Bioscience Journal | 2022 | vol. 38, e38006 | ISSN 1981-3163 1 André Lucas Domingos da SILVA1 , Jônatas Chagas de OLIVEIRA2 , Tatiana de CAMPOS3 1 Department of Education, State Department of Education, Culture and Sports, Rio Branco, Acre, Brazil. 2 Center for Biological and Natural Sciences, Federal University of Acre, Rio Branco, Acre, Brazil. 3 Brazilian Agricultural Research Corporation, Embrapa, Rio Branco, Acre, Brazil. Corresponding author: Tatiana de Campos Email: tatiana.campos@embrapa.br How to cite: SILVA, A.L.D., OLIVEIRA, J.C. and CAMPOS, T. Evaluation of microsatellite allelic patterns to DNA fingerprinting in rubber tree clones. Bioscience Journal. 2022, 38, e38006. https://doi.org/10.14393/BJ-v38n0a2022-54400 Abstract The rubber tree (Hevea brasiliensis) is native to the Amazon region, and it is widely exploited due to natural rubber produced from latex. There are many clonal varieties, without certification tests. In order to determine a genetic certification, 15 clones were genotyped to identify their genetic pattern. Ten microsatellites were used to determine a subset of alleles exclusive for each genetic profile. The genetic estimates obtained were: number of alleles per locus (N), expected (HE) and observed (HO) heterozygosity, Polymorphic Information Content (PIC) and Discriminatory Power (DP). The number of alleles (N) ranged from five to 14, with an average of 9.2. The HE mean (0.80) was higher than HO (0.60), indicating a selection for homozygotes. The locus informativeness was verified with PIC (0.77) and DP (0.90) means showing high polymorphism. The dendrogram represented the formation of three groups related to geographical origin. Clone MDF 180 presented the highest genetic divergence. Two genic pools represented the genetic composition of genotypes. Based on allelic profiles, a set of two microsatellites (A2365 and A2368) was able to distinguish all examined clones. The genetic certification using microsatellite fingerprinting proved to be an alternative to morphological traits. Keywords: Genetic Certification. SSR. Variety Identification. 1. Introduction The rubber tree [Hevea brasiliensis (Will. Ex Adr. De Juss.) Muell. Arg.] is a plant native to Amazon (Gonçalves et al. 1989). Natural rubber has unique physico-chemical characteristics, making it a strategic and irreplaceable material for more than 50 thousand industrialized products (Jain et al. 2009; Rippel and Bragança 2009). The natural rubber of H. brasiliensis has made this species the most used for commercial exploration, as this culture has desirable levels of quality and quantity of rubber polymers in the composition of latex (Gonçalves and Fontes 2009). However, phytosanitary problems caused by the fungus Microcyclus ulei (Henn.) Arx brought many damages to the crop. In an attempt to overcome productivity problems, studies were carried out to select genotypes that had promising characteristics for genetic improvement programs, such as resistance to climatic stress and diseases. For this purpose, many plants have been removed from their naturally occurring site to compose ex-situ collections, serving as sources of genetic resources for breeding programs (Souza et al. 2015). Ex-situ collections have redundancies due to duplicate accessions (Shan et al. 2007). These collections have operational costs for their maintenance, making it crucial to correctly characterize their accessions, EVALUATION OF MICROSATELLITE ALLELIC PATTERNS TO DNA FINGERPRINTING IN RUBBER TREE CLONES https://orcid.org/0000-0002-1553-2005 https://orcid.org/0000-0001-7421-718X https://orcid.org/0000-0002-1487-517X Bioscience Journal | 2022 | vol. 38, e38006 | https://doi.org/10.14393/BJ-v38n0a2022-54400 2 Evaluation of microsatellite allelic patterns to DNA fingerprinting in rubber tree clones both to differentiate and exclude duplicates safely and to identify genotypes. Molecular characterization is a tool capable of optimizing the set of accessions in a collection, grouping a sufficient number of genotypes that clearly represent the genetic diversity of the analyzed sample (Souza et al. 2015). To access genetic diversity within germplasm banks it is necessary to collect, evaluate, characterize, conserve and document to assist breeding programs with the germplasm necessary for the development of new varieties (Gimenes et al. 2000). The evaluation and characterization of germplasm are essential activities to adequately conserve genetic resources. These