Jacqueline H. A. Barker

An analysis of genetic diversity in coconut (Cocos nucifera) populations from across the geographic range using sequence-tagged microsatellites (SSRs) and AFLPs.

Abstract Genetic diversity in 31 individuals from 14 coconut populations across the entire geographic range (2–3 individuals per population) was assessed using sequence-tagged microsatellites (or simple sequence repeats, SSRs) and amplified fragment length polymorphism (AFLP). From the 39 SSR primer sets tested, only two gave patterns that could not be scored and used in the data analysis. The remainder included five SSRs that gave double-locus profiles in which one locus could still be scored separately. The 37 SSRs revealed between 2 and 16 alleles per locus and a total of 339 alleles in the 14 populations. Gene diversity (D = 1-Σpi2) ranged from 0.47 to 0.90. Two of the four Dwarf populations were homozygous at all 37 loci, which is consistent with their autogamous (self-fertilising) reproduction. One Dwarf population was heterozygous at one locus but the other (Niu Leka Dwarf), which is known to be cross-pollinating, showed high levels of heterozygosity. Generally, diversity was higher in populations from the South Pacific and South East Asia. Three SSR loci (CNZ46, CN2A5, CN11E6) gave distinct genotypes for all but two populations. The East African populations had higher heterozygosities than those from West Africa, and the populations from Tonga and Fiji generally had distinct alleles from those of the South Pacific. AFLP analysis with 12 primer combinations gave a total of 1106 bands, of which 303 were polymorphic (27%). Similarity matrices were constructed from the two data sets using the proportion of shared alleles for SSRs and a Jaccard coefficient for AFLPs. In each case cluster and principal co-ordinates analyses were performed, with the resultant dendrograms and plots revealing similar relationships among the populations for both approaches. There was generally a good separation of populations, and phenetic relationships were in agreement with those previously shown by RFLPs. The use of SSRs and AFLPs in genetic-diversity analysis for the establishment of germplasm collections is discussed.

Evidence that barley 3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase is a member of the sucrose nonfermenting-1-related protein kinase family

A protein kinase was partially purified from barley (Hordeum vulgare L. cv Sundance) endosperm by ammonium sulfate fractionation, followed by ion-exchange, Reactive Blue, Mono-Q, and phosphocellulose chromatography. It was shown to phosphorylate Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and a synthetic peptide that was shown previously to act as a substrate for HMG-CoA reductase kinase purified from cauliflower, confirming it to be barley HMG-CoA reductase kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation showed the presence of a polypeptide with an approximate relative molecular weight (Mr) of 60,000, which is the size predicted for the barley sucrose nonfermenting-1 (SNF1)-related protein kinases BKIN2 and BKIN12. Antisera were raised to a rye (Secale cereale L.) SNF1-related protein kinase (RKIN1) expressed in Escherichia coli as a fusion with maltose-binding protein and to a synthetic peptide with a sequence that is conserved in, and specific to, plant members of the SNF1-related protein kinase family. The maltose-binding protein-RKIN1 fusion protein antiserum recognized a doublet of polypeptides with an approximate Mr of 60,000 in crude endosperm extracts and a single polypeptide in root extracts, which co-migrated with the smaller polypeptide in the endosperm doublet. Both antisera recognized a polypeptide with an approximate Mr of 60,000 in the partially purified protein kinase preparation, suggesting strongly that barley HMG-CoA reductase kinase is a member of the SNF1-related protein kinase family.

The Di- and Polyamine Oxidases of Plants

Although the di- and polyamine oxidases of plants are apparently limited in their distribution, in some species they are remarkably active. Earlier work on these enzymes has been summarized by Smith (1985a), Morgan (1985), Rinaldi et al., (1986), and Mondovi and Avigliano (1987).

