Glutathione S-transferase: A Candidate Gene for Berry Color in Muscadine Grapes (Vitis rotundifolia)

Muscadine grapes (Vitis rotundifolia Michx.) are a specialty crop cultivated in the southern United States. Muscadines (2n=40) belong to the Muscadinia subgenus of Vitis, while all other cultivated grape species belong to the subgenus Euvitis (2n=38). The berry color locus in muscadines has been mapped to a 0.8 Mbp region syntenic with chromosome 4 of V. vinifera. In this study, we identified glutathione S-transferase4 (GST4) as a likely candidate gene for anthocyanin transport within the berry color locus. PCR and KASP genotyping identified a single intragenic SNP (C/T) marker corresponding to a proline to leucine mutation within the muscadine GST4 (VrGST4) that differentiated black (CC and CT) from bronze (TT) muscadines in 65 breeding selections, 14 cultivars, and 320 progeny from two mapping populations. Anthocyanin profiling on a subset of the progeny indicated a dominant VrGST4 action, with no allele dosage effect on total anthocyanin content or composition of individual anthocyanins. Proanthocyanidin content was similar in the seeds of both black and bronze genotypes, and seeds had much higher VrGST3 expression and lower VrGST4 expression than skins. VrGST4 expression was higher in post-veraison berries of black muscadines compared to pre-veraison berries, but no changes in gene expression in pre- and post-veraison berries were observed in the bronze muscadine cultivar. VrMybA1 expression was higher in post-veraison berries of both black and bronze muscadines. These results suggest that berry pigmentation in muscadines is regulated by a mechanism distinct from the MybA gene cluster that is responsible for berry color variation in V. vinifera.

). This is an interesting finding, as the berry color locus in V. vinifera was mapped to 165 chromosome 2 where MybA genes were identified as candidate genes (Fournier-Level et al. 166 2009) and suggests that other genes in the anthocyanin biosynthesis pathway possibly determine 167 berry color in V. rotundifolia. Therefore, the objective of this study was to identify potential   182 The annotated genes within the 11.1 to 11.9 Mbp interval on chromosome 4 of the V. vinifera 183 reference genome were explored to identify genes associated with anthocyanin biosynthesis and 184 compared with known sequences in the NCBI database using BLAST. The GST4 candidate gene 185 from the four muscadine cultivars was amplified using primers designed from the VvGST4 186 sequence. For initial PCR, genomic DNA was used to amplify the GST4 sequence using forward 187 primer 5' ATATCAAGCAGCGAGCTCCA 3' and reverse primer 5' 188 CCTCTTGGGAAAAAGCTTGG 3'. To isolate the full-length GST4 sequence, forward primer    233 File S1 contains a list with detailed information for all the supplementary tables and figures.

234
More detailed information about the materials and methods used in this study can be found in  Twenty-one genes annotated with known or putative function were identified between 11.1 to 245 11.9 Mbp on chromosome 4 of the 12X.0 version of the PN40024 V. vinifera reference genome.

246
Among those, only one full-length gene, annotated as VvGST4, was associated with the  (Table S1).  Figure 1A). Figure 1B shows the chromatograms of the nonsynonymous SNP region in the 260 bronze and black muscadine sequences with CCG codon identified in the black muscadines and 261 CTG codon in the bronze muscadine. The overlapping signals for C or T at position 512 in the 262 three black muscadines indicates the presence of both CCG and CTG alleles in these cultivars. 263 The deduced protein from the full-length VrGST4 sequence of both bronze and black-   286 In order to further validate the association of the intragenic C/T SNP with berry color in diverse 287 muscadine genotypes, KASP genotyping assay was performed on 320 progeny from the two   302 A qPCR assay was performed to detect the level of VrGST4 expression in berry skins from three 303 black muscadine cultivars and one bronze cultivar ( Figure 5). VrGST4 expression was depicted 304 as fold expression relative to VvUbiquitin reference. In all three black muscadines, VrGST4 305 expression was significantly higher in the post-veraison berries than the pre-veraison berries.  followed by petunidin (9.9 to 12.7%), peonidin (7.1 to 9.6%), cyanidin (6.4 to 9.3%), and 349 malvidin (2.7 to 5.0%). Petunidin, peonidin, and cyanidin content were similar among the three respectively. In all four genotypes, VrGST3 expression was higher in seeds compared to berry 367 skins with no difference in the expression between bronze and black muscadine seeds. In 368 contrast, as observed in Figure 5, VrGST4 expression was higher in berry skins than in seeds. A 369 similar trend was observed for VrMybA1, with higher expression observed in berry skins 370 compared to seeds. In general, VrGST4 expression had the greatest fold difference between seeds 371 and skins of both bronze and black genotypes, followed by VrGST3 and VrMybA1 expression.

372
VrGST3 expression, averaged across the two bronze and two black genotypes, was 33-and 14.5-373 fold higher in seeds than in berry skins, respectively ( Figure 9A), while average VrGST4 374 expression was ~500-and ~600-fold higher in berry skins than seeds of the bronze and black 375 genotypes, respectively ( Figure 9B). On the contrary, VrMybA1 showed only 2 to 3-fold higher 376 expression in berry skins compared to seeds in both bronze and black genotypes ( Figure 9C).

377
Results suggest a differential expression pattern with these three genes in seeds and berry skins 378 of bronze and black muscadines.

418
In this study, we initially isolated a partial genomic DNA sequence of VrGST4 from 419 muscadine cultivars. Hence, we could not determine the intron:exon structure directly by 420 comparing the VrGST4 genomic DNA with VrGST4 cDNA. However, based on the grouping of 421 VrGST4 protein sequence deduced from the full-length cDNA, it appears that VrGST4 from 422 muscadine has a gene structure similar to Type I GSTs, which contain three exons and two 423 introns. Although the Tau GSTs have a gene structure that is different from Phi GSTs, they were 424 found to complement each other functionally in terms of anthocyanin transport. For example,

425
ZmBZ2 and PhAN9 reciprocally complemented an9 and bz2 tissues in particle gun 426 bombardment assays (Alfenito et al. 1998), even though they have only 12% amino acid identity. 427 This functional complementation between monocot and dicot GST proteins suggests that they 428 share a common ancestral gene before evolution into species-specific GSTs that transport and VvGST4 were associated with anthocyanin transport in berries, VvGST3 was found to play a 520 major role in PA transport in seeds. In this study, we measured PA content in seeds of a  Figure 9C). There was no significant difference in the PA content among the three genotype 526 classes, indicating that bronze and black muscadines do not differ in PA content. There was 527 differential expression of VrGST3 and VrGST4 in seeds and berry skins. The high expression of 528 VrGST3 in seeds and weak or total absence of expression in berry skins of both bronze and black 529 muscadine genotypes, suggests that VrGST3 is almost exclusively involved in PA transport. In In this study, we isolated and characterized the candidate gene, VrGST4, responsible for 541 berry color variation in muscadine grapes for the first time. We identified a non-synonymous 542 SNP (C/T) within VrGST4 that corresponded to a proline to leucine mutation in the bronze 543 muscadines. A diagnostic KASP marker was developed from the intragenic SNP which co-