## 4 Discussion
Although we were generally able to amplify the analyzed fragment in a one-step PCR reaction when using DNA obtained from fresh specimens and isolated using column-based protocols, such conditions limit the analysis of historical samples and significantly increase its cost. Consequently, we established a protocol utilizing a two-step nested-PCR reaction with exonuclease I digestion performed between the steps. Although this protocol admittedly consumes more time, both the amplification and digestion steps can be completed in a single day. We hope that a more efficient protocol can be developed in the future. The amplification of the microsatellite markers used in this study was efficiently achieved in a one-step PCR reaction, regardless of the method of sample storage and DNA isolation. The overall lower efficiency of _Csd_ amplification, combined with the wide spectrum of allele lengths, can also lead to differences in the efficiency of amplifying the two alleles present in the analyzed diploid specimen if these alleles happen to vary significantly in length. The shorter allele amplifies much more readily, often at the expense of the longer allele's amplification efficiency. This discrepancy can be observed in the peak intensity during capillary electrophoresis. While, in extreme cases, this effect may pose challenges in reliably detecting signals from the longer allele, it generally proves beneficial as it facilitates the straightforward pairing of the 5\({}^{\prime}\) and 3\({}^{\prime}\) fragments of the analyzed alleles. Our results show that, although patriline identification using the _Csd_ genotyping offers a higher resolution (\(\times\)1.1-\(\times\)1.6) than using standard sets of microsatellites, it is not sufficient to identify all patrillines in a given colony. This issue could arise due to the limited number of restriction patterns compared to the variety of _Csd_ sequences. Consequently, relying solely on the restriction patterns of _Csd_ amplicons for assigning paternal alleles to individual groups may result in erroneously combining two or more distinct paternal alleles into a single group. Our initial assessment, which was performed in silico (using sets of _Csd_ sequences extracted from a database, see [26]), in order to estimate the extent of this error, showed that between 1 and 2 _Csd_ alleles in a colony consisting of 20 patrillines would be incorrectly assigned to a group with an identical restriction pattern but different sequences. The analysis was performed under the assumption that the queen was inseminated by 10-20 drones, which, in the light of our present results and studies performed by other groups, seems to be a substantially underestimated number [13]. Admittedly, using more microsatellite _loci_ may improve the capability to score all the patrillines within a single colony. However, the techniques to genotype via microsatellite _loci_ are time-consuming and prone to errors (null alleles, misinterpretation of allele banding patterns, contamination) [33]; thus, each additional _locus_ that needs to be amplified represents a significant effort. An adequate number of microsatellite _loci_ to infer all the patrillines depends largely on the diversity of each specific locus, the non-sampling error, and the size of the population and can thus vary between the studies [34; 35]. Using _Csd_ as a substitute marker for patriline estimation in the evaluation of mating control success instead of using multiple microsatellite _loci_ may still bring an advantage in the reduction in molecular and downstream analyses and a slightly enhanced resolution. Recent research proved that _Csd_ gene variants may also be efficiently determined from a complex mixed sample such as honey. Their technique targeted the HVR region of the _Csd_ gene and was compatible with high-throughput sequencing technologies [36].