Genetic stability of in vitro conserved germplasm of Humulus lupulus L .

Elena L. Peredo1*, Rosa Arroyo-García2, Barbara M. Reed3, M. Ángeles Revilla1 1Universidad de Oviedo, Department of Plant Physiology, Catedrático Rodrigo Uría s/n, 33071 Oviedo, Spain 2Departamento de Biotecnología, Instituto Nacional de Investigación Agraria y Alimentaria, Carretera de A Coruña, km 7.5, 28040 Madrid, Spain 3United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, Corvallis, OR 97333-2521, USA *e-mail: elperedo.uo@uniovi.es


Introduction
Humulus lupulus L. is a dioecious climbing perennial of the Cannabaceae family indigenous to Europe, Asia, and North America.The female inflorescences (cones) are widely used to preserve beer and to give it a characteristic aroma and flavour.The appearance of downy mildew in the northern hemisphere has increased the need to breed new resistant varieties.Breeding programmes continue to focus on increasing hop alpha acid content and aroma profiles.Several cultivars were developed by crossing traditional European cultivars with wild plants from America, with the aim at combining the aroma qualities of the European varieties and the high yielding capacity of the American hops (Moir 2000).
It is extremely important to develop effective methods to store the many cultivars, as well as breeding lines and diverse wild material needed for developing new cultivars.Currently the most useful techniques to store plant material are refrigerated cold storage and cryopreservation at ultra-low temperatures, usually in liquid nitrogen.It is important to keep in mind the risk of genetic and epigenetic instabilities caused by any storage methods.Several factors associated with in vitro culture procedures, such as the medium composition (Viterbo et al. 1994), may result in somatic variation in cold stored plants.During cryopreservation, exposing tissues to physical, chemical and physiological stresses may result in cryoinjury, which ultimately may have effects at genome level (Harding 2004).Several molecular marker techniques are available to analyze genetic stability in plants.Two commonly used techniques are Amplified Fragment Length Polymorphism (AFLP) (Vos et al. 1995) and Random Amplified Polymorphic DNA (RAPD) (Williams et al. 1990).Epigenetic changes due to the stress generated during in vitro culture could possibly generate altered phenotypes in the recovered plants.This epigenetic instability is implicated in the timing of the DNA replication, in determination of chromatin structure, in increasing mutation frequency; as a causal agent for some human diseases; and as a basis for epigenetic phe-nomena (Finnegan et al. 1998).Methylation Sensitive Amplified Polymorphism technique (MSAP) can be used to identify methylation changes in anonymous CCGG regions using the isoschizomeric HpaII/MspI restriction enzymes.
The aim of this study was to evaluate the genetic stability of several hop accessions, representative of those growing around the world, in order to assess the efficacy of standard cryopreservation and/ or cold storage protocols for the diverse cultivars and wild species in genebank collections.

Plant material
Hop accessions (51) representing wild hops, breeding lines, and cultivars supplied by the USDA, National Clonal Germplasm Repository (Corvallis, OR, USA) in February 2005 were analyzed.Each accession (cryopreserved or cold stored) included a control sample and at least two replicated sample treatments.Cold storage and cryopreservation were performed according Reed et al. (2003).Potted greenhouse-grown plants from the USDA core collection were used as control samples.Cold storage was applied for in vitro plants at 4 ºC with a 12-h photoperiod (10 µmolm -2 s -1 ) for one year.For cryopreservation, in vitro shoot tips were dissected from 2-week cold-acclimated plantlets and kept in liquid nitrogen (LN) for 3 years and regrown in the same in vitro conditions as the parent shoots for 4 months.

