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Genetic Diversity and Population Structure of Capirona (Caly- Cophyllum Spruceanum Benth.) from the Peruvian Amazon As- Sessed by RAPD Markers

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Genetic Diversity and Population Structure of Capirona (Caly- Cophyllum Spruceanum Benth.) from the Peruvian Amazon As- Sessed by RAPD Markers Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 June 2021 doi:10.20944/preprints202106.0162.v1 Article Genetic Diversity and Population Structure of Capirona (Caly- cophyllum spruceanum Benth.) from the Peruvian Amazon As- sessed by RAPD Markers Carla L. Saldaña 1, Johan D. Cancan 1, Wilbert Cruz 1, Mirian Y. Correa 2, Miriam Ramos 3, Eloy Cuellar 4, and Carlos I. Arbizu 1,* 1 Dirección de Desarrollo Tecnológico Agrario, Instituto Nacional de Innovación Agraria (INIA), Av. La Mo- lina 1981, Lima 15024, Peru; [email protected] (C.L.S); [email protected] (J.D.C); wil- [email protected] (W.C) 2 Universidad Nacional Pedro Ruíz Gallo, Facultad de Ciencias Biológicas, Juan XXIII No 391, Lambayeque, Perú; [email protected]. 3 Universidad Nacional Agraria La Molina, Facultad de Ciencias Forestales, Av. La Molina s/n, Lima, Lima 15024, Peru; [email protected]. 4 Universidad Nacional Agraria La Molina, Av. La Molina s/n, La Molina, Lima, Lima, 15024, Peru; eloycue- [email protected]. * Correspondence: [email protected] Abstract: Capirona (Calycophyllum spruceanum Benth) is a tree species of commercial importance widely distributed in South American forests and is traditionally used for its medicinal properties and wood quality. Studies on this tree species have been focused mainly on wood properties, prop- agation and growth. Genetic studies on capirona are very limited to date. Today it is possible to explore genetic diversity and population structure in a fast and reliable manner by using molecular markers. We here used 10 Random Amplified Polymorphic DNAs (RAPDs) markers to analyze ge- netic diversity and population structure of 59 samples of capirona that were sampled from four provinces located in the eastern region of the Peruvian amazon. A total of 186 bands were manually scored, generating a 59 x 186 presence/absence matrix. We used R software to calculate genetic dis- tances based on provesti coefficient. A dendrogram was generated using the UPGMA clustering algorithm and showed four groups that correspond to the geographic origin of the capirona sam- ples. Similarly, a discriminant analysis of principal components (DAPC) confirmed that capirona is grouped into four clusters. However, we also noticed few accessions are intermingled. Genetic di- versity estimation was conducted considering the four groups (populations) identified by adegenet package in R. Nei's genetic diversity estimate varied from 0.26 to 0.39 and Shannon index ranged from 2.48 to 2.83. AMOVA analysis revealed the greatest variation exist within populations (69.7%) and indicated that variability among populations is 31.5%. To our best knowledge, this is the first investigation employing molecular markers in capirona in Peru considering their natural distribu- tion, and sheds light towards its modern genetic improvement and for the sustainable management of forests in Peru. Keywords: Amazon forest; capirona; molecular markers; genetic diversity; population structure. 1. Introduction Calycophyllum spruceanum (Benth) (Rubiaceae) is a fast-growing forest specie [1] with a natural origin in the Amazon basin, covering the territories of Bolivia, Peru, Colombia, and Brazil [2]. Capirona is a valuable timber forest species in the Peruvian Amazon, widely used by local communities and is exported around the world. It has a high-density, durable wood, therefore, it is used for the construction of economically valuable products [3]. Capirona has a high potential for cultivation in agroforestry systems, which has coun- tered unsustainable land use due to activities such as slash-and-burn agriculture, and this © 2021 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 June 2021 doi:10.20944/preprints202106.0162.v1 2 of 12 specie represents an alternative to increase rural incomes and development [4]. Thus, ca- pirona is currently the object of research in the Peruvian Amazon basin in order to estab- lish participatory research with local communities, optimizing genetic and silvicultural management strategies including community conservation methods [1] since it is known that the indiscriminate use of forest species is generating deforestation in various regions of the planet [5-7]. Genetic diversity studies are indispensable for conducting conservation programs and sustainable management. Moreover, these studies based on molecular markers provide important information on the genetic makeup of the population because they are independent of the flexing effects of the environmental factors [8]. A demo- graphic study would not