Abstract
Nasuta-albomicans complex (NAC) of Drosophila is an artificial hybrid zone comprising of Drosophila nasuta nasuta, Drosophila nasuta albomicans and 16 Cytoraces, which are the evolutionary products of a long range hybridization experiment conducted in the laboratory environment. Occurrence of centric fission in the X3 chromosome of Cytorace 1 led to the derivation of Fissioncytorace-1. Molecular techniques have emerged as powerful and valuable tools for detection and exploitation of genetic polymorphism. In the present study, Cytorace 1 and Fissioncytorace-1 were subjected to Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) analyses to determine the introgression of D. n. nasuta and D. n. albomicans genomes. It was found that Cytorace 1 and Fissioncytorace-1 exhibit similarities in RAPD and ISSR profiles although different combinations of genomic regions could have favoured Fissioncytorace-1, for better morphophenotypes and fitness, when compared to Cytorace 1, which has existed for over 15 years from the time of its evolution in the laboratory environment.
Introduction
The nasuta-albomicans complex (NAC) of Drosophila is an artificial hybrid zone with “allo-sympatric” populations, showing variation in their cytogenetic differentiation, mating preference, body size and fitness (Ramachandra & Ranganath Citation1996; Tanuja et al. Citation2001; Harini & Ramachandra Citation2003). This is an assemblage comprising of Drosophila nasuta nasuta (♂ 2n = 8: 2n2n3n3nXnYn4n4n, ♀ 2n = 8: 2n2n3n3nXnXn4n4n), Drosophila nasuta albomicans (♂ 2n = 6: 2a2aX3aY3a4a4a, ♀ 2n = 6: 2a2aX3aX3a4a4a) and 16 Cytoraces, which are the evolutionary product of a long range hybridization experiment. Cytorace 1 (♂ 2n = 7: 2a2nYnX3a3n4n4n, ♀ 2n = 6: 2a2nX3aX3a4n4n), is the product of hybridization between the males of D. n. nasuta (Coorg, South India) and females of D. n. albomicans of Okinawa strain (Ramachandra & Ranganath Citation1986). The occurrence of a rare event of centric fission in a laboratory population of Drosophila, Cytorace 1, has been reported by Tanuja et al. (Citation1999). Within the karyotype of Cytorace 1, there is no freely available X-chromosome and it always existed as a part of the X3a chromosome. This centric fission has occurred in the metacentric X3 chromosome of D. n. albomicans, which is phylogenetically a product of three centric fusions (Ranganath & Hagele Citation1981). The derivative race of this centric fission event is known as the Fissioncytorace-1 () with a diploid number of 2n = 8, both in males (2a2n3n3nXsmYn4n4n) and females (2a2n3n3nXsmXsm4n4n), wherein the Xsm indicates the new submetacentric X-chromosome. The uniqueness of this fission race is the presence of a submetacentric X-chromosome (Xsm), while such a chromosome is absent in its parents, Cytorace 1 and also in its grandparents, D. n. nasuta and D. n. albomicans.