Immunological evidence that HMG-CoA reductase kinase-A is the cauliflower homologue of the RKIN1 subfamily of plant protein kinases

Three different antibodies against the RKIN1 and BKIN12 gene products from rye and barley recognized the 58 kDa subunit of HMG‐CoA reductase kinase‐A (HRK‐A) from Brassica oleracea on Western blots. HRK‐A was also detected by an antipeptide antibody in enzyme‐linked immunoassays, and this was competed by the peptide antigen. HRK‐A was not recognized by antibodies against plant, mammalian and Saccharomyces cereviseae relatives of RKIN1, i.e. wheat PKABA1, rat AMP‐activated protein kinase and S. cerevisiae Snf1p. RKIN1/HMG‐CoA reductase kinase‐A are now among the first protein kinases in plants to be well characterized at both the molecular and biochemical levels.

Differential expression of two barley SNF1-related protein kinase genes

We have amplified and cloned DNA sequences derived from a gene encoding a SNF1 (sucrose-non-fermenting 1)-related protein kinase which differs from that previously reported from barley. Northern blot and polymerase chain reaction (PCR) analysis of RNA populations, using specific probes and oligonucleotide primers, indicated that the two SNF1-related genes are differentially regulated. One is expressed in all tissues, whereas the other is expressed at high levels in the seed endosperm and aleurone, but at levels undetectable by northern blot analysis in other tissues. Comparisons with other plant SNF1-related protein kinase genes suggest that the form which is expressed at greatly enhanced levels in the seed is less similar to the other plant homologues which have been reported and may be unique to cereals.

Effect of enzyme-activated inhibitors on ornithine decarboxylase and growth of Botrytis cinerea

Abstract The enzyme ornithine decarboxylase (ODC) from Botrytis cinerea has been purified 68-fold with 74% recovery. The optimum pH was 7.3 and the Km for ornithine was 70μM. Using this preparation, the effects of several potential enzyme-activated inhibitors of ODC have been studied, comparing their properties with those of difluoromethylornithine (DFMO) which is a known growth inhibitor for Botrytis and other fungi. Difluoromethylarginine and dehydromonofluoromethylarginine, which are at least as effective as DFMO for the inhibition of growth of Botrytis cinerea, are relatively poor inhibitors of ODC. R,R-Methylacetylenicputrescine and dehydromonofluoromethylornithine, which are relatively poor inhibitors of Botrytis growth, are very efficient inhibitors of the ODC.

Growth inhibition of Botrytis cinerea by compounds interfering with polyamine metabolism

Summary: Several inhibitors of ornithine and arginine decarboxylases reduced growth of the fungus Botrytis cinerea cultured on Czapek Dox agar. Of these, the most effective were difluoromethylornithine (DFMO), dehydromonofluoro-methylornithine, difluoromethylarginine and dehydromonofluoromethylarginine. The growth inhibition due to 1 mM-DFMO could be partially reversed with 1 nM-putrescine. Other compounds causing significant reversal of DFMO-mediated growth inhibition included diaminopentane (cadaverine), diaminoheptane, spermidine, 7,7-difluorospermidine, spermine, bis(2-aminoethyl)amine, 2-hydroxy-1,3-diaminopropane, monoacetylputrescine, butenediamine and aminoguanidine. Some compounds, which were relatively innocuous by themselves, increased growth inhibition due to DFMO. Notably effective compounds were methylacetylenicputrescine, aminooxyaminopropane, butynediamine, 2,2-difluoroputrescine, diacetylputrescine, methylglyoxal bis(guanylhydrazone), streptomycin, certain methylated amines, and cyclohexylamine and related compounds. Growth inhibition due to a homologous series of diguanidines [NH2C(=NH)NH(CH2) x NHC(=NH)NH2] was also tested. These were especially effective when x = 12, and when x = 5 or 6. In general, the results suggest that amino-acid-based inhibitors of ornithine decarboxylase have a greater permeability than amine-based inhibitors.

