Patent Application: US-71482003-A

Abstract:
described is a method for analysing dna of a sweet potato , characterised in by the following steps : providing dna of a sweet potato , physically breaking said dna into dna pieces , introducing known sequences at at least one of the two ends of each dna piece , providing at least two primers , a first primer according to the formula n agtcctaacan 1 n 2 n 3 wherein n x is selected from a , c , g and t ; n is 0 to 20 ; n 1 is g , t , a or not present ; n 2 is a , c , g or not present ; n 3 is a , c , g or not present ; or a complementary sequence thereto ; and a second primer being able to anneal to the introduced sequence , amplifying dna of the dna pieces with said primers and analysing said amplified dna .

Description:
str187 retrotransposon sequence was found and cloned with a known method ( pearce et al . 1999 ). after having sequenced the str187 clones ( see seq . id . no . 1 ) oligonucleotide primer sequences have been designed capable in different methods to fingerprint and distinguish sweet potato genomes . during the procedure as outlined in the present examples two types of primers are used : the ltr primer or first primers are designed after the retrotransposon sequence optionally with features preventing amplification of 3 ′ ltr . the other primers or second primers may be any sequence which makes an adapter to the restriction site used , including a primer site . this adapter primer should match the pcr parameters of the first primer . both primers may be extended on the 3 ′ end with preferably 1 - 3 optional nucleotides . in the method according to the present examples the primers are used in pcr reactions with sweet potato dna templates . the nucleic acid polymerase used in the reactions is a commercially available thermostable dna polymerase from the thermophilic bacterium thermus aquaticus ( taq polymerase ) or other thermostable polymerases . the nucleotide triphosphate substrates are employed as described in pcr protocols , a guide to methods and applications , m . a . innis et al . 1989 and u . s . pat . nos . 4 , 683 , 195 and 4 , 683 , 204 . the substrates can be modified for a variety of experimental purposes in ways known to those skilled in the art . in the first step ( 1 .) of the present process sweet potato genomic dna as template dna is fragmented with sequence specific restriction endonucleases . it is possible to use one , two or even three different restriction endonucleases . fragmented genomic dna is ligated with restriction size compatible adapter sequences with designed adapter specific primer binding sites . one or more pcr reactions are performed with adapter specific and ltr specific primers . both primers can be extended with extra nucleotides to reduce the number of the amplified fragments . in the last pcr reaction the ltr primers are labelled so that the ltr - adapter primer amplified pcr product is distinguishable from the adapter - adapter primers . such labelling may be performed by any method known in the art . preferably , labelling by isotopes or non - isotopic methods such as biotinglation , fluorescent dyes or other methods . pcr - products may be separated by agarose or acryl amide gel - electrophoresis , manual or automatic , and visualised depending on the labelling of the ( ltr ) primer . similar procedures have been presented from waugh et al . ( 1997 ), ellis et al . ( 1998 ) and pearce et al . ( 2000 ). the electrophoresis of the amplified genomic fragments with the same flanking ltr sequence separates the different length of fragments according to the mobility . smaller fragments have higher mobility as the longer ones . different sweet potato samples turned out to have different electrophoresis pattern in consequence with the place and number of the retrotransposon insertions . automated gel - electrophoresis systems ( sequencer equipment , genotyper programme ) can compare more than hundred fragments of different length , but it is of course also possible to evaluate the result with manual methods . conversion of these electrophoresis patterns to a presence / absence ( yes / no ) per variety matrix is possible with genotyper or genographer programmes mentioned above or can of course be done manually . a clustering analysis of this matrix is possible using such methods as unweighted pair group method using arithmetic averages ( upgma ) ( sneath and sokal , 1973 ) or neighbour joining ( saitou and nei , 1987 ) with programmes such as treecon . other ordination analysis are also possible such as multidimension scaling ( mds ) or principal component analysis ( pco ) with programmes such as spss , systat , statistika or sas . further it is possible for the analysis of the geographical origin of the tested sweet potato sample to compare the number of the retrotransposon insertions in the related genotypes . plants growing on the same area are liable to the same stress effect which could induce among others retrotransposon activation further new insertions . lyophilised leave samples were used for all the analysis . sixty seven landraces were obtained from the kenya agricultural research institute gene banks at the university