Li Locus Research Articles

Development of SCAR markers linked to male sterility and very high linoleic acid content in safflower

Practically no molecular tools have been developed so far for safflower (Carthamus tinctorius L.) breeding. The objective of the present research was to develop molecular markers for the closely linked genes Li, controlling very high linoleic acid content, and Ms, controlling nuclear male sterility (NMS). A mapping population of 162 individuals was developed from the NMS line CL1 (64–79% linoleic acid) and the line CR-142 (84–90%), and phenotyped in the F2 and F3 generations. Bulked segregant analysis with random amplified polymorphic (RAPD) markers revealed linkage of five RAPD bands to the Li and Ms genes. RAPD fragments were converted into sequence-characterized amplified region (SCAR) markers. A linkage map including the five SCAR markers and the Li and Ms genes was constructed. SCAR markers flanked both loci at minimum distances of 15.7 cM from the Li locus and 3.7 cM from the Ms locus. These are the first molecular markers developed for trait selection in safflower.

The molecular biology of cyanogenesis.

The cyanogenic polymorphism in Trifolium repens L. (white clover) has been used as the basis of a study of the genetic control of cyanogenesis. The Ac locus controls the presence of two cyanoglucosides in white clover. Biochemical characterization of cyanoglucoside biosynthesis in plants containing different Ac alleles has shown that this is a complex locus which affects more than one step in the pathway. A study of the in vivo synthesis and processing of the cyanogenic beta-glucosidase (linamarase) of white clover led to the isolation of cDNA clones for this enzyme. The cloning strategy and structure of the cDNA clones is described. Together with biochemical and genetic data, these clones have been used to characterize the Li locus which controls linamarase activity in white clover. It has been shown that 'null' alleles of the Li locus result in very reduced levels of transcription of homologous mRNA sequences. The use of these cDNA clones to investigate the genomic organization of cyanogenesis genes in both white clover and other cyanogenic species is described, and their use in structural analysis of the cyanogenic beta-glucosidase is discussed.

Genetic Map Location of the Barley Developmental Mutant Liguleless in Relation to RFLP Markers

Barley plants homozygous for the li mutation have no ligule or auricle structures on their leaves. The li locus was previously mapped to the long arm of chromosome 2(2H) by linkage to other morphological mutants. In this paper the genetic map location of li in relation to RFLP markers was determined by bulk segregant analysis and analysis of individual mutant F2 plants. The li locus was 1.3 cM distal to XBCD266 on the long arm of chromosome 2(2H). The advantages of this approach for rapidly determining the genetic map location of barley mutants are discussed.

Simple sequence repeat (SSR) markers linked to the Ligon lintless (Li(1)) mutant in cotton.

Ligon lintless (Li(1)) is a monogenic, dominant mutant in cotton, whose expression results in extreme reductions in fiber length on mature seed. The objectives of this research were to compare fiber initiation between the Li(1) mutant and TM-1 to reveal the fiber initiation differences between normal and mutant phenotypes, to develop a linkage map of simple sequence repeat (SSR) markers with the Li(1) locus, and to identify the chromosomal location of the Li(1) locus. Comparative scanning electron microscopy studies of fiber development in a normal TM-1 genotype and the near-isogenic Li(1) mutant at 1 and 3 days postanthesis revealed little differences between the two during early stages of development, suggesting that Li(1) gene expression occurs later, probably during the elongation phase. Thirty-eight SSR loci were found to be polymorphic between TM-1 and Li(1) and were used for mapping in an F(2) population. Twenty-two SSR loci, along with Li(1), were located on eight linkage groups, covering a total genetic distance of 218.3 cM. Analysis of individual monosomic and monotelodisomic plants indicated that two SSR loci (MP4030 and MP673) from the Li(1) linkage group were located on chromosome 22.