Phenotypic traits often fail to serve as an unambiguous marker for systematic and diversity analysis because of the environmental influences (Wang & Tanksley Citation1989; Awasthi et al. Citation2004). Over the last 20 years, polymerase chain reaction (PCR) technology has offered new marker systems for detection and exploitation of genetic polymorphism. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) are two simple and quick techniques widely used for the diagnosis of genetic diversity. The former detects nucleotide sequence polymorphisms, using a single primer of arbitrary nucleotide sequence, and the latter permits detection of polymorphisms in inter-microsatellite loci, using a primer designed from dinucleotide or trinucleotide simple sequence repeats (Wu et al. Citation1994; Zietkiewics et al. Citation1994). ISSR analysis involves the PCR amplification of regions between adjacent, inversely oriented microsatellites, using a single simple sequence repeat (SSR) motifs (di-, tri-, tetra-, or pentanucleotides) containing primers at the 3′ or 5′ end by two to four arbitrary, often degenerate nucleotides (Zietkiewics et al. Citation1994). The potential supply of ISSR depends on the variety and frequency of microsatellites, which changes with species and the SSR motifs that are targeted (Depeiges et al. Citation1995). ISSR-PCR is simpler to use as prior knowledge of the target genome sequences flanking the repeat regions is not required and is more reliable than RAPD, as the primers used are longer, allowing for more stringent annealing temperatures and provides a higher reproducibility of bands than in RAPD (Hantula et al. Citation1996; Tsumura et al. Citation1996; Wolfe et al. Citation1998; Nagaraju et al. Citation2002). Although RAPD and ISSRs have been extensively used by plant biologists for a variety of applications such as cultivar identification and protection of plant variety rights, phylogenetic and diversity analysis, hybrid confirmation, genome mapping and gene tagging for marker assisted selections (Abbot Citation2001; Kumar et al. Citation2001; Pandit et al. Citation2007; Arif et al. Citation2009; Ikbal et al. Citation2010), they have been rarely used for animal studies (Reddy et al. Citation1999; Kostia et al. Citation2000). Molecular techniques have emerged as powerful and valuable tools in genetic fidelity analysis (Martins et al. Citation2004) in in vitro propagated plants and in germplasm study (Saravanakumar et al. Citation2010) and are the subject of many publications and reviews.
In view of this, the hybrid races, Cytorace 1 and Fissioncytorace-1, were subjected to RAPD and ISSR analysis to determine the introgression of parental genomes.
Materials and methods
Four members of the nasuta-albomicans complex of Drosophila were used in the present study;
| i. | D. n. nasuta (Coorg, South India), |
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| ii. | D. n. albomicans (Okinawa, University of Texas collections, 3045.11), |
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| iii. | Cytorace 1 (Ramachandra & Ranganath Citation1986). Cytorace 1 has been maintained since 25 years from their evolution in the laboratory environment, |
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| iv. | Fissioncytorace-1 (Tanuja et al. Citation1999). The Fissioncytorace-1 has been maintained since 15 years from their evolution in the laboratory environment. |
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All the stocks and the experimental cultures were maintained on standard wheat cream agar medium seeded with yeast at a temperature of 22 ± 1°C and at a relative humidity of 70–80%.
Genomic DNA isolation
Total genomic DNA (gDNA) pooled from 30 adult male and female flies was isolated (30 together of each sex) separately for the four races. DNA was extracted according to the standard procedure from Berkeley Drosophila Genome Project (BDGP) with slight modifications. Briefly, 30 anesthetized flies were collected in 1.5 ml microcentrifuge tubes and were frozen to −80°C. The flies were ground in 200 μl homogenizing buffer (100 mM Tris-HCl pH 7.5, 100 mM EDTA, 100 mM NaCl and 1% SDS) with a disposable micro pestle. An additional 200 μl homogenizing buffer was added and grinding was continued until only the cuticle remained. The homogenate was incubated at 60°C for 30 mins. The homogenate was added to 800 μl of a solution consisting 1 part of 5 M KAc stock and 2.5 parts 6 M LiCl stock solution and further kept for incubation on ice for at least 10 mins. Then the homogenate was centrifuged (10,000 rpm) for 15 mins at room temperature (RT). 1 ml of the supernatant was transferred to a new tube avoiding floating crud. The tube was respun if crud transfered. 500 μl of isopropanol was then added, mixed and spun (10,000 rpm) for 15 mins at RT. The supernatant was discarded and the pellet was given a wash with chilled 70% ethanol and kept for drying. The pellets were resuspended in 100 μl 10 mM TE (Tris/EDTA). The gDNA was then treated with RNase (200 μg/ml) at 37°C for 90 mins.
All the chemicals used in extraction were of analytical grade and were procured from SRL, India and Bangalore Genei, India.
