Azuma, A.

Fukai, E.

Hirochika, H.

Kobayashi, S.

Mitani, N.

English

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Vitis 48 (1), 55–56 (2009)Research NoteA retrotransposon-inserted VvmybA1a allele has been spread among cultivars of Vitis vinifera but not North Ameri-can or East Asian Vitis speciesN. MITANI1), A. AZUMA1), E. FUKAI2), H. HIROCHIKA2) and S. KOBAYASHI1)1) Grape and Persimmon Research Station, National Institute of Fruit Tree Science, Higashi-Hiroshima, Hiroshima, Japan2) Molecular Genetics Department, National Institute of Agrobio-logical Sciences, Tsukuba, Ibaraki, JapanK e y   w o r d s :  anthocyanin, myb, retrotransposon, grape, skin colour.Introduction: The colour of grape skins is deter-mined by the accumulation of anthocyanins. Previously, we showed that a Myb-related gene, VvmybA1, regulates anthocyanin biosynthesis in grapes (KOBAYASHI et al. 2004, 2005). Furthermore, LIJAVETZKY et al. (2006) and AZUMA et al. (2007) showed that the colouring of grape skin de-pends on the genotype of VvmybA1. The allele VvmybA1a contains a retrotransposon, Gret1, upstream of the Vvmy-bA1 coding sequences, and the expression of VvmybA1 is blocked. In contrast, the VvmybA1c allele is functional. VvmybA1c completely lacks Gret1 and most likely repre-sents the original sequence of VvmybA1 before Gret1 was inserted (VvmybA1c is dominant to VvmybA1a).A strong association between the VvmybA1a allele and white-fruited phenotypes has been detected in many culti-vars of the grapevine Vitis vinifera, although a small number of exceptions have been observed (LIJAVETZKY et al. 2006, THIS et al. 2007, WALKER et al. 2007). In addition, WALKER et al. (2007) showed that the locus houses two very similar adjacent genes (VvMYBA1 and VvMYBA2), either of which could control berry colour.It is thought that cultivation of V. vinifera began dur-ing the Neolithic era (6000-5000 BCE) along the east-ern shores of the Black Sea, and the cultivated grape is believed to have been domesticated from the wild grape V. vinifera subsp. sylvestris (hereafter denoted as sylves-tris) (MULLINS et al. 1992). Since the VvmybA1a allele is widely distributed among cultivars of V. vinifera, we hy-pothesized that Gret1 was originally inserted upstream of the VvmybA1 coding sequence of a black-skinned ancestor, and that subsequently a white-skinned grape was produced by spontaneous crossing (KOBAYASHI et al. 2004). To date, the VvmybA1a allele has been detected in over 200 acces-sions of V. vinifera (LIJAVETZKY et al. 2006, THIS et al. 2007, WALKER et al. 2007).Determination of the presence and distribution of the VvmybA1a allele in the wild grape and other Vitis species would provide new information on the genomic relation-Correspondence to: Dr. S. KOBAYASHI, Grape and Persimmon Research Station, National Institute of Fruit Tree Science, Akitsu 301-2, Higashi-Hiroshima, Hiroshima 739-2494, Japan. Fax: +81-846-45-5370; E-mail: skobaya@affrc.go.jpships among these species and on the evolutionary differ-entiation of the genus Vitis. Here we report on the distribu-tion of the VvmybA1a allele in typical cultivars of V. vini- fera and in North American and East Asian Vitis species.Material and Methods: Forty accessions (Table) were analysed. Cultivars of V. vinifera were eco-geographically classified into three proles: pontica, occidentalis, and ori-T a b l eDistribution of the VvmybA1a allele in Vitis speciesSpecies Cultivar name Skin colorGret1 insertionV. vinifera   subsp. sylvestris (♀)   subsp. sylvestris (♂)black---++     pontica Black HamburgAlphonse LavaléeFurmintPlavajblackblackwhitewhite+-++     occidentalis Cabernet    SauvignonMerlotPinot NoirChardonnayMelonRieslingblackblackblackwhitewhitewhite++++++     orientalis Muscat HamburgRizamatFlame TokajBajan SirejItaliaThompson       SeedlessNiunaiblackredredwhitewhitewhitewhite+++++++NA1) V. aestivalisV. cinereaV. doanianaV. labruscaV. longiiV. palmataV. ripariaV. rubraV. rupestrisGloire de    Montpellier (♂)Constantiablackblackblackblackblackblack---blackblack---------EA2) V. amurensisV. coignetiae (♀)V. coignetiae (♂)V. quingquangularisV. shiragaiV. thunbergiiblackblack---blackblackblack------V. vinifera x V. labrusca ConcordSteubenBronx SeedlessUrbanaGolden MuscatSenecablackblackredredwhitewhite+-++++                                1) NA = North America; 2) EA = East Asia. 56 N. MITANI et al.4904 in the 5′LTR region is a T, but the nucleotide in this position in the genome of 'Italia' was revealed to be a C by direct sequencing of the PCR product of Italia in this study. From the above sequence data, the MEGA4 software esti-mated the Gret1 insertion date to be roughly 0.20 ± 0.09 (s.d.) million years ago by the method described by TA-MURA et al. (2007). In this estimation, we used the rate of 1.5 × 10-8 nucleotide substitutions per synonymous site per year, which was estimated by analysis of sequence varia-tion in chalcone synthase and alcohol dehydrogenase loci in dicots (KOCH et al. 2000).Our findings suggest that Gret1 was inserted upstream of the VvmybA1 coding sequences in sylvestris or an un-known ancestor of sylvestris after the North American and East Asian species had diverged from the common ancestor (Figure). The VvmybA1a allele that appeared in