Gordon C. Machray

Polymorphism revealed by simple sequence repeats

Simple sequence repeats (SSRs) are a group of repetitive DNA sequences that represent a significant portion of higher eukaryote genomes. They can serve as highly informative genetic markers, and in conjunction with the use of polymerase chain reaction (PCR) technology enable the detection of length variation. This novel means of detecting polymorphism targets highly variable regions of the genome, and has revolutionized human and mammalian research. It is now poised to have a significant impact in plant science.

Tuberization in potato involves a switch from apoplastic to symplastic phloem unloading

Phloem unloading was studied in potato plants in real time during the early stages of tuberization using carboxyfluorescein (CF) as a phloem-mobile tracer, and the unloading pattern was compared with autoradiography of tubers that had transported 14C assimilates. In stolons undergoing extension growth, apoplastic phloem unloading predominated. However, during the first visible signs of tuberization, a transition occurred from apoplastic to symplastic transport, and both CF and 14C assimilates subsequently followed identical patterns of phloem unloading. It is suggested that the switch to symplastic sucrose unloading may be responsible for the upregulation of several genes involved in sucrose metabolism. A detailed analysis of sugar levels and 14C sugar partitioning in tuberizing stolons revealed a distinct difference between the apical region of the tuber and the subapical region. Analysis of invertase activity in nontuberizing and tuberizing stolons revealed a marked decline in soluble invertase in the subapical region of swelling stolons, consistent with the switch from apoplastic to symplastic unloading. However, cell wall–bound invertase activity remained high in the apical 1 to 2 mm of tuberizing stolons. Histochemical analysis of potato lines transformed with the promoter of an apoplastic invertase gene (invGE) linked to a reporter gene also revealed discrete gene expression in the apical bud region. Evidence is presented that the apical and lateral tuber buds function as isolated domains with respect to sucrose unloading and metabolism.

An extreme cytoplasmic bottleneck in the modern European cultivated potato (Solanum tuberosum) is not reflected in decreased levels of nuclear diversity

We have used the polymorphic chloroplast (cp) and nuclear simple sequence repeats (SSRs) to analyse levels of cytoplasmic and nuclear diversity in the gene pool of the European cultivated potato (Solanum tuberosum ssp. tuberosum). Primers designed from the complete chloroplast sequence of tobacco (Nicotiana tabacum) were used to amplify polymorphic products in a range of potato cultivars. Combining the data from seven polymorphic cpSSR loci gave 26 haplotypes, one of which (haplotype A) accounted for 151 out of the 178 individuals studied and corresponded to the T–type cytoplasm previously identified in cultivated potatoes using chloroplast restriction fragment length polymorphism analysis. Phylogenetic and diversity analyses of the relationships between cpSSR haplotypes confirmed much higher levels of cytoplasmic diversity outwith the T–type group. Diversity levels at eight nuclear SSR loci, however, were not significantly different between cytoplasmic groups, suggesting a severe maternal bottleneck in the evolution of the modern cultivated potato. These results highlight the importance in quantifying levels of cytoplasmic as well as nuclear diversity and confirm the need for a change in breeding practices to increase levels of non–T–type cytoplasm in the cultivated gene pool, thus helping reduce problems associated with pollen sterility. This may be facilitated by germplasm analysis using cpSSRs, which will allow efficient selection of diverse cytoplasm donors.

Expression of tandem invertase genes associated with sexual and vegetative growth cycles in potato.

The organisation of two invertase genes (invGE and invGF) linked in direct tandem repeat within the potato genome is detailed. The genes exhibit a similar intron/exon structure which differs from previously described plant invertase genes; while intron locations are conserved between the genes, minor differences in exon length are seen. Both genes encode enzymes with putative extracellular location. Biochemical analysis of gene expression showed expression in floral tissues for both genes, with expression of the upstream gene (invGE) also detected in leaf tissue. Promoter sequences from both genes have been fused to the β-glucuronidase (GUS) reporter gene (uidA) and transformed into potato. One promoter-GUS reporter construct was also transformed into tobacco. Histochemical analysis of transgenic lines defined specific expression from the downstream (invGF) promoter in potato and tobacco pollen, with expression first detected in the late uninucleate stage of tobacco microspore development. The invGE promoter determined expression in pollen and other floral tissues, but also at lateral nodes in stem, root and tuber. An association of invertase expression with generative tissue, both in vegetative and sexual modes of growth, is indicated.

