## 4 Discussion  
The presented results show that there are several advantages to using _Csd_ genotyping as a means of establishing patrilines in honeybee colonies. Compared to the microsatellite markers used in this study, _Csd_ alleles exhibit greater variability, providing a significantly higher resolution in genotyping. In addition to the previously mentioned influence of balancing selection on the diversity of _Csd_ alleles, this is attributed to the structure of _Csd_, which involves amplifying a gene fragment consisting of two variable elements--encoding the HVR and the proline-rich domain [15]. These elements are subsequently separated through restricted enzyme digestion and represented by HEX- and 6-FAM-labelled fragments, respectively. There is no correlation between the lengths of these aforementioned fragments, allowing them to be considered as two independent markers. The relatively high number of _Csd_ alleles identified in each colony simplifies the process of identifying maternal alleles, as these two maternal alleles appear with a much higher frequency (0.25 each) against the background of several paternal alleles. Consequently, we found that genotyping drones to identify maternal alleles is usually unnecessary. A relatively low number of distinct microsatellite alleles within a given population also gives rise to issues in reconstructing paternal genotypes. In situations where the worker bee's genotype matches that of the queen, unambiguously identifying the paternal allele becomes impossible. The scarcity of distinct alleles also elevates the probability of homozygous genotype formation. While these can be identified as such, they may also be interpreted as a consequence of the presence of a null allele that has not been amplified, possibly due to incompatible PCR primers. Furthermore, the most commonly used microsatellite markers consist of repeat units consisting of only two nucleotides, which can lead to errors in scoring their sizes [10,11]. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Colony ID/Marker** & _Csd_ & **A113** & **Ap43** & **Ap55** & **B124** & **A7** \\ \hline
2019-15 & 15 & 4 & 4 & 5 & 7 & 6 \\
2019-20 & 18 & 5 & 4 & 6 & 8 & 4 \\
2019-3 & 20 & 6 & 3 & 5 & 10 & 6 \\ ASP3 & 17 & 6 & 5 & 5 & 8 & 5 \\ ASP1 & 13 & 3 & 4 & 5 & 6 & 6 \\ \hline mean number of distinct alleles & 16.6 & 4.8 & 4 & 5.2 & 7.8 & 5.4 \\ \hline number of distinct alleles in total & 47 & 9 & 5 & 7 & 12 & 11 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Number of distinct marker _loci_ (maternal and paternal) found in each of the five analyzed colonies. \begin{table}
\begin{tabular}{c c c} \hline \hline  & **Microsatellite** & _Csd_ & **Microsatellite + _Csd_** \\ \hline
2019-15 & 8 & 13 & 17 \\
2019-20 & 11 & 16 & 24 \\
2019-3 & 11 & 18 & 24 \\ ASP3 & 12 & 17 & 28 \\ ASP1 & 10 & 11 & 21 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Number of patrilines identified in the analyzed colonies using microsatellites, _Csd_ or microsatellites combined with _Csd_ genotyping. Some microsatellite markers consist of two repeat units of different sizes, resulting in the uneven spacing of the amplicon lengths, further complicating the scoring process. In contrast to commonly used microsatellite markers, the variable region of the _Csd_ gene encodes a protein [15]. Therefore, the length of variation in _Csd_ alleles occurs in multiples of three nucleotides, resulting in evenly spaced amplicon fragments. Sizes can be predicted from sequencing data that are already available in publicly accessible databases. The only exception from the 3N rule is the length diversity of the sequence of the intron (62 nucleotides) localized between exons 7 and 8; however, in the described dataset, we found only two _Csd_ alleles differing in the length of the intron by one nucleotide (61 and 63 nucleotides in length). However, the use of _Csd_ genotyping has some drawbacks, the most significant of which is the relatively inefficient amplification of the _Csd_ HVR fragment, at least in our experience.