Restriction fragment length polymorphism segregation analysis of the Li locus in Trifolium repens L.

Abstract

The Li locus in white clover controls the presence of cyanogenic beta-glucosidase (linamarase) activity in leaf tissue, such that plants homozygous for the 'null' allele (li) have no linamarase activity in this tissue. The isolation of a cDNA clone from linamarase mRNA is described. The cDNA clone is used to further characterise alleles of the Li locus. Northern blot analysis shows that plants homozygous for the 'null' allele (li li) produce very reduced levels of mRNA which hybridises to the cDNA. Heterozygous plants (Li li), which have intermediate levels of enzyme activity, produce intermediate levels of mRNA. Southern blot analysis of Hind III digested genomic DNA shows that the white clover genome contains three genes with homology to the linamarase cDNA and that at least two of these genes segregate independently. Analysis of the cosegregation of linamarase activity and the presence of genomic restriction fragments identifies the genomic sequence specifying linamarase structure and indicates either a structural or cis acting control function of the Li locus.

The cyanogenic polymorphism in Trifolium repens L. (white clover)

“…The biochemical difference between Li Li and liii plants has been used to select linamarase clones from white clover developing leaf cDNA libraries (Hughes et at., 1990). The identity of the clones was established using hybrid select translation and immunoprecipitation of the polypeptide product.…”

“…The cDNA clones have also been used to analyse genomic DNA using Southern blot analysis (Hughes et at., 1990). Considerable restriction fragment length polymorphism exists in white clover for genomic sequences homologous to linamarase cDNA (Fig.…”

“…3). This polymorphism has been used to analyse the genomic organization of these sequences in the Li Li plant used as a source of mRNA for cloning (Hughes et at., 1990). The studies show that the white clover genome contains three genes with homology to linamarase cDNA and that at least two of these genes segregate independently.…”

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“…Cloned segments of the conser\-ative parts of ribosomal genes hybridize to homologous regions trom many species and have been used extensi\'ely to demonstrate restriction site variation in the rDNA within and among populations (Schaal & Learn, 1988). Otherwise, probes have been specifically cloned from the genome that is to be analyzed ('homologous probes'; Keim et al, 1989: Hughes et al. 1990).…”

“…Breeding genetics had shown that cyanogenesis in white clover depends on active alleles at two independently segregating loci. Recently, a specific molecular probe for one of these, the gene for the hvdrolvtic enzvme linamarase has heen isolated (Hughes et al 1990). Ecoiogically, cyanogenesis seems to be primarily a defence against herb ivory.…”