Analysis of genetic diversity in two plantations of Pinus caribaea var. hondurensis

Análise da diversidade genética em dois plantios de Pinus caribaea var. hondurensis

ABSTRACT

Forest tree breeding activities in plantations with exotic species implies several instances of material selection where genetic variation can be affected. The objective of the work was to verify the genetic variability present in two plantations of Pinus caribaea var. hondurensis by microsatellite markers, initially designed for Pinus taeda fluorescent-labeled. Thus, 299 individuals were analyzed using eight polymorphic microsatellite markers. The results indicate that both plantations have adequate levels of genetic diversity that are representative of the Pinus genus. Through the Bayesian method, it was possible to detect two different population genetic structures (K = 2) between both plantations analyzed. In conclusion, this study suggests that microsatellites markers are useful tools to monitor genetic variation in genetic breeding programs. The genetic diversity estimated in both plantations is similar and typical for Pinus plantations, and as expected, there was a slight decrease in genetic variability in the commercial plantation in comparation with the base plantation.

Keywords:
SSRs (Short Sequence Repeats); Caribbean pine; Agroforestry; Biotechnology

RESUMO

As atividades de melhoramento genético florestal em plantios com espécies exóticas envolvem várias instâncias de seleção de material, onde a variação genética pode ser afetada. O objetivo do trabalho foi verificar a variabilidade genética presente em duas plantações de Pinus caribaea var. hondurensis através de marcadores microssatélites, inicialmente desenhados para Pinus taeda. Assim, 299 indivíduos foram analisados utilizando oito marcadores microssatélites polimórficos. Os resultados indicam que ambas plantações têm níveis adequados de diversidade genética que são representativos do gênero Pinus. Através do método Bayesiano, foi possível detectar duas populações genéticas estruturadas (K = 2) entre as duas plantações analisadas. Em conclusão, este estudo sugere que os marcadores microssatélites são ferramentas úteis para monitorar a variação genética nos programas de melhoramento genético. A diversidade genética estimada em ambas plantações é semelhante e típica das plantações de Pinus, e como se esperava verificou-se uma ligeira diminuição da variabilidade genética na plantação comercial, em comparação com a plantação base.

Palavras-chave:
SSRs (Repetições de Sequência Curta); Pinheiro do Caribe; Agrofloresta; Biotecnologia

1 INTRODUCTION

Multiples species of the genus Pinus are widely used in homogenous reforestation programs in different parts of the world, due to their great adaptation to different climatic regimes and the application of their products (wood, fibers, cellulose) (AGUIAR et al., 2011). Pinus caribaea Morelet is an important and widely planted tropical conifer, its natural range covers from northern Mexico to southern Nicaragua, including the islands of Cuba and the Bahamas. This specie has been extensively used as an industrial planting in tropical and subtropical areas around the world (TAMBARUSSI et al., 2010). Nevertheless, forest breeding activity with introduced species involves several instances of material selection, in which genetic variation can be reduced. Inbreeding is a likely result in progeny produced in plantations of a newly introduced exotic species and could be increased in advanced generations. Inbreeding reduces genetic variability and low levels of genetic variation damage the evolutionary adaptation processes in a changing environment. Populations with low genetic variability are not adequate as a basis for breeding program populations (NEALE; WHEELER, 2019).

The incorporation of molecular markers in breeding programs seeks to optimize the structure of improvement/production plantations, increase the information needed to decide on the infusion of new materials and improve the selection strategies (MARCUCCI POLTRI et al., 2010). Among them, microsatellite markers have been indicated for genetic variability and population genetics studies, since they are co-dominant and constitute one of the most polymorphic molecular marker currently available. The use of selectively neutral molecular markers allows quantifying characteristics of genetic variability within and between populations (GRATTAPAGLIA; RESENDE 2011). Neutral markers have proved to be useful to study phylogeographic and gene flow patterns in conifers (BAGNOLI et al., 2009; GONZÁLEZ-MARTÍNEZ et al., 2010) and are increasingly being used to infer demographic history in tree species (DAÏNOU et al., 2010).

This study aimed to compare the genetic variability present in a base plantation of Pinus caribaea var. hondurensis included in a genetic improvement program with a commercial plantation by microsatellites molecular markers. These results will contribute to the knowledge of the genetic diversity variability through different practical instances of a forest breeding program.

