Selections that isolate recombinant mitochondrial genomes in animals
Homologous recombination operates in organisms from bacteriophage to human . This includes the mitochondrial genomes in many plant and fungal species ( Rank and Bech-Hansen , 1972; Dujon et al . , 1974; André et al . , 1992; Zassenhaus and Denniger , 1994; Shedge et al . , 2007 ) . Nonetheless , there is very little support for recombination in animal mitochondria ( Elson et al . , 2001; Berlin et al . , 2004; Hagström et al . , 2013 ) ; lack of an identified mitochondrial RecA homolog , evidence of continuous lineages of mitochondrial haplotypes and a failure to detect recombinants in propagated heteroplasmic lines are taken as indications that it does not occur . Despite evidence arguing against recombination of animal mitochondrial genomes , a variety of exceptional reports suggest that it can occur . The remarkable ‘double uniparental inheritance’ pattern of mitochondrial genomes in some bivalve mollusks has been associated with rare recombination events on at least an evolutionary time scale ( Ladoukakis and Zouros , 2001; Ladoukakis et al . , 2011 ) . One human patient was reported to carry recombinant genomes ( Kraytsberg , 2004 ) , and there have been reports of recombinant mitochondrial genotypes in some species like lizard and fish ( Guo et al . , 2006; Ciborowski et al . , 2007; Ujvari et al . , 2007 ) , but these reports are based on single individuals without documentation of parents or origin of the genomes presumed to have recombined . Given opposing observations , such as the introgression of intact genomes from one species into another ( Solignac , 2004 ) , it is not clear whether the cases reported are exceptional , or whether we have simply lacked the experimental power to directly demonstrate recombination in animals . Recombination between small regions of nonallelic homology has been proposed to underlie deletion and insertion mutations ( Mita et al . , 1990; Bacman et al . , 2009; Fukui and Moraes , 2009 ) . However , when precise , recombination can generate favorable combinations of alleles , which , when coupled to the action of purifying selection , could increase in abundance to restore function ( Muller , 1932 ) . These positive or negative impacts on gene function could influence evolution , the behavior of disease mutations , and the age-associated degenerative changes of the mitochondrial genome . Until recently , several factors have hindered detection of recombination of mitochondrial sequences . Chief among these , uniparental inheritance largely limits exposure of mitochondrial genomes to sibling genomes differing only at newly mutated sites ( Birky , 1995; DeLuca and O'Farrell , 2012; Sato and Sato , 2013 ) . Additionally , rare recombinant genomes can be difficult to detect: they can be stochastically lost during the random segregation , and if transmitted , they can be hard to track amid the chaotically segregating genomes . Finally , there are few markers suitable for design of conditions that would select for a rare recombinant genome . Previous work in Drosophila showed that germline expression of a restriction enzyme targeted to mitochondria results in potent selection against mitochondrial genomes carrying a cognate cleavage site ( Xu et al . , 2008 ) . Using this selection , a number of variant genomes lacking a particular site have been selected . In addition to removing a restriction site , these selected changes often also alter an encoded gene product ( Figure 1A ) . One of the variants that lost a XhoI site is a temperature-sensitive lethal mutation of mt:CoI that can be counter-selected at high temperature ( Hill et al . , 2014; Ma et al . , 2014 ) . Moreover , one can transfer cytoplasm between early Drosophila embryos of different mitochondrial genotypes to create heteroplasmic lines that carry both the recipient and donor genomes for multiple generations ( Matsuura et al . , 1989; Ma et al . , 2014 ) . In addition to these tools , characterization of diverged mitochondrial genomes has revealed marked differences in their abilities to compete for transmission when combined in heteroplasmic combinations , a feature that we have been able to use as another selectable trait . In this work , we have combined these tools to create a powerful system in which we can test for the existence of homologous recombination and select for recombinant genomes . 10 . 7554/eLife . 07247 . 003Figure 1 . Without selection , no recombination was detected in a stable heteroplasmic line after 60 generations . ( A ) Mutants at the BglII and XhoI sites of mtDNA used in this study ( Xu et al . , 2008; Ma et al . , 2014 ) . The mitochondrial genome of Drosophila resembles that of mammals . It has little intergenic spacing , and encodes 13 polypeptides , all of which are involved in oxidative phosphorylation as well as 22 tRNAs and two rRNAs required for mtDNA translation . A single non-coding region ( ∼5 kb ) called the ‘AT-rich region’ ( dark brown ) as it contains >90% A and T residues includes origins of replication and some repeated sequences of unknown function ( Lewis et al . , 1994 ) . The genome contains one BglII and one XhoI site in the coding regions of mt:ND2 and mt:CoI , respectively . ( B ) No wild-type genome was detected by Southern blotting analysis in heteroplasmic lines where mt:ND2del1 and mt:CoIT300I were maintained in the same population for more than 60 generations at 29°C . In the heteroplasmic lane , 40 adults were sacrificed and their mtDNA were cut with both BglII and XhoI , and probed by a DIG-labeled sequence that hybridizes to mt1579–mt2369 . The sensitivity of the Southern analysis was measured by loading a series of dilutions of wild-type mtDNA cut with both enzymes from 40 adult flies . DOI: http://dx . doi . org/10 . 