GRET1 RETROTRANSPOSON AND VVMYBA1 GENE SEQUENCES IN SOMATIC MUTANTS OF NEW

Somatic mutations in grapes are relatively frequent and associated with diversity in grape skin color and berry morphology. Mutations that occur on a side branch of the ‘Benitaka’ cultivar with rosy-red berry skin color generated the ‘Brasil’ cultivar, and mutations that occurred on a side branch of ‘Brasil’ generated the ‘Black Star’ cultivar, both showing a black color in the berry skin. Therefore, genetic characterization of the Gret1 retrotransposon and the VvmybA1 gene in ‘Italia’, ‘Rubi’, ‘Benitaka’, ‘Brasil’, and ‘Black Star’ was started to find whether the altered coloration of berries in ‘Brasil’ and ‘Black Star’ is a product of different mutation patterns in the investigated sequences. Six primer combinations were used for the amplification of different sequences of the Gret1 retrotransposon and VvmybA1 gene of the five cultivars. Polymerase chain reaction (PCR) of the Gret1 retrotransposon and the VvmybA1 gene and sequencing of the amplified products using six primer combinations showed no different alleles or different nucleotide sequences in ‘Brasil’ and ‘Black Star’. The sequencing of the VvmybA1 gene in the present study showed that the mutations that occurred in the cultivar ‘Italia’ for generating the ‘Benitaka’ cultivar persisted in the ‘Brasil’ and ‘Black Star’ cultivars.


INTRODUCTION
The diversity of grape skin color is relevant to the preference and marketing of table grapes and wine grapes. Somatic mutations in grapes are relatively frequent events associated with the diversity of grape skin color and berry morphology, which are economically important characteristics. Somatic mutations in the 'Italia' cultivar [Piróvano 65 (VIVC 5582: Vitis International Variety Catalogue (www.vivc.de) database)] and its somatic mutants have been the biological source for at least five new varieties of table grape. 'Italia' (white berries) is a hybrid of the cross between 'Bicane' (white berries; VIVC nº 1341) and 'Muscat Hamburg' (black berries; VIVC nº 8226) bred by Ângelo Pirovano in Italy. Independent mutations that occurred on side branches of the 'Italia' cultivar generated the 'Rubi' (VIVC nº 22689; Kishino and Mashima, 1980), 'Benitaka' (VIVC nº 19816;Sousa, 1996), and 'Redimeire' (Pires et al., 2003) cultivars. Mutations that occurred on side branches of the 'Benitaka' cultivar generated the 'Brasil' cultivar (VIVC nº 19817;Gonçalves, 1995), and mutations that occurred on side branches of the 'Brasil' cultivar generated the 'Black Star' cultivar (Roberto et al., 2012). The light rosy-red color of the berry skin and a more intense and uniform rosy-red color are observed in the 'Rubi' and 'Benitaka' cultivars, respectively, while black and red colors have been observed for the berry skin and pulp, respectively, of the 'Brasil' cultivar. The most striking feature that distinguishes the 'Black Star' grape as a new variety from the 'Brasil' cultivar is the ellipsoid and elongated shape of its berries.
Several biochemical and molecular markers have been used to assess the genetic divergence between the 'Italia' cultivar, showing the green color of the berry skin and its colored somatic mutants (Oliveira-Collet et al., 2005;Orasmo et al., 2007;Maia et al., 2009Maia et al., , 2018Strioto et al., 2019a,b). However, the biochemical and molecular divergence analysis could not explain the differences between the colors and shapes of the grape berries of the different cultivars. Berry skin color differences may reflect the polymorphism of genes controlling pigment distribution in them but not of genes associated with intermediary metabolism or with determined DNA segments distributed in the genome of the cultivars. However, Xia et al. (2021) have suggested that the methylation levels of the VvmybA1 promoter play a crucial role in regulating grape skin coloration. The high methylation level of the VvmybA1 promoter was positively correlated with low concentration of anthocyanins. The color of red and black grapes may result from the formation, accumulation, and distribution of anthocyanins, which exist in the berry skin. Expression of the gene encoding UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) is essential for anthocyanin synthesis in grapes (Boss et al., 1996a,b;Ford et al., 1998). UFGT is expressed in all colored cultivars but is not expressed in white cultivars (Kobayashi et al., 2001). UFGT transcription is regulated by two MybA transcription factors that are encoded by the genes VvmybA1 and VvmybA2, organized in a cluster on chromosome 2 (Kobayashi et al., 2002) while a set of three functional genes (VvmybA1, VvmybA2, and