activities, in addition to providing better knowledge of germplasm available, generate information that helps the collection management, facilitate researchers' access to new gene pools and increase the efficient use of materials. Molecular markers have been successfully used in rubber tree. Microsatellites have been widely used, both with clonal varieties and for natural rubber tree populations (Saha et al. 2005; Le Guen et al. 2009; Gouvêa et al. 2010; Souza et al. 2015; Souza 2018). They are ideal markers for studies of genetic diversity, as they are highly polymorphic, multi-allelic and codominant (Kalia et al. 2011; Fortes et al. 2016). Thus, the objective of this work was to perform the molecular identification of rubber tree clones for genetic certification. 2. Material and Methods Plant material Fifteen clones of H. brasiliensis were collected from Embrapa Acre (Rio Branco/AC) collection (Table 1). Genomic DNA was extracted from young leaflets according to the method described by Doyle and Doyle (1990). The DNA quantification was determined according to Qubit® 4.0 (Thermo Fisher Scientific Inc) fluorometric quantifications system. Table 1. Accessions of H. brasiliensis per geographical and genitor cross origin. Genotype Country Source Cross origin 1 CD 1174 Guatemala Avros 1581 x MDF 180 2 FDR 5802 Brazil Harbel 67 x CD 47 3 CDC 312 Guatemala Avros 308 x MDX 40 4 CDC 56 Guatemala MDX 91 x RRIM 614 5 FX 3864 Brazil PB 86 x FB 38 6 PMB 01 Brazil Primary clone 7 FDR 5240 Brazil Harbel 68 x TU 42-525 8 FDR 5597 Brazil Harbel 68 x TU 42-525 9 FDR 5788 Brazil Harbel 8 x MDF180 10 MDX 624 Brazil Avros 1581 x MDF* 11 FDR 5665 Brazil Harbel 62 x MDX 25 12 MDF 180 Peru Primary clone 13 FDR 4575 Brazil Harbel 8 x IAN 3893 14 MDX 607 Brazil Avros 1581 x MDF* 15 RRIM600 Malaysia TJIR 1 x PB 86 * = unknown accession. Analysis of microsatellites Ten microsatellites developed by Le Guen et al. (2009) (Table 2) were used. Amplification reactions were performed as described by Souza et al. (2015). The amplification products were genotyped in an automatic AB 3500xL sequencer (Applied Biosystems). The samples were multiplexed, organized based on the size of the fragments and the type of fluorophore (Souza 2018). The readings of the samples were performed using the GeneScan-600 LIZ® (Thermo Fisher Scientific Inc) standard in the GeneMarker program version 2.7.4 (SoftGenetics, State College, Pennsylvania, USA). Genotyping failures were repeated following the genotyping protocol on polyacrylamide gels (Creste et al. 2001). Bioscience Journal | 2022 | vol. 38, e38006 | https://doi.org/10.14393/BJ-v38n0a2022-54400 3 SILVA, A.L.D., OLIVEIRA, J.C. and CAMPOS, T. Table 2. Description of the ten microsatellite loci, including the primer sequence, repetitive motif and allelic amplitude. Loci Sequence F (5´-3´) Sequence R (5´-3´) Repetitive motif Amplitude allelic (bp) A2365 CACGACGTTGTAAAACGACGCTATCC ATCAGGCAA CTCCTTCTTTCTCCCC CT 190–228 A2368 CTATTCTACATTCTCCATGTC CACGACGTTGTAAAACGACCTTCTTA TTTTACTGGGCT CT 229–271 A2389 CTTTCTTTTGGTCTTTCTC CACGACGTTGTAAAACGACGCAACT CATCCACCAC CT 188–228 A2406 GTCCACAGAAATAAAACTCA CACGACGTTGTAAAACGACAGCCAT TTTCTCACCTC CT 113–171 A2413 ATCCAAACCTGCTCATAC CACGACGTTGTAAAACGACGACCCC TATCCAAAAGA CT 286–322 A2684 CACGACGTTGTAAAACGACTATGCG AACAAGGAAAG ACAGGGATTTACACATACAA CT 258–300 A2736 CACGACGTTGTAAAACGACGCAACC TGATGAATAAAGA AAATGAGAAACAAGAAGACC CT 102–148 BAC55 B02 GATGCCCTTGATTATGTT CACGACGTTGTAAAACGACACTTAAT GGGCTTTTCC CT 153–177 TA2163 ATGCAACAGAGTAGGAGA CACGACGTTGTAAAACGACTCAAAG CAAATGAAGTG (CT) (CA) 194–242 TAs2558 ACTCAATACAAAGGAAGGT CACGACGTTGTAAAACGACAGTTCC AAAGGTCGTG (CT) (CA) 218–256 The parameters to select the loci to fingerprint analysis considered: A – absence of “stutter” bands; B - markers with high discriminatory power and C - minimum number of markers that can discriminate varieties. Statistical analysis Microsatellites were characterized in terms of the number of alleles per locus (N), expected (HE) and observed (HO) heterozygosity, Polymorphic Information Content (PIC) for each locus and the average of these estimates for all loci, using the following equations, present in the Tools for Population Genetic Analyzes - TFPGA version 1.3 software (Miller 1997). To compare the efficiency of the markers for identifying the varieties, the Discriminatory Power (DP) was estimated for each locus (Tessier et al. 1999). The genetic distance analyzes were calculated using Rogers' modified distance (Wright 