microsatellites or 
 ssrs ( simple sequence repeats ) are 
 sequences in which one or few bases are tandemly repeated for varying numbers of 
 times . variations in ssr regions originate mostly from errors during the replication process , frequently dna 
 polymerase slippage , generating insertion or deletion of base pairs , resulting , 
 respectively , in larger or smaller regions [ 2 , 3 ] . ssr assessments in the human 
 genome have shown that many diseases are caused by mutation in these sequences . ssrs can be found in different regions of genes , that is , coding sequences , untranslated 
 sequences ( 5-utr and 3-utr ) , and introns , where the expansions and/or 
 contractions can lead to gene gain or loss of function . also , there are 
 evidences that genomic distribution of ssrs is related to chromatin 
 organization , recombination , and dna repair . ssrs are found throughout the 
 genome , in both protein - coding and noncoding regions . genome fractions as low 
 as 0.85% ( arabidopsis thaliana ) , 0.37% 
 ( zea mays ) , 0.21% ( caenorhabtis elegans ) , 0.30% ( sacharomyces cerevisae ) and as high as 3.0% 
 ( homo sapiens ) and 3.21% ( fugu rubripes ) have been found . some 
 bias for defined genomic locations has also been reported [ 6 , 7 ] . this class of 
 markers is broadly applied in genetics and plant breeding , due to its 
 reproducibility , multiallelic , codominant nature , and genomic abundance . its use 
 for integrating genetic maps , physical mapping , and anchoring gives geneticists 
 and plant breeders a pathway to link genotype and phenotype variations . the protocols for 
 isolating ssr loci for a new species were always very labor - intensive . currently , 
 with the accumulation of biological data originating from whole genome sequence 
 initiatives , the use of bioinformatics tools helps to maximize the 
 identification of these sequences and consequently , the efficiency in the 
 number of generated markers . the first in silico studies of ssrs were developed 
 using fasta   and blast   packages . later , more specific algorithms , such as sputinick , 
 repeatmasker , trf - tandem repeat find , troll , misa   and 
 ssrit ( simple sequence repeat tool ) , 
 were obtained . ssr detection is generally followed by the use of another program for primer design , to be 
 anchored on flanking sequences . also , in some applications , a third step using e - pcr 
  is added , with the goal of 
 verifying primer redundancy . building such a pipeline can be a very difficult task 
 for research groups not familiar with programming tools . in the present work , the 
 application integrates the following functions : ( i ) detection and 
 characterization of ssrs and minisatellite motifs between 1 and 10 base pairs ; ( ii ) 
 primer design for each locus found ;   ( iii ) 
 simulation of pcr ( polymerase chain reaction ) , amplifying fragments 
 with different primer pairs from a given set of fasta files ; ( iv ) global alignment 
 between amplicons generated by the same primer pair ; and ( v ) estimation of global 
 alignment scores and identities between amplicons , generating information on 
 primer specificity and redundancy . the algorithms used for the searches , alignment , and homology estimates are described separately . the algorithm used 
 for perfect and imperfect micro-/minisatellite searches was written in perl and 
 consists of the generation of a matrix that mixes a(adenine ) , t(thymine ) , 
 c(cytosine ) , and g(guanine ) in all possible composite arrangements between 1 
 and 10 nucleotides . the script instructions perform readings on fasta files , 
 searching all possible arrangements in each database sequence . several instructions in the algorithm used in ssrlocator resemble those from misa 
 and ssrit . however , additional instructions have been inserted in ssrlocator 's code . instead of allowing the overlap of a few nucleotides 
 when two ssrs are adjacent to each other and one of them is shorter than the 
 minimum size for a given class as found in misa and ssrit , a module written in delphi language records the data and eliminates such 
 overlaps . the ssr locator 
 software contains windows focused on the selection and configuration of ssr and 
 minisatellite types ( mono- to 10-mers ) and a 
 minimum number of repeats for each one of the selected types . the algorithm 
 calls a perfect repeat when one locus is present with 
 adjacent loci at an up or downstream distance higher than 100 bp . the algorithm calls 
