Mendelian inheritance, genetic linkage, and genotypic disequilibrium for nine microsatellite loci in Cariniana estrellensis (Raddi) Kuntze (Lecythidaceae)

Abstract

Cariniana estrellensis is one of the largest trees found in Brazilian tropical forests. The species is typical of advanced stages of succession, characteristic of climax forests, and essential in genetic conservation and environmental restoration plans. In this study, we assessed Mendelian inheritance, genetic linkage, and genotypic disequilibrium in nine microsatellite loci for a C. estrellensis population. We sampled and genotyped 285 adult trees and collected seeds from 20 trees in a fragmented forest landscape in Brazil. Based on maternal genotypes and their seeds, we found no deviation from the expected 1:1 Mendelian segregation and no genetic linkage between pairwise loci. However, for adults, genotypic disequilibrium was detected for four pairs of loci, suggesting that this result was not caused by genetic linkage. Based on these results, we analyzed microsatellite loci that are suitable for use in population genetic studies assessing genetic diversity, mating system, and gene flow in C. estrellensis populations.

Introduction

Cariniana estrellensis (Raddi) Kuntze (Lecythidaceae), or jequitibá-branco, has a wide geographic range distributed across Brazil, Bolivia, Paraguay, and Peru. The species is a priority for genetic conservation due to its ecological (reforestation) and commercial (wood and pulp) importance. It is currently threatened with extinction due to intense exploitation of the species as a timber resource (FAO, 2002). The species is hermaphroditic and pollinated mainly by bees of the genus Melipona and Trigona. Its winged seeds are dispersed by wind and periods of flowering and fruiting vary greatly across its range (Prance and Mori, 1979; Carvalho, 2003; Leite, 2007).

Microsatellite markers (simple sequence repeats, SSR) are a useful tool to analyze the genetics of forest species due to their high degree of polymorphism in terms of numbers of alleles. However, in order to use molecular markers as genetic markers, it is important to determine whether their inheritance follows the rules of Mendelian segregation, and whether the loci are genetically linked (Tambarussi et al., 2013; Manoel et al., 2015; Moraes et al., 2016). Studies assessing linkage among loci are also necessary because the detected loci are used to calculate averages among loci in population genetic studies; therefore, linked loci can create bias in the estimates (Guidugli et al., 2010). To enable the analysis of genetic diversity and structure, mating system, and gene flow for C. estrellensis herein we assess the Mendelian inheritance, genetic linkage, and genotypic disequilibrium for nine microsatellite loci developed for the species.

Materials and Methods

The study was based on samples collected from a highly fragmented forest landscape situated in a transition zone between the Savanna and Atlantic Forest biomes. The study area covers 448.2 ha and is located in the city of Bataguassu (Mato Grosso do Sul State, Brazil), alongside the Pardo River (21°38'00''S, 52°14'02''W). All 285 adult trees found in the area were sampled and 32 seeds were collected from each of 20 selected seed trees. For the molecular analyses, we used foliar or cambial tissue from adult trees and foliar samples from seeds germinated in a nursery.

Multilocus genotyping of the C. estrellensis samples was performed at the HERÉDITAS/GENOMAX laboratory. We used nine microsatellite loci chosen from the 15 loci previously developed for the species (Guidugli et al., 2009). The loci were analyzed in an ABI 3100XL automatic sequencer. Based on these genotypes, a multilocus profile was defined that allows for the identification of each sample individually and enables the analyses of population genetic parameters.

The study of microsatellite locus inheritance was based on the method described by Gillet and Hattemer (1989), which compares the genotype of a heterozygous maternal tree with the segregation of its open-pollinated progenies. This method assumes that all loci have regular segregation and their alleles follow Mendelian inheritance patterns based on the following conditions: i) regular meiotic segregation during ovule production; ii) random ovule fertilization by a type of pollen; and iii) no selection between the moment of fertilization and the genotyping of seeds. The model also assumes a co-dominant relationship between all alleles. The method further requires that all progeny of a tree must possess a maternal allele, and in cases of a heterozygous mother tree (e.g., A_iA_j, i ≠ j, the following are required: a) each individual within progeny must have one allele of the maternal tree, A_i or A_j; b) the number of heterozygous progeny A_iA_j (n_ij) must be equal to the sum of the number of homozygous progeny A_iA_i (n_ii) and A_jA_j (n_jj): n_ij = n_ii + n_jj; and c) the number of heterozygous progeny A_iA_k (n_ik) must be equal to the number of heterozygous progeny A_jA_k (n_jk), or n_ik = n_jk, in other words k ≠ i, j. The observed segregation of each progeny of the heterozygous maternal tree for a given locus was statistically compared to that expected for the segregation hypothesis of 1:1, using the G-test (Sokal and Rohlf, 1981):

(Equation 1)

where ln is the natural logarithm, E(n1) is the expected number of offspring genotypes A_iA_j

(Equation 2)

where E(n2) is the expected number of genotypes for alleles A_iA_k (n_ik) and A_jA_k (n_jk): E(n2) = 0.5(n_ik + n_jk). To avoid false positives, the G-test was determined only when n1 and n2 was ≥10, and deviation from the G-test between the observed and expected segregation was determined as statistically significant using the Bonferroni correction for multiple comparisons (95%, α = 0.05).

