Development of molecular markers for the trait of formation to improve breeding efforts

L. Hausmann; K. Neumann; R. Eibach; E. Zyprian; R. Töpfer

BAZ – Federal Centre for Breeding Research on Cultivated Plants

Institute for Grapevine Breeding Geilweilerhof

D-76833 Siebeldingen

Email: [email protected]

Summary -3,5-diglycoside, shortly malvin, early gained the interest of viticultural research. It was Ribéreau-Gayon who placed malvin within the focus being considered as indicative for hybrid grapevine varieties (Ribéreau-Gayon 1960). However, simultaneously he indicated that not all hybrids show this coloured compound. Despite this fact, for a long time malvin was used as an analytical marker for hybrid wines which were discriminated for their poor wine quality. Recent success of resistance breeding in grapevine gave clear cut evidence that malvin and wine quality show no correlation of any kind. Up to know the biosynthesis of malvin in the genus has not been elucidated in great detail. Recently, evidence was gained from the ornamental plant Perilla frutescens pointing to an 5-O-glucosyltransferase (5-GT) as the catalysing enzyme. However, an additional pathway may exist using -3-glucoside as a precursor and requiring methyltransferase reactions for modification of the intermediates. First analysis was conducted to identify 5-GT sequences in Vitis in order to develop a specific molecular marker. Thus, five different gene sequences were isolated and their expression pattern was monitored. The expression pattern of one of these genes was found to correlate with malvin formation.

Introduction The quality of wine is influenced by numerous compounds in particular sugar, alcohol, acid content, different aroma components (Rapp 1996, 1999; Wüst & Mosandl 2002; Amann 2003), phenols, tannins, etc. and in red wines by (Hesford & Ruffner). Furthermore, colour intensity of red wines depends on (a) the type and (b) the concentration of anthocyanins. For decades grapevine breeders in Germany paid much attention to this characteristic when selecting for new cultivars. Thus, it is self-explanatory that breeding strains and new cultivars like ‘Regent’ have been obtained which behave excellent in colour formation and colour intensity. A comparison of two red wine cultivars, the traditional cultivar ‘Lemberger’ and the new bred ‘Regent’, is given in Fig. 1 showing the difference in anthocyanin profiles as monitored by HPLC analysis. Both cultivars differ in regard of type and concentration of anthocyanins. Most obvious are differences in (malvidin-3- glucoside, red) and malvin (malvidin-3,5-diglucoside, dark red), respectively. Malvin can only be detected in ‘Regent’ while oenin as the presumed precursor of malvin occurs in both genotypes. Compared with oenin, malvin carries an additional glucose residue increasing its solubility, stability and colour intensity. -diglucosides in grapevine have been investigated since a long time. Especially malvin was analysed by Ribereau-Gayon and coworkers, because they considered malvin to be typical for hybrid vines (Ribéreau-Gayon 1960). However, Ribéreau-Gayon ascertained simultaneously that not every hybrid contains this colour type (Ribéreau-Gayon 1960). Nevertheless, for a long time malvin had been considered to be an indicator for hybrid wines which were proscribed due to their poor wine quality. Since this correlation was true for just a particular type of vines but not generally, progress in grapevine breeding puts further question marks on it. Recently, newly bred cultivars derived from resistance breeding were classified showing clearly that malvin content and wine quality are not correlated at all. Further evidence is coming from genetic analysis and is presented below. The biosynthesis of malvin in Vitis has not been elucidated completely. Recent tests on an ornamental plant (Perilla frutescens) have shown an UDP-glucose:anthocyanin 5-O- glucosyltransferase (5-GT) being the catalysing enzyme (Fig. 2). Biosynthesis of malvin seems to be also possible via -3,5-diglucoside catalysed by a methyltransferase (MT) (Fig. 3). In a first approach we initiated experiments in order to identify candidate genes for 5- GT from Vitis in order to develope a specific molecular marker.

Lemberger

Malvin Oenin

Regent

Malvin Oenin

Fig. 1: Chromatograms of red wines from the cultivars ‘Lemberger’ and ‘Regent’ analysed by HPLC. Major differences are the concentrations of oenin (malvidin-3-glucoside) and malvin (malvidin-3,5-diglucoside), the latter being absent in cv. ‘Lemberger’.

