Effie M. Ablett

Analysis of SSRs derived from grape ESTs

Abstract One hundred and twenty four microsatellites were isolated from analysis of 5000 Vitis expressed sequence tags (ESTs). A diversity of dinucleotide and trinucleotide simple sequence repeat (SSR) motifs were present. Primers were designed for 16 of these SSRs and they were tested on seven accessions. Ten of the sixteen primer pairs resulted in PCR products of the expected size. All ten functional primers were polymorphic across the accessions studied. Polymorphisms were evident at the level of cultivars, Vitis species, and between related genera. SSRs that were from the 3′ untranslated region (3′UTR) were most polymorphic at the cultivar level, the 5′ untranslated region (5′ UTR) SSRs were most polymorphic between cultivars and species, and those SSRs within coding sequence were most polymorphic between species and genera. These results show that EST-derived SSRs in Vitis are useful as they are polymorphic and highly transferable. With EST SSRs being applicable to studies at several taxonomic levels, the large number of SSRs (approximately 1000) that will be available from an expanded EST database of 45 000 will have many potential applications in mapping and identity research.

cDNA microarray analysis of developing grape (Vitis vinifera cv. Shiraz) berry skin.

Microarray analysis of Vitis vinifera cv. Shiraz developing berries has revealed the expression patterns of several categories of genes. Microarray slides were constructed from 4,608 PCR-amplified cDNA clones derived from a ripening grape berry cDNA library. The mRNA expression levels of the genes represented by these cDNAs were measured in flowers, week 2 post-flowering whole berries, week 5, week 8, week 10 (véraison, green berries), week 12 and week 13 berry skin. In addition, a comparison of RNA expression in pigmented and unpigmented berry skin at véraison (week 10) was undertaken. Image and statistical analysis revealed four sets of genes with distinctive and similar expression profiles over the course of berry development. The first set was composed of genes which had maximum RNA expression in flowers, followed by a steady decrease in expression. The most prominent group within this set were genes which have a role in photosynthesis. The second set of cDNAs was dominated by genes involved in flavonoid biosynthesis and had a peak of expression week 2 post-flowering. The data indicate co-ordinate regulation of flavonoid biosynthetic genes which code for the enzymes 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, flavonone hydroxylase, anthocyanidin reductase and cytochrome b5. The third set of cDNAs exhibited maximum expression week 5 post-flowering, midway between flowering and véraison, a period of rapid berry growth. This set of cDNAs is dominated by genes which code for structural cell wall proteins. The fourth set of genes was dramatically up-regulated at véraison and remained up-regulated until 13 weeks post-flowering. This set of genes was composed of a diverse range of genes, a reflection of the complexity of ripening, most with no known function.

Analysis of grape ESTs: global gene expression patterns in leaf and berry.

Analysis of 2479 ESTs from Vitis vinifera berry tissue and 2438 from leaf revealed that 1% of the ESTs match to known Vitis proteins, 72% to plant proteins, 11% to non-plant, and 16% had no match (P[N]>0.5). The levels of redundancy were similar in the leaf and berry libraries. Only 12% of the genes matched by the ESTs were common to both libraries indicating marked differences in the genes expressed in the two tissues. The abundance of transcripts with predicted cellular roles in leaf and berry were estimated by classifying the primary BLAST matches to known proteins (score >80) into functional categories. Thirty-six percent of the leaf transcripts were involved in photosynthesis, compared to 3% in the berry. This is a much higher proportion of transcripts involved with a function limited to specialized cells, than was found when transcripts of 33 human tissues were compared using a similar approach, suggesting plant cells may involve their cellular machinery to a greater extent in specialized activities than animal cells. Relatively enhanced expression of specific transcription factors, and genes involved in defense, detoxification, stress response, proteolysis, trafficing, and signal transduction, suggests berry tissue is actively engaged in responding to environmental stimuli.

AFLP markers distinguishing an early mutant of Flame Seedless grape

Molecular markers have been frequently used to differentiate grape species and cultivars. There are fewer cases where molecular markers have been used to differentiate grape clones within a cultivar, or for the demarcation of somatic mutants from parental clones. This study reports the first successful utility of AFLPs for the differentiation of somatic mutants from their parental grapevine line, and discusses the potential for similar AFLP applications. The somatic mutant analysed demonstrates earlier budburst characteristics than the Flame Seedless line from which is arose. Analysis of 64 AFLP primer combinations in silver stained polyacrylamide produced in excess of 3000 markers in Vitis vinifera, and provided two markers which differentiated the somatic mutant, from its parental line. One marker was 440 bpin length and was produced with primer combinationEcoR1-AT and Mse1-CTT. The second marker was 340 bp in length and generated with primer combination EcoR1-TC and Mse1-CAC.