 an imperfect repeat when the same motif is present on both sides of a fragment containing 
 up to 5 base pairs . the algorithm 
 identifies a composite locus when two or more adjacent 
 loci were found at distances between 6 and 100 bp . in this study , only these repeats 
 have been described as the most efficient loci for use as molecular markers 
 . the software ssrlocator was configured to locate a minimum of 20 bp ssrs : 
 monomers(x20 ) , 2-mers(x10 ) , 3-mers(x7 ) , 4-mers(x5 ) , 5-mers(x4 ) , 6-mers(x4 ) , and 
 minissatellites : 7-mers(x3 ) , 8-mers(x3 ) , 9-mers(x3 ) , and 10-mers(x3 ) . in order to 
 validate the efficiency of ssrlocator in finding ssrs and minisatellites , the 
 same database was analyzed withmisa 
 and ssrit , using the same parameters for minimum number of repeats . an algorithm written in delphi language performs calls to primer3 , which execute primer designs . these 
 results are fed to a module that performs virtual - pcrs and allocates individual 
 identification , forward and reverse primer sequences , and a sequence fragment 
 corresponding to the region flanked by the primers ( original amplicon ) to each 
 ssr locus . a window allows the selection of primer3 parameters , such as range 
 of primer and amplicon sizes , as well as optimum primer size , ranges of melting temperature 
 ( tm ) ( minimum , maximum , and optimum ) and gc content ( minimum and optimum ) . for 
 primer searches , the software automatically looks for five base pair distances from 
 both ssr ( 5 and 3 ) 
 flanking sites .   in this study , the 
 following parameters were used : amplicon size 
 between 100 and 280 bp ; minimum , optimum , and maximum annealing temperature 
 ( tm ) of 45 , 50 , and 55 , respectively , minimum , optimum , and maximum primer size 
 of 15 , 20 , and 25 bp , respectively . the algorithm consists in reading the file generated by the previous module 
 ( ssr locus , forward and reverse primers , and original amplicon ) , followed by a 
 search of sequences containing primer annealing sites . when annealing sites are found for the two primers , the flanked 
 region and the primer sequences are copied to a new variable called  paralog amplicon .  for the global alignment between paralog and original amplicon sequences 
 and score calculations ( match , mismatch , gaps ) , a routine was written in delphi language using 
 the algorithms of needleman and wunsch ( 1970 )   and smith and waterman 
 ( 1981 ) . also , in the same module , amplicon identities were calculated 
 according to waterman ( 1994 )   and vingron and waterman ( 1994 ) . the strategy of creating a two - language 
 hybrid program was established as a function of : ( i ) the higher speed achieved 
 by handling large text files with perl as compared to delphi , and ( ii ) the better 
 fitness of perl for generating combinatory strings to be located . the perl 
 module was transformed into an executable file , making unnecessary to install perl libraries during program installing . the graphic interface built , integrating input and output windows to the 
 windows operational system , was obtained using the suite turbo delphi , where a 
 menu system executes calls for each of the previously described modules . nipponbare ) nonredundant full length nonredundant 
 cdna 
 sequences , sequenced by the rice full - length cdna consortium , mapped on the databases derived from the sequencing of japonica ( japonica draft 
 genome , bac / pac clones irgsp ) and indica ( indica draft genome ) subspecies   were used for the 
 analyses . these sequences are deposited in ncbi as two groups , the 
 first comprising accesses from ak058203 to ak074028 , and 
 the second comprising accesses from ak98843 to ak111488 . all these sequences 
 can be also found in kome ( knowledge - based oryza molecular biological 
 encyclopedia ) . a flow chart representing the different steps performed by the software is shown in figure 1 . a total of 3907 micro- and minisatellites were detected by ssrlocator in the 28 469 
 analyzed cdna sequences . the same database searched with misa and ssrit presented 
 3913 and 3917 loci , respectively . the mono- , 4-mer , 6-mer , 7-mer , 8-mer , 9-mer , 
 and 10-mer repeats were identical for the three programs . in the case of 2-mer repeats , 594 elements were detected by ssrlocator and 596 elements were detected by misa 
 and ssrit . 3-mer repeats were differently scored by ssrlocator ( 1990 ) and the 
 other two ( 1994 ) algorithms . for 5-mer repeats , ssrlocator and misa found the 