VvmybA3) for UFGT transcription factors are associated with the grape berry pigmentation pathway (Walker et al., 2007). Kobayashi et al. (2005) showed that VvmybA1 plays an important role in regulating anthocyanin biosynthesis in grapes. The emergence of the white berry genotype is a result of silencing the VvmybA1 gene (Kobayashi et al., 2005;Walker et al., 2007) because of the insertion of the retrotransposon Gret1 into its promoter (Lijavetzky et al., 2006;This et al., 2006;Azuma et al., 2009). The bud-mutation from white to red berry skin in grapes is caused by the deletion of the retrotransposon inserted into VvmybA1. Cultivars with Gret1 inserted into the promoter region of the VvmybA1 gene (VvmybA1a allele) in homozygosity do not produce anthocyanins, as in the case of the white cultivar 'Italia', whereas colored cultivars have at least one functional VvmybA1 allele (VvmybA1b or VvmybA1c). An intra-long terminal repeat (LTR) recombination between the 5′-LTR and 3′-LTR in the Gret1 gene led to the loss of an internal part of the retrotransposon and caused the recovery of VvmybA1 expression (allele VvmybA1b) and anthocyanins production in the colored berries of 'Ruby Okuyama' (Kobayashi et al., 2004(Kobayashi et al., , 2005. Kobayashi et al. (2004) showed that the cultivar 'Ruby Okuyama' is heterozygous for the VvmybA1a and VvmybA1b alleles. However, Azuma et al. (2009) reported that the recovery of the pink skin color in 'Benitaka' was determined by the occurrence of the functional allele VvmybA1 BEN at the VvmybA1 locus, supposedly produced by homologous recombination between VvmybA1a and VvmybA3. 'Benitaka' has the VvmybA1 locus in heterozygous with the VvmybA1a allele (non-functional) and a new VvmybA1 BEN allele that restores the transcription of the VvmybA1 gene.
The events involved in restoring the transcriptional activity of the VvmybA1 gene have been reported for the 'Ruby Okuyama' and 'Benitaka' cultivars. However, reports that address any changes in the VvmybA1 gene related to the change in color from black detected in the berry skin of the 'Brasil' and 'Black Star' cultivars were not found in the literature. Black grape cultivars stand out for their high content of phenolic compounds and antioxidant properties (Hadidi et al., 2014). Furthermore, the antibacterial properties (Georgiev et al., 2014) and the highest nutraceutical properties (Crupi et al., 2015) reported for black grapes encourage researchers to investigate and understand their generation. The current study investigates sequences of the Gret1 retrotransposon and the VvmybA1 gene in the 'Italia', 'Rubi', 'Benitaka', 'Brasil', and 'Black Star' cultivars to find if the altered coloration of berries in the 'Brasil' and 'Black Star' cultivars is a product of different mutation patterns in the Gret1 retrotransposon and VvmybA1 sequences.

Polymerase chain reaction (PCR) amplification of the Gret1 retrotransposon and the VvmybA1 gene in cultivars
The origin of the 'Brasil' and 'Black Star' cultivars and striking features of the shape and color of the berries that distinguish the 'Rubi', 'Benitaka', 'Brasil', and 'Black Star' grapes as new varieties from the 'Italia' cultivar are shown in Figure 1. Partially expanded contaminant-free leaves were collected from four plants of each vineyard of 'Italia', 'Rubi', 'Benitaka', 'Brasil', and 'Black Star' cultivars, established at 23° 30′ 56″ S/51° 47′ 57″ W, in Marialva, Paraná state, Brazil. Samples were individually stored in labeled plastic screen bags to avoid mixing of grape varieties, maintained on ice (at 4 °C), transferred to the laboratory, frozen in liquid nitrogen, and stored at -80 °C until DNA extraction. DNA was extracted from leaf tissues following Thomas et al. (1993), with minor modifications reported by Strioto et al. (2019a), which specified using 100 mg leaves from individual plants instead of 2.0 g. Leaf samples from each plant were centrifuged for 10 min at 3000 rpm at room temperature (~22 °C). DNA quantity was determined using a Picodrop Spectrophotometer (Pico 100; version 4.0/21/03/11). The average concentration of DNA was between 33 ng/L and 450 ng/L per sample. After quantification, DNA samples were diluted to a final concentration of 10 ng/L until use. Amplification reactions were performed using DNA extracted from 20 young leaves obtained from five cultivars, and the "F" (forward) and "R" (reverse) primers a, d3, b, e1, c, and d (Table I).  (Lijavetzky et al., 2006), a (F) -c (R) , b (F) -c (R) (Kobayashi et al., 2004;Azuma et al., 2007), and b (F) -d (R)     recombination between the 5′-LTR and 3′-LTR in the Gret1 gene in 'Ruby Okuyama' (according to Kobayashi et al., 2004).

