Using flow cytometry and microsatellite DNA analysis (MSDA), we studied hybrids between Rubus caesius and various other Rubus species, with an emphasis on Sweden. We show that hybrids between Rubus caesius and Rubus sect. Corylifolii arise easily. They show a large variation in morphology, but can normally be recognised by a number of characters. They are typically hexaploids, but ~13% of the hybrids are pentaploids and ~8% tetraploids. With MSDA, they are harder to identify than hybrids with R. idaeus, partly because all Corylifolii species have themselves arisen from R. caesius hybrids and therefore share many alleles with R. caesius, partly because hybridisation with R. caesius seems to give rise to some variation in microsatellite regions. Hybrids with species of R. sect. Rubus are much rarer in Sweden and we have only identified one such case. We show that R. cyclomorphus and R. tiliaster are not proper apomictic species, at least not in Sweden, but rather a collection of genetically unrelated hybrids between R. caesius and R. raduloides or R. tiliaster. MSDA analysis of R. slesvicensis and R. firmus shows that these species are problematic. We identify two distinct taxa, one from Skåne in Sweden, which we describe as a new species, R. ruboculus, and another from the neighbourhood of Viborg and Schleswig, although the latter is probably not identical with R. slesvicensis s.s. All the other samples form a diverse group of putative R. caesius hybrids at three different ploidy levels. Rubus glauciformis is uniform in Småland, Öland and Blekinge, but becomes harder to distinguish from various hybrids in Skåne. We conclude that once R. caesius and R. idaeus hybrids are properly identified, along with a small number of new apomictic species (often with a local distribution), the genus Rubus does not pose any serious taxonomical problems in Sweden.

Five hundred seventy-six samples (young leaves from the tip of the annual shoot) of R. caesius, several Corylifolii species and putative R. caesius hybrids were collected, mainly from Sweden, but also from Denmark, Norway and Germany. The localities are specified in Table S1 in the Supplementary Material. Vouchers are deposited in LD. The samples were stored in a freezer at –80˚C until they were used. 

DNA was extracted with the CTAB method (Doyle and Doyle 1987). The concentration of the DNA was determined by a fluorometer and the samples were diluted with ddH2O to ~14 ng/µL. Four pairs of microsatellite primers, described in Table S2, were used to analyse variable microsatellite loci. The three loci Ru105b, Ru117b and Ru275a were described in R. idaeus (Graham et al. 2004), whereas the locus mRaCIRRIV2A8 was described in R. alceifolius Poir. (Ansellem et al. 2001). They will here be referred to as 105b, 117b, 275a and 2A8, respectively. They have been successfully used also for other Rubus species (Stafne et al. 2005, Zhou 2014, Hedrén et al. 2020, Ryde et al. 2021). One primer in each pair was tagged with a fluorescing molecule (Cy5-) to enable the identification of the polymerase chain reaction (PCR) products in an automated sequencer (ALF Express II). 

The PCR amplification consisted of 35 cycles of denaturation at 94℃ for 30 s, annealing at varying temperatures (see Table S2) for 30 s and polymerisation at 72℃ for 1 min (10 min in the last cycle). Each PCR was performed in a solution consisting of (in a total volume of ca. 6.4 µL): 4.4 µL ddH2O, 0.66 µL 10´ reaction buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl and 15 mM MgCl2), 0.56 µL dNTPs (2.5 mM for each nucleotide), 0.25 µL Cy5-tagged primer (10 µg/mL), 0.1 µL untagged complementary primer (25 µmol/mL), 0.03 µL Taq polymerase (5 u/µL; Applied Biosystems) and 0.6 µL template DNA (14 ng/µL).

The PCR products were mixed with formamide and appropriate size markers and were then heated to 94℃ for 25 min before they were separated by electrophoresis in a polyacrylamide gel. The DNA fragments were detected and analysed with the help of an ALF Express II analysis system. Samples that resulted in poor PCR reactions were purified with a Qiagen purification kit, after which the PCR and electrophoresis were redone. Samples that still resulted in poor amplification (unspecific amplification, dominance of short fragments etc.) were excluded altogether.

The relative height of the amplified fragments on the resulting electropherogram was recorded on a five-graded scale for each sample to obtain an estimate of band intensity. Differences in relative band intensity between plants with overlapping banding patterns were matched with information on ploidy levels and interpreted as possibly due to variation in allele copy numbers. Similarly, differences in relative band intensity between plants of the same ploidy expressing different numbers of bands were also interpreted as due to variation in allele copy numbers. Several samples were re-amplified by PCR and rerun to verify the patterns of relative band intensity. Such analyses gave rise to closely similar differences in band intensity as the original runs, with only minor variation in peak height (typically, at most by a single unit along the five-graded scale used by us).

Data can be accessed using Ms Excel.