**2. Materials and methods**

Fully matured berries were collected from Pinot gris (clone B. 10) stocks in September 2016. Seeds were removed from the berries, washed in tap water and dried in room temperature. Only fully matured, brown-coloured seeds were used in the analyses. Pinot noir, Pinot gris (clone B. 10.), Chardonnay and Cabernet sauvignon DNA (extracted formerly from leaves) were also used as controls.

DNA was extracted from 12 single seeds. Prior to extraction the seeds were crushed in a mortar to powder; then, this powder was moved to a tube. Qiagen Plant Mini Kits were used for DNA extraction following the instructions of the manufacturer. The amount and quality of DNA were determined spectrophotometrically. The DNA was diluted to a concentration of 10 ng/ml.

The SSR analysis was performed at 19 loci (see **Table 1**). PCR reaction mix was the following: 0.2 mM of each primer, 12.5 ml of Hot Start Master Mix (Quiagen) and 50 ng of template DNA, completed to the total volume of 25 ml with DNA- and

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**3. Results**

*a*

**Table 1.**

*List of SSR loci [6].*

*Reconstruction of Parental SSR Haplotypes from a Single Grape Seed*

**Linkage groupa SSR locus Annealing temp.** VMC8A7 64°C VMC7G3 60°C VVMD28 62°C VrZag21 62°C VrZag79 60°C VMC4G6 50°C VVMD7 50°C VMC1F10 57°C VMC1C10 60°C VrZag25 67°C VVS2 60°C VMC2H4 57°C VMC3D12 57°C VMCNG1E1 58°C VMC5G8 58°C VVMD5 53°C Scu06vv 60°C VVIM10 57°C VMC5E9 58°C

RNA-free distilled water. The following thermal profile was used: (1) 94°C for 45 min; 35 cycles of: (2) 94°C for 1 min, (3) annealing temperature (see **Table 1**) for 1 min, 73°C for 1 min; and (4) 73°C for 7 min. Each forward primer of the primer pairs was fluorescently labelled with 6FAM on the 5′ end of the DNA chain. Separation of the amplified products was carried out through capillary electrophoresis in a PE-Applied Biosystems 3100 Automated Capillary DNA Sequencer; the molecular sizes of the products were determined using Peak Scanner Software (v. 1.0; Applied Biosystems) [8]. Allele sizes and peak area were also recorded for

DNA extraction and amplification in 18 loci (out of the 19) were successful in all of the 12 seeds. In VVMD5 the amplification was weak, so the results were unevaluated. In all of the remaining 18 loci, the maternal and parental alleles were determined (**Figure 2**); according to our presumption, the allele with the higher area value was supposed to be maternal. Ratio of the quantity of maternal and paternal alleles was computed based on area values. In some loci (where 'Pinot gris' has a homozygote genotype and the majority of the seeds showed also homozygote genotype— VMC4G6 and VMCNG1E1), this ration was excluded from the further analyses.

Based on the remaining 16 loci, the average ratio of maternal and paternal alleles ranged from 1.89 (VMC5E9) to 2.58 (VrZag25), which confirms our presumption.

every single allele. Data were stored in Microsoft Excel [8].

*Linkage group numbers according to Adam-Blondon et al. [7].*

*DOI: http://dx.doi.org/10.5772/intechopen.85685*


#### *Reconstruction of Parental SSR Haplotypes from a Single Grape Seed DOI: http://dx.doi.org/10.5772/intechopen.85685*

#### *Linkage group numbers according to Adam-Blondon et al. [7].*