 same number of repeats ( 426 ) , while ssrit ( 430 ) found a different value . the results obtained with ssrlocator 
 indicate that out of 28 469 cdna sequences , 3765 ( 13.22% ) presented one or 
 more micro-/minisatellite loci . in other studies , microsatellites were found in 
 the following proportions in ests : 3% in arabidopsis   , 4% in rosaceae , 8.11% in barley , 2.9% in sugarcane , and values ranging between 611%   and 1.54.7% 
   for cereals in general ( maize , 
 barley , rye , sorghum , rice , and wheat ) . considering the 3765 fl - cdna 
 sequences , in 3632 ( 92.96% ) only a single micro-/minisatellitelocus was detected . in 125 sequences , two loci were detected , in seven 
 sequences three lociandonly one sequence had four loci , adding 
 up to 3907 occurrences . among the types analyzed , ssrs ( mono to 6-mer repeats ) 
 and minisatellites ( 7- to 10-mer repeats ) comprised 96.98% and 4.12% of 
 detected loci , respectively . the distribution of occurrences detected by ssrlocator was consisted of 138 monomers , 
 594 2-mers , 1990 3-mers , 251 4-mers , 426 5-mers , 390 6-mers , 82 7-mers ,   6 8-mers , 25 9-mers , and 5 10-mers , corresponding to rates of 3.53% , 15.20% , 50.93% , 
 6.42% , 10.90% , 9.98% , 2.10% , 0.15% , 0.64% , and 0.13% , respectively ( see table 1 ) . for the 
 remaining ssrs , average percentage values have been reported as between 17 and 40% for 2-mer , 
 5478% for 3-mer , 2.66.6% for 4-mer , 0.41.3% for 5-mer , and less than 1% for 6-mer 
 repeats   and 26.5% 
 for 2-mer , 65.4% 3-mer , 6.8% 4-mer , 0.77% 5-mer , and 0.45% for 6-mer repeats   for barley , maize , wheat , sorghum , rye , and rice , respectively . 
 in nonredundant transcripts from the tigr database , 15.6% 2-mer , 61.6% 3-mer , 
 8.5% 4-mer , and 14.4% 5-mer repeats were found in rice . the frequency of micro / minisatellite 
 locus occurrence for each million nucleotides ( loci / mb )   in this study was 2.94 , 
 12.64 , 42.34 , 5.34 , 9.06 , 8.30 , 1.74 , 0.13 , 0.53 , and 0.11 for mono to 10-mer 
 repeats / mb , respectively . overall occurrences of 83.13 loci / mb were found ( see table 1 ) . in other studies , different taxa were described in analyses of est databases , 
 such as 133 loci / mb ( barley ) , 161 loci / mb ( wheat , sorghum and rye ) , and 256 loci / mb 
 for rice . also , for nonredundant ests in rice , sorghum , barley , wheat , and 
 arabidopsis , frequencies of 277 , 169 , 112 , 94 and 133 loci / mb were found , 
 respectively . frequencies closer to those found in this study were 
 described for cds regions of rosaceaespecies , with an average of 40.978 loci / mb for 
 rose , almond and peach , while 39 loci / mb were found for arabidopsis . on table 2 , the contents and percentage values for different micro-/minisatellite motifs 
 are shown . for monomer , 2-mer and 3-mer repeats , all possible arrangements are 
 shown , while for 4-mer to 10-mer repeats , only the ten most frequent motifs are 
 shown . the a / t monomer 
 repeats were found in 125 loci , with 111 ( 88.80% ) and 14 ( 11.20% ) loci formed 
 by a and t nucleotides , respectively . the c / g motifs were found in 13 loci , 
 with ten ( 76.92% ) and three ( 23.08% ) loci formed by c and g , respectively . motifs 
 ag / ct and ga / tc were the most frequent and added up to 8.52% of 2-mer ssrs , and 
 6.89% and 5.96% of all 3907 detected occurrences . the motifs ct , ga , and tc were 
 the most abundant adding up to 172 , 143 , and 90 loci , respectively . in maize , 
 barley , rice , sorghum , and wheat ests , the motif ag was described as the most 
 frequent [ 6 , 16 , 28 , 29 , 31 , 32 ] . however , in some studies , the most frequent 
 motif was ga [ 30 , 33 ] . repeats composed by guanine and cytosine were the most 
 abundant among trimers , with occurrences of 18.44% , 17.89% , and 10.60% , 
 respectively , for the motifs ccg / cgg , cgc / gcg , and gcc / ggc , adding up to 23.9% of 
 the overall frequencies of micro-/minisatellites in the analysis . the motifs 
 cgc , ccg , and cgg were the most frequent comprising 218 , 197 , and 170 loci , 
 respectively . many reports indicate the 3-mer ccg as the most frequent in 
 maize , barley , wheat , sorghum and rye [ 6 , 16 , 28 , 32 ] , sugarcane   and rice [ 29 , 31 ] . among 4-mers , 100 different 
 arrangements were found , where the motifs gatc ( 7.17% ) , atta / aat ( 6.77% ) , and 
 atcg / cgat ( 5.98% ) were the most frequent . these motifs add up to 19.92% of 4-mer 
 repeats found and represent 1.28% of the overall content of micro-/minisatellites . 
