Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique

Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique 1 first-author †E-mail:mtoral@uchile.cl †E-mail:mtoral@uchile.cl mtoral@uchile.cl 2 3 1 4 1 3 Department of Forest Management, Faculty of Forestry, University of Chile, Santa Rosa 11315, La Pintana, Santiago, Chile Department of Forest Management, Faculty of Forestry, University of Chile, Santa Rosa 11315, La Pintana, Santiago, Chile Department of Ecological Sciences, Faculty of Sciences, University of Chile, Santa Rosa 11315, La Pintana, Santiago, Chile Department of Ecological Sciences, Faculty of Sciences, University of Chile, Santa Rosa 11315, La Pintana, Santiago, Chile Department of Forestry, School of Agriculture and Forestry, University of Córdoba, 14080 Córdoba, Spain Department of Forestry, School of Agriculture and Forestry, University of Córdoba, 14080 Córdoba, Spain Department of Genetic, School of Agriculture and Forestry, University of Córdoba, 14080 Córdoba, Spain Department of Genetic, School of Agriculture and Forestry, University of Córdoba, 14080 Córdoba, Spain A protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) was established to analyse the gene diversity and genotype identification for clones of Sequoia sempervirens (D. Don) Endl. in Chile. Ten (out of 34) clones from introduction trial located in Voipir-Villarrica, Chile, were studied. The PCR-RAPDs technique and a modified hexadecyltrimethylammonium bromide (CTAB) protocol were used for genomic DNA extraction. The PCR tests were carried out employing 10-mer random primers. The amplification products were detected by electrophoresis in agarose gels. Forty nine polymorphic bands were obtained with the selected primers (BG04, BF07, BF12, BF13, and BF14) and were ordered according to their molecular size. The genetic similarity between samples was calculated by the Jaccard index and a dendrogram was constructed using a cluster analysis of unweighted pair group method using arithmetic averages (UPGMA). Of the primers tested, 5 (out of 60) RAPD primers were selected for their reproducibility and high polymorphism. A total of 49 polymorphic RAPD bands were detected out of 252 bands. The genetic similarity analysis demonstrates an extensive genetic variability between the tested clones and the dendrogram depicts the genetic relationships among the clones, suggesting a geographic relationship. The results indicate that the RAPD markers permitted the identification of the assayed clones, although they are derived from the same geographic origin. Sequoia sempervirens (D. Don) Endl. Random amplified polymorphic DNAs (PCR-RAPDs) Molecular markers Clone assays Chile Genetic relationships INTRODUCTION 1 Sequoia sempervirens (D. Don) Endl. is a conifer originating from the west coast of California (USA) and the southwestern coast of Oregon (USA). Characteristics of S . sempervirens biology, such as seed germination, seedling survival, phenology, pollen dispersal, and seed crop production, have been reported elsewhere (Noss, 2000 2000 ). S . sempervirens is the only hexaploid (2 n =6 x =66) amongst the members of Taxodiaceae (Ahuja and Neale, 2002 2002 ), which has been challenging genetic studies of the species. In Sequoia , the existence of a high degree of polymorphism has been reported using chemical indicators (Hall and Langenheim, 1987 1987 ), allozyme (Rogers, 1999 1999 ; Rogers and Westfall, 2007 2007 ), and microsatellite markers (Bruno and Brinegar, 2004 2004 ), within the same population as well as among different populations. A study of allozyme diversity in the species (Rogers, 1999 1999 ) revealed genetically distinct groups corresponding largely to natural regions. Although this allozyme analysis has been an effective tool in the study of redwood inheritance (Rogers, 1997 1997 ; 1999 1999 ) and diversity (Rogers, 2000 2000 ), new DNA-based genetic markers have been applied (Bruno and Brinegar, 2004 2004 ). Sequoia was introduced into