of nairobi , field station , kabete 59 landraces were obtained from the ugandan national agricultural research institute , namulongeand . forty four landraces were obtained from the tanzania agricultural research institute , tengeru . individual pathogen tested clones from columbia , peru , mexico , brasil and papua new guinea were obtained from the international potato centre germplasm collection at kabete , kenya . from this total sample 9 genotypes from different countries were selected for primer comparisons and for comparison of the s - sap system with other molecular markers ( aflps and rapds ). details of these genotypes are given in table 1 . names , country of origin and type of genotype of the nine selected varieties used for testing primer combinations and for comparing s - sap molecular markers with rapd and aflp markers all the kari ( kenya agricultural research institute ) and cip ( international potato centre ) germplasm was sampled from field collections . for most of the ugandan and tanzania germplasm , vine cuttings were sampled from the field collection and planted in pots in a green house . four weeks later , fresh leaves were sampled for freeze drying . in all cases , 5 - 7 very young leaves were cut from vigorously growing plants , immediately dipped in liquid nitrogen . freeze dried leaves were stored at 4 ° c . until dna was isolated . about 20 mg of freeze dried plant material in liquid nitrogen was ground in a bead mill for 5 minutes . total dna was isolated and purified with a ‘ dneasy plant minikit ’ ( qiagen ) following the original protocol . after extraction , 4 μl of 10 mg / ml rnase a was added and the sample incubated at 37 ° c . for one hour . dna was quantified with a ‘ tko 100 ’ mini - fluorimeter ( hoefer scientific instruments ) and quality assessed on a 0 . 8 % agarose gel stained with 0 . 5 μg / μl ethidium bromide in a 1 × tbe buffer . msei or ecori restriction enzyme digested genomic dna was amplified with degenerate rnaseh gene specific and enzyme cutting site specific flanked pcr primers as written by pearce et al . 1999 . separation of the biotinilated first pcr products was made on streptavidin coated magnetic dynabeads particles . 5 ′ biotinilated rnaseh primer : 5 ′ mgnacnaarcayathga ( seq id . no : 16 ) nested rnaseh primer : 5 ′ gcngayatnytnacnaa ( seq id . no : 17 the degenerated rnaseh primers are kindly gift from the laboratory of aj flavell ( department of biochemistry , univ . dundee ) and were designed by sequence homologies of known retrotransposon origin rnaseh genes . the amplified fragments were cloned into topo 4 ta cloning vector ( topo ta cloning kit , clontech k4575 - 01 ) and sequenced . approximately one hundred clones with variable degree of homology to ty1 - copia rnaseh gene were identified but only three ( str6 , str85 , str187 ) showed the characteristic rnaseh gene , stop codon , polypurine track and putative 3 ′ ltr sequence elements ( fig2 ). the str6 and str187 sequences proved to be homologue with the ty1 - copia retrotransposons . the str85 clone was not recognised by blast search as copia type retrotransposon sequence despite the copia homologue primer site in the rnaseh similar sequence and the polypurine track region . the putative inverted repeat region ( ir ) of the ltr region is different in the three sweet potato sequences , only the str187 clone contains the characteristic tgtt sequences . although with lower frequencies other ir sequences occur ( picea abies tpa8 tagtt ) it is believed already as a mutation . furthermore , in the putative ltr region of the str6 clone , after the tatt inverted repeat sequence a 34 bp long direct repeat was recognised which provided another proof for the unusually high mutation rate in the sweet potato retrotransposon population . the starting point of the 3 ′ ltr sequences for the rest of the sequenced clones could not be determined , since they did not contain a recognisable polypurine - track after the rnaseh gene stop codon . in many cases the sequence was interrupted with the msei restriction cutting site , however using the rare cutting ecori enzyme to fragment the genomic dna longer clones have been got , but the identification of the ltr sequence was further not possible . the ltr sequence detected in the str6 and str187 clones proved to be functional in the s - sap analysis while str85 did not produce an amplified polymorphic banding pattern . fig2 shows the list of the ltr and eco adapter primers tested in s - sap reactions . sweet potato dna sequences were isolated with degenerate oligonucleotide primers corresponding to conserved domains of the ty1 - copia retrotransposon rnaseh gene fragment and flanked adapter primers . the amplified clones were cloned as written in methods . 