionin precursor genes (pKG1348) using the ditelosomic addition lines.

B=Betzes, CS=Chinese Spring

The locations of the thionin precursor and high pI a-amylase genes on the short and long arms respectively of chromosome 6 are consistent with the assignment of the thionin precursor genes to chromosome 6 by Bohlmann et al. (1988) and with recent RFLP mapping (Kleinhofs et al., 1988; Wettstein-Knowles, 1989). Similarly Hejgaard et al. (1984) have previously mapped the gene for BASI to chromosome 2 using isoenzyme analysis of the wheat/barley addition lines. Loi et al. (1988) showed that the cDNA encoding (1-3, 1-4) b-glucanase isoenzymne II hybridized to two restriction fragments in EcoRI digested genomic DNA from cv Betzes, one of which was located on chromosome 1. The second fragment was not present in any of the available addition lines of Betzes chromosomes into Chinese Spring wheat, and was therefore assumed to be located on chromosome 5. The location of the gene for glutamine synthetase (chloroplast form) has not been reported previously.

Acknowledgements:

This research was supported by Shell Research Ltd. and Nickerson R.P.B. Ltd.

We are indebted to all the workers referred to above who generously made their clones available for this study.

References:

Bohlmann, H., S. Clausen, S. Behnke, S. Giese, C. Hiller, U. Reimann-Philipp, G. Schrader, V. Barkholt, and K. Apel. 1988. Leaf specific thionins of barley - a novel class of cell wall proteins toxic to plant-pathogenic fungi and possibly involved in the defense mechanism of plants. EMBO J. 7:1559-1565.

Chandler, P.M., J.A. Zwar, J.V. Jacobsen, T.J.V. Higgins, and A.S. Inglis. 1984. The effects of gibberellic acid and abscisic acid on a-amylase mRNA levels in barley aleurone layers studied using an a-amylase cDNA clone. Plant. Mol. Biol. 3:407-418.

Fincher, G.B., P.A. Lock, M.M. Morgan, K. Lingelbach, R.E.H. Wettenhall, J.F.B. Mercer, A. Brandt, and K.K. Thomsen. 1986. Primary structure of the (1-3, 1-4)-b-D-glucan 4-glucanohydrolase from barley aleurone. Proc. Natl. Acad. Sci. USA 83:2081-2085.

Gausing, K. 1987. Thionin genes specifically expressed in barley leaves. Planta 171:241-246.

Hejgaard, J., S.E. Bjorn, and G. Nielsen. 1984. Localization to chromosomes of structural genes for the major protease inhibitors of barley grain. Theor. Appl. Genet. 68:127-130.

Islam, A.K.M.R. and K.W. Shepherd. 1983. Ditelosomic additions of barley chromosomes to wheat. In Proc. 6th Int. Wheat Genet. Symp., Kyoto. pp. 233-238.

Kleinhofs, A., S. Chao, and P.J. Sharp. 1988. Mapping of nitrate reductase genes in barley and wheat. In Proc. 7th Int. Wheat Genet. Symp. Vol. 1 IPSR, Cambridge. pp 541-546.

Loi, L., B. Ahluwalia, and G.B. Fincher. 1988. Chromosomal location of genes encoding barley (1-3, 1-4)-,b-glucan 4-glucanohydrolases. Plant Physiol. 87:300-302.

Wettstein-Knowles, P. von. 1989. Facets of the barley genome. In Science for Plant Breeding. Proc. XII. Eucarpia Conf. "kkVortrage für Pflanzenzutung" P. Parey Sci. Publ. Berlin. Vol. 16 (In press).


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