 in barley ests , acgt was reported as the most abundant motif [ 16 , 28 ] . for 
 other species , aaag / cttt and aagg / cctt in lolium 
 perene , aaag / cttt and aaac / gttt in arabidopsis utrs [ 6 , 35 ] , and aaat and 
 aaag in citrus [ 36 , 37 ] were described as most abundant . among 5-mers , 188 
 different arrangements were detected and the most frequent were ctcct , ctctc , and 
 cctcc with 17 , 17 , and 12 occurrences , respectively . in the analysis of cds 
 regions , the acccg motif was the most frequent in arabidopsis , aaaag in s. cerevisae , c. elegans , 
 and aaaac in different primates . also , the motifs 
 aaaat , aaaac , and aaaag were described as the most frequent in eukaryotes . 
 in rice , repeats of 
 type 6-mer were detected in 230 different arrangements , where cgcctc and tcgccg 
 were the most frequent , occurring in 12 and 10 loci , respectively . other 
 studies have shown higher frequencies for the motifs aagatg , aaaaat in arabidopsis 
 , aaaaag in citrus , aacacg in s. cerevisae , accagg in c. elegans and ccccgg in primates . for 
 all remaining repeats ( minisatellites ) , the occurrences are widely distributed 
 with low - percentage values for each arrangement . for 7-mer , 8-mer , 9-mer , and 10-mer 
 repeats , the totals of occurrences were 57 , 5 , 23 , and 5 , respectively . the design of 
 primers for the 3907 detected micro-/minisatellites resulted in 3329 primer 
 pairs , covering 85.20% of loci . the running of  virtual pcr  generated a total of 
 4610 amplicons . a module in ssrlocator checks for primer redundancy . a total 
 of 2397 primer pairs amplified only the fragment from its original locus ( specific 
 amplicons ) and 932 pairs amplified one or more regions besides the original 
 locus . from these , 692 pairs amplified two fragments , one from the original 
 site and a second from another region ( paralogous ) . in this case , 692 specific amplicons 
 plus 692 redundant amplicons , were detected . a total of 143 , 90 , 2 , and 5 primer 
 pairs generated three ( two redundancies ) , four ( three redundancies ) , five ( four 
 redundancies ) , and six ( five redundancies ) fragments , respectively . the final 
 product of 932 primers with more than one anchoring region resulted in 932 specific 
 amplicons and 1281 redundant amplicons , adding up to 2213 fragments . to investigate the ability of these 
 primers in amplifying genomic sequences , an extra experiment was performed 
 against the whole rice genomic sequence available at ncbi . the different groups 
 of redundant and nonredundant primer sets , that is , amplifying one , two , three , 
 or more times in the cdna database , were tested against the genomic sequence . 