Chile by Albert ( 1908 1908 ) in 1908 to stabilize the dunes of Chanco-Constitución and recommended as a promising species for the development of commercial forest plantations in this country. However, plantations for commercial use have started only in 1952, with yields ranging between 17 and 28 m 3 /ha, according to stand quality (Toral et al., 2005 2005 ). There is evidence that some of the seed sources used in Chile have markedly different performances in the field in terms of growth and other commercial traits of interest (Toral et al., 2005 2005 ). Information on the levels and patterns of genetic diversity, based on both molecular markers and quantitative traits, facilitates the development of used strategies for forest tree species (Moran and Bell, 1987 1987 ). Such strategies also highlight the plant material that is potentially used in plantation programs. Most of the plantations of Sequoia in Chile arise from uncontrolled seed sources. Morphological evidence indicates that poor selection material was present in forest plantations (Toral et al., 2005 2005 ). These data, together with the evidence that some seedlings perform poorly in plantations (Osses, 2004 2004 ), are consistent with an inadequate provenance maybe used. Selected clones are superior for growth and hence have been the main source of material for breeding populations in recent years (Kuser et al., 1995 1995 ; Kuser, 1996 1996 ). However, there are other requisites for material in a breeding program of S . sempervirens , such as disease resistance, good form, and pulp and wood properties. The Kuser collection comprises 180 clones, obtained from 90 Sequoia -growing areas and natural regions in California (USA). This collection is being tested in assays of provenances and clones from several regions and countries, among which the following can be cited: four trials in USA, two in France, one in Spain and one in New Zealand (Kuser et al., 1995 1995 ). In Chile, the two assays have been established in Villarrica and Lanco, respectively (Toral et al., 2005 2005 ). The selection of the provenances or clones with the best aptitude, highest yield, and best timber quality will permit the future establishment of the best-adapted genetic material for specific objectives such as gain in volume or straightness of the wood. In recent years, biotechnological methods, the application of molecular markers such as RAPD markers, have been fairly successfully used for identifying genetic patterns of specific clones selected in improved populations. These techniques allow the complementation or, even, the replacement of plant or clone identification based on other analytical techniques. RAPD markers have been successfully employed in several plant genera for the reconstruction of phylogenetic relationships (Furman et al., 1997 1997 ; Nkongolo et al., 2002 2002 ; Syring et al., 2005 2005 ). Moreover, RAPDs have also been used for quantifying the intra- and interpopulation gene diversities in several forest species (Grotkopp et al., 2004 2004 ). In Chile, there is a lack of information about the genetic pattern and field response of the Sequoia clones selected from the Kuser collection (Toral et al., 2005 2005 ). Therefore, in the present study we aimed to test an effective protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) for Sequoia sempervirens (D. Don) Endl. using 10 clones that are representative of those used in Sequoia plantations in Chile, and to apply the results to the analysis of gene diversity and genotype identification in clones of the species introduced into Chile. MATERIALS AND METHODS 1 Plant material 2 The material used was a fraction of the Kuser collection that comprises 180 clones representing 90 sites of Sequoia provenances (Fig. 1 1 ) (Kuser et al., 1995 1995 ). For this study, 10 from 34 clones used in the assay located in Voipir-Villarrica, Chile (39°19′20″ S, 72°17′08″ W) were analyzed by PCR-RAPDs (Table 1 1 ). The 10 clones selected had the best