provide this kind of information, influencing decision making. In recent years, molecular markers such as DNA-based markers like random-amplified polymorphic DNA (RAPD), microsatellite or simple sequence repeat (SSR) and ISSR have been used in studies for the analysis of phylogeny, inter-species relationships, and genetic diversity for forest species like Pinus leucodermis, Cedrela Odorata Eucalyptus globulus, Swietenia macrophylla, Bertholletia excels and Populus deltoides [9-14]. Previous research determined the genetic variation of capirona by using amplified fragment length polymorphisms (AFLP) among nine populations from the Peruvian Am- azon [1]. They demonstrated that variation among individuals within populations is pre- dominant. However, they did not employed samples from Madre de Dios region, which is considered a place where capirona exists naturally [15]. On the other hand, Tauchen et al., [3] identified the genetic variability of capirona at the level of DNA polymorphism evaluated by non-specific ITS primers of plant materials from Pucallpa, department of Ucayali in the Peruvian Amazon. Their results showed that morphological diversity is greater than genetic diversity of capirona. They also resolved that the environmental fac- tor had a greater impact on the phenotype in those accessions. Finally, their results are in line with the claims of previous studies on C. spruceanum, suggesting greater variation within accessions than between accessions. RAPDs is an especially useful DNA-based method for initial research of genetic diversity in plant species [16, 17] as capirona. Also, RAPDs have demonstrated great potential in the analysis of polymorphism and genetic diversity as they are fast, easy to test and require low concentrations of DNA [17]. The objective of this study was to determine the genetic diversity and population structure of capirona from primary forests located in the regions of Ucayali, San Martín and Madre de Dios located in the Peruvian Amazon to establish in the near future a mod- ern breeding program, and a sustainable conservation of this tree species. 2. Materials and Methods 2.1. Plant material We collected 59 samples of capirona from Ucayali, San Martín and Madre de Dios regions in the Peruvian Amazon considering their natural range of distribution. Leaf sam- ples were collected in paper envelopes, stored in airtight container with silicone gel, and transported to the National Institute of Agricultural Innovation (INIA for its acronym in Spanish) for genomic DNA extraction. Further details of the samples examined in this study are available in Table 1. 2.2. DNA amplification We extracted genomic DNA by using the CTAB method with minor modifications [18,19]. All procedures were performed in 1.5 mL plastic microfuge tubes. About 0.1 g dry leaves were grounded in liquid nitrogen and suspended in 600 mL of 2x CTAB buffer containing 0.2% b-mercaptoethanol, followed by incubating at 65 °C for 60 min, then equal volume of chloroform: isoamylalcohol (24:1, v/v) was added, and the sample was shaken gently and then centrifuged. The supernatant was extracted, then 10X CTAB buffer and chloro- form: isoamylalcohol (24:1, v/v) was added again. The supernatant was extracted and then Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 June 2021 doi:10.20944/preprints202106.0162.v1 3 of 12 mixed with ice cold isopropanol. DNA was recovered as a pellet by centrifugation, washed with ice-cold ethanol twice (70 y 90%), and then DNA was dried in the air. Finally, DNA was resuspended in nuclease-free water. The RNA contamination in all the samples was removed by digesting the extract with RNase-A (100 µg ml-1) for 30 min at 37ºC. DNA quality and quantity were checked in 1% agarose gels using Gelred (Biotium®, USA) and by standard spectrophotometry. Table 1. The 59 samples of capirona characterized in this study, code, and geographic information. Number Code Location Region Country Number Code Location Region Country 1 Cap 018 Irazola Ucayali Peru 31 Cap 057 LBS San Martín Peru 2 Cap 019 Irazola Ucayali Peru 32 Cap 058 LBS San Martín Peru 3 Cap 020 Irazola Ucayali Peru 33 Cap 059 LBS San Martín Peru 4 Cap 021 Irazola Ucayali Peru 34 Cap 060 LBS San Martín Peru 5 Cap 022 Irazola Ucayali Peru 35 Cap 061 LBS San Martín Peru 6 Cap 023 Irazola Ucayali Peru 36 Cap 062 LBS San Martín Peru 7 Cap 024 Irazola Ucayali Peru 37 Cap 063 LBS San Martín Peru 8 Cap 030 Masisea Ucayali Peru 38 Cap 064 LBS San Martín Peru 9 Cap 031 Masisea Ucayali Peru 39 Cap 065 LBS San Martín Peru 10 Cap 032 Masisea Ucayali Peru 40 Cap 066 LBS San Martín Peru 11 Cap 033 Masisea Ucayali Peru 41 Cap 067 LBS San Martín Peru 12 Cap 034 Masisea Ucayali Peru 42 Cap 068 LBS San Martín Peru 13 Cap 035 Masisea Ucayali Peru 43 Cap 069 LBS San Martín Peru 14 Cap 037 Masisea Ucayali Peru 44 Cap 070 Iñapari Madre de Dios Peru 15 Cap 038 Masisea Ucayali
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