interspe-cific hybrids was presumably transferred from V. vinifera by crossing. We analysed only the VvmybA1a allele and only in a limited number of species or cultivars. Further analyses using the other VvmybA1 alleles and a wide range of species and accessions in the genus Vitis, and especially in sylvestris, will be necessary to clarify the genomic rela-tionships and evolutionary differentiation of Vitis species.This work was supported by Grant-in-Aid for Scientific Re-search (C: 19580041).AZUMA, A.; KOBAYASHI, S.; YAKUSHIJI, H.; YAMADA, M.; MITANI, N.; SATO, A.; 2007: VvmybA1 genotype determines grape skin color. Vitis 46, 154-155.CADLE-DAVIDSON, M. M.; OWENS, C. L.; 2008: Genomic amplifica-tion of the Gret1 retroelement in white-fruited accessions of wild Vitis and interspecific hybrids. Theor. Appl. Genet. 116, 1079-1094.KOBAYASHI, S.; GOTO-YAMAMOTO, N.; HIROCHIKA, H.; 2004: Retrotrans-poson-induced mutations in grape skin color. Science 304, 982.KOBAYASHI, S.; GOTO-YAMAMOTO, N.; HIROCHIKA, H.; 2005: Associa-tion of VvmybA1 gene expression with anthocyanin production in grape (Vitis vinifera) skin-color mutants. J. Jpn. Soc. Hort. Sci. 74, 196–203.KOCH, M. A.; HAUBOLD, B.; MITCHELL-OLDS, T.; 2000: Comparative evolutionary analysis of chalcone synthase and alcohol dehy-drogenase loci in Arabidopsis, Arabis, and related genera. Mol. Biol. Evol. 17, 1483-1498.LEWIN, B.; 1997: Genes VI, 603-604. Oxford University Press, New York.LIJAVETZKY, D.; RUIZ-GARCIA, L.; CABEZAS, J. A.; DE ANDRES, M. T.; BRAVO, G.; IBANEZ, A.; CARRENO, J.; CABELLO, F.; IBANEZ, J.; MARTINEZ-ZAPATER, J. M.; 2006: Molecular genetics of berry col-our variation in table grape. Mol. Gen. Genom. 276, 427-435.MULLINS, M. G.; BOUQUET, A.; WILLIAMS, L. E.; 1992: Biology of the Grapevine, 4-16. Cambridge University Press, New York.TAMURA, K.; DUDLEY, J.; NEI, M.; KUMAR, S.; 2007: MEGA4: Molecu-lar evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599.THIS, P.; LACOMBE, T.; CADLE-DAVIDSON, M.; OWENS, C. L.; 2007: Wine grape (Vitis vinifera L.) color associates with allelic variation in the domestication gene VvmybA1. Theor. Appl. Genet. 114, 723-730.WALKER, A. R.; LEE, E.; BOGS, J.; MCDAVID, D. A. J.; THOMAS, M. R.; ROBINSON, S. P.; 2007: White grapes arose through the muta-tion of two similar and adjacent regulatory genes. Plant J. 49, 772-785.Received April 23, 2008entalis. We selected several cultivars with different skin colours from these proles. All these species and cultivars are part of the germplasm collection maintained in the vineyards of the Grape and Persimmon Research Station, National Institute of Fruit Tree Science, Japan. (The syl-vestris, the American species, and the East Asian species came from the collections of the Turkmenistan Experimen-tal Station for Plant Genetic Resources, the US Department of Agriculture, and the University of Osaka Prefecture, re-spectively.)Total DNA was extracted from young leaves of the plants as described (KOBAYASHI et al. 2005) and used as a template for the polymerase chain reaction (PCR). The primers for the 3′LTR region of Gret1 were ‘a’ and ‘c’ (Figure, KOBAYASHI et al. 2004), and PCR reactions and electrophoresis were performed as reported (KOBAYASHI et al. 2004). The 5′LTR regions of Gret1 were amplified by using primers ‘b’ (Figure, KOBAYASHI et al. 2004) and ‘d’ (5′-CACAGACAGCGCCAATGTTGT-3′) as above, and sequenced directly. Then the sequences of the two LTRs were compared in each cultivar.Figure: A model of evolutional differentiation of Vitis species.Results and Discussion: The VvmybA1a allele, which contains Gret1, was detected in almost all of the cultivars of V. vinifera that we analysed, including sylvestris and the interspecific hybrids (V. vinifera × V. labrusca), with the exception of 'Alphonse Lavallée' and 'Steuben' (Table). In contrast, it was not detected in any of the North American or East Asian Vitis species. Most recently, CADLE-DAVID-SON and OWENS (2008) showed that the VvmybA1a allele was not detected even in white-fruited wild Vitis species (V. aestivalis and V. riparia).The two LTRs of a retrotransposon are usually identi-cal at the time of its insertion into the host genome (LEWIN 1997). Therefore, the date of insertion can be estimated from the sequence divergence between the two LTRs. We analysed the two LTR sequences of Gret1 in the VvmybA1a alleles of the above cultivars. The sequences of the 5′LTR differed from those of the 3′LTR at only five bases in each cultivar; however, no differences were observed within either sequence among cultivars, including sylvestris. Al-though we earlier reported that the sequences of the two LTRs of VvmybA1a differed at four bases (KOBAYASHI et al. 2004), five is correct. That is, in the sequence data of clone It22 registered as VvmybA1a in the DNA Data Bank of Ja-pan (accession no. AB111100), the nucleotide at position 

A retrotransposon-inserted VvmybA1a allele has been spread among cultivars of Vitis vinifera but not North American or East Asian Vitis species

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A retrotransposon-inserted VvmybA1a allele has been spread among cultivars of Vitis vinifera but not North American or East Asian Vitis species

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