percentages of nursery rooting, growth rate, and survival after plantations (Toral et al., 2005 2005 ). Source: forestry databases, Simpson Korbel-Arcata-California, 2002; masl: metre above sea level Original code Latitude Longitude Altitude (masl) PLUS C 40°56′ S 123°58′ W 91~140 RB-236-10 41°45′ S 123°56′ W 90~140 RB2-238-16 40°01′ S 123°49′ W 91~140 RB54-225-17 40°01′ S 123°49′ W 91~140 RB54-225-14 40°01′ S 123°49′ W 91~140 RB54-225-19 40°56′ S 123°58′ W 91~140 COMMERCIAL 41°45′ S 123°56′ W 90~140 RB2-236-18 40°01′ S 123°51′ W 91~140 PLUS B 41°08′ S 123°56′ W 91~140 RB3-57-77-7 41°09′ S 123°56′ W 91~140 Genomic DNA extraction process and amplification by PCR 2 Needles from the clones in the last growth period were collected in November 2004, flash-frozen in liquid nitrogen, and ground in a mortar to a fine powder. Total genomic DNA was extracted following a modified hexadecyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1990 1990 ; Tsumina et al., 1995 1995 ). For DNA extraction, the needles were exhaustively rinsed with distilled sterile water, homogenized with a mini-grinder in TENC buffer (100 mmol/L Tris, 1.4 mol/L NaCl, 20 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.055 mol/L CTAB, 0.038 mol/L mercaptoethanol), preheated at 60 °C with 10 mg/ml polyvinylpyrrolidone (PVP), and incubated at 60 °C for 1 h. Then, 500 µl of chloroform-octanol (24:1, v/v) was added and centrifuged at 6000 r/min for 5 min. The aqueous phase was extracted and DNA was precipitated by adding 1/10 volume of 3 mol/L sodium acetate and 2 volumes of 95% (v/v) ethanol, incubated at −20 °C for 1 h, and then centrifuged at 13 000 r/min for 5 min. After drying, the pellet was resuspended in 200 µl of TE buffer (10 mmol/L Tris, 1 mmol/L EDTA; pH 8), treated with 300 µl of chloroform-isoamilic alcohol, and then homogenized. DNA was rinsed with 70% (v/v) ethanol, resuspended in TE buffer, and then incubated with RNase at 37 °C for 1 h. Finally, DNA was cleaned with 1/2 volume of 8 mol/L LiCl and then left at room temperature for 1 h. DNA was precipitated with 1 volume of 95% (v/v) ethanol at −20 °C and then resuspended in 50 µl of TE buffer. PCR reactions were conducted according to Williams et al.( 1990 1990 ) using 10-mer random primers (Operon Technologies, Alameda, CA, USA). 30~50 µg of chromosomic DNA was used in the amplification reaction, which contained 2.0 mmol/L MgCl 2 , 0.1 mmol/L dNTP, 0.4 µmol/L of the primer, 2.5 U of Taq polymerase (GibcoBRL, USA), and 1 mg/ml bovine serum albumin (BSA) in PCR buffer (10 mmol/L Tris-HCl (pH 8.8), 50 mmol/L KCl), with a final reaction volume of 25 µl. The amplification reaction was performed in a GeneAmp PCR 2400 thermocycler (Perkin Elmer, USA) programmed for 5 min of pre-denaturation at 94 °C, followed by 45 cycles with a denaturation at 92 °C for 1 min and a hybridization for 5 min, and next, 45 cycles with a denaturation at 92 °C for 1 min, an annealing at 36 °C for 1.5 min, and an extension at 72 °C for 1 min. The final extension was done at 72 °C for 5 min. In order to verify the reproductibility of RAPD reaction, 2~3 DNA samples independent of each clone were assayed with selected primers that, for RAPD analysis, were assayed in at least 3 independent PCR reactions. The amplification products were detected by electrophoresis in 1.2% agarose gels (v/v) in TAE buffer (40 mmol/L Tris-acetate, 1 mmol/L EDTA; pH 8), and stained with ethidium bromide. Statistic analysis 2 With the primers selected, the bands obtained for each clone were identified and ordered in accordance with their molecular weight, the one with the largest size being the first. The band pattern obtained was translated into a binary matrix of 0 s and 1 s, where 0 corresponded to the absence and 1 to the presence of this fragment size. The degree of similarity between the clones was obtained by the Jaccard ( 1908 1908 ) index, which considers the number of shared bands over the total number of the bands compared. The similarity was