Kerekes, A.

Kiss, E.

Kozma, P.

Roznik, D.

Szőke, A.

Tóth-Lencsés, K.

Veres, A.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 56, 53–54 (2017)© The author(s).                               This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License                               (http://creative-commons.org/licenses/by-sa/4.0/).Correspondence to: Dr. E. Kiss, Institute of Genetics, Microbiology and Biotechnology, Páter Károly str 1, Gödöllő, 2100 Hungary, Fax: +36-28-522069. E-mail: Kiss.Erzsebet@mkk.szie.huDOI: 10.5073/vitis.2017.56.53-54Research NoteOrigin of 'Csillám', a promising source for black rot resistanceE. Kiss1), K. TóTh-LEncsés1), A. Szőke1), A. KErEKEs1), A. VErEs1), D. rozniK2) and P. KozmA2)1) Szent István University, Institute of Genetics, Microbiology and Biotechnology, Gödöllő, Hungary2) Department of Grape Breeding, Research Institute for Viticulture and Enology, University of Pécs, Pécs, HungaryK e y  w o r d s :  microsatellite (SSR) analysis; parentage; berry colour; VvMybA1 and VvMybA2 genes.Introduction: French-American grape hybrids and their derivatives produced in the last 100 years are im-portant resistance gene sources against powdery, downy mildews and black rot. Previously we reported on the parentage analysis of Hungarian grapevine cultivars of 'Seibel-Seyve-Villard' origin among them 'Csillám', lineage of which ('Csabagyöngye' x 'Seyve Villard 12375') has been disproven (TóTh-LEncsés et al. 2015). Deciphering the true origin of varieties is interesting in itself, but in the case of 'Csillám' another remarkable observation made it even more stimulating; in a survey aiming at the comparison of the resistance against black rot - (BR) [(Guignardia bidwellii (Ellis) Viala & Ravaz, anamorph Phyllosticta ampelicida (Engleman)] a new severely threatening grapevine disease - 'Csillám' displayed a surprisingly high level of tolerance among 74 accessions, exceeding all the 'Seyve Villard' de-scendents, only 'Seibel 7053', 'Merzling' and 'Börner' (Vitis riparia Gm x Vitis cinerea Arnold) gave similar excellent results (rozniK et al. unpubl.).This directed our attention to decipher the true parentage of 'Csillám'. For this purpose, we applied two approaches: SSR genotyping at 31 loci and determining the genetic background of berry pigmentation both in the white berried 'Csillám' and its "novel" candidate black berried parents. The white grapes arose from the ancient black ones by in-dependent mutations causing the inhibition of anthocyanin biosynthesis. The insertion of the Gret1 retrotransposon in the promoter of the VvMybA1 transcription factor gene is one of the reasons for colourless phenotype (KobAyAshi et al. 2004); two SNPs in the coding region of VvMybA2 gene also block the pigment biosynthesis (WALKEr et al. 2007; cArrAsco et al. 2015). Material and Methods:  P l a n t  m a t e r i a l :  Young leaves of the cultivars listed in the Table and reference varie-ties for berry colour analysis, 'Barbera', 'Chardonnay', 'Pinot noir' were collected from the gene bank of the Institute of Viticulture and Enology of Pécs (PTE) and Eger.P C R ,  S S R  a n d  C A P S  a n a l y s i s :  DNA isolation and microsatellite analyses were carried out ac-cording to hALász et al. (2005). First, 9 nuclear SSR primer pairs VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZAG62 and VrZAG79 were chosen according to the GrapeGen06 EU project. For parentage