7554/eLife . 07247 . 003 Here , we provide clear evidence for homologous genetic exchange between Drosophila mitochondrial genomes under various conditions . The complete genomic sequence of parental and recombinant molecules details exchange events , and several recombinants are shown to involve transfer of a substantial segment of sequence from one genome to the other . We also show that exchange is stimulated by double-strand breaks ( DSBs ) , as is recombination in many systems . Importantly , the success of the selections that we have applied shows that production of favorable combinations of alleles by recombination , even if rare , can have a profound benefit .
We used Southern analysis to test for recombination between genomes distinguished by differences in restriction sites . A line heteroplasmic for mt:ND2del1 , which lacks the BglII site , and mt:CoIT300I , which lacks the XhoI site , carries both genomes stably at 29°C ( Figure 1B ) ( Ma et al . , 2014 ) . Each of the genomes of this heteroplasmic line has a defect complemented by the other so that the line is healthy but neither genomes is lost , a balancing selection ( Ma et al . , 2014 ) . Recombination should produce wild-type genomes distinguished by the presence of both restriction sites and consequent production of a ∼1 . 6-kb fragment upon cutting with both BglII and XhoI . Even after maintenance of this line for more than 60 generations , this 1 . 6 kb band was not detected ( Figure 1B ) . Reconstruction shows that this Southern assay can detect recombinant molecules at the level of 1 in 1000 ( Figure 1B ) . Thus , like related experiments by others ( Hagström et al . , 2013 ) , this physical assay failed to detect recombination . We conclude that recombination in this situation is not frequent ( Sato et al . , 2005 ) . We then developed methods to genetically select for recombinant mitochondrial genomes in the hopes that such approaches would both detect its occurrence and allow isolation of the recombination product . To give recombinant genomes an advantage , we produced a heteroplasmic line wherein one genome was compromised by a temperature-sensitive mutation and the other was compromised in its ability to compete for transmission . A temperature-sensitive genome with two mutations , mt:ND2del1 + mt:CoIT300I , was introduced into a strain with a genome named ATP6[1] ( Celotto et al . , 2006 , 2011 ) . High temperature , 29°C , selected against the mt:CoIT300I allele , and in previously analyzed heteroplasmic lines where the partner genome was a closely related wild-type genome , this selection resulted in a multi-generational decline of the temperature-sensitive genome and its eventual elimination ( Ma et al . , 2014 ) . To our surprise , when in competition with the ATP6[1] genome , which is distinguished by numerous sequence polymorphisms and a shorter AT-rich region , the temperature-sensitive genome displaced the ATP6[1] genome over a few generations at 29°C , even though the flies homoplasmic for ATP6[1] are relatively healthy and apparently more robust than mt:ND2del1 + mt:CoIT300I flies at both temperatures . The decline of ATP6[1] leaves the temperature-sensitive genome without complementing mt:CoI activity , and the entire population dies after several generations ( Figure 2A ) . 10 . 7554/eLife . 07247 . 004Figure 2 . Selection revealed homologous recombination in a heteroplasmic line containing the ATP6[1] genome and the temperature-sensitive double-mutant: mt:ND2del1 + mt:CoIT300I . ( A ) The abundance the ATP6[1] genome declined when co-resident with mt:ND2del1 + mt:CoIT300I at 29°C . After several generations , the flies at 29°C started to die . ( B ) A combination of a restriction fragment length polymorphism and a restriction site difference reveals the emergence of a recombinant genome . mtDNA isolated from 40 adults from each generation was cut with EcoRI in the presence or absence of XhoI . The schematics show the distribution of the EcoRI and XhoI sites on the whole parental genomes ( left ) and a detail of the largest EcoRI fragment with the position ( purple bar ) of a hybridization probe ( center ) . Southern analysis shows single and double cut samples taken at different generations during the selection . Only the two parental bands were detected early , from G0 to G5 ( shown for G3 ) . From G6 onward , a third EcoRI fragment appeared that had a length characteristic of the mt:ND2del1 + mt:CoIT300I genome but with a XhoI site . By G7 , the ATP6[1] specific fragment was not detected while a new apparently recombinant genome dominated the population . ( C ) Detailed maps of three genomes sequenced by PacBio SMRT . Red lines indicate mismatches between ATP6[1] and mt:ND2del1 + mt:CoIT300I sequences . The ATP6[1] genome also lacks ∼1 . 