The analysis of genetic structure and parentage of Croatian autochthonous grape varieties

Način kako male vinogradarske zemlje mogu doskočiti uniformizaciji vinskog tržišta jest da trend univerzalnosti u vinogradarskoj proizvodnji mijenjaju u korist autohtonosti. O dosadašnjem uspjehu spašavanja i revitalizacije hrvatskog autohtonog sortimenta govori činjenica da sve više raste interes i potražnja za udomaćenim sortimentom, kako u potrošnji tako i u proizvodnji. Dokazivanje autohtonosti jedan je od bitnih poteza u brendiranju proizvoda. U idealnom slučaju, dokazi da je sorta autohtona, tj. nastala na nekom području jesu otkriće njenih roditelja koji su također tamo udomaćeni te duga tradicija uzgoja sorte koju je također potrebno dokazati kroz opširno proučavanje dostupnih povijesnih izvora. Znanstveni pristup i dostupni moderni alati mogu barem donekle dati odgovor da li je nešto autohtono, a to je dosad urađeno za svega nekolicinu hrvatskih sorata. Cilj istraživanja bio je stoga za sve potencijalno autohtone sorte unutar hrvatskih kolekcija utvrditi njihov status (bez ili sa sinonimima/homonima u drugim državama), pripadnost nekom skupu gena te analizirati njihove srodstvene odnose (s prvenstvenim naglaskom na analizu roditeljstva) zajedno s dostupnim genotipovima najveće svjetske kolekcije u Vassalu (INRA, Francuska). Ovo je prvenstveno odrađeno korištenjem za to danas najmodernijih genetičkih alata - analizom nuklearne DNA s 36 mikrosatelitskih (SSR) i dva seta SNP markera te mikrosatelitskim utvrđivanjem klorotipova. Dio ispitivanih SSR lokusa odnosio se na novu generaciju tri- i tetra- nukleotidnih lokusa koje se preporuča zbog manjih pogrešaka u designacija alela, ali isti se zbog pojave nul alela i/ili slabije informativnosti nisu pokazali kao idealni set za genotipizaciju. Genotipizacijom na devet standardnih SSR lokusa i usporedbom s formiranom bazom podataka koja je sadržavala preko 4200 genetskih profila utvrđen je status za ukupno 212 prikupljenih primki. Dvije trećine sorata (95 primki) unutar kolekcija nema sinonima u niti jednoj drugoj zemlji, egzistiraju samo na prostoru Hrvatske i mogu se zasad smatrati autohtonima. Globalna analiza genetske strukture (k=5) pokazala je da je polovina hrvatskog sortimenta dio inferiranog balkanskog genskog skupa ekvivalentno Negruljevoj ekološko geografskoj klasifikaciji. Mikro analiza genetske strukture pratila je utvrđene srodstvene odnose i razdvojila dalmatinski sortiment kao najhomogeniji podskup unutar Hrvatske. Utvrđena je razlika u bogatstvu i distribuciji klorotipova između hrvatskih regija, a pojava klorotipa A isključivo u priobalnom dijelu upućuje na potencijalnu introgresiju divlje loze pri formiranju sortimenta. Kompletna rekonstrukcija roditeljstva bila je moguća za svega 19% sorata, a suprotno očekivanjima, u mnogo njih sudjelovale su strane sorte koje nisu evidentirane unutar hrvatske germplazme poput Bombina bianca. Ključna sorta za formiranje hrvatskog sortimenta je Plavac mali za kojeg je ujedno odbačeno ranije predlagano roditeljstvo zbog nepodudaranja na 4 SSR, 3 SNP te kloroplastne distribucije. Za ukupno 45% sorata detektirana je barem jedna roditelj-potomak veza s nekom od pretpostavljeno autohtonih sorata, odnosno drugi roditelj nije rekonstruiran, te za neke postoje moguće roditeljpotomak veze sa stranim sortama. Stoga ne možemo potvrditi hipotezu da je većina sorata uključenih u istraživanje nastala na području današnje Hrvatske (iako većina egzistira isključivo ovdje). Dodatnih 8 sorata možemo smatrati autohtonima (također zasad) jer kao potencijalnog roditelja imaju barem jednu autohtonu sortu, a drugi istraživači za iste nisu rekonstruirali roditelj potomak veze. U konačnici ukupan broj autohtonih sorata iznosi, zasad, 103. Brojnost utvrđenih sinonima, stranih sorata u roditeljstvima te posljedično veliki broj sorata miješanog porijekla posljedica je kompleksnih pedigrea, migracije i erozije sorata vinove loze na području Hrvatske.The way that small wine-growing countries can cope with globalization and uniformity of the wine market is orientation to autochthonous varieties which is in agreement with rural development and ecological balance. Modern consumers value quality and originality, which leads to different approaches in the development of viticulture and enology. Developed winegrowing countries invested great resources in new technologies, as well as in the rescue and revitalization of indigenous cultivars. Due to the importance and potential of wine-growing and wine making Croatia did the same. Efforts to save native grape germplasm started twenty years ago, through a series of professional and scientific projects. Increasing interest and demand for planting material is the evidence those efforts are successful. Due to the fragmented vineyards big scale production of massive, cheap and competitive wines is impossible. One of the ways to justify higher price is to offer unique product, and proving its authenticity is one of the key steps. Ideally, the proof that a variety is autochthonous is the discovery of its also local parents as well as the long tradition of local cultivation. The latter has to be supported through time-consuming and extensive studies of available historical records. Grapevine can be vegetatively and sexually propagated so the are many different breeding scenarios and levels of autochthonousness which are sometimes hard to reconstruct. Scientific approach and modern genetic tools can, to a certain extent, help defining if a variety is or is not indigenous, which so far has been done only for few Croatian varieties. Therefore the aim of this research was: to define status (with or without synonyms/homonyms) for all potentially indigenous accessions which can be found within Croatian grapevine collections; to assign them to