2 MATERIALS AND METHODS

2.1 Plant material

Pine needles were obtained from different plantations of Pinus caribaea var. hondurensis planted in different trials in the Argentinean Northeast (NEA). The trials were established with seeds collected from individuals growing in natural populations and came from the National School of Forestry Science (ESNACIFOR, Honduras) in the year 1980, and from the current Oxford Forestry Institute (England) former CFI Oxford, in the year 1973. Both trials were installed in Bella Vista, province of Corrientes, and belong to the Experimental Station of the National Institute of Agricultural Technology (INTA EEA Bella Vista) (28° 30' 23.82'' S and 59° 2' 32.11'' O). The design was a Randomized Complete Block with five repetitions, plots of 9- 25 per plot (a distance of 3.5 m). From these two trials, 169 individuals (N = 169) were collected for further studies, called from now BASE PLANTATION (the plantations belonging to INTA). The commercial tree plantation, from now on referred as: COMMERCIAL PLANTATION, corresponds to a plantation of select phenotypic of commercial origin belonging to the Company PINDO S.A. (N = 130), located in Puerto Esperanza, Misiones, Argentina (26° 1' 35.48'' S and 54° 1' 45.38'' O).

2.2 DNA Extraction

Total genomic DNA was extracted from adult leaf tissue following a CTAB (Hexadecyltrimethylammonium bromide) modified protocol and kit: Nucleo Spin plant II Macherey-Nagel®, based in the SDS buffer. DNA quantification was performed with a spectrophotometer at 280 nm (Nanodrop ND-1000®). Twelve of twenty-four microsatellites loci of the PtTX series (designed for Pinus taeda) have been selected based on the amplification products, unequivocal and congruent with the data presented in the bibliographical antecedents (VILLALBA, 2010). The polymerase chain reaction (PCR) has been conducted in a GenePro® Thermal Cycler E-96G.

2.3 PCR and Molecular markers

Reaction mixture: 10x Invitrogen buffer (1x), 10 mM each of dNTPs, 10 mM of each primer, and 5 units/ul of Taq polymerase enzyme (Invitrogen®). The MgCl₂ concentrations varied according to the needs of each pair of oligonucleotides. As template, 20 ng/ul of DNA dissolved in sterile bidistilled water were used. A typical touchdown PCR program consisted of the following steps: five minutes at 94° C, followed by 10 or 20 touchdown cycles, depending on the need for each pair of oligonucleotides, with the following conditions: 94° C (1 Min), drop in temperature to 1° C or 0.5° C (10 or 20 touchdown cycles, respectively) and 72° C (1 min). This step was continued with 30 cycles of amplification at 94 °C (1 min), followed by 1 min annealing, according to the annealing temperature specific for each primer, 72° C (1 min), and a final extension of 10 min at 72° C. The amplification products were analyzed using an ABI 3130xl Automatic Capillary Sequencer (Applied Biosystems®, USA), belonging to the Genomic Platform of the Institute of biotechnology (INTA Castelar, Hurlingham, Buenos Aires, Argentina). The electropherogram was analyzed using GeneMapper® 4.0 program.

2.4 Genetic Diversity analysis

The genetic diversity parameters of polymorphic loci were calculated by GenAlEx 6.4 (PEAKALL; SMOUSE, 2006). The polymorphic information content (PIC) for each locus was estimated. The genetic structure of Pinus caribaea var. hondurensis was determined using the Bayesian clustering approach implemented in STRUCTURE v 2.3 (PRITCHARD et al., 2000). This software uses multilocus genotype data to assign individuals to genetically divergent clusters. For each simulation, was used ten independent runs for each K value (K= 1-10 populations) to obtain L(K) with LOCPRIOR assigned (HUBISZ et al., 2009) under the admixture model, allele frequencies correlated, with a burn-in of 100,000 steps, and Markov Chain Monte Carlo for 200,000 steps. The optimal number of K clusters was determined using the ΔK criteria. Additionally, we conducted the hierarchical analysis of molecular variance (AMOVA) to assess the level of differentiation among both plantations with GenAlEx 6.4.

3 RESULTS AND DISCUSSION

3.1 Microsatellite amplification

From the PtTX series of microsatellites markers, twelve have been transferred to Pinus caribaea var. hondurensis originally designed for Pinus taeda (Table 1). All the amplified loci correspond to triplet repeat and low copy number motifs; this type of microsatellites markers are the ideal candidates to be transferred between conifer species (KALIA et al., 2011). They are highly conserved due to their possible functional role in eukaryotic transcription, replication, gene expression, and regulation.