6 kb of the AT-rich region ( two type I repeats and two type II repeats , see Figure 2—figure supplement 1A for details ) . Pink arrows indicate approximate points of exchange with the given range defined by the nearest neighboring polymorphisms ( see deposited full sequences in GenBank as KT174472 , KT174473 and KT174474 ) . ( D ) Proposed progression leading to the recombinant . The original ATP6[1] genome is still displaced , but the newly emerged recombinant competes effectively and persists . By generation 6 , the abundance of the recombinant genome is sufficient to complement the temperature-sensitive genome so that some viable flies sustain the line . Over subsequent generations at 29°C , the recombinant genome increases in relative abundance because of selection against the temperature-sensitive genome . DOI: http://dx . doi . org/10 . 7554/eLife . 07247 . 00410 . 7554/eLife . 07247 . 005Figure 2—figure supplement 1 . mtDNA maps for the two parental genotypes ( ATP6[1] , mt:ND2del1 + mt:CoIT300I ) and two other recombinants . ( A ) Schematic illustration of repeat structure of the AT-rich regions of ATP6[1] and mt:ND2del1 + mt:CoIT300I genomes . ( B ) mtDNA map of two other recombinants . The size of the recombinant genomes was determined by Southern analysis as described in Figure 2B . The coding region was amplified by PCR followed by standard sequencing . We do not have accurate sequence for the non-coding region . The pink arrow indicates approximate points of exchange with the given range defined by the nearest neighboring polymorphisms . Black arrows indicate the two ends of the coding region that has been sequenced . DOI: http://dx . doi . org/10 . 7554/eLife . 07247 . 005 To select for possible recombinant genomes , we followed five independently established lines with a high starting proportion of ATP6[1] genomes ( 50–90% ) at 29°C . Initially , all the lines were healthy and grew productively , but with decline in ATP6[1] abundance , the health of the lines held at 29°C declined abruptly after five or six generations and died out within the next few generations . One line went through a similar crisis with diminished survival , recovered and continued to produce viable progeny in subsequent generations . Southern analysis showed emergence of a new genotype , which was cut by XhoI ( like the ATP6[1] genome ) , but had a long AT-rich region ( like the double mutant ) ( Figure 2B ) . To map recombination sites , we sequenced the parental and recombinant genomes using the PacBio single molecule real-time sequencing technique ( SMRT ) , whose long reads gave us unambiguous sequence that included the repeats of the 5 kb highly AT-rich noncoding region ( Figure 1A and Figure 2 ) . Complete sequences of these genomes revealed that the two parental genomes differed by more than 100 SNPs plus >20 indels , and the ATP6[1] genome lacked ∼1 . 6 kb of the AT-rich region that was present in the mt:ND2del1 + mt:CoIT300I genome ( Figure 2C and Figure 2—figure supplement 1A ) . The recombinant genome was the result of an exchange of a large continuous segment to produce an ∼60%/40% chimera of the ATP6[1] and mt:ND2del1 + mt:CoIT300I genomes that lacks the mt:ND2 and mt:CoI mutations but contains the non-coding region of the mt:ND2del1 + mt:CoIT300I genome ( Figure 2C ) . Southern analysis also showed the abundance of the recombinant genome at later stages of the selection . After generation 6 , the original ATP6[1] genome was no longer detected ( Figure 2B ) , and a new heteroplasmic line was formed with the other parental genome mt:ND2del1 + mt:CoIT300I and the recombinant genome . This line was viable at 29°C as the temperature-sensitive defect of the double mutant is complemented by the ATP6[1] mt:CoI of the recombinant genome ( Figure 2D ) . Over subsequent generations , a multigenerational selection for function caused an increase in the proportion of the recombinant genome ( Figure 2B ) , showing that it had lost the transmission disadvantage of the parental ATP6[1] genome . This led us to conclude that the ability to compete for transmission is localized to the sequences of the recombinant that came from the temperature-sensitive genome . This includes the entire AT-rich regulatory region and some flanking sequences distinguished by three SNPs ( Figure 2C ) . We later isolated two other recombinant genomes by following another 46 heteroplasmic lines as above . Both recombinants had a size ( ∼19 . 