An AFLP estimation of the outcrossing rate of Spondias tuberosa (Anacardiaceae), an endemic species to the Brazilian semiarid region Una estimación AFLP de la tasa de fecundación cruzada de Spondias tuberosa (Anacardiaceae), una especie endémica de la región semiárida de Brasil
Carlos Antonio Fernandes Santos1* & Renata Natália Cândido de Souza Gama2*
The umbu tree (Spondias tuberosa) is one of the most important endemic species to the Brazilian tropical semiarid region. The umbu tree has edible fruits with a peculiar flavor that are consumed in natura or in a semi-industrialized form, such as jams, candies and juices. The majority of endemic species to Brazilian semiarid region have not been studied or sampled to form germ- plasm collections, which increases the risk of losing genetic variability of the adapted species to xerophytic conditions. The aim of this study was to estimate outcrossing rates in S. tuberosa using a multilocus mixed model in order to guide genetic resources and breeding programs of this species. DNA samples were extracted from 92 progenies of umbu trees, which were distributed among 12 families. These trees were planted by seed in 1991 in Petrolina, PE, Brazil. The experimental design was a randomized block, with a total of 42 progenies sampled in three regions. The experimental units were composed by five plants and five replications. The out- crossing rate was estimated by the multilocus model, which is available in the MLTR software, and was based on 17 polymorphic AFLP bands obtained from AAA_CTG and AAA_CTC primer combinations. The observed heterozygotes ranged from 0.147 to 0.499, with a maximum frequency estimated for the AAA_CTC_10 amplicon. The multilocus outcrossing estimation ( ) was 0.804±0.072, while the single-locus ( ) was 0.841±0.079, which suggests that S. tuberosa is predominantly an outcrossing species. The difference between and was -0.037±0.029, which indicates that biparental inbreeding was nearly absent. The mean inbreeding coefficient or fixation index ( ) among maternal plants was - 0.103±0.045, and the expected was 0.108, which indicates that there was no excess of heterozygotes in the maternal population. The outcrossing estimates obtained in the present study indicate that S. tuberosa is an open-pollinated species. Biometrical models applied to this species should therefore take into account the deviation from random outcrossing to estimate genetic parameters and the constitution of broad germplasm samples to preserve the genetic variability of the species. Outcrossing rates based on AFLP and the mixed-mating model should be applied to other studies of plant species in the Brazilian semiarid region.
Key words: outcrossing rate, MLTR, heterozygosis.
Resumen
El árbol de umbu (Spondias tuberosa) es una de las especies endémicas más importantes de la región semiárida del Brazil. El mismo tiene frutos comestibles con sabor distinto y puede ser consumido fresco o semiindustrializado, como mermeladas y zumos. La mayoría de las especies endémicas de la región ]]>
S. tuberosa basada en el modelo multi-locus mixto, con el fin de orientar los recursos genéticos y los programas de mejoramiento de esta especie. Muestras de ADN fueron extraídas de 92 progenies de árboles umbuzeiro, distribuidos en 12 familias, que se ]]>
) fue 0.804±0.072, mientras que el locus de uno-locus ( ) fue 0.841±0.079, lo que sugiere que S. tuberosa es predominantemente una especie de polinización cruzada. La diferencia entre el
y fue de -0.037± 0,029, lo que indica que la endogamia bi-parental fue casi inexistente. La media del coeficiente de fijación ( ) ]]>
esperada fue 0.108, lo que indica que no hubo un exceso de heterocigotos en la población materna. Las estimaciones obtenidas en este trabajo indican que S. tuberosa es una especie de polinización cruzada. Los modelos biométricos aplicados a esta especie deben tener en cuenta la desviación del cruce aleatorio para estimar los parámetros genéticos y la ]]>
Palabras clave: tasa de fecundación cruzada, MLTR, heterocigosis. ]]>
The umbu tree (Spondias tuberosa Arruda - Anacardiaceae) is a xerophytic tree species that is endemic to the tropical Brazilian semiarid region (Prado & Gibbs 1993). The high drought tolerance of this species is due to its specialized root structure (xylopodium), which plays a key role in long-term water storage. The tree has edible fruits with a peculiar flavor that are consumed
in natura or semi-industrialized in different forms, such as jams, candies and juices.
This species has a greater potential for agronomic cultivation in environments with minimal rainfall (Santos 1999) or degraded soil. The umbu tree was considered an endangered species because of the harvesting of the modified roots of ]]>
Understanding the mating system of a species is fundamentally important for genetic improvement and ]]>
et al. 2004).
