R. Potter

Validation of molecular markers for wheat breeding

Five sets of markers were assessed for their usefulness in breeding, two linked to wheat stem rust gene Sr2, several markers linked to a chromosome segment conferring Yr17/Lr37/Sr38 resistance, two reported markers for the linked genes Lr35 andSr39, one for Lr28, and one linked to flour colour. The gene for Sr2 confers adult plant resistance to stem rust (Puccinia graminis f.sp. tritici) and was originally transferred to bread wheat from the tetraploid emmer (‘Yaroslav’) to the cultivars Hope and H-44. The gene is located on the short arm of chromosome 3B and confers a durable adult plant resistance to stem rust usually expressed only in the field. The chromosome segment carrying the Lr37, Sr38, Yr17 resistance genes is located on 2AS and was originally introduced into wheat through an Aegilops ventricosa Triticum persicum cross, followed by a cross to the cultivar Marne (VPM1). The flour colour quantitative trait locus was originally described in a Yarralinka Schomburg cross and is located on chromosome 7A. The primers as originally developed required optimisation for more routine use in a breeding program.

A PCR-based marker for selection of starch and potential noodle quality in wheat

A strong association between the absence of the granule-bound starch synthase (GBSS) protein for the 4A chromosome of wheat and Japanese Udon noodle quality has been previously described. The aim of this study was to identify a molecular marker linked to the GBSS 4A locus which could be used to identify wheat with the desired texture for Udon noodles. PCR primers were designed to target this gene which gave a 440 bp PCR band, corresponding to the presence or absence of the 4A GBSS gene. Of the 268 genotypes screened with these primers, 267 were correctly identified using the PCR primers. The remaining genotype was shown to be heterogeneous for the marker. The PCR marker test developed has advantages over existing methods used to screen for Udon noodle starch quality as it enables high throughput, accurate tests to be carried out on leaves of young seedlings or mature seed and identify breeding lines that are heterogeneous for the 4A allele which will allow for reselections. Application of this PCR test will speed up selection for Udon noodle quality genotypes and reduce breeding costs for production of noodle wheat varieties. Abbreviations: CTAB, cetyltrimethlammonium bromide; FSV, flour swelling volume; GBSS, granule-bound starch synthase; IEF, isoelectric focusing; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.

An assessment of genetic stability of potato in vitro by molecular and phenotypic analysis

It has generally been assumed that plants micropropagated by multiplication of organised meristems or by serial subculture of stem nodes are genetically stable. This assumption has been tested for potato plants, all of which originated from one shoot culture of cv Desiree, that have been propagated or stored under different regimes in vitro, by two methods. The first is by developing DNA restriction fragment analysis and using it to study changes in DNA banding patterns of regenerated plants, and the second is morphological analysis of the same plants grown in the field. Potato plants regenerated via callus can exhibit somaclonal variation, and so plants regenerated via a brief callus phase from the same stock plant of Desiree were also analysed. Molecular analysis involved combinations of 4 single or low copy DNA probes and 4 restriction enzymes. Variation in DNA fragment patterns was not found in any of the 128 plants micropropagated through 6 rounds of nodal subculture, nor in 18 plants that had been stored at low temperature or in 21 plants stored by osmotic reduction of growth. In contrast, 6 of 46 plants regenerated from leaf callus exhibited variations in banding pattern. In the morphological analysis, variants were also only identified in the population of plants regenerated via leaf callus. These results support the concensus view that the plants obtained from micropropagation of meristems are stable genetically, in contrast to those regenerated via callus. However, with the combinations of DNA probes and restriction enzymes used in the analysis, it is not possible to state that variation was not present. Restriction fragment analysis is a suitable approach for analysis of genetic stability of stock plants in vitro, but more plants can be screened for stability by morphological analysis in the field if time delays of 1–2 years are acceptable.

Differential display analysis of gene expression in the cytoplasm of giant cells induced in tomato roots by Meloidogyne javanica.