5 kb ) similar to the temperature-sensitive genome , implying that mt:ND2del1 + mt:CoIT300I was the source of the regulatory region . By sequencing the coding region , we show that one recombinant has the entire coding sequence of the ATP6[1] genome , whereas the other contains a much smaller segment of ATP6[1] extending at least from mt671 to mt5978 , with the rest of the coding region belonging to the mt:ND2del1 + mt:CoIT300I genome ( Figure 2—figure supplement 1B ) . We conclude that our selection has isolated recombinant genomes including extensive stretches of sequence originating from the parental genomes and exhibiting functional traits of these parental genomes . Normal meiotic recombination is induced by DSBs and studies in many contexts have suggested that DSBs , whether experimentally produced or secondary to DNA damage , greatly stimulates recombination . Indeed , DSBs are thought to be the key initiators of homologous exchange . To test the importance of DSBs for mtDNA recombination , we set up a condition to select for recombination in conjunction with restriction cutting of the mtDNA . We had produced genomes that lacked the XhoI site , mt:CoIR301Q , or the BglII site , mt:ND2del1 . While each genome is resistant to one enzyme , expression of mito-BglII and mito-XhoI simultaneously in the germline should cut either of these genomes . Indeed , germline expression of both enzymes in flies that were homoplasmic for either mt:ND2del1 or mt:CoIR301Q led to sterility in females ( Figure 3A ) . As previously shown , resistance ought to emerge at a low frequency due to mutations at the second restriction site ( Xu et al . , 2008 ) . Indeed , upon selection ∼1% of the females were weakly fertile , giving a few escaper F1 progeny harboring a variety of mutant alleles that inactivate the remaining restriction site . In contrast , even though the combination of enzymes was able to select against both genotypes , when flies were heteroplasmic for the two genomes ( in a 50:50 ratio ) , fertile females were very frequent ( >90% ) , and most ( 60% ) were as fecund as wild type . PCR , DNA sequencing , and phenotypic analysis showed that these progeny carried a single genome with the restriction-resistant alleles found in the two parents ( Figure 3A ) . 10 . 7554/eLife . 07247 . 006Figure 3 . Introducing DSBs into both parental genomes vastly induces homologous recombination in two heteroplasmic lines . ( A ) Expression of both mito-BglII and mito-XhoI enzymes in the germline effectively sterilizes most females carrying either mt:ND2del1 or mt:CoIR301Q genome , resistant to only one of the enzymes , but resistant progeny appear at a much higher frequency if the female carries both types of sensitive genomes . The resistant progeny from the heteroplasmic parent are homoplasmic for a newly generated recombinant genotype carrying both parental alleles: mt:ND2del1 + mt:CoIR301Q . ( B ) The same level of rescue was observed when a different heteroplasmic line was used , which had a different starting ratio of two parental genomes . Results are means ±SD ( n = 4 × 100 for each heteroplasmic line ) . DOI: http://dx . doi . org/10 . 7554/eLife . 07247 . 006 A second heteroplasmic line where the mt:CoIR301Q allele was replaced with the mt:CoIT300I allele gave a similar frequency of recombinant progeny , even though the starting ratio for the two parental genomes was not equal ( Figure 3B ) . The resulting recombinant lines exhibited the phenotypes expected for the input alleles . For instance , mt:ND2del1 + mt:CoIR301Q flies were male sterile , and mt:ND2del1 + mt:CoIT300I flies were temperature lethal . The success of a second and very different selection confirms that homologous exchange between mitochondrial genomes can occur . Most females exposed to the restriction enzyme selection were fertile , while in the selection without DSBs ( above , Figure 2 ) , the recombinant emerged from a population over several generations of selection . The difference suggests that the frequency of recombination upon expression of the two restriction enzymes is much higher . We propose that DSBs produced by restriction enzyme cutting induces exchange as well as selecting for the products that are resistant to cutting . To test whether recombination could also occur in somatic tissues , we examined the consequence of expressing both restriction enzymes in the developing eye under the control of ey:GAL4 driver . The high level of expression that occurs at 29°C results in pupal lethality ( headless phenotype ) in 99% flies that are homoplasmic for mitochondrial genomes mutant at only one site and the few survivors are eyeless or have a small eye phenotype . Similar expression in flies heteroplasmic for the two mutant genomes gave survivors at 10% with most survivors showing well-developed eyes ( Supplementary file 1 ) , suggesting that recombination of mtDNA also occurred in somatic tissues exposed to the double restriction enzyme selection . Somatically , active recombination could impact the stability of the mitochondrial genome in the soma , a factor thought to be important in aging ( Cortopassi et al . , 1992; Liu et al . , 1998; Cao et al . , 2001; Bender et al . , 2006 ) . Given the high frequency of recovered recombinants when both genomes of a heteroplasmic strain were cut , we asked whether we could detect recombination if DSBs were introduced into only one genome . To do this , we made a heteroplasmic line containing the wild-type genome and the temperature-sensitive double-mutant mt:ND2del1 + mt:CoIT300I , and used expression of restriction enzyme to select against wild-type genome and used the temperature-dependent selection against the double mutant . When we expressed mito-BglII in the germline while keeping the flies at 29°C , about 14 . 