DOI: 10.5061/dryad.3j9kd51j3
title: Hybrids between Rubus idaeus and Rubus sect. Corylifolii and their relation to R. pruinosus and R. rosanthus

description: We have studied hybrids between Rubus idaeus and various members of R.
 Sect. Corylifolii, primarily in Sweden. With the help of DNA-ploidy level
 determinations using flow cytometry and microsatellite DNA analysis of
 over 500 samples, we show that the material can be divided into four
 stable apomictic species (belonging to Subsect. Subidaei) and a large
 number of primary hybrids. Stable species can be recognised by a
 distribution that is distinct from the Corylifolii parent, a uniform
 morphology and an almost invariant genetic pattern. We confirm that R.
 cordatiformis has arisen from the hybrid R. eluxatus idaeus. Moreover, our
 study shows that R. lagerbergii has arisen from the hybrid R. dissimulans
 idaeus. For R. onsalaënsis, we unexpectedly identify the Corylifolii
 ancestor as a previously unidentified blackberry with a very restricted
 distribution in the northern part of the province of Halland, which is
 here described as R. antecedens. Furthermore, we show that R. pruinosus s.
 str. has arisen from the hybrid R. aureolus idaeus and that it is known
 only from a rather restricted area in the provinces of Småland,
 Östergötland and Södermanland in eastern Sweden. On the other hand, the
 great majority of what has been called R. pruinosus is a diverse
 collection of primary hybrids between R. idaeus and various Corylifolii
 species, which show a large variation both genetically and
 morphologically. The same applies to R. rosanthus, which represents R.
 norvegicus idaeus hybrids: The microsatellite analysis shows that they
 have arisen independently and therefore should be considered as primary
 hybrids. We have identified primary hybrids between R. idaeus and R.
 gothicus (by far the most common hybrid in Sweden), R. aureolus, R.
 camptostachys, R. decurrentispinus, R. eluxatus, R. friesianus, R.
 hallandicus, R. lidforssii and R. norvegicus.
description: Samples (young leaves from the tip of the annual shoot) were
 collected from <i>R. idaeus</i>,
 several <i>Corylifolii</i> species and putative
 hybrids<i> </i>were collected from Sweden. The localities are
 specified in the Supplementary Material and a number of vouchers are
 deposited in LD. The samples were stored in a freezer at –80˚C until they
 were used.  DNA was extracted with the 2´ CTAB
 method (Doyle and Doyle 1987). The concentration of the DNA was determined
 by a fluorometer and the samples were diluted with
 ddH<sub>2</sub>O to ~14 ng/µL. Four pairs of microsatellite
 primers, described in Table 1, were used to analyse variable
 microsatellite loci. The three loci Ru105b, Ru117b and Ru275a were
 described for <i>R. idaeus</i> (Graham et al. 2004),
 whereas locus mRaCIRRIV2A8 was described from <i>Rubus
 alceifolius</i>(Ansellem et al. 2001). They will be called 105b,
 117b, 275a and 2A8, respectively, in the following. They have also been
 successfully used also for other <i>Rubus</i> species (Stafne
 et al. 2005, Zhou 2014, Lewin 2016, Hedrén et al. 2019). One primer in
 each primer pair was tagged with a fluorescing molecule (Cy5-) to enable
 the identification of the polymerase chain reaction (PCR) products in the
 automatic sequencing machine (ALF Express II).  The PCR
 reactions consisted of 35 cycles of denaturation at 94 ℃ for 30 s,
 hybridisation at varying temperatures (see Table 1) for 30 s and
 polymerisation at 72 ℃ for 1 min (10 min in the last cycle). Each PCR
 reaction was performed in a solution consisting of (in a total volume of
 ca. 6.4 µL): 4.4 µL ddH<sub>2</sub>O, 0.66 µL 10´ reaction
 buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl and 15 mM
 MgCl<sub>2</sub>), 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 proper size markers, and were then heated to 94 ℃ for
 25 min before they were separated with electrophoresis in a polyacrylamide
 gel. The DNA fragments were detected and analysed with the help of an ALF
 Express II analysis system. The relative height of the bands was estimated
 on a five-graded scale. Samples that resulted in poor PCR reactions were
 purified with a Qiagen purification kit, after which the PCR and
 electrophoresis were redone.
description: The readme file contains an explanation of each of the
 variable in the dataset (i.e. in the uploaded Excel file).