RAPD analysis
Annealing temperature of each of the ten random primers used for RAPD analysis and four ISSR primers were optimized through gradient PCR (). For RAPD analysis, PCR amplifications were carried out in a reaction volume of 25 μl containing 100 mM Tris (pH 9.0), 500 mM KCl and 1% Triton X-100, 3 mM MgCl2, 20 pM primer, 200 μM dNTPs, 1.0 U of Taq DNA polymerase and 20 ng of genomic DNA. RAPD reactions were carried out using the following cycle: initial denaturation at 94°C for 2 min, followed by 40 cycles at 94°C for 1 min, 1 min of subsequent annealing temperature for each of the primers used, 2 min at 72°C followed by a final extension for 15 min at 72°C.
Table I. List of ISSR and RAPD primers used for the present study
ISSR analysis
Out of seven ISSR primers screened, three di-nucleotide and one tetra-nucleotide primer amplified the DNA of the four races in the present study (). Each reaction mixture of 25 μl contained 30 ng of genomic DNA, 10 pM primer, 100 mM Tris (pH 9.0), 500 mM KCl and 1% Triton X-100, 1.5 mM MgCl2, 200 μM dNTPs and 1.0 U of Taq DNA polymerase. ISSR amplifications consisted of an initial denaturation step at 94°C for 5 min, followed by 40 cycles of denaturing at 94°C for 1 min, annealing for 1 min at 55°C, extension at 72°C for 2 min and a final extension at 72°C for 15 min. Amplifications were performed in Corbett Research (Australia) and Eppendorf (Germany) Thermal Cyclers. At least two PCR amplifications were performed for each sample with ISSR and RAPD primers to evaluate the reproducibility of the amplified fragments. Amplified products were separated in a 1.5% agarose gel containing ethidium bromide (1μg/ml) in1X TBE (Tris/Borate/EDTA) buffer at a constant voltage of 60 V. Molecular weight markers, 100 bp and 500 bp (Bangalore Genei, India) were used for estimating the band size. Gel images were recorded and the band sizes were quantified by Mega bioprint 1000 system (Vilber Lourmat, France). All chemicals used in the experiment were of molecular biology grade and were procured from Bangalore Genei, India.
Data analysis
Each amplification product was considered as a marker. Only distinct, reproducible and well-resolved fragments were scored for presence (1) or absence (0) for each of the ISSR and RAPD primers separately with the four analyzed samples. If a relevant band was present in one or more races, but not in all the four races analyzed, then it was considered as a polymorphic locus. A two dimensional matrix was generated for both the marker systems. Genetic distance (Nei Citation1972) among the four races was computed with Tools for Population Genetics Analysis (TFPGA) program. To study the divergence among the races, genetic distance by TFPGA and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method of clustering were used to generate a dendrogram. The fit of dendrograms obtained were checked by bootstrapping conducted for 1000 permutations.
Results
In the present study, a set of ten random oligonucleotide and seven ISSR primers were used for initial screening of Fissioncytorace-1, its parents (Cytorace1) and grandparents (D. n. nasuta ♂ × D. n. albomicans ♀). Out of the seven ISSR primers used, three primers did not give any amplification while the ten RAPD primers gave amplifications. Amplifications with the RAPD primers yielded 76 fragments of different size ranging from 200–1800 bp. Out of the 76 fragments generated, 56 were polymorphic fragments (73.68%) and 20 were shared with 7.6 average numbers of amplified fragments per primer and 5.6 average numbers of polymorphic fragments per primer. Some of the representative RAPD amplified fragments are shown in Percent polymorphism ranged from 14.28% to a maximum of 100% (). Overall D. n. nasuta DNA produced 43 fragments, D. n. albomicans DNA produced 48, and Cytorace 1 and Fissioncytorace-1 DNA produced 47 fragments each. Of the 43 fragments of D. n. nasuta, 20 were shared with all the other races, five fragments were inherited in the two hybrid races, Cytorace 1 and Fissioncytorace-1; four were shared only with D. n. albomicans and the remaining 14 were specific to D. n. nasuta. Twelve fragments of D. n. albomicans were inherited in Cytorace 1 and Fissioncytorace-1, 20 were shared with all the races, however, four fragments were shared with D. n. nasuta and the remaining 12 fragments out of the 48 fragments generated with the 10 RAPD primers were specific to D. n. albomicans. Of the 47 fragments of Cytorace 1 and Fissioncytorace-1, 20 were shared with D. n. nasuta and D. n. albomicans, five fragments were inherited from D. n. nasuta, and 12 fragments were inherited from D. n. albomicans and 10 fragments were unique to the two hybrid races. Cytorace 1 and Fissioncytorace-1 showed similar fragments generated with the representative 10 RAPD primers. Genetic distance (Nei Citation1972) among the four races was computed with TFPGA program. To study the divergence between the races, genetic distance by TFPGA and UPGMA method of clustering were used to generate a dendrogram. The distance value ranged from 0.0001–0.7402.