To determine if the loci were genetically linked, a test was carried out between pairs of loci using genetic information from mother trees that were doubly heterozygous for two loci (A_iA_j, B_lB_m). The segregation was recorded in their progeny. In this case, the null hypothesis (H_O) was regular Mendelian segregation of 1:1:1:1. The regular segregation hypothesis between pairs of loci was accepted or rejected based on a maximum likelihood G-test (Sokal and Rohlf, 1981), performed for each progeny:

(Equation 3)

where n_il, n_im, n_jl, and n_jm are the observed numbers of the phenotypes A_iB_l, A_iB_m, A_jB_l, and A_jB_m, respectively, and E(n) is the expected number of each genotype A_iB_l, A_iB_m, A_jB_l, and A_jB_m, calculated by E(n) = 0.25 (n_il + n_im + n_jl + n_jm). We again applied the Bonferroni correction for multiple comparisons (95%, a = 0.05) to avoid false positives.

The genotypic disequilibrium test between pairwise loci was only performed with adult samples. Estimates of gene frequencies based on open-pollinated progeny arrays are biased because each progeny has at least one maternal allele, resulting in a genotypic disequilibrium. This analysis was carried out using the FSTAT software (Goudet, 1995). The probabilities of the significance test were obtained by permutation of alleles among individuals, associated with the Bonferroni correction for multiple comparisons (95%, α = 0.05).

Results

After Bonferroni correction, we found no deviation from 1:1 Mendelian segregation for the nine loci analyzed for C. estrellensis heterozygous trees (Table 1). Furthermore, after Bonferroni correction, we detected no deviation from 1:1:1:1 Mendelian segregation between pairwise loci, indicating that the nine loci analyzed in this study are not linked (Table 2). However, significant genotypic disequilibrium was detected between four pairs of loci among adult trees after Bonferroni correction: Ces01 x Ces02, Ces01 x Ces04, Ces01 x Ces11, and Ces04 x Ces11 (Table 3).

Table 1: Mendelian inheritance tests for nine microsatellite loci in Cariniana estrellensis.

Table 2. Values of maximum likelihood G-test for the hypothesis of independent segregation between pairwise loci (1:1:1:1) of Cariniana estrellensis.

Table 3: Genotypic disequilibrium between pairwise microsatellite loci for Cariniana estrellensis adult trees.

Discussion

Confirmation of Mendelian segregation for individual loci was confirmed based on the expected 1:1 Mendelian segregation test. Independent segregation of alleles between different loci was performed through the linkage test based on 1:1:1:1 segregation, using genetic information of doubly heterozygous mother trees and observed segregation in progenies. We found that the nine loci assessed herein present Mendelian segregation and are not linked. Thus, these molecular markers developed by Guidugli et al. (2010) can be considered as genetic markers and our results support the hypothesis that they are not located in the same chromosome linkage group. However, genotypic disequilibrium was detected between four pairs of loci for adults. Genotypic disequilibrium is largely caused by genetic linkage, natural and artificial selection, genetic bottleneck, founder effect, and genetic drift (Hartl and Clark, 2010). Among these, genetic bottleneck and genetic drift can be the result of forest fragmentation due to decreases in effective population size, resulting in a limited number of pollen donors participating in reproduction. In studying a different population of the same species, Guidugli et al. (2010) found no significant genotypic disequilibrium between the same pairwise loci assessed herein. Thus, the genotypic disequilibrium that we detected may be attributed to genetic drift caused by forest fragmentation in the study region. Studies on other tree species have also found an absence of genetic linkage with a presence of genotypic disequilibrium, including Araucaria angustifolia (Medina-Macedo et al., 2014), Copaifera langsdorffii (Tarazi et al., 2010), Cariniana legalis (Tambarussi et al., 2013), and Genipa americana (Manoel et al., 2015). The nine microsatellite loci evaluated in this study exhibit Mendelian inheritance, are not linked, and segregate independently. These loci are therefore suitable for population genetic analyses, which can generate precise estimates of genetic diversity, spatial genetic structure, mating system, and contemporary gene flow for C. estrellensis.

Acknowledgments

Research supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; Project #2014/02675-8) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors would like to thank FAPESP for financial support provided to T.Y.K. Kubota (Scholarship #2014/02675-8) and CNPq for research funding to A.M. Sebbenn. We also extend a special thank to Universidade Estadual Paulista/UNESP and to Dr. Evelyn Nimmo for her correction of the English in the manuscript.