Morphology and Molecular Analysis of Ancient Grape Seeds

Abstract

the morphometry of Vitis vinifera seeds from different archaeological sites was studied. preservation status differed between sites. non-invasive and quantitative image analysis based on elliptic Fourier analysis (eFa) established different morphological variations between populations. Molecular analysis was performed to study genetic relationships, using nuclear microsatellite ssrs markers with high polymorphism. Morphometric analysis of archaeological endocarp outlines and allelic profiles of endocarp tegument delineated the general species-specific qualities of the modern cultivar. IANSA 2011 ● II/2 ● 95–100 Claudio Milanesi, francesca Antonucci, Paolo Menesatti, Corrado Costa, Claudia faleri, Mauro Cresti: Morphology and Molecular Analysis of Ancient Grape Seeds 96 2.2 Morphometric analysis the shape analysis of ancient grape seeds for each site, (Firenze, Miranduolo and poggio Bacherina, three samples) was compared with fresh tuscany cultivars from 11 varietals (sangiovese casciano, sangiovese Montalcino, cabernet Bossi, Merlot Bossi, canaiolo Monti in chianti, canaiolo Montalbuccio, trebbiano Montalbuccio, trebbiano tognazza, Malvasia Monti in chianti, albano Montalbuccio and silevstris) and a total of 498 samples. the morphometric method was described in Milanesi et al. 2011 and antonucci et al. 2012. 2.3 DNA analysis ancient seeds were quickly released from the matrix by hcl and hF treatment, which cleaned and removed organic matter (Milanesi et al. 2006), and subsequently exposed overnight to uV-light. specimens were immediately processed for dna extraction under controlled sterile conditions. small pieces of archaeological and modern seeds were divided in two and processed separately for dna extraction and pcr amplification under different sterile laminar-flow hoods. genomic dna was extracted using a method reported by Mulcahy et al. (1993). seven different microsatellite markers (VVMd7, VVMd21, VVMd25, VVMd27, VVMd31, VVMd36, VVs2), generated by Bowers et al. (1999), were amplified. PCR amplification and amplicons detection were performed according to Masi et al. (2001). The amplified loci were cloned using the topo ta clonig kit version F (InVItrogen). plasmids were extracted using the QIAGEN Plasmid Purification kit, and sequenced using the alFexpress autoread sequencing Kit, and analyzed on a polyacrylamide paa gel run and the alFexpressII semi-automated dna sequencer. post run data analysis was performed with the alFwin sequence analyzer 2.00. the generation of the consensus sequence and the pairwise sequence was carried out with dnasys 2.10 software. the analysis was performed in order to observe VVMd7, VVMd21, VVMd25, VVMd27, VVMd31, VVMd36 and VVs2 at least three times.