Table II. Total number of fragments amplified with ten RAPD primers in four members of the nasuta-albomicans complex of Drosophila
Figure 2. RAPD profile of D. n. nasuta, D. n. albomicans, Cytorace 1 and Fissioncytorace- 1 generated with the primers (A) OPD-08 (B) OPE-02 (C) OPE-06 and (D) OPE-09, M1 = 100 bp DNA marker and M2 = 500 bp DNA marker (arrow indicates polymorphic fragments).
The genetic distance between Cytorace 1 and Fissioncytorace-1 was 0.0001. D. n. albomicans, Cytorace 1 and Fissioncytorace-1 form a cluster in the dendrogram while D. n. nasuta is an out-group (). This indicates that the hybrid races were closer to D. n. albomicans than D. n. nasuta. The genetic distance recorded between the two hybrid races and D. n. albomicans was 0.5241, whereas the genetic distance between the two hybrid races and D. n. nasuta was 0.7402.
Figure 3. Cluster dendrogram showing the relationship among D. n. nasuta, D. n. albomicans, Cytorace 1 and Fissioncytorace-1 using RAPD primers.
shows the total number of amplified fragments and polymorphic fragments in the four races of the NAC of Drosophila with the four ISSR primers. The primers generated 37 differently sized fragments ranging from 300 to 1600 bp in total of which, 22 (59.45%) were polymorphic. Some of the representative PCR amplified ISSR fragments are shown in The number of bands varied from seven to 11 with an average of 9.25 bands per primer. Each of the three di-nucleotide primers generated 10 fragments on average, whereas, the tetra-nucleotide primer could amplify only seven fragments. The highest number of polymorphic fragments was eight, generated with UBC-811 and the minimum number was three, generated with (ACTG) 4. UBC-810 and UBC-842 primers generated six and five polymorphic fragments respectively.
Table III. Total number of fragments amplified in four members of the nasuta-albomicans complex of Drosophila using four ISSR primers
Figure 4. ISSR profile of D. n. nasuta (♂), D. n. albomicans (♀), Cytorace 1 and Fissioncytorace- 1 generated with the primers (A) UBC-810 (B) UBC-811 (C) UBC-842 and (D) (ACTG) 4, M1 = 100 bp DNA marker and M2 = 500 bp DNA marker (arrow indicates the species-specific unique fragments).
The four ISSR primers generated 28 fragments in D. n. nasuta, 24 in D. n. albomicans and 27 fragments each in Cytorace 1 and Fissioncytorace-1. Of the 28 fragments of D. n. nasuta, 15 were shared with the other races, eight fragments were inherited in the two hybrid races, Cytorace 1 and Fissioncytorace-1; and four fragments were found to be unique and not inherited in the hybrid races and one fragment was shared with D. n. albomicans. Three fragments of D. n. albomicans were inherited in Cytorace 1 and Fissioncytorace-1, 15 were shared with all the races and remaining five fragments were specific to D. n. albomicans and one fragment was shared with D. n. nasuta. Of the 27 fragments in Cytorace 1 and Fissioncytorace-1, 15 were shared with D. n. nasuta and D. n. albomicans, eight fragments were inherited exclusively from D. n. nasuta and three from D. n. albomicans, and one fragment was specific and novel to the hybrid races only. D. n. albomicans specific fragments were inherited more in the hybrids than D. n. nasuta specific fragments. As in RAPD analysis, the four representative ISSR primers also generated similar fragments in Cytorace 1 and Fissioncytorace-1.