estimated based on the following formula: J = a /( a + b + c ), where J is Jaccard index; a is the number of bands that are common to the two compared genotypes; b is the number of bands present in the 1st genotype and absent in the 2nd one; c is the number of bands absent in the 1st genotype and present in the 2nd one. The sum of a , b , and c will give the total number of the characters measured. The dendrogram was constructed by a cluster analysis using the unweighted pair group method using arithmetic averages (UPGMA) with bootstrap values over 1000 replicates using the packages FreeTree (Manly, 1991 1991 ; Pavlicek et al., 1999 1999 ). RESULTS 1 RAPD analysis 2 To identify primers that detect polymorphism, 60 primers corresponding to the BF, BG, and BH series (Operon Technologies, Alameda, CA, USA) were screened using genomic DNA from the clones (Table 2 2 ). From the set of primers assayed, five were selected, which gave a reproducible positive reaction and polymorphic bands. The sequence of the primers selected and the number of bands amplified are shown in Table 3 3 . With these primers, a total of 252 bands was obtained, 49 of which were sufficiently polymorphic to allow the differentiation of the clones under study. Depending on the primer, 6~16 polymorphic bands were obtained with an average of 9.8. PS 5′–3′: primer sequence 5′–3′ Primer PS 5′–3′ OPBG-01 GTGGCTCTCC OPBG-02 GGAAAGCCCA OPBG-03 GTGCCACTTC OPBG-04 GTTCCCGACA OPBG-05 CAAGCCGTGA OPBG-06 GTGGATCGTC OPBG-07 CAGAGGTTCC OPBG-08 GACCAGAGGT OPBG-09 GGCTCTGGGT OPBG-10 GGGATAAGGG OPBG-11 ACGGCAATGG OPBG-12 CCCGAGAAAC OPBG-13 GGTTGGGCCA OPBG-14 GACCAGCCCA OPBG-15 ACGGGAGAGA OPBG-16 TGCTTGGGTG OPBG-17 TCCGGGACTC OPBG-18 TGGCGCTGGT OPBG-19 GGTCTCGCTC OPBG-20 TGGTACCTGG 2 OPBH-01 CCGACTCTGG OPBH-02 GTAAGCCGAG OPBH-03 GGAGCAGCAA OPBH-04 ACCTGCCAAC OPBH-05 GTAGGTCGCA OPBH-06 TCGTGGCACA OPBH-07 TGTACGGCAC OPBH-08 ACGGAGGCAG OPBH-09 GTCTTCCGTC OPBH-10 GTGTGCCTGG OPBH-11 AGCCCAAAGG OPBH-12 TCGCCTTGTC OPBH-13 AGTTGGGCAG OPBH-14 ACCGTGGGTG OPBH-15 GAGAACGCTG OPBH-16 CTGCGGGTTC OPBH-17 CTCTTACGGG OPBH-18 GACGCTTGTC OPBH-19 GTCGTGCGGA OPBH-20 CACCGACATC 2 OPBF-01 GGAGCTGACT OPBF-02 GACACACTCC OPBF-03 TCCCTTGACC OPBF-04 GACAGGTTGG OPBF-05 CACCCCGAAA OPBF-06 TCCACGGGCA OPBF-07 CACCATCGTG OPBF-08 CCTGGGTCCA OPBF-09 ACCCAGGTTG OPBF-10 GTGACCAGAG OPBF-11 GACGACCGCA OPBF-12 CTTCGCTGTC OPBF-13 CCGCCGGTAA OPBF-14 CCGCGTTGAG OPBF-15 ACGCGAACCT OPBF-16 AGGGTCCGTG OPBF-17 CAAGCTCGTG OPBF-18 AGCCAAGGAC OPBF-19 TTCCCGCACT OPBF-20 ACCCTGAGGA PS 5′–3′: primer sequence 5′–3′; n 1 : number of amplified bands; n 2 : number of polymorphic bands Primer PS 5′–3′ n 1 n 2 BG 04 GTTCCCGACA 38 8 BF 07 CACCATCGTG 27 6 BF 12 CTTCGCTGTC 71 10 BF 13 CCGCCGGTAA 64 16 BF 14 CCGCGTTGAG 52 9 4 2 Overall 252 49 Fig. 2 2 shows, as an example, the RAPD markers obtained with primer BF14 in the PCR reaction. The size of each polymorphic fragment amplified with the five primers and for all the clones studied is given in Table 4 4 . Molecular weights are ordered decreasedly 2 Clone 5 Molecular weight (bp) BF07 BF13 BF12 BF14 BG04 PLUS B 650 2300-1900-1700-1400-1300-1100-950-850-650 1400-1300-1200-1031-800-700 1500-1100-1031-950-850-700 1300-1200 COMMERCIAL 1500-950-650 2300-1400-1200-950-850-700-650 1600-1400-1300-1200-1100-950-800-700 1500-1300-1031-800 950-850 RB54-225-17 1500-1200-950 2300-1400-1300-1100-950-850-750-700-650 1600-1300-1200-1100-1031-800-700 1500-1300-1100-1031-900-850-700 1300-1200-850 PLUS C 1500-1200-950 2300-1400-1200-850-750-450 1600-1400-1300-1200-800-700 1300-900-850-700 1200-950-850-550 RB2-238-16 1500-1200-950-500 2300-1400-1300-1200-1100-950-850 1600-1400-1300-1200-950-800-700 1500-1300-1031-850-700 1300-1200-950-850-550 RB3-57-77-7 1500-700-650 2300-1700-1400-1200 1300-1100-800-700-600 1500-1100-900-850-700 1300-1200-950 RB-236-10 1500-950 2300-1900-1500-1200-950-850 1600-1400-1300-1200-950-800-700 1500-1300-1100-1031-850-700 1300-1200-950-850-550 RB54-225-14 1500-950 2300-1700-1400-1200-1100-750 