Molecular techniques
Altogether 51 hop accessions were analysed, seven accessions were cryopreserved and 36 were kept under cold storage conditions.In addition, samples of four accessions were independently conserved under both treatments.For each accession one control plant and at least two controls were analysed.
In total 169 samples were analysed.The RAPD reactions were carried out according to Pillay and Kenny (1996) with slight modifications.Twelve annealing temperatures were tested for each primer and the most appropriate annealing temperature was selected to avoid repetition problems and to increase the number of easy-scored DNA fragments.In all the tested primers, the highest temperature which produced the most suitable band profile was selected (see Table 1 for list of primers and annealing temperatures).In the AFLP assay the genetic stability of five cold-stored and five cryopreserved accessions was tested.We consider that these 10 selected accessions (Table 2) are representative of the total variation within Humulus lupulus as they include wild hops, females, males, diploid and triploids.AFLP analysis was performed according to Cervera et al. (1998).The primers used for the selective amplification were Eco AGC, AGA, AAC, and Mse CAT, CTT.A standard silver staining protocol was used to reveal the bands.
Three hop accessions representative of commercially cultivated hops were selected for the evaluation of methylation stability (Table 3).The MSAP analysis was performed following the general steps according to Cervera et al. (2002).For the selective amplification fluorochrome-labelled primers were used, HpaII/MspI +AAC, +ACT, +ACG (Applied Biosystem, CA, USA) combined with EcoRI +ACT, and AAT.Samples were electrophoresed in an automatic sequencer ABI PRISM® 3100 Genetic Analyzer (Applied Biosystem, CA, USA).The loci were considered polymorphic when differences in the presence/absence of bands in the EcoRI/MspI and EcoRI/HpaII patterns among the control and the treated samples were detected.

Results
Eleven RAPD primers were selected for the assay.A total of 125 loci were detected with a mean of 11 loci per primer, ranging from 0.3 kb to 1.5 kb.Nearly 20,000 bands were scored in the 169 analysed plants.There were clear differences in the banding patterns among accessions but no differences were detected between the control and the treated samples from the same accession, no matter which storage protocol was used (Fig. 1).Table 2. List of accessions analysed using AFLP, primer combination assayed, and number of DNA fragments detected.
In the AFLP analysis, clear and specific patterns were detected for each of the 10 accessions tested.The average number of loci per sample and primer combination was 70.5 ± 6.05 (Table 2), ranging in size from 130 bp to 450 bp.The maximum number of detected DNA fragments per accession and primer combination was 86 (EcoAAC/ MseCTT, 'USDA 21120' and 'Calicross') and the minimum 54 (EcoAGA/MseCAT, 'Vojvodina').Primer combinations EcoACC/MseCAT and ACC/ CTT did not produce differing patterns among accessions.The most bands were detected in all the accessions with EcoAAC/Mse CTT (79.4±5.2) while the fewest was with AGA/ACT (60.0±3.5).Although nearly 19,000 bands were scored in total, no differences between the band patterns of the controls and the cryopreserved or cold stored samples were detected.
Six primer combinations were used in the MSAP analysis which produced a total of 617 clearly detected loci.Clear and repetitive peaks were detected with the automatic sequencer (Fig. 2).The mean number of observed loci per primer combination and cultivar was 34.94 and over 6200 bands were scored.For each accession, and in all the primer combinations tested, variations in the epigenetic profiles were detected between the treated samples and the control.The percentage of monomorphic loci detected in each accession was 61.7% ('USDA 21055'), 63.3% ('Calicross'), and 52.6% ('Tardif de Bourgogne') (Table 3).Each polymorphic locus was assigned to one of the following categories: polymorphism present in both treatments, exclusively in the cryopreserved samples, only in cold stored samples, and singleton (change present in just one plant under the same Table 3. Percentages of monomorphic and polymorphic MSAP loci detected in each cultivar.The polymorphic loci are arranged by behaviour category: variation present all analysed samples of both treatments, in all samples of one treatment (cryopreservation or cold storage), and present only in a single plant (no specific pattern of variation detected).In each group of three: control, and two treated samples.
treatment).Most of the detected variation was shared by both treatments, ranging from 23.7% to 26.7% depending on the accession.The amount of variation that could be specifically related to either of the treatments was relatively low ranging from 2.6 to 9.8% of the total detected MSAP loci for the cryopreserved plants, and from 2.6 to 8.6% in cold-stored plants.The variation that could not be attributed to either or both of the treatments ranged from 2.7 to 5.19%.