Three out of four microsatellites amplified not reported previously for Pinus caribaea var. hondurensis were polymorphic (PtTX4018, PttX4092, and PtTX4098), and eight microsatellites were successfully used to compare both populations of 299 individuals.

Table 1
Characteristics of eleven polymorphic microsatellites loci and one monomorphic used in this study in Pinus caribaea var. hondurensis plantations

	Pinus caribaea var. hondurensis
Locus	Library Type	Repeat motif	Allele size (bp)	Reference
PtTX2123	Genomic	(AGC)8	190 - 199	Elsik et al. 2000
PtTX2128	Genomic	(GAC)8	232 - 245
PtTX3013	Low Copy	(GTT)10	132 - 145
PtTX3025	Low Copy	(CAA)10	250 - 311
PtTX3030	Low Copy	(TA)4 (GGT)10	300 - 359
PtTX3098	Low Copy	(GTT)8	136 - 185	Kutil et al. 2001
PtTX3107	Low Copy	(CAT)14	152 - 172	Elsik et al. 2001
PtTX3116	Low Copy	(TTG)7 (TTG)5	113 - 168
PtTX4018	UnderMethilated	(GAC)15	161 - 216	Zhou et al. 2002
PtTX4090	UnderMethilated	(CTT)6	188
PtTX4092	UnderMethilated	(GAA)21	106 - 153
PtTX4098	UnderMethilated	(GAA)7	161 - 172

• Source: Authors (2019)

3.2 Genetic diversity

The genetic diversity was characterized by different parameters (Table 2). The total number of alleles (Na) were 61 for the BASE PLANTATION, and 58 for the COMMERCIAL PLANTATION, with a minimum of three alleles (PtTX2123-PtTX2128) and a maximum of 16 for the most polymorphic marker (PtTX3116) in both plantations.

Table 2
Genetic diversity of two studied plantations of Pinus caribaea var. hondurensis.

BASE PLANTATION						COMMERCIAL PLANTATION
N	Na	Ho	UHe	PIC	Locus	N	Na	Ho	UHe	PIC
160	3	0,55	0,51	0,44	PtTX2123	116	3	0,45	0,53	0,46
161	3	0,32	0,27	0,24	PtTX2128	117	4	0,46	0,5	0,43
159	7	0,55	0,62	0,56	PtTX3013	125	10	0,55	0,68	0,63
163	9	0,61	0,51	0,46	PtTX3098	120	7	0,65	0,76	0,72
169	9	0,62	0,61	0,57	PtTX3107	117	10	0,44	0,55	0,51
165	16	0,81	0,86	0,84	PtTX3116	118	16	0,72	0,86	0,84
165	9	0,77	0,64	0,61	PtTX4018	124	4	0,29	0,39	0,37
164	5	0,37	0,35	0,31	PtTX4098	113	4	0,33	0,52	0,41
163,25 (1,14)	7,62 (1,49)	0,58 (0,05)	0,55 (0,06)	0,50 (0,18)	MEAN (SD)	118,75 (1,43)	7,25 (1,58)	0,49 (0,05)	0,60 (0,05)	0,54 (0,16)

• Source: Authors (2019). In where: N: number of analyzed individuals, Na: numbers of alleles, Ho: Observed Heterozygosity, UHe: Unbiased Heterozygosity, PIC: polymorphism index context.

The mean number of alleles was 7.6 (SD 1.5) and 7.2 (SD 1.5), respectively. These results are similar to those reported for other pine species: in Pinus radiata were six alleles (N = 96); in Pinus resinosa and Pinus oocarpa (N = 502) nine alleles (N = 518) (SOTO et al., 2010). In Pinus patula (N = 60) and Pinus tecunumanii (N = 108), a total of five alleles were detected, although the sample size was smaller (DVORAK et al., 2009).

For populations from Mexico, three (N = 14) and four alleles (N = 60) were detected (DELGADO et al., 2011). The results obtained in this work could indicate a comparable genetic diversity in both plantations of Pinus caribaea var. hondurensis. The conservation of genetic diversity is essential for many reasons; these include adaptation to environmental changes, the risks of inbreeding depression in the viability of the population, and the need to maintain genetic resources for possible future use (NEALE and WHEELER, 2019).