The plant species that result from out- crossing maintain the majority of their distributed genetic variability within the population, in contrast to a predominantly self-pollinated species in which the majority of the genetic variability is distributed between populations (Hamrick & Godt 1989). Reproductive studies have revealed that a large number of plant ]]>
et al. 2006).
The development and application of isozymes was a breakthrough in estimating plant population outcrossing rates and was mainly explored in perennial trees. Subsequently, DNA molecular markers, such as RAPD and AFLP, have been used to estimate the outcrossing rates of many ]]>
et al. 1997, Muluvi et al. 2004). Because of their dominance, RAPD and AFLP markers have limitations. To solve the dominance limitations, Ritland (2002) has presented a statistical estimation that is based on multilocus procedures for genes that occur in intermediate frequencies.
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This study aimed to determine the dominant reproductive system of S. tuberosa using AFLP markers based on multilocus estimation in order to aid the development of strategies to sample, preserve and initiate breeding efforts in S. tuberosa.
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Materials and methods
Tree samples and DNA extractions: The leaves of 92 adult S. tuberosa trees, which were planted in 1991, were sampled in the Caatinga Experimental Station of Embrapa tropical semiarid, in ]]>
S. tuberosa progenies originated from 12 families, which numbered from five to 12 progenies/family. The collected leaves were frozen at -80ºC until DNA extraction.
Genomic DNA was ]]>
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Approximately 200ng of genomic DNA of each progeny was double digested for 2.5h with the endonuclease enzymes EcoRI and MseI. The digested DNA was ligated with T4 DNA ligase to a final volume of 7.25μL. Pre- amplification reactions were performed to generate a final volume of 15µL (1.5µM of each EcoRI and MseI primer, ]]>
2, 0.5 units of Taq DNA Polymerase and 2.0µL of diluted 5-fold ligated DNA solution) and were thermocycled for 20 cycles at 94oC for 30s, 56oC for 1min and then at 72oC for 1min.
After pre-amplification, the samples were diluted 20-fold in TE buffer. Selective amplifications ]]>
EcoRI primer, 0.3µM of MseI primer, 0.2mM of dNTPs, 1x PCR buffer (100mM Tris-HCl (pH 8.3) and 500mM KCl), 2.5mM MgCl2, 0.2 units of Taq DNA polymerase and 2µL of pre-amplified DNA). The touchdown thermocycling conditions involved (a) 1 cycle at 94oC for 30s, 65oC for 30 s and 72oC for 60s; (b) 13 cycles with an initial annealing temperature of 65oC and reductions of 0.7oC for every subsequent cycle; ]]>
et al. (2001). All reactions were performed at the genetic laboratory of the Embrapa tropical semiarid unit. ]]>
For data analysis, the appearance of AFLP polymorphic bands was scored as a 1 for present and 0 for absent. Chi-square tests were performed for all AFLP amplicons using Mendelian inheritance ratios of 3:1, 1:1 or 1:3.
Single-locus (
) and multilocus out- crossing rates, AFLP marker-allele frequencies, observed heterozygote frequencies, ovule and pollen frequencies and the coefficient of parental inbreeding (
) were estimated with the mixed-mating model (mixed outcrossing and selfing) using the software MLTR (Ritland 2002). This model specifies that both selfing and outcrossing occur in the population. One hundred bootstraps were used in the analysis. Maximum-likelihood estimates were obtained for all AFLP marker loci employed in the estimation of outcrossing rates and
coefficients.
Results
A total of seventeen polymorphic ]]>
A chi-square test at the 5% significance level indicated that of the seventeen AFLP bands, four ]]>
The observed heterozygotes ranged from 0.147 to 0.499 (Table 1). The maximum frequency of heterozygotes is 0.50 (Falconer & Mackay 1996), estimated for the AFLP AAA_ CTG_3 and AAA_CTC_10 amplicons. ]]>
The multilocus outcrossing estimation ( ) based on all AFLP loci was 0.804±0.072, while the single-locus estimation (
) was 0.841±0.079. Using only the Thirteen AFLP loci that fit the Mendelian segregation (Table 1), the multilocus outcrossing estimation (
) was 0.869±0.059, while the single-locus estimation ( ) was 0.933±0.063.
The difference between and
was -0.037±0.029, and the mean inbreeding coefficient or fixation index ( ) among the maternal plants was - 0.103±0.045.
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Discussion
The studied S. tuberosa population is the oldest experimental population established for genetic studies, and it contains an important gene pool for the conservation of S. ]]>
. This study matched the current efforts to conserve this important endemic species in the Brazilian semiarid region (Santos et al. 2008) as an in vivo gene bank.