Discussion
Cryopreservation and cold storage are two of the most appropriate techniques to conserve large collections of plants (Ashmore 1997).Both techniques involve in vitro culture manipulation of the stored tissues, that could be considered a potential risk for the generation of genetic instability (Brar and Jain 1998).An increasing number of studies indicate that plants recovered from cold storage or slow growth have no genetic alterations (Hao et al. 2004, Renau-Morata et al. 2006).This is also the case observed for these cold-stored hops; no RAPD or AFLP variation could be detected in any of analysed accessions.Similar data were obtained for cryopreserved hop accessions.No changes attributed to somaclonal variation were detected in the eleven accessions subjected to cryopreservation when analysed either with RAPD or AFLP.Based on these observations, it is reasonable to deduce that both cold storage and cryopreservation can be used for the routine storage of hops.Similar results are reported for other species in the current literature: no somaclonal variation was observed in cryopreserved apple (Hao et al. 2001, Lui et al. 2004), or grape and kiwi (Zhaj et al. 2003).However, it is important to note that somaclonal variation was reported in Dendrathema grandiflora (Martín and Gonzalez-Benito 2005), and Hypericum perforatum (Urbanová et al. 2006) Epigenetic changes are a common cause for somaclonal variation, due to the stresses generated during in vitro culture, and these changes could possibly generate altered phenotypes in the recovered plants.In our study, clear epigenetic changes were detected in each accession when compared to the potted plants used as controls.Over 26% of the detected MSAP loci shared some sort of modification after cold storage or cryopreservation.In any of the cultivars, the variation explained by the storage method itself was higher than the amount of variation shared by both treatments.This might be explained by the epigenetic changes related to physiological alterations produced by in vitro establishment.DNA methylation is a dynamic mechanism by means of which plasticity is induced by environmental and/or ontogenic signals (Ramchandani et al. 1999).Therefore, it is not surprising that there is a correlation between the physiological changes produced due to in vitro growth and epigenetic alterations detected in all the in vitro plants.Similar results were found in previous studies of in vitro establishment in hop in which nearly 30% of the detected MSAP loci were polymorphic (Peredo et al. 2009).However, as there are exclusive methylation changes in the cold-stored and cryopreserved plants, we can assume that each protocol is an additional source of epigenetic variation.Methylation changes were also reported in cryopreserved apple and strawberries (Hao et al. 2001 and2002) and citrus callus under slow growth (Hao et al. 2004).Few studies on the genetic stability of hop plants are available.Patzak (2003) described an increased frequency of genetic changes after thermotherapy of in vitro hop meristems.MSAP polymorphisms were detected in hop plants regenerated from callus (Peredo et al. 2006).In summary, methylation changes were detected in both cold stored and cryopreserved plants, although no genetic changes were identified using RAPD and AFLP.We can assume that the genotypes behave similarly when cryopreserved or cold stored as no major differences were found in a range of hop accessions.Both protocols are suitable for use as standard storage methods, but it is important to take into account that a considerable amount of epigenetic change can be induced, particularly during the in vitro process.

Fig. 1 .
Fig. 1.Examples of RADP patterns detected in several hop accession with RAPD primer OPA 01.For each accession no differences were found between the control and treated samples (cold stored or cryopreserved).In each group of three: control, and two treated samples.

Fig. 2 .
Fig. 2. Example of methylation changes detected with MSAP in accession 'Tardif de bourgone'.From top to bottom: control plant, two cryopreseved plants, two cold stored plants.

Table 1 .
List of primers selected for the RAPD assay, annealing temperature selected for each RAPD primer, and number of RAPD loci detected in the 51 hop accessions analysed.