Nei's (Citation1972) genetic distance between Cytorace 1 and Fissioncytorace-1 was 0.0001. D. n. nasuta, Cytorace 1 and Fissioncytorace-1 form a cluster in the dendrogram, while D. n. albomicans is an out-group (). This indicates that the hybrids were closer to D. n. nasuta than D. n. albomicans. The genetic distance between the two hybrid races and D. n. nasuta was 0.2787 whereas the genetic distance between the two hybrid races and D. n. albomicans was 0.6058.
Figure 5. Cluster dendrogram showing the relationship among D. n. nasuta, D. n. albomicans, Cytorace 1 and Fissioncytorace-1 using ISSR primers.
Combined RAPD and ISSR cluster analysis of D. n. nasuta; D. n. albomicans, Cytorace 1 and Fissioncytorace-1 showed two distinct groups, with Cytorace 1 and Fissioncytorace-1 forming one group with a genetic distance (Nei Citation1972) of 0.0001, D. n. nasuta and D. n. albomicans are placed in another group ().
Figure 6. Combined RAPD and ISSR cluster analysis of D. n. nasuta, D. n. albomicans, Cytorace 1 and Fissioncytorace-1 showing two distinct groups.
Discussion
Hybrid zones in any natural population contain diverse genotypes that are resultant of several generations of recombination (Harrison Citation1990). Polymorphism in a given population is often due to the existence of genetic variants represented by the number of alleles at a locus and their frequency of distribution in a population (Gupta et al. Citation2008). Available genetic mapping tools have made the complete exploitation of the genotypic diversity in hybrid zones and the utility of hybrid zones for analyzing the genetic architecture has been reported (Rieseberg et al. Citation1999). Hybridization plays important creative roles in the evolution of both plants and animals however; limited work has been done in animals (Ramachandra & Ranganath Citation1986, Citation1990, 1996; Rieseberg et al. Citation1999; Harini & Ramachandra Citation2003). Because of the open genetic system of D. n. nasuta and D. n. albomicans brought about by force sympatry followed by interracial hybridization, has resulted in the formation of new hybrid lineages which are unique but represent a differential composition of the parental chromosome so they are also an admixture of the parental genomes. ISSR and RAPD markers have been used as tools for population discrimination and genetic variation studies in plants (Fernandez et al. Citation2002) and animals (Roux et al. Citation2007). At present, they have emerged as powerful and valuable tools in screening the genetic stability of in vitro propagation in cultivated plantlets like banana, Musa acuminate L., (Lakshmanan et al. Citation2007) and in monitoring genetic fidelity in Bombax mori germplasm (Saravanakumar et al. Citation2010).
In the present study a high level of polymorphism was detected with both RAPD and ISSR primers. RAPD markers were more efficient than ISSR as they detected 100% polymorphism in the four races studied, as compared with ISSR markers. RAPD primers are more GC rich than the ISSR primers. The numbers of total polymorphic and discriminant fragments are higher for RAPD because of the high frequency of annealing sites of the RAPD primers in the genomic DNA (Virk et al. Citation1995). However, Zietkiewics et al. (Citation1994) reported that ISSRs have a high capacity to reveal polymorphism and offer great potential than other arbitrary primers like RAPD in determining intra- and inter-genomic diversity.
The 10 RAPD primers generated 47 fragments in total in Cytorace 1 and Fissioncytorace-1. Twelve fragments of D. n. albomicans and five fragments of D. n. nasuta were inherited in Cytorace 1 and Fissioncytorace-1, 20 fragments were shared with D. n. nasuta and D. n. albomicans and 10 were unique to Cytorace 1 and Fissioncytorace-1.