the inferred genepools and to preform parentage and kinship analysis (through defining primarily parent offspring and full sib connections) together with available genotypes from the worlds' biggest grape collection in Vassal (INRA, France). This was performed using state of the art genetic tools - analysis of nuclear DNA via 36 microsatellite (SSR) markers, two sets of SNP (Single Nucleotide Polymorphism) markers as well as chlorotype determination. Part of the tested SSR loci referred to the new generation of tri- and tetra- nucleotide loci recommended for their accuracy in allele designation. Due to the high recorded null allele frequency and partially low polymorphism they are not recommended as substitution for standard set of microsatellite markers. This was also the case for potentially functional SNP markers from SNP set 2 that showed serious deviations from Hardy-Weinberg equilibrium. 45 selected SNPs from SNP set 1 showed high amplification rates and informativeness in accordance with other studies. The main drawback of these SNP markers which are also being recommended as an alternative for nine standard microsatellites is absence of publicly available SNP profiles and hence they cannot be used for the comparison with international varieties. Reference varieties from Vassal collection were used for synchronization of microsatellite data. Status for a total of 212 sampled accessions was determined after genotyping of nine standard SSR loci and comparing them with created internal SSR database which contained more than 4200 grape genetic profiles. For two thirds (95 accessions) synonyms were not found in any other country, they exist only in Croatian vineyards and can, for now, be regarded as autochthonous varieties. The highest synonyms occurrence was with Italy, Slovenia, Bosnia and Herzegovina and with German speaking countries which can be correlated to our common history. The difference in abundance and chlorotype distribution was detected among Croatian winegrowing regions. The emergence of chlorotype A only along Adriatic coast suggests possible introgression with wild grape. They proved as a valuable tool for defining mother/father identity but also for rejecting falsely proposed parentages. Analysis of global genetic structure using Structure program (k=5) inferred that half of the varieties can be grouped in Balcan gene pool in accordance with Negrul's ecogeographical distribution of grape varieties. Analysis of genetic structure on micro level followed kinship patterns and defined Dalmatian pool as the richest and most homogeneous gene pool. Parentage and kinship analysis was done using 20 SSR markers common to Vassal genotypes, and addition 14 tri- and tetra nucleotide SSR loci as well as 45 SNP loci were visually screened and used when it was possible as additional affirmation of the proposed parentage trio. The complete parentage reconstruction was possible for only 19% of the varieties. Unexpectedly, in many those cases one of the parents was a foreign variety that is a genotype that was not recorded in any of the Croatian grape collections. This refers the most to Bombino bianco, high- yielding variety from Italian Apuglia region. Besides Bombino b., other key genitors to Croatian varieties were defined. In the first place this is Plavac mali, the most important red variety. Previously proposed parentage where Dobričić and Tribidrag were parents of Plavac mali was rejected due to the inconsistencies on 4 analyzed SSR loci, 3 SNP loci and chlorotype distribution. This case illustrates complexity and sometimes inaccuracies in genealogical reconstructions. Since the exact time of cultivars' emergence is unknown, as is with traditional cultivars, possibly the true parent(s) are extinct and some varieties are product of complex pedigrees the number of genotyped loci used in parentage reconstructions should be much larger and comprehensive then it was the case so far. The fact that the majority of Croatian germplasms’ key genitors are today critically endangered or have disappeared highlights the importance and justification for preserving locally neglected varieties, although they maybe have no economic potential. Revealing the Croatian names and their possible existence in old vineyards is a future challenge for varieties like Bombino bianco or Alba imputotato. The reconstructed kinship group of Heunisch Weiss, consisting of varieties grown in northwestern part of Croatia, but also in Primorje and Istria, as well as the presented historical documents supports the hypothesis about its Pannonian origin. Records from 19th century talking about winegrowers' affinity to high yielding but low quality varieties and the number of those in reconstructed parentages witness the socio-economic circumstances that shaped the Croatian grapevine assortment. For 45% of the varieties at least one parent-offspring connection with some supposedly autochthonous variety was detected. Since the other parent was not found so the complete parentage therefore can't be reconstructed and for some there are possible parent-offspring connections with foreign varieties, hypothesis that the majority of supposedly autochthonous variety in this research originated in Croatia cannot be confirmed. Additional 8 varieties can be considered autochthonous (also for now) since potential parent offspring relationships were reconstructed with at least one autochthonous variety and there are no parent offspring relationships with foreign germplasm. Overall, until proven otherwise, 103 varieties can be considered autochthonous. The number of defined and confirmed synonyms, number of nonindigenous varieties in parentages and the according amount of admixed genotypes is a direct consequence of complex grape pedigrees, cultivars migration and genetic erosion