The genetic diversity was analyzed with the observed (Ho) and the Unbiased Expected Heterocigozity (Ho and UHe, respectively) (Table 2). The mean levels of genetic diversity observed in the analyzed plantations of Pinus caribaea var. hondurensis (BASE PLANTATION Ho: 0.58; COMMERCIAL PLANTATION Ho: 0.49) are typical of predominantly cross-pollinated forest species (NEALE; WHEELER, 2019). They agree with the values obtained in other plantations of the same species, analyzed with microsatellites molecular marker (8 SSR).

According to Andrade Furlan et al., (2007), the genetic variability of trees from the natural range revealed similar Ho values in the base plantation (0.24) and commercial plantations (N = 25, Ho = 0.30). Shepherd et al. (2002) reported a Ho of 0.49 (29 SSR) in plantations of the same species in Australia. The data obtained by Delgado et al. (2011) in two natural populations (N = 14 and N = 60) remaining and marginal of Pinus caribaea var. hondurensis in Mexico (6 SSR), showed values of Ho: 0.42. Some particularities could explain the levels of genetic diversity found in conifers: long generational intervals allow less exposure to potential bottleneck processes during the reproductive period (SANCHEZ et al., 2014). Furthermore, high levels of crossbreeding are a particularly important factor in maintaining genetic diversity in the species. Another factor is that most of the forest species are monoecious, and this prevents self-fertilization because the sexes are spatially separated (in different structures and sections of the tree) and temporarily (pollen release occurs at different times of female maturation) (SEBASTIANI et al., 2012). The PICs obtained were similar for each plantation. Four microsatellite markers had values greater than 0.5, and one of them: PtTX3116, showed the highest value (0.81), being the most informative marker in both plantations.

3.3 Genetic structure

Based on the results of the Bayesian clustering, the Pinus caribaea var. hondurensis plantations were separated into two clusters (K=2) using STRUCTURE. The BASE PLANTATION was assigned to the first genetic cluster, whereas the COMMERCIAL PLANTATION formed the second cluster (Figure 1). This result is consistent with the AMOVA (Phipt 0.28) (Table 3). Most of the genetic variation (72%) observed in this study was due to the genetic variation of individuals within populations.

Table 3
Analysis of Molecular Variance (AMOVA)

Source of variation	Degree of freedom	Sum of aquares	Variance components	Variation (%)	p value
Among populations	1	343,9	2,2	28%	0,0001
Within populations	297	1.717,9	5,7	72%
Total	298	2.061,1	8,08	100%

• Source: Authors (2019)

Figure 1
Bar plot showing STRUCTURE results for K= 2

The genetic differentiation between the Pinus caribaea var. hondurensis plantations was 28% (between the BASE PLANTATION and COMMERCIAL PLANTATION). This result could be due to the different origins of the seed material. The COMMERCIAL PLANTATION, as a consequence of the phenotypic selection, could present new allelic combinations. The amount of variation may well differ between quantitative traits and neutral marker loci. The level of differentiation between populations at neutral loci depends on a balance between migration and genetic drift (NEALE; WHEELER, 2019). Even low levels of migration will equalize gene frequencies between populations at such loci. When there is diversifying selection, the balance between selection and migration can result in considerable genetic differences between populations. While migration rates are equal for all genes, selection acts differently on different parts of the genome. The artificial selection changes the genetic structures of populations in manifold ways. Successful selection for a trait changes the allelic structures at the controlling gene loci and has some correlated effects on other traits. Measuring levels of genetic variation within and among populations is a critical first step in evaluating the evolutionary biology and tree improvement potential of a species (ISIK, 2014).

4 CONCLUSION

Microsatellite markers that come from low copy number or low methylation genomic areas are highly transferable in species within the genus Pinus.

The genetic diversity estimated in both plantations is typical of Pinus plantations. The commercial plantation has a slight decrease in genetic diversity compared with the base plantation.

The Base plantation and commercial plantation have different genetic structures.

The microsatellite markers are useful tools for the monitoring of genetic variation in successive stages of forest genetic improvement.

Acknowledgement

To Jonathan von Below for his valuable contributions to the revision of the English language. Pamela Villalba for her laboratory experience in Pinus taeda SSR amplification.

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• This research was funded by the Project: Application of Molecular Tools for the Use and Conservation of Forest Genetic Diversity (PNFOR1104064- INTA)

Publication Dates

• Publication in this collection
  20 Feb 2023
• Date of issue
  Oct-Dec 2022

History

• Received
  04 Nov 2019
• Accepted
  04 May 2021
• Published
  23 Nov 2022