2 - 300 random clones have been sequenced but only three clones with recognisable ltr sequences were found . in these three clones the stop codon of the rnaseh genes , the characteristic polypurine tracks and the putative 3 ′ ltr regions could be distinguished . every retrotransposon class has a different ltr region , which is homologue in the class , but not between classes . the fact that only two working ltr region were found between more hundred sequenced clones one can suppose , that in the sweet potato the mutation rate of the retrotransposons are very high , and also that only few classes of retrotransposon class exist . otherwise it has to be considered , that the sweet potato are propagated mainly vegetatively , which means , that a retrotransposon insertion in the vegetative cells has longer “ life time ” furthermore bigger chance for mutations . it is known that different biotic and abiotic stresses can induce the mobility of the retrotransposon ( mhiri et al ., 1997 ; grandbastien et al ., 1997 ). however plant genomes have evolved mechanisms to repress uncontrolled retrotransposon expansion , such as dna methylation ( liu and wendel 2000 ) deleterious mutations ( nuzhdin 1999 ; heslop - harrison et al . 1997 ), unequal crossing over and / or intrachromosomal recombination between ltrs ( shirasu et al . 2000 ). the high variability of the str187 retrotransposon insertion between different sweet potato clones alludes to the mobility of these retrotransposon . after preliminary experiments the str187 retrotransposon ltr sequences were used to design s - sap primers . increasing the number of the selective nucleotide on the adapter or ltr primers the number of the detected insertions were reduced as expected . however the reduction was much more effective ( 4 - 5 times per nucleotide ) if more selective nucleotides on the ltr primer were increased the best scoring was achieved with only one nucleotide extension on the ltr primer but it has to be considered that only a 6 bp cutting enzyme was used to fragment the genomic dna . waugh et al . fragmented the barley genomic dna with a 6 and a 4 bp cutting enzyme accordingly to the aflp procedure , generating more and shorter genomic fragments , but they had to reduce the number of the amplified fragments to a scorable amount with increasing the number of the selective nucleotides . furthermore , they did not use the selective nucleotide on the ltr primer accordingly they amplified not only the plant specific genomic dna but possibly the internal retrotransposon sequences too . the procedure from waugh et al . ( 1997 ) was adopted to sweet potato with some modification . genomic dna was digested only with one rear - cutting enzyme ( ecori ) and ligated with specific adapter in one reaction . two pcr reactions were performed . the first pre - selective pcr amplification was made with dynazyme taq polymerase in 50 μl reactions during 30 cycles on 52 ° c . annealing temperature . ltr specific primers without any extension and the e01 - adapter primer ( table 2 ) were used . second , selective pcr amplification was made with quiagen hot taq dna polymerase in 25 μl reactions . touch down from 70 ° c . (− 0 . 7 ° c ./ cycle ) to 55 ° c . than another 20 cycles at 55 ° c . annealing temperature . with selective nucleotide extended fam labelled transposon primer ( str187g ) was combined with the e01 adapter primer ( table 2 ). reactions were loaded on acrylamide gel and separated on abi 373 automated sequencer . in the original s - sap protocol ( waugh et al .) the genomic dna are cut with two enzymes as it is usual in aflps , a rare cutter and a frequent cutter ( vos et al .). however adapting the s - sap technique for sweet potato digesting the genomic dna with only one rare cutting enzyme instead of two improved the number and length of the polymorphic bands . further improvement was achieved by pre - amplifying the adapted dna with the adapter and non - labelled ltr primers . the second specific amplification was carried out with the adapter primer and selective nucleotide extended ltr specific primer . these modifications resulted in a high number of amplified products both polymorphic and monomorphic . in preliminary experiments the three sweet potato ltr primers were tested in s - sap analysis and the str187 showed the highest level of polymorphism . the str6 primer produced a moderate number of polymorphic patterns , but no amplification products were obtained with str85 . nine sweet potato varieties were selected from africa , south and central america and papua new guinea and tested with the different ltr / adapter primer combinations . table 3 shows the results of these comparisons . table 3 : comparison of the different primer combinations in s - sap analysis of nine sweet potato varieties . frequencies ( freq .) means that the tested str187 retrotransposon has insertion into one , two or all of the nine genome . column n represents the total number of the insertions , which are present in the nine genome one , two or nine times . dates are shown also in percentage . in the row insertion ( ins .) are shown the total number of the insertions amplified with the given primer pair . the e44 adapter primer in combination with the str187gc or g primers gave 36 and 173 polymorphic bands respectively , representing individual retrotransposon insertions . reducing the number of the selective nucleotide on the ltr primer significantly elevate the number of the amplified insertions . the same relation was observed in case of the e01 / 187gc and e01 / 187g primers . reducing the selective nucleotide