6% of the F1 females were viable and fertile ( Figure 4A ) . PCR analysis and restriction digestion showed that the progeny had lost the wild-type allele of mt:ND2 , which was targeted by the restriction enzyme , but they were heteroplasmic for a new mt:ND2del1 genome and the double-mutant parental mt:ND2del1 + mt:CoIT300I genome ( Figure 4A ) . Apparently , the wild-type mtDNA was cut and homologous repair or exchange using the mutant mt:ND2del1 sequence led to loss of the site . Initially , the abundance of the newly generated mt:ND2del1 genome was very low in all the progeny ( Figure 4B ) , probably because only a small fraction of the wild-type genomes underwent homologous exchange . However , because a low level of the recombinant genome is sufficient to rescue the temperature-sensitive phenotype of mt:ND2del1 + mt:CoIT300I , this low level of recombinant genome supported production of fertile heteroplasmic flies . Since the more functional mt:ND2del1 genome has a selective advantage at 29°C , its abundance increased over subsequent generations ( Figure 4B ) , and in some lineages , the mt:ND2del1 + mt:CoIT300I genome was completely eliminated after 18 generations ( Ma et al . , 2014 ) . 10 . 7554/eLife . 07247 . 007Figure 4 . Homology-dependent conversion of the wild-type BglII site into the sequence of the mt:ND2del1 allele following cutting of the BglII site . ( A ) Expression of mito-BglII in the germline of flies heteroplasmic for wild-type and mt:ND2del1 + mt:CoIT300I genomes at 29°C led to isolation of progeny with repaired wild-type genome at the BglII site and converted it to mt:ND2del1 . ( B ) The starting abundance for the newly generated mt:ND2del1 was low , but it increased over generations as the genome possessed a selective advantage at 29°C . The abundance of mt:ND2del1 was measured by PCR amplifying a mtDNA region ( mt1826–mt2496 ) using mtDNA from 40 adults as template followed by restriction digestion using XhoI in four heteroplasmic lineages from generation 1 to 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 07247 . 007 Again , we isolated recombinant genomes at a relatively high frequency , which leads us to propose that a single DSB is sufficient to promote recombination ( see ‘Discussion’ ) . However , we note that without additional markers , we could do little to characterize the nature of the exchange events . In order to study whether DSB-induced exchanges involve restricted local repair events , or exchange of longer stretches of DNA , we needed more markers . To achieve this , we examined recombination between the diverged mitochondrial genomes of Drosophila melanogaster and Drosophila yakuba . We made a heteroplasmic D . melanogaster line in which the D . yakuba mtDNA is sensitive to BglII and the D . melanogaster genome is sensitive to XhoI ( see ‘Materials and methods’ ) ( Figure 5A ) . Within the coding sequences , the D . yakuba genome shared about 93% sequence identity with mt:ND2del1 , while the non-coding AT-rich sequence was highly diverged and reduced to about 1 kb in length . We introduced DSBs into both genomes by germline expression of mito-BglII and mito-XhoI and found that about 10% of the females were fertile . Progeny homoplasmic for recombinant genomes were recovered . All propagating recombinants contained the AT-rich region of the D . melanogaster genome ( assessed by Southern analysis , Figure 5—figure supplement 1 ) , which we expected because D . melanogaster genomes outcompete D . yakuba genomes very quickly when co-resident ( H Ma and PH O'Farrell , unpublished ) . We characterized the mitochondrial genomes of three progeny lines by PCR and standard sequencing . All the recombinant genomes had one crossover point very close to but upstream of the XhoI site of the mt:ND2del1 genome ( Figure 5B ) . A second crossover is considerably downstream: in two of the recombinants , the other crossover occurred between markers at mt3124 and 3184 , and between markers at mt3379 and 3445 . In the third case , the other crossover is further downstream and we obtained mixed sequencing signals for a region between mt5524 and mt6291 ( Figure 5C ) . Flies were likely to be heteroplasmic for that particular region as if multiple recombinants are carried in this line . Based on these recombinants , we concluded that homology-based repair of DSBs promoted exchange of a substantial uninterrupted stretch of sequence . We also note that there were no discontinuities in the mapped region of exchange as might occur if repair randomly converted mismatches in heteroduplex in one direction or the other ( see below for more discussion ) . 10 . 7554/eLife . 07247 . 008Figure 5 . Exchange between two mitochondrial genomes of different species that share 93% sequence homology . ( A ) A heteroplasmic line containing both a Drosophila yakuba ( mt:ND1R274W ) genome and a Drosophila melanogaster ( mt:ND2del1 ) genome was made . 