1600-1400-1300-1200-1100-1031-950-800-700 1031-900-850-700 1300-1200-950-850 RB54-225-19 1500-1200-950 1200-1100-750-650-550 1600-1400-1300-1200-1100-1031-800-700 1500-1300-1031-900-850-700 1300-1200-950-850-550 RB2-236-18 1500-950-700 2300-1400-1200-850-350 1600-1400-1300-1200-1100-950-800-700 1300-1031-950-900-700 1700-1200-850-700-450 The amplicons obtained with the different primers were distributed between 350 and 2300 bp. All the clones exhibited at least one polymorphic band with each primer, e.g., PLUS B with BF07, although in the majority of the cases the primers amplified between 4~9 polymorphic bands. Some bands were common to majority of clones, such as the 2300-bp and 14000-bp fragments obtained with BF13, which were common to 8 clones from all assayed clones. The results show that the RAPD markers, polymorphic bands, obtained with the primers selected produced a single pattern of bands for each clone assayed, permitting their identification. Genetic similarity and clustering of genotypes 2 RAPD marker differences among the pooled DNA samples were analyzed to infer genetic relationships among populations (Table 5 5 ). The similarity coefficients among clones varied from maximum 0.719 between RB-236-10 and RB2-238-16 clones to minimum 0.306 between PLUS C and PLUS B clones, with the average of 0.502, indicating the high level of genetic variation that exists in the Sequoia clones selected. Likewise, the similarity coefficients within the most frequent clones used in Chile ranged from 0.306 to 0.559 and 0.333 to 0.594 for PLUS B and COMMERCIAL clones, respectively. Taking the similarity matrix, a dendrogram was set up by means of a UPGMA cluster analysis (Fig. 3 3 ). The reliability of the dendrogram was determined by bootstrap analysis over 1000 replicates. The results of the cluster analysis show that RAPD profiles obtained from RB-236-10 and RB2-238-16 clones had the highest similarity degree (close to 0.72) forming a cluster with a high bootstrap value (70%). In addition, this cluster appeared related to PLUS C clone, although the association was not supported by the bootstrap value. Another cluster was formed by genotypes RB54-225-19, RB54-225-14, and RB54-225-17, which had a similarity degree of over 0.6 but a bootstrap value of lower than 50%. Clone: 1: PLUS B; 2: COMMERCIAL; 3: RB54-225-17; 4: PLUS C; 5: RB2-238-16; 6: RB3-57-77-7; 7: RB-236-10; 8: RB54-225-14; 9: RB54-225-19; 10: RB2-236-18 Clone * * 1 2 3 4 5 6 7 8 9 10 1 1 2 0.371 1 3 0.559 0.514 1 4 0.306 0.469 0.529 1 5 0.486 0.576 0.629 0.645 1 6 0.419 0.333 0.400 0.387 0.371 1 7 0.457 0.594 0.556 0.563 0.719 0.424 1 8 0.441 0.485 0.588 0.600 0.606 0.500 0.576 1 9 0.378 0.457 0.647 0.613 0.618 0.382 0.588 0.677 1 10 0.316 0.515 0.447 0.531 0.500 0.353 0.514 0.545 0.432 1 Both clusters formed a separated group from the rest of the clones with a bootstrap value of 51%. Although the clones assayed derived the localities geographically closed, they exhibited enough variability, detected by RAPD marker, to its molecular identification. With the data available, it is not possible to correlate the genetic variability observed with the geographical origin of the clones. DISCUSSION 1 Diverse comparative studies have been conducted to assess the genetic variability and population relationships in Sequoia sempervirens (Rogers, 1999 1999 ; 2000 2000 ; Bruno and Brinegar, 2004 2004 ; Rogers and Westfall, 2007 2007 ). Specifically, genetic relationships between old-growth populations have shown a considerable fine-scale spatial genetic structure (Rogers and Westfall, 2007 2007 ). However, the delimitation of clone variability still is an important problem in forest species (Hansen, 2008 2008 ). In this study, RAPD markers were applied. The value of RAPD analysis for efficient germplasm management in plants is already known (Krutovskii et al., 1998 1998 ; Nkongolo et al., 2002 2002 ; Allnutt et al., 2003 2003 ). The use of RAPD markers for phylogeny has been questioned