Nenad Malenica

Evolution of the tetraploid Anemone multifida (2n = 32) and hexaploid A. baldensis (2n = 48) (Ranunculaceae) was accompanied by rDNA loci loss and intergenomic translocation: evidence for their common genome origin.

BACKGROUND AND AIMS In the genus Anemone two small groups of taxa occur with the highest ploidy levels 2n = 6x = 48, belonging to the closely related clades: the montane/alpine Baldensis clade and the more temperate Multifida clade. To understand the formation of polyploids within these groups, the evolution of allohexaploid A. baldensis (AABBDD, 2n = 6x = 48) from Europe and allotetraploid Anemone multifida (BBDD, 2n = 4x = 32) from America was analysed. METHODS Internal transcribed spacer and non-transcribed spacer sequences were used as molecular markers for phylogenetic analyses. Cytogenetic studies, including genomic in situ hybridization with genomic DNA of potential parental species as probe, fluorescence in situ hybridization with 5S and 18S rDNA as probes and 18S rDNA restriction analyses, were used to identify the parental origin of chromosomes and to study genomic changes following polyploidization. KEY RESULTS This study shows that A. multifida (BBDD, 2n= 4x = 32) and A. baldensis (AABBDD, 2n = 6x = 48) are allopolyploids originating from the crosses of diploid members of the Multifida (donor of the A and B subgenomes) and Baldensis groups (donor of the D subgenome). The A and B subgenomes are closely related to the genomes of A. sylvestris, A. virginiana and A. cylindrica, indicating that these species or their progeny might be the ancestral donors of the B subgenome of A. multifida and A and B subgenomes of A. baldensis. Both polyploids have undergone genomic changes such as interchromosomal translocation affecting B and D subgenomes and changes at rDNA sites. Anemone multifida has lost the 35S rDNA loci characteristic of the maternal donor (B subgenome) and maintained only the rDNA loci of the paternal donor (D subgenome). CONCLUSIONS It is proposed that A. multifida and A. baldensis probably had a common ancestor and their evolution was facilitated by vegetation changes during the Quaternary, resulting in their present disjunctive distribution.

Cytogenetic and phylogenetic studies of diploid and polyploid members of Tribe Anemoninae (Ranunculaceae)

The ancestry, phylogenetic differentiation and systematic classification of the worldwide-distributed genus Anemone have been debated for many years. In this paper 11 Anemone, three Pulsatilla species and Hepatica nobilis were subjected to detailed karyotype analysis with the aim of obtaining new cytogenetic data that will contribute to karyotype evolutionary studies of the tribe Anemoninae. The results are interpreted in a phylogenetic context, established from the intergenic nontranscribed spacer (NTS) of 5S rDNA and internal transcribed spacer (ITS) of 35S rDNA. One to three 35S and one to three 5S rDNA loci are present in diploid and polyploid taxa. The 35S rDNA loci are located terminally on the short arm of acrocentric chromosomes, while for 5S rDNA there is no preferential chromosomal position as it exhibits terminal, subterminal, interstitial or pericentromeric positions, and is located either on acrocentric or metacentric chromosomes. The karyotype of hexaploid A. baldensis (2n = 6x = 48) is presented for the first time, and A. sylvestris is proposed as one of its putative parental species. Chromosome fusion/translocation is proposed as the key mechanism involved in reduction of the basic chromosome number from 8 in the Anemone subgenus to 7 in the Anemonidium subgenus. The cytogenetic data obtained are mainly supported by ITS and NTS phylogeny. Diversification of the genus Anemone was accompanied by a large reduction of heterochromatin, from the Mediterranean anemones that have large amounts of heterochromatin to the New World anemones without any detectable heterochromatic blocks.

Whole genome amplification and microsatellite genotyping of herbarium DNA revealed the identity of an ancient grapevine cultivar

Reconstruction of the grapevine cultivation history has advanced tremendously during the last decade. Identification of grapevine cultivars by using microsatellite DNA markers has mostly become a routine. The parentage of several renowned grapevine cultivars, like Cabernet Sauvignon and Chardonnay, has been elucidated. However, the assembly of a complete grapevine genealogy is not yet possible because missing links might no longer be in cultivation or are even extinct. This problem could be overcome by analyzing ancient DNA from grapevine herbarium specimens and other historical remnants of once cultivated varieties. Here, we present the first successful genotyping of a grapevine herbarium specimen and the identification of the corresponding grapevine cultivar. Using a set of nine grapevine microsatellite markers, in combination with a whole genome amplification procedure, we found the 90-year-old Tribidrag herbarium specimen to display the same microsatellite profile as the popular American cultivar Zinfandel. This work, together with information from several historical documents, provides a new clue of Zinfandel cultivation in Croatia as early as the beginning of fifteenth century, under the native name Tribidrag. Moreover, it emphasizes substantial information potential of existing grapevine and other herbarium collections worldwide.

Grapevine variety determination from herbarium and archeological specimens

Genotyping old grapevine samples, in particular cultivar identification via microsatellite profiling, is not yet routine. Success depends on several factors, the age of the investigated material being the most obvious. In addition, the amount and integrity of DNA depends on specimen storage/preservation history. Moreover, conta-mination and fragmentation processes make the isolated DNA a difficult template for PCR amplification. Three old grapevine specimens were investigated in this study. Two derived from a 90-year-old Croatian grapevine herbarium collection: cv. ?Tribidrag? and cv. ?Brzamin?. Both are indigenous Croatian grapevine cultivars not present in contemporary germplasm collections. The third was a 2000-year-old archeological underwater grapevine specimen. Several different approaches for DNA isolation were tested. A commercial plant DNA isolation kit was the best choice in terms of avoiding contamination with contemporary grapevine DNA. Initial attempts to PCR-amplify standard grapevine microsatellite loci from isolated DNA did not work satisfactory. Therefore, a whole genome amplification (WGA) kit was applied to overcome this problem. The WGA performed well with the cv. ?Tribidrag? herbarium sample and the 2000-year-old archeological specimen. While the amplified, cloned and sequenced DNA from the archeological specimen was from origins other than grapevine, indicating contamination of the specimen, clones deriving from the cv. ?Tribidrag? corresponded to different grapevine chromosomes. Therefore, we used the cv. ?Tribidrag? DNA for successful amplification of the VVS2 microsatellite locus by PCR, thereby demonstrating the proof of principle. Eventually we genotyped the cv. ?Tribidrag? specimen at six standard microsatellite loci. The SSR profile obtained was identical to that of ?Zinfandel?/?Primitivo?/?Crljenak kastelanski?. These results are in line with the previously proposed Croatian origin of ?Zinfandel?. This time we identi¬fied the name ?Tribidrag? as the most ancient name for cv. ?Zinfandel?, which has been cultivated in Croatia, according to several historical documents, since the beginning of the 15th century.