90% of heteroplasmic females induced to express both mito-BglII and mito-XhoI enzymes in the germline were sterile , but the escaper progeny from the fertile females contained newly generated recombinant genotypes . ( B ) Sequences of the parental and three recombinant genomes reveal that one recombination junction is close to the XhoI site of the D . melanogaster genome . The XhoI site ( yellow highlight ) present in the D . melanogaster parent sequence ( top line ) is absent in all the other sequences . Pink rectangles indicate the interval in which recombination occurred . ( C ) A schematic illustration of where the second crossover was for the recombinant genomes . Arrows indicate the approximate position and the numbers , which are based on the D . melanogaster sequence , given the positions of bounding polymorphisms . DOI: http://dx . doi . org/10 . 7554/eLife . 07247 . 00810 . 7554/eLife . 07247 . 009Figure 5—figure supplement 1 . Southern Blot analysis of the long EcoRI segment covering the AT-rich region of two parental genomes ( D . yakuba mt:ND1R274W and D . melanogaster mt:ND2del1 ) and three recombinant genomes . None of the recombinant genomes ( three left lanes ) carry the short ( 7 . 3 kb ) EcoRI fragment produced from the AT-rich region of the D . yakuba mtDNA ( rightmost lane ) . We conclude that all the recombinants carry the D . melanogaster AT-rich region and attribute the shift in the size of the fragment ( from 11 . 5 to 13 . 3 kb ) in recombinant 3 to loss of the EcoRI site at position 5343 as demonstrated by sequencing . The probe used recognizes mt1579–mt2369 . DOI: http://dx . doi . org/10 . 7554/eLife . 07247 . 009
Multiple modes of homology-dependent exchange are used in different organisms and different situations . Though differing in their details , these different modes often involve DNA breaks of one or both strands , resection of ends , heteroduplex formation , local replication , and resolution . Some features of the processes contributing to mtDNA recombination can be inferred from the sequence of three D . melanogaster/D . yakuba recombinants that we isolated following introduction of DSBs . In all cases , one crossover point was very close to the cleavage site of XhoI , indicating stimulation of exchange by DSBs . Some resection ( 8–44 bp ) must have occurred as the sites of exchange are located two or three SNPs upstream of the cleavage site ( Figure 5B ) . Surprisingly , the actual exchange events occur in sequences with frequent interruptions of homology such that one of the exchanges occurred within a stretch of only 11 bases of homology . In contrast , some of the more thoroughly characterized homology-dependent mechanisms such as those catalyzed by RecB-C and RecF require significantly greater homology ( 23–27 bp and 44–90 bp , respectively ) ( Shen and Huang , 1986 ) . This discordance favors other mechanisms such as break-induced recombination ( BIR ) , a template switching mechanism of recombination , which can use very short regions of homology in an exchange process referred to as microhomology-mediated BIR ( Hastings et al . , 2009; Anand et al . , 2014 ) , to be the underlying mechanism . The second site of exchange is far from either cleavage site , but seems to occur in regions with high homology . It is not clear whether both exchanges occur at the same time: since the genomes are circular , some types of exchange will produce a dimer circle that might resolve by an intramolecular recombination event at a later time . Our finding that one of our recombinant lines carried more than one recombinant genome with a common point of exchange near the XhoI cleavage site but different downstream points of exchange is consistent with this possibility . If separated in time , propagation of the unresolved product of the first recombination could give a population of molecules and independent resolution events could give a mixed population . While these analyses give us only a limited perspective on the mechanism of recombination , it is notable that the abundant SNPs suggest that each of the characterized events involved exchange of a substantial segment without interruptions ( at least 1 kb ) , rather than restricted local repair . When no DSBs were introduced by restriction enzymes in lines heteroplasmic for ATP6[1] and mt:ND2del1 + mt:CoIT300I , all of the recombinants again involved the exchange of a substantial and continuous stretch of sequence ( at least 5 kb ) , indicating either the exchange of extended region of duplex or priming of an extended stretch of replication template by a second genome . We also noted that two of the three recombinants had a crossover site very close to where the Drosophila mTERF ( DmTTF ) binds ( mt6314–mt6341 and mt11698–mt11725 , respectively [Roberti et al . , 2003] ) . Although a relatively weak association , we call attention to it because mtDNA replication pauses at the DmTTF binding sites ( Jõers et al . , 2013 ) , and such pauses might destabilize forks and promote exchange . Homologous recombination plays major roles in repairing DSBs and in genetic exchange . Below , we discuss the significance of these two roles for maintaining mitochondrial genomes in natural populations . Our finding