A Molecular Method for the Identification of Honey Bee Subspecies Used by Beekeepers in Russia

Apis mellifera L. includes several recognized subspecies that differ in their biological properties and agricultural characteristics. Distinguishing between honey bee subspecies is complicated. We analyzed the Folmer region of the COX1 gene in honey bee subspecies cultivated at bee farms in Russia and identified subspecies-specific SNPs. DNA analysis revealed two clearly distinct haplogroups in A. mellifera mellifera. The first one was characterized by multiple cytosine-thymine (thymine-cytosine) transitions, one adenine-guanine substitution, and one thymine-adenine substitution. The nucleotide sequence of the second haplogroup coincided with sequences from other subspecies, except the unique C/A SNP at position 421 of the 658-bp Folmer region. A. mellifera carnica and A. mellifera carpatica could be distinguished from A. mellifera mellifera and A. mellifera caucasica by the presence of the A/G SNP at position 99 of the 658-bp Folmer region. The G/A SNP at position 448 was typical for A. mellifera carnica. A. mellifera caucasica COX1 sequence lacked all the above-mentioned sites. We developed a procedure for rapid identification of honey bee subspecies by PCR with restriction fragment length polymorphism (RFLP) using mutagenic primers. The developed molecular method for honey bee subspecies identification is fast and inexpensive.