 from the 2397 nonredundant primers , only 924 amplified a locus in the genomic 
 sequence . this difference was already expected because of difficulties in 
 amplifying genomic regions , that is , if some primers anneal to a boundary 
 region between two exons in the cdna , the presence of introns would make this 
 annealing site no more available . it is interesting to note that from the 924 
 amplicons detected , 914 ( 99% ) did amplify only one locus in the genomic region , 
 agreeing with the cdna results . when the primer sets that amplified two 
 different cdnas were run against the genomic sequence , only 294/692 ( 42.5% ) did 
 amplify , having 14.5% 
 been able to amplify two different loci . these results indicate that ssr 
 locator performance was consistent between the two databases regarding the 
 nonredundant loci , that is , from those loci that were able to be amplified in 
 both databases , their status of nonredundant was maintained . the changes 
 observed for the redundant loci can be attributable to many causes , including 
 redundancy in the cdna database , but also to biological reasons due to primer 
 positioning . results of a global 
 alignment between amplicons from original and redundant sites are shown in table 3 . among the 1281 redundant amplifications , 787 ( 61.44% ) resulted in a perfect 
 alignment between both loci ( identity equal to 100 ) . for redundant amplicons 
 with identity levels of 9699% , and 9095% , 452 
 ( 35.28% ) and 8 ( 0.62% ) loci were found , respectively . the fact that such a high 
 percentage of redundant loci show high identity is probably a consequence of 
 the genome fraction chosen , that is , expressed sequences . this fraction is 
 under tight selection pressure and should not accumulate variations such as 
 substitutions or indels at a high rate . as expected , comparisons to whole 
 genome , generated a great deal of polymorphism , due to the inclusion of 
 intronic regions in the alignments ( data not shown ) . the software ssrlocator was successfully implemented , adding steps for ( 1 ) ssr 
 discovery , ( 2 ) primer design , and ( 3 ) pcr simulation between the primers obtained 
 from original sequences and other fasta files . also , the software produces 
 reports for frequency of occurrence , nucleotide arrangement , primer lists with 
 all standard information needed for pcr and global alignments . from the pcr 
 simulation , it was possible to point out which primer pairs were nonredundant , 
 suggesting that these primers are more appropriate for mapping purposes . in 
 this case , however , wet lab experiments should be performed to confirm the 
 advantage of nonredundant over redundant primers for mapping . it is possible that the results for micro-/minisatellite frequencies ( loci / mb ) obtained 
 in this study diverge from the results found in the literature . this can be 
 explained by the different databases used ( redundant ests , nonredundant ests and/or 
 fl - cdna ) , different algorithm configurations and minimum requirements set for 
 counting motifs . the results showed that 932 ( 27.99% ) primers presented 
 amplifications in more than one gene sequence . this could be mostly due to the 
 fact that primer pairs derived from a specific gene ( cdna ) anchored in similar 
 sites in other duplicated genes , since 5,607/28,469 ( 19.70% ) genes were described as paralogs in the 
 annotation of the database used . gene duplication along with polyploidy 
 and transposon amplification are the major driving forces in genome evolution . also , a 
 second possibility is that some primers were generated from protein domain 
 regions within the analyzed cdnas . these domains could be found in protein 
 families with many genome copies , resulting in the observed redundancies . a 
 validation of the redundancies of cdna results was obtained through a virtual - pcr against the whole 
 rice genome sequence . from the nonredundant primers that generated an amplicon , 
 ca . finally , 
 this tool can be used successfully for data mining strategies to find ssr 
 primers in genomic or expressed sequences ( ests / cdnas ) . also , this software can 
 be a tool for microsatellite discovery in databanks of related species , 
 anchoring primers in ortholog or paralog regions contained between databases 
 from two different species .
microsatellites or ssrs ( simple sequence repeats ) are ubiquitous short tandem duplications occurring in eukaryotic organisms . 
 these sequences are among the best marker technologies applied in plant genetics and breeding . 
 the abundant genomic , bac , and est sequences available in databases allow the survey regarding presence and location of ssr loci . 
 additional information concerning primer sequences is also the target of plant geneticists and breeders . in this paper , we describe a utility that integrates ssr searches , frequency of occurrence of motifs and arrangements , primer design , and pcr simulation against other databases . 
 this simulation allows the performance of global alignments and identity and homology searches between different amplified sequences , that is , amplicons . in order to validate the tool functions , 
 ssr discovery searches were performed in a database containing 28 469 nonredundant rice cdna sequences .