that restriction cutting gives frequent recombinant products demonstrates that this process is active in the repair of these DSBs in mtDNA . While genetically distinct genomes in heteroplasmic flies provide the means to detect homologous exchange , sibling molecules ought to have a greater opportunity to guide homology-dependent repair as they will occur , at least transiently , in the same mitochondrion or nucleoid . In homoplasmic flies , accurate repair using sibling molecules will leave no trace . Only misaligned intra-molecular recombination between repeats would give a clear signature . Occurrence of recombination junctions in regions of low homology in this study supports suggestions that non-allelic recombination can contribute to formation of deletions and duplications in the mitochondrial genomes—a frequent cause of mitochondrial defects during aging . Such a recombination could also explain several observations found in natural population ( Rand and Harrison , 1989; La Roche et al . , 1990; Lunt and Hyman , 1997; Ludwig et al . , 2000 ) . For instance , mtDNA of European rabbit ( Oryctolagus councils ) has repeated 153-bp motifs in the vicinity of the replication origin of H strand and very individual carries genomes with different numbers of these repeats ( Casane et al . , 1994 ) . In Drosophila , mtDNA varies in size from 16 to 20 kb ( Solignac et al . , 1986; Townsend and Rand , 2004; Rand , 2011 ) , mainly due to the variation in the length of the noncoding region , which contains two types of tandemly repeated elements ( Lewis et al . , 1994 ) . Rand has shown that spontaneous changes in length in populations as if repeats can come and go at a relatively rapid pace ( Rand , 2011 ) . While accurate repair using sibling molecules is likely to be the main purpose of mitochondrial recombination , more rare non-allelic events can have a major impact on the production of variants , and exchanges between distinctly marked genomes , as reported here , provide a means to characterize the process . Recombination is detected by and well known for its ability to promote genetic exchange . However , animal mtDNA is restricted in the types of exchange that can occur because uniparental inheritance prevents intermingling of genomes . The mitochondrial genome of each female is sheltered from encounters with foreign genomes and will be passed on in an isolated lineage or clone . Genetic exchange between distantly related genomes might occur as a result of occasional paternal transmission that introduces a foreign genome ( e . g . , Kraytsberg , 2004 ) , but the significance of such violations of uniparental inheritance are matters of an ongoing debate . For the most part , the integrity of mitochondrial haplotypes suggests that the contribution of exchange among distantly related genomes is relatively small ( Elson et al . , 2001 ) . The limitations on genetic exchange imposed by uniparental inheritance , which are similar to those in asexually propagating organisms , do not mean that there is no meaningful genetic exchange ( Hurst and Peck , 1996 ) . It has become increasingly apparent that organisms can carry more than one mitochondrial genotype , that is , they are naturally heteroplasmic because of new mutations , stably transmitting heteroplamic combinations , or a breakdown of uniparental inheritance ( Solignac et al . , 1983; Ladoukakis et al . , 2011; Tsang and Lemire , 2011; Payne et al . , 2012; Ma et al . , 2014; Ye et al . , 2014 ) . In this sense , the situation we created by introducing the temperature-sensitive genome into the ATP6[1] line , might be a model for things that can happen naturally . Emergence of a new genome with strong selfish drive , but with a defect in function , or introduction of such genome from another lineage will create a natural situation analogous to our experiment . The outcome of our experimental manipulation shows that this situation has generally detrimental consequences . Indeed , the temperature-sensitive mutation together with the drive advantage created a sort of ‘population time bomb’: the lineage remained healthy for multiple generations allowing the population to expand greatly , and then it collapsed upon elimination of the functional mt genome . Even rare recombination can uncouple a positively selected drive mutation from detrimental mutations , and as in our experiment , selection can then restore health . Thus , even occasional genetic exchange would prevent rogue genomes from wiping out lineages . In conclusion , we show that recombination among mitochondrial genomes occurs in Drosophila and that this recombination can be used to manipulate genomes for functional mapping . We suggest that recombination will influence evolution of the mitochondrial genome in animals and impact the genetic behavior of mitochondrial disease mutations .