analysis, additional 22 primers were involved into microsat-ellite profile determination shown in the supplemental Table. A CAPS marker 20D18CB9 linked to the VvMybA1 gene coding a transcription factor which regulates the an-thocyanin biosynthesis was applied. The PCR amplicons generated with the 5'-GAT GAC CAA ACT GCC ACT GA-3' forward and the: 5'- ATC ACC TTG TCC CAC CAA-3' reverse primers were digested with DdeI enzyme as it was described by WALKEr et al. (2006). The same protocol was applied for the allele-specific primers of VvMybA1 gene and VvMybA2 locus linked CAPS marker. Fragments amplified with VvMybA1 specific primers were separated in 1.5 % ethidium-bromide stained (0.5 μg·mL-1) agarose gel. S N a P s h o t   a n a l y s i s : Two SNPs (VvMybA2R44/K980, VvMybA2C22) found in the VvMybA2 gene were examined with SNaPshot method (ABI PRISM SNaPshot Multiplex kit) according to the manufacturer's manual (KErEKEs et al. 2015).S t a t i s t i c a l  a n a l y s i s : Parentage was analyzed by the Identity 1.0 statistics (WAgnEr and sEfc 1999).Results and Discussion: The 'Seyve-Villard 12375' x 'Csabagyöngye' pedigree of  'Csillám' (CSFT194) published in the literature (KozmA 1986, KozmA jr. 2002, bényEi and Lőrincz 2005), has already been disproven by applying the 9 GrapeGen SSR primer pairs (TóTh-LEncsés et al. 2015). T a b l e    SNP genotypes of the three reference cultivars ('Barbera', 'Chardonnay', 'Pinot noir') and the 'Seibel 4643', 'Kék-frankos' and 'Csillám' in the VvMybA2 gene VarietiesSNP in VvMybA2 geneVvMybA2R44 / K980VvMy-bA2C22References     Barbera G/G G/G     Chardonnay T/T T/T     Pinot noir G/T G/TCultivars of interest     Seibel 4643 G/T G/T     Kékfrankos G/T G/T     Csillám T/T T/TExplanation: G/G, homozygous black: 'Barbera';T/T, homozygous white: 'Chardonnay'; two loss-of-func-tion alleles; G/T, heterozygous black: 'Pinot noir', one functional allele. 54 E. Kiss et al.Analysis and evaluation of 383 genotypes (data not shown), including 45 interspecific hybrids of 'Seibel' and 'Seyve Villard' origin at the same 9 loci made it possible to predict that 'Kékfrankos' and 'Seibel 4643' are the putative parents of 'Csillám'. To confirm the new parentage, we used more 22 SSR primer pairs and genotyped 'Csillám' together with its disput-ed and "novel" parents at 31 microsatellite loci (supplemen-tal Table). Additional 7-7 loci excluded the 'Seyve-Villard 12375' x 'Csabagyöngye' pedigree and all the 31 loci verified the 'Kékfrankos' x 'Seibel 4643' origin of 'Csillám'.'Csillám' is a white berried cultivar, while the varieties we found to be its new "candidate" parents ('Kékfrankos' and 'Seibel') bear black fruits (Figure).For providing further proof that 'Kékfrankos' x 'Seibel 4643' is the real pedigree of 'Csillám' we tested the varieties of interest for the presence or absence of Gret1 retrotrans-poson in the promoter of the VvMybA1 gene.The CAPS analysis proved that 'Kékfrankos' is a hete-rozygous black genotype (restriction digestion resulted in three DNA fragments 329, 248, 213 bp), but the also black berried 'Seibel 4643' showed homozygous white genotype (329, 248 bp) for the Gret1 insertion similarly to the really white 'Csillám' (supplemental Figure). Since the blackness of the 'Seibel 4643' cannot be explained by the lack of Gret1 we examined the allele pro-file of VvMybA2 transcription factor gene with SNaPshot method based on reference varieties. We determined