1978), based on the distance matrix generated by the TFPGA program version 1.3 (Miller 1997), using the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) for the dendrogram construction in the MEGAX software (Kumar et al. 2018). The number of groups (K) was obtained using the STRUCTURE version 2.3.4 program (Pritchard et al. 2000), based on Bayesian statistics. These groups are made up of individuals who share the same gene pool. The program was run with K ranging from K = 1 to K = 10. Twenty independent simulations were made for each K, using the “admixture” model, independent allele frequencies, 10,000 burn-in and 100,000 Monte Carlo simulations of Markov Chains (MCMC). The determination of the most likely K number was performed using the Structure Harvester v. 0.6.9 (Earl and Vonholdt 2012), based on ΔK values according to Evanno et al. (2005). The allocation of accessions within the groups was carried out according to the probability of each individual belonging to each of the groups. 3. Results and Discussion All the ten tested microsatellites were polymorphic (Table 3). The number of alleles per locus (N) ranged from five (BAC55-B02) to 14 (TA2163), with an average of 9.2 (Table 3). Other clones presented similar estimates when genotyped also with microsatellites (Silva 2019). Bioscience Journal | 2022 | vol. 38, e38006 | https://doi.org/10.14393/BJ-v38n0a2022-54400 4 Evaluation of microsatellite allelic patterns to DNA fingerprinting in rubber tree clones Table 3. Characterization of ten microsatellites in 15 clones of Hevea brasiliensis with the genetic parameters: number of alleles per locus (N); rare alleles (A) expected heterozygosity (HE); observed heterozygosity (HO); polymorphic information content (PIC) and discriminatory power (DP). Locus N A (<0,05%) HE HO PIC DP A2365 9 3 0.83 0.80 0.80 0.96 A2368 9 5 0.73 0.53 0.71 0.84 A2389 8 5 0.59 0.46 0.57 0.78 A2406 9 3 0.88 0.26 0.85 0.93 A2413 9 1 0.87 0.73 0.84 0.96 A2684 7 2 0.62 0.40 0.60 0.83 A2736 9 3 0.83 0.60 0.80 0.92 BAC55-B02 5 0 0.76 0.73 0.74 0.89 TA2163 14 6 0.93 0.60 0.90 0.97 TAs2558 13 8 0.88 0.86 0.86 0.97 Total 92 36 - - - - Mean 9.2 3.6 0.80 0.60 0.77 0.90 The expected heterozygosity (HE) ranged from 0.59 (A2389) to 0.93 (TA2163), with an average of 0.80 (Table 3). The values of observed heterozygosity (HO) varied between 0.26 (A2406) and 0.86 (TAs2558) with an average of 0.60. All loci had HO values lower than HE. This result indicates a deficit of heterozygotes. This genetic profile had also been reported in other studies (Le Guen et al. 2009; Souza et al. 2015; Souza 2018). H. brasiliensis has a mixed reproductive system and preferentially cross-pollinating with an inbreeding rate above 20% (Paiva 1992). The deficit in heterozygotes may be the result of the pressure for homozygotes exerted by a natural crossing of related plants due to the longevity of a perennial species with a long breeding cycle, as well as the controlled crossing to obtain cultivars (Laurance et al. 2004; Secco 2008). This fact is consistent with the origin of the analyzed genotypes, with only two primary clones (PMB 01 and MDF 180). The primary clones presented rare alleles, except for PMB 01, which shared an allele with clone RRIM 600 at locus A2736 and with clones FX 3864 and RRIM 600 with locus BAC55-B02. It is important to note that these three individuals were collected close to Brazil. The values of the polymorphic information content (PIC) ranged from 0.57 (A2389) to 0.90 (TA2163), with an average of 0.77 (Table 3). These values indicate that the microsatellites used in the study are highly informative. Botstein et al. (1980), defined those markers with PIC values greater than 0.5 are highly informative. In addition, PIC can be used to establish relationships between closely related materials, such as paternity or the probability that parents are heterozygous as well as to know the parents from the progeny genotype (Botstein et al. 1980). Perseguini et al. (2012) obtained an average of 0.59 (between 0.23 to 0.86), values slightly lower than the present study. This may be related to the EST-SSR marker used. These loci are more conserved and tend to access less polymorphism. On the other hand, they can be more transferable between species within the genus (Scott et al. 2000; Eujayl et al. 2001). The set of microsatellites analyzed showed Discriminatory Power (DP) between 0.78 (locus A2389) to 0.97 (locus TA2163 and TAs2558), with an average of 0.93 (Table 