Agricultural and Food Science, vol. 18 (2009): 144-151 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 144–151. 144 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 144–151. 145 © Agricultural and Food Science Manuscript received April 2008 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 The genetic and epigenetic stability of hop accessions cryopreserved for one year or cold stored for three years was evaluated using several molecular markers (RAPD, AFLP, and MSAP). Clear, repetitive patterns were obtained among accessions and between control and treated samples. Although no genetic changes were detected among the control plants grown in the greenhouse and in vitro plants regenerated from slow-cooling cryopreserved shoot tips or cold stored in vitro shoots, MSAP analysis detected methylation changes in 36% of the loci. Nevertheless, only 2.6 to 9.8% of the detected changes could be ascribed to the conservation procedure and most of them seemed to be generated as a result of the in vitro introduction. Due to the number of accessions analysed (51) we can cautiously deduce that the genetic behaviour described in this work after cryopreservation or cold-storage protocols is common to most hop genotypes and these storage procedures are suitable for standard use. However, it is important to keep in mind the epigenetic changes produced, particularly during any in vitro processes. Key-words: AFLP (Amplified Fragment Length Polymorphism), cold storage, cryopreservation, MSAP (Methylation Sensitive Amplified Polymorphism), RAPD (Random Amplified Polymorphic DNA), soma- clonal variation. A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 144–151. 144 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 144–151. 145 Introduction Humulus lupulus L. is a dioecious climbing perennial of the Cannabaceae family indigenous to Europe, Asia, and North America. The female inflores- cences (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 re- frigerated 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 composi- tion (Viterbo et al. 1994), may result in somatic variation in cold stored plants. During cryopreser- vation, 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 tech- niques are available to analyze genetic stability in plants. Two commonly used techniques are Ampli- fied 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 increas- ing mutation frequency; as a causal agent for some human diseases; and as a basis for epigenetic phe- nomena (Finnegan et al. 1998). Methylation Sensi- tive Amplified Polymorphism technique (MSAP) can be used to identify methylation changes in anonymous CCGG regions using the isoschizo- meric 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 as- sess the efficacy of standard cryopreservation and/ or cold storage protocols for the diverse cultivars and wild species in genebank collections. Material and methods Plant material Hop accessions (51) representing wild hops, breed- ing 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 stor- age was applied for in vitro plants at 4 ºC with a 12-h photoperiod (10 µmolm-2s-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 con- trol plant and at least two controls were analysed. A G R I C U L T U R A L A N D F O O D S C I E N C E Peredo, E.L. et al. In vitro genetic stability of hops 146 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 144–151. 147 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 anneal- ing 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 accord- ing 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 com- mercially cultivated hops were selected for the evaluation of methylation stability (Table 3). The MSAP analysis was performed following the gen- eral 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 electro- phoresed 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). Primer Sequence Annealing T° Detected bands OPA 01 CAGGCCCTTC 42 °C 12 OPA 02 TGCCGAGCTG 42 °C 11 OPA 03 AGTCAGCCAC 51 °C 10 OPA 04 AATCGGGCTG 40 °C 13 OPA 05 AGGGGTCTTG 44 °C 10 OPA 06 GGTCCCTGAC 45 °C 9 OPA 07 GAAACGGGTG 39 °C 11 OPA 08 GTGACGTAGG 42 °C 13 OPA 09 GGGTAACGCC 42 °C 15 OPA 10 GTGATCGCAG 40 °C 12 OPA 11 CAATCGCCGT 42 °C 9 Total 125 Mean 11.36±1.85 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. A G R I C U L T U R A L A N D F O O D S C I E N C E Peredo, E.L. et al. In vitro genetic stability of hops 146 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 144–151. 