that Figure: Bunch and leaf of 'Csillám' (Pál KozmA's photo), 'Kékfrankos' and 'Seibel 4643' (http://lescepages.free.fr/roi_noirs.html).'Seibel 4643' carries functional SNP in heterozygous form in both SNP positions, therefore the anthocyanin biosynthesis operates even if it is homozygous for the Gret1 insertion in the VvMybA1 locus (Table). Our results not only confirm the true origin of 'Csillám' from the berry colour side, but also prove that it inherited the VvMybA1+Gret1 and VvMybA2 SNP (VvMybA2R44 / K980; VvMybA2C22 mutant 'white' alleles from both parents ('Kékfrankos'/'Blaufränkisch' and 'Seibel 4643'/'Roy de noirs'/'Pannonhalmi kék'). Astonishingly excellent behaviour of 'Csillám' in black rot resistance survey may be due to the 'Seibel 4643' parent. This fact calls our attention to find those cultivars which also derive from crosses including 'Seibel 4643'. 'Csillám' itself is a promising BR resistant cultivar so we think that it is well worth taking it into breeding programs as a perspective BR resistance source.  This research was supported by the grants of Research Centre of Excellence 1476-4/2016/FEKUT/SZIU and the EU INNOVINE (FP7-KBBE-2012-6-311775). Bényei, F.; Lőrincz, A.; 2005: Borszőlőfajták, csemegeszőlő-fajták és alan-yok. Wine, table grape varieties, and rootstocks, 110-111. Mezőgazda Kiadó, Budapest.cArrAsco, D.; DE LorEnzis, g.; mAghrADzE, D.; rEViLLA, E.; bELLiDo, A.; fAiLLA, o.; Arroyo-gArciA, r.; 2015: Allelic variation in the VvMybA1 and VvMybA2 domestication genes in natural grapevine populations (Vitis vinifera subsp. sylvestris). Plant Syst. Evol. 301, 1613-1624.HALáSz, G.; VereS, A.; kozmA, P.; kiSS, e.; BALoGH, A.; GALLi, z.; Szőke, A.; hoffmAnn, s.; hEszKy, L.; 2005: Microsatellite fingerprinting of grapevine (Vitis vinifera L.) varieties of Carpathian Basin. Vitis 44, 173-180.kerekeS, A.; De LorenziS, G.; Szőke, A.; kiSS, e.; FAiLLA, o.; 2015: Anal-ysis of VvMybA1 and VvMybA2 genes in grape bud sports. Vitis 54 (Special Issue), 45-48.KobAyAshi, s.; goTo-yAmAmoTo, n.; hirochiKA, h.; 2004: Retrotranspos-on-induced mutations in grape skin color. Science 304, 982. KozmA P.; 1986: Qualité de raisin et resistance de la vigne dans les pop-ulations hybrides interspecifiques, 242-246. Atti del 4. Symposio Internationale di Genetica della Vite, April 13-18, 1985. Verona, Italy.KozmA P. jr.; 2002: Resistant Grape Varieties Originating from Fran-co-American Hybrids in Hungary. Int. J. Hortic. Sci. 8, 47-50.TóTH-LencSéS, A. k.; kozmA, P.; Szőke, A.; kerekeS, A.; VereS, A.; kiSS, E.; 2015: Parentage analysis in Hungarian grapevine cultivars of 'Seibel'-'Seyve-Villard' origin. Vitis 54 (Special Issue), 27-29. WAgnEr, h. W.; sEfc, K. m.; 1999: IDENTITY 1.0. Centre for Applied Genetics. University of Agricultural Sciences. Vienna.WALKEr, A. r.; LEE, E.; bogs, J.; mcDAViD, D. A. J.; ThomAs, m. r.; rob-inson, s. P.; 2007: White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J. 49, 772-785.WALKEr, A. r.; LEE, E.; robinson s. P.; 2006: Two new grape cultivars, bud sports of Cabernet Sauvignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus. Plant Mol. Biol. 62, 623-635.Received November 11, 2016Accepted February 3, 2017

Origin of 'Csillám', a promising source for black rot resistance

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Origin of 'Csillám', a promising source for black rot resistance

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