3). DP estimates allow you to select loci that are most likely to differentiate between two individuals. Lower DP values (0.07 to 0.90, with an average of 0.40 per locus) were also detected with EST-SSRs (Perseguini et al. 2012). Microsatellites A2365 and A2368 with DP 0.96 and 0.84, respectively, presented a unique allelic profile to identify the accessions. Locus A2365 discriminated nine individuals with distinct profiles and two shared profiles: one for individuals CDC 56, FDR 5597 and FDR 5788 and another for accessions MDF 180 and MDX 607. These genotypes were discriminated by locus A2368, which presented nine exclusive profiles and a common profile among six individuals (CDC 312, FDR 5240, FDR 5597, MDX 624, FDR 5665 and MDX 607) (Table 4). Bioscience Journal | 2022 | vol. 38, e38006 | https://doi.org/10.14393/BJ-v38n0a2022-54400 5 SILVA, A.L.D., OLIVEIRA, J.C. and CAMPOS, T. Table 4. Genotype based on microsatellite fingerprints for locus A2365 e A2368. Allele+ 1 9 3 -1 9 6 1 9 4 -1 9 8 1 9 4 -2 3 0 1 9 6 -1 9 6 1 9 6 -1 9 8 1 9 6 -2 1 2 1 9 6 -2 3 0 1 9 8 -1 9 8 1 9 8 -2 1 2 1 9 8 -2 1 3 2 0 2 -2 1 9 2 4 8 -2 5 5 2 5 0 -2 5 0 2 5 0 -2 5 5 2 5 0 -2 5 7 2 5 5 -2 5 5 2 5 5 -2 6 0 2 5 5 -2 6 9 2 5 5 -2 7 4 2 5 7 -2 6 9 2 6 7 -2 7 0 Clones CD 1174 + + FDR 5802 + + CDC 312 + + CDC 56 + + FX 3864 + + PMB 01 + + FDR 5240 + + FDR 5597 + + FDR 5788 + + MDX 624 + + FDR 5665 + + MDF 180 + + FDR 4575 + + MDX 607 + + RRIM 600 + + Besse et al. (1993) identified 73 Wickham clones with minisatellites probes, but this analysis is laborious and evolves manipulation of radioactive phosphorous. The alleles accessed by microsatellites A2365 and A2368 were effective for varietal identification of the 15 clones tested and have methodological advantages for routine use, as safe security without radioactive residue. This microsatellite set can be widely applied in studies with rubber tree clones, allowing a direct, fast, low-cost analysis, capable of guaranteeing the correct discrimination and certification of this set of genotypes. No redundant accessions were identified (Figure 1). The most divergent pair were MDX 624 vs FDR 5802 clones with a genetic distance of 0.85. Bioscience Journal | 2022 | vol. 38, e38006 | https://doi.org/10.14393/BJ-v38n0a2022-54400 6 Evaluation of microsatellite allelic patterns to DNA fingerprinting in rubber tree clones Figure 1. Dendrogram with 15 genotypes of H. brasiliensis, according to Rogers modified genetic distance for grouping in the UPGMA method using ten microsatellites. Dotted lines highlight the three formed groups (A, B and C). The closest individuals were CDC 312 and FDR 5665 clones, which presented 0.52 of genetic distance. Their origins as germplasm are documented as Guatemala and Brazil, respectively. It can be explained by ancestry composition, because both clones have one parent from the same MDX line and consequently share a common genetic base. Clone clusters were formed relating to the origin and parental (Figure 1). Group A formed only by clone MDF 180, Group B with varieties CDC 1174, FDR 5802 and FDR 5788 and Group C with clones CDC 312, FDR 5665, MDX 624, MDX 607, FDR 5240, RRIM 600, FDR 5597, CDC 56, FDR 4575, FX 3864 and PMB 01. When verifying the source and parental data, relationships were found that explained the groupings. Group A has its origins in Madre de Dios - Peru, one of the species' naturally occurring sites, and its clonal lineage is widely used in controlled crosses to obtain new varieties. Group B varieties originate in Brazil and Guatemala with parents of the Harbel and MDF clonal line, justifying the similarity of these genotypes. Group C individuals have origins in Brazil, Guatemala, and Malaysia, however individuals from Guatemala and Malaysia are products of plant crosses originating from a naturally occurring area, which corroborates their similarity (Table 1). The similarity of individuals within the group may be related to the restricted genetic base formed by plants collected by Henry Wickham in Pará, which gave rise to several clonal strains such as RRIM and Harbel that participated in crossings of most common commercial clones (Webster and Baulkwill 1989). IAN, MDX and MDF clones are varieties created in the native region of the species, and the crossing of these parents can rescue a diversity similar to the natural individuals of the Amazon region, becoming genetically close (Webster and Baulkwill 1989). The proximity between the varieties within the