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.

Potato (Solanum tuberosum) invertase-encoding cDNAs and their differential expression.

A full-length cDNA clone encoding a potato invertase (Inv) has been isolated. It is highly related (77% nucleotide identity) to a previously characterised potato cDNA clone encoding a putative extracellular Inv. These Inv genes encode a subfamily of apoplastic enzymes which are shown to be distinct, on the basis of sequence similarity, from the related subfamily of vacuolar enzymes. In order to differentiate between the expression of the two potato genes encoding apoplastic Inv, a single-stranded conformational polymorphism (SSCP) assay was developed for products generated by reverse transcription-polymerase chain reaction (RT-PCR) utilising primers designed to amplify both potato sequences. Using this approach, we have shown that these two identified Inv from potato are expressed in a tissue-specific and developmentally regulated manner.

Exploiting plant somatic radiation hybrids for physical mapping of expressed sequence tags.

Methods are described for the optimisation of the generation of radiation hybrids suitable for physical mapping of a plant (barley) genome. A combination of PCR-based technologies, involving the use of whole genome, mixed primer and hemi-nested primer amplifications, can greatly extend their utility for the physical mapping of expressed sequence tags (ESTs). Using panels of hybrids and ESTs, donor DNA retention and individual marker retention frequencies for the expressed portion of the barley genome in the hybrids were estimated.

Characterisation of a complementary DNA encoding a novel plant enzyme with sucrolytic activity.

The cloning of a 1332 bp cDNA from a potato (Solanum tuberosumL.) cv. Cara leaf cDNA expression library, using an antibody raised against a purified tuber protein preparation with sucrolytic activity, is described. The corresponding gene in potato is of low copy number, is expressed in a variety of tissues, and encodes a protein which includes several domains with similarity to database sequences, including ferredoxin from Clostridium pasteurianum. Expression of the cDNA in E. coli yields a fusion protein with sucrolytic activity.

Organisation and expression of a potato (Solanum tuberosum) protein kinase gene

Abstract A 4.3 kb genomic clone from potato ( Solanum tuberosum ) cv. Saturna has been isolated which contains the full coding sequence of a protein kinase gene ( Stpk1 ), 1 kb of 3′ sequence, and the first exon of a neighbouring gene encoding a sucrolytic enzyme. An identical organisation could be detected in a genomic clone carrying a gene that encodes a protein kinase homologue in Arabidopsis thaliana (Hayashida et al., Gene, 121 (1992) 325–330). Southern and RT-PCR analyses indicate that Stpk1 is a single copy gene which is expressed throughout the plant with an increase in transcript abundance in leaf after infection with Phytophthora infestans . While comparisons using the deduced amino acid sequence for STPK1 indicate that it is most closely related to cyclic nucleotide-dependent protein kinases, it contains an additional plant-specific insert of 85 amino acids in the catalytic domain.

Processing of a 58 × 103Mr invertase from potato tubers

Abstract Processing of a 58 × 10 3 M r acid invertase to a 48 × 10 3 M r polypeptide has been demonstrated in potato tuber extracts using antibodies raised against a synthetic N -terminal peptide. Processing of the 58 × 10 3 M r protein occurred during the preparation of extracts in acetate buffer, pH 5.2, but not in Tris-HCI buffer, pH 7.5. A modified rapid extraction procedure suggested that the processing event also occurred in planta . Processing apparently increased the activity of acid invertase after foaming, a technique previously reported to dissociate the potato tuber invertase from an endogenous inhibitor. Western blot analysis revealed that the relative abundance of the 48 × 10 3 M r polypeptide was correlated with acid invertase activity.