Species specific primer sets design
Candidate regions for designing monk seal specific primers were
identified within the mtDNA regions targeted by MarVer primers
(Valsecchi et al ., 2020), as the amplicons produced by the three
primer sets -MarVer1, MarVer2 and MarVer3 - were all highly polymorphic
also between the two sister species of the Mediterranean (Monachus
monachus ) and the Hawaiian (Neomonachus schauinslandi ) monk
seals: 12 (out of 199bp), 14 (out of 87bp) and 24 (out of 237bp)
variable sites were found in the fragments amplified by MarVer1, MarVer2
and MarVer3 respectively. Internally to each amplicon, one monk-seal
specific primer was designed from the stretch of sequence showing the
largest number of diagnostic sites. Each monk-seal specific primer was
paired for amplification to the corresponding MarVer universal primer
(Valsecchi et al ., 2020) on the opposite strand. In those
instances where the universal primer presented one degenerate base, the
mammalian variant was used (Valsecchi et al ., 2020). For the
reasons exposed in the Discussion, another prerequisite we included in
the search for adequate priming sites was that of aiming at producing
amplicons sufficiently different in size between the three loci.
Hereafter we use the terms “MarVer1”, “MarVer2” and “MarVer3” to
designate the three loci, rather than the primer sets.