Due to a long repetitive sequence in the AT-rich non-coding region of Drosophila mtDNA , PacBio SMRT was used to sequence the whole mitochondrial genome for three genotypes . SMRT sequencing can generate extraordinary long reads ( >30 , 000 bp ) with extremely high consensus accuracy ( >99 . 999% ) . Thus , the whole AT rich region could be covered in a single read without the trouble of re-assembly . Purified mtDNA ( isolated as described above ) was linearized by restriction cutting at the PstI site and a ∼20-kb band was isolated and gel purified after electrophoresis . About 500 ng of gel purified linear mtDNA sample was used for library preparation using a modified 10 kb Template Preparations Protocol ( PacBio , University of Washington , Seattle ) . Basically , the blunt hairpin SMRTBell adaptors were ligated to the repaired ends of the double-strand DNA fragments . Failed ligation products were removed by adding ExoIII and ExoVII exonucleases . The attached templates were further purified with 0 . 5× and 0 . 45× bead-washes for sequencing . We conducted the sequencing reactions on a PacBio RSII system using one SMRT cell for each genome . All libraries were sequenced using P4/C2 chemistry . mtDNA samples from mt:ND2del1 + mt:CoIT300I , and the recombinants were run with 120 min movie time and ATP6[1] sample was run with 180 min . Raw reads were analyzed following either HGAP ( for de novo assembly ) and BLASR protocol ( for resequencing ) in SMRT Portal 2 . 2 . The coverage for each samples ranged from 1000× to 35 , 000× . When only the coding region of some genomes were sequenced , two long-range PCR reactions using Expand Long Template PCR system ( Roche ) was performed: mt186–mt7502 , mt6905–mt14797 with the following program: 1 cycle of 93°C for 3 min , 30 cycles of 93°C 15 s , 50°C 30 s , 60°C 8 min , and 1 cycle of 60°C for 10 min . Primers were designed all around the coding region ( Supplementary file 2 ) for sequencing by QuintaraBio ( Albany , CA ) .
Homologous recombination is widespread and catalyzes evolution . Nonetheless , its existence in animal mitochondrial DNA is questioned . We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines . In four experimental settings , recombinant genomes became the sole or dominant genome in the progeny . Thus , selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit . Double-strand breaks enhanced recombination in the germline and revealed somatic recombination . When the recombination partner was a diverged Drosophila melanogaster genome or a genome from a different species such as Drosophila yakuba , sequencing revealed long continuous stretches of exchange . In addition , the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome . Thus , recombination can be harnessed to dissect function and evolution of mitochondrial genome .
Animals store the main part of their DNA—including all of the genes that are required to build and maintain an individual—inside their cells in a structure called the nucleus . Most of the information stored in the DNA is stored in duplicate , with one copy inherited from the individual's mother via the egg , and the other from the father via the sperm . This duplicate storage allows a very important damage repair process to occur , where undamaged sequences in one copy can be used to repair damage in the other . This process of homologous repair uses mechanisms that are also used in another important genetic process . When sperm and egg cells are formed , the parental DNA goes through a process called homologous recombination , in which DNA molecules are cut and reassembled into new arrangements . This recombination process ‘shuffles’ genetic combinations , making every individual unique—a process of great evolutionary importance that allows natural selection to act on distinct traits . The structures inside cells that generate energy—called mitochondria—also contain DNA , which is inherited only from mothers . Little is known about whether recombination is possible in the mitochondrial DNA of animals . Ma and O'Farrell used genetics techniques to investigate recombination in the mitochondria of fruit flies . One experiment tracked how a mutation that makes flies less healthy at high temperatures spread as flies were bred for several generations . When the mutation was associated with a mitochondrial genome that had a strong drive towards replication , the mutation became more widespread over time , and in most cases , this eventually resulted in the mutation killing the flies . In rare cases , however , a few flies survived , giving rise to a healthy population . Molecular analyses revealed that , in these survivors , the defective genome had recombined with the other mitochondrial DNA to produce a new genome that lacked the mutation but retained the high replicative drive . This new recombinant genome worked normally and was able to resist the spread of the defective genomes . In addition , by artificially cutting mitochondrial DNA , Ma and O'Farrell show that such ‘double-strand breaks’ lead to recombination , signaling a role for homologous repair in the repair of damaged , in this case broken , DNA . Recombination is also possible between the mitochondrial DNA of two different fruit fly species , and this recombination process can assemble long stretches of DNA . Now that the recombination of animal mitochondrial DNA is known to be possible , future work will be required to understand how it works and how it affects evolution .