Combinations of primers a
PCR was performed with 20 μL, containing 20 ng of genomic DNA, 1× buffer (10 mM Tris-HCl pH 8.8), 2.0 mM of MgCl 2 , 0.2 mM each of dATP, dGTP, dCTP, and dTTP, 0.3 μM of each primer, one unit of Taq polymerase platinum (Invitrogen; Lonza Bioscience, Morrisville, NC, USA), and Milli-Q water to bring the reaction to the final volume. PCR was performed on a Veriti thermal cycler (Applied Biosystems), and the cycling conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 30 s, annealing at 60-65 °C for 30 s, and extension at 72 °C for 60 s. A final extension step was performed at 72 °C for 5 min. After DNA amplification, 4 μL loading buffer (0.25% bromophenol blue and 30% glycerol) was added to each amplification product, and electrophoresis was performed on a 1.2% agarose gel with 0.5× TBE buffer (44.5 mM Tris-borate and 1 mM EDTA) at 60 V for 4 h. After electrophoresis, the gels were stained with ethidium bromide at 0.5 μg/mL, and images were acquired with a Molecular Image LOCCUS L-PIX-HE system using Picasa 3. The size of the PCR products was determined using a 1-kb DNA ladder (Invitrogen).  Basic local alignment search tool (Altschul et al., 1990) was used to compare the sequences obtained with the sequences available in the NCBI database. The AB111100.1 sequence of the 'Italia', which is 20,286-bp long comprising the Gret1 retrotransposon and the VvmybA1 gene, was used to align the sequences, amplified using the six primer combinations. BioEdit (Hall, 1999) was used to align the sequencing results with the AB111100.1 sequence available in the database (https://www.ncbi.nlm.nih.gov/nuccore/AB111100. 1).
The primer combination b (F) -e1 (R) used to amplify the 5′-LTR of the Gret1 retrotransposon (Lijavetzky et al., 2006) revealed an allele of 1,100 bp in the five cultivars, showing that 'Italia', 'Rubi', 'Benitaka', 'Brasil', and 'Black Star' cultivars have the Gret1 retrotransposon inserted into the promoter of the VvmybA1 gene at one of the homologous chromosomes ( Figure 3C). The primer combination b (F) -d (R) used to amplify the 5′-LTR of Gret1 retrotransposon ) revealed an allele of 1,000 bp in the five cultivars, supporting the evidence that the 'Italia', 'Rubi', 'Benitaka', 'Brasil', and 'Black Star' cultivars have the Gret1 retrotransposon inserted into the promoter of the VvmybA1 gene at one of the homologous chromosomes ( Figure 3D).
The a (F) -c (R) primer combination used to amplify the VvmybA1 locus and the 3′-LTR region of the Gret1 retrotransposon (Azuma et al., 2007) revealed an allele of 1,600 bp in the five cultivars and an allele of 2,000 bp in the 'Benitaka', 'Brasil', and 'Black Star' cultivars ( Figure 3E). The b (F) -c (R) primer combination used to amplify the VvmybA1 locus and 3′-LTR of the Gret1 retrotransposon (Azuma et al., 2007) revealed an allele of 900 bp in the five cultivars and an allele of 1,600 bp in the 'Rubi' cultivar ( Figure 3F). The use of the six primer combinations to amplify specific sequences of the Gret1 retrotransposon and the VvmybA1 gene in the 'Italia', 'Rubi', 'Benitaka', 'Brasil', and 'Black Star' cultivars showed that the 'Italia' cultivar is homozygous at the VvmybA1 gene (VvmybA1 ITA /VvmybA1 ITA ) and the 'Rubi' and 'Benitaka' cultivars with the VvmybA1 gene in heterozygous (VvmybA1 ITA /VvmybA1 RUB and VvmybA1 ITA /VvmybA1 BEN , respectively); this is consistent with the findings of Kobayashi et al. (2004) and Azuma et al. (2009). The six primer combinations also showed the VvmybA1 ITA /VvmybA1 BEN genotype in the 'Brasil' and 'Black Star' cultivars. No additional alleles were observed in the new cultivars as 'Brasil' generated from a somatic mutation in 'Benitaka', and 'Black Star' generated from the 'Brasil' cultivar.
