The Journal of Heredity 2001:92(3)
© 2001 The American Genetic Association 92:279-282
Brief Communication |
Inheritance Pattern of RAPD Markers in Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae)
From the Departamento de Biologia Geral, Universidade Federal de Viçosa. Av. PH Rolfs, s/n, 36.571-000, Viçosa, MG, Brazil (Tavares, Ribeiro, Campos, Barros) and Departamento de Biologia, Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto (IBILCE/UNESP), São José do Rio Preto, SP, Brazil (Oliveira).
Address correspondence to Mara Garcia Tavares at the address above.
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Abstract |
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Melipona quadrifasciata is an important pollinator agent in several regions of Brazil. Data concerning the genetics of this species are scarce in the literature. In this work we used the random amplified polymorphic DNA (RAPD) technique to determine the degree of polymorphism and the inheritance pattern of these molecular markers in this species. Our ultimate goal is to establish tools to be used in the study of the genomic organization of M. quadrifasciata. Genomic DNA from progenies F1 and BC1 were assayed with 79 different primers, yielding an average of 6.67 bands and 1.68 polymorphisms per primer. Three types of polymorphisms were detected: band presence/absence, band intensity, and fragment-length polymorphisms. Most of the observed polymorphisms were band presence/absence, typical of RAPD-dominant markers. The number of observed polymorphisms and their segregation in accordance with a Mendelian proportion confirm the importance of this technique for genome analysis of species like M. quadrifasciata that are poorly studied at the genetic level.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Melipona quadrifasciata, a species of stingless bee popularly known as "mandaçaia," is an important pollinator agent in several Brazilian ecosystems. Many scientific studies have characterized the biology and the behavior of these bees, however, very little effort has been devoted to the development and use of molecular genetic markers for this species. In spite of this, M. quadrifasciata has a great potential to become a model organism for genetic studies because of its lack of sting and the possibility of making controlled crosses in laboratory, and so its genomic organization must be known.
In order to have markers to construct a linkage map for this species, this work aimed at the evaluation of the frequency of random amplified polymorphic DNA (RAPD) polymorphisms and the determination of their inheritance pattern within M. quadrifasciata. This map will be very useful for further genome analysis in Melipona and will also facilitate the mapping of quantitative trait loci (QTLs) and the characterization of the system for sex determination in this species, allowing comparisons with maps already known for other Hymenoptera species.
The polymerase chain reaction (PCR)-RAPD technique is based on the amplification of genomic DNA using decamer primers of random sequence in a PCR (Welsh and McClelland 1990; Williams et al. 1990). The amplification products are separated electrophoretically and can be directly visualized without using specific radioactive probes. Polymorphisms due to base changes at the primer annealing sites or due to deletions or insertions in the sequence flanked by the primers are frequent among genotypes and can be used as molecular markers.
Many articles described this technique as a new tool to analyze the genome of insect species. Most of them dealt with the identification of species and subspecies, including mosquitoes (Flavia et al. 1994; Wilkerson et al. 1993), aphids (Black et al. 1992; Puterka et al. 1993), white fly (Gawel and Bartlett 1993), grasshoppers (Chapco et al. 1992), fruit fly (Baruffi et al. 1995; Haymer and McInnis 1994), and several Hymenoptera parasites (Edwards and Hoy 1993; Landry et al. 1993).
The simplicity of the RAPD analysis, and the small amount of DNA required for the reactions, makes this technique a powerful and efficient tool for genetic analysis of various species. However, most RAPD markers are inherited in a dominant fashion. For this reason, RAPDs provide less information than codominant markers, such as microsatellites (Williams et al. 1990). Nevertheless, here we demonstrate that M. quadrifasciata is quite suitable for analysis with RAPD markers because it is a haplodiploid insect. The dominance of RAPD markers, in this case, can be overcome by performing the analyses in haploid drones, which supply information about the loci present in the parental female, distinguishing the homozygous loci from the heterozygous.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Genetic Material
A heterozygous virgin queen for the enzymatic marker hydroxybutyrate dehydrogenase (Hbdh, E.C. 1.1.1.30) was crossed with a drone that had been previously sterilized by treatment with gamma-ray irradiation (60,000r) emitted by a cobalt-60 pump. Subsequently the queen was introduced in a colony from which the original queen had been removed. Sixty haploid adult drones originated from this cross (F1) were used to determine the type and frequency of RAPD polymorphisms.
One of the haploid drones was backcrossed to the parental queen, resulting in the BC1 progeny. These progeny consisted of diploid males and females (workers and queens) at a frequency of 1:1 and a small amount of haploid males originated from nonfertilized eggs. The haploid males were detected by cytogenetic analysis of bright-eyed pupae (data not shown).
Enzymatic analyses of Hbdh (Alfenas et al. 1991) were performed in the BC1 progeny to select heterozygous diploid drones and workers for this locus which were used for the DNA analysis. In this way the inheritance pattern of RAPD markers in the haploid and diploid progeny could be accessed.