with one , the number of the amplified insertions elevated from 51 to 261 . the number of the selective nucleotide on the adapter specific primer has only a minor effect on the insertion amplification . in table 3 there are presented the frequencies of the insertions amplified from only one , two or even all of the nine plan genomes . it can be seen that the polymorphism is very high ; the percentage of the monomorph bands comparing with the total number of the insertions is only 1 - 2 %. however the number of the unique insertions — amplified from only one plant genome — is very high 33 - 69 % of the total insertions . a phylogenetic analysis of the nine sweet potato varieties with the e01 - str187 / g and e44 - str187 / g primer combinations are shown in fig5 . both primer combinations distinguish the south american varieties from the african ones . the clones from mexico and papua new guinea were associated to the african types . with the two other primer combinations where the ltr primer is extended with two nucleotides , the south american and african varieties were not differentiated from each other ( data not shown ). the aflp methodology was essentially as described by vos et al . ( 1995 ) but adapted for sweet potato with fluorescent labelling and sequencer running of the gel . two restriction enzymes , msei and ecori were used to fragment the genomic dna . the restriction - digested dna was subsequently ligated to two different synthesised double - stranded oligonucleotides that consists of a short dna strand and the restriction enzyme recognition site ( table 4 ). pre - amplification was done using primers e01 and m01 . an annealing temperature of 60 ° c . was used for 45 cycles . selective amplification of the pcr products of the pre - amplification was done with primers identical to the pre - amplification primers with an additional 2 selective nucleotides at their 3 ′ ends ( table 4 ). ecori selective primers were abi - fam fluorescent labelled to prevent occurrence of ‘ doublets ’ on the gels due to unequal mobility of the two strands of the amplified fragments ( vos et al ., 1995 ). the samples were loaded on a 6 % polyacrylamide denaturing gel and run with an abi prism 373 sequencer for 10 hours . the gel was scanned and samples extracted using genescan 3 . 1 programme . the pcr products of selective amplification were visualised . an internal size standard was incorporated into the sample . visualised peaks indicating position of amplified fragments were analysed with genotyper 2 . 5 programme to develop a 0 / 1 ( absence / presence ) fragment by sample matrix . peak filter conditions were set to include only peaks with scaled height of at least 30 . selection of categories was done as described above for the s - sap procedure . informative products typically fall within 50 - 450 bp , ( sharbel 1999 ). only categories between 50 - 400 bp were utilised for data analysis . rapd amplifications were carried out as described by williams et al . ( 1991 ) with a few modifications as described in gichuki et al ., ( 2001 ). data were analysed with genotyper 2 . 5 programme . peaks , corresponding to an amplified retrotransposon insertion were designated into categories . the tolerance of a category was chosen to be ± 0 . 25 - 0 . 5 bp , which means if two amplified fragments show bigger difference than 0 . 5 or 1 bp , then they were selected as two different categories . data representing insertions in bp were converted with the genotyper programme to a presence / absence ( 1 / 0 ) of insertion per variety matrix for use in other phylogenetic programmes such as treecon . the ty1 - copia transposon based s - sap analysis is a dominant marker system yielding a multiband pattern . each individual band of this pattern represents a unique retrotransposon integration site ( fig3 ). the objective was to test whether a genotyping system based on the consecutive integration of retrotransposon elements results in a similar genetic relatedness of accessions compared to those generated using for rapds and aflps , which are based on the alterations of the dna sequence . therefore nine sweet potato genotypes representing different geographic regions already identified by rapd analysis were analysed by aflp and s - sap techniques respectively ( table 1 ). the banding patterns were compared with upgma dendograms using nei , 1979 genetic distance ( fig4 ). total number of amplification products obtained per analysis type , number that were polymorphic , mean number of products per assay ( primer or primer product ) and overall percentage of polymorphic loci * only distinct bands which demonstrated polymorphism were scored for rapds table 5 shows the details of the three analysis methods . the percentage of the polymorphic loci was the highest in s - sap analysis ( 97 . 7 %) where 260 insertions were amplified with only one primer pair . in the barley genome , a 25 - 30 % increase in the rate of polymorphism has been observed with retrotransposon - based s - sap , as compared to standard aflp ( kumar 1996 ; waugh et al . 