The four ISSR primers generated 27 fragments in Cytorace 1 and Fissioncytorace-1 each, of which 15 were shared with all the other races, three fragments were inherited from D. n. albomicans and eight from D. n. nasuta, one fragment was specific and novel to the hybrid race only. D. n. nasuta specific fragments were inherited more in the hybrids than D. n. albomicans specific fragments. Earlier studies on the chromosomes (Ramachandra & Ranganath Citation1986), body size and fitness (Harini & Ramachandra Citation1999, 2003), and the longevity (Harini & Ramachandra Citation2003; Ranjini & Ramachandra Citation2009) revealed that, Cytorace 1 is closer to D. n. nasuta than D. n. albomicans. In the present study, even though the chromosome of D. n. nasuta origin is represented as a higher number in the Cytorace 1 and Fissioncytorace-1, the dendrogram generated using RAPD data revealed that Cytorace 1 and Fissioncytorace-1 are closer to D. n. albomicans whereas the dendrogram generated using the ISSR data placed D. n. nasuta, Cytorace 1 and Fissioncytorace-1 in one cluster with D. n. albomicans as out-group indicating the hybrids were closer to D. n. nasuta. This clearly indicates that the genome of the two hybrid races is an admixture of the two parental races. When we look at the chromosomes of these races, of the 13 chromosomes in Cytorace 1, eight chromosomes are derived from D. n. nasuta and five chromosomes are derived from D. n. albomicans. Similarly, in Fissioncytorace-1, 11 out of the 16 chromosomes are inherited from D. n. nasuta and five are inherited from D. n. albomicans (Ramachandra & Ranganath Citation1986; Tanuja et al. Citation1999).
Fissioncytorace-1, which has existed for 15 years from its evolution in the laboratory environment, is a product of centric fission of the X3 chromosome of Cytorace 1; wherein the functional centromere of the metacentric X3 chromosome of D. n. albomicans origin is split into an acrocentric chromosome 3 and a sub-metacentric X chromosome, each containing a functional centromere. This has resulted in the diploid number of 2n = 8 in both males and females instead of 2n = 7 in males and 2n = 6 in females as in its parent, Cytorace 1.
Cytorace 1 and Fissioncytorace-1 have similar DNA profiles even though their chromosome number is different. The generation of novel fragments in the hybrid races could possibly be a result of introgression. Thus, during the evolution of Cytorace 1, introgression produced unique combinations of the sequences and maintained them suggesting genomic stability in Cytorace 1 as well as Fissioncytorace-1.
Evolutionary genetics of Fissioncytorace-1 and Cytorace 1 dealt at the level of morphophenotypes, fitness and inter-genotypic competitive ability assessment revealed that Fissioncytorace-1 showed increased body size, sternopleural bristle number, ovarioles number, lifetime fecundity and lifetime fertility with reduced inter-specific competitive ability and hatching success when compared to Cytorace 1 (Bijaya & Ramachandra Citation2010). But RAPD and ISSR analysis with 10 RAPD representative primers and four ISSR representative primers generated similar fragments in Cytorace 1 and Fissioncytorace-1. In the dendrogram generated with RAPD cluster analysis, D. n. albomicans, Cytorace 1 and Fissioncytorace-1 forms a cluster while D. n. nasuta is an out-group. The dendrogram based on ISSR cluster analysis placed D. n. nasuta, Cytorace 1 and Fissioncytorace-1 in one group and D. n. albomicans is an out-group. However all the three cluster analysis based on RAPD, ISSR and combined RAPD and ISSR have agreeably placed Cytorace 1 and Fissioncytorace-1 in one group with a genetic distance (Nei Citation1972) of 0.0001. This indicates that although both Cytorace 1 and Fissioncytorace-1 differ in their chromosome number at the genomic level they are identical. This suggests that introgressed chromosomes of Cytorace 1 in Fissioncytorace-1 stay stable even after the 15 years of its origin; however, its morphophenotypes and fitness are superior to Cytorace 1. In view of this, the phenotypic and fitness traits evolve more rapidly than the genotypic divergence in Cytorace 1 and Fissioncytorace-1.
Acknowledgements
The authors thank the Department of Science and Technology, New Delhi for the financial assistance to establish “Unit on Evolution and Genetics” under the IRHPA scheme (No. SR/SO/LU-01/2003, dt 16-11-2005), the Chairman, DOS in Zoology for providing facilities and Prof. H. A. Ranganath for his encouragement.
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