SUMMARY Giant cells induced by root-knot nematodes are highly specialized cells which function as transfer cells and provide nutrients to support the growth and reproduction of the nematode. Changes in the overall pattern of gene expression in giant cells occur during the formation and maintenance of the nematode feeding cells. Differential display analysis has been carried out to detect changes in gene expression in giant cells induced in tomato roots by Meloidogyne javanica, using mRNA isolated directly from mature giant cell cytoplasm, compared to non-infected root tissue. Eighty-one differential displayed bands were generated, and of these, 73 were up-regulated and 8 were down-regulated. Twenty-seven sequences were obtained by direct sequencing of the bands, and 16 fragments were further analysed by real-time quantitative RT-PCR. The most highly up-regulated transcript increased 56-fold in giant cells, and the greatest down-regulation was 11-fold. A time course of expression of the highest and lowest expressed transcripts was also undertaken by quantitative RT-PCR using giant cell enriched tissue. These showed similar changes in expression, but values were dramatically reduced. This result shows the importance of analysing giant cell cytoplasm directly, rather than starting with giant cell enriched tissue, to obtain accurate information on changes in gene expression in nematode feeding cells. Sequenced transcripts showed significant homology to mitogen-activated protein kinase, S-adenosylmethionine decarboxylase, cysteine synthase, cytochrome c reductase subunit, and ribosomal proteins. The expression analysed reflects the high metabolic rate in mature giant cells rather than processes of giant cell induction.

Typing Mlo alleles for powdery mildew resistance in barley by single nucleotide polymorphism analysis using MALDI-ToF mass spectrometry

Single nucleotide polymorphisms (SNPs) have been identified in a range of plant genomes. Development of rapid, low-cost methods to enable their validation and implementation as molecular markers is now required for practical applications. We report the development of single and multi-nucleotide primer extension assays to genotype co-dominant SNPs from small quantities of barley leaf tissue. In the single nucleotide primer extension assay, a genotyping primer with its 3′ end directly flanking a SNP was annealed to a target sequence and extended by a single dideoxynucleotide triphosphate complementary to the polymorphic base. In the multi-nucleotide primer extension assay, designed to facilitate allele calling, the genotyping primer with its 3′ end flanking the SNP was extended by either 1 or 2 nucleotides, depending on the allele encountered. Extension products were analysed using MALDI-ToF mass spectrometry and, making use of the molecular weight difference between DNA bases, the incorporated nucleotides were identified by the increase in mass of the extended primers. Based on a SNP identified in the barley Mlo gene, primer extension assays were designed and used for co-dominant marker-assisted selection of barley seedlings segregating for mlo-mediated resistance to powdery mildew. This allowed accurate selection of progeny lines carrying alleles for resistance to powdery mildew, including heterozygotes. Doubled haploid barley progenies were screened for Mlo alleles and a complete correlation between mlo/mlo genotype and resistant phenotype was found The method has been used by barley breeders for routine selection of barley genotypes resistant to powdery mildew.

Implementation of probes for tracing chromosome segments conferring barley yellow dwarf virus resistance

Two PCR-based assays were examined for tracing the presence of a Thinopyrum chromosome segment (Tc6 or Tc14) conferring barley yellow dwarf virus (BYDV) resistance in wheat breeding lines. The microsatellite gwm37 was used to assay the Thinopyrum chromosome segment or its wheat, Group 7, homoeologous segment, and was effective in characterising breeders material since heterozygous lines could be identified. A new set of primers derived from a Thinopyrum-specific DNA segment (csTiB1) provided a dominant marker that was readily scored by agarose gel electrophoresis. It was also demonstrated that the csTiB1 primers could be used to establish a solid phase PCR assay that avoided the requirement for gel electrophoresis and was amenable to use in a high-throughput, microtitre plate format. Depending on the number of DNA samples to be assayed, both primer pairs appear to have a place in breeding programs.

Subtractive hybridization of cDNA from small amounts of plant tissue

A subtractive hybridization method is described that allows the generation of a subtractive gene library from small amounts of plant or other eukaryotic tissues. The method uses paramagnetic oligo-dT beads to capture poly-adenylated mRNA and to synthesize the complementary cDNA on a solid support. The use of magnetic beads facilitates the change of reaction buffers and the removal of primers and minimizes yield losses. Subtracted material obtained from this method can either be cloned directly or used to screen a specific library.