Material and methods Analysis of anthocyanins berries were crushed in a mortar and boiled for 2-3 minutes in a microwave. After chilling on ice, berries were pressed and juice was centrifuged at 2150 x g for 10 minutes to remove turbidity. Prior to HPLC analysis for anthocyanins according to O.I.V. recommendations (http://www.oiv.int) the juice was passed through a filter of 0.45 µm pore size. PCR-/3’-RACE Using degenerate primers based on published 5-GT sequences fragments of genomic DNA of various genotypes were PCR-amplified. Primers 04034, ACITTYCCIGCICARGGICAYAT HAAYCC and 04032 TTCCAICCRCARTGIGTIACRAARCAICC were used. RNA for gene expression analysis was extracted from berry skin according to Boss et al. (1996a). 3’-RACE- PCR was performed using the BD Smart RACE cDNA Amplification Kit from Biosciences Clonetech (Heidelberg). The following gene specific primers were used, which were deduced from cloned and sequenced 5GT-candidate genes: gene 5: 04095, GGTCCTAGCTCACAAAGCCG; gene 6: 05026, TCGTGGATTGCTGGACAGTGGCCG; gene 7: 05027, GCTCGCGGGTTGTTAGAAAGCGGAAGAC; gene 8: 05028, GCACGAG GTTTGCTAGATTGTGGCCAGC; gene 9: 05034, GCCTGTGGTCTGCTAAATAGTGAC CGAC; 3-GT: 04121, TTGGAGTTTCAGGCATTCAAGG. Touchdown PCR was performed according to the suppliers recommendations. Mapping and linkage/recombination analysis For linkage analysis the program JoinMap 3.0 (Van Ooijen and Voorrips 2001) was used according to Fischer et al. (2004).

Results and discussion Cultivars ‘Lemberger’ and ‘Regent’ show clear-cut differences concerning the anthocyanin profiles (Fig. 1). Both cultivars produce anthocyanines (= 3-glucoside) of delphinidin, , petunidin, and malvidin (see Fig. 1 and Fig. 3) although in different concentrations. Oenin is the most abundant anthocyanin found in ‘Lemberger’, entirely lacking diglucosides. Cultivar ‘Regent’, giving deep coloured red wines, in addition shows high amounts of 3,5-diglucosids particularly malvin. It is interesting to note that significant

OH

OH

+ HO O OH

OH OCH3

OH OH

Delphinidin + HO O OCH3

O-Gluc OCH3 OH Malvidin-3-glucosid OH

+ HO UDP-glucose: O 5-GT anthocyanin OCH3 5-O-glucosyltransferase O-Gluc Malvidin-3,5-diglucosid O-Gluc

Fig. 2: Structure of the anthocyanidin body (delphinidin, blue violet) and a derivative thereof, malvin-3-glucoside (oenin, red). The formation of malvidin-3,5 diglucoside (malvin, dark red) is catalysed by a 5-GT (=UDP-D-glucose:anthocyanin 5-O-β-D-glucosyltransferase). An alternative biosynthesis is presented in Fig. 3. amounts of other anthocyanidin-diglucosides then malvin can not be observed in ‘Regent’. They would be expected if malvin-synthesis would take the route via delphinidin- and petunidin-3,5-diglucoside as indicated in Fig. 3. If this observation is true, at least for ‘Regent’ the major biosynthesis of malvin is catalysed by an UDP-D-glucose:anthocyanin 5- O-β-D-glucosyltransferase (5-GT).

