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Hybrids between Rubus idaeus and Rubus Sect. Corylifolii and their relation to R. pruinosus and R. rosanthus

Citation

Ryde, Ulf et al. (2021), Hybrids between Rubus idaeus and Rubus Sect. Corylifolii and their relation to R. pruinosus and R. rosanthus, Dryad, Dataset, https://doi.org/10.5061/dryad.3j9kd51j3

Abstract

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.

Methods

Samples (young leaves from the tip of the annual shoot) were collected from R. idaeus, several Corylifolii species and putative hybrids 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 ddH2O 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 R. idaeus (Graham et al. 2004), whereas locus mRaCIRRIV2A8 was described from Rubus alceifolius(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 Rubus 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 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 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.

Usage Notes

The readme file contains an explanation of each of the variable in the dataset (i.e. in the uploaded Excel file).

Funding

Lunds botaniska förening

Botaniska föreningen i Göteborg

Magnus Bergvalls Stiftelse

Lunds botaniska förening

Botaniska föreningen i Göteborg