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

The cyanogenic polymorphism in white clover is controlled by alleles of two independently segregating loci. Biochemical studies have shown that non-functional alleles of the Ac locus, which controls the level of cyanoglucoside produced in leaf tissue, result in the loss of several steps in the biosynthetic pathway. Alleles of the Li locus control the synthesis of the hydrolytic enzyme, linamarase, which is responsible for HCN release following tissue damage. Studies on the selective forces and the distribution of the cyanogenic morphs of white clover are discussed in relation to the quantitative variation in cyanogenesis revealed by biochemical studies. Molecular studies reveal considerable restriction fragment length polymorphism for linamarase homologous genes.

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

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.

Biochemical characterisation of the Li locus, which controls the activity of the cyanogenic ?-glucosidase in Trifolium repens L.

The cyanogenic β-glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300-103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.

Manifold effects of the cyanogenic loci in white clover

In white clover alternate alleles Ac/ac and Li/li at two unlinked loci determine the presence/absence of cyanogenic glucosides and the enzyme linamarase respectively. Plants possessing both cyanogenic glucosides and enzyme are cyanogenic. Morphological measurements were made on a random sample of plants from a population polymorphic at both loci. Individuals possessing linamarase had larger leaves than those lacking the enzyme. Competition experiments between all phenotypes showed a significant effect of Li phenotype frequency on total yield. Competitive interactions were such as to maintain polymorphism at the Li locus. It is concluded that the pattern of variation at the Ac and Li loci cannot be adequately explained by selection which discriminates between cyanogenic and acyanogenic phenotypes.

The production of β-glucosidase in cultured cells of Trifolium repens L.

Evidence is presented that β-glucosidase activity in white clover cells suspended in a liquid medium containing glucose, is lower than the β-glucosidase activity of cells from a non-sugar medium. This difference is not due to differences in the extraction of enzyme activity. The production of β-glucosidase in cultured cells is demonstrated in a number of genetically different clones of tissue and is not dependent on the presence of functional alleles at the Li locus.

Studies on the nature of the Li locus in Trifolium repens L. I. Purification and properties of the enzyme components.

Evidence has been sought on the possible existence of multiple forms of the enzyme controlled by the Li locus in white clover. During purification of enzyme from LiLi plants, there was no separation of activities against the β-glucosides, p-nitrophenyl β-d-glucoside, salicin, and linamarin-lotaustralin, and the β-galactoside, p-nitrophenyl β-d-galactoside. In addition, tests on mixtures of these four substrates provided no evidence for the existence of more than one enzyme. Immunological tests have shown that plants homozygous for the recessive li allele do not contain an enzymatically inactive protein, antigenically related to the normal enzyme. This suggests that li alleles either specify a low-activity immunologically altered protein or control the synthesis of very low levels of normal enzyme.

Studies on the nature of the Li locus in Trifolium repens L. II. The effect of genotype on enzyme activity and properties.

Various reagents which prevent enzyme inhibition by phenolic compounds were tested in attempts to improve the medium used to extract the Li-controlled enzyme activities from white clover leaves. The addition of 50 mm diethyldithiocarbamate to the extraction medium gave a fivefold increase in the enzyme activity of LiLi white clover extracts against p-nitrophenyl β-d-glucoside, linamarin-lotaustralin, and p-nitrophenyl β-d-galactoside. These three substrates were used in tests on the effect of genotype on enzyme activity. An absence of dominance at the Li locus was demonstrated, with a dosage effect of Li alleles on enzyme activity. A new Li allele was identified in the Lili clone, C11, which had low levels of enzyme activity. In crosses with two lili clones, Li(C11)li progeny were produced with activity levels similar to those of the C11 parent. Inhibition and heat-inactivation tests suggest that the lili clone, D4, produces an altered form of β-glucosidase which may also be present in LiLi plants. The nature of the Li locus is discussed.

Studies on the β-glucosidase System ofTrifolium repensL.

Using DEAE-cellulose two distinct /3-glucosidase enzymes have been identified in white clover; one form is primarily associated with the seed, the other with young leaves. Evidence is presented for the separate nature of /3-glucosidase and /3-galactosidase activity in dry seed flour and in young leaf tissue. These results are considered in relation to the function of the Li locus in white clover, which is shown to control both /3-glucosidase and /3-galactosidase activity in expanding leaves.