Phenylpropanoid Pathway

Flavonoid Pathway

3-GT 3-GT

Cyanidin-3-G Delphinidin-3-G MT 5-GT Peonidin-3-G MT 5-GT Petunidin-3-G Malvidin-3-G MT

5-GT Cyanidin-3,5-DG 5-GT Delphinidin-3,5-DG 5-GT

MT Peonidin-3,5-DG MT Petunidin-3,5-DG Malvidin-3,5-DG MT

Fig. 3. Biosynthesis of anthocyanidin-3,5-diglucosides. Precursors are derived from the phenylpropanoid/ pathway. Malvidin-3,5-diglucoside (malvin, dark red) is being synthesised either by UDP-glucose:anthocyanin 5-O-glucosyltransferase (5-GT) or alternatively via petunidin-3,5-diglucoside and its methylation. Diglucosides are highlighted in grey. 3-GT = UDP-D-glucose:anthocyanidin 3-O-β-D-glucosyltransferase (EC 2.4.1.115); 5-GT = UDP-D-glucose:anthocyanin 5-O-β-D-glucosyltransferase; MT = methyltransferase.

Cluster I 5-GT

Cluster II 3-GT

Cluster III (apparently unrelated sequences)

Fig. 4: Cluster analysis of amplified and published GT-gene sequences. The primers used preferentially amplified 5-GT-related sequences. Consequently we looked for 5-GT-genes in ‘Regent’ as well as in other genotypes. Degenerate primers and genomic DNA were used for amplification of 5-GT -sequences which significantly differ from 3-GT-sequences as described by Yamazaki et al. (1999). Sequence analysis of the cloned amplification products revealed five different GT-homologous genes. Altogether these and the published GT-sequences form three major gene-clusters (see Fig. 4). Cluster I consists of 5-GT-genes, cluster II goups 3-GT-genes and cluster III covers sequences showing rather unrelated GT-genes (Vogt & Jones 2000). Four GT-genes obtained from ‘Regent’ (gene 6, 7, 8, and 9) are closely related to 5-GT-genes found in other plants. In contrast gene 5 is located slightly separated (Fig. 4, arrow). This gene shows higher homology with an UDP-glucose:salicylic acid glucosyltransferase from tobacco. The sequence information obtained was used to deduce gene specific primers for 3’-RACE- analysis in order to monitor the gene expression of the 5-GT gene family (see Fig. 5 and Table 1). Different genotypes including some found in the pedigree of ‘Regent’ were analysed for candidate gene expression in the skin of maturing berries. Fig. 5 shows as an example the expression of 3-GT, a prerequisite for colour formation resulting in the first coloured anthocyanin metabolites (Boss et al. 1996b, Ford et al. 1998). Expression of 3-GT in berries is indicative for red cultivars. Thus, it was expected that the red cultivars from ‘Regent’ to ‘Munson’ (Fig. 5) show expression of the 3-GT. ‘Seibel 880’ is considered as a white

er in ss ei M . 1 . 2 l. in p p n a c nz re re ci 3 sc ia a 8 p p r or 6 u r G 6 0 0 l u ll x 1 r a e 4 8 8 u t bo e eu 5 ab ip t n n 6 8 8 o n c r el l r an to so el el el ep a ge am an e b is is c n n b b b ah u n e h h ub ei it it li li u ei ei ei o iq ia __R __C __C __S __S __V __V __A __C __M __S __S __S __N __P __D 5-GT Å 700 bp

D D D D D D D D D D ______3-GT

Å 1050 bp

Fig. 5: Comparative expression analysis of gene 7, a presumed candidate for UDP-D- glucose:anthocyanin 5-O-β-D-glucosyltransferase (5-GT), and UDP-D- glucose:anthocyanidin 3-O-β-D-glucosyltransferase (3-GT) in berry skin of various genotypes (‘Regent’, its parents ‘Diana’ and ‘Chambourcin’, as well as other genotyopes found in the pedigree ‘Regent’). The tested genotypes of Vitis wild species are just representatives of the species described in the ‘Regent’ pedigree. Grey bar = vines showing red berries, white bar = vines developing white berries. D = diglucoside forming vines. The expression of candidate gene 7 is correlated with diglucoside formation. ‘Seibel 880’ a white berry genotype indicates low expression of gene 7 and 3-GT only in berries of advanced ripening stages. The first RNA preparation includes skin from berries with slightly reddish areas resulted in a gene expression signal while a second RNA preparation from white berries of normal ripening stags did not result in GT-expression. genotype, however, berries show slightly reddish areas upon advanced berry ripening. Thus, low 3-GT-expression within a first RNA preparation can be explained. A second RNA preparation from ‘Seibel 880’, avoiding berries with reddish skin, showed no 3-GT expression indicating 3-GT again as the prerequisite for colour formation. Cultivar ‘Piquepoul’ is a traditional cultivar typically forming red berries and thus showing 3-GT expression. A look on the gene expression of candidate genes 5 and 6 as summarized in table 1 reveales gene expression in all genotypes. No correlation with malvin formation exists. Similarly candidate genes 8 and 9 show no correlation, since both are not expressed in the berry skin. In contrast, candidate gene 7 is expressed in all genotypes showing malvin formation. Even in ‘Seibel 880’ transcripts of gene 7 can be detected if berry skins with slightly reddish areas were analysed. White berry skins of ‘Seibel 880’, however, fail to result in an amplification product (Fig. 5). Furthermore, gene 7 correlates also with 3-GT expression except for the malvin free cultivar ‘Piquepoul’ which does not carry pieces of wild Vitis genomes. Thus, gene 7 shows a clear-cut correlation with (a) formation of anthocyanins and (b) formation of diglucosides.