Characterisation and inheritance of nuclear microsatellite loci for use in population studies of the allotetraploid Salix alba–Salix fragilis complex

We present nine polymorphic di- and tri-nucleotide repeat nuclear microsatellite markers selected specifically for their use in high throughput studies concerning the dioecious allotetraploid Salix alba–Salix fragilis willow complex. These taxa and their hybrids are difficult to discriminate using morphological characters. Thus, multiplex reactions were developed for these microsatellite loci and their effectiveness to distinguish individuals, especially hybrids, and their inheritance patterns in controlled crosses were determined. All loci displayed disomic–monogenic inheritance which allowed for the genotype data to be analysed as for a diploid organism. The nine loci produced a total of 67 alleles (mean, 7.4 alleles per locus; range, 3–11 alleles) in a reference panel of 57 individuals from two germplasm collections and natural populations. Gene diversity values (as measured by the expected heterozygosity) ranged from 0.000–0.820. A total of 53 distinct multilocus genotypes were observed, and ordination analysis revealed three separate clusters corresponding to S. alba, S. fragilis and hybrids. The microsatellite loci described here will be used in population genetic studies to investigate genetic variation, gene flow, levels of hybridisation and the extent of introgression in natural populations of the S. alba–S. fragilis complex. They are also useful for clonal identification, conservation and sustainable management of germplasm collections, genetic mapping and the selection of individuals and/or certification of controlled crosses for breeding programmes.

Genetic mapping of rust resistance loci in biomass willow

Rust diseases caused by Melampsora spp. represent a major threat to the productivity of short rotation coppice (SRC) willows grown for biomass, causing yield losses of up to 40%. The routine use of fungicide in SRC plantations is not a viable option because of economic and environmental considerations; thus, breeding for rust resistance is a major target for willow breeding programmes. To characterise the genetic basis of rust resistance in willow and provide targets for use in future marker-assisted selections, quantitative trait analyses were performed using a large full-sib mapping population (K8) which segregates for rust resistance and several other important agronomic traits. Rust resistance in field conditions was assessed in three consecutive years. For a more detailed genetic dissection, laboratory inoculation tests using isolates of two distinct and prevalent pathotypes (LET1 and LET5) were also performed. For field-based resistance, a major quantitative resistance locus, designated SRR1 (Salix Rust Resistance 1), was detected in addition to several quantitative trait loci (QTL) of more modest effect. Inoculation test data also supported an important role for SRR1. Specific interactions between particular rust isolates and different QTL were detected, and QTL that only influenced resistance in field conditions were identified. The QTL reported here represent an important basis for the future development of markers for use in willow breeding programmes. As the linkage map for the K8 population is anchored to the Populus trichocarpa genome sequence, a more efficient marker development for future fine-scale mapping and candidate gene identification is possible.

Insensitivity of Septoria tritici and Ustilago maydis to inhibitors of ornithine decarboxylase

Growth of Septoria tritici and Ustilago maydis is relatively insensitive to 1 m m difluoromethylomithine (DFMO), and other potent enzyme-activated inhibitors of ornithine decarboxylase (ODC). For the ODC from S. tritici , the K i s for DFMO, dehydromonofluoromethylornithine (ΔFMO) and methylacetylenicputrescine (MAP) are 22, 5, and 35 μ m respectively, and the ODC from U. maydis appears to be similarly sensitive. On growing in 1 m m -DFMO, the polyamine content, which consisted mainly of spermidine in both fungi, is reduced to 30–40%. DFMO appears to penetrate freely into both fungi. The maximum ornithine concentration is about 12 m m in S. tritici and about 0·6 m m in U. maydis when grown in DFMO. In S. tritici neither arginine, citrulline nor acetylornithine appeared to serve as precursors for putrescine, and growth was not inhibited by 1 m m difluoromethylarginine (DFMA), or -citrulline (DFMC). In U. maydis no evidence was found for the decarboxylation of arginine or citrulline.