DNA Extraction and PCR Amplification
Each individual of the F1 and BC1 progenies was frozen in liquid nitrogen and immediately separated in two parts: head and mesosome, and metasome. The metasome of the BC1 individuals was used to determine the Hbdh phenotype, and the head and mesosome were used for DNA extraction. In both techniques a different pestle and mortar were used for each sample to eliminate cross-contamination.
The genomic DNA was extracted as described by Waldschmidt et al. (1997). The amplification reaction mixture (25 µl) contained 3.5 mM MgCl2, 10 mM/50 mM Tris-KCl (pH 8.3), 0.1 mM of each dNTP (dATP, dTTP, dGTP, dCTP), 0.4 µM of a decamer primer (Operon Technologies, Alameda, CA), 1 U of Taq DNA polymerase, and 12.5 ng of genomic DNA. The mixture was placed in a thermocycler model PTC-100 (MJ Research) programmed for 40 cycles. Each cycle consisted of 15 s at 94°C, 30 s at 35°C, and 1 min at 72°C. After the 40th cycle a final extension step of 7 min at 72°C was performed. The amplification products were resolved in 1.2% agarose gels immersed in TBE (90 mM Tris-borate, 10 mM EDTA), stained with ethidium bromide (10 µg/ml), visualized, and documented under ultraviolet (UV) light.
Preliminary tests (not shown) defined 79 primers to be used in the RAPD analyses. These primers produced consistent bands that could be easily scored. The polymorphic bands were scored as 1 (presence) and 0 (absence). The scoring process was performed twice by two different people. The two readings were compared and data considered ambiguous were removed. The bands were then analyzed for 1:1 segregation using the chi-squared test (P > .05).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Amplification of the genomic DNA of M. quadrifasciata with 79 random primers yielded 527 bands, with an average of 6.67 bands per primer. Three hundred ninety-four bands were monomorphic and 133 were polymorphic, an average polymorphism of 1.68 bands per primer.
Most RAPD markers are inherited in a dominant way. In general, it is not possible to distinguish heterozygous- from homozygous-dominant individuals at such loci; both have the "band present" phenotype. In this work, three types of polymorphisms were observed: band presence/absence, band intensity, and fragment length polymorphisms (Figure 1). These types of polymorphisms occurred 99, 12, and 22 times, respectively. The majority of the markers (95.5%) for which the queen was heterozygous segregated 1:1 in the F1 drones (P > .05). These types of polymorphisms have also been observed during the construction of linkage maps for Apis mellifera (Hunt and Page 1992), Neurospora crassa (Williams et al. 1990), Helianthus (Rieseberg et al. 1993), and Eucalyptus (Grattapaglia and Sederoff 1994).
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Considering the three possible types of polymorphisms, some primers were selected to verify the inheritance pattern of these markers in the BC1 progeny. For this purpose, we analyzed gels containing the amplification products of the parental drone, haploid drones, diploid drones, and workers (Figures 2 and 3).
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As observed in Figure 2A, the presence/absence polymorphism generated by primer OPH-19 segregated 1:1 in the F1 individuals, revealing that the queen was heterozygous for this locus. As the drone used to generate the BC1 progeny possessed the marker, each of the progeny also presented it.
On the other hand, DNA analysis of the parental drone with primer OPR-6 (Figure 2B, lane 1), which also generated presence/absence polymorphism in the F1, revealed that this individual did not show the band. Consequently this marker showed a Mendelian segregation within the diploid BC1 progeny, as expected for a cross involving a heterozygous queen and a hemizygous recessive male (not showing the corresponding band).
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Twelve markers (9.02%) segregated in the haploid drones of the F1 as band intensity polymorphisms, showing that the queen was heterozygous for these loci. It was also observed that these markers segregated in the BC1 progeny independent of the parental drone phenotype (Figure 3). The segregation of these polymorphisms in the BC1 progeny may be due to intrinsic characteristics of this type of marker which may lead to differential amplifications. The differences could be the result of a different number of sequences in tandem or the degree of mismatches between the primer and its binding site.
One of the amplification products generated with primer OPC-1 in the F1 segregated for band size (Figure 4). Codominant alleles must be amplified with the same primer and be present simultaneously in heterozygous individuals. As expected, both alleles for this locus were present in half of the workers and half of the diploid drones in the BC1 progeny. The other half of the progeny and the parental drone showed only the small-size band (Figure 4). These results clearly show that the queen was heterozygous for this locus. Additional hybridization experiments should be performed to confirm the homology between the two "alleles" detected in this locus.
The clear segregation pattern present in the F1 and BC1 progenies demonstrate the usefulness of RAPD markers in genetic analysis of M. quadrifasciata. Genetic linkage analyses among the markers detected in this work are presently under way to establish a linkage map for this species to be used in future genetic studies.
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Acknowledgments |
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We are grateful to M. A. Del Lama for suggestions and assistance. This research was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPEMIG (Fundação de Amparo à Pesquisa de Minas Gerais).
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Footnotes |
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Corresponding Editor: Ross MacIntyre
Received June 12, 1999
Accepted October 31, 2000
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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