Table 1: Overview about the gene expression of candidate genes for 5-GT (UDP-D- glucose:anthocyanin 5-O-β-D-glucosyltransferase). Three genes showed expression in berry skins, two of them did not. Gene 8 and 9 might be expressed in a different tissue or represent pseudogenes. N (noir) = red berries; B (blanc) = white berries; genotypes showing diglucosides in the must are highlighted in light grey. Red berries are labelled dark grey.

5-GT candidate genes

Cultivar/Genotype 3-GT gene gene gene gene gene 5 6 7 8 9 '‘Regent’' N ++ ++ + ++ - - 'Chambourcin' N ++ ++ ++ + - - 'Chancellor' N ++ ++ ++ ++ - - 'Subereux' N ++ ++ ++ ++ - - 'Seibel 5163' N ++ ++ + ++ - - 'Vitis labrusca' N ++ ++ ++ ++ - - 'Vitis riparia Cl.M.' N ++ ++ ++ ++ - - 'Alicante Ganzin' N ++ ++ + ++ - - 'Clinton' N ++ ++ ++ ++ - -

'Munson' N + + - + - - 'Seibel 6468' B - ++ ++ - - - 'Seibel 880' 1.prep. B (+) ++ + (+) - - 'Seibel 880' 2.prep. B - ++ + - - -

'Noah' B - + + - - - 'Piquepoul' N ++ ++ + - - - 'Diana' B - ++ ++ - - -

In parallel to the characterisation of candidate genes for anthocyanidin-diglucoside synthesis the trait malvin formation was mapped in a population of ‘Regent’ x ‘Lemberger’ (Fischer et al. 2004). The mapping population consists of 144 individuals, 97 of them produce red berries. The anthocyanin pattern of the must of red F1 descendents was determined by HPLC analysis in three subsequent years. Despite quantitative differences over the years, which are caused by environmental influences, the results were qualitatively consistent. Thus, a clear 1:1 segregation was determined. Within the integrated consensus-map the phenotypical marker malvin was located at the margin of linkage group 12 (corresponding to linkage group 9 according to the internationalen mapping nomenclature). The next marker (microsatellite marker VMC1c10) was found in a distance of 12 cM (Fig. 6). Most important is the finding that malvin formation is not linked to resistance traits. Additionally, traits like berry colour or 3-GT are inherited independently since they are located on different linkage groups in the genetic map of ‘Regent’ (Fischer et al. 2004, Zyprian, unpublished).

12 (IGGP-9)

0 Malvi n

122222 VMC1c1 0 19 R14199

30 N171400

42 O1670

Fig. 6: Linkage group 12 of the integrated map of ‘Regent’ X ‘Lemberger’ (corresponding to linkage group IGGP 9). The malvin locus is found at the margin of linkage group 12 12 cM apart from the next marker.

Perspective Gene 7 seems to be a good candidate for a 5-GT and its mapping is in progress. Currenly Marker VMC1c10 shows a resonably good correlation with malvin formation. Functional studies need to be done to proof the presumed function of gene 7.

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