native region of the species is due to the distribution of its genetic structure through dispersion mechanisms such as the hydrographic network of the Amazon basin that cross countries like Peru, Bolivia, Colombia, and Brazilian states like Pará and Amazonas. The possible differences are related to the natural isolation of plants in key areas (Le Guen et al. 2009). The Bayesian analysis of the STRUCTURE program and the statistics of Evanno et al. (2005) indicated that the highest delta K value has two distinct groups (clusters) (K = 2). From this data it was possible to obtain a histogram showing the genetic composition of each individual and their respective grouping by similarity to ancestry (Figures 2 and 3). A C B Bioscience Journal | 2022 | vol. 38, e38006 | https://doi.org/10.14393/BJ-v38n0a2022-54400 7 SILVA, A.L.D., OLIVEIRA, J.C. and CAMPOS, T. Figure 2. Delta chart (K) with K = 2, obtained by applying the statistical model of Evanno et al. (2005), in the STRUCTURE program, based on the analysis of 15 clones of H. brasiliensis. Figure 3. Histogram relating the 15 genotypes of H. brasiliensis according to the Bayesian analysis generated by the STRUCTURE program. Columns represent individuals and the colors represent the two gene pools (K = 2). The histogram (Figure 3) shows the genetic composition and the proportion of each pool per clone. The red pool predominated the composition of individuals CDC 56, FX 3864, PMB 01, FDR 5788, MDX 624, FDR 5665, MDF 180, and FDR 4575. Clones with common parental origin had a similar genetic composition. FDR 5240 and FDR 5597 have the same parents and showed a predominance of the green pool in their genetic composition. Clone FDR 5788 has as its parent the primary clone MDF 180, justifying its similar composition pool. Individuals CDC 1174, MDX 624 and MDX 607 also had parents of the MDF source but were grouped in the green pool. This is due to the second parent of their crosses, belonging to the clonal lineage Avros (Indonesia), which have a restricted genetic base and are closely related to the Amazon region. RRIM 600 from Malaysia was found, which also has a strong genetic relationship with Amazonian genotypes due to ancestry (Gonçalves et al. 1999). This genetic relatedness was also verified in other genetic studies (Souza et al. 2015; Souza 2018). This analysis represents a robust complementary tool for certifying the clones' authenticity, enabling the tracking of their geographical origin by relating other individuals based on their ancestry. It satisfactorily complements traditional morphological characterization and could be adopted in breeding programs. Bioscience Journal | 2022 | vol. 38, e38006 | https://doi.org/10.14393/BJ-v38n0a2022-54400 8 Evaluation of microsatellite allelic patterns to DNA fingerprinting in rubber tree clones 4. Conclusions Microsatellites provided a robust fingerprint analysis. The genotypic profiles allowed to access molecular diversity to characterize, identify, and provided genetic certification in 15 rubber tree clones. It is a powerful tool for breeding programs. Authors' Contributions: SILVA, A.L.D.: conception and design, acquisition of data, analysis and interpretation of data, drafting the article; OLIVEIRA, J.C.: acquisition of data, drafting the article; CAMPOS, T.: conception and design, analysis and interpretation of data, drafting the article. All authors have read and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Ethics Approval: Not applicable. Acknowledgments: The authors would like to thank the funding for the realization of this study provided by the Brazilian agencies CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil), Finance Code 001, and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brasil). The authors would also like to thank Embrapa (Empresa Brasileira de Pesquisa Agropecuária - Brasil) for providing the structure, and Dr. Rivadalve Coelho Gonçalves for his guidance in collecting leaf samples. 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Received: 1 May 2020 | Accepted: 2 March 2021 | Published: 16 February 2022 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1590/S0100-204X2012000800008 https://doi.org/10.1093/genetics/155.2.945 https://doi.org/10.1590/S0100-40422009000300024 https://doi.org/10.1111/j.1439-0523.2004.01053.x https://doi.org/10.1007/s001220051344 https://doi.org/10.1007/s10722-006-0008-2 https://doi.org/10.1371/journal.pone.0134607 https://doi.org/10.1007/s001220051054