1997 ; gong - xin yu and r . p . wise 2000 ). in the present case this ration is smaller , 19 % comparing with the aflp method . although rapds showed a high level of polymorphism only distinct banding patterns which showed polymorphism in an earlier study of 74 genotypes were included ( gichuki et al ., 2001 in paper ). therefore polymorphism of the rapd analysis is over - estimated , therefore it is not comparative with the aflp and s - sap data ( table 5 ). the high polymorphism observed in the three methods may be due to the vegetative propagation of the sweet potato . all the three different genotyping method clearly identified two south american clones zapallo ( peru )/ and camote amarillo ( colombia ) as a separate group ( see fig4 ). the four african clones were also identified as another group . the mexican clone , no . 221 and the papua new guinea , naveto , were in all three cases related to the african clones . the brazilian clone , santo amaro , was related to the south american clones in both the s - sap and the rapd and with the african clones in the aflp analysis . the important factors in choice of a genetic marker includes , development time and cost , capital outlay , amount and quality of dna required , prior knowledge of dna sequence , required technical expertise , robustness , informativeness , genome coverage and reproducibility ( vos et al ., 1995 ; milbourne et al ., 1997 ; milbourne et al ., 1998 ; powell et al ., 1996 ). the s - sap markers require a higher initial cost of development than both rapds and aflps due to the need to isolate the ltr repeat sequence of the retrotransposon . on the other hand the ltr sequence adaptation costs to specific genomes is comparable to that of aflps . the s - sap was demonstrated to be superior to both rapd and aflp in terms of number of amplification products revealed and number of polymorphic loci ( table 5 ). to select the 12 rapd random primers more than 100 primers were screened and only about half produced any amplification products . considering that 12 rapd assay and 2 aflp assays were required to achieve approximately the same level of analysis , it is evident that on per assay basis the s - sap procedure may be the fastest of the three methods for genetic analysis and characterisation of the sweet potato at a comparable cost . compared to fluorescent aflps it was found that the s - sap peaks were more distinct . though both the aflp and s - sap markers are dominant , the high multiplex ration of the s - saps indicates that they are more informative . aflp and rapd markers target random regions of the genome . however some concerns have been expressed by some writers regarding centrometric - clustering of aflp markers particularly for linkage studies . most aflp primers seem to target the at - rich centromere region of the chromosome . the ty1 - copia retrotransposon is widely distributed throughout the genome ( pearce 1996 , schmidt 1996 , heslop - harrison 1997 ). this would mean that the ty - 1 copia ltr s - sap markers are also widely distributed since they are anchored to the retrotransposon . reproducibility of a marker system is quite important especially for germplasm characterisation , mapping and where results have to be exchanged between different labs and scientists . the aflps have been shown to be more reproducible than rapds ( jones et al ., 1995 ). the sequence - specific nature of the s - sap analysis may improve this reproducibility . preliminary results indicated a high level of reproducibility using different pcr equipments ( data not shown ). considering all these factors it is clear that the ty - 1 copia s - sap marker system is a powerful method for genetic analysis in sweet potato . the usefulness of retrotransposon s - sap markers has already been demonstrated in barley ( waugh et al ., 1997 ) and in peas ( elliot et al .). hundred seventy - one east - african accessions from uganda , tanzania and kenya were analysed using the e01 — 187g primer combination in the s - sap analysis . this primer combination yielded the highest number of polymorphic bands . the pcr amplification and the analysis of the fragments by size were done as described in materials and methods . from different areas of east africa a total of 61 varieties from kenya , 44 from tanzania and 61 from uganda , were selected . kenyan varieties came from the central and western highlands and the nyanza region of the victoria lake basin . from tanzania the varieties came from three areas , the east coast , the north - central highlands and the lake zone . ugandan varieties were grouped into those originating from the north - east ugandan and the rest originating from central and western uganda the geographical areas of origin are shown in the fig6 . in the s - sap analysis of all the samples 242 insertions category of the str187 retrotransposon were found . fig7 present all the varieties in a dendogram based the upgma analysis . to simplify the analysis the samples in accordance with the geographical origin or as a member of a given monophyletic group established by treecon upgma analysis were compared . the 172 varieties were first grouped by geographical origin summarised to the given country part then the analysis result was scored and established a phylogenetic tree ( see fig1 ). the phylogenetic tree shows separation of the east - african sources . east and north tanzania are separated from the lake part of tanzania , which is closely related to the central / west ugandan samples . these results are corresponding to the geographical position . interestingly the northeast ugandan samples are mapped closer to the kenyan one than the central / west ugandan varieties , but taking considering the geographical localisation it is also feasible . although the central kenyan samples grouped together with the other kenyan varieties on the phylogenetic tree it is separated from western and nyanza part of the country . comparing the 172 tested varieties with each other by upgma cluster analysis ten subgroups have been identified . the subgroups are listed in table 6 . this type of analysis shows similar results , but divergence not only between but also in the different country partd to can be observed . the details are shown in table 7 . table z distribution of the varieties in the clustered groups no . of possible groups percentage insertion sites central kenya 1 43 % 203 2 36 % 164 western kenya 7 25 % 162 1 18 % 203 nyanza 7 48 % 162 1 20 % 203 central / western uganda 3 84 % 161 e / ne - uganda 1 30 % 203 2 14 % 164 7 39 % 162 tanzania - east 8 66 % 82 6 28 % 93 tanzania - lake 3 60 % 161 8 30 % tanzania - north 3 100 % the kenyan varieties are grouped mostly into the group 1 , 2 and 7 together with the northeast ugandan ones . for example , 43 % of the central kenyan clones are in the group 1 and 33 % of them in the group 2 . similarly the nyanza clones distributed mainly into the group 7 but with smaller percent also present in the group 1 and 2 . western kenyan samples show the highest diversity , the highest representation is in the group 7 with 23 %, but they can be found also in the group 1 , 2 and 5 . the northeast ugandan clones show similarity with the kenyan one , they are mapped into the group 7 , 1 and 2 , 39 %, 30 % and 14 % respectively . much more conserved the central - western ugandan clones , eighty - four percent of them are in the group 3 together with the three north tanzanian varieties and 60 % of the lake - tanzanian samples . another thirty percent of the lake tanzanian varieties are together with the 66 % of the east tanzanian samples in the group 8 . the rest 28 % of the east tanzanian varieties were separated into the group 6 . analysing the number of the insertions in the different groups an increasing number of possible insertion sites from the coast part of tanzania ( east ) to central kenya has been found . the highest possible insertion number was found in the group 1 ( 203 ). around 16 % of the investigated clones were found in that group , with the highest representation of the central kenyan samples ( 43 %). in the group 2 , 3 , 7 and 9 the number of the possible insertion sites were 164 , 161 , 162 , and around 60 - 90 possible insertion sites and the most predominant are the east tanzanian samples in the group 6 and 8 ( see table 7 and fig9 ). table 7 shows only the most characteristic two groups ( 6 , 7 ), because the others ( 4 , 5 and 10 ) are too small or too diverse ( see also table 6 ). as already mentioned , retrotransposons transpose via an rna intermediate , which means , that the parental insertion remains fixed in the genome . therefore every further insertion must have happened later , meaning a recent change in the genome . continuing this theory the spread of a retrotransposon in the geographical distribution can be followed . in that case one is able to follow the spread of the str187 retrotransposon in space and time . it is supposed that where the number of the insertion of the given retrotransposon is lower there is the starting point of its spread on a given area . following this theory and based on the results about the increasing number of the insertions , it is proposed that the sweet potato in east - africa occurred first in east - tanzania ( insertions 80 - 90 ) and spread further to lake -, north - tanzania , central / western uganda ( ins . 161 ), east / northeast uganda and kenya ( fig1 ), coming round the victoria lake . in kenya and northeast uganda three distribution areas with different insertions rates were found . varieties from central , western and nyanza area of kenya are grouped into the groups 1 , 2 or 7 together with the northeast - ugandan clones , where the number of retrotransposon insertions is 203 , 164 or 162 respectively ( see table 7 .). these results could suggest that in kenya one part of the varieties were exposed to different biotic and abiotic effects , which could induce the retrotransposon expression resulting in new insertions . considering the fact , that the sweet potato was introduced into africa not longer than five hundred years ago and during this time the retrotransposon insertion could increase 2 - 3 times in the african resources it can be supposed that the str187 retrotransposon is a still mobile retrotransposon . boeke j d , and corces v g ( 1989 ) transcription and reverse transcription of retrotransposons . annu rev microbiol 43 : 403 - 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