The six PCR products obtained using the b (F) -c (R) primer combination were sequenced to identify mutation sites in the nucleotide sequences of the VvmybA1 gene. Sequences amplified by the b (F) -c (R) primer combination showed changes in nucleotides at three positions in the first intron of VvmybA1. Sequences amplified using the b (F) -c (R) primer combination also showed changes in the nucleotides in the first and third exons of VvmybA1. The ATC codon in the first exon of the VvmybA1 gene encoding the amino acid isoleucine in the 'Italia' and 'Rubi' cultivars was replaced by ACC encoding the amino acid threonine in the 'Benitaka', 'Brasil', and 'Black Star' cultivars ( Figure 4A).
The dinucleotide "TA" corresponding to the 15,437 th and 15,438 th positions in the "Italia" and "Rubi" cultivars was replaced by the dinucleotide "AG" in the 'Benitaka', 'Brasil', and 'Black Star' cultivars ( Figure 4B). The "A" nucleotide at position 15,477 of the first intron of VvmybA1 in 'Italia' and 'Rubi' was replaced by "G" in the 'Benitaka', 'Brasil', and 'Black Star' cultivars. The deletion of "T" was observed in the third exon of VvmybA1 in the 'Brasil' cultivar ( Figure 4C). The replacement of "T" of the ATC codon in the 'Italia' cultivar by "C" in 'Benitaka', conferring the exchange of the codon for ACC in the first exon of VvmybA1, has also been reported by Azuma et al. (2009). PCR amplification of the Gret1 retrotransposon and the VvmybA1 gene showed no different alleles in 'Benitaka', 'Brasil', and 'Black Star'. Except for the deletion of a nucleotide "T" at position 15,768 of the VvmybA1 gene in the 'Brasil' cultivar that was present in 'Black Star', the sequencing of the amplified products using the six primer combinations showed no difference in the nucleotide sequences in 'Benitaka', 'Brasil', and 'Black Star'. The deletion of "T" at position 15,768 of the VvmybA1 gene in the 'Brasil' cultivar that was present in the 'Black Star' cultivar may be due to a somatic mutation that occurred in the 'Brasil' cultivar after the generation of 'Black Star'. It seems that this phenomenon was not associated with the diversity of grape skin color and berry morphology. Somatic mutations in grapes appear to be relatively frequent events that may or may not be associated with the diversity in grape skin color and berry morphology. A higher number of anthocyanins has been reported in black-berried grape cultivars (Conner and Maclean, 2013;Fan et al., 2018). Röckel et al. (2020) showed that repetitive DNA elements (grapevine color enhancer, GCE) in the promoter of VvmybA1 were strongly associated with an increase in the expression of VvmybA1 itself, the VvUFGT gene regulated by it, and the anthocyanins content. However, anthocyanins profile variations may be due to the differential accumulation of anthocyanins types (He et al., 2010). High levels of cyanidin-based (red) or delphinidin-based anthocyanins (blue to purple) may be determinants of berry skin color (Mu et al., 2014;Azuma et al., 2015). Thus, the transcript levels of flavonoid 3′-hydroxylase and flavonoid 3′, 5′-hydroxylase may be involved in the biosynthesis of cyanidin-based and delphinidin-based anthocyanins. Furthermore, several alleles of the VvmybA1 and VvmybA2 genes are associated with the regulation of anthocyanins and tannins biosynthesis in grapes (Jiu et al., 2021). A detailed functional characterization of the VvmybA2r and VvmybA2w genes as well as their potential regulatory networks involved in the formation of berry color has been reported by Jiu et al. (2021). Investigation of the VvmybA2 gene and the epigenetic events in the promoter of the VvmybA1 gene, as suggested by Xia et al. (2021), are potential focus areas for future studies to identify events related to changes in color to black, detected in the berry skin of the 'Brasil' and 'Black Star' cultivars.

CONCLUSIONS
Changes detected in the VvmybA1 gene, which plays an important role in regulating anthocyanins biosynthesis in grapes, did not explain the color change to black in the berries of the 'Brasil' and 'Black Star' cultivars. The sequencing of the VvmybA1 gene in the present study showed that the mutations that occurred in the cultivar 'Italia' for generating the cultivar 'Benitaka' (replacement of "TA" by "AG" at the 15,437 and 15,438 positions, "A" by "G" nucleotides at position 15,477 of the first intron, and "ATC" by "ACC" codon in the first exon of the VvmybA1 gene) persisted in the 'Brasil' and 'Black Star' cultivars. Despite the detected differences in the VvmybA1 gene, specific mutations that occurred only in the 'Brasil' and 'Black Star' cultivars with black berries could not be determined.