147 Primer combinations (*)Accessions agc/cat agc/ctt aga/cat aga/ctt aac/ctt acc/ cat acc/ ctt Total (1) Mean (1) USDA 21055 Cold (2)/Cryo (2) F 2n Oregon, USA -Breeding USDA 62013 ×USDA 6616-35M 74 72 60 70 78 70 74 498 71.1 ±5.6 USDA 21120 Cold (2)/Cryo (2) F 2n Oregon, USA -Breeding 19005 ×19046M 69 67 62 72 86 70 74 500 71.4 ±7.5 Calicross Cold (2)/Cryo (2) F 2n New Zealand -Cultivar California Cluster × Fuggle seedling 71 75 65 78 86 70 74 519 74.1 ±6.7 Tardif de Bourgogne Cold (2)/Cryo (2) F 2n France Cultivar Clonal selection landrace, Alsace 62 71 61 80 73 70 74 491 70.1 ±6.7 Missouri 3 Cryo (4) F 2n Missouri, USA -Wild Selected from wild in Missouri 70 70 66 79 74 70 74 503 71.8 ±4.2 USDA 64033M Cold(2) M 2n Oregon, USA -Breeding German female7k491 × OP 68 62 61 72 72 70 74 479 68.4 ±5.1 Colorado 3-1 Cold (2) F 2n Colorado, USA -Wild Selected from wild in Colorado 67 63 57 74 78 70 74 483 69 ±7.3 Willamette Cold (2) F 3n Oregon, USA -Cultivar USDA 21003 × Fuggle 64 68 61 71 82 70 74 490 70 ±6.8 Vojvodina Cold (2) F 2n Yugoslavia -Cultivar Northern Brewer × male 63 67 54 72 82 70 74 482 68.8 ±8.8 Hallertauer Magnum Cold (2) F 2n Germany -Cultivar Galena × German male 75/5/3 65 65 59 74 83 70 74 490 70 ±7.9 Total (2) 673 680 606 742 794 700 740 4935 70.5 ±6.1 Mean (2) 67.3±3.8 68±4.1 60.6±3.5 74.2±3.5 79.4±5.2 70 74 (1) Total and mean of bands per primer combination. (2) Total and mean of bands per accession. (*) Information supplied: Accession name. Treatment and number of treated samples (cold, cold storage; cryo, cryopreservation). Sex: F, female; M, male. Ploidy: 2n, diploid; 3n, triploid. Origin. State: breeding (line); wild; cultivar. Pedigree. Table 2. List of accessions analysed using AFLP, primer combination assayed, and number of DNA fragments detected. A G R I C U L T U R A L A N D F O O D S C I E N C E Peredo, E.L. et al. In vitro genetic stability of hops 148 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 144–151. 149 In the AFLP analysis, clear and specific pat- terns 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 maxi- mum number of detected DNA fragments per ac- cession 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 ac- cessions. 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 to- tal, 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 prim- er 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 fol- lowing categories: polymorphism present in both treatments, exclusively in the cryopreserved sam- ples, only in cold stored samples, and singleton (change present in just one plant under the same Monomorphic loci Polymorphic loci In both treatments Only in cryopreservated Only cold stored Singletons USDA 21055 61.72 26.23 5.07 4.29 2.73 Calicross 63.3 26.59 2.66 2.66 4.78 Tardif de Bourgogne 52.6 23.7 9.82 8.67 5.19 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). Fig. 1. Examples of RADP pat- terns detected in several hop ac- cession with RAPD primer OPA 01. For each accession no dif- ferences were found between the control and treated samples (cold stored or cryopreserved). In each group of three: control, and two treated samples. A G R I C U L T U R A L A N D F O O D S C I E N C E Peredo, E.L. et al. In vitro genetic stability of hops 148 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 144–151. 149 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 tech- niques 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 at- tributed 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 cryopre- served 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 330320 340 350 360 370 380 360 370 380 2000 0 4000 330320 340 350 2000 0 4000 360 370 380330320 340 350 2000 0 4000 360 370 380330320 340 350 2000 0 4000 360 370 380330320 340 350 2000 0 4000 Fig. 2. Example of methylation changes detected with MSAP in accession ‘Tardif de bourgone’. From top to bottom: con- trol plant, two cryopreseved plants, two cold stored plants. A G R I C U L T U R A L A N D F O O D S C I E N C E Peredo, E.L. et al. In vitro genetic stability of hops 150 A G R I C U L T U R A L A N D F O O D S C I E N C E Vol. 18 (2009): 144–151. 151 during in vitro culture, and these changes could possibly generate altered phenotypes in the recov- ered 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 modi- fication 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 phys- iological alterations produced by in vitro establish- ment. DNA methylation is a dynamic mechanism by means of which plasticity is induced by environ- mental 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 al- terations 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 methyla- tion changes in the cold-stored and cryopreserved plants, we can assume that each protocol is an ad- ditional source of epigenetic variation. Methylation changes were also reported in cryopreserved apple and strawberries (Hao et al. 2001 and 2002) 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 detect- ed in both cold stored and cryopreserved plants, although no genetic changes were identified using RAPD and AFLP. 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