The number of polymorphic bands obtained in this study was in accord with the
S. tuberosa results reported by Santos et al. (2008), who reported 3 to 16 polymorphic bands from 12 AFLP PC.
The deviations from Mendelian inheritance detected in the present study were expected because all of the 92 pooled individuals were considered to be within the frequencies, and no family estimations were obtained because of the small number of ]]>
According to Ritland (2002), when many loci are used for the estimation, it does not matter if a few loci show significant deviations because the ]]>
The difference between and ]]>
) among maternal plants was -0.103±0.045, and the expected was 0.108, as calculated by
(Gaiotto et al. 1997). These results indicate that there was no excess of heterozygotes in the maternal population. An excess of heterozygotes was reported by Moraes et al. (2005), however, in Myracrodruon urundeuva, with values ranging from -0.252 to ]]>
The multilocus model (Ritland 2002) has a few assumptions, as revised by Sebbenn et al. (1998): (1) the pollen pool should be homogeneous in the crossing with maternal plants, (2) there should be independent segregation among the loci, (3) there should be an absence of selection and mutation, and (4) the loci should be in Hardy-Weinberg equilibrium. An assumption of intermediate allele ]]>
et al. 1997). Departures from these assumptions, mainly in pollen pool homogeneity, have been reported by Gaiotto et al. (1997), Sebbenn et al. (1998), Muluvi et al. (2004), Gusson et al. (2006) and Muchugi et al. (2008).
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The multilocus estimation of 0.804 is close to the 0.74 value reported by Souza (2000) using three isozyme systems. in the present study, the frequencies of ovules and pollen were constrained to be equal for all AFLP markers, which suggest an absence of departures in the mixed model. These results suggest that S. tuberosa is an open-pollinated species, with a selfing rate of 0.196. S. tuberosa is ]]>
As reviewed by Muchugi et al. ]]>
Eucalyptus grandis ( =0.84), Platypodium elegan (
=0.92) and Shorea congestiflora ( =0.87). According to the authors, outcrossing is a reasonable explanation for the high genetic variability observed in tropical trees. In an isolated study, Stacy et al. (1996) used five isozyme systems to determine that S. mombin was a ]]>
Based on the proportion of selfing, adjustments can be made to the coefficient of additive genetic variance that is estimated from open-pollinated families when the assumption of complete half-sib relationship is not met (Gaiotto et al. 1997), as observed ]]>
S. tuberosa in the present study. The high levels of heterozygosity detected in the present study should be relevant for in situ conservation because a large number of new genetic recombinations could occur and maintain the evolutionary capacity of this species to adapt to climate changes and colonize new areas.
The outcrossing ]]>
S. tuberosa is a predominantly open-pollinated species. Therefore, biometrical models applied to this species should take into account deviation from random outcrossing to estimate genetic parameters. As recommended by Freitas et al. (2004), while evaluating Myracrodruon urundeuva, biometrical models should also account for the constitution of broad germplasm samples to ]]>
The high heterozygosity levels observed in the analyzed umbu tree families were also an important variable for the in situ conservation of this species because new allelic recombination can occur, thus making it possible for the appearance of new genotypes that are readily adapted to environmental ]]>
More than 932 savanna-like plant species, including 380 endemic species, have been identified in the Brazilian semiarid Region (Brasil 2002). The majority of these species have not been studied or sampled to form a germplasm collection, which increases the risk of losing genetic variability of the adapted species to xerophytic conditions. information about the reproductive system is ]]>
Acknowledgments
We are grateful with the Bank of Northeast of Brazil for their financial support.
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*Correspondencia: ]]>
Carlos Antonio Fernandes Santos:Embrapa Semiárido, Caixa Postal 23. 56300-970. Petrolina, PE. Brazil; casantos@cpatsa.embrapa.br Renata Natália Cândido de Souza Gama: Universidade Estadual de Feira de Santana. Avenida Transnordestina, SN, Novo Horizonte, 44.036-900, Feira de Santana-BA, Brazil; renata.natalia@hotmail.com 1. Embrapa Semiárido, Caixa Postal 23. 56300-970. Petrolina, PE. Brazil; casantos@cpatsa.embrapa.br 2. Universidade Estadual de Feira de Santana. Avenida Transnordestina, SN, Novo Horizonte, 44.036-900, Feira de Santana-BA, Brazil; renata.natalia@hotmail.com
) was 0.804±0.072, while the single-locus ( 
) was 0.841±0.079, which suggests that S. tuberosa is predominantly an outcrossing species. The difference between 
) among maternal plants was - 0.103±0.045, and the expected