mainly because of the possible lack of homology of comigrating bands (Harris, 1999 1999 ). Nevertheless, RAPD data have been very helpful for the inference of genetic relationships among closely related species and subspecific taxa (Furman et al., 1997 1997 ; Nkongolo et al., 2002 2002 ). Apparently, the present work constitutes the first application of RAPD markers for the study of relationships among Sequoia clones in Chile. The RAPD profiles were easy to reproduce by use of a standardized protocol, and consistency was shown in producing identical bands. Genetic differences among clones were successfully identified with the DNA pooling strategy, and an average of 9.8 polymorphic markers was obtained per primer, which is consistent with the number of markers identified per primer in plants (5~20). Our results show the morphological similarities observed between Sequoia populations, which is consistent with previous report (Millar et al., 1985 1985 ; Anekonda et al., 1993 1993 ; 1994 1994 ). The 49 polymorphic markers obtained in this study represent 19.4% of the total number of bands amplified. It has been possible to find five primers from the Operon Technologies that provided polymorphic bands for Sequoia in a RAPD analysis (BG04, BF07, BF12, BF13, and BF14). These molecular markers permit the characterization and detection of the differences and/or similarities between the clones evaluated. We have found that reliable RAPD data can be generated by repeating each amplification reaction three times and using conservative criteria of band selection. Similarity index and clustering analysis reveal a high degree of differentiation between Sequoia clones; however, a larger number of clones are necessary to determine genetic structure. The cluster analysis showed that RAPD markers permitted the characterization of the clones assayed and the quantification of similarities between them (Rogers, 2000 2000 ). The RAPD grouping of the clones per similarity degree seems to be related to their geographical origin. Namely, the clones RB-236-10 and RB2-238-16 formed a cluster, which had a higher similarity degree between them and was supported by a high bootstrap value. Another cluster was formed by the clones RB54-225-19, RB54-225-14, and RB54-225-17. Both clusters came from Humboldt County and from localities close by. Additionally, two clones (PLUS B and RB-236-10) are clustered more externally in the dendrogram with high bootstrap value, but with a low similarity degree between them. One explanation for this dissimilarity could be that although both clones derived from Humboldt, they are derived from places farther apart (Table 1 1 ). Since the clones studied derived from collection of Humboldt County (California, USA) with the same geographical origin, a high similarity between them would be expected; however, the values of similarity obtained suggest that they have enough genetic variability detectable with RAPD markers. However, the small number of clones assayed and their uncertain genetic relationship to one another do the “resolving power” of the RAPD method unknown on this trial, which should be taken into account for future applications (Rogers, 1999 1999 ). We conclude that RAPD variation is useful in establishing genetic relationships between Sequoia clones. In the future, the inclusion of more clones in the analysis with RAPD markers and the complementing of molecular data with other techniques will improve the resolution of genetic relationships and the potential use in Sequoia plantations in Chile. 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DNA polymorphisms amplified by arbitrary primers are useful as genetic markers 10.1631/jzus.B0820162 Project (No. CONICYT-FONDEF PROYECT D01 I 1008) supported by the National Commission of Science and Research of Chile 2008-05-05 2008-12-25 2008-10-28 2022-01-05 Copyright © 2009, Journal of Zhejiang University Science 2009 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 10